Female genital tract immunization: Evaluation of candidate immunoadjuvants on epithelial cell...

Vaccine 24 (2006) 5744–5754

Female genital tract immunization: Evaluation of candidateimmunoadjuvants on epithelial cell secretion of CCL20 and

dendritic/Langerhans cell maturation

Magali Cremel, Hind Hamzeh-Cognasse, Christian Genin ∗, Olivier DelezayGroupe Immunite des Muqueuses et Agents Pathogenes (GIMAP, EA3064), St Etienne University, France

Received 22 February 2006; received in revised form 25 April 2006; accepted 25 April 2006Available online 9 May 2006

Abstract

The female genital tract is an important site for numerous pathogens entry. Local immunization, generating specific mucosal IgA andsystemic IgG, represents an interesting alternative immunization pathway. However, such a vaccine strategy needs mucosal adjuvants toobtain the best immune response. Considering that the immunization process is mainly dependent on the capture and on the transport of theaeaa©

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ntigen by Langerhans cells, we evaluated potential adjuvant molecules by analysing their effects on the CCL20 secretion by endocervical andxocervical/vaginal epithelial cells as well as on dendritic cell and Langerhans cell maturation. We demonstrated that DC-Chol and Zymosanre the most efficient mucosal candidate immunoadjuvants that generate a strong increase of CCL20 secretion by the two epithelial cell linesnd the maturation of dendritic and Langerhans cells, respectively.

2006 Elsevier Ltd. All rights reserved.

eywords: Female genital tract; Local immunization; Candidate mucosal immunoadjuvants

. Introduction

The female genital tract represents an alternative immu-ization pathway (compared to systemic one) since this non-raumatic route could promote the production of antibodies inarious sites [3]. Indeed, it has been previously demonstratedhat vaginal immunization was able to generate local IgA andystemic IgG production [6–9]. However, immune responseowards the antigen is highly dependent on its capacity toe captured and processed by the immature dendritic cells inenital mucosa: the Langerhans cells (LC). LC are selectivelyecruited by a main chemokine, CCL20, secreted by vaginalpithelial cells in response to local inflammation or mucosalamage [12]. This chemokine acts through the CCR6 recep-or, which is highly expressed in Langerhans cell precursors

∗ Correspondence to: GIMAP, Faculte de Medecine, 15, rue Ambroiseare, 42023 Saint Etienne Cedex 2, France. Tel.: +334 77 82 83 79;ax: +334 77 82 84 93.

E-mail address: [email protected] (C. Genin).

(LCp) [13]. Such a mechanism has been demonstrated fornumerous epithelial structures including skin, intestinal andgenital tract [20–23]. LC are preferentially found inside mul-tilayer epithelia, but dendritic cells located in chorion underthe monostratified epithelia could also participate in the firststeps of antigen capture [24]. The involvement of CCL20in the migration of Langerhans/dendritic cells has been alsodemonstrated in mono or pluristratified epithelia [27,28]. Itseems that an efficient mucosal immunization has to promotethe selective recruitment of immune cells by generating alocal secretion of CCL20 dependent on the adjuvant proper-ties of the vaccine formulation. The recent development ofnew potential adjuvants able to promote CCL20 secretion,such as Flagellin, clearly argues in favour of such a mech-anism [33,34]. Numerous compounds isolated from viruses,bacteria or fungi have been evaluated for immunomodulatingproperties. The great majority of these molecules acts throughspecific receptors, the toll-like receptors (TLR), involved inthe innate immune response [35–38]. The expression of suchreceptors on epithelial cells allows the permanent sensing of

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M. Cremel et al. / Vaccine 24 (2006) 5744–5754 5745

the mucosal micro-environment and TLR dysfunction leadsto susceptibility to bacterial or viral invasion [39].

However, LCp recruitment at the epithelial cells con-tact site may not be sufficient to induce a specific immuneresponse. LC have to mature after antigen capture in order toactivate T lymphocytes in the lymph nodes. The LC matura-tion process is associated with a co-receptor switch (CCR6to CCR7) allowing the migration of LC from epithelial struc-ture to lymph nodes [12,13] as well as with the acquisition ofa set of molecules (CD80, CD83 and CD86) [40] required foroptimal antigen presentation. In addition, antigen-presentingcells have been shown to secrete cytokines after antigen con-tact or infection [41], such IL-6 and IL-8, who participateactively in the immune response process [42,43].

In order to evaluate the efficiency of various compoundssusceptible to be used as mucosal adjuvants in female genitaltract, we have analyzed the effects of these molecules on twoparameters, i.e. CCL20 secretion and LC/DC activation.

This study was performed using two epithelial cell lines,SiHa and HEC1A, representative of pluristratified exocervi-cal/vaginal and monostratified endocervical/uterine epithe-lial structures, respectively [22,44–48]. This approach couldbe relevant for the screening of new vaginal mucosalcandidate immunoadjuvants to develop local vaccinationstrategy.

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more than 80% of CD1a and variable proportions of Lan-gerin (20–50%) and were very similar to normal Langerhanscells [49]. Dendritic cells derived from mononuclear cellswere obtained from peripheral blood of healthy donors bycentrifugation on Histopaque®1077 (Sigma-Aldrich). Mono-cytes were depleted of T and B cells using mouse anti-CD2,CD7, CD16, CD19, CD56 and CD235a mAbs and anti-mouseIg coupled to magnetic microbeads according to the manu-facturer’s instructions (Dynal Biotech, Compiegne, France).The technique routinely resulted in more than 80% purity, asassessed by flow cytometry experiments. Purified monocytes(106 cells/ml) were cultured for 5 days in six-well tissue cul-ture plates in RPMI 1640 culture medium supplemented withl-glutamine (Cambrex BioScience), penicillin-streptomycinsolution (Sigma-Aldrich) and 10% heat-inactivated FBS.To induce dendritic cell differentiation, recombinant humanGM-CSF (500 U/ml, Peprotech) and Interleukine-4 (IL-4,50 U/ml, Peprotech) were added to the medium. On Days2 and 4, cells were fed with fresh medium and cytokines. AtDay 5, dendritic cells obtained expressed more than 60% ofCD1a, more than 40% of DC-Sign and 0% of CD 14 andLangerin.

2.2. Cell stimulations

When confluent, SiHa and HEC1A cells, cultured in 48-wasrw(((a1Cg1b2(csdd

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. Materials and methods

.1. Cell culture

The SiHa and HEC1A cell lines were routinely grown inMEM-F12 medium (Cambrex BioScience, Verviers, Bel-ium), supplemented with 10% heat inactivated fetal bovineerum (FBS) and penicillin-streptomycin solution (Sigma-ldrich, St. Louis, MO, USA). SiHA cells were very similar

o normal vaginal epithelial cells [22].LC were obtained from umbilical cord blood CD34+

ematopoietic progenitor cells. First, cord blood mononu-lear cells were separated from granulocytes and erythrocytesy Ficoll density gradient centrifugation. Monocyte depletionas performed by differential cell adherence in a 75-cm2 cul-

ure flask with RPMI 1640 medium (Cambrex BioScience)ompleted with penicillin, streptomycin, amphotericin B and0% heat-inactivated FBS. CD34+ progenitors were iso-ated through a positive immunoselection procedure (CD34+

ultiSort kit, Miltenyi Biotech, Bergisch Gladbach, Ger-any). Cells were cultured during 6 days in the presence

f stem cell factor (SCF, 10 U/ml, Preprotech, London, UK),ecombinant human granulocyte macrophage-colony stimu-ating factor (GM-CSF, 200 U/ml, Peprotech), tumour necro-is factor-� (TNF-�, 50 U/ml, Peprotech) and transformingrowth factor-� (TGF-�, 100 U/ml, Peprotech). At Day 6,ells were cultured with only GM-CSF and TGF-� to preventpontaneous maturation of the cells. At Day 10, Langer-ans cells obtained expressed more than 90% of HLA-DR,

ell plates, were treated for 24 h with various moleculest optimal concentration to obtain no toxicity, mea-ured with XTT test, and cell stimulations using dose-esponses for each compound (data not shown). IL-1�as used as positive control for activation at 25 ng/ml

R&D Systems, Abingdon, UK), Pam3CSK4 at 20 �g/mlInvivogen, San Diego, CA, USA), FSL-1 at 2 �g/mlInvivogen), Zymosan at 20 �g/ml (Invivogen), Poly(I:C)t 25 �g/ml (Invivogen), LPS from E. coli 0111:B4 at0 �g/ml (Sigma-Aldrich), Flagellin at 100 ng/ml (Apotechorporation, Switzerland), Loxoribine at 34 �g/ml (Invivo-en), DNA derived from E. faecalis, Cholera toxin at0 �g/ml (Sigma-Aldrich), DC-Chol at 35 �g/ml (providedy sanofi pasteur, Marcy l’Etoile, France) and Chitosan at00 �g/ml (provided by sanofi pasteur). DC-Chol (3�[N-N′,N′-dimethylaminoethane)-carbamoyl] cholesterol) is aationic lipid derived from cholesterol which mimics thetructure of liposome and Chitosan (� (1 → 4) 2-amino 2-eoxy �-d glucan) is a cationic bioadhesive polysaccharideerived from crustacean shells.

Cells were also pre-treated with Bay 11-7085 at 2.5 �g/mlTebu, Le Perray en Yvelines, France), an inhibitor of the NF-B pathway, before activation.

Langerhans cells and dendritic cells cultured in six-welllates were treated for 48 h at Days 10 and 5, respectively,ith the same concentrations of all compounds used for

pithelial cell stimulation in order to homogenize the study.PS was used as positive control. DC-Chol was cytotoxic forC and LC in culture at a concentration of 35 �g/ml and wassed at 7 �g/ml. Cholera toxin (CT) concentration used was

5746 M. Cremel et al. / Vaccine 24 (2006) 5744–5754

selected as the most efficient for inducing the maturation ofdendritic cells.

2.3. Flow cytometry

The detection of the TLRs on the cell membrane(viable cells) or in intracellular compartments (fixed, per-meabilized cells) was evaluated by flow cytometry experi-ments. For intracellular staining, cells were fixed with 3.6%paraformaldehyde (15 min) and permeabilized with 0.2%Triton X-100 (20 min). After two washes, the cells were incu-bated for 1 h at 4 ◦C with 2 �g of antibodies directed againstTLR1, TLR2, TLR4, TLR6, TLR7, TLR9 (Imgenex, SanDiego, USA), TLR3 (Diaclone, France), TLR5 (Apotech)and CD14 (BD Biosciences, San Jose, USA). After washes,the cells were incubated (30 min at 4 ◦C) with 5 �g ofphycoerythrin (PE) or fluorescein isothiocianate (FITC)-conjugated antibodies (DakoCytomation, Glostrup, Den-mark). An anti-HLA-I antibody (DakoCytomation, France)served as positive control.

Flow cytometry was also used to assess the maturation sta-tus of both DC and LC after the treatments with molecules.Cells were incubated for 1 h at 4 ◦C with 2 �g phycoerythrin(PE)-conjugated antibodies directed against CD80, CD83and CD86 (BD Biosciences). In all the experiments, isotypicantibodies were used as negative controls.

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2.5. Statistics

Values were analyzed by Student’s t-test usingSTATISTICA

TMdata analysis software system (Stat-

Soft Inc., Tulsa, OK, USA). Values of p < 0.05 (black stars)were considered significant.

3. Results

3.1. Expression of toll-like receptors in HEC1A andSiHa cell lines

Since most of compounds tested in this study have beenshown to interact with the toll-like receptors (TLR; seeTable 1), their expression was evaluated by flow cytometryon HEC1A and SiHa cells (Fig. 1).

HEC1A cells (Fig. 1A, plain histograms) were shown tohighly express TLR2 (72 ± 11.4% of the cells) and TLR9(65.4 ± 9%) on their plasma membrane. TLR4 and TLR5were found on 23.5 ± 12.7 and 14.3 ± 0.5% of the cells,respectively, and TLR7 was detected only on 6.4 ± 0.6% ofthe cells. TLR1, TLR3 or TLR6 and CD14 molecule werenot detected on the HEC1A plasma membrane.

SiHa cells (Fig. 1A, open histograms) were shown toh(foTp

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.4. Cytokine and chemokine detection

The concentration of CCL20 in cell supernatants wasuantified by using an enzyme-linked immunosorbent assayELISA) detection kit (Quantikine, R&D Systems), accord-ng to the manufacturer’s instruction. Optical density was

easured at 450 nm. All the assays were performed inriplicate.

IL-1�, IL-6 and IL-8 levels were measured in cellupernatants by a chemiluminescent technique with anmmulit automate (Diagnostic Product Corporation, Losngeles, CA, USA). RPMI medium was used as negative

ontrol.

able 1ist of the compounds tested and the respective receptors

ompounds Receptors S

am3CSK4 TLRs 2/1 SSL-1 TLRs 2/6 Symosan TLRs 2/6; CD 14 Coly(I:C) TLR3 SPS TLR4; CD 14 Elagellin TLR5 Soxoribine TLR7 GNA TLR9 EC-Chol Chitosan Bholera toxin GM1 V

L-1� IL-1R

ighly express TLR2 (87.7 ± 12% of the cells) and TLR962.3 ± 12.4%) but also CD14 (70.2 ± 12.5%) on their sur-ace. Like HEC1A cells, TLR5 and TLR7 were detected onlyn 11.7 ± 0.5 and 7.9 ± 0.9% of the SiHa cells, respectively.LR1, TLR3, TLR4 and TLR6 were not detected on the SiHalasma membrane.

To complete the study of TLRs and CD14 expression byhe two cell lines, HEC1A and SiHa cells were analyzed byow cytometry after fixation and permeabilization allowinglasma membrane and intracellular protein detection.

As shown in Fig. 1B, HEC1A cells (plain histograms)ighly expressed TLR2 (73.7 ± 6.8% of the cells), TLR782.5 ± 4.3%) and TLR9 (93 ± 2.8%). The cells also sig-ificantly expressed TLR3 (53 ± 2% of the cells), TLR444.1 ± 22.3%) and TLR5 (47.8 ± 0.5). TLR1, TLR6 and

References

c lipoprotein [1,2]c mycoplasmal lipoprotein [4,5]

ll from S. cerevisiae [10,11]c analogue of double-stranded viral RNA [14,15]111:B4 [16,17]urium [18,19]

ne analog [25,26]lis [29,30]lipid

merrae [31,32]


Fig. 1. Analysis of TLR expression by flow cytometry on cell membranes(A) and in whole cells (B) of HEC1A (plain bars) and SiHa (open bars)epithelial cell lines. Irrelevant antibodies or secondary antibodies served asnegative control (NC). The negative control showed in the figure representsthe control for TLR1–4, TLR6, TLR9 and CD14. For TLR5 and TLR7,negative controls were in the same values. The results are expressed as themean of three different experiments ± S.D.

CD14 were weakly detected on, respectively, 6 ± 2.7,12.7 ± 2.1 and 10.1 ± 2.5% of the HEC1A cells.

SiHa cells (Fig. 1B, open histograms) were shownto highly express TLR2 (95 ± 3.4% of the cells), TLR3(74.6 ± 23.5%), TLR5 (87.4 ± 0.2%), TLR7 (96.4 ± 3.4%)and TLR9 (99.3 ± 0.6%). TLR1, TLR4 and TLR6 were alsodetected on 23.1 ± 6.8, 49.6 ± 11.6 and 8 ± 2.5% of the SiHacells, respectively. Surprisingly, CD14 detection on perme-abilized cells (3 ± 1.9% of the cells) was lower than thoseobtained for live cells, suggesting a protein degradation bythe treatment of fixation and permeabilization.

3.2. CCL20 secretion by epithelial cells after mucosalcandidate immunoadjuvant stimulation

Various molecules were tested for their capacity to stim-ulate the secretion of CCL20 by SiHa and HEC1A epithelialcells. The cells were incubated for 24 h with the optimal con-centration (which was evaluated as non-toxic by XTT assay,data not shown) of all compounds listed in Table 1 and thesupernatants were evaluated for CCL20 content by ELISA.

As shown in Fig. 2A, Flagellin and DC-Chol were ableto strongly stimulate CCL20 secretion by the HEC1A cells,at the same level as the positive control, IL-1�. The basal

Fig. 2. Effect of the candidate mucosal adjuvants on CCL20 secretion by thetwo epithelial cell lines after a 24 h treatment: HEC1A cells (A) and SiHacells (B) were stimulated with Pam3CSK4 (20 �g/ml), FSL-1 (2 �g/ml),Zymosan (20 �g/ml), Poly(I:C) (25 �g/ml), LPS (10 �g/ml), Flagellin(100 ng/ml), Loxoribine (34 �g/ml), E. faecalis DNA (1/1000), DC-Chol(35 �g/ml), Chitosan (200 �g/ml) and Cholera toxin (CT, 10 �g/ml). IL-1�

(25 ng/ml) served as positive control. Results are represented as the foldof untreated cell secretion. The results are expressed as the mean of threeindependent experiments ± S.D. Values were analyzed by Student’s t-test.Values of p < 0.05 (black stars) were considered significant.

secretion of CCL20 was significantly increased by morethan 50-fold after Flagellin, DC-Chol and IL-1� treatments.Poly(I:C) significantly increased the basal production ofCCL20 by more than nine-fold. CCL20 secretion reached2116.8 ± 19.6 pg/ml and 2161.6 ± 45.5 pg/ml, respectively,after 100 ng/ml of Flagellin and 25 ng/ml of IL-1� treatmentsversus 35.1 ± 6.4 pg/ml for untreated cells. After 35 �g/mlof DC-Chol and 25 �g/ml of Poly(I:C) treatments, CCL20concentration reached, respectively, 594.5 ± 240.2 pg/ml and112.3 ± 19.8 pg/ml versus 11.5 ± 2.7 pg/ml for untreatedHEC1A cells. All the other components tested were unableto stimulate CCL20 secretion by HEC1A cells.

SiHa cells could be stimulated by some compoundstested (Fig. 2B). DC-Chol, like the IL-1� positive control,strongly increased the basal CCL20 secretion by more than70-fold. Poly(I:C), FSL-1, Flagellin and Pam3CSK4 werealso able to stimulate CCL20 secretion by more than 10-fold and Chitosan by more than 4-fold. The basal CCL20secretion (8.3 ± 1.6 pg/ml) reached 682.9 ± 2.9 pg/ml and663.9 ± 21.9 pg/ml after, respectively, 35 �g/ml of DC-Chol


Fig. 3. Inhibition of CCL20 secretion by SiHa and HEC1A cells using theNF-�B inhibitor Bay 11-7085: (A) HEC1A cells were stimulated over 24 hwith Poly(I:C) (25 �g/ml), DC-Chol (35 �g/ml) and Flagellin (100 ng/ml) inthe absence or presence of 2.5 �g/ml of Bay 11-7085; and (B) the same exper-iments were performed with SiHa cells stimulated with Poly(I:C) (25 �g/ml),DC-Chol (35 �g/ml), Flagellin (100 ng/ml), Chitosan (200 �g/ml), FSL-1(2 �g/ml) and Pam3CSK4 (20 �g/ml). Supernatants of untreated HEC1Aand SiHa cells were tested as controls. The results are expressed as the meanof three different experiments ± S.D.

and 25 ng/ml of IL-1� treatments. The CCL20 secretionwas also significantly increased by 25 �g/ml of Poly(I:C)(157 ± 14 pg/ml), 2 �g/ml of FSL-1 (124.4 ± 22.7 pg/ml),100 ng/ml of Flagellin (105.5 ± 4.3 pg/ml) and 20 �g/mlof Pam3CSK4 (92.7 ± 0.5 pg/ml). SiHa cells were sig-nificantly stimulated by Chitosan treatment at 200 �g/ml(241.4 ± 6.7 pg/ml versus 58.9 ± 2.2 pg/ml for untreatedcells). All the other components tested were unable to stim-ulate CCL20 secretion by SiHa cells.

3.3. Inhibition of CCL20 secretion by the NF-κBpathway inhibitor Bay 11-7085

To evaluate the intracellular mechanisms involved inCCL20 gene activation by the different compounds, a specificinhibitor of the NF-�B pathway (Bay 11-7085) was used. Thecells were incubated with Bay 11-7085 (2.5 �g/ml) and thentested for the capacity to secrete CCL20 under the differ-ent stimulations described above. Cell viability controls didnot evidence significant cell death by Bay 11-7085 treatment(data not shown).

As shown in Fig. 3A, a complete inhibition of CCL20production by HEC1A cells was observed for all the com-

pounds able to stimulate CCL20 secretion. The CCL20 con-centrations after Poly(I:C) (316.3 ± 25.3 pg/ml), DC-Chol(904.8 ± 225.9 pg/ml) or Flagellin (2575.3 ± 85.5 pg/ml)treatments decreased to levels of basal secretion (35.1 ±6.4 pg/ml).

The same results were obtained for the SiHa cell line(Fig. 3B), since all the stimulations for CCL20 secretionwere totally abolished by Bay 11-7085. For example, theeffect of DC-Chol, the most potent stimulation factor forSiHa cells, was inhibited by Bay 11-7085 by more than 90%(422.8 ± 9.4 pg/ml versus 10.7 ± 2.2 pg/ml of CCL20 in theabsence or presence of the inhibitor, respectively). These dataindicated that epithelial CCL20 secretion was mediated bythe NF-�B intracellular pathway.

3.4. Effect of mucosal candidate immunoadjuvants ondendritic cell/Langerhans cell activation

The various molecules were evaluated for their capacity toinduce dendritic or Langerhans cell activation. DC, derivedfrom monocytes, or LC, derived from hematopoietic CD34+cord blood precursors, were incubated for 48 h with the vari-ous compounds before being analyzed for activation markerexpression (CD80, CD83 and CD86).

As shown in Fig. 4A, in the absence of any treatment,CD80 and CD83 were expressed on 25.5 and 3.3% of the DC,ra((sFaam(saCqtwL

Leeo(bshPboL

espectively. CD80 expression was increased by LPS useds positive control (71.9% of the cells), CT (52.2%), FSL-162.2%), Poly(I:C) (53.8%), Zymosan (50.9%) and Flagellin44.7%). The same results were observed for CD83 expres-ion, which was highly enhanced by LPS (58% of the cells),SL-1 (58%) and Zymosan (52%). CD83 expression waslso stimulated after CT (37% of the cells), Poly(I:C) (16%)nd Flagellin (37%) treatments. The CD86 co-activationarker was already highly expressed on untreated cells

86% of the cells). However, stimulation of CD86 expres-ion was measured by membrane expression intensity (MFI)nd we observed the same results obtained with CD80 andD83. For these markers, the membrane expression fre-uency on LC was also correlated with the variation ofhe membrane expression intensity (Fig. 4B). No effectas observed by using Pam3CSK4, DC-Chol, Chitosan oroxoribine.

As shown in Fig. 4A, similar results were obtained withC. In the absence of any treatment, CD80 and CD83 werexpressed, respectively, on 48.1 and 34.4% of LC. CD80xpression was increased after LPS (positive control, 76.9%f the cells), FSL-1 (70.7%), Poly(I:C) (68.9%) and Zymosan90.5%) treatments. This marker was less highly enhancedy CT (58.8% of the cells) and Loxoribine (62.9%). Theame results were obtained for CD83 expression which wasighly increased by LPS (62% of the cells), FSL-1 (65%),oly(I:C) (64.7%) and Zymosan (88.6%) and less highlyy CT (42.4% of the cells) and Loxoribine (47.9%). Liken DC, CD86 was already highly expressed on untreatedC cells (78.4% of the cells). A significant increase of the


membrane expression intensity was observed with all thetreatments performed with compounds able to increase CD80and CD83 expression (Fig. 4B). The CD80 and CD83 mem-brane expression frequencies were also correlated with thevariation of their membrane expression intensities. No effectwas observed by using Pam3CSK4, DC-Chol, Flagellin orChitosan.

Taken together, these data indicated that LC and DC couldbe selectively activated by FSL-1, Poly (I:C) and Zymosan.Flagellin and Loxoribine were able to specifically activateDC and LC, respectively.

3.5. Effect of mucosal candidate immunoadjuvants onIL-6 and IL-8 production by dendritic and Langerhanscells

The analysis of cytokine production by DC and LC wasevaluated. IL-1�, IL-6 and IL-8 contents were measured insupernatants of DC and LC cultured for 48 h with the variousmolecules.

There was no detectable production of IL-1� even afterthe different stimulations (Table 2). DC and LC secretedIL-8 before stimulation (567 pg/ml and 823 pg/ml, respec-

FeC(U

ig. 4. Measurement of dendritic cells (DCs) and Langerhans cells (LCs) maturatxpression of maturation markers (CD80, CD83 and CD86) was analyzed by flowT (10 �g/ml), FSL-1 (2 �g/ml), Poly(I:C) (25 �g/ml), Zymosan (20 �g/ml), Flag

7 �g/ml) and Pam3CSK4 (20 �g/ml). Results are represented as membrane expnstimulated cells served as negative control. Results are representative of three in

ion induced by candidate mucosal adjuvants after 48 h treatment. Surfacecytometry after incubation with LPS used as positive control (10 �g/ml),

ellin (100 ng/ml), Loxoribine (34 �g/ml), Chitosan (200 �g/ml), DC-Cholression frequency (%, A) and membrane expression intensity (MFI, B).dependent experiments.


Fig. 4. (Continued ).

tively) but not IL-6. LPS, FSL-1, Poly(I:C), Zymosan andPam3CSK4 were able to increase IL-6 and IL-8 secretionby both DC and LC. DC-Chol and Chitosan were only ableto weakly stimulate IL-8 secretion by DC. In the same way,Flagellin could stimulate the secretion of IL-6 and IL-8 onlyin DC but not in LC. CT and Loxoribine were not able tostimulate the secretion of these two cytokines in LC or inDC.

4. Discussion

In this study, we have analyzed in vitro the properties ofvarious compounds that could be potentially used as mucosaladjuvants in vivo. We focused on their ability to modulatethe secretion of the LC precursors chemoattractive cytokine

CCL20 by epithelial cells at the female genital tract level andon their capacity to promote LC or DC maturation.

We first show that TLR expression profiles of endocervi-cal/uterine (HEC1A) and vaginal/exocervical (SiHa) humancell lines were widely comparable for most TLR. The cellularlocalization of TLR has been already much described and itis commonly admitted that TLR1, TLR2, TLR4, TLR5 andTLR6 are preferentially found on the cell surface while TLR3,TLR7, and TLR9 are mainly found inside the cells [50].However, our results are not in perfect agreement with thesedata for TLR1, TLR6 (detected intracellularly) and TLR9(evidenced on cell plasma membrane). Previous publisheddata are consistent with a cell plasma membrane targetingdepending on the cell type used and/or the origin of the tis-sue considered [51–54]. In addition to cellular localization,the expression of TLR in the female genital tract is also con-


Table 2Effect of candidate mucosal adjuvants on IL-6 and IL-8 production by DC and LC after 48 h incubation

Dendritic cells Langerhans cells

IL-6 (pg/ml) IL-8 (pg/ml) IL-6 (pg/ml) IL-8 (pg/ml)

Medium <2 <5 <2 <5Unstimulated <2 567 <2 823LPS (10 �g/ml) 196 7500 21.3 3551CT (10 �g/ml) <2 895 4.2 730FSL-1 (2 �g/ml) 19.8 7500 7.7 2961Poly(I:C) (25 �g/ml) 8.1 2128 14.4 1711Zymosan (20 �g/ml) 25.3 7500 28.9 7500Flagellin (100 ng/ml) 19.4 7500 <2 785Loxoribine (34 �g//ml) <2 750 <2 869Chitosan (200 �g/ml) 4.1 1233 <2 1166DC-Chol (7 �g/ml) <2 1169 <2 1014Pam3CSK4 (20 �g/ml) 21.7 7500 8.9 3889

Cytokine quantification was performed with Immulit automate by chemiluminescence. Results are representative of at least three independent experiments.

troversial [55–57]. All these results highly suggest a verycomplex regulation of TLR synthesis and/or expression lead-ing to functionality.

We demonstrate that DC-Chol was the most efficientmucosal candidate immunoadjuvant able to generate anincrease in CCL20 secretion by both HEC1A and SiHa celllines. It could be noted that cholera toxin (known as the mostpotent mucosal adjuvant) was unable to stimulate CCL20secretion by the two epithelial cell lines tested. This point isnot in agreement with a previous in vivo study, which demon-strated that CT was able to mobilize immature DC in themouse intestinal epithelium through stimulation of CCL20secretion [58]. Our model allows to test only the direct effectof CT on epithelial and dendritic cells in vitro; however, dur-ing infection, mucosa often contain also inflammatory cells asmacrophages. Several studies showed that macrophages cansecrete several cytokines such as IL-1� after CT treatment[59] which suggests that CCL20 production by epithelial cellsin vivo could be induced by indirect activation.

The use of the NF-�B pathway inhibitor (Bay 11-7085)demonstrates that all of the products tested are able toenhance CCL20 secretion through this intracellular pathway,in agreement with previous results obtained with IL-1� onvaginal [22] or on intestinal [60] epithelial cells. We alsoobserve that CCL20 stimulation of the HEC1A and SiHacell lines by the different compounds is not always in cor-r

results indicate that TLR plasma membrane expression isnot always sufficient for inducing CCL20 secretion. The firstexplanation could be that we evaluated the cell activationby studying only one chemokine. The second one was theimplication of secondary molecules to enhance the recog-nition and/or the stimulation signal of selected PAMPs. Forexample, in the case of TLR4/LPS interaction, LPS-bindingprotein (LBP) and CD14 were necessary to “present” LPSto TLR4 and MD-2 in order to induce TLR4 activation.“Accessory molecules” such as CD36 and Dectin-1 seemedalso necessary for some TLR2-ligands recognition [61–63].Such results could also be explained by the cooperationbetween TLR as previously described for TLR1/TLR2 andTLR6/TLR2. More, TLR9/TLR2 association was recentlydiscovered which allowed to imagine the presence of otherTLR cooperations [64]. The last explanation was the regula-tion of TLR activities through expression of TLR-antagoniststo suppress the activation of these TLR present at the cellsurface, as already described in the gastro-intestinal mucosa[65]. All these data suggest that complex mechanisms couldbe involved in epithelial cell responses after PAMPs acti-vation, at least for CCL20 secretion. However, mucosalimmunological responses is not associated only with thesecretion of CCL20 which attracts CCR6+ immune cells inmucosa since DC and LC have to migrate from the epithelialstructures to lymph nodes in order to present the captureda

TS on CCLo

LPS

E−−

D+++++

elation with the presence of the related receptors. These

able 3ummary table representing the effect of each candidate mucosal adjuvantn the maturation of the dendritic and Langerhans cells

Candidate mucosal adjuvants

Pam3CSK4 FSL-1 Zymosan Poly(I:C)

pithelial cellsHEC1A cells − − − +SiHa cells + + − +

endritic cellsDendritic cells ± +++ +++ ++Langerhans cells ± ++ +++ ++

ntigen to T lymphocytes.

20 secretion by the two epithelial cell lines of the female genital tract and

Flagellin Loxoribine DC-Chol Chitosan Cholera toxin

+++ − ++ − −+ − +++ ++ −

++ − ± ± ++− + − − +


Our results demonstrated that Zymosan was the most effi-cient compound for DC and LC activation as evidenced bythe increase of CD80 and CD83 expression [66,67]. Thisresult underlines the fact that mucosal candidate immunoad-juvants could act differentially on cytokine/chemokine secre-tion stimulation by epithelial cells and on DC/LC activation(see Table 3). For example, CT is unable to stimulate CCL20secretion but could promote the activation of DC and LC. Incontrast, DC-Chol, which is a potent CCL20 inducing com-pound on epithelial cells of the female genital tract, is unableto promote DC or LC maturation. In addition to phenotypicchanges, DC maturation could be associated with the secre-tion of cytokines in order to amplify immune responses. Inthis way, IL-6 can be produced by presenting cells after anti-gen contact and acts as a “co-stimulatory” signal to stimulateT cells. This cytokine was reported to polarize naıve CD4+ Tcells to effector Th2 cells [68]. In addition, IL-8 secretionby DC after stimulation [69] promoted neutrophil migra-tion, representing another component of the innate immuneresponse [70]. Zymosan was the most efficient stimulatorymolecule since it was able to induce IL-6 secretion andstrongly increase the production of IL-8 by DC and LC.

Taken together, endocervical/uterine and exocervi-cal/vaginal epithelial cell lines and generated DC and LCcould be valuable tools for studying the properties of mucosalcandidate immunoadjuvants and appeared of particular inter-eicjfilrTlfs

bcebgmtc

A

EioC

these studies, and Phillip Lawrence and Willy Berlier forassistance in preparing the manuscript.

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