Research ArticleProcoagulatory State in Inflammatory Bowel DiseasesIs Promoted by Impaired Intestinal Barrier Function

Luca Pastorelli12 Elena Dozio2 Laura Francesca Pisani2 Massimo Boscolo-Anzoletti3

Elena Vianello2 Nadia Munizio1 Luisa Spina1 Gian Eugenio Tontini1 Flora Peyvandi34

Massimiliano Marco Corsi Romanelli25 and Maurizio Vecchi12

1Gastroenterology and Gastrointestinal Endoscopy Unit IRCCS Policlinico San Donato 20097 San Donato Milanese Italy2Department of Biomedical Sciences for Health University of Milan 20131 Milan Italy3Angelo Bianchi Bonomi Hemophilia and Thrombosis Centre IRCCS Carsquo Granda Ospedale Maggiore Policlinico 20122 Milan Italy4Department of Medical and Surgical Pathophysiology and Transplantations University of Milan 20122 Milan Italy5Operative Unit of Laboratory Medicine IRCCS Policlinico San Donato 20097 San Donato Milanese Italy

Correspondence should be addressed to Luca Pastorelli lucapastorellimecom

Received 29 October 2014 Revised 27 January 2015 Accepted 1 February 2015

Academic Editor Gerassimos Mantzaris

Copyright copy 2015 Luca Pastorelli et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Inflammatory and immune mediated disorders are risk factors for arterial and venous thromboembolism Inflammatory boweldiseases (IBD) confer an even greater risk of thromboembolic events than other inflammatory conditions It has been shown thatIBD patients display defective intestinal barrier functions Thus pathogen-associated molecular patterns (PAMPs) coming fromthe intestinal bacterial burden might reach systemic circulation and activate innate immunity receptors on endothelial cells andplatelets promoting a procoagulative state Aim of the study was to test this hypothesis correlating the presence of circulatingPAMPs with the activation of innate immune system and the activation of the coagulatory cascade in IBD patients Specificallywe studied lipopolysaccharide (LPS) Toll-like receptor (TLR) 2 TLR4 and markers of activated coagulation (ie D-Dimer andprothrombin fragment F1+2) in the serum and plasma of IBD patientsWe found that LPS levels are increased in IBD and correlatewith TLR4 concentrations although a mild correlation between LPS and CRP levels was detected clinical disease activity does notappear to influence circulating LPS Instead serum LPS correlates with both D-Dimer and F1 + 2 measurements Taken togetherour data support the role of an impairment of intestinal barrier in triggering the activation of the coagulatory cascade in IBD

1 Introduction

Cardiovascular events including both arterial and venousthromboembolism are a major cause of death and morbidityin Western countries [1] Several cofactors influence the riskof cardiovascular events and among these inflammationseems to play a relevant role [2] In fact systemic and local-ized inflammatory processes can accelerate atherosclerosisand cause a hypercoagulative state [3] As a matter of fact theatherosclerotic plaque is ultimately an inflammatory lesion[4] where activated macrophages sustain the damage [5]and the inflammatory and coagulative molecular cascadesare strictly linked and share several common mediators [3]As such it is not surprising that a wealth of epidemiologic

data demonstrates an increased prevalence of thromboem-bolic complications in inflammatory and immune-mediateddisorders [6] Remarkably it has been shown that amongmany inflammatory andor immune-mediated conditionsinflammatory bowel diseases (IBD) namely ulcerative colitis(UC) and Crohnrsquos disease (CD) confer the most prominentrisk of thromboembolism [7]

IBD are chronic and relapsing inflammatory diseases ofthe gut and their exact etiology is still obscure [8] howeverseveral key events in the pathogenesis of IBD are wellrecognized and studied It is commonly accepted that theonset of IBD is linked to the presence of an altered intestinalpermeability [9] a reduced mucosal innate immunity func-tion [9] and an exaggerated adaptive immune response [10]

Hindawi Publishing CorporationGastroenterology Research and PracticeVolume 2015 Article ID 189341 10 pageshttpdxdoiorg1011552015189341

2 Gastroenterology Research and Practice

The increased incidence of thromboembolic events inIBD patients is likely to be multifactorial and the inflam-matory process may have itself a role together with vitamindeficiencies (ie vitamin B12 vitamin B6 and folate) andother acquired factors [11ndash13]Moreover a defective intestinalmucosa barrier function may as well contribute to theprocoagulative state specifically in IBD patients In facta leaky epithelial layer andor a defective innate immuneresponse impair gut capability to control intestinal bacterialburden allowing bacterial components to penetrate intothe intestinal mucosa [9] reach the systemic circulationand come into contact with endothelial cells and plateletsThose bacterial components such as lipopolysaccharide(LPS) may work as pathogen-associated molecular patterns(PAMPs) that is molecules associated with different groupsof microorganisms and after being recognized by cells theyactivate a prompt innate immune response [14] In factimmune and nonimmune cells present on their membranespecific receptors deputed to recognize PAMPs such as Toll-like receptors (TLRs) [14] Apart from membrane-boundTLRs soluble TLRs were also described These moleculescan be found in body fluids upon bacterial sensing [15ndash17]and acting as decoy receptors for PAMPs they negativelyregulatemembrane-boundTLR activation in order to preventexaggerated innate immune activation [18ndash20] Interestinglysoluble TLRs have been proposed as potential biomarkers forseveral inflammatoryinfectious conditions including IBD[21ndash26]

Both endothelial cells and platelets possess innate immu-nity receptors such as TLR2 and TLR4 [27ndash29] that afterbeing activated by PAMPs promote cell activation and thesubsequent release of different procoagulative molecules allof them triggering the molecular pathways of coagulation

In the present paper we evaluated the potential linkbetween decreased intestinal barrier function and activationof coagulation in IBD To this aimwe explored the correlationbetween circulating LPS soluble TLR2 and TLR4 serumlevels and sensitive markers of activated coagulation (ieD-Dimer and prothrombin fragment F1 + 2) [30] thusevaluating the final steps of the coagulatory cascade

2 Materials and Methods

21 Patients After having read and signed a specificinformed consent 58 consecutive IBD patients (35 CD and23 UC) followed up at the Gastroenterology and Gastroin-testinal EndoscopyUnit of the IRCCS Policlinico SanDonatoand 20 healthy controls were enrolled in the study Blood wascollected from all IBD patients and all control subjects AllIBD diagnoses had been confirmed by standardized clinicalendoscopic and histologic criteria [31 32] Disease activitywas assessed using the Harvey-Bradshaw Index (HBI) for CDpatients [33] and the Mayo scores for UC [34] Patients withproctitis were excluded from the studyThe demographic andclinical characteristics of the patients are reported in Table 1The study was designed to respect the ethical guidelines ofthe Declaration of HelsinkiThe Internal Review Board of thelocal Ethical Committee approved the study protocol (Ethical

Committee Protocol number 2025ASLMilano-2 approvedon June 14 2007)

22 Blood Sampling and Serum and Plasma CollectionPeripheral blood was collected by venipuncture of an ante-cubital vein without any blood stasis using sterilized needlesin BD vacutainer SST II Advance for serum collection and inBD vacutainer with sodium citrate for plasma collection A3mL blood tube was used for serum and plasma separationafter centrifugation at 2370 g for 15min at room temperatureSerum and plasma were stored at minus80∘C in 05mL aliquotsuntil the time of assays

23 C Reactive Protein Measurement C reactive protein(CRP) levels were measured by a commercially availableimmunoturbidimetric assay (Roche Diagnostic Germany)

24 TLR2 and TLR4 Protein Quantification in Serum Themeasurement of TLR2 and TLR4 was performed by meansof commercially available sandwich enzyme immunoassay(RampD System USA and USCN Life Sciences Inc Chinaresp) following manufacturersrsquo instructions

25 LPS Quantification The Endpoint Chromogenic Limu-lus Amebocyte Lysate (LAL) Test (Lonza) was used as aquantitative test for LPS according tomanufacturerrsquos instruc-tions Briefly serum was diluted 1 10 with LAL reagent water(LRW) heat inactivated in awater bath for 15minutes at 70∘Cand then diluted 1 2 in LRW The test was performed in amicroplate at 37∘C in heating block Each sample was testedin duplicate Fifty 120583L of diluted sample was mixed with 50120583Lof the LAL supplied in the test kit and incubated at 37∘C(plusmn1∘C) for 10 minutes One hundred 120583L of substrate solutionwas then mixed with the LAL sample and incubated at 37∘C(plusmn1∘C) for an additional 6 minutes The reaction was stoppedwith 100 120583L acetic acid 25 vv in water The absorbance ofthe sample was determined spectrophotometrically at 405ndash410 nm

26 D-Dimer and F1 + 2 Dosage D-Dimer HS kit (Instru-mentation Laboratory Spain) was used for the quantitativedetermination of D-Dimer in human citrated plasma fol-lowing manufacturerrsquos instructions Briefly the D-dimer HS500 latex reagent is a suspension of polystyrene latex particlesof uniform size coated with the F(ab1015840)

2fragment of a mon-

oclonal antibody highly specific for the D-Dimer domainincluded in fibrin soluble derivatives Plasma containing D-Dimer was mixed with the latex reagent and the reactionbuffer obtaining agglutination directly proportional to theD-Dimer concentration

ENzygnost F1 + 2 sandwich ELISA test (Siemens Ger-many) was used to measure prothrombin fragment F1 + 2according to manufacturerrsquos instructions

27 Statistical Analysis Data were analyzed by use of a com-puterized program (GraphPad Prism GraphPad SoftwareInc San Diego CA) Statistical methods employed includedthe use of Mann-Whitney Test and Spearman Correlation

Gastroenterology Research and Practice 3

Table 1 Demographic and clinical characteristics of enrolled IBD patients

Diagnosis CD UC Controls of patients 35 23 20Age (mean plusmn SD) 4079 plusmn 1668 4539 plusmn 1514 3979 plusmn 1324Sex (malefemale) 1619 158 128Location CD

Ileal (L1) 11 mdash mdashColonic (L2) 17 mdash mdashIleocolonic (L3) 7 mdash mdashUpper GI (L4) 0 mdash mdash

Extent UCLeft sided colitis mdash 6 mdashExtensive colitis mdash 17 mdash

Disease behavior CDNonstricturing nonpenetrating (B1) 21 mdash mdashStricturing (B2) 8 mdash mdashPenetrating (B3) 1 mdash mdashPerianal disease 5 mdash mdash

Previous resective surgery 2 mdash mdashDisease activity indexes

Harvey-Bradshaw Index (mean plusmn SD) 537 plusmn 402 mdash mdashMayo score (mean plusmn SD) mdash 696 plusmn 385 mdash

Folate serum levels (mcgL) 598 plusmn 202 656 plusmn 328Vitamin B12 serum levels (ngL) 3984 plusmn 2300 3308 plusmn 1245

Test Data are presented as the median and interquartilerange The statistical significance was set at 119875 lt 005

3 Results

31 Circulating LPS Is Increased in IBD Patients and Correlateswith TLR2 and TLR4 Serum Concentration in UC In orderto evaluate the penetration of PAMPs in the blood flowas a result of a defective intestinal barrier function in IBDwe measured circulating LPS in the sera of IBD patientsversus control subjects LPS levels were more elevated in CD(0400 (95 CI 0333ndash0540) EUmL) and UC (0430 (95CI 0276ndash0677) EUmL) patients compared with controls(0325 (95 CI 0264ndash0411) EUmL 119875 = 0044 and 119875 =0205 resp) (Figure 1(a)) When performing a subanalysis ofCD patients according to their disease location as describedby the Montreal classification [35] we detected significantlyincreased LPS levels in patients with colonic disease (0420(95 CI 0350ndash0620) EUmL) compared to controls (119875 =0031) (Figure 1(a))Then wemeasured serum concentrationof TLR2 as putative aspecific marker of systemic innateimmune activation Similarly to LPS we found more abun-dant TLR2 in CD (51721 (95 CI 41207ndash89073) pgmL)and UC (44760 (95 CI 33990ndash70441) pgmL) sera versuscontrols (28115 (95 CI 12223ndash41205) pgmL 119875 = 0002and 119875 = 0040 resp) (Figure 1(b)) Interestingly onlyCD patients with colonic or ileocolonic involvement showedincreased TLR2 levels (51280 (95 CI 43692ndash77827) and65672 (95 CI 57843ndash119555) pgmL resp 119875 = 0003 and119875 = 0003 resp) compared to controls We next analyzed

the correlation between LPS and TLR2 levels although wedid not find any correlation between these two variableswhen considering the whole IBD patients (Figure 1(c)) orCD patients (Figure 1(d)) a significant correlation (119903 =0495 119875 = 0016) was observed when we considered UCpatients separated from CD ones (Figure 1(d)) In additionsubanalysis of CD patients according to disease location didnot reveal any further association (data not shown) We alsoevaluated the correlation between serum TLR4 (Figure 2(a))that is the principal innate immune receptor for LPS andcirculating LPS and we detected a significant correlation(119903 = 0421 119875 = 0001) (Figure 2(b)) interestingly whenwe repeated the analysis dividing patients according to theirdisease we found more robust correlation for UC (119903 = 0512119875 = 0008) and CD with colonic involvement (L2 + L3)(119903 = 0468 119875 = 0024) than for CD as a whole (119903 = 0332119875 = 0054) (Figure 2(c))

32 LPS Levels Correlate with Biochemical but Not ClinicalActivity in IBD Patients Whereas TLR2 and TLR4 Levels AreIndependent of Disease Activity In order to evaluate whetheror not circulating LPS TLR2 and TLR4 were merely markersof systemic inflammation or disease activity we analyzedthe correlation between those variables with a biochemicalactivity marker that is CRP and two commonly used clinicalactivity indexes such as the Mayo score for UC and theHarvey-Bradshaw Index for CD Our analysis was able todemonstrate only a weak correlation between circulatingLPS and CRP concentrations (119903 = 0295 119875 = 0035)(Figure 3)Weobserved no correlation at all when subanalysis

4 Gastroenterology Research and Practice

00

05

10

15

20

25

LPS

(EU

mL)

00

05

10

15

20

25

LPS

(EU

mL)

CON

T

UC

CD

CON

T

CD L

1

CD L

2

CD L

3

P = 0044 P = 0031

(a)

0

2000

4000

6000

8000

TLR2

(pg

mL)

0

2000

4000

6000

8000

TLR2

(pg

mL)

CON

T

UC

CD

CON

T

CD L

1

CD L

2

CD L

3

P = 0002

P = 004P = 0003

P = 0003

(b)

00 05 10 15 20 250

2000

4000

6000

8000

LPS (EUmL)

TLR2

(pg

mL)

(c)

00 05 10 15 20 250

2000

4000

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LPS (EUmL)

TLR2

(pg

mL)

00 05 10 15 20 250

2000

4000

6000

8000

LPS (EUmL)

TLR2

(pg

mL)

r = 0495

P = 0016

(d)

Figure 1 Circulating LPS and TLR2 are increased in IBD and correlate with each other in UC Concentrations of LPS and TLR2 weremeasured in the sera of IBD (both UC and CD) patients and healthy controls (CONT) (a) LPS serum levels in CONT UC and CD patients(left panel) and CD locations according to Montreal classification (right panel) (b) TLR2 serum levels in CONT UC and CD patients (leftpanel) and CD locations according to Montreal classification (right panel) (c) Correlation between circulating levels of LPS and TLR2 inIBD patients (d) Correlation between circulating levels of LPS and TLR2 in UC (left panel) and CD (right panel) patients Horizontal barsin (a) and (b) represent median and interquartile range Statistical analysis was performed by means of Mann-Whitney Test and SpearmanCorrelation Test 119875 lt 005 was considered statistically significant

for disease and disease location was performed (data notshown) suggesting that LPS TLR2 and TLR4 levels aremostly independent of disease activity No correlations havebeen observed with the clinical indexes (ie Mayo score andHarvey-Bradshaw Index) (Figure 3)

33 Concentrations of Circulating LPS Correlate with PlasmaLevels of Markers of Activated Coagulation We also mea-sured the plasma concentrations of D-Dimer and prothrom-bin fragment F1 + 2 which are well known markers ofactivated coagulation and are increased in procoagulativestates LPS levels significantly correlated with both D-Dimer(119903 = 0422 119875 = 0001) and F1 + 2 concentrations (119903 =0440 119875 = 00008) (Figure 4) however no correlationwas found between TLR2 or TLR4 and those coagulationmarkers (Figure 4) On the other hand when we analyzedonly data obtained in UC patients we not only confirmedthe correlation between LPS and both D-Dimer (119903 = 0467119875 = 0028) and F1 + 2 (119903 = 0521 119875 = 0012) butalso detected good correlations between TLR2 and D-Dimer(119903 = 0776 119875 lt 00001) TLR4 and D-Dimer (119903 = 0560119875 = 0006) and TLR4 and F1 + 2 (119903 = 0573 119875 = 0005)(Figure 5)When all CDpatientswere considered for analysisno significant correlation was found remarkably when only

patients with colonic disease (L2 and L3) were taken intoaccount correlations between D-Dimer and F1 + 2 and LPSlevels were confirmed (119903 = 0435 119875 = 0042 and 119903 = 0590119875 = 0003 resp) (Figure 6)

4 Discussion

Since 1972 when Shorter et al postulated that a primarydefect in gut permeability and barrier function may leadto the onset of persistent inflammation in the gut and tothe development of IBD [36] a growing body of evidencehas demonstrated that the impairment of intestinal barrierfunction is a common feature in IBD patients leading toderangements in both epithelial permeability to gut antigensand early mucosal innate immune responses protectingfrommicroorganisms penetration andbacterial translocation[8 9] Interestingly genetic studies demonstrated a strongcorrelation between the carriage of polymorphisms of genesregulating intestinal epithelial paracellular permeability andthe risk of developing UC [37 38] whereas being carrier ofinnate immunity-related gene polymorphisms increases thesusceptibility to CD [38]

Patients suffering from IBD and particularly UC [39]are more prone to incur into thromboembolic events [40]

Gastroenterology Research and Practice 5

0

2000

4000

6000

8000

TLR4

(pg

mL)

0

2000

4000

6000

8000

TLR4

(pg

mL)

UC CD CD L1 CD L2 CD L3

(a)

00 05 10 15 20 250

2000

4000

6000

8000

LPS (EUmL)

TLR4

(pg

mL) r = 0421

P = 0001

(b)

00 05 10 15 20 250

1000

2000

3000

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5000

LPS (EUmL)

TLR4

(pg

mL)

00 05 10 15 20 250

2000

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TLR4

(pg

mL)

00 05 10 15 200

1000

2000

3000

4000

5000

LPS (EUmL)

TLR4

(pg

mL)

r = 0512

P = 0008

r = 0332

P = 0054

r = 0468

P = 0024

(c)

Figure 2 TLR4 serum correlates with circulating LPS in IBD patients Concentrations of LPS and TLR4 were measured in the sera of IBD(both UC and CD) patients (a) TLR4 serum levels in UC and CD patients (left panel) and CD locations according to Montreal classification(right panel) (b) Correlation between circulating levels of LPS and TLR4 in IBD patients (c) Correlation between circulating levels of LPSand TLR4 in UC (left panel) CD (central panel) patients and CD patients with colonic and ileocolonic involvement (L2 + L3 according toMontreal classification) (right panel) Horizontal bars in (a) represent median and interquartile range Statistical analysis was performed bymeans of Spearman Correlation Test 119875 lt 005 was considered statistically significant

similarly to patients affected by other inflammatory andimmune-related disorders [6] however the only presence ofan ongoing inflammatory process does not explain by itselfthis phenomenon In fact it has been shown that the riskof thromboembolism is more elevated in IBD than rheuma-toid arthritis a disease often characterized by a prominentsystemic inflammation [7] thus suggesting that some gutspecific mechanisms may be involved Besides deficienciesof vitamins involved in the proper regulation of coagulatoryhomeostasis such as vitamin B6 vitamin B12 and folic acid[11ndash13] the alteration of intestinal barrier function may be acofactor promoting a procoagulative state in IBD

The data presented in this paper strongly support thisnovel hypothesis In fact in order to assess intestinal perme-ability to PAMPs and bacterial translocation from the gut wemeasured circulating LPS and found higher concentrationsof this bacterial component in the sera of IBD patients (Fig-ure 1(a)) Consistent with the data previously presented byCandia et al [26] IBD patients also presented higher serumlevels of TLR2 as a sign of innate immune activation (Figures1(b) 1(c) and 1(d)) Remarkably serum TLR2 was recentlydescribed to correlate with the presence of prosthetic jointinfection in patients undergoing revision of joint arthroplastybecause of suspected local infection [23] thus circulatingTLR2may be considered amarker of the presence of bacterial

components in the blood flow Moreover high levels ofsoluble TLR2 were shown in the sera of patients affected bypsoriasis [21] and systemic lupus erythematosus [25] diseasesin which innate immunity plays a major pathogenetic role[41 42] As such high levels of soluble TLR2 may reflect theactivation of innate inflammatory responses

LPS levels correlated with the serum concentrations ofTLR4 which is the innate immune receptor deputed to LPSrecognition (Figure 2) This may be a sign of an increasedexpression and activation of TLR4 because of LPS binding

Apart from a very modest correlation between LPSand CRP levels LPS TLR2 and TLR4 concentrations didnot appear to be influenced by biochemical and clinicaldisease activity (Figure 3) suggesting that their levels may beinfluenced for the great part by intestinal permeability

Both TLR2 and TLR4 are expressed by platelets andendothelial cells [27ndash29] moreover the binding of theirrespective ligands causes the procoagulatory activation ofthese cell populations More in detail TLR2 signaling inplatelets leads to a thromboinflammatory response throughthe activation of phosphoinositide 3-kinase [43] cyclooxy-genase and purinergic P2Y1 and P2Y12 receptors [44]and alpha-granule release [45] whereas LPS-TLR4 bindingenhances classical agonist-induced platelet aggregation [4647] The TLR2 and TLR4 signaling on endothelial cells

6 Gastroenterology Research and Practice

00 05 10 15 20 250

2

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CRP

(mg

dL)

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CRP

(mg

dL)

0 2000 4000 6000 80000

2

4

6

8

10

TLR4 (pgmL)

CRP

(mg

dL)r = 0295

P = 0035

(a)

00 05 10 15 20 250

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LPS (EUmL)

May

o sc

ore

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TLR2 (pgmL)

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(b)

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Brad

shaw

Inde

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vey-

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shaw

Inde

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Har

vey-

Brad

shaw

Inde

x

(c)

Figure 3 Circulating LPS correlates with biochemical but not clinical activity in IBD patients Concentrations of LPS TLR2 TLR4 andCRP were measured in the sera of IBD (both UC and CD) patients Clinical activity of UC and CD was evaluated using Mayo score andHarvey-Bradshaw Index (a) Correlation between CRP serum levels and circulating LPS TLR2 and TLR4 in IBD patients (b) Correlationbetween Mayo score and circulating LPS TLR2 and TLR4 in UC patients (c) Correlation between Harvey-Bradshaw Index and circulatingLPS TLR2 and TLR4 in CD patients Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 was consideredstatistically significant

strongly activates NF-120581B [5 29] leading to the release ofproinflammatory mediators which can activate the coagula-tory cascade Nonetheless the pathogenesis of atheroscleroticplaques ismediated by the presence of activatedmacrophageswithin the plaque which also respond vigorously to TLRstimulation releasing proinflammatory cytokines [48]

Given themultiplicity of cell types and pathways involvedin the activation of coagulation mediated by TLRs wedecided tomeasure the activation products of the coagulationand fibrinolysis that is the fibrin degradation product D-Dimer and prothrombin fragment F1 + 2 as they describeclosely the overall activation of the coagulatory cascade [30]Remarkably LPS levels significantly correlated with bothD-Dimer and F1 + 2 (Figure 4) clearly suggesting thatcirculating PAMPsmay function as triggers for the activationof coagulation

It may be worthy to note that the correlation betweencirculating LPS and markers of activated coagulation wasstronger when considering only UC patients (Figures 1(d)2(c) and 5) and CD patients with colonic or ileocolonicdisease (L2 and L3 according to Montreal classification [35])(Figure 6)These data suggest that the presence of colonic dis-ease is the key risk factor for coagulatory unbalance that mayfollow bacterial translocation from the gut indeed the colonhas to cope with a significantly greater bacterial burden thanthe rest of the gastrointestinal tract as such it is rational thata break-down of intestinal barrier function in this site mightlead to amore prominent bacterial penetrationtranslocation

Moreover in order to explain the differences we found inUC versus CD other issues should be taken into account CDpatients often display greater signs of systemic inflammationrather than UC as such data coming from CD patients may

Gastroenterology Research and Practice 7

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(EU

mL)

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TLR2

(pg

mL)

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TLR4

(pg

mL)

r = 0422

P = 0001

(a)

0 200 400 600 800 100000

05

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LPS

(EU

mL)

0 200 400 600 800 10000

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8000TL

R2 (p

gm

L)

0 200 400 600 800 10000

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TLR4

(pg

mL)

r = 0440

P = 00008

F1 + 2 (pmolL) F1 + 2 (pmolL) F1 + 2 (pmolL)

(b)

Figure 4 Circulating LPS correlates with markers of activated coagulation in IBD patients Concentrations of LPS TLR2 and TLR4 andlevels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of IBD patients (a) Correlationbetween D-Dimer plasma levels and serum LPS TLR2 and TLR4 in IBD patients (b) Correlation between prothrombin fragment F1+2 andcirculating LPS TLR2 and TLR4 in IBD patients Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 wasconsidered statistically significant

0 500 1000 150000

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LPS

(EU

mL)

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TLR2

(pg

mL)

0 500 1000 15000

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TLR4

(pg

mL)

r = 0467

P = 0028

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P = 0006

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(a)

0 200 400 600 800 100000

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(EU

mL)

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mL)

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(pg

mL)

F1 + 2 (pmolL) F1 + 2 (pmolL) F1 + 2 (pmolL)

r = 0521

P = 0012

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P = 0005

(b)

Figure 5 Circulating LPS TLR2 and TLR4 correlate with markers of activated coagulation in UC patients Concentrations of LPS TLR2and TLR4 and levels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of UC patients(a) Correlation between D-Dimer plasma levels and serum LPS TLR2 and TLR4 in UC patients (b) Correlation between prothrombinfragment F1 + 2 and circulating LPS TLR2 and TLR4 in UC patients Statistical analysis was performed by means of Spearman CorrelationTest 119875 lt 005 was considered statistically significant

8 Gastroenterology Research and Practice

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(EU

mL)

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(EU

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(a)

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P = 0003

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(b)

Figure 6 Circulating LPS correlates with markers of activated coagulation in CD patients with colonic involvement Concentrations of LPSand levels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of CD patients (a) Correlationbetween D-Dimer plasma levels and serum LPS in all CD patients (left panel) CD patients with exclusive ileal involvement (L1 accordingto Montreal classification) (central panel) and CD patients with colonic involvement (L2 + L3 according to Montreal classification) (b)Correlation between prothrombin fragment F1 + 2 and circulating LPS in all CD patients (left panel) CD patients with exclusive ilealinvolvement (L1 according to Montreal classification) (central panel) and CD patients with colonic involvement (L2 + L3 according toMontreal classification) Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 was considered statisticallysignificant

suffer from the noise resulting from inflammation-drivenactivation of coagulation as an alternative PAMPs-mediatedcoagulatory activation may have a different relevance orfollow different pathways in UC versus CD also consideringthat genetic data suggest different defects in intestinal barrierfunction in those two diseases [38]

Taken together our data support the novel hypothesisthat intestinal barrier defects contribute to the developmentof a procoagulatory state in IBD Our results need to beconfirmed with future studies in fact the correlations weobserved in human patients do not demonstrate the bio-logical importance of the phenomenon we are postulatingA further limitation of our study is that we are usingvery simple markers in order to explore extremely complexsystems Indeed experiments on animal models of intestinalinflammation are warranted in order to provide the finaldemonstration of our hypothesis and mechanistically dissectit and evaluate its real biologic relevance

Conflict of Interests

None of the authors has any conflict of interests related to thispaperwork to declare

Acknowledgments

The authors acknowledge Dr Rossella Bader for her impor-tant technical support This work was supported by theItalian Ministry of University and Research PRIN Grant2007K4HZEJ 004 (MV)

References

[1] W Rosamond K Flegal K Furie et al ldquoHeart disease andstroke statisticsmdash2008 update a report from the Americanheart association statistics committee and stroke statistics sub-committeerdquo Circulation vol 117 no 4 pp e25ndashe46 2008

[2] C T Esmon ldquoInflammation and thrombosisrdquo Journal ofThrom-bosis and Haemostasis vol 1 no 7 pp 1343ndash1348 2003

[3] J Petaja ldquoInflammation and coagulation An overviewrdquoThrom-bosis Research vol 127 supplement 2 pp S34ndashS37 2011

[4] J Frostegard ldquoImmunity atherosclerosis and cardiovasculardiseaserdquo BMCMedicine vol 11 no 1 article 117 2013

[5] K J Moore F J Sheedy and E A Fisher ldquoMacrophages inatherosclerosis a dynamic balancerdquo Nature Reviews Immunol-ogy vol 13 no 10 pp 709ndash721 2013

[6] B Zoller X Li J Sundquist and K Sundquist ldquoAutoimmunediseases and venous thromboembolism a review of the litera-turerdquo American Journal of Cardiovascular Disease vol 2 no 3pp 171ndash183 2012

Gastroenterology Research and Practice 9

[7] W Miehsler W Reinisch E Valic et al ldquoIs inflammatorybowel disease an independent and disease specific risk factorfor thromboembolismrdquo Gut vol 53 no 4 pp 542ndash548 2004

[8] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[9] L Pastorelli C D Salvo J R Mercado M Vecchi and T TPizarro ldquoCentral role of the gut epithelial barrier in the patho-genesis of chronic intestinal inflammation iessons learned fromanimal models and human geneticsrdquo Frontiers in Immunologyvol 4 article 280 Article ID Article 280 2013

[10] M Zaeem Cader and A Kaser ldquoRecent advances in inflam-matory bowel disease mucosal immune cells in intestinalinflammationrdquo Gut vol 62 no 11 pp 1653ndash1664 2013

[11] S Saibeni L Spina andM Vecchi ldquoExploring the relationshipsbetween inflammatory response and coagulation cascade ininflammatory bowel diseaserdquo European Review for Medical andPharmacological Sciences vol 8 no 5 pp 205ndash208 2004

[12] S Saibeni M Cattaneo M Vecchi et al ldquoLow vitamin B6

plasma levels a risk factor for thrombosis in inflammatorybowel disease role of inflammation and correlation with acutephase reactantsrdquoThe American Journal of Gastroenterology vol98 no 1 pp 112ndash117 2003

[13] M Cattaneo M Vecchi M L Zighetti et al ldquoHigh prevalenceof hyperhomocysteinemia in patients with inflammatory boweldisease a pathogenic link with thromboembolic complica-tionsrdquoThrombosis and Haemostasis vol 80 no 4 pp 542ndash5451998

[14] T H Mogensen ldquoPathogen recognition and inflammatorysignaling in innate immune defensesrdquo Clinical MicrobiologyReviews vol 22 no 2 pp 240ndash273 2009

[15] E LeBouder J E Rey-Nores N K Rushmere et al ldquoSolubleforms of Toll-like receptor (TLR)2 capable of modulating TLR2signaling are present in human plasma and breast milkrdquo Journalof Immunology vol 171 no 12 pp 6680ndash6689 2003

[16] K I Iwami T Matsuguchi A Masuda T Kikuchi TMusikacharoen and Y Yoshikai ldquoCutting edge naturallyoccurring soluble form of mouse toll-like receptor 4 inhibitslipopolysaccharide signalingrdquo Journal of Immunology vol 165no 12 pp 6682ndash6686 2000

[17] I Munoz M P Sepulcre J Meseguer and V Mulero ldquoMolecu-lar cloning phylogenetic analysis and functional characteriza-tion of soluble Toll-like receptor 5 in gilthead seabream Sparusauratardquo Fish amp Shellfish Immunology vol 35 no 1 pp 36ndash452013

[18] S L Zunt L V Burton L I Goldblatt E E Dobbins and MSrinivasan ldquoSoluble forms of Toll-like receptor 4 are present inhuman saliva and modulate tumour necrosis factor-120572 secretionby macrophage-like cellsrdquo Clinical and Experimental Immunol-ogy vol 156 no 2 pp 285ndash293 2009

[19] A-C Raby E le Bouder C Colmont et al ldquoSoluble TLR2reduces inflammation without compromising bacterial clear-ance by disrupting TLR2 triggeringrdquo Journal of Immunologyvol 183 no 1 pp 506ndash517 2009

[20] R-S Chang Y-C Wang and S-T Kao ldquoSoluble toll-likereceptor 4 reversed attenuating effect of Chinese herbal Xiao-Qing-Long-Tang on allergen induced nerve growth factor andthymic stromal lymphopoietinrdquo Experimental and TherapeuticMedicine vol 6 no 5 pp 1199ndash1207 2013

[21] K Kondelkova J Krejsek L Borska et al ldquoMembrane andsoluble Toll-like receptor 2 in patients with psoriasis treatedby Goeckerman therapyrdquo International Journal of Dermatologyvol 53 no 11 pp e512ndashe517 2014

[22] H-B Yang K-Q Xie J-M Deng and S-M Qin ldquoExpressionof soluble toll-like receptors in pleural effusionsrdquo ChineseMedical Journal vol 123 no 16 pp 2225ndash2230 2010

[23] E Galliera L Drago C Vassena et al ldquoToll-like receptor 2in serum a potential diagnostic marker of prosthetic jointinfectionrdquo Journal of Clinical Microbiology vol 52 no 2 pp620ndash623 2013

[24] A Zhao C Blackburn J Chin and M Srinivasan ldquoSoluble tolllike receptor 2 (TLR-2) is increased in saliva of children withdental cariesrdquo BMC Oral Health vol 14 no 1 article 108 2014

[25] M E Houssen R H El-Mahdy and D A Shahin ldquoSerumsoluble toll-like receptor 2 a novel biomarker for systemic lupuserythematosus disease activity and lupus-related cardiovasculardysfunctionrdquo International Journal of Rheumatic Diseases 2014

[26] E Candia D Dıaz-Jimenez P Langjahr et al ldquoIncreasedproduction of soluble TLR2 by lamina propria mononuclearcells from ulcerative colitis patientsrdquo Immunobiology vol 217no 6 pp 634ndash642 2012

[27] F Cognasse H Hamzeh P Chavarin S Acquart C Geninand O Garraud ldquoEvidence of Toll-like receptor molecules onhuman plateletsrdquo Immunology and Cell Biology vol 83 no 2pp 196ndash198 2005

[28] F X Zhang C J Kirschning R Mancinelli et al ldquoBacteriallipopolysaccharide activates nuclear factor-kappaB throughinterleukin-1 signaling mediators in cultured human dermalendothelial cells and mononuclear phagocytesrdquo The Journal ofBiological Chemistry vol 274 no 12 pp 7611ndash7614 1999

[29] E Faure O Equils P A Sieling et al ldquoBacterial lipopolysac-charide activates NF-120581B through toll-like receptor 4 (TLR-4) in cultured human dermal endothelial cells Differentialexpression of TLR-4 andTLR-2 in endothelial cellsrdquoThe Journalof Biological Chemistry vol 275 no 15 pp 11058ndash11063 2000

[30] M D Boisclair H Ireland and D A Lane ldquoAssessment ofhypercoagulable states bymeasurement of activation fragmentsand peptidesrdquo Blood Reviews vol 4 no 1 pp 25ndash40 1990

[31] E F Stange S P L Travis S Vermeire et al ldquoEuropean evi-dence-based Consensus on the diagnosis and management ofulcerative colitis definitions and diagnosisrdquo Journal of Crohnrsquosamp Colitis vol 2 no 1 pp 1ndash23 2008

[32] A Dignass G van Assche J O Lindsay et al ldquoThe secondEuropean evidence-based Consensus on the diagnosis andmanagement of Crohnrsquos disease current managementrdquo Journalof Crohnrsquos amp Colitis vol 4 no 1 pp 28ndash62 2010

[33] R F Harvey and J M Bradshaw ldquoA simple index of Crohnrsquos-disease activityrdquoThe Lancet vol 1 no 8167 p 514 1980

[34] KW SchroederW J Tremaine andDM Ilstrup ldquoCoated oral5-aminosalicylic acid therapy for mildly to moderately activeulcerative colitis a randomized studyrdquoTheNewEngland Journalof Medicine vol 317 no 26 pp 1625ndash1629 1987

[35] J Satsangi M S Silverberg S Vermeire and J-F ColombelldquoThe Montreal classification of inflammatory bowel diseasecontroversies consensus and implicationsrdquo Gut vol 55 no 6pp 749ndash753 2006

[36] R G Shorter K A Huizenga and R J Spencer ldquoA workinghypothesis for the etiology and pathogenesis of nonspecificinflammatory bowel diseaserdquoTheAmerican Journal of DigestiveDiseases vol 17 no 11 pp 1024ndash1032 1972

[37] A I Thompson and C W Lees ldquoGenetics of ulcerative colitisrdquoInflammatory Bowel Diseases vol 17 no 3 pp 831ndash848 2011

[38] C W Lees J C Barrett M Parkes and J Satsangi ldquoNew IBDgenetics common pathways with other diseasesrdquo Gut vol 60no 12 pp 1739ndash1753 2011

[39] G C Nguyen and J Sam ldquoRising prevalence of venous throm-boembolism and its impact on mortality among hospitalized

10 Gastroenterology Research and Practice

inflammatory bowel disease patientsrdquo The American Journal ofGastroenterology vol 103 no 9 pp 2272ndash2280 2008

[40] H Yuhara C Steinmaus D Corley et al ldquoMeta-analysis therisk of venous thromboembolism in patients with inflammatorybowel diseaserdquoAlimentary Pharmacology andTherapeutics vol37 no 10 pp 953ndash962 2013

[41] W R Swindell P E Stuart M K Sarkar et al ldquoCellular dis-section of psoriasis for transcriptome analyses and the post-GWAS erardquo Acta Veterinaria Scandinavica vol 7 article 272014

[42] D Salem R Subang P Laplante J Levine and J Rauch ldquoThedual role of innate immunity in antiphospholipid syndrome andsystemic lupus erythematosusrdquo Lupus vol 23 no 12 pp 1327ndash1331 2014

[43] P Blair S Rex O Vitseva et al ldquoStimulation of Toll-likereceptor 2 in human platelets induces a thromboinflammatoryresponse through activation of phosphoinositide 3-kinaserdquoCirculation Research vol 104 no 3 pp 346ndash354 2009

[44] H Kalvegren C Skoglund C Helldahl M Lerm MGrenegard and T Bengtsson ldquoToll-like receptor 2 stimulationof platelets is mediated by purinergic P2X1-dependent Ca2+mobilisation cyclooxygenase and purinergic P2Y1 and P2Y12receptor activationrdquo Thrombosis and Haemostasis vol 103 no2 pp 398ndash407 2010

[45] S Rex L M Beaulieu D H Perlman et al ldquoImmune versusthrombotic stimulation of platelets differentially regulates sig-nalling pathways intracellular protein-protein interactions and120572-granule releaserdquo Thrombosis and Haemostasis vol 102 no 1pp 97ndash110 2009

[46] G Zhang J Han E J Welch et al ldquoLipopolysaccharide stim-ulates platelet secretion and potentiates platelet aggregationvia TLR4MyD88 and the cGMP-dependent protein kinasepathwayrdquoThe Journal of Immunology vol 182 no 12 pp 7997ndash8004 2009

[47] G T Brown and T MMcIntyre ldquoLipopolysaccharide signalingwithout a nucleus kinase cascades stimulate platelet sheddingof proinflammatory IL-1120573-rich microparticlesrdquo The Journal ofImmunology vol 186 no 9 pp 5489ndash5496 2011

[48] L Chavez-Sanchez J E Espinosa-Luna K Chavez-Rueda MV Legorreta-Haquet E Montoya-Dıaz and F Blanco-FavelaldquoInnate immune system cells in atherosclerosisrdquo Archives ofMedical Research vol 45 no 1 pp 1ndash14 2014

Gastroenterology Research and Practice 3

Table 1 Demographic and clinical characteristics of enrolled IBD patients

Diagnosis CD UC Controls of patients 35 23 20Age (mean plusmn SD) 4079 plusmn 1668 4539 plusmn 1514 3979 plusmn 1324Sex (malefemale) 1619 158 128Location CD

Ileal (L1) 11 mdash mdashColonic (L2) 17 mdash mdashIleocolonic (L3) 7 mdash mdashUpper GI (L4) 0 mdash mdash

Extent UCLeft sided colitis mdash 6 mdashExtensive colitis mdash 17 mdash

Disease behavior CDNonstricturing nonpenetrating (B1) 21 mdash mdashStricturing (B2) 8 mdash mdashPenetrating (B3) 1 mdash mdashPerianal disease 5 mdash mdash

Previous resective surgery 2 mdash mdashDisease activity indexes

Harvey-Bradshaw Index (mean plusmn SD) 537 plusmn 402 mdash mdashMayo score (mean plusmn SD) mdash 696 plusmn 385 mdash

Folate serum levels (mcgL) 598 plusmn 202 656 plusmn 328Vitamin B12 serum levels (ngL) 3984 plusmn 2300 3308 plusmn 1245

Test Data are presented as the median and interquartilerange The statistical significance was set at 119875 lt 005

3 Results

31 Circulating LPS Is Increased in IBD Patients and Correlateswith TLR2 and TLR4 Serum Concentration in UC In orderto evaluate the penetration of PAMPs in the blood flowas a result of a defective intestinal barrier function in IBDwe measured circulating LPS in the sera of IBD patientsversus control subjects LPS levels were more elevated in CD(0400 (95 CI 0333ndash0540) EUmL) and UC (0430 (95CI 0276ndash0677) EUmL) patients compared with controls(0325 (95 CI 0264ndash0411) EUmL 119875 = 0044 and 119875 =0205 resp) (Figure 1(a)) When performing a subanalysis ofCD patients according to their disease location as describedby the Montreal classification [35] we detected significantlyincreased LPS levels in patients with colonic disease (0420(95 CI 0350ndash0620) EUmL) compared to controls (119875 =0031) (Figure 1(a))Then wemeasured serum concentrationof TLR2 as putative aspecific marker of systemic innateimmune activation Similarly to LPS we found more abun-dant TLR2 in CD (51721 (95 CI 41207ndash89073) pgmL)and UC (44760 (95 CI 33990ndash70441) pgmL) sera versuscontrols (28115 (95 CI 12223ndash41205) pgmL 119875 = 0002and 119875 = 0040 resp) (Figure 1(b)) Interestingly onlyCD patients with colonic or ileocolonic involvement showedincreased TLR2 levels (51280 (95 CI 43692ndash77827) and65672 (95 CI 57843ndash119555) pgmL resp 119875 = 0003 and119875 = 0003 resp) compared to controls We next analyzed

the correlation between LPS and TLR2 levels although wedid not find any correlation between these two variableswhen considering the whole IBD patients (Figure 1(c)) orCD patients (Figure 1(d)) a significant correlation (119903 =0495 119875 = 0016) was observed when we considered UCpatients separated from CD ones (Figure 1(d)) In additionsubanalysis of CD patients according to disease location didnot reveal any further association (data not shown) We alsoevaluated the correlation between serum TLR4 (Figure 2(a))that is the principal innate immune receptor for LPS andcirculating LPS and we detected a significant correlation(119903 = 0421 119875 = 0001) (Figure 2(b)) interestingly whenwe repeated the analysis dividing patients according to theirdisease we found more robust correlation for UC (119903 = 0512119875 = 0008) and CD with colonic involvement (L2 + L3)(119903 = 0468 119875 = 0024) than for CD as a whole (119903 = 0332119875 = 0054) (Figure 2(c))

32 LPS Levels Correlate with Biochemical but Not ClinicalActivity in IBD Patients Whereas TLR2 and TLR4 Levels AreIndependent of Disease Activity In order to evaluate whetheror not circulating LPS TLR2 and TLR4 were merely markersof systemic inflammation or disease activity we analyzedthe correlation between those variables with a biochemicalactivity marker that is CRP and two commonly used clinicalactivity indexes such as the Mayo score for UC and theHarvey-Bradshaw Index for CD Our analysis was able todemonstrate only a weak correlation between circulatingLPS and CRP concentrations (119903 = 0295 119875 = 0035)(Figure 3)Weobserved no correlation at all when subanalysis

4 Gastroenterology Research and Practice

00

05

10

15

20

25

LPS

(EU

mL)

00

05

10

15

20

25

LPS

(EU

mL)

CON

T

UC

CD

CON

T

CD L

1

CD L

2

CD L

3

P = 0044 P = 0031

(a)

0

2000

4000

6000

8000

TLR2

(pg

mL)

0

2000

4000

6000

8000

TLR2

(pg

mL)

CON

T

UC

CD

CON

T

CD L

1

CD L

2

CD L

3

P = 0002

P = 004P = 0003

P = 0003

(b)

00 05 10 15 20 250

2000

4000

6000

8000

LPS (EUmL)

TLR2

(pg

mL)

(c)

00 05 10 15 20 250

2000

4000

6000

8000

LPS (EUmL)

TLR2

(pg

mL)

00 05 10 15 20 250

2000

4000

6000

8000

LPS (EUmL)

TLR2

(pg

mL)

r = 0495

P = 0016

(d)

Figure 1 Circulating LPS and TLR2 are increased in IBD and correlate with each other in UC Concentrations of LPS and TLR2 weremeasured in the sera of IBD (both UC and CD) patients and healthy controls (CONT) (a) LPS serum levels in CONT UC and CD patients(left panel) and CD locations according to Montreal classification (right panel) (b) TLR2 serum levels in CONT UC and CD patients (leftpanel) and CD locations according to Montreal classification (right panel) (c) Correlation between circulating levels of LPS and TLR2 inIBD patients (d) Correlation between circulating levels of LPS and TLR2 in UC (left panel) and CD (right panel) patients Horizontal barsin (a) and (b) represent median and interquartile range Statistical analysis was performed by means of Mann-Whitney Test and SpearmanCorrelation Test 119875 lt 005 was considered statistically significant

for disease and disease location was performed (data notshown) suggesting that LPS TLR2 and TLR4 levels aremostly independent of disease activity No correlations havebeen observed with the clinical indexes (ie Mayo score andHarvey-Bradshaw Index) (Figure 3)

33 Concentrations of Circulating LPS Correlate with PlasmaLevels of Markers of Activated Coagulation We also mea-sured the plasma concentrations of D-Dimer and prothrom-bin fragment F1 + 2 which are well known markers ofactivated coagulation and are increased in procoagulativestates LPS levels significantly correlated with both D-Dimer(119903 = 0422 119875 = 0001) and F1 + 2 concentrations (119903 =0440 119875 = 00008) (Figure 4) however no correlationwas found between TLR2 or TLR4 and those coagulationmarkers (Figure 4) On the other hand when we analyzedonly data obtained in UC patients we not only confirmedthe correlation between LPS and both D-Dimer (119903 = 0467119875 = 0028) and F1 + 2 (119903 = 0521 119875 = 0012) butalso detected good correlations between TLR2 and D-Dimer(119903 = 0776 119875 lt 00001) TLR4 and D-Dimer (119903 = 0560119875 = 0006) and TLR4 and F1 + 2 (119903 = 0573 119875 = 0005)(Figure 5)When all CDpatientswere considered for analysisno significant correlation was found remarkably when only

patients with colonic disease (L2 and L3) were taken intoaccount correlations between D-Dimer and F1 + 2 and LPSlevels were confirmed (119903 = 0435 119875 = 0042 and 119903 = 0590119875 = 0003 resp) (Figure 6)

4 Discussion

Since 1972 when Shorter et al postulated that a primarydefect in gut permeability and barrier function may leadto the onset of persistent inflammation in the gut and tothe development of IBD [36] a growing body of evidencehas demonstrated that the impairment of intestinal barrierfunction is a common feature in IBD patients leading toderangements in both epithelial permeability to gut antigensand early mucosal innate immune responses protectingfrommicroorganisms penetration andbacterial translocation[8 9] Interestingly genetic studies demonstrated a strongcorrelation between the carriage of polymorphisms of genesregulating intestinal epithelial paracellular permeability andthe risk of developing UC [37 38] whereas being carrier ofinnate immunity-related gene polymorphisms increases thesusceptibility to CD [38]

Patients suffering from IBD and particularly UC [39]are more prone to incur into thromboembolic events [40]

Gastroenterology Research and Practice 5

0

2000

4000

6000

8000

TLR4

(pg

mL)

0

2000

4000

6000

8000

TLR4

(pg

mL)

UC CD CD L1 CD L2 CD L3

(a)

00 05 10 15 20 250

2000

4000

6000

8000

LPS (EUmL)

TLR4

(pg

mL) r = 0421

P = 0001

(b)

00 05 10 15 20 250

1000

2000

3000

4000

5000

LPS (EUmL)

TLR4

(pg

mL)

00 05 10 15 20 250

2000

4000

6000

8000

LPS (EUmL)

TLR4

(pg

mL)

00 05 10 15 200

1000

2000

3000

4000

5000

LPS (EUmL)

TLR4

(pg

mL)

r = 0512

P = 0008

r = 0332

P = 0054

r = 0468

P = 0024

(c)

Figure 2 TLR4 serum correlates with circulating LPS in IBD patients Concentrations of LPS and TLR4 were measured in the sera of IBD(both UC and CD) patients (a) TLR4 serum levels in UC and CD patients (left panel) and CD locations according to Montreal classification(right panel) (b) Correlation between circulating levels of LPS and TLR4 in IBD patients (c) Correlation between circulating levels of LPSand TLR4 in UC (left panel) CD (central panel) patients and CD patients with colonic and ileocolonic involvement (L2 + L3 according toMontreal classification) (right panel) Horizontal bars in (a) represent median and interquartile range Statistical analysis was performed bymeans of Spearman Correlation Test 119875 lt 005 was considered statistically significant

similarly to patients affected by other inflammatory andimmune-related disorders [6] however the only presence ofan ongoing inflammatory process does not explain by itselfthis phenomenon In fact it has been shown that the riskof thromboembolism is more elevated in IBD than rheuma-toid arthritis a disease often characterized by a prominentsystemic inflammation [7] thus suggesting that some gutspecific mechanisms may be involved Besides deficienciesof vitamins involved in the proper regulation of coagulatoryhomeostasis such as vitamin B6 vitamin B12 and folic acid[11ndash13] the alteration of intestinal barrier function may be acofactor promoting a procoagulative state in IBD

The data presented in this paper strongly support thisnovel hypothesis In fact in order to assess intestinal perme-ability to PAMPs and bacterial translocation from the gut wemeasured circulating LPS and found higher concentrationsof this bacterial component in the sera of IBD patients (Fig-ure 1(a)) Consistent with the data previously presented byCandia et al [26] IBD patients also presented higher serumlevels of TLR2 as a sign of innate immune activation (Figures1(b) 1(c) and 1(d)) Remarkably serum TLR2 was recentlydescribed to correlate with the presence of prosthetic jointinfection in patients undergoing revision of joint arthroplastybecause of suspected local infection [23] thus circulatingTLR2may be considered amarker of the presence of bacterial

components in the blood flow Moreover high levels ofsoluble TLR2 were shown in the sera of patients affected bypsoriasis [21] and systemic lupus erythematosus [25] diseasesin which innate immunity plays a major pathogenetic role[41 42] As such high levels of soluble TLR2 may reflect theactivation of innate inflammatory responses

LPS levels correlated with the serum concentrations ofTLR4 which is the innate immune receptor deputed to LPSrecognition (Figure 2) This may be a sign of an increasedexpression and activation of TLR4 because of LPS binding

Apart from a very modest correlation between LPSand CRP levels LPS TLR2 and TLR4 concentrations didnot appear to be influenced by biochemical and clinicaldisease activity (Figure 3) suggesting that their levels may beinfluenced for the great part by intestinal permeability

Both TLR2 and TLR4 are expressed by platelets andendothelial cells [27ndash29] moreover the binding of theirrespective ligands causes the procoagulatory activation ofthese cell populations More in detail TLR2 signaling inplatelets leads to a thromboinflammatory response throughthe activation of phosphoinositide 3-kinase [43] cyclooxy-genase and purinergic P2Y1 and P2Y12 receptors [44]and alpha-granule release [45] whereas LPS-TLR4 bindingenhances classical agonist-induced platelet aggregation [4647] The TLR2 and TLR4 signaling on endothelial cells

6 Gastroenterology Research and Practice

00 05 10 15 20 250

2

4

6

8

10

LPS (EUmL)

CRP

(mg

dL)

0 2000 4000 6000 80000

2

4

6

8

10

TLR2 (pgmL)

CRP

(mg

dL)

0 2000 4000 6000 80000

2

4

6

8

10

TLR4 (pgmL)

CRP

(mg

dL)r = 0295

P = 0035

(a)

00 05 10 15 20 250

5

10

15

LPS (EUmL)

May

o sc

ore

0 2000 4000 6000 80000

5

10

15

TLR2 (pgmL)

May

o sc

ore

0 2000 4000 6000 80000

5

10

15

TLR4 (pgmL)

May

o sc

ore

(b)

00 05 10 15 20 250

5

10

15

LPS (EUmL)

Har

vey-

Brad

shaw

Inde

x

0 2000 4000 6000 80000

5

10

15

TLR2 (pgmL)

Har

vey-

Brad

shaw

Inde

x

0 1000 2000 3000 4000 50000

5

10

15

TLR4 (pgmL)

Har

vey-

Brad

shaw

Inde

x

(c)

Figure 3 Circulating LPS correlates with biochemical but not clinical activity in IBD patients Concentrations of LPS TLR2 TLR4 andCRP were measured in the sera of IBD (both UC and CD) patients Clinical activity of UC and CD was evaluated using Mayo score andHarvey-Bradshaw Index (a) Correlation between CRP serum levels and circulating LPS TLR2 and TLR4 in IBD patients (b) Correlationbetween Mayo score and circulating LPS TLR2 and TLR4 in UC patients (c) Correlation between Harvey-Bradshaw Index and circulatingLPS TLR2 and TLR4 in CD patients Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 was consideredstatistically significant

strongly activates NF-120581B [5 29] leading to the release ofproinflammatory mediators which can activate the coagula-tory cascade Nonetheless the pathogenesis of atheroscleroticplaques ismediated by the presence of activatedmacrophageswithin the plaque which also respond vigorously to TLRstimulation releasing proinflammatory cytokines [48]

Given themultiplicity of cell types and pathways involvedin the activation of coagulation mediated by TLRs wedecided tomeasure the activation products of the coagulationand fibrinolysis that is the fibrin degradation product D-Dimer and prothrombin fragment F1 + 2 as they describeclosely the overall activation of the coagulatory cascade [30]Remarkably LPS levels significantly correlated with bothD-Dimer and F1 + 2 (Figure 4) clearly suggesting thatcirculating PAMPsmay function as triggers for the activationof coagulation

It may be worthy to note that the correlation betweencirculating LPS and markers of activated coagulation wasstronger when considering only UC patients (Figures 1(d)2(c) and 5) and CD patients with colonic or ileocolonicdisease (L2 and L3 according to Montreal classification [35])(Figure 6)These data suggest that the presence of colonic dis-ease is the key risk factor for coagulatory unbalance that mayfollow bacterial translocation from the gut indeed the colonhas to cope with a significantly greater bacterial burden thanthe rest of the gastrointestinal tract as such it is rational thata break-down of intestinal barrier function in this site mightlead to amore prominent bacterial penetrationtranslocation

Moreover in order to explain the differences we found inUC versus CD other issues should be taken into account CDpatients often display greater signs of systemic inflammationrather than UC as such data coming from CD patients may

Gastroenterology Research and Practice 7

0 500 1000 150000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR2

(pg

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR4

(pg

mL)

r = 0422

P = 0001

(a)

0 200 400 600 800 100000

05

10

15

20

25

LPS

(EU

mL)

0 200 400 600 800 10000

2000

4000

6000

8000TL

R2 (p

gm

L)

0 200 400 600 800 10000

2000

4000

6000

8000

TLR4

(pg

mL)

r = 0440

P = 00008

F1 + 2 (pmolL) F1 + 2 (pmolL) F1 + 2 (pmolL)

(b)

Figure 4 Circulating LPS correlates with markers of activated coagulation in IBD patients Concentrations of LPS TLR2 and TLR4 andlevels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of IBD patients (a) Correlationbetween D-Dimer plasma levels and serum LPS TLR2 and TLR4 in IBD patients (b) Correlation between prothrombin fragment F1+2 andcirculating LPS TLR2 and TLR4 in IBD patients Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 wasconsidered statistically significant

0 500 1000 150000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR2

(pg

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR4

(pg

mL)

r = 0467

P = 0028

r = 0776

r = 0560

P = 0006

P lt 00001

(a)

0 200 400 600 800 100000

05

10

15

20

25

LPS

(EU

mL)

0 200 400 600 800 10000

2000

4000

6000

8000

TLR2

(pg

mL)

0 200 400 600 800 10000

2000

4000

6000

8000

TLR4

(pg

mL)

F1 + 2 (pmolL) F1 + 2 (pmolL) F1 + 2 (pmolL)

r = 0521

P = 0012

r = 0573

P = 0005

(b)

Figure 5 Circulating LPS TLR2 and TLR4 correlate with markers of activated coagulation in UC patients Concentrations of LPS TLR2and TLR4 and levels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of UC patients(a) Correlation between D-Dimer plasma levels and serum LPS TLR2 and TLR4 in UC patients (b) Correlation between prothrombinfragment F1 + 2 and circulating LPS TLR2 and TLR4 in UC patients Statistical analysis was performed by means of Spearman CorrelationTest 119875 lt 005 was considered statistically significant

8 Gastroenterology Research and Practice

0 100 200 300 40000

05

10

15

20

D-dimer (ngmL)

LPS

(EU

mL)

0 200 400 600 800 100000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

CD (L1 only)

0 200 400 600 800 100000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

r = 0435

P = 0042

CD (L2 + L3 only)CD (L1 + L2 + L3)

(a)

0 200 400 600 80000

05

10

15

20

LPS

(EU

mL)

0 100 200 30000

05

10

15

20

25LP

S (E

Um

L)

CD (L1 only)

0 200 400 600 80000

05

10

15

20

25

LPS

(EU

mL)

F1 + 2 (pmolL)F1 + 2 (pmolL)F1 + 2 (pmolL)

r = 0590

P = 0003

CD (L2 + L3 only)CD (L1 + L2 + L3)

(b)

Figure 6 Circulating LPS correlates with markers of activated coagulation in CD patients with colonic involvement Concentrations of LPSand levels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of CD patients (a) Correlationbetween D-Dimer plasma levels and serum LPS in all CD patients (left panel) CD patients with exclusive ileal involvement (L1 accordingto Montreal classification) (central panel) and CD patients with colonic involvement (L2 + L3 according to Montreal classification) (b)Correlation between prothrombin fragment F1 + 2 and circulating LPS in all CD patients (left panel) CD patients with exclusive ilealinvolvement (L1 according to Montreal classification) (central panel) and CD patients with colonic involvement (L2 + L3 according toMontreal classification) Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 was considered statisticallysignificant

suffer from the noise resulting from inflammation-drivenactivation of coagulation as an alternative PAMPs-mediatedcoagulatory activation may have a different relevance orfollow different pathways in UC versus CD also consideringthat genetic data suggest different defects in intestinal barrierfunction in those two diseases [38]

Taken together our data support the novel hypothesisthat intestinal barrier defects contribute to the developmentof a procoagulatory state in IBD Our results need to beconfirmed with future studies in fact the correlations weobserved in human patients do not demonstrate the bio-logical importance of the phenomenon we are postulatingA further limitation of our study is that we are usingvery simple markers in order to explore extremely complexsystems Indeed experiments on animal models of intestinalinflammation are warranted in order to provide the finaldemonstration of our hypothesis and mechanistically dissectit and evaluate its real biologic relevance

Conflict of Interests

None of the authors has any conflict of interests related to thispaperwork to declare

Acknowledgments

The authors acknowledge Dr Rossella Bader for her impor-tant technical support This work was supported by theItalian Ministry of University and Research PRIN Grant2007K4HZEJ 004 (MV)

References

[1] W Rosamond K Flegal K Furie et al ldquoHeart disease andstroke statisticsmdash2008 update a report from the Americanheart association statistics committee and stroke statistics sub-committeerdquo Circulation vol 117 no 4 pp e25ndashe46 2008

[2] C T Esmon ldquoInflammation and thrombosisrdquo Journal ofThrom-bosis and Haemostasis vol 1 no 7 pp 1343ndash1348 2003

[3] J Petaja ldquoInflammation and coagulation An overviewrdquoThrom-bosis Research vol 127 supplement 2 pp S34ndashS37 2011

[4] J Frostegard ldquoImmunity atherosclerosis and cardiovasculardiseaserdquo BMCMedicine vol 11 no 1 article 117 2013

[5] K J Moore F J Sheedy and E A Fisher ldquoMacrophages inatherosclerosis a dynamic balancerdquo Nature Reviews Immunol-ogy vol 13 no 10 pp 709ndash721 2013

[6] B Zoller X Li J Sundquist and K Sundquist ldquoAutoimmunediseases and venous thromboembolism a review of the litera-turerdquo American Journal of Cardiovascular Disease vol 2 no 3pp 171ndash183 2012

Gastroenterology Research and Practice 9

[7] W Miehsler W Reinisch E Valic et al ldquoIs inflammatorybowel disease an independent and disease specific risk factorfor thromboembolismrdquo Gut vol 53 no 4 pp 542ndash548 2004

[8] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[9] L Pastorelli C D Salvo J R Mercado M Vecchi and T TPizarro ldquoCentral role of the gut epithelial barrier in the patho-genesis of chronic intestinal inflammation iessons learned fromanimal models and human geneticsrdquo Frontiers in Immunologyvol 4 article 280 Article ID Article 280 2013

[10] M Zaeem Cader and A Kaser ldquoRecent advances in inflam-matory bowel disease mucosal immune cells in intestinalinflammationrdquo Gut vol 62 no 11 pp 1653ndash1664 2013

[11] S Saibeni L Spina andM Vecchi ldquoExploring the relationshipsbetween inflammatory response and coagulation cascade ininflammatory bowel diseaserdquo European Review for Medical andPharmacological Sciences vol 8 no 5 pp 205ndash208 2004

[12] S Saibeni M Cattaneo M Vecchi et al ldquoLow vitamin B6

plasma levels a risk factor for thrombosis in inflammatorybowel disease role of inflammation and correlation with acutephase reactantsrdquoThe American Journal of Gastroenterology vol98 no 1 pp 112ndash117 2003

[13] M Cattaneo M Vecchi M L Zighetti et al ldquoHigh prevalenceof hyperhomocysteinemia in patients with inflammatory boweldisease a pathogenic link with thromboembolic complica-tionsrdquoThrombosis and Haemostasis vol 80 no 4 pp 542ndash5451998

[14] T H Mogensen ldquoPathogen recognition and inflammatorysignaling in innate immune defensesrdquo Clinical MicrobiologyReviews vol 22 no 2 pp 240ndash273 2009

[15] E LeBouder J E Rey-Nores N K Rushmere et al ldquoSolubleforms of Toll-like receptor (TLR)2 capable of modulating TLR2signaling are present in human plasma and breast milkrdquo Journalof Immunology vol 171 no 12 pp 6680ndash6689 2003

[16] K I Iwami T Matsuguchi A Masuda T Kikuchi TMusikacharoen and Y Yoshikai ldquoCutting edge naturallyoccurring soluble form of mouse toll-like receptor 4 inhibitslipopolysaccharide signalingrdquo Journal of Immunology vol 165no 12 pp 6682ndash6686 2000

[17] I Munoz M P Sepulcre J Meseguer and V Mulero ldquoMolecu-lar cloning phylogenetic analysis and functional characteriza-tion of soluble Toll-like receptor 5 in gilthead seabream Sparusauratardquo Fish amp Shellfish Immunology vol 35 no 1 pp 36ndash452013

[18] S L Zunt L V Burton L I Goldblatt E E Dobbins and MSrinivasan ldquoSoluble forms of Toll-like receptor 4 are present inhuman saliva and modulate tumour necrosis factor-120572 secretionby macrophage-like cellsrdquo Clinical and Experimental Immunol-ogy vol 156 no 2 pp 285ndash293 2009

[19] A-C Raby E le Bouder C Colmont et al ldquoSoluble TLR2reduces inflammation without compromising bacterial clear-ance by disrupting TLR2 triggeringrdquo Journal of Immunologyvol 183 no 1 pp 506ndash517 2009

[20] R-S Chang Y-C Wang and S-T Kao ldquoSoluble toll-likereceptor 4 reversed attenuating effect of Chinese herbal Xiao-Qing-Long-Tang on allergen induced nerve growth factor andthymic stromal lymphopoietinrdquo Experimental and TherapeuticMedicine vol 6 no 5 pp 1199ndash1207 2013

[21] K Kondelkova J Krejsek L Borska et al ldquoMembrane andsoluble Toll-like receptor 2 in patients with psoriasis treatedby Goeckerman therapyrdquo International Journal of Dermatologyvol 53 no 11 pp e512ndashe517 2014

[22] H-B Yang K-Q Xie J-M Deng and S-M Qin ldquoExpressionof soluble toll-like receptors in pleural effusionsrdquo ChineseMedical Journal vol 123 no 16 pp 2225ndash2230 2010

[23] E Galliera L Drago C Vassena et al ldquoToll-like receptor 2in serum a potential diagnostic marker of prosthetic jointinfectionrdquo Journal of Clinical Microbiology vol 52 no 2 pp620ndash623 2013

[24] A Zhao C Blackburn J Chin and M Srinivasan ldquoSoluble tolllike receptor 2 (TLR-2) is increased in saliva of children withdental cariesrdquo BMC Oral Health vol 14 no 1 article 108 2014

[25] M E Houssen R H El-Mahdy and D A Shahin ldquoSerumsoluble toll-like receptor 2 a novel biomarker for systemic lupuserythematosus disease activity and lupus-related cardiovasculardysfunctionrdquo International Journal of Rheumatic Diseases 2014

[26] E Candia D Dıaz-Jimenez P Langjahr et al ldquoIncreasedproduction of soluble TLR2 by lamina propria mononuclearcells from ulcerative colitis patientsrdquo Immunobiology vol 217no 6 pp 634ndash642 2012

[27] F Cognasse H Hamzeh P Chavarin S Acquart C Geninand O Garraud ldquoEvidence of Toll-like receptor molecules onhuman plateletsrdquo Immunology and Cell Biology vol 83 no 2pp 196ndash198 2005

[28] F X Zhang C J Kirschning R Mancinelli et al ldquoBacteriallipopolysaccharide activates nuclear factor-kappaB throughinterleukin-1 signaling mediators in cultured human dermalendothelial cells and mononuclear phagocytesrdquo The Journal ofBiological Chemistry vol 274 no 12 pp 7611ndash7614 1999

[29] E Faure O Equils P A Sieling et al ldquoBacterial lipopolysac-charide activates NF-120581B through toll-like receptor 4 (TLR-4) in cultured human dermal endothelial cells Differentialexpression of TLR-4 andTLR-2 in endothelial cellsrdquoThe Journalof Biological Chemistry vol 275 no 15 pp 11058ndash11063 2000

[30] M D Boisclair H Ireland and D A Lane ldquoAssessment ofhypercoagulable states bymeasurement of activation fragmentsand peptidesrdquo Blood Reviews vol 4 no 1 pp 25ndash40 1990

[31] E F Stange S P L Travis S Vermeire et al ldquoEuropean evi-dence-based Consensus on the diagnosis and management ofulcerative colitis definitions and diagnosisrdquo Journal of Crohnrsquosamp Colitis vol 2 no 1 pp 1ndash23 2008

[32] A Dignass G van Assche J O Lindsay et al ldquoThe secondEuropean evidence-based Consensus on the diagnosis andmanagement of Crohnrsquos disease current managementrdquo Journalof Crohnrsquos amp Colitis vol 4 no 1 pp 28ndash62 2010

[33] R F Harvey and J M Bradshaw ldquoA simple index of Crohnrsquos-disease activityrdquoThe Lancet vol 1 no 8167 p 514 1980

[34] KW SchroederW J Tremaine andDM Ilstrup ldquoCoated oral5-aminosalicylic acid therapy for mildly to moderately activeulcerative colitis a randomized studyrdquoTheNewEngland Journalof Medicine vol 317 no 26 pp 1625ndash1629 1987

[35] J Satsangi M S Silverberg S Vermeire and J-F ColombelldquoThe Montreal classification of inflammatory bowel diseasecontroversies consensus and implicationsrdquo Gut vol 55 no 6pp 749ndash753 2006

[36] R G Shorter K A Huizenga and R J Spencer ldquoA workinghypothesis for the etiology and pathogenesis of nonspecificinflammatory bowel diseaserdquoTheAmerican Journal of DigestiveDiseases vol 17 no 11 pp 1024ndash1032 1972

[37] A I Thompson and C W Lees ldquoGenetics of ulcerative colitisrdquoInflammatory Bowel Diseases vol 17 no 3 pp 831ndash848 2011

[38] C W Lees J C Barrett M Parkes and J Satsangi ldquoNew IBDgenetics common pathways with other diseasesrdquo Gut vol 60no 12 pp 1739ndash1753 2011

[39] G C Nguyen and J Sam ldquoRising prevalence of venous throm-boembolism and its impact on mortality among hospitalized

10 Gastroenterology Research and Practice

inflammatory bowel disease patientsrdquo The American Journal ofGastroenterology vol 103 no 9 pp 2272ndash2280 2008

[40] H Yuhara C Steinmaus D Corley et al ldquoMeta-analysis therisk of venous thromboembolism in patients with inflammatorybowel diseaserdquoAlimentary Pharmacology andTherapeutics vol37 no 10 pp 953ndash962 2013

[41] W R Swindell P E Stuart M K Sarkar et al ldquoCellular dis-section of psoriasis for transcriptome analyses and the post-GWAS erardquo Acta Veterinaria Scandinavica vol 7 article 272014

[42] D Salem R Subang P Laplante J Levine and J Rauch ldquoThedual role of innate immunity in antiphospholipid syndrome andsystemic lupus erythematosusrdquo Lupus vol 23 no 12 pp 1327ndash1331 2014

[43] P Blair S Rex O Vitseva et al ldquoStimulation of Toll-likereceptor 2 in human platelets induces a thromboinflammatoryresponse through activation of phosphoinositide 3-kinaserdquoCirculation Research vol 104 no 3 pp 346ndash354 2009

[44] H Kalvegren C Skoglund C Helldahl M Lerm MGrenegard and T Bengtsson ldquoToll-like receptor 2 stimulationof platelets is mediated by purinergic P2X1-dependent Ca2+mobilisation cyclooxygenase and purinergic P2Y1 and P2Y12receptor activationrdquo Thrombosis and Haemostasis vol 103 no2 pp 398ndash407 2010

[45] S Rex L M Beaulieu D H Perlman et al ldquoImmune versusthrombotic stimulation of platelets differentially regulates sig-nalling pathways intracellular protein-protein interactions and120572-granule releaserdquo Thrombosis and Haemostasis vol 102 no 1pp 97ndash110 2009

[46] G Zhang J Han E J Welch et al ldquoLipopolysaccharide stim-ulates platelet secretion and potentiates platelet aggregationvia TLR4MyD88 and the cGMP-dependent protein kinasepathwayrdquoThe Journal of Immunology vol 182 no 12 pp 7997ndash8004 2009

[47] G T Brown and T MMcIntyre ldquoLipopolysaccharide signalingwithout a nucleus kinase cascades stimulate platelet sheddingof proinflammatory IL-1120573-rich microparticlesrdquo The Journal ofImmunology vol 186 no 9 pp 5489ndash5496 2011

[48] L Chavez-Sanchez J E Espinosa-Luna K Chavez-Rueda MV Legorreta-Haquet E Montoya-Dıaz and F Blanco-FavelaldquoInnate immune system cells in atherosclerosisrdquo Archives ofMedical Research vol 45 no 1 pp 1ndash14 2014

4 Gastroenterology Research and Practice

00

05

10

15

20

25

LPS

(EU

mL)

00

05

10

15

20

25

LPS

(EU

mL)

CON

T

UC

CD

CON

T

CD L

1

CD L

2

CD L

3

P = 0044 P = 0031

(a)

0

2000

4000

6000

8000

TLR2

(pg

mL)

0

2000

4000

6000

8000

TLR2

(pg

mL)

CON

T

UC

CD

CON

T

CD L

1

CD L

2

CD L

3

P = 0002

P = 004P = 0003

P = 0003

(b)

00 05 10 15 20 250

2000

4000

6000

8000

LPS (EUmL)

TLR2

(pg

mL)

(c)

00 05 10 15 20 250

2000

4000

6000

8000

LPS (EUmL)

TLR2

(pg

mL)

00 05 10 15 20 250

2000

4000

6000

8000

LPS (EUmL)

TLR2

(pg

mL)

r = 0495

P = 0016

(d)

Figure 1 Circulating LPS and TLR2 are increased in IBD and correlate with each other in UC Concentrations of LPS and TLR2 weremeasured in the sera of IBD (both UC and CD) patients and healthy controls (CONT) (a) LPS serum levels in CONT UC and CD patients(left panel) and CD locations according to Montreal classification (right panel) (b) TLR2 serum levels in CONT UC and CD patients (leftpanel) and CD locations according to Montreal classification (right panel) (c) Correlation between circulating levels of LPS and TLR2 inIBD patients (d) Correlation between circulating levels of LPS and TLR2 in UC (left panel) and CD (right panel) patients Horizontal barsin (a) and (b) represent median and interquartile range Statistical analysis was performed by means of Mann-Whitney Test and SpearmanCorrelation Test 119875 lt 005 was considered statistically significant

for disease and disease location was performed (data notshown) suggesting that LPS TLR2 and TLR4 levels aremostly independent of disease activity No correlations havebeen observed with the clinical indexes (ie Mayo score andHarvey-Bradshaw Index) (Figure 3)

33 Concentrations of Circulating LPS Correlate with PlasmaLevels of Markers of Activated Coagulation We also mea-sured the plasma concentrations of D-Dimer and prothrom-bin fragment F1 + 2 which are well known markers ofactivated coagulation and are increased in procoagulativestates LPS levels significantly correlated with both D-Dimer(119903 = 0422 119875 = 0001) and F1 + 2 concentrations (119903 =0440 119875 = 00008) (Figure 4) however no correlationwas found between TLR2 or TLR4 and those coagulationmarkers (Figure 4) On the other hand when we analyzedonly data obtained in UC patients we not only confirmedthe correlation between LPS and both D-Dimer (119903 = 0467119875 = 0028) and F1 + 2 (119903 = 0521 119875 = 0012) butalso detected good correlations between TLR2 and D-Dimer(119903 = 0776 119875 lt 00001) TLR4 and D-Dimer (119903 = 0560119875 = 0006) and TLR4 and F1 + 2 (119903 = 0573 119875 = 0005)(Figure 5)When all CDpatientswere considered for analysisno significant correlation was found remarkably when only

patients with colonic disease (L2 and L3) were taken intoaccount correlations between D-Dimer and F1 + 2 and LPSlevels were confirmed (119903 = 0435 119875 = 0042 and 119903 = 0590119875 = 0003 resp) (Figure 6)

4 Discussion

Since 1972 when Shorter et al postulated that a primarydefect in gut permeability and barrier function may leadto the onset of persistent inflammation in the gut and tothe development of IBD [36] a growing body of evidencehas demonstrated that the impairment of intestinal barrierfunction is a common feature in IBD patients leading toderangements in both epithelial permeability to gut antigensand early mucosal innate immune responses protectingfrommicroorganisms penetration andbacterial translocation[8 9] Interestingly genetic studies demonstrated a strongcorrelation between the carriage of polymorphisms of genesregulating intestinal epithelial paracellular permeability andthe risk of developing UC [37 38] whereas being carrier ofinnate immunity-related gene polymorphisms increases thesusceptibility to CD [38]

Patients suffering from IBD and particularly UC [39]are more prone to incur into thromboembolic events [40]

Gastroenterology Research and Practice 5

0

2000

4000

6000

8000

TLR4

(pg

mL)

0

2000

4000

6000

8000

TLR4

(pg

mL)

UC CD CD L1 CD L2 CD L3

(a)

00 05 10 15 20 250

2000

4000

6000

8000

LPS (EUmL)

TLR4

(pg

mL) r = 0421

P = 0001

(b)

00 05 10 15 20 250

1000

2000

3000

4000

5000

LPS (EUmL)

TLR4

(pg

mL)

00 05 10 15 20 250

2000

4000

6000

8000

LPS (EUmL)

TLR4

(pg

mL)

00 05 10 15 200

1000

2000

3000

4000

5000

LPS (EUmL)

TLR4

(pg

mL)

r = 0512

P = 0008

r = 0332

P = 0054

r = 0468

P = 0024

(c)

Figure 2 TLR4 serum correlates with circulating LPS in IBD patients Concentrations of LPS and TLR4 were measured in the sera of IBD(both UC and CD) patients (a) TLR4 serum levels in UC and CD patients (left panel) and CD locations according to Montreal classification(right panel) (b) Correlation between circulating levels of LPS and TLR4 in IBD patients (c) Correlation between circulating levels of LPSand TLR4 in UC (left panel) CD (central panel) patients and CD patients with colonic and ileocolonic involvement (L2 + L3 according toMontreal classification) (right panel) Horizontal bars in (a) represent median and interquartile range Statistical analysis was performed bymeans of Spearman Correlation Test 119875 lt 005 was considered statistically significant

similarly to patients affected by other inflammatory andimmune-related disorders [6] however the only presence ofan ongoing inflammatory process does not explain by itselfthis phenomenon In fact it has been shown that the riskof thromboembolism is more elevated in IBD than rheuma-toid arthritis a disease often characterized by a prominentsystemic inflammation [7] thus suggesting that some gutspecific mechanisms may be involved Besides deficienciesof vitamins involved in the proper regulation of coagulatoryhomeostasis such as vitamin B6 vitamin B12 and folic acid[11ndash13] the alteration of intestinal barrier function may be acofactor promoting a procoagulative state in IBD

The data presented in this paper strongly support thisnovel hypothesis In fact in order to assess intestinal perme-ability to PAMPs and bacterial translocation from the gut wemeasured circulating LPS and found higher concentrationsof this bacterial component in the sera of IBD patients (Fig-ure 1(a)) Consistent with the data previously presented byCandia et al [26] IBD patients also presented higher serumlevels of TLR2 as a sign of innate immune activation (Figures1(b) 1(c) and 1(d)) Remarkably serum TLR2 was recentlydescribed to correlate with the presence of prosthetic jointinfection in patients undergoing revision of joint arthroplastybecause of suspected local infection [23] thus circulatingTLR2may be considered amarker of the presence of bacterial

components in the blood flow Moreover high levels ofsoluble TLR2 were shown in the sera of patients affected bypsoriasis [21] and systemic lupus erythematosus [25] diseasesin which innate immunity plays a major pathogenetic role[41 42] As such high levels of soluble TLR2 may reflect theactivation of innate inflammatory responses

LPS levels correlated with the serum concentrations ofTLR4 which is the innate immune receptor deputed to LPSrecognition (Figure 2) This may be a sign of an increasedexpression and activation of TLR4 because of LPS binding

Apart from a very modest correlation between LPSand CRP levels LPS TLR2 and TLR4 concentrations didnot appear to be influenced by biochemical and clinicaldisease activity (Figure 3) suggesting that their levels may beinfluenced for the great part by intestinal permeability

Both TLR2 and TLR4 are expressed by platelets andendothelial cells [27ndash29] moreover the binding of theirrespective ligands causes the procoagulatory activation ofthese cell populations More in detail TLR2 signaling inplatelets leads to a thromboinflammatory response throughthe activation of phosphoinositide 3-kinase [43] cyclooxy-genase and purinergic P2Y1 and P2Y12 receptors [44]and alpha-granule release [45] whereas LPS-TLR4 bindingenhances classical agonist-induced platelet aggregation [4647] The TLR2 and TLR4 signaling on endothelial cells

6 Gastroenterology Research and Practice

00 05 10 15 20 250

2

4

6

8

10

LPS (EUmL)

CRP

(mg

dL)

0 2000 4000 6000 80000

2

4

6

8

10

TLR2 (pgmL)

CRP

(mg

dL)

0 2000 4000 6000 80000

2

4

6

8

10

TLR4 (pgmL)

CRP

(mg

dL)r = 0295

P = 0035

(a)

00 05 10 15 20 250

5

10

15

LPS (EUmL)

May

o sc

ore

0 2000 4000 6000 80000

5

10

15

TLR2 (pgmL)

May

o sc

ore

0 2000 4000 6000 80000

5

10

15

TLR4 (pgmL)

May

o sc

ore

(b)

00 05 10 15 20 250

5

10

15

LPS (EUmL)

Har

vey-

Brad

shaw

Inde

x

0 2000 4000 6000 80000

5

10

15

TLR2 (pgmL)

Har

vey-

Brad

shaw

Inde

x

0 1000 2000 3000 4000 50000

5

10

15

TLR4 (pgmL)

Har

vey-

Brad

shaw

Inde

x

(c)

Figure 3 Circulating LPS correlates with biochemical but not clinical activity in IBD patients Concentrations of LPS TLR2 TLR4 andCRP were measured in the sera of IBD (both UC and CD) patients Clinical activity of UC and CD was evaluated using Mayo score andHarvey-Bradshaw Index (a) Correlation between CRP serum levels and circulating LPS TLR2 and TLR4 in IBD patients (b) Correlationbetween Mayo score and circulating LPS TLR2 and TLR4 in UC patients (c) Correlation between Harvey-Bradshaw Index and circulatingLPS TLR2 and TLR4 in CD patients Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 was consideredstatistically significant

strongly activates NF-120581B [5 29] leading to the release ofproinflammatory mediators which can activate the coagula-tory cascade Nonetheless the pathogenesis of atheroscleroticplaques ismediated by the presence of activatedmacrophageswithin the plaque which also respond vigorously to TLRstimulation releasing proinflammatory cytokines [48]

Given themultiplicity of cell types and pathways involvedin the activation of coagulation mediated by TLRs wedecided tomeasure the activation products of the coagulationand fibrinolysis that is the fibrin degradation product D-Dimer and prothrombin fragment F1 + 2 as they describeclosely the overall activation of the coagulatory cascade [30]Remarkably LPS levels significantly correlated with bothD-Dimer and F1 + 2 (Figure 4) clearly suggesting thatcirculating PAMPsmay function as triggers for the activationof coagulation

It may be worthy to note that the correlation betweencirculating LPS and markers of activated coagulation wasstronger when considering only UC patients (Figures 1(d)2(c) and 5) and CD patients with colonic or ileocolonicdisease (L2 and L3 according to Montreal classification [35])(Figure 6)These data suggest that the presence of colonic dis-ease is the key risk factor for coagulatory unbalance that mayfollow bacterial translocation from the gut indeed the colonhas to cope with a significantly greater bacterial burden thanthe rest of the gastrointestinal tract as such it is rational thata break-down of intestinal barrier function in this site mightlead to amore prominent bacterial penetrationtranslocation

Moreover in order to explain the differences we found inUC versus CD other issues should be taken into account CDpatients often display greater signs of systemic inflammationrather than UC as such data coming from CD patients may

Gastroenterology Research and Practice 7

0 500 1000 150000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR2

(pg

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR4

(pg

mL)

r = 0422

P = 0001

(a)

0 200 400 600 800 100000

05

10

15

20

25

LPS

(EU

mL)

0 200 400 600 800 10000

2000

4000

6000

8000TL

R2 (p

gm

L)

0 200 400 600 800 10000

2000

4000

6000

8000

TLR4

(pg

mL)

r = 0440

P = 00008

F1 + 2 (pmolL) F1 + 2 (pmolL) F1 + 2 (pmolL)

(b)

Figure 4 Circulating LPS correlates with markers of activated coagulation in IBD patients Concentrations of LPS TLR2 and TLR4 andlevels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of IBD patients (a) Correlationbetween D-Dimer plasma levels and serum LPS TLR2 and TLR4 in IBD patients (b) Correlation between prothrombin fragment F1+2 andcirculating LPS TLR2 and TLR4 in IBD patients Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 wasconsidered statistically significant

0 500 1000 150000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR2

(pg

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR4

(pg

mL)

r = 0467

P = 0028

r = 0776

r = 0560

P = 0006

P lt 00001

(a)

0 200 400 600 800 100000

05

10

15

20

25

LPS

(EU

mL)

0 200 400 600 800 10000

2000

4000

6000

8000

TLR2

(pg

mL)

0 200 400 600 800 10000

2000

4000

6000

8000

TLR4

(pg

mL)

F1 + 2 (pmolL) F1 + 2 (pmolL) F1 + 2 (pmolL)

r = 0521

P = 0012

r = 0573

P = 0005

(b)

Figure 5 Circulating LPS TLR2 and TLR4 correlate with markers of activated coagulation in UC patients Concentrations of LPS TLR2and TLR4 and levels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of UC patients(a) Correlation between D-Dimer plasma levels and serum LPS TLR2 and TLR4 in UC patients (b) Correlation between prothrombinfragment F1 + 2 and circulating LPS TLR2 and TLR4 in UC patients Statistical analysis was performed by means of Spearman CorrelationTest 119875 lt 005 was considered statistically significant

8 Gastroenterology Research and Practice

0 100 200 300 40000

05

10

15

20

D-dimer (ngmL)

LPS

(EU

mL)

0 200 400 600 800 100000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

CD (L1 only)

0 200 400 600 800 100000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

r = 0435

P = 0042

CD (L2 + L3 only)CD (L1 + L2 + L3)

(a)

0 200 400 600 80000

05

10

15

20

LPS

(EU

mL)

0 100 200 30000

05

10

15

20

25LP

S (E

Um

L)

CD (L1 only)

0 200 400 600 80000

05

10

15

20

25

LPS

(EU

mL)

F1 + 2 (pmolL)F1 + 2 (pmolL)F1 + 2 (pmolL)

r = 0590

P = 0003

CD (L2 + L3 only)CD (L1 + L2 + L3)

(b)

Figure 6 Circulating LPS correlates with markers of activated coagulation in CD patients with colonic involvement Concentrations of LPSand levels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of CD patients (a) Correlationbetween D-Dimer plasma levels and serum LPS in all CD patients (left panel) CD patients with exclusive ileal involvement (L1 accordingto Montreal classification) (central panel) and CD patients with colonic involvement (L2 + L3 according to Montreal classification) (b)Correlation between prothrombin fragment F1 + 2 and circulating LPS in all CD patients (left panel) CD patients with exclusive ilealinvolvement (L1 according to Montreal classification) (central panel) and CD patients with colonic involvement (L2 + L3 according toMontreal classification) Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 was considered statisticallysignificant

suffer from the noise resulting from inflammation-drivenactivation of coagulation as an alternative PAMPs-mediatedcoagulatory activation may have a different relevance orfollow different pathways in UC versus CD also consideringthat genetic data suggest different defects in intestinal barrierfunction in those two diseases [38]

Taken together our data support the novel hypothesisthat intestinal barrier defects contribute to the developmentof a procoagulatory state in IBD Our results need to beconfirmed with future studies in fact the correlations weobserved in human patients do not demonstrate the bio-logical importance of the phenomenon we are postulatingA further limitation of our study is that we are usingvery simple markers in order to explore extremely complexsystems Indeed experiments on animal models of intestinalinflammation are warranted in order to provide the finaldemonstration of our hypothesis and mechanistically dissectit and evaluate its real biologic relevance

Conflict of Interests

None of the authors has any conflict of interests related to thispaperwork to declare

Acknowledgments

The authors acknowledge Dr Rossella Bader for her impor-tant technical support This work was supported by theItalian Ministry of University and Research PRIN Grant2007K4HZEJ 004 (MV)

References

[1] W Rosamond K Flegal K Furie et al ldquoHeart disease andstroke statisticsmdash2008 update a report from the Americanheart association statistics committee and stroke statistics sub-committeerdquo Circulation vol 117 no 4 pp e25ndashe46 2008

[2] C T Esmon ldquoInflammation and thrombosisrdquo Journal ofThrom-bosis and Haemostasis vol 1 no 7 pp 1343ndash1348 2003

[3] J Petaja ldquoInflammation and coagulation An overviewrdquoThrom-bosis Research vol 127 supplement 2 pp S34ndashS37 2011

[4] J Frostegard ldquoImmunity atherosclerosis and cardiovasculardiseaserdquo BMCMedicine vol 11 no 1 article 117 2013

[5] K J Moore F J Sheedy and E A Fisher ldquoMacrophages inatherosclerosis a dynamic balancerdquo Nature Reviews Immunol-ogy vol 13 no 10 pp 709ndash721 2013

[6] B Zoller X Li J Sundquist and K Sundquist ldquoAutoimmunediseases and venous thromboembolism a review of the litera-turerdquo American Journal of Cardiovascular Disease vol 2 no 3pp 171ndash183 2012

Gastroenterology Research and Practice 9

[7] W Miehsler W Reinisch E Valic et al ldquoIs inflammatorybowel disease an independent and disease specific risk factorfor thromboembolismrdquo Gut vol 53 no 4 pp 542ndash548 2004

[8] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[9] L Pastorelli C D Salvo J R Mercado M Vecchi and T TPizarro ldquoCentral role of the gut epithelial barrier in the patho-genesis of chronic intestinal inflammation iessons learned fromanimal models and human geneticsrdquo Frontiers in Immunologyvol 4 article 280 Article ID Article 280 2013

[10] M Zaeem Cader and A Kaser ldquoRecent advances in inflam-matory bowel disease mucosal immune cells in intestinalinflammationrdquo Gut vol 62 no 11 pp 1653ndash1664 2013

[11] S Saibeni L Spina andM Vecchi ldquoExploring the relationshipsbetween inflammatory response and coagulation cascade ininflammatory bowel diseaserdquo European Review for Medical andPharmacological Sciences vol 8 no 5 pp 205ndash208 2004

[12] S Saibeni M Cattaneo M Vecchi et al ldquoLow vitamin B6

plasma levels a risk factor for thrombosis in inflammatorybowel disease role of inflammation and correlation with acutephase reactantsrdquoThe American Journal of Gastroenterology vol98 no 1 pp 112ndash117 2003

[13] M Cattaneo M Vecchi M L Zighetti et al ldquoHigh prevalenceof hyperhomocysteinemia in patients with inflammatory boweldisease a pathogenic link with thromboembolic complica-tionsrdquoThrombosis and Haemostasis vol 80 no 4 pp 542ndash5451998

[14] T H Mogensen ldquoPathogen recognition and inflammatorysignaling in innate immune defensesrdquo Clinical MicrobiologyReviews vol 22 no 2 pp 240ndash273 2009

[15] E LeBouder J E Rey-Nores N K Rushmere et al ldquoSolubleforms of Toll-like receptor (TLR)2 capable of modulating TLR2signaling are present in human plasma and breast milkrdquo Journalof Immunology vol 171 no 12 pp 6680ndash6689 2003

[16] K I Iwami T Matsuguchi A Masuda T Kikuchi TMusikacharoen and Y Yoshikai ldquoCutting edge naturallyoccurring soluble form of mouse toll-like receptor 4 inhibitslipopolysaccharide signalingrdquo Journal of Immunology vol 165no 12 pp 6682ndash6686 2000

[17] I Munoz M P Sepulcre J Meseguer and V Mulero ldquoMolecu-lar cloning phylogenetic analysis and functional characteriza-tion of soluble Toll-like receptor 5 in gilthead seabream Sparusauratardquo Fish amp Shellfish Immunology vol 35 no 1 pp 36ndash452013

[18] S L Zunt L V Burton L I Goldblatt E E Dobbins and MSrinivasan ldquoSoluble forms of Toll-like receptor 4 are present inhuman saliva and modulate tumour necrosis factor-120572 secretionby macrophage-like cellsrdquo Clinical and Experimental Immunol-ogy vol 156 no 2 pp 285ndash293 2009

[19] A-C Raby E le Bouder C Colmont et al ldquoSoluble TLR2reduces inflammation without compromising bacterial clear-ance by disrupting TLR2 triggeringrdquo Journal of Immunologyvol 183 no 1 pp 506ndash517 2009

[20] R-S Chang Y-C Wang and S-T Kao ldquoSoluble toll-likereceptor 4 reversed attenuating effect of Chinese herbal Xiao-Qing-Long-Tang on allergen induced nerve growth factor andthymic stromal lymphopoietinrdquo Experimental and TherapeuticMedicine vol 6 no 5 pp 1199ndash1207 2013

[21] K Kondelkova J Krejsek L Borska et al ldquoMembrane andsoluble Toll-like receptor 2 in patients with psoriasis treatedby Goeckerman therapyrdquo International Journal of Dermatologyvol 53 no 11 pp e512ndashe517 2014

[22] H-B Yang K-Q Xie J-M Deng and S-M Qin ldquoExpressionof soluble toll-like receptors in pleural effusionsrdquo ChineseMedical Journal vol 123 no 16 pp 2225ndash2230 2010

[23] E Galliera L Drago C Vassena et al ldquoToll-like receptor 2in serum a potential diagnostic marker of prosthetic jointinfectionrdquo Journal of Clinical Microbiology vol 52 no 2 pp620ndash623 2013

[24] A Zhao C Blackburn J Chin and M Srinivasan ldquoSoluble tolllike receptor 2 (TLR-2) is increased in saliva of children withdental cariesrdquo BMC Oral Health vol 14 no 1 article 108 2014

[25] M E Houssen R H El-Mahdy and D A Shahin ldquoSerumsoluble toll-like receptor 2 a novel biomarker for systemic lupuserythematosus disease activity and lupus-related cardiovasculardysfunctionrdquo International Journal of Rheumatic Diseases 2014

[26] E Candia D Dıaz-Jimenez P Langjahr et al ldquoIncreasedproduction of soluble TLR2 by lamina propria mononuclearcells from ulcerative colitis patientsrdquo Immunobiology vol 217no 6 pp 634ndash642 2012

[27] F Cognasse H Hamzeh P Chavarin S Acquart C Geninand O Garraud ldquoEvidence of Toll-like receptor molecules onhuman plateletsrdquo Immunology and Cell Biology vol 83 no 2pp 196ndash198 2005

[28] F X Zhang C J Kirschning R Mancinelli et al ldquoBacteriallipopolysaccharide activates nuclear factor-kappaB throughinterleukin-1 signaling mediators in cultured human dermalendothelial cells and mononuclear phagocytesrdquo The Journal ofBiological Chemistry vol 274 no 12 pp 7611ndash7614 1999

[29] E Faure O Equils P A Sieling et al ldquoBacterial lipopolysac-charide activates NF-120581B through toll-like receptor 4 (TLR-4) in cultured human dermal endothelial cells Differentialexpression of TLR-4 andTLR-2 in endothelial cellsrdquoThe Journalof Biological Chemistry vol 275 no 15 pp 11058ndash11063 2000

[30] M D Boisclair H Ireland and D A Lane ldquoAssessment ofhypercoagulable states bymeasurement of activation fragmentsand peptidesrdquo Blood Reviews vol 4 no 1 pp 25ndash40 1990

[31] E F Stange S P L Travis S Vermeire et al ldquoEuropean evi-dence-based Consensus on the diagnosis and management ofulcerative colitis definitions and diagnosisrdquo Journal of Crohnrsquosamp Colitis vol 2 no 1 pp 1ndash23 2008

[32] A Dignass G van Assche J O Lindsay et al ldquoThe secondEuropean evidence-based Consensus on the diagnosis andmanagement of Crohnrsquos disease current managementrdquo Journalof Crohnrsquos amp Colitis vol 4 no 1 pp 28ndash62 2010

[33] R F Harvey and J M Bradshaw ldquoA simple index of Crohnrsquos-disease activityrdquoThe Lancet vol 1 no 8167 p 514 1980

[34] KW SchroederW J Tremaine andDM Ilstrup ldquoCoated oral5-aminosalicylic acid therapy for mildly to moderately activeulcerative colitis a randomized studyrdquoTheNewEngland Journalof Medicine vol 317 no 26 pp 1625ndash1629 1987

[35] J Satsangi M S Silverberg S Vermeire and J-F ColombelldquoThe Montreal classification of inflammatory bowel diseasecontroversies consensus and implicationsrdquo Gut vol 55 no 6pp 749ndash753 2006

[36] R G Shorter K A Huizenga and R J Spencer ldquoA workinghypothesis for the etiology and pathogenesis of nonspecificinflammatory bowel diseaserdquoTheAmerican Journal of DigestiveDiseases vol 17 no 11 pp 1024ndash1032 1972

[37] A I Thompson and C W Lees ldquoGenetics of ulcerative colitisrdquoInflammatory Bowel Diseases vol 17 no 3 pp 831ndash848 2011

[38] C W Lees J C Barrett M Parkes and J Satsangi ldquoNew IBDgenetics common pathways with other diseasesrdquo Gut vol 60no 12 pp 1739ndash1753 2011

[39] G C Nguyen and J Sam ldquoRising prevalence of venous throm-boembolism and its impact on mortality among hospitalized

10 Gastroenterology Research and Practice

inflammatory bowel disease patientsrdquo The American Journal ofGastroenterology vol 103 no 9 pp 2272ndash2280 2008

[40] H Yuhara C Steinmaus D Corley et al ldquoMeta-analysis therisk of venous thromboembolism in patients with inflammatorybowel diseaserdquoAlimentary Pharmacology andTherapeutics vol37 no 10 pp 953ndash962 2013

[41] W R Swindell P E Stuart M K Sarkar et al ldquoCellular dis-section of psoriasis for transcriptome analyses and the post-GWAS erardquo Acta Veterinaria Scandinavica vol 7 article 272014

[42] D Salem R Subang P Laplante J Levine and J Rauch ldquoThedual role of innate immunity in antiphospholipid syndrome andsystemic lupus erythematosusrdquo Lupus vol 23 no 12 pp 1327ndash1331 2014

[43] P Blair S Rex O Vitseva et al ldquoStimulation of Toll-likereceptor 2 in human platelets induces a thromboinflammatoryresponse through activation of phosphoinositide 3-kinaserdquoCirculation Research vol 104 no 3 pp 346ndash354 2009

[44] H Kalvegren C Skoglund C Helldahl M Lerm MGrenegard and T Bengtsson ldquoToll-like receptor 2 stimulationof platelets is mediated by purinergic P2X1-dependent Ca2+mobilisation cyclooxygenase and purinergic P2Y1 and P2Y12receptor activationrdquo Thrombosis and Haemostasis vol 103 no2 pp 398ndash407 2010

[45] S Rex L M Beaulieu D H Perlman et al ldquoImmune versusthrombotic stimulation of platelets differentially regulates sig-nalling pathways intracellular protein-protein interactions and120572-granule releaserdquo Thrombosis and Haemostasis vol 102 no 1pp 97ndash110 2009

[46] G Zhang J Han E J Welch et al ldquoLipopolysaccharide stim-ulates platelet secretion and potentiates platelet aggregationvia TLR4MyD88 and the cGMP-dependent protein kinasepathwayrdquoThe Journal of Immunology vol 182 no 12 pp 7997ndash8004 2009

[47] G T Brown and T MMcIntyre ldquoLipopolysaccharide signalingwithout a nucleus kinase cascades stimulate platelet sheddingof proinflammatory IL-1120573-rich microparticlesrdquo The Journal ofImmunology vol 186 no 9 pp 5489ndash5496 2011

[48] L Chavez-Sanchez J E Espinosa-Luna K Chavez-Rueda MV Legorreta-Haquet E Montoya-Dıaz and F Blanco-FavelaldquoInnate immune system cells in atherosclerosisrdquo Archives ofMedical Research vol 45 no 1 pp 1ndash14 2014

Gastroenterology Research and Practice 5

0

2000

4000

6000

8000

TLR4

(pg

mL)

0

2000

4000

6000

8000

TLR4

(pg

mL)

UC CD CD L1 CD L2 CD L3

(a)

00 05 10 15 20 250

2000

4000

6000

8000

LPS (EUmL)

TLR4

(pg

mL) r = 0421

P = 0001

(b)

00 05 10 15 20 250

1000

2000

3000

4000

5000

LPS (EUmL)

TLR4

(pg

mL)

00 05 10 15 20 250

2000

4000

6000

8000

LPS (EUmL)

TLR4

(pg

mL)

00 05 10 15 200

1000

2000

3000

4000

5000

LPS (EUmL)

TLR4

(pg

mL)

r = 0512

P = 0008

r = 0332

P = 0054

r = 0468

P = 0024

(c)

Figure 2 TLR4 serum correlates with circulating LPS in IBD patients Concentrations of LPS and TLR4 were measured in the sera of IBD(both UC and CD) patients (a) TLR4 serum levels in UC and CD patients (left panel) and CD locations according to Montreal classification(right panel) (b) Correlation between circulating levels of LPS and TLR4 in IBD patients (c) Correlation between circulating levels of LPSand TLR4 in UC (left panel) CD (central panel) patients and CD patients with colonic and ileocolonic involvement (L2 + L3 according toMontreal classification) (right panel) Horizontal bars in (a) represent median and interquartile range Statistical analysis was performed bymeans of Spearman Correlation Test 119875 lt 005 was considered statistically significant

similarly to patients affected by other inflammatory andimmune-related disorders [6] however the only presence ofan ongoing inflammatory process does not explain by itselfthis phenomenon In fact it has been shown that the riskof thromboembolism is more elevated in IBD than rheuma-toid arthritis a disease often characterized by a prominentsystemic inflammation [7] thus suggesting that some gutspecific mechanisms may be involved Besides deficienciesof vitamins involved in the proper regulation of coagulatoryhomeostasis such as vitamin B6 vitamin B12 and folic acid[11ndash13] the alteration of intestinal barrier function may be acofactor promoting a procoagulative state in IBD

The data presented in this paper strongly support thisnovel hypothesis In fact in order to assess intestinal perme-ability to PAMPs and bacterial translocation from the gut wemeasured circulating LPS and found higher concentrationsof this bacterial component in the sera of IBD patients (Fig-ure 1(a)) Consistent with the data previously presented byCandia et al [26] IBD patients also presented higher serumlevels of TLR2 as a sign of innate immune activation (Figures1(b) 1(c) and 1(d)) Remarkably serum TLR2 was recentlydescribed to correlate with the presence of prosthetic jointinfection in patients undergoing revision of joint arthroplastybecause of suspected local infection [23] thus circulatingTLR2may be considered amarker of the presence of bacterial

components in the blood flow Moreover high levels ofsoluble TLR2 were shown in the sera of patients affected bypsoriasis [21] and systemic lupus erythematosus [25] diseasesin which innate immunity plays a major pathogenetic role[41 42] As such high levels of soluble TLR2 may reflect theactivation of innate inflammatory responses

LPS levels correlated with the serum concentrations ofTLR4 which is the innate immune receptor deputed to LPSrecognition (Figure 2) This may be a sign of an increasedexpression and activation of TLR4 because of LPS binding

Apart from a very modest correlation between LPSand CRP levels LPS TLR2 and TLR4 concentrations didnot appear to be influenced by biochemical and clinicaldisease activity (Figure 3) suggesting that their levels may beinfluenced for the great part by intestinal permeability

Both TLR2 and TLR4 are expressed by platelets andendothelial cells [27ndash29] moreover the binding of theirrespective ligands causes the procoagulatory activation ofthese cell populations More in detail TLR2 signaling inplatelets leads to a thromboinflammatory response throughthe activation of phosphoinositide 3-kinase [43] cyclooxy-genase and purinergic P2Y1 and P2Y12 receptors [44]and alpha-granule release [45] whereas LPS-TLR4 bindingenhances classical agonist-induced platelet aggregation [4647] The TLR2 and TLR4 signaling on endothelial cells

6 Gastroenterology Research and Practice

00 05 10 15 20 250

2

4

6

8

10

LPS (EUmL)

CRP

(mg

dL)

0 2000 4000 6000 80000

2

4

6

8

10

TLR2 (pgmL)

CRP

(mg

dL)

0 2000 4000 6000 80000

2

4

6

8

10

TLR4 (pgmL)

CRP

(mg

dL)r = 0295

P = 0035

(a)

00 05 10 15 20 250

5

10

15

LPS (EUmL)

May

o sc

ore

0 2000 4000 6000 80000

5

10

15

TLR2 (pgmL)

May

o sc

ore

0 2000 4000 6000 80000

5

10

15

TLR4 (pgmL)

May

o sc

ore

(b)

00 05 10 15 20 250

5

10

15

LPS (EUmL)

Har

vey-

Brad

shaw

Inde

x

0 2000 4000 6000 80000

5

10

15

TLR2 (pgmL)

Har

vey-

Brad

shaw

Inde

x

0 1000 2000 3000 4000 50000

5

10

15

TLR4 (pgmL)

Har

vey-

Brad

shaw

Inde

x

(c)

Figure 3 Circulating LPS correlates with biochemical but not clinical activity in IBD patients Concentrations of LPS TLR2 TLR4 andCRP were measured in the sera of IBD (both UC and CD) patients Clinical activity of UC and CD was evaluated using Mayo score andHarvey-Bradshaw Index (a) Correlation between CRP serum levels and circulating LPS TLR2 and TLR4 in IBD patients (b) Correlationbetween Mayo score and circulating LPS TLR2 and TLR4 in UC patients (c) Correlation between Harvey-Bradshaw Index and circulatingLPS TLR2 and TLR4 in CD patients Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 was consideredstatistically significant

strongly activates NF-120581B [5 29] leading to the release ofproinflammatory mediators which can activate the coagula-tory cascade Nonetheless the pathogenesis of atheroscleroticplaques ismediated by the presence of activatedmacrophageswithin the plaque which also respond vigorously to TLRstimulation releasing proinflammatory cytokines [48]

Given themultiplicity of cell types and pathways involvedin the activation of coagulation mediated by TLRs wedecided tomeasure the activation products of the coagulationand fibrinolysis that is the fibrin degradation product D-Dimer and prothrombin fragment F1 + 2 as they describeclosely the overall activation of the coagulatory cascade [30]Remarkably LPS levels significantly correlated with bothD-Dimer and F1 + 2 (Figure 4) clearly suggesting thatcirculating PAMPsmay function as triggers for the activationof coagulation

It may be worthy to note that the correlation betweencirculating LPS and markers of activated coagulation wasstronger when considering only UC patients (Figures 1(d)2(c) and 5) and CD patients with colonic or ileocolonicdisease (L2 and L3 according to Montreal classification [35])(Figure 6)These data suggest that the presence of colonic dis-ease is the key risk factor for coagulatory unbalance that mayfollow bacterial translocation from the gut indeed the colonhas to cope with a significantly greater bacterial burden thanthe rest of the gastrointestinal tract as such it is rational thata break-down of intestinal barrier function in this site mightlead to amore prominent bacterial penetrationtranslocation

Moreover in order to explain the differences we found inUC versus CD other issues should be taken into account CDpatients often display greater signs of systemic inflammationrather than UC as such data coming from CD patients may

Gastroenterology Research and Practice 7

0 500 1000 150000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR2

(pg

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR4

(pg

mL)

r = 0422

P = 0001

(a)

0 200 400 600 800 100000

05

10

15

20

25

LPS

(EU

mL)

0 200 400 600 800 10000

2000

4000

6000

8000TL

R2 (p

gm

L)

0 200 400 600 800 10000

2000

4000

6000

8000

TLR4

(pg

mL)

r = 0440

P = 00008

F1 + 2 (pmolL) F1 + 2 (pmolL) F1 + 2 (pmolL)

(b)

Figure 4 Circulating LPS correlates with markers of activated coagulation in IBD patients Concentrations of LPS TLR2 and TLR4 andlevels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of IBD patients (a) Correlationbetween D-Dimer plasma levels and serum LPS TLR2 and TLR4 in IBD patients (b) Correlation between prothrombin fragment F1+2 andcirculating LPS TLR2 and TLR4 in IBD patients Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 wasconsidered statistically significant

0 500 1000 150000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR2

(pg

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR4

(pg

mL)

r = 0467

P = 0028

r = 0776

r = 0560

P = 0006

P lt 00001

(a)

0 200 400 600 800 100000

05

10

15

20

25

LPS

(EU

mL)

0 200 400 600 800 10000

2000

4000

6000

8000

TLR2

(pg

mL)

0 200 400 600 800 10000

2000

4000

6000

8000

TLR4

(pg

mL)

F1 + 2 (pmolL) F1 + 2 (pmolL) F1 + 2 (pmolL)

r = 0521

P = 0012

r = 0573

P = 0005

(b)

Figure 5 Circulating LPS TLR2 and TLR4 correlate with markers of activated coagulation in UC patients Concentrations of LPS TLR2and TLR4 and levels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of UC patients(a) Correlation between D-Dimer plasma levels and serum LPS TLR2 and TLR4 in UC patients (b) Correlation between prothrombinfragment F1 + 2 and circulating LPS TLR2 and TLR4 in UC patients Statistical analysis was performed by means of Spearman CorrelationTest 119875 lt 005 was considered statistically significant

8 Gastroenterology Research and Practice

0 100 200 300 40000

05

10

15

20

D-dimer (ngmL)

LPS

(EU

mL)

0 200 400 600 800 100000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

CD (L1 only)

0 200 400 600 800 100000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

r = 0435

P = 0042

CD (L2 + L3 only)CD (L1 + L2 + L3)

(a)

0 200 400 600 80000

05

10

15

20

LPS

(EU

mL)

0 100 200 30000

05

10

15

20

25LP

S (E

Um

L)

CD (L1 only)

0 200 400 600 80000

05

10

15

20

25

LPS

(EU

mL)

F1 + 2 (pmolL)F1 + 2 (pmolL)F1 + 2 (pmolL)

r = 0590

P = 0003

CD (L2 + L3 only)CD (L1 + L2 + L3)

(b)

Figure 6 Circulating LPS correlates with markers of activated coagulation in CD patients with colonic involvement Concentrations of LPSand levels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of CD patients (a) Correlationbetween D-Dimer plasma levels and serum LPS in all CD patients (left panel) CD patients with exclusive ileal involvement (L1 accordingto Montreal classification) (central panel) and CD patients with colonic involvement (L2 + L3 according to Montreal classification) (b)Correlation between prothrombin fragment F1 + 2 and circulating LPS in all CD patients (left panel) CD patients with exclusive ilealinvolvement (L1 according to Montreal classification) (central panel) and CD patients with colonic involvement (L2 + L3 according toMontreal classification) Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 was considered statisticallysignificant

suffer from the noise resulting from inflammation-drivenactivation of coagulation as an alternative PAMPs-mediatedcoagulatory activation may have a different relevance orfollow different pathways in UC versus CD also consideringthat genetic data suggest different defects in intestinal barrierfunction in those two diseases [38]

Taken together our data support the novel hypothesisthat intestinal barrier defects contribute to the developmentof a procoagulatory state in IBD Our results need to beconfirmed with future studies in fact the correlations weobserved in human patients do not demonstrate the bio-logical importance of the phenomenon we are postulatingA further limitation of our study is that we are usingvery simple markers in order to explore extremely complexsystems Indeed experiments on animal models of intestinalinflammation are warranted in order to provide the finaldemonstration of our hypothesis and mechanistically dissectit and evaluate its real biologic relevance

Conflict of Interests

None of the authors has any conflict of interests related to thispaperwork to declare

Acknowledgments

The authors acknowledge Dr Rossella Bader for her impor-tant technical support This work was supported by theItalian Ministry of University and Research PRIN Grant2007K4HZEJ 004 (MV)

References

[1] W Rosamond K Flegal K Furie et al ldquoHeart disease andstroke statisticsmdash2008 update a report from the Americanheart association statistics committee and stroke statistics sub-committeerdquo Circulation vol 117 no 4 pp e25ndashe46 2008

[2] C T Esmon ldquoInflammation and thrombosisrdquo Journal ofThrom-bosis and Haemostasis vol 1 no 7 pp 1343ndash1348 2003

[3] J Petaja ldquoInflammation and coagulation An overviewrdquoThrom-bosis Research vol 127 supplement 2 pp S34ndashS37 2011

[4] J Frostegard ldquoImmunity atherosclerosis and cardiovasculardiseaserdquo BMCMedicine vol 11 no 1 article 117 2013

[5] K J Moore F J Sheedy and E A Fisher ldquoMacrophages inatherosclerosis a dynamic balancerdquo Nature Reviews Immunol-ogy vol 13 no 10 pp 709ndash721 2013

[6] B Zoller X Li J Sundquist and K Sundquist ldquoAutoimmunediseases and venous thromboembolism a review of the litera-turerdquo American Journal of Cardiovascular Disease vol 2 no 3pp 171ndash183 2012

Gastroenterology Research and Practice 9

[7] W Miehsler W Reinisch E Valic et al ldquoIs inflammatorybowel disease an independent and disease specific risk factorfor thromboembolismrdquo Gut vol 53 no 4 pp 542ndash548 2004

[8] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[9] L Pastorelli C D Salvo J R Mercado M Vecchi and T TPizarro ldquoCentral role of the gut epithelial barrier in the patho-genesis of chronic intestinal inflammation iessons learned fromanimal models and human geneticsrdquo Frontiers in Immunologyvol 4 article 280 Article ID Article 280 2013

[10] M Zaeem Cader and A Kaser ldquoRecent advances in inflam-matory bowel disease mucosal immune cells in intestinalinflammationrdquo Gut vol 62 no 11 pp 1653ndash1664 2013

[11] S Saibeni L Spina andM Vecchi ldquoExploring the relationshipsbetween inflammatory response and coagulation cascade ininflammatory bowel diseaserdquo European Review for Medical andPharmacological Sciences vol 8 no 5 pp 205ndash208 2004

[12] S Saibeni M Cattaneo M Vecchi et al ldquoLow vitamin B6

plasma levels a risk factor for thrombosis in inflammatorybowel disease role of inflammation and correlation with acutephase reactantsrdquoThe American Journal of Gastroenterology vol98 no 1 pp 112ndash117 2003

[13] M Cattaneo M Vecchi M L Zighetti et al ldquoHigh prevalenceof hyperhomocysteinemia in patients with inflammatory boweldisease a pathogenic link with thromboembolic complica-tionsrdquoThrombosis and Haemostasis vol 80 no 4 pp 542ndash5451998

[14] T H Mogensen ldquoPathogen recognition and inflammatorysignaling in innate immune defensesrdquo Clinical MicrobiologyReviews vol 22 no 2 pp 240ndash273 2009

[15] E LeBouder J E Rey-Nores N K Rushmere et al ldquoSolubleforms of Toll-like receptor (TLR)2 capable of modulating TLR2signaling are present in human plasma and breast milkrdquo Journalof Immunology vol 171 no 12 pp 6680ndash6689 2003

[16] K I Iwami T Matsuguchi A Masuda T Kikuchi TMusikacharoen and Y Yoshikai ldquoCutting edge naturallyoccurring soluble form of mouse toll-like receptor 4 inhibitslipopolysaccharide signalingrdquo Journal of Immunology vol 165no 12 pp 6682ndash6686 2000

[17] I Munoz M P Sepulcre J Meseguer and V Mulero ldquoMolecu-lar cloning phylogenetic analysis and functional characteriza-tion of soluble Toll-like receptor 5 in gilthead seabream Sparusauratardquo Fish amp Shellfish Immunology vol 35 no 1 pp 36ndash452013

[18] S L Zunt L V Burton L I Goldblatt E E Dobbins and MSrinivasan ldquoSoluble forms of Toll-like receptor 4 are present inhuman saliva and modulate tumour necrosis factor-120572 secretionby macrophage-like cellsrdquo Clinical and Experimental Immunol-ogy vol 156 no 2 pp 285ndash293 2009

[19] A-C Raby E le Bouder C Colmont et al ldquoSoluble TLR2reduces inflammation without compromising bacterial clear-ance by disrupting TLR2 triggeringrdquo Journal of Immunologyvol 183 no 1 pp 506ndash517 2009

[20] R-S Chang Y-C Wang and S-T Kao ldquoSoluble toll-likereceptor 4 reversed attenuating effect of Chinese herbal Xiao-Qing-Long-Tang on allergen induced nerve growth factor andthymic stromal lymphopoietinrdquo Experimental and TherapeuticMedicine vol 6 no 5 pp 1199ndash1207 2013

[21] K Kondelkova J Krejsek L Borska et al ldquoMembrane andsoluble Toll-like receptor 2 in patients with psoriasis treatedby Goeckerman therapyrdquo International Journal of Dermatologyvol 53 no 11 pp e512ndashe517 2014

[22] H-B Yang K-Q Xie J-M Deng and S-M Qin ldquoExpressionof soluble toll-like receptors in pleural effusionsrdquo ChineseMedical Journal vol 123 no 16 pp 2225ndash2230 2010

[23] E Galliera L Drago C Vassena et al ldquoToll-like receptor 2in serum a potential diagnostic marker of prosthetic jointinfectionrdquo Journal of Clinical Microbiology vol 52 no 2 pp620ndash623 2013

[24] A Zhao C Blackburn J Chin and M Srinivasan ldquoSoluble tolllike receptor 2 (TLR-2) is increased in saliva of children withdental cariesrdquo BMC Oral Health vol 14 no 1 article 108 2014

[25] M E Houssen R H El-Mahdy and D A Shahin ldquoSerumsoluble toll-like receptor 2 a novel biomarker for systemic lupuserythematosus disease activity and lupus-related cardiovasculardysfunctionrdquo International Journal of Rheumatic Diseases 2014

[26] E Candia D Dıaz-Jimenez P Langjahr et al ldquoIncreasedproduction of soluble TLR2 by lamina propria mononuclearcells from ulcerative colitis patientsrdquo Immunobiology vol 217no 6 pp 634ndash642 2012

[27] F Cognasse H Hamzeh P Chavarin S Acquart C Geninand O Garraud ldquoEvidence of Toll-like receptor molecules onhuman plateletsrdquo Immunology and Cell Biology vol 83 no 2pp 196ndash198 2005

[28] F X Zhang C J Kirschning R Mancinelli et al ldquoBacteriallipopolysaccharide activates nuclear factor-kappaB throughinterleukin-1 signaling mediators in cultured human dermalendothelial cells and mononuclear phagocytesrdquo The Journal ofBiological Chemistry vol 274 no 12 pp 7611ndash7614 1999

[29] E Faure O Equils P A Sieling et al ldquoBacterial lipopolysac-charide activates NF-120581B through toll-like receptor 4 (TLR-4) in cultured human dermal endothelial cells Differentialexpression of TLR-4 andTLR-2 in endothelial cellsrdquoThe Journalof Biological Chemistry vol 275 no 15 pp 11058ndash11063 2000

[30] M D Boisclair H Ireland and D A Lane ldquoAssessment ofhypercoagulable states bymeasurement of activation fragmentsand peptidesrdquo Blood Reviews vol 4 no 1 pp 25ndash40 1990

[31] E F Stange S P L Travis S Vermeire et al ldquoEuropean evi-dence-based Consensus on the diagnosis and management ofulcerative colitis definitions and diagnosisrdquo Journal of Crohnrsquosamp Colitis vol 2 no 1 pp 1ndash23 2008

[32] A Dignass G van Assche J O Lindsay et al ldquoThe secondEuropean evidence-based Consensus on the diagnosis andmanagement of Crohnrsquos disease current managementrdquo Journalof Crohnrsquos amp Colitis vol 4 no 1 pp 28ndash62 2010

[33] R F Harvey and J M Bradshaw ldquoA simple index of Crohnrsquos-disease activityrdquoThe Lancet vol 1 no 8167 p 514 1980

[34] KW SchroederW J Tremaine andDM Ilstrup ldquoCoated oral5-aminosalicylic acid therapy for mildly to moderately activeulcerative colitis a randomized studyrdquoTheNewEngland Journalof Medicine vol 317 no 26 pp 1625ndash1629 1987

[35] J Satsangi M S Silverberg S Vermeire and J-F ColombelldquoThe Montreal classification of inflammatory bowel diseasecontroversies consensus and implicationsrdquo Gut vol 55 no 6pp 749ndash753 2006

[36] R G Shorter K A Huizenga and R J Spencer ldquoA workinghypothesis for the etiology and pathogenesis of nonspecificinflammatory bowel diseaserdquoTheAmerican Journal of DigestiveDiseases vol 17 no 11 pp 1024ndash1032 1972

[37] A I Thompson and C W Lees ldquoGenetics of ulcerative colitisrdquoInflammatory Bowel Diseases vol 17 no 3 pp 831ndash848 2011

[38] C W Lees J C Barrett M Parkes and J Satsangi ldquoNew IBDgenetics common pathways with other diseasesrdquo Gut vol 60no 12 pp 1739ndash1753 2011

[39] G C Nguyen and J Sam ldquoRising prevalence of venous throm-boembolism and its impact on mortality among hospitalized

10 Gastroenterology Research and Practice

inflammatory bowel disease patientsrdquo The American Journal ofGastroenterology vol 103 no 9 pp 2272ndash2280 2008

[40] H Yuhara C Steinmaus D Corley et al ldquoMeta-analysis therisk of venous thromboembolism in patients with inflammatorybowel diseaserdquoAlimentary Pharmacology andTherapeutics vol37 no 10 pp 953ndash962 2013

[41] W R Swindell P E Stuart M K Sarkar et al ldquoCellular dis-section of psoriasis for transcriptome analyses and the post-GWAS erardquo Acta Veterinaria Scandinavica vol 7 article 272014

[42] D Salem R Subang P Laplante J Levine and J Rauch ldquoThedual role of innate immunity in antiphospholipid syndrome andsystemic lupus erythematosusrdquo Lupus vol 23 no 12 pp 1327ndash1331 2014

[43] P Blair S Rex O Vitseva et al ldquoStimulation of Toll-likereceptor 2 in human platelets induces a thromboinflammatoryresponse through activation of phosphoinositide 3-kinaserdquoCirculation Research vol 104 no 3 pp 346ndash354 2009

[44] H Kalvegren C Skoglund C Helldahl M Lerm MGrenegard and T Bengtsson ldquoToll-like receptor 2 stimulationof platelets is mediated by purinergic P2X1-dependent Ca2+mobilisation cyclooxygenase and purinergic P2Y1 and P2Y12receptor activationrdquo Thrombosis and Haemostasis vol 103 no2 pp 398ndash407 2010

[45] S Rex L M Beaulieu D H Perlman et al ldquoImmune versusthrombotic stimulation of platelets differentially regulates sig-nalling pathways intracellular protein-protein interactions and120572-granule releaserdquo Thrombosis and Haemostasis vol 102 no 1pp 97ndash110 2009

[46] G Zhang J Han E J Welch et al ldquoLipopolysaccharide stim-ulates platelet secretion and potentiates platelet aggregationvia TLR4MyD88 and the cGMP-dependent protein kinasepathwayrdquoThe Journal of Immunology vol 182 no 12 pp 7997ndash8004 2009

[47] G T Brown and T MMcIntyre ldquoLipopolysaccharide signalingwithout a nucleus kinase cascades stimulate platelet sheddingof proinflammatory IL-1120573-rich microparticlesrdquo The Journal ofImmunology vol 186 no 9 pp 5489ndash5496 2011

[48] L Chavez-Sanchez J E Espinosa-Luna K Chavez-Rueda MV Legorreta-Haquet E Montoya-Dıaz and F Blanco-FavelaldquoInnate immune system cells in atherosclerosisrdquo Archives ofMedical Research vol 45 no 1 pp 1ndash14 2014

6 Gastroenterology Research and Practice

00 05 10 15 20 250

2

4

6

8

10

LPS (EUmL)

CRP

(mg

dL)

0 2000 4000 6000 80000

2

4

6

8

10

TLR2 (pgmL)

CRP

(mg

dL)

0 2000 4000 6000 80000

2

4

6

8

10

TLR4 (pgmL)

CRP

(mg

dL)r = 0295

P = 0035

(a)

00 05 10 15 20 250

5

10

15

LPS (EUmL)

May

o sc

ore

0 2000 4000 6000 80000

5

10

15

TLR2 (pgmL)

May

o sc

ore

0 2000 4000 6000 80000

5

10

15

TLR4 (pgmL)

May

o sc

ore

(b)

00 05 10 15 20 250

5

10

15

LPS (EUmL)

Har

vey-

Brad

shaw

Inde

x

0 2000 4000 6000 80000

5

10

15

TLR2 (pgmL)

Har

vey-

Brad

shaw

Inde

x

0 1000 2000 3000 4000 50000

5

10

15

TLR4 (pgmL)

Har

vey-

Brad

shaw

Inde

x

(c)

Figure 3 Circulating LPS correlates with biochemical but not clinical activity in IBD patients Concentrations of LPS TLR2 TLR4 andCRP were measured in the sera of IBD (both UC and CD) patients Clinical activity of UC and CD was evaluated using Mayo score andHarvey-Bradshaw Index (a) Correlation between CRP serum levels and circulating LPS TLR2 and TLR4 in IBD patients (b) Correlationbetween Mayo score and circulating LPS TLR2 and TLR4 in UC patients (c) Correlation between Harvey-Bradshaw Index and circulatingLPS TLR2 and TLR4 in CD patients Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 was consideredstatistically significant

strongly activates NF-120581B [5 29] leading to the release ofproinflammatory mediators which can activate the coagula-tory cascade Nonetheless the pathogenesis of atheroscleroticplaques ismediated by the presence of activatedmacrophageswithin the plaque which also respond vigorously to TLRstimulation releasing proinflammatory cytokines [48]

Given themultiplicity of cell types and pathways involvedin the activation of coagulation mediated by TLRs wedecided tomeasure the activation products of the coagulationand fibrinolysis that is the fibrin degradation product D-Dimer and prothrombin fragment F1 + 2 as they describeclosely the overall activation of the coagulatory cascade [30]Remarkably LPS levels significantly correlated with bothD-Dimer and F1 + 2 (Figure 4) clearly suggesting thatcirculating PAMPsmay function as triggers for the activationof coagulation

It may be worthy to note that the correlation betweencirculating LPS and markers of activated coagulation wasstronger when considering only UC patients (Figures 1(d)2(c) and 5) and CD patients with colonic or ileocolonicdisease (L2 and L3 according to Montreal classification [35])(Figure 6)These data suggest that the presence of colonic dis-ease is the key risk factor for coagulatory unbalance that mayfollow bacterial translocation from the gut indeed the colonhas to cope with a significantly greater bacterial burden thanthe rest of the gastrointestinal tract as such it is rational thata break-down of intestinal barrier function in this site mightlead to amore prominent bacterial penetrationtranslocation

Moreover in order to explain the differences we found inUC versus CD other issues should be taken into account CDpatients often display greater signs of systemic inflammationrather than UC as such data coming from CD patients may

Gastroenterology Research and Practice 7

0 500 1000 150000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR2

(pg

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR4

(pg

mL)

r = 0422

P = 0001

(a)

0 200 400 600 800 100000

05

10

15

20

25

LPS

(EU

mL)

0 200 400 600 800 10000

2000

4000

6000

8000TL

R2 (p

gm

L)

0 200 400 600 800 10000

2000

4000

6000

8000

TLR4

(pg

mL)

r = 0440

P = 00008

F1 + 2 (pmolL) F1 + 2 (pmolL) F1 + 2 (pmolL)

(b)

Figure 4 Circulating LPS correlates with markers of activated coagulation in IBD patients Concentrations of LPS TLR2 and TLR4 andlevels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of IBD patients (a) Correlationbetween D-Dimer plasma levels and serum LPS TLR2 and TLR4 in IBD patients (b) Correlation between prothrombin fragment F1+2 andcirculating LPS TLR2 and TLR4 in IBD patients Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 wasconsidered statistically significant

0 500 1000 150000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR2

(pg

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR4

(pg

mL)

r = 0467

P = 0028

r = 0776

r = 0560

P = 0006

P lt 00001

(a)

0 200 400 600 800 100000

05

10

15

20

25

LPS

(EU

mL)

0 200 400 600 800 10000

2000

4000

6000

8000

TLR2

(pg

mL)

0 200 400 600 800 10000

2000

4000

6000

8000

TLR4

(pg

mL)

F1 + 2 (pmolL) F1 + 2 (pmolL) F1 + 2 (pmolL)

r = 0521

P = 0012

r = 0573

P = 0005

(b)

Figure 5 Circulating LPS TLR2 and TLR4 correlate with markers of activated coagulation in UC patients Concentrations of LPS TLR2and TLR4 and levels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of UC patients(a) Correlation between D-Dimer plasma levels and serum LPS TLR2 and TLR4 in UC patients (b) Correlation between prothrombinfragment F1 + 2 and circulating LPS TLR2 and TLR4 in UC patients Statistical analysis was performed by means of Spearman CorrelationTest 119875 lt 005 was considered statistically significant

8 Gastroenterology Research and Practice

0 100 200 300 40000

05

10

15

20

D-dimer (ngmL)

LPS

(EU

mL)

0 200 400 600 800 100000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

CD (L1 only)

0 200 400 600 800 100000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

r = 0435

P = 0042

CD (L2 + L3 only)CD (L1 + L2 + L3)

(a)

0 200 400 600 80000

05

10

15

20

LPS

(EU

mL)

0 100 200 30000

05

10

15

20

25LP

S (E

Um

L)

CD (L1 only)

0 200 400 600 80000

05

10

15

20

25

LPS

(EU

mL)

F1 + 2 (pmolL)F1 + 2 (pmolL)F1 + 2 (pmolL)

r = 0590

P = 0003

CD (L2 + L3 only)CD (L1 + L2 + L3)

(b)

Figure 6 Circulating LPS correlates with markers of activated coagulation in CD patients with colonic involvement Concentrations of LPSand levels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of CD patients (a) Correlationbetween D-Dimer plasma levels and serum LPS in all CD patients (left panel) CD patients with exclusive ileal involvement (L1 accordingto Montreal classification) (central panel) and CD patients with colonic involvement (L2 + L3 according to Montreal classification) (b)Correlation between prothrombin fragment F1 + 2 and circulating LPS in all CD patients (left panel) CD patients with exclusive ilealinvolvement (L1 according to Montreal classification) (central panel) and CD patients with colonic involvement (L2 + L3 according toMontreal classification) Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 was considered statisticallysignificant

suffer from the noise resulting from inflammation-drivenactivation of coagulation as an alternative PAMPs-mediatedcoagulatory activation may have a different relevance orfollow different pathways in UC versus CD also consideringthat genetic data suggest different defects in intestinal barrierfunction in those two diseases [38]

Taken together our data support the novel hypothesisthat intestinal barrier defects contribute to the developmentof a procoagulatory state in IBD Our results need to beconfirmed with future studies in fact the correlations weobserved in human patients do not demonstrate the bio-logical importance of the phenomenon we are postulatingA further limitation of our study is that we are usingvery simple markers in order to explore extremely complexsystems Indeed experiments on animal models of intestinalinflammation are warranted in order to provide the finaldemonstration of our hypothesis and mechanistically dissectit and evaluate its real biologic relevance

Conflict of Interests

None of the authors has any conflict of interests related to thispaperwork to declare

Acknowledgments

The authors acknowledge Dr Rossella Bader for her impor-tant technical support This work was supported by theItalian Ministry of University and Research PRIN Grant2007K4HZEJ 004 (MV)

References

[1] W Rosamond K Flegal K Furie et al ldquoHeart disease andstroke statisticsmdash2008 update a report from the Americanheart association statistics committee and stroke statistics sub-committeerdquo Circulation vol 117 no 4 pp e25ndashe46 2008

[2] C T Esmon ldquoInflammation and thrombosisrdquo Journal ofThrom-bosis and Haemostasis vol 1 no 7 pp 1343ndash1348 2003

[3] J Petaja ldquoInflammation and coagulation An overviewrdquoThrom-bosis Research vol 127 supplement 2 pp S34ndashS37 2011

[4] J Frostegard ldquoImmunity atherosclerosis and cardiovasculardiseaserdquo BMCMedicine vol 11 no 1 article 117 2013

[5] K J Moore F J Sheedy and E A Fisher ldquoMacrophages inatherosclerosis a dynamic balancerdquo Nature Reviews Immunol-ogy vol 13 no 10 pp 709ndash721 2013

[6] B Zoller X Li J Sundquist and K Sundquist ldquoAutoimmunediseases and venous thromboembolism a review of the litera-turerdquo American Journal of Cardiovascular Disease vol 2 no 3pp 171ndash183 2012

Gastroenterology Research and Practice 9

[7] W Miehsler W Reinisch E Valic et al ldquoIs inflammatorybowel disease an independent and disease specific risk factorfor thromboembolismrdquo Gut vol 53 no 4 pp 542ndash548 2004

[8] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[9] L Pastorelli C D Salvo J R Mercado M Vecchi and T TPizarro ldquoCentral role of the gut epithelial barrier in the patho-genesis of chronic intestinal inflammation iessons learned fromanimal models and human geneticsrdquo Frontiers in Immunologyvol 4 article 280 Article ID Article 280 2013

[10] M Zaeem Cader and A Kaser ldquoRecent advances in inflam-matory bowel disease mucosal immune cells in intestinalinflammationrdquo Gut vol 62 no 11 pp 1653ndash1664 2013

[11] S Saibeni L Spina andM Vecchi ldquoExploring the relationshipsbetween inflammatory response and coagulation cascade ininflammatory bowel diseaserdquo European Review for Medical andPharmacological Sciences vol 8 no 5 pp 205ndash208 2004

[12] S Saibeni M Cattaneo M Vecchi et al ldquoLow vitamin B6

plasma levels a risk factor for thrombosis in inflammatorybowel disease role of inflammation and correlation with acutephase reactantsrdquoThe American Journal of Gastroenterology vol98 no 1 pp 112ndash117 2003

[13] M Cattaneo M Vecchi M L Zighetti et al ldquoHigh prevalenceof hyperhomocysteinemia in patients with inflammatory boweldisease a pathogenic link with thromboembolic complica-tionsrdquoThrombosis and Haemostasis vol 80 no 4 pp 542ndash5451998

[14] T H Mogensen ldquoPathogen recognition and inflammatorysignaling in innate immune defensesrdquo Clinical MicrobiologyReviews vol 22 no 2 pp 240ndash273 2009

[15] E LeBouder J E Rey-Nores N K Rushmere et al ldquoSolubleforms of Toll-like receptor (TLR)2 capable of modulating TLR2signaling are present in human plasma and breast milkrdquo Journalof Immunology vol 171 no 12 pp 6680ndash6689 2003

[16] K I Iwami T Matsuguchi A Masuda T Kikuchi TMusikacharoen and Y Yoshikai ldquoCutting edge naturallyoccurring soluble form of mouse toll-like receptor 4 inhibitslipopolysaccharide signalingrdquo Journal of Immunology vol 165no 12 pp 6682ndash6686 2000

[17] I Munoz M P Sepulcre J Meseguer and V Mulero ldquoMolecu-lar cloning phylogenetic analysis and functional characteriza-tion of soluble Toll-like receptor 5 in gilthead seabream Sparusauratardquo Fish amp Shellfish Immunology vol 35 no 1 pp 36ndash452013

[18] S L Zunt L V Burton L I Goldblatt E E Dobbins and MSrinivasan ldquoSoluble forms of Toll-like receptor 4 are present inhuman saliva and modulate tumour necrosis factor-120572 secretionby macrophage-like cellsrdquo Clinical and Experimental Immunol-ogy vol 156 no 2 pp 285ndash293 2009

[19] A-C Raby E le Bouder C Colmont et al ldquoSoluble TLR2reduces inflammation without compromising bacterial clear-ance by disrupting TLR2 triggeringrdquo Journal of Immunologyvol 183 no 1 pp 506ndash517 2009

[20] R-S Chang Y-C Wang and S-T Kao ldquoSoluble toll-likereceptor 4 reversed attenuating effect of Chinese herbal Xiao-Qing-Long-Tang on allergen induced nerve growth factor andthymic stromal lymphopoietinrdquo Experimental and TherapeuticMedicine vol 6 no 5 pp 1199ndash1207 2013

[21] K Kondelkova J Krejsek L Borska et al ldquoMembrane andsoluble Toll-like receptor 2 in patients with psoriasis treatedby Goeckerman therapyrdquo International Journal of Dermatologyvol 53 no 11 pp e512ndashe517 2014

[22] H-B Yang K-Q Xie J-M Deng and S-M Qin ldquoExpressionof soluble toll-like receptors in pleural effusionsrdquo ChineseMedical Journal vol 123 no 16 pp 2225ndash2230 2010

[23] E Galliera L Drago C Vassena et al ldquoToll-like receptor 2in serum a potential diagnostic marker of prosthetic jointinfectionrdquo Journal of Clinical Microbiology vol 52 no 2 pp620ndash623 2013

[24] A Zhao C Blackburn J Chin and M Srinivasan ldquoSoluble tolllike receptor 2 (TLR-2) is increased in saliva of children withdental cariesrdquo BMC Oral Health vol 14 no 1 article 108 2014

[25] M E Houssen R H El-Mahdy and D A Shahin ldquoSerumsoluble toll-like receptor 2 a novel biomarker for systemic lupuserythematosus disease activity and lupus-related cardiovasculardysfunctionrdquo International Journal of Rheumatic Diseases 2014

[26] E Candia D Dıaz-Jimenez P Langjahr et al ldquoIncreasedproduction of soluble TLR2 by lamina propria mononuclearcells from ulcerative colitis patientsrdquo Immunobiology vol 217no 6 pp 634ndash642 2012

[27] F Cognasse H Hamzeh P Chavarin S Acquart C Geninand O Garraud ldquoEvidence of Toll-like receptor molecules onhuman plateletsrdquo Immunology and Cell Biology vol 83 no 2pp 196ndash198 2005

[28] F X Zhang C J Kirschning R Mancinelli et al ldquoBacteriallipopolysaccharide activates nuclear factor-kappaB throughinterleukin-1 signaling mediators in cultured human dermalendothelial cells and mononuclear phagocytesrdquo The Journal ofBiological Chemistry vol 274 no 12 pp 7611ndash7614 1999

[29] E Faure O Equils P A Sieling et al ldquoBacterial lipopolysac-charide activates NF-120581B through toll-like receptor 4 (TLR-4) in cultured human dermal endothelial cells Differentialexpression of TLR-4 andTLR-2 in endothelial cellsrdquoThe Journalof Biological Chemistry vol 275 no 15 pp 11058ndash11063 2000

[30] M D Boisclair H Ireland and D A Lane ldquoAssessment ofhypercoagulable states bymeasurement of activation fragmentsand peptidesrdquo Blood Reviews vol 4 no 1 pp 25ndash40 1990

[31] E F Stange S P L Travis S Vermeire et al ldquoEuropean evi-dence-based Consensus on the diagnosis and management ofulcerative colitis definitions and diagnosisrdquo Journal of Crohnrsquosamp Colitis vol 2 no 1 pp 1ndash23 2008

[32] A Dignass G van Assche J O Lindsay et al ldquoThe secondEuropean evidence-based Consensus on the diagnosis andmanagement of Crohnrsquos disease current managementrdquo Journalof Crohnrsquos amp Colitis vol 4 no 1 pp 28ndash62 2010

[33] R F Harvey and J M Bradshaw ldquoA simple index of Crohnrsquos-disease activityrdquoThe Lancet vol 1 no 8167 p 514 1980

[34] KW SchroederW J Tremaine andDM Ilstrup ldquoCoated oral5-aminosalicylic acid therapy for mildly to moderately activeulcerative colitis a randomized studyrdquoTheNewEngland Journalof Medicine vol 317 no 26 pp 1625ndash1629 1987

[35] J Satsangi M S Silverberg S Vermeire and J-F ColombelldquoThe Montreal classification of inflammatory bowel diseasecontroversies consensus and implicationsrdquo Gut vol 55 no 6pp 749ndash753 2006

[36] R G Shorter K A Huizenga and R J Spencer ldquoA workinghypothesis for the etiology and pathogenesis of nonspecificinflammatory bowel diseaserdquoTheAmerican Journal of DigestiveDiseases vol 17 no 11 pp 1024ndash1032 1972

[37] A I Thompson and C W Lees ldquoGenetics of ulcerative colitisrdquoInflammatory Bowel Diseases vol 17 no 3 pp 831ndash848 2011

[38] C W Lees J C Barrett M Parkes and J Satsangi ldquoNew IBDgenetics common pathways with other diseasesrdquo Gut vol 60no 12 pp 1739ndash1753 2011

[39] G C Nguyen and J Sam ldquoRising prevalence of venous throm-boembolism and its impact on mortality among hospitalized

10 Gastroenterology Research and Practice

inflammatory bowel disease patientsrdquo The American Journal ofGastroenterology vol 103 no 9 pp 2272ndash2280 2008

[40] H Yuhara C Steinmaus D Corley et al ldquoMeta-analysis therisk of venous thromboembolism in patients with inflammatorybowel diseaserdquoAlimentary Pharmacology andTherapeutics vol37 no 10 pp 953ndash962 2013

[41] W R Swindell P E Stuart M K Sarkar et al ldquoCellular dis-section of psoriasis for transcriptome analyses and the post-GWAS erardquo Acta Veterinaria Scandinavica vol 7 article 272014

[42] D Salem R Subang P Laplante J Levine and J Rauch ldquoThedual role of innate immunity in antiphospholipid syndrome andsystemic lupus erythematosusrdquo Lupus vol 23 no 12 pp 1327ndash1331 2014

[43] P Blair S Rex O Vitseva et al ldquoStimulation of Toll-likereceptor 2 in human platelets induces a thromboinflammatoryresponse through activation of phosphoinositide 3-kinaserdquoCirculation Research vol 104 no 3 pp 346ndash354 2009

[44] H Kalvegren C Skoglund C Helldahl M Lerm MGrenegard and T Bengtsson ldquoToll-like receptor 2 stimulationof platelets is mediated by purinergic P2X1-dependent Ca2+mobilisation cyclooxygenase and purinergic P2Y1 and P2Y12receptor activationrdquo Thrombosis and Haemostasis vol 103 no2 pp 398ndash407 2010

[45] S Rex L M Beaulieu D H Perlman et al ldquoImmune versusthrombotic stimulation of platelets differentially regulates sig-nalling pathways intracellular protein-protein interactions and120572-granule releaserdquo Thrombosis and Haemostasis vol 102 no 1pp 97ndash110 2009

[46] G Zhang J Han E J Welch et al ldquoLipopolysaccharide stim-ulates platelet secretion and potentiates platelet aggregationvia TLR4MyD88 and the cGMP-dependent protein kinasepathwayrdquoThe Journal of Immunology vol 182 no 12 pp 7997ndash8004 2009

[47] G T Brown and T MMcIntyre ldquoLipopolysaccharide signalingwithout a nucleus kinase cascades stimulate platelet sheddingof proinflammatory IL-1120573-rich microparticlesrdquo The Journal ofImmunology vol 186 no 9 pp 5489ndash5496 2011

[48] L Chavez-Sanchez J E Espinosa-Luna K Chavez-Rueda MV Legorreta-Haquet E Montoya-Dıaz and F Blanco-FavelaldquoInnate immune system cells in atherosclerosisrdquo Archives ofMedical Research vol 45 no 1 pp 1ndash14 2014

Gastroenterology Research and Practice 7

0 500 1000 150000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR2

(pg

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR4

(pg

mL)

r = 0422

P = 0001

(a)

0 200 400 600 800 100000

05

10

15

20

25

LPS

(EU

mL)

0 200 400 600 800 10000

2000

4000

6000

8000TL

R2 (p

gm

L)

0 200 400 600 800 10000

2000

4000

6000

8000

TLR4

(pg

mL)

r = 0440

P = 00008

F1 + 2 (pmolL) F1 + 2 (pmolL) F1 + 2 (pmolL)

(b)

Figure 4 Circulating LPS correlates with markers of activated coagulation in IBD patients Concentrations of LPS TLR2 and TLR4 andlevels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of IBD patients (a) Correlationbetween D-Dimer plasma levels and serum LPS TLR2 and TLR4 in IBD patients (b) Correlation between prothrombin fragment F1+2 andcirculating LPS TLR2 and TLR4 in IBD patients Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 wasconsidered statistically significant

0 500 1000 150000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR2

(pg

mL)

0 500 1000 15000

2000

4000

6000

8000

D-dimer (ngmL)

TLR4

(pg

mL)

r = 0467

P = 0028

r = 0776

r = 0560

P = 0006

P lt 00001

(a)

0 200 400 600 800 100000

05

10

15

20

25

LPS

(EU

mL)

0 200 400 600 800 10000

2000

4000

6000

8000

TLR2

(pg

mL)

0 200 400 600 800 10000

2000

4000

6000

8000

TLR4

(pg

mL)

F1 + 2 (pmolL) F1 + 2 (pmolL) F1 + 2 (pmolL)

r = 0521

P = 0012

r = 0573

P = 0005

(b)

Figure 5 Circulating LPS TLR2 and TLR4 correlate with markers of activated coagulation in UC patients Concentrations of LPS TLR2and TLR4 and levels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of UC patients(a) Correlation between D-Dimer plasma levels and serum LPS TLR2 and TLR4 in UC patients (b) Correlation between prothrombinfragment F1 + 2 and circulating LPS TLR2 and TLR4 in UC patients Statistical analysis was performed by means of Spearman CorrelationTest 119875 lt 005 was considered statistically significant

8 Gastroenterology Research and Practice

0 100 200 300 40000

05

10

15

20

D-dimer (ngmL)

LPS

(EU

mL)

0 200 400 600 800 100000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

CD (L1 only)

0 200 400 600 800 100000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

r = 0435

P = 0042

CD (L2 + L3 only)CD (L1 + L2 + L3)

(a)

0 200 400 600 80000

05

10

15

20

LPS

(EU

mL)

0 100 200 30000

05

10

15

20

25LP

S (E

Um

L)

CD (L1 only)

0 200 400 600 80000

05

10

15

20

25

LPS

(EU

mL)

F1 + 2 (pmolL)F1 + 2 (pmolL)F1 + 2 (pmolL)

r = 0590

P = 0003

CD (L2 + L3 only)CD (L1 + L2 + L3)

(b)

Figure 6 Circulating LPS correlates with markers of activated coagulation in CD patients with colonic involvement Concentrations of LPSand levels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of CD patients (a) Correlationbetween D-Dimer plasma levels and serum LPS in all CD patients (left panel) CD patients with exclusive ileal involvement (L1 accordingto Montreal classification) (central panel) and CD patients with colonic involvement (L2 + L3 according to Montreal classification) (b)Correlation between prothrombin fragment F1 + 2 and circulating LPS in all CD patients (left panel) CD patients with exclusive ilealinvolvement (L1 according to Montreal classification) (central panel) and CD patients with colonic involvement (L2 + L3 according toMontreal classification) Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 was considered statisticallysignificant

suffer from the noise resulting from inflammation-drivenactivation of coagulation as an alternative PAMPs-mediatedcoagulatory activation may have a different relevance orfollow different pathways in UC versus CD also consideringthat genetic data suggest different defects in intestinal barrierfunction in those two diseases [38]

Taken together our data support the novel hypothesisthat intestinal barrier defects contribute to the developmentof a procoagulatory state in IBD Our results need to beconfirmed with future studies in fact the correlations weobserved in human patients do not demonstrate the bio-logical importance of the phenomenon we are postulatingA further limitation of our study is that we are usingvery simple markers in order to explore extremely complexsystems Indeed experiments on animal models of intestinalinflammation are warranted in order to provide the finaldemonstration of our hypothesis and mechanistically dissectit and evaluate its real biologic relevance

Conflict of Interests

None of the authors has any conflict of interests related to thispaperwork to declare

Acknowledgments

The authors acknowledge Dr Rossella Bader for her impor-tant technical support This work was supported by theItalian Ministry of University and Research PRIN Grant2007K4HZEJ 004 (MV)

References

[1] W Rosamond K Flegal K Furie et al ldquoHeart disease andstroke statisticsmdash2008 update a report from the Americanheart association statistics committee and stroke statistics sub-committeerdquo Circulation vol 117 no 4 pp e25ndashe46 2008

[2] C T Esmon ldquoInflammation and thrombosisrdquo Journal ofThrom-bosis and Haemostasis vol 1 no 7 pp 1343ndash1348 2003

[3] J Petaja ldquoInflammation and coagulation An overviewrdquoThrom-bosis Research vol 127 supplement 2 pp S34ndashS37 2011

[4] J Frostegard ldquoImmunity atherosclerosis and cardiovasculardiseaserdquo BMCMedicine vol 11 no 1 article 117 2013

[5] K J Moore F J Sheedy and E A Fisher ldquoMacrophages inatherosclerosis a dynamic balancerdquo Nature Reviews Immunol-ogy vol 13 no 10 pp 709ndash721 2013

[6] B Zoller X Li J Sundquist and K Sundquist ldquoAutoimmunediseases and venous thromboembolism a review of the litera-turerdquo American Journal of Cardiovascular Disease vol 2 no 3pp 171ndash183 2012

Gastroenterology Research and Practice 9

[7] W Miehsler W Reinisch E Valic et al ldquoIs inflammatorybowel disease an independent and disease specific risk factorfor thromboembolismrdquo Gut vol 53 no 4 pp 542ndash548 2004

[8] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[9] L Pastorelli C D Salvo J R Mercado M Vecchi and T TPizarro ldquoCentral role of the gut epithelial barrier in the patho-genesis of chronic intestinal inflammation iessons learned fromanimal models and human geneticsrdquo Frontiers in Immunologyvol 4 article 280 Article ID Article 280 2013

[10] M Zaeem Cader and A Kaser ldquoRecent advances in inflam-matory bowel disease mucosal immune cells in intestinalinflammationrdquo Gut vol 62 no 11 pp 1653ndash1664 2013

[11] S Saibeni L Spina andM Vecchi ldquoExploring the relationshipsbetween inflammatory response and coagulation cascade ininflammatory bowel diseaserdquo European Review for Medical andPharmacological Sciences vol 8 no 5 pp 205ndash208 2004

[12] S Saibeni M Cattaneo M Vecchi et al ldquoLow vitamin B6

plasma levels a risk factor for thrombosis in inflammatorybowel disease role of inflammation and correlation with acutephase reactantsrdquoThe American Journal of Gastroenterology vol98 no 1 pp 112ndash117 2003

[13] M Cattaneo M Vecchi M L Zighetti et al ldquoHigh prevalenceof hyperhomocysteinemia in patients with inflammatory boweldisease a pathogenic link with thromboembolic complica-tionsrdquoThrombosis and Haemostasis vol 80 no 4 pp 542ndash5451998

[14] T H Mogensen ldquoPathogen recognition and inflammatorysignaling in innate immune defensesrdquo Clinical MicrobiologyReviews vol 22 no 2 pp 240ndash273 2009

[15] E LeBouder J E Rey-Nores N K Rushmere et al ldquoSolubleforms of Toll-like receptor (TLR)2 capable of modulating TLR2signaling are present in human plasma and breast milkrdquo Journalof Immunology vol 171 no 12 pp 6680ndash6689 2003

[16] K I Iwami T Matsuguchi A Masuda T Kikuchi TMusikacharoen and Y Yoshikai ldquoCutting edge naturallyoccurring soluble form of mouse toll-like receptor 4 inhibitslipopolysaccharide signalingrdquo Journal of Immunology vol 165no 12 pp 6682ndash6686 2000

[17] I Munoz M P Sepulcre J Meseguer and V Mulero ldquoMolecu-lar cloning phylogenetic analysis and functional characteriza-tion of soluble Toll-like receptor 5 in gilthead seabream Sparusauratardquo Fish amp Shellfish Immunology vol 35 no 1 pp 36ndash452013

[18] S L Zunt L V Burton L I Goldblatt E E Dobbins and MSrinivasan ldquoSoluble forms of Toll-like receptor 4 are present inhuman saliva and modulate tumour necrosis factor-120572 secretionby macrophage-like cellsrdquo Clinical and Experimental Immunol-ogy vol 156 no 2 pp 285ndash293 2009

[19] A-C Raby E le Bouder C Colmont et al ldquoSoluble TLR2reduces inflammation without compromising bacterial clear-ance by disrupting TLR2 triggeringrdquo Journal of Immunologyvol 183 no 1 pp 506ndash517 2009

[20] R-S Chang Y-C Wang and S-T Kao ldquoSoluble toll-likereceptor 4 reversed attenuating effect of Chinese herbal Xiao-Qing-Long-Tang on allergen induced nerve growth factor andthymic stromal lymphopoietinrdquo Experimental and TherapeuticMedicine vol 6 no 5 pp 1199ndash1207 2013

[21] K Kondelkova J Krejsek L Borska et al ldquoMembrane andsoluble Toll-like receptor 2 in patients with psoriasis treatedby Goeckerman therapyrdquo International Journal of Dermatologyvol 53 no 11 pp e512ndashe517 2014

[22] H-B Yang K-Q Xie J-M Deng and S-M Qin ldquoExpressionof soluble toll-like receptors in pleural effusionsrdquo ChineseMedical Journal vol 123 no 16 pp 2225ndash2230 2010

[23] E Galliera L Drago C Vassena et al ldquoToll-like receptor 2in serum a potential diagnostic marker of prosthetic jointinfectionrdquo Journal of Clinical Microbiology vol 52 no 2 pp620ndash623 2013

[24] A Zhao C Blackburn J Chin and M Srinivasan ldquoSoluble tolllike receptor 2 (TLR-2) is increased in saliva of children withdental cariesrdquo BMC Oral Health vol 14 no 1 article 108 2014

[25] M E Houssen R H El-Mahdy and D A Shahin ldquoSerumsoluble toll-like receptor 2 a novel biomarker for systemic lupuserythematosus disease activity and lupus-related cardiovasculardysfunctionrdquo International Journal of Rheumatic Diseases 2014

[26] E Candia D Dıaz-Jimenez P Langjahr et al ldquoIncreasedproduction of soluble TLR2 by lamina propria mononuclearcells from ulcerative colitis patientsrdquo Immunobiology vol 217no 6 pp 634ndash642 2012

[27] F Cognasse H Hamzeh P Chavarin S Acquart C Geninand O Garraud ldquoEvidence of Toll-like receptor molecules onhuman plateletsrdquo Immunology and Cell Biology vol 83 no 2pp 196ndash198 2005

[28] F X Zhang C J Kirschning R Mancinelli et al ldquoBacteriallipopolysaccharide activates nuclear factor-kappaB throughinterleukin-1 signaling mediators in cultured human dermalendothelial cells and mononuclear phagocytesrdquo The Journal ofBiological Chemistry vol 274 no 12 pp 7611ndash7614 1999

[29] E Faure O Equils P A Sieling et al ldquoBacterial lipopolysac-charide activates NF-120581B through toll-like receptor 4 (TLR-4) in cultured human dermal endothelial cells Differentialexpression of TLR-4 andTLR-2 in endothelial cellsrdquoThe Journalof Biological Chemistry vol 275 no 15 pp 11058ndash11063 2000

[30] M D Boisclair H Ireland and D A Lane ldquoAssessment ofhypercoagulable states bymeasurement of activation fragmentsand peptidesrdquo Blood Reviews vol 4 no 1 pp 25ndash40 1990

[31] E F Stange S P L Travis S Vermeire et al ldquoEuropean evi-dence-based Consensus on the diagnosis and management ofulcerative colitis definitions and diagnosisrdquo Journal of Crohnrsquosamp Colitis vol 2 no 1 pp 1ndash23 2008

[32] A Dignass G van Assche J O Lindsay et al ldquoThe secondEuropean evidence-based Consensus on the diagnosis andmanagement of Crohnrsquos disease current managementrdquo Journalof Crohnrsquos amp Colitis vol 4 no 1 pp 28ndash62 2010

[33] R F Harvey and J M Bradshaw ldquoA simple index of Crohnrsquos-disease activityrdquoThe Lancet vol 1 no 8167 p 514 1980

[34] KW SchroederW J Tremaine andDM Ilstrup ldquoCoated oral5-aminosalicylic acid therapy for mildly to moderately activeulcerative colitis a randomized studyrdquoTheNewEngland Journalof Medicine vol 317 no 26 pp 1625ndash1629 1987

[35] J Satsangi M S Silverberg S Vermeire and J-F ColombelldquoThe Montreal classification of inflammatory bowel diseasecontroversies consensus and implicationsrdquo Gut vol 55 no 6pp 749ndash753 2006

[36] R G Shorter K A Huizenga and R J Spencer ldquoA workinghypothesis for the etiology and pathogenesis of nonspecificinflammatory bowel diseaserdquoTheAmerican Journal of DigestiveDiseases vol 17 no 11 pp 1024ndash1032 1972

[37] A I Thompson and C W Lees ldquoGenetics of ulcerative colitisrdquoInflammatory Bowel Diseases vol 17 no 3 pp 831ndash848 2011

[38] C W Lees J C Barrett M Parkes and J Satsangi ldquoNew IBDgenetics common pathways with other diseasesrdquo Gut vol 60no 12 pp 1739ndash1753 2011

[39] G C Nguyen and J Sam ldquoRising prevalence of venous throm-boembolism and its impact on mortality among hospitalized

10 Gastroenterology Research and Practice

inflammatory bowel disease patientsrdquo The American Journal ofGastroenterology vol 103 no 9 pp 2272ndash2280 2008

[40] H Yuhara C Steinmaus D Corley et al ldquoMeta-analysis therisk of venous thromboembolism in patients with inflammatorybowel diseaserdquoAlimentary Pharmacology andTherapeutics vol37 no 10 pp 953ndash962 2013

[41] W R Swindell P E Stuart M K Sarkar et al ldquoCellular dis-section of psoriasis for transcriptome analyses and the post-GWAS erardquo Acta Veterinaria Scandinavica vol 7 article 272014

[42] D Salem R Subang P Laplante J Levine and J Rauch ldquoThedual role of innate immunity in antiphospholipid syndrome andsystemic lupus erythematosusrdquo Lupus vol 23 no 12 pp 1327ndash1331 2014

[43] P Blair S Rex O Vitseva et al ldquoStimulation of Toll-likereceptor 2 in human platelets induces a thromboinflammatoryresponse through activation of phosphoinositide 3-kinaserdquoCirculation Research vol 104 no 3 pp 346ndash354 2009

[44] H Kalvegren C Skoglund C Helldahl M Lerm MGrenegard and T Bengtsson ldquoToll-like receptor 2 stimulationof platelets is mediated by purinergic P2X1-dependent Ca2+mobilisation cyclooxygenase and purinergic P2Y1 and P2Y12receptor activationrdquo Thrombosis and Haemostasis vol 103 no2 pp 398ndash407 2010

[45] S Rex L M Beaulieu D H Perlman et al ldquoImmune versusthrombotic stimulation of platelets differentially regulates sig-nalling pathways intracellular protein-protein interactions and120572-granule releaserdquo Thrombosis and Haemostasis vol 102 no 1pp 97ndash110 2009

[46] G Zhang J Han E J Welch et al ldquoLipopolysaccharide stim-ulates platelet secretion and potentiates platelet aggregationvia TLR4MyD88 and the cGMP-dependent protein kinasepathwayrdquoThe Journal of Immunology vol 182 no 12 pp 7997ndash8004 2009

[47] G T Brown and T MMcIntyre ldquoLipopolysaccharide signalingwithout a nucleus kinase cascades stimulate platelet sheddingof proinflammatory IL-1120573-rich microparticlesrdquo The Journal ofImmunology vol 186 no 9 pp 5489ndash5496 2011

[48] L Chavez-Sanchez J E Espinosa-Luna K Chavez-Rueda MV Legorreta-Haquet E Montoya-Dıaz and F Blanco-FavelaldquoInnate immune system cells in atherosclerosisrdquo Archives ofMedical Research vol 45 no 1 pp 1ndash14 2014

8 Gastroenterology Research and Practice

0 100 200 300 40000

05

10

15

20

D-dimer (ngmL)

LPS

(EU

mL)

0 200 400 600 800 100000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

CD (L1 only)

0 200 400 600 800 100000

05

10

15

20

25

D-dimer (ngmL)

LPS

(EU

mL)

r = 0435

P = 0042

CD (L2 + L3 only)CD (L1 + L2 + L3)

(a)

0 200 400 600 80000

05

10

15

20

LPS

(EU

mL)

0 100 200 30000

05

10

15

20

25LP

S (E

Um

L)

CD (L1 only)

0 200 400 600 80000

05

10

15

20

25

LPS

(EU

mL)

F1 + 2 (pmolL)F1 + 2 (pmolL)F1 + 2 (pmolL)

r = 0590

P = 0003

CD (L2 + L3 only)CD (L1 + L2 + L3)

(b)

Figure 6 Circulating LPS correlates with markers of activated coagulation in CD patients with colonic involvement Concentrations of LPSand levels of D-Dimer and prothrombin fragment F1 + 2 were measured in the sera and plasma respectively of CD patients (a) Correlationbetween D-Dimer plasma levels and serum LPS in all CD patients (left panel) CD patients with exclusive ileal involvement (L1 accordingto Montreal classification) (central panel) and CD patients with colonic involvement (L2 + L3 according to Montreal classification) (b)Correlation between prothrombin fragment F1 + 2 and circulating LPS in all CD patients (left panel) CD patients with exclusive ilealinvolvement (L1 according to Montreal classification) (central panel) and CD patients with colonic involvement (L2 + L3 according toMontreal classification) Statistical analysis was performed by means of Spearman Correlation Test 119875 lt 005 was considered statisticallysignificant

suffer from the noise resulting from inflammation-drivenactivation of coagulation as an alternative PAMPs-mediatedcoagulatory activation may have a different relevance orfollow different pathways in UC versus CD also consideringthat genetic data suggest different defects in intestinal barrierfunction in those two diseases [38]

Taken together our data support the novel hypothesisthat intestinal barrier defects contribute to the developmentof a procoagulatory state in IBD Our results need to beconfirmed with future studies in fact the correlations weobserved in human patients do not demonstrate the bio-logical importance of the phenomenon we are postulatingA further limitation of our study is that we are usingvery simple markers in order to explore extremely complexsystems Indeed experiments on animal models of intestinalinflammation are warranted in order to provide the finaldemonstration of our hypothesis and mechanistically dissectit and evaluate its real biologic relevance

Conflict of Interests

None of the authors has any conflict of interests related to thispaperwork to declare

Acknowledgments

The authors acknowledge Dr Rossella Bader for her impor-tant technical support This work was supported by theItalian Ministry of University and Research PRIN Grant2007K4HZEJ 004 (MV)

References

[1] W Rosamond K Flegal K Furie et al ldquoHeart disease andstroke statisticsmdash2008 update a report from the Americanheart association statistics committee and stroke statistics sub-committeerdquo Circulation vol 117 no 4 pp e25ndashe46 2008

[2] C T Esmon ldquoInflammation and thrombosisrdquo Journal ofThrom-bosis and Haemostasis vol 1 no 7 pp 1343ndash1348 2003

[3] J Petaja ldquoInflammation and coagulation An overviewrdquoThrom-bosis Research vol 127 supplement 2 pp S34ndashS37 2011

[4] J Frostegard ldquoImmunity atherosclerosis and cardiovasculardiseaserdquo BMCMedicine vol 11 no 1 article 117 2013

[5] K J Moore F J Sheedy and E A Fisher ldquoMacrophages inatherosclerosis a dynamic balancerdquo Nature Reviews Immunol-ogy vol 13 no 10 pp 709ndash721 2013

[6] B Zoller X Li J Sundquist and K Sundquist ldquoAutoimmunediseases and venous thromboembolism a review of the litera-turerdquo American Journal of Cardiovascular Disease vol 2 no 3pp 171ndash183 2012

Gastroenterology Research and Practice 9

[7] W Miehsler W Reinisch E Valic et al ldquoIs inflammatorybowel disease an independent and disease specific risk factorfor thromboembolismrdquo Gut vol 53 no 4 pp 542ndash548 2004

[8] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[9] L Pastorelli C D Salvo J R Mercado M Vecchi and T TPizarro ldquoCentral role of the gut epithelial barrier in the patho-genesis of chronic intestinal inflammation iessons learned fromanimal models and human geneticsrdquo Frontiers in Immunologyvol 4 article 280 Article ID Article 280 2013

[10] M Zaeem Cader and A Kaser ldquoRecent advances in inflam-matory bowel disease mucosal immune cells in intestinalinflammationrdquo Gut vol 62 no 11 pp 1653ndash1664 2013

[11] S Saibeni L Spina andM Vecchi ldquoExploring the relationshipsbetween inflammatory response and coagulation cascade ininflammatory bowel diseaserdquo European Review for Medical andPharmacological Sciences vol 8 no 5 pp 205ndash208 2004

[12] S Saibeni M Cattaneo M Vecchi et al ldquoLow vitamin B6

plasma levels a risk factor for thrombosis in inflammatorybowel disease role of inflammation and correlation with acutephase reactantsrdquoThe American Journal of Gastroenterology vol98 no 1 pp 112ndash117 2003

[13] M Cattaneo M Vecchi M L Zighetti et al ldquoHigh prevalenceof hyperhomocysteinemia in patients with inflammatory boweldisease a pathogenic link with thromboembolic complica-tionsrdquoThrombosis and Haemostasis vol 80 no 4 pp 542ndash5451998

[14] T H Mogensen ldquoPathogen recognition and inflammatorysignaling in innate immune defensesrdquo Clinical MicrobiologyReviews vol 22 no 2 pp 240ndash273 2009

[15] E LeBouder J E Rey-Nores N K Rushmere et al ldquoSolubleforms of Toll-like receptor (TLR)2 capable of modulating TLR2signaling are present in human plasma and breast milkrdquo Journalof Immunology vol 171 no 12 pp 6680ndash6689 2003

[16] K I Iwami T Matsuguchi A Masuda T Kikuchi TMusikacharoen and Y Yoshikai ldquoCutting edge naturallyoccurring soluble form of mouse toll-like receptor 4 inhibitslipopolysaccharide signalingrdquo Journal of Immunology vol 165no 12 pp 6682ndash6686 2000

[17] I Munoz M P Sepulcre J Meseguer and V Mulero ldquoMolecu-lar cloning phylogenetic analysis and functional characteriza-tion of soluble Toll-like receptor 5 in gilthead seabream Sparusauratardquo Fish amp Shellfish Immunology vol 35 no 1 pp 36ndash452013

[18] S L Zunt L V Burton L I Goldblatt E E Dobbins and MSrinivasan ldquoSoluble forms of Toll-like receptor 4 are present inhuman saliva and modulate tumour necrosis factor-120572 secretionby macrophage-like cellsrdquo Clinical and Experimental Immunol-ogy vol 156 no 2 pp 285ndash293 2009

[19] A-C Raby E le Bouder C Colmont et al ldquoSoluble TLR2reduces inflammation without compromising bacterial clear-ance by disrupting TLR2 triggeringrdquo Journal of Immunologyvol 183 no 1 pp 506ndash517 2009

[20] R-S Chang Y-C Wang and S-T Kao ldquoSoluble toll-likereceptor 4 reversed attenuating effect of Chinese herbal Xiao-Qing-Long-Tang on allergen induced nerve growth factor andthymic stromal lymphopoietinrdquo Experimental and TherapeuticMedicine vol 6 no 5 pp 1199ndash1207 2013

[21] K Kondelkova J Krejsek L Borska et al ldquoMembrane andsoluble Toll-like receptor 2 in patients with psoriasis treatedby Goeckerman therapyrdquo International Journal of Dermatologyvol 53 no 11 pp e512ndashe517 2014

[22] H-B Yang K-Q Xie J-M Deng and S-M Qin ldquoExpressionof soluble toll-like receptors in pleural effusionsrdquo ChineseMedical Journal vol 123 no 16 pp 2225ndash2230 2010

[23] E Galliera L Drago C Vassena et al ldquoToll-like receptor 2in serum a potential diagnostic marker of prosthetic jointinfectionrdquo Journal of Clinical Microbiology vol 52 no 2 pp620ndash623 2013

[24] A Zhao C Blackburn J Chin and M Srinivasan ldquoSoluble tolllike receptor 2 (TLR-2) is increased in saliva of children withdental cariesrdquo BMC Oral Health vol 14 no 1 article 108 2014

[25] M E Houssen R H El-Mahdy and D A Shahin ldquoSerumsoluble toll-like receptor 2 a novel biomarker for systemic lupuserythematosus disease activity and lupus-related cardiovasculardysfunctionrdquo International Journal of Rheumatic Diseases 2014

[26] E Candia D Dıaz-Jimenez P Langjahr et al ldquoIncreasedproduction of soluble TLR2 by lamina propria mononuclearcells from ulcerative colitis patientsrdquo Immunobiology vol 217no 6 pp 634ndash642 2012

[27] F Cognasse H Hamzeh P Chavarin S Acquart C Geninand O Garraud ldquoEvidence of Toll-like receptor molecules onhuman plateletsrdquo Immunology and Cell Biology vol 83 no 2pp 196ndash198 2005

[28] F X Zhang C J Kirschning R Mancinelli et al ldquoBacteriallipopolysaccharide activates nuclear factor-kappaB throughinterleukin-1 signaling mediators in cultured human dermalendothelial cells and mononuclear phagocytesrdquo The Journal ofBiological Chemistry vol 274 no 12 pp 7611ndash7614 1999

[29] E Faure O Equils P A Sieling et al ldquoBacterial lipopolysac-charide activates NF-120581B through toll-like receptor 4 (TLR-4) in cultured human dermal endothelial cells Differentialexpression of TLR-4 andTLR-2 in endothelial cellsrdquoThe Journalof Biological Chemistry vol 275 no 15 pp 11058ndash11063 2000

[30] M D Boisclair H Ireland and D A Lane ldquoAssessment ofhypercoagulable states bymeasurement of activation fragmentsand peptidesrdquo Blood Reviews vol 4 no 1 pp 25ndash40 1990

[31] E F Stange S P L Travis S Vermeire et al ldquoEuropean evi-dence-based Consensus on the diagnosis and management ofulcerative colitis definitions and diagnosisrdquo Journal of Crohnrsquosamp Colitis vol 2 no 1 pp 1ndash23 2008

[32] A Dignass G van Assche J O Lindsay et al ldquoThe secondEuropean evidence-based Consensus on the diagnosis andmanagement of Crohnrsquos disease current managementrdquo Journalof Crohnrsquos amp Colitis vol 4 no 1 pp 28ndash62 2010

[33] R F Harvey and J M Bradshaw ldquoA simple index of Crohnrsquos-disease activityrdquoThe Lancet vol 1 no 8167 p 514 1980

[34] KW SchroederW J Tremaine andDM Ilstrup ldquoCoated oral5-aminosalicylic acid therapy for mildly to moderately activeulcerative colitis a randomized studyrdquoTheNewEngland Journalof Medicine vol 317 no 26 pp 1625ndash1629 1987

[35] J Satsangi M S Silverberg S Vermeire and J-F ColombelldquoThe Montreal classification of inflammatory bowel diseasecontroversies consensus and implicationsrdquo Gut vol 55 no 6pp 749ndash753 2006

[36] R G Shorter K A Huizenga and R J Spencer ldquoA workinghypothesis for the etiology and pathogenesis of nonspecificinflammatory bowel diseaserdquoTheAmerican Journal of DigestiveDiseases vol 17 no 11 pp 1024ndash1032 1972

[37] A I Thompson and C W Lees ldquoGenetics of ulcerative colitisrdquoInflammatory Bowel Diseases vol 17 no 3 pp 831ndash848 2011

[38] C W Lees J C Barrett M Parkes and J Satsangi ldquoNew IBDgenetics common pathways with other diseasesrdquo Gut vol 60no 12 pp 1739ndash1753 2011

[39] G C Nguyen and J Sam ldquoRising prevalence of venous throm-boembolism and its impact on mortality among hospitalized

10 Gastroenterology Research and Practice

inflammatory bowel disease patientsrdquo The American Journal ofGastroenterology vol 103 no 9 pp 2272ndash2280 2008

[40] H Yuhara C Steinmaus D Corley et al ldquoMeta-analysis therisk of venous thromboembolism in patients with inflammatorybowel diseaserdquoAlimentary Pharmacology andTherapeutics vol37 no 10 pp 953ndash962 2013

[41] W R Swindell P E Stuart M K Sarkar et al ldquoCellular dis-section of psoriasis for transcriptome analyses and the post-GWAS erardquo Acta Veterinaria Scandinavica vol 7 article 272014

[42] D Salem R Subang P Laplante J Levine and J Rauch ldquoThedual role of innate immunity in antiphospholipid syndrome andsystemic lupus erythematosusrdquo Lupus vol 23 no 12 pp 1327ndash1331 2014

[43] P Blair S Rex O Vitseva et al ldquoStimulation of Toll-likereceptor 2 in human platelets induces a thromboinflammatoryresponse through activation of phosphoinositide 3-kinaserdquoCirculation Research vol 104 no 3 pp 346ndash354 2009

[44] H Kalvegren C Skoglund C Helldahl M Lerm MGrenegard and T Bengtsson ldquoToll-like receptor 2 stimulationof platelets is mediated by purinergic P2X1-dependent Ca2+mobilisation cyclooxygenase and purinergic P2Y1 and P2Y12receptor activationrdquo Thrombosis and Haemostasis vol 103 no2 pp 398ndash407 2010

[45] S Rex L M Beaulieu D H Perlman et al ldquoImmune versusthrombotic stimulation of platelets differentially regulates sig-nalling pathways intracellular protein-protein interactions and120572-granule releaserdquo Thrombosis and Haemostasis vol 102 no 1pp 97ndash110 2009

[46] G Zhang J Han E J Welch et al ldquoLipopolysaccharide stim-ulates platelet secretion and potentiates platelet aggregationvia TLR4MyD88 and the cGMP-dependent protein kinasepathwayrdquoThe Journal of Immunology vol 182 no 12 pp 7997ndash8004 2009

[47] G T Brown and T MMcIntyre ldquoLipopolysaccharide signalingwithout a nucleus kinase cascades stimulate platelet sheddingof proinflammatory IL-1120573-rich microparticlesrdquo The Journal ofImmunology vol 186 no 9 pp 5489ndash5496 2011

[48] L Chavez-Sanchez J E Espinosa-Luna K Chavez-Rueda MV Legorreta-Haquet E Montoya-Dıaz and F Blanco-FavelaldquoInnate immune system cells in atherosclerosisrdquo Archives ofMedical Research vol 45 no 1 pp 1ndash14 2014

Gastroenterology Research and Practice 9

[7] W Miehsler W Reinisch E Valic et al ldquoIs inflammatorybowel disease an independent and disease specific risk factorfor thromboembolismrdquo Gut vol 53 no 4 pp 542ndash548 2004

[8] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[9] L Pastorelli C D Salvo J R Mercado M Vecchi and T TPizarro ldquoCentral role of the gut epithelial barrier in the patho-genesis of chronic intestinal inflammation iessons learned fromanimal models and human geneticsrdquo Frontiers in Immunologyvol 4 article 280 Article ID Article 280 2013

[10] M Zaeem Cader and A Kaser ldquoRecent advances in inflam-matory bowel disease mucosal immune cells in intestinalinflammationrdquo Gut vol 62 no 11 pp 1653ndash1664 2013

[11] S Saibeni L Spina andM Vecchi ldquoExploring the relationshipsbetween inflammatory response and coagulation cascade ininflammatory bowel diseaserdquo European Review for Medical andPharmacological Sciences vol 8 no 5 pp 205ndash208 2004

[12] S Saibeni M Cattaneo M Vecchi et al ldquoLow vitamin B6

plasma levels a risk factor for thrombosis in inflammatorybowel disease role of inflammation and correlation with acutephase reactantsrdquoThe American Journal of Gastroenterology vol98 no 1 pp 112ndash117 2003

[13] M Cattaneo M Vecchi M L Zighetti et al ldquoHigh prevalenceof hyperhomocysteinemia in patients with inflammatory boweldisease a pathogenic link with thromboembolic complica-tionsrdquoThrombosis and Haemostasis vol 80 no 4 pp 542ndash5451998

[14] T H Mogensen ldquoPathogen recognition and inflammatorysignaling in innate immune defensesrdquo Clinical MicrobiologyReviews vol 22 no 2 pp 240ndash273 2009

[15] E LeBouder J E Rey-Nores N K Rushmere et al ldquoSolubleforms of Toll-like receptor (TLR)2 capable of modulating TLR2signaling are present in human plasma and breast milkrdquo Journalof Immunology vol 171 no 12 pp 6680ndash6689 2003

[16] K I Iwami T Matsuguchi A Masuda T Kikuchi TMusikacharoen and Y Yoshikai ldquoCutting edge naturallyoccurring soluble form of mouse toll-like receptor 4 inhibitslipopolysaccharide signalingrdquo Journal of Immunology vol 165no 12 pp 6682ndash6686 2000

[17] I Munoz M P Sepulcre J Meseguer and V Mulero ldquoMolecu-lar cloning phylogenetic analysis and functional characteriza-tion of soluble Toll-like receptor 5 in gilthead seabream Sparusauratardquo Fish amp Shellfish Immunology vol 35 no 1 pp 36ndash452013

[18] S L Zunt L V Burton L I Goldblatt E E Dobbins and MSrinivasan ldquoSoluble forms of Toll-like receptor 4 are present inhuman saliva and modulate tumour necrosis factor-120572 secretionby macrophage-like cellsrdquo Clinical and Experimental Immunol-ogy vol 156 no 2 pp 285ndash293 2009

[19] A-C Raby E le Bouder C Colmont et al ldquoSoluble TLR2reduces inflammation without compromising bacterial clear-ance by disrupting TLR2 triggeringrdquo Journal of Immunologyvol 183 no 1 pp 506ndash517 2009

[20] R-S Chang Y-C Wang and S-T Kao ldquoSoluble toll-likereceptor 4 reversed attenuating effect of Chinese herbal Xiao-Qing-Long-Tang on allergen induced nerve growth factor andthymic stromal lymphopoietinrdquo Experimental and TherapeuticMedicine vol 6 no 5 pp 1199ndash1207 2013

[21] K Kondelkova J Krejsek L Borska et al ldquoMembrane andsoluble Toll-like receptor 2 in patients with psoriasis treatedby Goeckerman therapyrdquo International Journal of Dermatologyvol 53 no 11 pp e512ndashe517 2014

[22] H-B Yang K-Q Xie J-M Deng and S-M Qin ldquoExpressionof soluble toll-like receptors in pleural effusionsrdquo ChineseMedical Journal vol 123 no 16 pp 2225ndash2230 2010

[23] E Galliera L Drago C Vassena et al ldquoToll-like receptor 2in serum a potential diagnostic marker of prosthetic jointinfectionrdquo Journal of Clinical Microbiology vol 52 no 2 pp620ndash623 2013

[24] A Zhao C Blackburn J Chin and M Srinivasan ldquoSoluble tolllike receptor 2 (TLR-2) is increased in saliva of children withdental cariesrdquo BMC Oral Health vol 14 no 1 article 108 2014

[25] M E Houssen R H El-Mahdy and D A Shahin ldquoSerumsoluble toll-like receptor 2 a novel biomarker for systemic lupuserythematosus disease activity and lupus-related cardiovasculardysfunctionrdquo International Journal of Rheumatic Diseases 2014

[26] E Candia D Dıaz-Jimenez P Langjahr et al ldquoIncreasedproduction of soluble TLR2 by lamina propria mononuclearcells from ulcerative colitis patientsrdquo Immunobiology vol 217no 6 pp 634ndash642 2012

[27] F Cognasse H Hamzeh P Chavarin S Acquart C Geninand O Garraud ldquoEvidence of Toll-like receptor molecules onhuman plateletsrdquo Immunology and Cell Biology vol 83 no 2pp 196ndash198 2005

[28] F X Zhang C J Kirschning R Mancinelli et al ldquoBacteriallipopolysaccharide activates nuclear factor-kappaB throughinterleukin-1 signaling mediators in cultured human dermalendothelial cells and mononuclear phagocytesrdquo The Journal ofBiological Chemistry vol 274 no 12 pp 7611ndash7614 1999

[29] E Faure O Equils P A Sieling et al ldquoBacterial lipopolysac-charide activates NF-120581B through toll-like receptor 4 (TLR-4) in cultured human dermal endothelial cells Differentialexpression of TLR-4 andTLR-2 in endothelial cellsrdquoThe Journalof Biological Chemistry vol 275 no 15 pp 11058ndash11063 2000

[30] M D Boisclair H Ireland and D A Lane ldquoAssessment ofhypercoagulable states bymeasurement of activation fragmentsand peptidesrdquo Blood Reviews vol 4 no 1 pp 25ndash40 1990

[31] E F Stange S P L Travis S Vermeire et al ldquoEuropean evi-dence-based Consensus on the diagnosis and management ofulcerative colitis definitions and diagnosisrdquo Journal of Crohnrsquosamp Colitis vol 2 no 1 pp 1ndash23 2008

[32] A Dignass G van Assche J O Lindsay et al ldquoThe secondEuropean evidence-based Consensus on the diagnosis andmanagement of Crohnrsquos disease current managementrdquo Journalof Crohnrsquos amp Colitis vol 4 no 1 pp 28ndash62 2010

[33] R F Harvey and J M Bradshaw ldquoA simple index of Crohnrsquos-disease activityrdquoThe Lancet vol 1 no 8167 p 514 1980

[34] KW SchroederW J Tremaine andDM Ilstrup ldquoCoated oral5-aminosalicylic acid therapy for mildly to moderately activeulcerative colitis a randomized studyrdquoTheNewEngland Journalof Medicine vol 317 no 26 pp 1625ndash1629 1987

[35] J Satsangi M S Silverberg S Vermeire and J-F ColombelldquoThe Montreal classification of inflammatory bowel diseasecontroversies consensus and implicationsrdquo Gut vol 55 no 6pp 749ndash753 2006

[36] R G Shorter K A Huizenga and R J Spencer ldquoA workinghypothesis for the etiology and pathogenesis of nonspecificinflammatory bowel diseaserdquoTheAmerican Journal of DigestiveDiseases vol 17 no 11 pp 1024ndash1032 1972

[37] A I Thompson and C W Lees ldquoGenetics of ulcerative colitisrdquoInflammatory Bowel Diseases vol 17 no 3 pp 831ndash848 2011

[38] C W Lees J C Barrett M Parkes and J Satsangi ldquoNew IBDgenetics common pathways with other diseasesrdquo Gut vol 60no 12 pp 1739ndash1753 2011

[39] G C Nguyen and J Sam ldquoRising prevalence of venous throm-boembolism and its impact on mortality among hospitalized

10 Gastroenterology Research and Practice

inflammatory bowel disease patientsrdquo The American Journal ofGastroenterology vol 103 no 9 pp 2272ndash2280 2008

[40] H Yuhara C Steinmaus D Corley et al ldquoMeta-analysis therisk of venous thromboembolism in patients with inflammatorybowel diseaserdquoAlimentary Pharmacology andTherapeutics vol37 no 10 pp 953ndash962 2013

[41] W R Swindell P E Stuart M K Sarkar et al ldquoCellular dis-section of psoriasis for transcriptome analyses and the post-GWAS erardquo Acta Veterinaria Scandinavica vol 7 article 272014

[42] D Salem R Subang P Laplante J Levine and J Rauch ldquoThedual role of innate immunity in antiphospholipid syndrome andsystemic lupus erythematosusrdquo Lupus vol 23 no 12 pp 1327ndash1331 2014

[43] P Blair S Rex O Vitseva et al ldquoStimulation of Toll-likereceptor 2 in human platelets induces a thromboinflammatoryresponse through activation of phosphoinositide 3-kinaserdquoCirculation Research vol 104 no 3 pp 346ndash354 2009

[44] H Kalvegren C Skoglund C Helldahl M Lerm MGrenegard and T Bengtsson ldquoToll-like receptor 2 stimulationof platelets is mediated by purinergic P2X1-dependent Ca2+mobilisation cyclooxygenase and purinergic P2Y1 and P2Y12receptor activationrdquo Thrombosis and Haemostasis vol 103 no2 pp 398ndash407 2010

[45] S Rex L M Beaulieu D H Perlman et al ldquoImmune versusthrombotic stimulation of platelets differentially regulates sig-nalling pathways intracellular protein-protein interactions and120572-granule releaserdquo Thrombosis and Haemostasis vol 102 no 1pp 97ndash110 2009

[46] G Zhang J Han E J Welch et al ldquoLipopolysaccharide stim-ulates platelet secretion and potentiates platelet aggregationvia TLR4MyD88 and the cGMP-dependent protein kinasepathwayrdquoThe Journal of Immunology vol 182 no 12 pp 7997ndash8004 2009

[47] G T Brown and T MMcIntyre ldquoLipopolysaccharide signalingwithout a nucleus kinase cascades stimulate platelet sheddingof proinflammatory IL-1120573-rich microparticlesrdquo The Journal ofImmunology vol 186 no 9 pp 5489ndash5496 2011

[48] L Chavez-Sanchez J E Espinosa-Luna K Chavez-Rueda MV Legorreta-Haquet E Montoya-Dıaz and F Blanco-FavelaldquoInnate immune system cells in atherosclerosisrdquo Archives ofMedical Research vol 45 no 1 pp 1ndash14 2014

10 Gastroenterology Research and Practice

inflammatory bowel disease patientsrdquo The American Journal ofGastroenterology vol 103 no 9 pp 2272ndash2280 2008

[40] H Yuhara C Steinmaus D Corley et al ldquoMeta-analysis therisk of venous thromboembolism in patients with inflammatorybowel diseaserdquoAlimentary Pharmacology andTherapeutics vol37 no 10 pp 953ndash962 2013

[41] W R Swindell P E Stuart M K Sarkar et al ldquoCellular dis-section of psoriasis for transcriptome analyses and the post-GWAS erardquo Acta Veterinaria Scandinavica vol 7 article 272014

[42] D Salem R Subang P Laplante J Levine and J Rauch ldquoThedual role of innate immunity in antiphospholipid syndrome andsystemic lupus erythematosusrdquo Lupus vol 23 no 12 pp 1327ndash1331 2014

[43] P Blair S Rex O Vitseva et al ldquoStimulation of Toll-likereceptor 2 in human platelets induces a thromboinflammatoryresponse through activation of phosphoinositide 3-kinaserdquoCirculation Research vol 104 no 3 pp 346ndash354 2009

[44] H Kalvegren C Skoglund C Helldahl M Lerm MGrenegard and T Bengtsson ldquoToll-like receptor 2 stimulationof platelets is mediated by purinergic P2X1-dependent Ca2+mobilisation cyclooxygenase and purinergic P2Y1 and P2Y12receptor activationrdquo Thrombosis and Haemostasis vol 103 no2 pp 398ndash407 2010

[45] S Rex L M Beaulieu D H Perlman et al ldquoImmune versusthrombotic stimulation of platelets differentially regulates sig-nalling pathways intracellular protein-protein interactions and120572-granule releaserdquo Thrombosis and Haemostasis vol 102 no 1pp 97ndash110 2009

[46] G Zhang J Han E J Welch et al ldquoLipopolysaccharide stim-ulates platelet secretion and potentiates platelet aggregationvia TLR4MyD88 and the cGMP-dependent protein kinasepathwayrdquoThe Journal of Immunology vol 182 no 12 pp 7997ndash8004 2009

[47] G T Brown and T MMcIntyre ldquoLipopolysaccharide signalingwithout a nucleus kinase cascades stimulate platelet sheddingof proinflammatory IL-1120573-rich microparticlesrdquo The Journal ofImmunology vol 186 no 9 pp 5489ndash5496 2011

[48] L Chavez-Sanchez J E Espinosa-Luna K Chavez-Rueda MV Legorreta-Haquet E Montoya-Dıaz and F Blanco-FavelaldquoInnate immune system cells in atherosclerosisrdquo Archives ofMedical Research vol 45 no 1 pp 1ndash14 2014