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. 2014 Oct 1;178(2):353–363. doi: 10.1111/cei.12405

Engagement of Toll-like receptor 2 enhances interleukin (IL)-17+ autoreactive T cell responses via p38 mitogen-activated protein kinase signalling in dendritic cells

R Wei *,1, L Dong *,1, Q Xiao *, D Sun †, X Li *, H Nian *
PMCID: PMC4233384  PMID: 24965660

Abstract

Functional analysis of single Toll-like receptors (TLRs) in vivo is necessary to understand how they shape the ocular inflammation involved in uveitis. In this study we explored the role and mechanisms of TLR-2 agonists on the autoreactive T helper type 17 (Th17) response in experimental autoimmune uveitis (EAU). Treatment by peptidoglycan (PGN), a specific TLR-2 agonist, remarkably increased mRNA levels of Th17-lineage genes interleukin (IL)-17A, IL-21 and RAR-related orphan receptor (ROR)γt and promoted antigen-specific Th17 response in EAU mice. A mixture of PGN and interphotoreceptor retinoid-binding protein peptide (IRBP161–180) could effectively induce EAU in the absence of complete Freund's adjuvant (CFA). PGN treatment also enhanced the pathogenic activities of activated antigen-specific Th17 cells in vivo. PGN significantly increased the production of IL-1β, IL-6 and IL-23 of dendritic cells (DCs) and enhanced their ability to promote IL-17+ uveitogenic T cells. Enhanced immunostimulatory activities of PGN-DCs depend upon p38 activation. Inhibition of p38 mitogen-activated protein kinase (MAPK) activity dramatically decreased IL-17 gene expression and antigen-specific Th17 responses stimulated by PGN-DCs. Our findings suggest that PGN treatment dramatically promotes the IL-17+ uveitogenic T cell responses via enhancing the immunostimulatory activities of DCs. This effect may be mediated, at least in part, by activation of the p38 signalling pathway in DCs.

Keywords: EAU, DCs, p38 MAPK, Th17 cells

Introduction

Autoreactive T cells producing IL-17 [T helper type 17 (Th17)] have been attributed to the pathogenesis of a number of autoimmune diseases, including uveitis and its murine model, experimental autoimmune uveitis (EAU) [1–3]. The development of Th17 cells from naive CD4+ T cells is controlled by the local cytokine milieu, including interleukin (IL)-6 and transforming growth factor (TGF)-β [4], as well as IL-1β [5] and IL-23 [6]. Although the factors that affect the development of Th17 cells from naive cells are well known, possible factors that influence the generation of pathogenic Th17 cells are still poorly understood.

Toll-like receptors (TLRs), the most important family of pattern-recognition receptors (RPRs), play essential roles in innate host defence as well as in the control of adaptive immune responses [7,8]. TLR engagement controls activation of the adaptive immune responses through inducing the production of inflammatory cytokines of dendritic cells (DCs) [9,10]. DCs activate T cells by supplying antigenic and co-stimulatory signals as well as an additional set of ‘third polarizing signals’ that can greatly affect T cell function [11]. It has been reported that the conditions during the initial exposure to antigen prime DCs, thus determining whether the effector phenotype is either a Th1 or a Th17 response [2,12,13].

It has been shown that many TLRs are involved in the development of clinical uveitis and EAU, and only TLR-2 is involved in the gene susceptibility to uveitis with Behçet's disease [14]. A pathogenic role for TLR-2 in uveitis is further suggested by its increased expression in patients with Behçet's disease [15]. A recent report demonstrated that a combination of TLR-2 plus TLR-4 agonists promote an antigen-specific Th17 response by activating γδ T cells in EAU mice [16]. However, little is known about the mechanism of single TLR-2 stimulation in the generation of the IL-17+ uveitogenic T cells in EAU.

Mitogen-activated protein kinases (MAPKs) play critical roles in the activation of inflammatory cells. MAPKs are composed of three major subgroups: extracellular signal-related kinase1/2 (ERK1/2), p38 and c-Jun N-terminal kinase (JNK). Among them, the p38 MAPK pathway has been demonstrated to play an important role in the development of autoimmunity [17]. Excessive activation of the p38 MAPK is associated with autoimmune diseases, and inhibitors of this pathway have been considered as new therapeutic strategies [18]. Recent work has identified the role of p38 in IL-17 production in EAU [19]. However, whether or not p38 MAPK regulates the cross-talk between innate and adaptive immunity in EAU remains unknown.

In the present study, we have investigated the role of TLR-2 stimulation in controlling TLR-mediated T cell responses and elucidated the underlying pathways. We provide direct evidence that peptidoglycan (PGN), a specific TLR-2 agonist, enhances EAU development by increasing effector antigen-specific Th17 cell generation. TLR-2 stimulation also increases IL-17 and RAR-related orphan receptor (RORγt) gene transcription. Importantly, we demonstrate that TLR-2 activation leads to an increased DC-dependent Th17 cell differentiation via the activation of p38 signalling in DCs.

Materials and methods

Animals and reagents

B10RIII mice (8 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and female C57BL/6 (B6) mice (10 weeks old) were purchased from Vital River Laboratory Animal Technology (Beijing, China). All procedures were carried out in accordance with the regulations stipulated by the Animal Use and Protection Committee at Tianjin Medical University and the EU Directive 2010/63/EU for animal experiments. A truncated form of interphotoreceptor retinoid-binding protein (IRBP) peptide1–20 was synthesized and purified by Sangon (Shanghai, China). Mycobacterium tuberculosis strain H37RA was obtained from Difco (Detroit, MI, USA). Pertussis toxin (PTX) was purchased from Sigma (St Louis, MO, USA). Recombinant murine IL-12 and IL-23 were purchased from R&D Systems (Minneapolis, MN, USA). Fluorescein isothiocyanate (FITC)-conjugated anti-IL-17 antibody and phycoerythrin (PE)-conjugated anti-IFN-γ were purchased from Biolegend (San Diego, CA, USA). The p38 inhibitor SB203580 was obtained from Sigma. The mouse TLR-1/2 agonist Pam3CSK4, TLR2/dectin-1 agonist Zymosan, TLR-2/4 agonist lipopolysaccharide (LPS), TLR-2 agonist lipoteichoic acid (LTA) and PGN were purchased from Invivogen (San Diego, CA, USA). Anti-phospho-p38 antibody (3D7), anti-phospho-SAPK/JNK (G9) and anti-phospho-ERK1/2 (E10) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Lymphocyte proliferation assay

IRBP-specific T cells (4 × 105) in a total volume of 200 μl were cultured at 37°C for 48 h in 96-well tissue culture plates with medium or IRBP1–20 and irradiated syngeneic spleen antigen-presenting cells (APCs) (1 × 105). In every experimental condition, each culture was performed in triplicate. T cell proliferation was studied thereafter by measurement of bromodeoxyuridine (BrdU) incorporation using a cell proliferation kit (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer's instructions.

Induction of EAU and adoptive transfer

Mice were immunized subcutaneously over six spots at the tail base and on the flank with 150 μl of emulsion containing uveitogenic peptide. The uveitogenic peptide used for B6 was IRBP1–20 (amino acids 1–20 of human IRBP, 150 μg/mouse) and that for B10RIII mice was IRBP161–180 (amino acids 161–180 of human IRBP, 75 μg/mouse). The peptides were emulsified in either complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) or IFA containing TLR-2 ligand PGN. The dose of PGN used for in-vivo immunization was 250 μg/mouse (the optimal dose for inducing EAU).

At day 13 after immunization, donor mice were killed and T cells were isolated from pooled spleen and draining lymph node cells by passing through nylon wool columns, and then 1 × 107 T cells/well were seeded into six-well plates, together with syngeneic APCs (irradiated spleen cells) and 10 μg/ml of IRBP1–20 under Th17 polarizing conditions (culture medium supplemented with IL-23). After 2 days, activated T cell blasts were separated on a centrifugation gradient (Ficoll; GE Health Care, Little Chalfont, UK) and injected [2 × 106, intraperitoneally (i.p.)] into naive B6 mice. Ten days after cell transfer, disease was assessed by funduscopy.

Scoring of EAU

The mice were examined three times a week for clinical signs of EAU by indirect funduscopy. The pupils were dilated with 0·5% tropicamide and 1·25% phenylephrine hydrochloride ophthalmic solutions, and funduscopic grading of disease was performed using the scoring system reported by Thurau et al. [20]. Eyes were processed for histopathology and stained with standard haematoxylin and eosin. Histopathological scores were assigned in a masked fashion on a scale of 0–4, based on the number, type and size of lesions [21].

Generation of bone marrow-derived DCs

Bone marrow-derived DCs were generated as described previously [22]. Bone marrow cells were flushed from the femurs and tibias of C57BL/6. The cells (1 × 106) were cultured in 24-well plates in medium supplemented with 10 ng/ml granulocyte–macrophage colony-stimulating factor (GM-CSF; R&D Systems, Minneapolis, MN, USA) and 10 ng/ml recombinant interleukin-4 (rIL-4; R&D Systems). Non-adherent cells were carefully removed, and fresh medium was added every 2 days. On day 7, non-adherent cells were collected for phenotyping. In some experiments, DCs were preincubated with SB203580 for 1 h and then with PGN. Because SB203580 was dissolved in dimethylsulphoxide (DMSO), DCs incubated with DMSO alone were used as a control.

Real-time quantitative reverse transcription–polymerase chain reaction (qRT–PCR)

Total RNA from cells was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). The first-strand cDNA was synthesized with a reverse transcription kit (Fermentas, Burlington, ON, Canada). All gene transcripts were analysed by quantitative PCR (qPCR) with SYBR Green Master Mix (ABI; Applied Biosystems, Foster City, CA, USA) using an ABI 7900 HT sequence Detection System. Gene-specific primers for real-time PCR are listed in Table 1. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as endogenous control in all experiments. For each sample, the relative abundance of target mRNA was calculated from the obtained CΔt values for both target and endogenous reference gene GAPDH by applying the following formula: relative mRNA expression = 2[CΔt(GAPDH) – CΔt(target)].

Table 1.

The sequences of primers used in this study for real-time reverse transcription–polymerase chain reaction (RT–PCR)

Gene name Forward primer sequence Reverse primer sequence
GAPDH 5′-CATGGCCTTCCGTGTTCCTA-3′ 5′-GCGGCACGTCAGATCCA-3′
IL-23p19 5′-CATAGCTGCCCGGGTCTTT-3′ 5′-GGCACTAAGGGCTCAGTCAGA-3′
IL-12p35 5′-GCGTGGGAGTGGGATGTG-3′ 5′-GCAAAACGATGGCAAACCA-3′
IL-1β 5′-AGTTGACGGACCCCAAAAGA-3′ 5′-GGACAGCCCAGGTCAAAGG-3′
TNF-α 5′-CAGCCGATGGGTTGTACCTT-3 5′-GGCAGCCTTGTCCCTTGA-3′
IL-6 5′-CCACGGCCTTCCCTACTTC-3′ 5′-TTGGGAGTGGTATCCTCTGTGA-3′
T-bet 5′-ACCTGTTGTGGTCCAAGTTCAA-3′ 5′-GCCGTCCTTGCTTAGTGATGA-3′
RORγt 5′-CCTCAGCGCCCTGTGTTTT-3′ 5′-GCATGCAGCTTTTGCCTGTT-3′
IL-21 5′-GCATGCAGCTTTTGCCTGTT-3′ 5′-GTCTTATTGTTTCCAGGGTTTGATG-3′
IL-17A 5′-CCTGGCGGCTACAGTGAAG-3′ 5′-TTTGGACACGCTGAGCTTTG-3′
IFN-γ 5′-TTGGCTTTGCAGCTCTTCCT-3′ 5′-TGACTGTGCCGTGGCAGTA-3′
CCL20 5′-CTGATGCTTTTTTGGGATGGA-3′ 5′-CCCCAGCTGTGATCATTTCC-3′
AhR 5′-AATCCCACATCCGCATGATT-3′ 5′-TTTGCAAGAAGCCGGAAAAC-3′
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GAPDH = glyceraldehyde 3-phosphate dehydrogenase; IL = interleukin; IFN = interferon; TNF = tumour necrosis factor; ROR = RAR-related orphan receptor; AhR = aryl hydrocarbon receptor.

Intracellular staining and enzyme-linked immunosorbent assay (ELISA)

For intracellular staining, cells were exposed to 50 ng/ml of phorbol myristate acetate (PMA), 1 μg/ml of ionomycin and 1 μg/ml of brefeldin A (Sigma) for 4 h and were then washed, fixed and permeabilized overnight with buffer (Cytofix/Cytoperm; eBioscience, San Diego, CA, USA), intracellularly stained with antibodies against IFN-γ and IL-17 and analysed on a flow cytometer (fluorescence activated cell sorter (FACS)Calibur; BD Biosciences, San Jose, CA, USA). The cytokines in the culture supernatants were detected with commercially available ELISA kits (R&D Systems).

Western blot analysis

DCs were cultured at 2 × 106/ml with PGN for the indicated time intervals. Cell lysates were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, and blotted with antibodies specific for phosphor-p38 (Cell Signal Technology, Danvers, MA, USA) and a horseradish peroxidase (HRP)-linked secondary antibody. The nitrocellulose was stripped and probed with antibodies specific for β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Statistical analysis

All experiments were performed at least three times. Data are presented as mean ± standard deviation (s.d.) and analysed for significance using a two-tailed Student's t-test. P-values of less than 0·05 were considered statistically significant.

Results

PGN-activated DCs induce stronger uveitogenic Th17 cell responses in vitro

We first investigated the role of TLR-2 agonists activated DCs in the priming of 17+ uveitogenic T cells. After treatment with or without various TLR-2 ligands, bone marrow-derived DCs were co-cultured with purified IRBP1–20-specific T cells isolated from immunized B6 mice in the presence of antigen. The ability of TLR-2-stimulated DCs to activate Th1 or Th17 cells was assessed by measuring intracellular expression of IFN-γ and IL-17 in activated T cells. As shown in Fig. 1a, the proportions of antigen-specific IL-17+ T cells were significantly higher in the LPS- (16·0%) or PGN-treated (17·8%) group, and slightly higher in the LTA (11·3%) or Pam3CSK4 (10·9%) group compared with the control group (7·3%), but no difference existed between the Zymosan and the control group (7·4 versus 7·3%, respectively). Further ELISA assay showed that the concentrations of IL-17 were significantly higher in the LPS, the PGN and the Pam3CSK4 groups, but not in the LTA and the Zymosan groups. Taken together, these results indicate that PGN-treated DCs generate a condition that significantly favours expansion of the antigen-specific Th17 cells. Because of the stronger effect of PGN-DCs on the antigen-specific Th17 cells, further experiments were performed with PGN, a specific TLR-2 agonist.

Fig. 1.

Fig. 1

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Peptidoglycan (PGN) treatment enhanced the T helper type 17 (Th17)-polarizing capacity of dendritic cells (DCs). (a) DCs were treated with various Toll-like receptor (TLR)-2 ligands for 24 h, and then were washed and cultured with uveitogenic T cells isolated from immunized B6 mice in the presence of antigen. Interleukin (IL)-17+ or interferon (IFN)-γ+ cells were determined by intracellular staining. (b) Enzyme-linked immunosorbent assay (ELISA) analysis of IL-17 levels in the culture supernatant 48 h after stimulation with antigen. Results are representative of three independent experiments. *P < 0·05; **P < 0·01.

PGN treatment affects mRNA and protein expression of Th17-polarizing cytokines in DCs

Because the cytokines IL-1β, IL-6 and IL-23 produced by innate cells co-operate to regulate the induction of Th17 cells [23,24], we examined the ability of PGN to stimulate production of these cytokines from DCs. Bone marrow-derived DCs were incubated with or without 10 μg/ml PGN for 4–24 h. We found that PGN treatment significantly enhanced IL-23, IL-1β, tumour necrosis factor (TNF)-α and IL-6 gene expression in DCs. The induction of IL-23, IL-1 and TNF-α gene expression in PGN-DCs reached peak level at 8 h; however, the gene expression of IL-6 increased gradually (Fig. 2a). Further ELISA assays showed that PGN-DCs produced significantly increased amounts of IL-23, IL-1β, IL-6 and TNF-α, but not IL-12 (Fig. 2b).

Fig. 2.

Fig. 2

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Effect of peptidoglycan (PGN) on dendritic cell (DC) gene expression and on cytokine release. (a) Kinetics of mRNA expression of PGN-activated DCs. DCs were stimulated for various times with PGN. Tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-12, IL-23 and IL-1 mRNA relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was measured by quantitative polymerase chain reaction (qPCR). (b) TNF-α, IL-6, IL-12, IL-23 and IL-1β release in the 48-h culture supernatants. *P < 0·05; **P < 0·01. Results are representative of three independent experiments.

PGN treatment enhances the expression of Th17 lineage-specific genes and the generation of antigen-specific Th17 cells in immunized mice

To further assess the effect of PGN treatment on the generation of antigen-specific Th1 and Th17 cells in vivo, B6 mice were immunized with IRBP1–20/CFA, IRBP1–20/IFA or IRBP1–20/IFA containing TLR-2 ligand PGN (IRBP1–20/IFA/PGN), then the in vivo-primed T cells were stimulated with IRBP1–20 and APCs under Th17 or Th1 polarizing conditions. As shown in Fig. 3a, both the IRBP1–20/CFA and the IRBP1–20/IFA/PGN groups had significantly higher percentages of IL-17+ T cells (22·9 versus 14·1%, respectively) compared with the IRBP1–20/IFA group (2·5%). Meanwhile, the levels of IL-17 in the supernatants were determined by ELISA analysis. Consistent with the results described above, the levels of IL-17 were significantly elevated in the IRBP1–20/IFA/PGN and the IRBP1–20/CFA groups when compared with those in the IRBP1–20/IFA group (Fig. 3b). In contrast, the frequencies of antigen-specific IFN-γ+ T cells and the production of IFN-γ appeared to be less affected by the PGN treatment.

Fig. 3.

Fig. 3

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Incomplete Freund's adjuvant (IFA) containing peptidoglycan (PGN) enhances the in-vivo priming of interleukin (IL)-17+ T cells in B6 mice. (a) interphotoreceptor retinoid binding protein (IRBP)-specific T cells from IRBP1–20/CFA, IRBP1–20/IFA or IRBP1–20/IFA containing PGN immunized mice were stimulated with IRBP1–20 in the presence of syngeneic antigen-presenting cells (APCs) for 5 days under T helper type 17 (Th17) or Th1 polarized conditions. IL-17+ or IFN-γ+ cells were determined by intracellular staining. (b) IL-17 and IFN-γ levels in the culture supernatants assessed by enzyme-linked immunosorbent assay (ELISA) 48 h after stimulation with antigen. (c) Relative mRNA expression of IL-17, IL-21, CCL20, RAR-related orphan receptor (ROR)γt, aryl hydrocarbon receptor (AhR) and T-bet in the uveitogenic T cells from IRBP/IFA or IRBP/IFA + PGN immunized mice. *P < 0·05; **P < 0·01. Data are representative of three individual experiments, using at least two mice per group.

We next investigated the underlying molecular mechanism by which PGN treatment enhances the observed IL-17 production. We compared the lineage-related gene expression of uveitogenic T cells isolated from immunized mice with and without PGN treatment. Consistent with the protein results shown above, the genes specific for the Th17 lineage [25], including IL-17, IL-21 and RORγt, were significantly up-regulated in IRBP1–20/IFA/PGN immunized mice compared to the IRBP1–20/IFA immunized mice (Fig. 3c). There were no significant differences among Th1-related genes, such as T-bet, between these two groups.

PGN effectively induces EAU as substitutes of CFA and increases the pathogenic activities of antigen-specific Th17 cells in vivo

To examine whether PGN influences susceptibility to EAU, we immunized the B10RIII mice with IRBP161–180 emulsified in IFA, CFA or IFA/PGN. Starting from 8 days after immunization, EAU development was monitored by funduscopy followed by pathological examination. As shown in Fig. 4a,b, mice immunized with IRBP161–180/CFA developed EAU, whereas mice immunized with IRBP161–180/IFA did not. Interestingly, at a dose of 250 μg/mouse, the PGN treatment significantly enhanced the EAU susceptibility of the recipient mice compared to the mice induced by IRBP/IFA alone. PGN at a dosage of 400 μg/mouse developed a comparable EAU score. However, a PGN dosage at 100 μg/mouse developed a much less severe EAU score than the group with a PGN dosage of 250 μg/mouse or 400 μg/mouse.

Fig. 4.

Fig. 4

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Toll-like receptor (TLR)-2 agonist peptidoglycan (PGN) enhances the experimental autoimmune uveitis (EAU)-inducing activity of interphotoreceptor retinoid binding protein (IRBP)161–180 in the B10RIII mouse. (a) B10RIII mice were immunized with 75 μg of uveitogenic peptide IRBP161–180 emulsified in incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA) or IFA containing PGN (100 μg, 250 μg, 400 μg/mouse), as indicated. Average disease scores (a) and histopathology of eyes (b) from the IFA, the IFA/PGN (250 μg/mouse) or the CFA-treated groups (haematoxylin and eosin, ×100). (c) IRBP-specific T cells from PBP/IFA or IRBP/IFA + PGN immunized B6 mice stimulated for 2 days in vitro with antigen under T helper type 17 (Th17) polarized conditions, were transferred to naive B6 mice. EAU was scored by funduscopy. (d) Proliferation analysis of IRBP-specific T cells derived from IRBP/IFA or IRBP/IFA + PGN immunized mice. (a,c) Data were from three independent experiments with at least three mice per group.

To determine whether PGN treatment also enhances the pathogenic activity of activated autoreactive T cells, we isolated T cells from spleen and draining lymph nodes of B6 mice immunized with or without PGN. The T cells were stimulated in vitro with the immunizing IRBP peptide for 48 h and were transferred to naive B6 mice. As shown in Fig. 4c, T cells from IRBP/IFA-immunized mice failed to induce EAU in recipient mice, whereas T cells from IRBP/IFA/PGN-immunized mice were effective in disease induction. To determine how PGN treatment increased the disease-inducing ability of IRBP-specific T cells, IRBP1–20-specific T cells were assessed for proliferation. As can be seen in Fig 4d, the BrdU incorporation assay showed an enhanced proliferation of the uveitogenic T cells derived from PGN-treated, immunized mice.

PGN activates p38 MAPK in DCs and p38 MAPK mediates PGN-stimulated gene expression of proinflammatory cytokines in DCs

The production of IL-23, IL-1β and IL-6 has been linked to MAPKs [26,27]. To determine if MAPKs are involved in the up-regulation of IL-1β, IL-23 and IL-6 expression by PGN stimulation in DCs, the phosphorylation levels of MAPKs in DCs treated with PGN were determined by Western blot. As shown in Fig. 5a, PGN stimulation led to phosphorylation of p38, indicating the activation of p38 by PGN. The phosphorylation of p38 was detectable after 15 min, was maximal at 3 h, but still detectable for up to 12 h (Fig. 5a). In contrast, there were no obvious changes in the levels of phosphorylated ERK1/2 and JNK at all time -points examined.

Fig. 5.

Fig. 5

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Phosphorylation of mitogen-activated protein kinases (MAPKs) in dendritic cells (DCs) and inhibition of p38 suppresses peptidoglycan (PGN)-induced interleukin (IL)-6, IL-23 and IL-1β gene expression. (a) Top, levels of phosphorylated p38, c-Jun terminal kinase (JNK) and extracellular-regulated kinase (ERK)1/2 in DCs incubated with PGN. Bottom, Band intensity ratio was measured using Quantity One software (Bio-Rad, Hercules, CA). (b) DCs were preincubated with vehicle only or the p38 inhibitor SB203580 for 1 h before the addition of PGN; 4 h later, IL-6, IL-23, IL-1β and IL-12 mRNA expression was determined by quantitiative polymerase chain reaction (qPCR). Data are representative of three independent experiments. *P < 0·05; **P < 0·01.

To further confirm the role of p38 MAPK in PGN-induced IL-23, IL-1β and IL-6 expression, the pharmacological inhibitor of p38 (SB203580) was used. DCs were pretreated with SB203580 for 1 h and subsequently stimulated with PGN for 4 h. As shown in Fig. 6, p38 inhibitor significantly inhibited PGN-induced IL-1β, IL-6 and IL-23 gene expression in DCs (Fig. 5b). These data demonstrate the importance of the p38 MAPK pathway in the regulation of PGN-induced cytokine expression in DCs.

Fig. 6.

Fig. 6

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Inhibition of p38 suppresses the ability of peptidoglycan (PGN) treated dendritic cells (DCs) to polarize T helper type 17 ( Th17) cells. (a) DCs were preincubated with vehicle only or the p38 inhibitor SB203580 for 1 h before the addition of PGN (10 μg/ml) for 24 h, followed by co-cultured with uveitogenic T cells from interphotoreceptor retinoid binding protein (IRBP)-immunized B6 mice in the presence of antigen. Expression of interleukin (IL)-17 and interferon (IFN)-γ in uveitogenic T cells were determined by intracellular staining. (b) The same cells were analysed by quantitiative polymerase chain reaction (qPCR) for expression of IL-17 and IFN-γ. Data are representative of three independent experiments.

Inhibition of p38 suppresses the ability of PGN-DCs to induce antigen-specific Th17 cell responses

We next explored the influence of the p38 inhibitor on the ability of PGN-DCs to induce antigen-specific Th17 cell responses in vitro. After DCs were pretreated with 10 μm SB203580 or vehicle (DMSO) for 1 h, DCs with or without PGN stimulation were co-cultured with purified IRBP1–20-specific T cells in the presence of antigen. As shown in Fig. 6a, PGN-induced antigen-specific Th17 cell responses were markedly suppressed by SB203580. Further qPCR assays showed that blocking of p38 inhibited the induction of IL-17 gene expression by PGN-DCs (Fig. 6b).

Discussion

Toll-like receptors sense invading microbial products and initiate the adaptive immune responses through activation of DCs. In the present study, we explored the impact of various TLR-2 ligand-activated DCs on the activation of uveitogenic T cells. We found that PGN, a specific TLR-2 ligand, is a potent inducer of antigen-specific Th17 cells.

Our findings regarding the promotion of antigen-specific Th17 cells by TLR-2 stimulation with PGN are in accordance with most recent findings in murine [28,29] and human [30–32] subjects. For example, Reynolds et al. and Summers et al. independently demonstrated that TLR-2 ligation led to enhanced Th17 cell responses [28,29]. However, contrasting evidence also exists. For example, Manicassamy et al. reported that TLR-2 signalling suppressed IL-23- and Th17-mediated autoimmune responses [33]. The differences between these studies might be due to distinct methods, including the use of different TLR-2 agonists.

Transcription factors play a critical role in driving T cells toward specific lineages. It is known that RORγt is critical for the induction and differentiation of Th17 cells. Our data indicated that PGN treatment significantly up-regulated transcription of RORγt in vivo, suggesting that PGN treatment affects Th17 cell lineage commitment in EAU. IFN-γ is known to inhibit the development of Th17 cells by paracine and cell-intrinsic actions [34]. Here, we observed that the generation of IFN-γ-producing cells were less affected by PGN treatment, nor was the mRNA expression of T-bet among activated uveitogenic T cells, suggesting that TLR-2 ligation by PGN may selectively target factors critical for Th17 cell development rather than inhibiting Th1 T cells. These results are supported further by a study by Reynolds et al. [28], which demonstrated that TLR-2 signalling had a high impact on Th17 cells but not Th1 cells. We also found, that upon immunization, PGN-treated mice display a reduced number of forkhead box protein 3 (FoxP3)-producing regulatory T cells (Treg) cells (data not shown) in the spleen and lymph nodes. We believe that this finding indicates a role for PGN treatment in the suppression of Treg cell differentiation. This is in line with previous studies by Lal et al. and Prinz et al. [35,36]. However, the relationship between reduced Treg cells and enhanced Th17 cell responses is not clear, given that Treg cells are not so effective in the control of Th17 responses as in the suppression of Th1 responses [37,38].

Consistent with our current report, Fang et al. [39] reported that the single TLR-1/2 agonist Pam3CSK4 exacerbated EAU with a suboptimal immunization protocol. However, there are some notable differences between the two studies. Fang et al. did not address the effect and mechanism of Pam3CSK4 on antigen-specific Th17 cells. In contrast, we demonstrated enhancement of the pathogenic Th17 response, inclusive of IL-17 and other Th17-related genes in vivo by PGN treatment (Fig. 4). Furthermore, we further confirmed the impact of PGN treatment on the function of antigen-specific Th17 cells using the adoptive transfer model.

For pathogenic Th17 cell development, they must receive signals from relevant proinflammatory cytokines such as IL-1β, IL-6 and IL-23, which are produced mainly by DCs [40]. IL-1β has been reported to enhance IL-6-mediated differentiation of Th17 cells [41]. IL-6 synergizes with IL-23 to enhance the differentiation, survival and expansion of Th17 cells [24]. In this study, we found that PGN treatment significantly increased the mRNA and protein expression of IL-1β, IL-6 and IL-23 in DCs. These data suggest that PGN treatment can trigger a Th17-polarizing programme in DCs. Indeed, we found that PGN-DCs showed an increased and more sustained capacity to promote the expansion and differentiation of Th17 cells in vitro. Our findings were supported further by the results in humans, which showed that TLR2 ligation promoted Th17 development via promoting the immunostimulatory activities of DCs [31,32,42,43].

We next explored the molecular mechanism underlying the altered expression of IL-1β, IL-6 and IL-23 of DCs in response to PGN. Several studies have shown a critical role for p38 MAPK in the regulation of multiple proinflammatory cytokines in human DCs [44,45]. A recent study demonstrated that TLR-2 signalling mediated activation of p38 MAPK in human monocytes [46,47]. We therefore propose that p38 MAPK is a key factor mediating the activation of DCs by PGN. Our results showed that p38 was phosphorylated rapidly in response to PGN in DCs. Preincubation of DCs with p38 inhibitor significantly decreased PGN-induced mRNA expression of IL-1β, IL-6 and IL-23. These findings suggest that increased Th17-polarizing cytokines produced by DCs involved primarily the activation of p38 MAPK. It was reported recently that p38 signalling programmes DCs to drive Th17 cell differentiation [48]. In the current study, we also found that PGN made use of the p38 signalling pathway to regulate IL-17 expression in vitro. Inhibition of p38 signalling significantly inhibited the ability of PGN-DCs to polarize Th17 cells, resulting in blocking PGN-DCs-induced antigen-specific Th17 responses in uveitogenic T cells. Blocking of p38 MAPK activation has also been reported to enhance the ability of TLR agonist-activated DCs to promote antigen-specific Th1 responses [49]. However, we did not observe a significant impact of p38 MAPK modulation on antigen-specific Th1 cells. Different TLR agonists and different cell types may explain the difference we observed. In addition to p38, MAPKs have two other members: ERK1/2 and JNK. A recent report by Liang et al. indicated that PGN induced the activation of ERK1/2, but not JNK, in monocyte-derived macrophages (MDMs) derived from active human patients [47]. In contrast, we observed that there were no significant changes in the phosphorylation levels of both ERK1/2 and JNK in DCs following PGN treatment at the different time-points examined. This difference may be ascribed to differences in the cell populations.

In summary, we found that PGN, a specific TLR-2 ligand, modulated EAU by concurrently promoting the commitment and lineage stability of antigen-specific Th17 cells. The effect of PGN on antigen-specific Th17 responses may be attributed in part to the activation of p38 signalling in DCs, which is involved in production of Th17-polarizing cytokines including IL-6, IL-1β and IL-23. These cytokines subsequently signal through signal transducer and activator of transcription 3 (STAT-3) activation to enhance the expression of Th17-related genes, including IL-17, IL-21 and RORγt [50].

Acknowledgments

We thank Dr Huanfa Yi (Institute of Immunology, The First Hospital of Jilin University) for critically reading the manuscript. This work was supported by funding from National Natural Science Foundation Grant (81100646, 31100991) and Tianjin Municipal Science and Technology Commission Grant (13JCYBJC23300).

Disclosures

The authors have no financial conflicts of interest.

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