Autoimmunity Reviews xxx (2014) xxx–xxx

AUTREV-01631; No of Pages 9

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Autoimmunity Reviews

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Review

The intestinal microbiota and microenvironment in liver☆,☆☆

Hong-Di Ma a, Yin-Hu Wang a, Christopher Chang b, M. Eric Gershwin b, Zhe-Xiong Lian a,c,⁎a Liver Immunology Laboratory, Institute of Immunology and The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center,University of Science and Technology of China, Hefei, Anhui 230027, Chinab Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, Davis, CA 95616, USAc Innovation Center for Cell Biology, Hefei National Laboratory for Physical Sciences at Microscale, Hefei, Anhui 230027, China

☆ Financial support: Financial support was providedProgram of China (973 Program-2013CB944900, 2010Natural Science Foundation of China (81130058, 8143003☆☆ There are no conflicts of interests regarding the pub

⁎ Corresponding author at: Liver Immunology LaboratoSchool of Life Sciences, University of Science andTechnologTel./fax: +86 551 63600317.

E-mail addresses:mahongdi@ustc.edu.cn (H.-D. Ma),H. Wang), c3chang@yahoo.com (C. Chang), megershwinzxlian1@ustc.edu.cn (Z.-X. Lian).

http://dx.doi.org/10.1016/j.autrev.2014.10.0131568-9972/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Ma H-D, et al, The10.1016/j.autrev.2014.10.013

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 October 2014Accepted 5 October 2014Available online xxxx

Keywords:Intestinal microbiotaGut–liver axisInflammatory bowel diseasePrimary sclerosing cholangitisPrimary biliary cirrhosisMucosal immunity

The intestinal microbiome plays a significant role in the development of autoimmune diseases, in particular, in-flammatory bowel diseases. But the interplay between the intestinal tract and the livermay explain the increasedassociation with autoimmune liver diseases and inflammatory bowel diseases. The gut–liver axis involves multi-ple inflammatory cell types and cytokines, chemokines and othermoleculeswhich lead to the destruction of nor-mal liver architecture. Triggers for the initiation of these events are unclear, but appear to include multipleenvironmental factors, including pathogenic or even commensal microbial agents. The variation in the gutmicrobiome has been cited as amajor factor in the pathogenesis of autoimmune liver disease and even other au-toimmune diseases. The unique positioning of the liver at the juncture of the peripheral circulation and the portalcirculation augments the interaction between naïve T cells and other hepatic cells and leads to the disruption inthe development of tolerance to commensal bacteria and other environmental agents. Finally, the innate immunesystem and in particular toll-like receptors play a significant role in the pathogenesis of autoimmune liverdisease.

© 2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Gut microbiota participates in the initiation and maintenance of autoimmune liver diseases by modulating the innate immune system . . . . . . . 0

2.1. Biliary innate immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.2. Innate immune cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3. Intestinal micro-environment affects T and B lymphocyte immunity in autoimmune liver disease . . . . . . . . . . . . . . . . . . . . . . 03.1. Molecular mimicry and production of autoantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.2. Activation of autoreactive T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4. The association of intestinal status with inflammatory cytokines in the context of autoimmune liver diseases . . . . . . . . . . . . . . . . . . . 05. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

by the National Basic ResearchCB945300) and the National4).lication of this article.ry, Institute of Immunology andyof China,Hefei 230027, China.

wangyh8@mail.ustc.edu.cn (Y.-@ucdavis.edu (M.E. Gershwin),

intestinal microbiota and m

1. Introduction

The liver and the gastrointestinal tract are intimately connected inthe context of metabolic activity and immune responses, primarilyresulting from their close anatomical and physiological relationship[1]. The liver has a dual blood supply. A quarter of the blood supply is de-rived from the systemic circulation which reaches the liver through thehepatic artery. The other three quarters are gut derived nutrient-richblood that enters the liver through the portal vein. The term “gut–liveraxis” has been coined to reflect the immunological phenomenon linkingthe two in health and disease [2].

icroenvironment in liver, Autoimmun Rev (2014), http://dx.doi.org/

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Microbes exist in the gut environment and play a significant role indigestion, and are a part of the mucosal immune system that helpsshape our ability to distinguish safe and danger signals [3,4]. Since theliver receives blood from both systemic circulation and the intestines,microbes in the intestines may also affect the immune environment inthe liver. Portal venous blood returning from the intestines containsthe products of digestion, along with antigens and microbial productsthat originate from the bacteria in the small and large intestines [5].The exposure of liver cells to antigens, and to microbial products de-rived from the intestinal bacteria, results in a distinctive local immuneenvironment that modulates immune tolerance in the liver [6–8].

To establish this tolerogenic environment, hepatic immune cellsincluding Kupffer cells, natural killer cells, dendritic cells and lympho-cytes, together with other nonparenchymal cells including endothelialcells and stellate cells orchestrate a controlled and organized responseto these potentially highly inflammatory factors from the intestines [9,10]. However, this tolerance is quite metastable and can be reversedby the right combination of signals, resulting in active local immunity[5]. For example, changes in the composition of the microbiome oralterations in gut permeability can promote translocation of microbesinto the portal circulation that delivers blood directly to the liver [2].These gut-derived microbial components represent danger signals forthe host cells in liver, activate the inflammatory cascade in immunecells and modulate the function of liver parenchymal cells [9].

There is a growing body of evidence showing that the intestinalmicrobiome possesses critical functions in liver physiology and meta-bolic homeostasis [11]. Gut-derived bacterial products aggravate hepat-ic fibrosis [12,13], whereas, gut sterilization helps prevent hepaticfibrosis after bile-duct ligation [12]. Moreover, disruption of the intesti-nal mucosal barrier facilitates bacterial translocation and promotes theprogression of liver fibrosis [13]. Prior studies also suggested that thegut microbiota may affect the progression of non-alcoholic fatty liverdisease (NAFLD)/non-alcoholic steatohepatitis (NASH) [14,15]. Thisdysbiosis in the digestive tract results in the exacerbation of hepaticsteatosis and obesity [16].

The relationship between intestinal homeostasis and autoimmuneliver diseases is summarized in this review by discussing the potentialinvolvement of intestinal homeostasis in the pathogenesis of autoim-mune liver diseases [17–19], the understanding ofwhichwill ultimatelysteer the development of novel clinical treatments.

2. Gut microbiota participates in the initiation and maintenance ofautoimmune liver diseases by modulating the innate immunesystem

As the first line of defense against pathogens, the innate immunesystemmediates interactions between the hosts and their intestinalmi-croflora [20]. Pattern recognition receptors widely expressed on innateimmune cells and parenchymal cell are essential for the recognition andclearance of commensal and pathogenic microflora [21]. The conflictbetween the innate immune system and gut microflora is not onlynecessary to repel the invasion of pathogenic microorganisms, butalso critical to maintain a balanced immune response that is reflectedin good overall health [22,23]. Evidence has been shown that thedysbiosis of innate immunity in the gastrointestinal tract is one of themost important factors in the initiation and perpetuation of autoim-mune liver injury [24]. The gut microflora thereby plays a significantrole in the pathogenesis of autoimmune liver diseases.

2.1. Biliary innate immunity

Bile ducts are a significant component of liver architecture, as biliaryepithelial cells (BECs) maintain contact with pathogen associated mo-lecular patterns (PAMPs) originating from intestinal microflora derivedfrom bile [25]. As part of the lining of the bile duct, BECs act as a barrierand express a series of molecules associated with immune recognition

Please cite this article as: Ma H-D, et al, The intestinal microbiota and m10.1016/j.autrev.2014.10.013

and response, including toll-like receptors (TLRs), histocompatibilitycomplex (MHC) antigens, adhesion molecules and co-stimulatorymolecules [26]. BECs thus play a role in both the innate and adaptiveimmune systems. This distinct characteristic of BECs facilitates theirrole in the defense against microbial infection, but also renders suchcells uniquely susceptible to autoimmune disorders such as PBC andPSC (Fig. 1A) [27–31].

Biliary epithelial cell antibodies (BEC-Abs), which have been foundto be present in high frequency in the bile ducts of patients with PSC,up-regulate expression of TLR4 and TLR9 as well as MyD88 on culturedBECs [32]. TLR-expressing BECs, when further stimulatedwith lipopoly-saccharide and CpG DNA, produce proinflammatory cytokines andchemokines including interleukin-1β, interleukin-8, interferon-γ,tumor necrosis factor-α, granulocyte-macrophage colony-stimulatingfactor, and transforming growth factor-β (Fig. 1A) [32,33]. Upregulationof TLRs as a result of binding of PSC BEC-Abs demonstrated that BEC-Abmay be a critical regulator of cholangitis in PSC. Specifically, bindingof lipopolysaccharide and CpG DNA to their respective TLRs inducesBECs to produce pro-inflammatory cytokines and chemokines, leadingto the further recruitment of inflammatory cells and resulting incholangitis in PSC. This indicates that environmental factors are essen-tial triggers that can initiate inflammation in the livers of patients withPSC [32]. Interestingly, PSC often coexists with ulcerative colitis [34],suggesting that alteration of the gut microbiome may also play a rolein inflammatory bowel disease. Furthermore, accentuated responsesof both TLR4 and TLR9 have been reported in the intestinal epitheliumof patients with ulcerative colitis [35,36]. These observations suggestthat stimulators of TLRs from the digestive tract in PSC patients withulcerative colitis may play a role in the pathogenesis of PSC.

Based on evidence, we can surmise that gut microbes render BECsactive participants and mediators of their own destruction in PSC(Fig. 1A). On the one hand, microbial pathogens from the gut can possi-bly circulate to bile ducts in the liver and activate an inflammatoryresponse via TLR activation. On other hand, the adaptive immuneresponse mediated by BEC-Ab may facilitate the innate immune re-sponses by induction of functional TLRs on the bile duct epithelium ofPSC patients. Thus, both the innate and adaptive immune responsescooperate to establish a pro-inflammatory loop, resulting in an en-hanced or sustained inflammatory signaling in BECs, leading to the au-toimmune injury found in BECs in PSC patients.

Bacterial products have also been implicated in the pathogenesis ofbile duct diseases other than PSC [37]. Several bacterial products havebeen detected in liver tissue from patients with PBC, suggesting anassociation with the development of PBC [38,39]. Similar to PSC, TLR4was found to be expressed at high levels on BECs in PBC patients. Thisaberrant expression of TLR4was also observed in periportal hepatocytesof PBC livers and extended to interlobular hepatocytes in advancedstages of PBC [40]. The deviant distribution of TLR4 in the livers of PBCpatients suggests the involvement of bacterial pathogens and TLR4 indifferent stages of PBC.

2.2. Innate immune cells

Liver innate immune cells are essential effector cells that undergovarious forms of activation in response to stimuli, including microbialsignals originating from the gut. Many autoimmune disorders havebeen linked to dysfunctional innate immune cells responding againstcommensal microflora. Such aberrant immune responses partially con-tribute to the pathogenesis of PBC.

The innate immune cells of patients with PBC demonstrate higherreactivity than controls (Fig. 1B). For example, the frequency and abso-lute number of blood and liver natural killer (NK) cells in patients withPBC, as well as their cytotoxic activity and perforin expression, are in-creased [41,42]. Moreover, monocyte-derived macrophages (MDMϕ)in patients with PBC polarize most likely to M1, and they exhibit strong

icroenvironment in liver, Autoimmun Rev (2014), http://dx.doi.org/

Fig. 1.Gutmicrobiota participates in the initiation andmaintenance of autoimmune liver diseases bymodulating the innate immune system. A. Biliary innate immunity. Bile ducts form anespecially critical architecture, in which BECs act as a barrier and are constantly exposed to bacterial products from enteric microflora. The microbial pathogens from the gut get into theintrahepatic bile ducts following circulation and activate inflammatory responses via TLR expressed on BECs. Moreover, in PSC, BEC-Ab facilitated this TLR activation and induced BECs toproducemore cytokines/chemokines, leading to the possible recruitment of inflammatory cells and causing cholangitis in PSC. B. Innate immune cells. On the one hand, there are increasedfrequency and number of innate immune cells (such as NK cells and monocytes) in autoimmune disorders. On the other hand, innate immune cells from patients with autoimmune liverdiseases demonstrate higher reactivity via TLR activation by potential gut derived stimuli.

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reactivity when they interact with AMAs and apoptotic bodies of BECs[41].

Toll-like receptors (TLRs), as evolutionarily conserved germline-encoded pattern recognition receptors, have a crucial role in earlyhost defense by recognizing pathogen associated molecular patterns(PAMPs) andmay serve as an important link between innate and adap-tive immunity [28,43–45]. While a robust TLR response is critical forsurvival and defense against invading pathogens, inappropriate signal-ing in response to alterations in the local microflora environment canbe detrimental [46,47]. Such “off-normal TLR responses” may form thebasis for a large number of gastrointestinal and liver disorders [48–50].

Different chemical and infectious agents derived from the outsideenvironment, or in vivo metabolism, can potentially activate variousTLRs on monocytes and amplify the inflammatory responses, leadingto the breakdown of immune tolerance and the initiation of autoimmu-nity [51]. In particular, liver autoimmunity, due to the close relationshipof the liver and gastrointestinal tract, can be associated with TLRs' mod-ulators from the gut. While receiving large volumes of blood via theportal vein, the liver is constantly exposed to bacterial products fromenteric microflora. Under normal circumstances, the immune systemof healthy individuals exhibits liver tolerance to TLR ligands. However,injuries, infections or other causes may lead to a breakdown of thistolerance and result in inflammatory disorders of the liver. In thesecases, pathogens from intestinal microbiota may inappropriately acti-vate TLRs on innate immune cells in liver which may contribute to thepathogenesis of autoimmune liver diseases.

Indeed, peripheral monocytes from patients with PBC secrete highlevels of cytokines via TLR activation [41]. In addition to BECs, mono-cytes from patients with PBC are also more susceptible to activation ofTLRs (Fig. 1B). Mao et al. reported that monocytes from patients with

Please cite this article as: Ma H-D, et al, The intestinal microbiota and m10.1016/j.autrev.2014.10.013

PBC were more sensitive to signaling via selective TLRs (TLR2, TLR3,TLR5 and TLR9) compared with healthy controls [41]. This resulted toenhanced secretion of pro-inflammatory cytokines by PBC monocyteswhich would be integral to the inflammatory response and critical inthe breakdown of self-tolerance [41]. It has also been demonstratedthat after LPS stimulation, the level of TLR4 expression is significantlyincreased in PBC monocytes. Further research has confirmed that theTLR4 signaling pathway is able to be highly activated in PBC patients[52,53].

In response to the TLR ligands, the activation of NF-κB and expres-sion of MyD88 mRNA have been shown to be significantly increasedin PBC PBMCs as compared with controls. Notably, the surface expres-sion of RP105, which is considered a negative regulator of TLR4 re-sponses, is decreased in PBC monocytes in comparison with controls[54]. The hypersensitivity to TLR4 signaling in PBC monocytes suggestsa role of TLR4 in the pathogenesis of PBC. In addition, TLR3 has alsobeen reported to be capable of aggravating PBC. Ambrosini et al. report-ed that the addition of polyI:C, a viral RNAmimetic and TLR3 agonist, toa PBC mouse model caused a profound exacerbation of autoimmunecholangitis, including a significant increase in infiltrating CD8+ T cells,a marked increase of proinflammatory cytokines and signs of fibrosisin liver tissues [55].

3. Intestinal micro-environment affects T and B lymphocyteimmunity in autoimmune liver disease

3.1. Molecular mimicry and production of autoantibodies

Serologically, PBC is characterized by the presence of anti-mitochondrial antibodies (AMAs) in 90–95% of patients [56]. The

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presence of autoantibodies may precede the occurrence of symptomsby many years [57]. AMAs react with mitochondrial antigens of the 2-oxoacid dehydrogenase complex (2-OADC), including an epitopeon the E2 subunit of the pyruvate dehydrogenase enzyme complex(PDC-E2) and the E2 subunit of functionally related 2-oxo-acid dehy-drogenase complexes, branched chain 2-oxo-acid dehydrogenase(BCOADC-E2), and 2-oxo-glutarate dehydrogenase (OGDC-E2) [58].PDC-E2 is a well-conserved molecule among various species, especiallyat the lipoic acid binding sites which is the major epitope for bothautoantibodies and for reactive CD4 and CD8 T cells in PBC [26,59,60].Because of its evolutionary conservation, sera from patients withPBC have been shown to react with both human and Escherichia coliPDC-E2 [61]. The reactivity of AMA to both human and gut bacterialmolecules has provided clues to the relationship between the onsetof PBC and intestinal microflora (Fig. 2A). Earlier work has shown thatPDC-E2 specific autoreactive CD4 T cell clones recognize E. coli PDC-E2[62]. In addition, bacterial homologous peptides have been shown tohave an agonistic effect on autoreactive CD8 T cells specific for PDC-E2[63].

Molecularmimicry is one of themechanisms bywhich an individualmay fail to discriminate between self and non-self [64], thus disruptingthe balance between a hypo- and hyperimmune state, leading to either

Fig. 2. Intestinal micro-environment affects T and B lymphocyte immunity in autoimmune livactivation of the immune system to cross-react with conservative PDC-E2 of self-evoking stronexposure to pathogen-associated molecular patterns (such as bacterial CpG-B, ligand of TLR9 oresponsiveness in B cells. C. Aberrantly increased chemokines guide intestinal primed T lymLiver sinusoidal endothelial cells (LSECs) are the link between the intestinal micro-environmenolite conversion, LSECs induce Treg differentiation leading to anergy and tolerance in liver.

Please cite this article as: Ma H-D, et al, The intestinal microbiota and m10.1016/j.autrev.2014.10.013

infectious diseases or autoimmunity respectively [65]. These non-specific reactions to PDC-E2 of gut bacteria such as E. coli may result inthe activation of the human immune system to cross-react with theconserved human PDC-E2, evoking strong autoreactive responses anddevelopment of the disease [62,66].

In addition to TLR3 and TLR4, activation of TLR9 has been demon-strated to contribute to the development of PBC (Fig. 2B). Kikuchiet al. reported that after exposure to bacterial CpG-B, the frequency ofintracellular IgM-positive B cells was markedly increased in PBMCsfrom PBC patients. These B cells produced high levels of IgM and wereidentified as functional CD27+ memory B cells [67]. The prevalenceand unusually high levels of antimitochondrial antibodies (AMAs) inpatients with PBC suggest a profound loss of B cell tolerance [68,69].Further studies have provided evidence that the AMA productionof these B cells is significantly promoted by CpG-B stimulation [70].

Since a number of studies have suggested a bacterial etiology in PBC,it can be hypothesized that the promotion of a hyper-IgM state and theincreased production of AMA by bacterial CpG-B may be a reflection ofthe role of the CpG ligand, TLR9, in the development of a chronic poly-clonal innate immune response in patients with PBC. As previouslymentioned, pathogens from enteric microflora may be considered tobe an important cause of inappropriate response of innate immune

er diseases. A. Reactions to PDC-E2 of gut bacteria such as E. coli may result in the cross-g autoreactive responses and initiate PBC by this molecular mimicry event. B. Long-termr other PAMPs from the gut) may sensitize TLRs and result in aberrant humoral immunephocytes homing to the liver, which causes T cell mediated hepatic disorders in PSC. D.t and the hepatic immune compartment. By gut-derived antigen presentation and metab-

icroenvironment in liver, Autoimmun Rev (2014), http://dx.doi.org/

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cells in PBC. Thus, microorganisms from the gut may sensitize TLRs oninnate immune cells and result in aberrant humoral immune respon-siveness in B cells of PBC patients [71].

3.2. Activation of autoreactive T cells

The liver stands at a hemodynamic confluence [5], where blood fromthe systemic circulation and the intestinal tract converge into hepaticsinusoids. This distinct architecture not only enables circulating naïveT cells to make direct contact with various cell types in the liver suchas hepatocytes, hepatic stellate cells (HSC), Kupffer cells as well asliver sinusoidal endothelial cells (LSECs) and bile duct epithelial cells,but also allows primed effector T cells from the intestinal tract to mi-grate into the liver.

As characterized by progressive bile duct destruction, primarysclerosing cholangitis (PSC) also develops as an extra-intestinal compli-cation of inflammatory bowel disease (IBD) [72,73]. The prevalence ofIBD (typically chronic ulcerative colitis (CUC)) among PSC patients isapproximately 70–80% while 2–7.5% of patients with CUC will developPSC [74]. An aberrant homing of intestinal T lymphocytes to the liveris thought to be an important factor for the development of T cell medi-ated hepatic disorders associated with gut inflammation (Fig. 2C) [75].Furthermore, PSC can develop for the first time many years after IBDhas become quiescent and even after previous proctocolectomy [76].In consideration of this fact, Eksteen et al. hypothesized that PSC is me-diated by long-lived memory T cells originally primed in the gut, andwere able to eventually cause extra-intestinal inflammation in theabsence of active IBD [77]. This is supported by the evidence thatmuco-sal T cells are recruited to the liver by the aberrant expression of the gut-specific chemokine CCL25 in the liver of PSC patients, which mediatesthe binding of α4β7

+ T cells with mucosal addressin cell adhesionmolecule 1 on the hepatic endothelium [77].

Sinusoids are specialized hepatic structures with thin-walled vesselsthat allow oxygenated blood from the arterial system tomixwith portalvenous blood returning from the intestine. They are the link betweenthe intestinal micro-environment and the hepatic immune compart-ment (Fig. 2D). Liver sinusoidal endothelial cells (LSECs) are believedto shift the hepatic immune response toward tolerance by presentingmajor histocompatibility complex (MHC) I-restricted antigens, therebyinducing anergy and tolerance [78,79]. In particular, Kruse et al. report-ed that priming of CD4+ T cells by liver sinusoidal endothelial cells in-duces CD25low FoxP3− regulatory T cells that suppress autoimmunehepatitis, as shown by their effects on reducing the levels of alanineaminotransferase and cellular infiltrates in a T cell-mediated autoim-mune hepatitis model in vivo [80].

In addition to various types of immune cells, metabolites includingantigens derived from food and biological molecules from the intestinaltract are also transferred into the liver through normal vascular chan-nels. Food antigens and microbiome brought from the intestinal tractare considered to be the main contributors to the development of livertolerance [81]. Products of metabolism from the intestinal tract suchas retinoic acid (RA), a derivative of vitamin A, have also been reportedas an important factor in the “gut–liver axis” [1,82,83].

T cells acquire a gut-homing phenotype with high levels of α4β7

integrin and of CC chemokine receptor 9 (CCR9) [84,85], which is de-pendent on retinoic acid (RA) provided by dendritic cells (DCs) fromthe gut-associated lymphoid tissue (GALT) [86,87]. RA is able to inducethe expression of gut homing molecules on T cells [88,89]. Neumannand colleagues found that LSECs expressed functional retinal dehydro-genases which can convert vitamin A to RA. Moreover, in addition toGALT-DCs, LSECs can also prime CD4+ T cell and promote gut tropismvia the action of vitamin A [82]. This liver–gut trafficking is a novel fea-ture of gut–liver axis and in that the interaction between the liver andgut is bidirectional [1].

The effect of RA on T-cell activation and differentiation is closely re-lated to liver immunity [90]. It has been reported that RAmay regulate a

Please cite this article as: Ma H-D, et al, The intestinal microbiota and m10.1016/j.autrev.2014.10.013

much broader range of CD4+ T cell priming and differentiation, includ-ing serving as a cofactor for the development of induced regulatory T(iTreg) cells [91,92]. Vitamin A is absorbed by the intestinal tract andtransported into the liver through the circulation. Lack of absorptionor low efficiency of conversion of vitamin A can potentially lead to adecreased number of iTreg cells, thereby breaking down the balanceof liver immunity and creating a precarious environment for thedevelopment of autoimmune liver disease. Mehal proposed that theloss of vitamin A may result in eventual loss of RA and a reduction inLSEC-mediated production of regulatory CD4+ T cells, opening up thepossibility that derangements in RA-based signaling have a role in auto-immune hepatitis [1].

It should be noted that although growing evidence supports thenotion that aberrant intestinal T lymphocytes and intestinal metabolicdisturbance trigger the development of intestinal and liver disorders,the intestinal tract and liver disorders are characterized by distinctpathogenesis.

4. The association of intestinal status with inflammatory cytokinesin the context of autoimmune liver diseases

Tumor necrosis factor (TNF), a major factor in the immune responseto bacterial infection, contributes to the pathogenesis of several autoim-mune diseases, including rheumatoid arthritis (RA) and Crohn's disease[93–96]. Proven as the most efficient therapy for RA thus far, TNFantagonists have attracted considerable attention in the treatment ofautoimmune diseases [97]. Likewise, circulating levels of TNF wasmost significantly correlated with hepatobiliary injury in autoimmunediseases [98]. Moreover, genetic studies have identified a significantassociation between possession of the TNF2 allele and susceptibility toPSC [99]. Although the role of TNF in the development and progressionof PSChas yet to be clearly defined, it has beenproposed that the inflam-matory responses in PSC may be initiated by the secretion of cytokineslike TNF by hepatic macrophages in response to exposure to intestinalbacterial antigens in the portal venous blood (Fig. 3A). TNF and endo-toxin may stimulate biliary epithelial cells to secrete chemokines andcytokines that would in turn attract and activate neutrophils, mono-cytes, macrophages, T cells and fibroblasts. The accumulation of theseeffector cells resulted in biliary damage [100].

In autoimmune liver diseases, it has been reported that the frequen-cy of IFN-γ+ cells is significantly increased in the cellular infiltrates inlivers of patients with autoimmune hepatitis [101]. In addition, thefrequency of IFN-γ expressing cells is predominantly found in associa-tion with damaged bile ducts in PBC patients [102]. At the same time,it has been reported that IFN-γ can increase epithelial permeabilityas indicated by markers of paracellular permeability and bacterialtranscytosis, with at least a portion of the bacteria using the transcellu-lar permeation pathway (Fig. 3B) [103,104]. Although IFN-γ appears tobe involved in both autoimmune liver diseases and gut bacterial trans-location [105–107], solid and reliable evidence is needed to prove thatIFN-γ induced gut bacterial translocation contributes to the develop-ment of these autoimmune liver diseases.

IL-17 is the signature cytokine of T helper (Th)17 cells,which play animportant role in defense against extracellular bacteria and fungi [108,109], particularly at epithelial and mucosal surfaces [110]. Both func-tional and genetic evidence suggests the involvement of IL-17 in thepathogenesis of mucosal immune disorders in gut colitis [111–113].For example, in both Crohn's disease (CD) and ulcerative colitis (UC),the two major forms of IBD in humans, increased expression of IL-17and IL-23 has been found in the intestinal lesions [113–116]. Moreover,IL-17 producing cells have also been implicated in the pathogenesis ofseveral other human autoimmune diseases, such as systemic lupuserythematosus (SLE), multiple sclerosis, psoriasis, and rheumatoidarthritis [111,117–120]. In particular, an increase in the frequency ofIL-17+ lymphocytic infiltration in liver tissues from PBC patients hasalso been reported [121–123]. Similar observations have been made in

icroenvironment in liver, Autoimmun Rev (2014), http://dx.doi.org/

Fig. 3. The association of intestinal status with inflammatory cytokines in the context of autoimmune liver diseases. A. Hepatic macrophages primed by intestinal bacterial antigens in theportal venous blood secrete cytokines like TNF and initiate inflammatory recruitment. The recruited effector cells destroy the bile duct in PSC. B. IFN-γ can increase epithelial permeabilityby increasing paracellular permeability and bacterial transcytosis. IFN-γ induced gut bacterial translocation may contribute to the inflammation in the liver. C. PAMPs derived from intes-tinal commensal bacteria are recognized by antigen presenting cells in the gut and presented to T cells. Primed T cells produce inflammatory cytokines such as IL-17 and IL-23 to amplifythe inflammatory responses and finally lead to disorders in the liver.

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IL-2Rα−/− mice, a murine model of human PBC, where marked aggre-gations of IL-17+ cells are foundwithin portal tracts and increased num-bers of Th17 cells in the liver may be found when compared to theperiphery [121].

In recent genome-wide association studies (GWAS), several locihave been found to be associated with PSC [124,125]. Among these,polymorphisms within genes which code for molecules involved inTh17 differentiation and transduction of signals received by toll-likereceptor (TLR) and dectin-1 have been found. These PPRs (pattern rec-ognition receptors), which recognize conserved molecules of bacterialand fungal species [126], are of particular interest. Katt et al. reportedthat IL-17A-expressing lymphocytes as well as bacterial RNA werefound within the portal tracts of PSC livers and demonstrated an in-creased Th17 response to microbial stimulation in patients with PSC[127]. Since IL-17 secreted by Th17 cells may link pathogen defense toautoimmunity [128], it is likely that an increased exposure to pathogensor a change in the microbial community in bile may induce an increasein IL-17 secretion by Th17 cells, which may then contribute to uncon-trolled portal and biliary inflammation in PSC [129].

It should also be mentioned that specific gut commensals, such assegmentedfilamentous bacteria (SFB), promote the generation of an in-testinal subset of Th17 cells and enhance the expression of interferon-γ(IFN-γ) or Foxp3 in gut T cell subsets [130–133]. In a T cell transfer

Please cite this article as: Ma H-D, et al, The intestinal microbiota and m10.1016/j.autrev.2014.10.013

model of arthritis, it has been shown that in the localmicroenvironmentof gut-associated lymphoid tissues, inflammatory cytokines elicited bythe commensal flora dynamically enhance the antigen responsivenessof T cells that were otherwise tuned down to a systemic self-antigen.This ultimately leads to an enhanced recruitment of pathogenic T cellsand the development of a more severe form of autoimmune arthritis[134]. Similarly, the severity of autoimmune liver diseases is associatedwith gut commensal flora and the predominant effector cells in autoim-mune liver diseases are also T cells. Antigens from intestinal commensalbacteria (such as SFBs) can be recognized by DCs ormacrophages in thegut and presented to T cells (Fig. 3C). Primed T cells proliferate and dif-ferentiate into effector cells which produce inflammatory cytokinessuch as IL-17 and IL-23, which amplify the inflammatory responsesand finally lead to the breakdown of tolerance in the liver. Likewise,whether inflammatory cytokines elicited by the commensal flora candynamically enhance the antigen responsiveness or affect the functionof effector T cells in autoimmune liver diseases deserves to be furtherstudied [51].

5. Conclusion

Autoimmune liver diseases are complex diseases arising from theinteraction of both environmental factors and genetic background. In

icroenvironment in liver, Autoimmun Rev (2014), http://dx.doi.org/

7H.-D. Ma et al. / Autoimmunity Reviews xxx (2014) xxx–xxx

the last few years, the incidence of autoimmune liver diseases hasbeen increasing [135]. The reasons for this observation are unclear, butmay reflect a change in our lifestyles and environments, which mayinclude changes in the microbiome to which we are exposed. Sincethe immune-modulating capabilities of microorganisms in the gut havebecome a significant topic for recent research, the role of intestinalmicro-environment factors in the pathogenesis of autoimmune liver dis-eases has been better appreciated and has taken on greater importance.Although the effect of the intestinal micro-environment on autoimmuneliver diseases has not yet been thoroughly investigated, strong evidencesuggested that pathogen-derived compounds and products of metabo-lism from the gut play an important role in modulating liver diseases.

In this review, we summarize the effects of the intestinal microbiotaon the liver immune microenvironment through the gut–liver axis. Onthe one hand, agents from the intestinal tract are transported to theliver through normal blood circulation and enter the hepatic sinusoidsthrough the portal vein. At this junction of blood flow, innate immunecells, such as DCs and Kupffer cells, present antigens to prime naïve Tcells near hepatic sinusoids. Intensive abnormal priming of hepatic infil-trated T cells by inappropriate stimulators from the gut can potentiallylead to disorders in liver immunity. Inside the liver, TLR-expressingBECs and hepatic infiltrating monocytes activated by abnormal signalsfrom the gut result in aggravated innate and adoptive immune re-sponses, which cause persistent inflammation in the liver.

On the other hand, the dysbiosis of gut microbiota leads to an im-mune imbalance in the intestinal tract. In this context, aberrantly acti-vated T cells, which expressed specific chemokine receptors as well asintegrins, can migrate from the gut into the liver, and further inducechronic liver inflammation.

Lastly, we review the critical effects of inflammatory cytokineson the initiation and development of autoimmune liver diseases.Pro-inflammatory cytokines link pathogen defense to liver autoim-munity by recruiting inflammatory immune effector cells into theliver. The accumulation of these “attackers” results in autoimmuneliver disease and chronic liver tissue damage. In addition, pro-inflammatory cytokines can increase intestinal epithelial permeabilityand lead to bacterial transcytosis, resulting in the breakdown of muco-sal immune balance and acting as a “fuse” toward the development ofliver autoimmunity.

To this date, critical questions remain unanswered regarding theinteraction between the intestinal mucosa and the hepatic micro-environment [136,137]. In particular, the relationship betweenintestinal homeostasis and autoimmune liver disease has yet to befully understood. Future studies directed at these questions are neededto identify predictors for autoimmune liver diseases and to provideimportant guidance for the development of new therapies with ap-plications in autoimmune liver disease and autoimmunity in general[138–143].

Take-home messages

• Autoimmune liver diseases are complex diseases arising from theinteraction of both environmental factors and genetic background.

• Gutmicrobiota participates in the initiation andmaintenance of auto-immune liver diseases by modulating the innate immune system.

• Multiple cell types and cytokine molecules may be involved in thepathogenesis of autoimmune liver disease.

• The gut–liver axis allows greater interaction between the peripheralcirculation and the liver, leading to an association between autoim-mune liver disease and inflammatory bowel diseases such as ulcera-tive colitis or Crohn's disease.

• In addition to bacteria and other microbes as potential triggers of au-toimmunity, a variety of other environmental exposures includingchemical agents may affect the development of liver immune diseasethrough disruption of normal tolerogenic physiologic mechanisms.

Please cite this article as: Ma H-D, et al, The intestinal microbiota and m10.1016/j.autrev.2014.10.013

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