Oral Escherichia coli Colonization Factor Antigen I (CFA/I) Fimbriae Ameliorate Arthritis via IL-35, not IL-27

Abstract

A Salmonella therapeutic expressing enterotoxigenic E. coli colonization factor antigen I (CFA/I) fimbriae protects against collagen-induced arthritis (CIA) by eliciting two regulatory T cell (Treg) subsets: TGF-β-producing Foxp3⁻CD39⁺CD4⁺ and IL-10-producing Foxp3⁺CD39⁺CD4⁺ T cells. However, it is unclear if CFA/I fimbriae alone are protective, and if other regulatory cytokines are involved especially in the context for the EBI3-sharing cytokines, Treg-derived IL-35 and APC-derived IL-27, both capable of suppressing Th17 cells and regulating autoimmune diseases. Subsequent evaluation revealed that a single oral dose of purified, soluble CFA/I fimbriae protected against CIA as effectively as Salmonella-CFA/I, and found Foxp3⁺CD39⁺CD4⁺ T cells as the source of secreted IL-35, whereas IL-27 production by CD11c⁺ cells was inhibited. Inquiring into their relevance, CFA/I fimbriae-treated IL-27 receptor-deficient (WSX-1^−/−) mice were equally protected against CIA as wild-type mice suggesting a limited role for IL-27. In contrast, CFA/I fimbriae-mediated protection was abated in EBI3^−/− mice accompanied by the loss of TGF-β- and IL-10-producing Tregs. Adoptive transfer of B6 CD39⁺CD4⁺ T cells to EBI3^−/− mice with concurrent CFA/I plus IL-35 treatment effectively stimulated Tregs suppressing proinflammatory CII-specific Th cells. Opposingly, recipients co-transferred with B6 and EBI3^−/− CD39⁺CD4⁺ T cells and treated with CFA/I plus IL-35 failed in protecting mice implicating the importance for endogenous IL-35 to confer CFA/I-mediated protection. Thus, CFA/I fimbriae stimulate IL-35 required for the co-induction of TGF-β and IL-10.

Introduction

Rheumatoid arthritis (RA) is an incurable, systemic inflammatory disease of the joints leading to cartilage loss, bone erosion, ankylosis, and consequent disability. A chronic inflammation involves a complex network of pathogenic cells from the innate and adaptive immune systems as well as a number of soluble factors that contribute to joint destruction (1, 2). TNF-α is a major facilitator of this disease progression (3, 4), and TNF-α antagonists can effectively diminish inflammation and attenuate destruction of cartilage and bone (5–8). However, prolonged treatment with TNF-α antagonists can have deleterious side-effects making patients more susceptible to opportunistic infections (9, 10).

In this same vein, efforts have also focused in adapting regulatory T cells (Tregs) and their cytokines, particularly IL-10 and TGF-β, to treat arthritis (11–14) and other autoimmune diseases (15–17). Recently, the spectrum of cytokines with regulatory properties has expanded to include the newly discovered CD4⁺ T cell cytokine, IL-35 (18), and the finding that the APC-derived cytokine, IL-27, can also be anti-inflammatory (19). IL-35 is formed by Epstein-Barr virus-induced gene 3 (EBI3) subunit dimerizing with IL-12p35 subunit (20). It is produced by Tregs and potently suppresses inflammatory diseases (18, 21). IL-35 binds to its receptor, the heterodimer IL-12Rβ2 and gp130, signaling through transcription factors STAT1 and STAT4 (22). In addition to stimulating forkhead box (Foxp3)⁺ CD25⁺ CD4⁺ T cells, we have shown IL-35 to stimulate alternative Treg subsets encompassed within CD39⁺CD4⁺ T cells and capable of conferring protection against collagen-induced arthritis (CIA) (23). It has been implicated that IL-35 can act independent of IL-10 or TGF-β (24), but our studies have shown that in the absence of IL-10, IL-35 was ineffective (23).

In contrast, dimerizing with p28, EBI3 subunit forms IL-27 which is primarily produced by APCs. IL-27 binds to its receptor, WSX-1, and can promote Th1 cell polarization as a consequence of T-bet and STAT1 activation (25, 26). Yet, WSX-1^−/− mice showed impaired resistance to Trypanosoma cruzi leading to a prolonged parasitemia and higher mortality rate (27) supported by greater TNF-α, IL-6, and Th2-type cytokines during established T. cruzi infection. Given these findings, IL-27 signaling also appeared important for regulation of antiparasitic immune responses in WSX-1^−/− mice (27). Initially found to support Th1 cell development, IL-27p28 neutralization diminished IFN-γ production resulting in reduced disease severity in adjuvant-induced arthritis in rats (28) as well as in experimental autoimmune encephalomyelitis (EAE) in mice (29). IL-27R^−/− mice were also resistant to proteoglycan-induced arthritis and showed reduced IFN-γ production (30). On the other hand, IL-27 is currently studied mostly for its described immunoregulatory properties (19, 31–34). Acting on activated CD4⁺ and CD8⁺ effector T cells, IL-27 suppresses Th17 cell-transferred EAE implicating the importance of stimulating IL-10-producing T cells (31).

Colonization factor antigen I (CFA/I) is a virulence factor for enterotoxigenic E. coli to enable intestinal colonization of humans (35). In an effort to generate a vaccine, this fimbria was expressed by an attenuated Salmonella vaccine vector, and showed protection in animals stimulating elevated mucosal IgA and serum IgG Abs subsequent oral vaccination (36, 37). Interestingly, this vaccine was found to inhibit proinflammatory cytokine production offering the possibility of serving as an anti-inflammatory vaccine (38). It was subsequently found to ameliorate such inflammatory diseases as EAE (15, 39) and CIA (40, 41). For CIA, two functionally distinct, but complementing subsets of regulatory T cells are induced with Salmonella-CFA/I: TGF-β-producing Foxp3⁻CD39⁺ and IL-10-producing Foxp3⁺CD39⁺ CD4⁺ T cells (41). Together these subsets suppress type II collagen (CII)-specific Th1 and Th17 cells (41). Stimulation of TGF-β by CFA/I fimbriae is also important for upregulation of CD39 apyrase in a cAMP response element binding protein (CREB)-dependent fashion by CD4⁺ T cells (41).

In this study, we demonstrate that purified soluble CFA/I fimbriae, when given orally, can recapitulate our findings using Salmonella-CFA/I by the stimulation of TGF-β⁺ Foxp3⁻ CD39⁺CD4⁺ and IL-10⁺ Foxp3⁺CD39⁺CD4⁺ T cells to suppress CIA. Aside from these relevant findings is the observation that soluble CFA/I fimbriae induces IL-35-producing Foxp3⁺ Tregs in mice protected from CIA, but also diminish IL-27 production by CD11c⁺ cells. Supporting the importance of endogenous IL-35 for protection against CIA, EBI3^−/− mice are unresponsive to oral CFA/I fimbriae treatment. In contrast, WSX-1^−/− mice, although showing less disease severity, were ameliorated from arthritis upon oral CFA/I fimbriae treatment evident by the enhanced TGF-β and IL-10 production and elevated presence of CD39⁺ Tregs. Furthermore, these data show that endogenous IL-35 is essential for resolving arthritis by CFA/I fimbriae since CIA EBI3^−/− recipients were responsive to adoptive transfer with wild-type CD39⁺CD4⁺ T cells and subsequent co-treatment with CFA/I fimbriae and recombinant IL-35. Exogenous IL-35 was only effective in EBI3^−/− mice so long as treatments continued, otherwise, upon ceasing its administration, arthritis resumed. Donor cells diluted with EBI3^−/− CD39⁺CD4⁺ T cells failed to confer protection. Thus, this work shows CFA/I fimbriae can stimulate IL-35, not IL-27, for protection against CIA, and an endogenous source of IL-35 is required for CFA/I fimbriae to abate disease.

Materials and Methods

Mice

Male C57BL/6 mice (Charles River Laboratories), breeding colonies of EBI3^−/− and WSX-1^−/− mice (The Jackson Laboratory) were maintained at Montana State University Animal Resources Center. All mice were kept in ventilated cages with high-efficiency particulate absorbing-filtered barrier. Sterile food and water were supplied ad libitum. All experimental protocols were concordant with institutional policies for animal health and well-being.

Purification of CFA/I fimbriae

The CFA/I fimbriae producing E. coli strain (H695) was grown on Minca agar in pans at 37° C for 48–60 h. Cells were harvested from the agar surface and sheared for 15 minutes on ice. Cell debris was removed by centrifugation at 10,000 rpm for 20 minutes. The fimbriae were precipitated overnight in a final concentration of 20% 4.1 M ammonium sulfate and 20 mM Tris-HCl, then resuspended in 5 ml of deionized/distilled water, and dialyzed overnight into deionized/distilled water to remove residual salts. The next day, insoluble proteins were separated via ultracentrifugation at 18,000 rpm (40,000 × g) for 1 h. CFA/I fimbriae were again precipitated from supernatant overnight using 20% final concentration of 4.1 M ammonium sulfate, resuspended in PBS, and quality was evaluated by SDS-PAGE and Western blot analyses. Western blots were probed with rabbit anti-CFA/I fimbriae Ab (developed in-house). Endotoxin was removed via anionic exchange chromatography using an Uno Q column (Bio-Rad Laboratories, Hercules, CA). Endotoxin levels were below biologically relevant levels using the Limulus Amebocyte Lysate assay (Associates of Cape Cod Inc., E. Falmouth, MA).

CIA

CIA was induced in C57BL/6, EBI3^−/−, or WSX-1^−/− mice with 100 µg of chicken CII (Chondrex, Redmond WA) emulsified in complete Freund’s adjuvant (Chondrex) (23, 41), and 100 µl of emulsion was given s.c. at approximately 0.5 cm from the base of tail. Mice were observed daily beginning on day 21 post-CII challenge at the onset of disease. Each limb was evaluated using a scale of 0–3 as previously described (23): 0, no clinical signs; 1, mild redness of a paw or swelling of single digits; 2, significant swelling of ankle or wrist with erythema; 3, severe swelling and erythema of multiple joints; maximum score per mouse is12.

CFA/I fimbriae and IL-35 treatments

Mice were orally gavaged with 200 µl of sterile 50% saturated sodium bicarbonate solution followed by a single dose of 80 µg of purified soluble CFA/I fimbriae or sterile PBS on day 14 post-CII challenge. Recombinant mouse IL-35 was expressed and purified as previously described (23). IL-35 treatments of EBI3^−/− mice consisted of 1 µg doses of rmIL-35 given i.p. from days 21 through 25 post-CII challenge.

Histopathology

Paws from front and hind limbs and knees were fixed in 10% neutral-buffered formalin, decalcified in 5% formic acid, processed and embedded in paraffin, and cut into 5 µm sections. Adjacent sections were stained with H&E to evaluate inflammation and bone destruction or with toluidine blue to evaluate cartilage matrix and chondrocyte loss. H&E stained sections were scored: 0, no changes in joint structure; 1, synovial hyperplasia and moderate inflammatory infiltration; 2, pannus formation with cartilage degeneration; 3, severe inflammatory infiltration, cartilage and bone degeneration. Toluidine blue stained sections were scored: 0, no cartilage loss; 1, superficial zone proteoglycan and chondrocytes loss; 2, proteoglycan and chondrocytes loss into the middle zone of the layer above the tidemark; 3, severe chondrocytes and proteoglycan matrix loss through tidemark (23). Paw and knee joint sections were evaluated, with a total maximum score of 18 possible per mouse.

Ab and cytokine ELISAs

To evaluate CII-specific or CFA/I fimbriae-specific serum IgG titers, flat-bottom, 96-wells (MaxiSorb, Nunc) were coated with 2 µg/ml ELISA Grade CII (Chondrex) or 2 µg/ml purified CFA/I fimbriae overnight at 4°C. Sera samples collected from individual mice on day 35 post-CII challenge were serially diluted and added to coated wells as previously described (40). Ag-specific reactivity was detected with goat anti-mouse HRP-labeled IgG1, IgG2a, IgG2b Abs (Southern Biotechnology Associates, Birmingham, AL), then developed with ABTS (Moss, Pasadena, CA). Endpoint titers represent the reciprocal dilutions of the last dilution yielding an absorbance at OD₄₁₅ ≥0.100 units above negative controls.

To evaluate CII-specific T cell responses, on day 40 or 42 post-CII challenge, CD4⁺ T cells were cell-sorted by negative selection on magnetic beads (Invitrogen) from popliteal, axillary, inguinal, and iliac lymph nodes (LNs). For all cell sorts, purity was >98%. 10⁶/ml of purified CD4⁺ T cells were restimulated with 50 µg/ml of T-Cell Proliferation CII (Chondrex) in the presence of syngenic irradiated (3000 rad) APCs for 4 days at 37°C and 5% CO₂. Supernatants were subjected to cytokine-specific ELISAs as previously described (15, 23, 41). Briefly, diluted standards and CD4⁺ T cell supernatants were added to wells pre-coated with cytokine-specific capture mAb (IFN-γ, IL-6, IL-10, IL-17, BD Pharmingen, San Diego, CA; TGF-β, R&D Systems, Inc., Minneapolis, MN). Reactions were detected with biotinylated cytokine-specific Abs, then, after incubation with anti-biotin-HRP (Vector Laboratories), developed with ABTS. To measure IL-35 production, wells were coated with anti-human/mouse p35 mAb (clone 27537; R&D Systems). Rabbit IgG anti-IL-35 Ab (developed in-house) was used as secondary Ab in a capture ELISA. Reaction was detected with HRP-conjugated donkey anti-rabbit IgG (adsorbed against mouse, rat, hamster, human, bovine serum proteins; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). To measure IL-27 secretion, whole LN lymphocytes from PBS- or CFA/I fimbriae-treated mice were restimulated with 50 µg/ml of T-Cell Proliferation Grade CII (Chondrex) for 3 days, then supernatants were subjected to capture IL-27 ELISA. Anti-mouse IL-27 EBI3 capture (R&D Systems) and biotinylated anti-mouse IL-27 p28 (R&D Systems) detection mAb pair was used in this ELISA.

Flow cytometry

Upon termination of the disease course, spleen, LN, and mesenteric lymph node (MLN) lymphocytes were stained with fluorochrome-labeled anti-CD39 and anti-CD4 mAbs (eBioscience, San Diego, CA). For analysis of surface expressed TGF-β, biotin-labeled anti-TGF-β Ab (R&D Systems) paired with fluorochrome-conjugated streptavidin (BD Pharmingen) were used. For Foxp3 intracellular expression analysis, after staining of surface markers, cells were fixed in 2% paraformaldehyde, permeabilized with ice-cold methanol, then stained with fluorochrome-labeled anti-Foxp3 mAb (eBioscience). For intracellular IL-35 and IL-27 detection, cells were incubated with 25 ng/ml PMA, 1 µg/ml ionomycin, and 10 µg/ml brefeldin A for 6 h. Following surface immunofluorescent staining, cells were fixed in 2% paraformaldehyde for 10 minutes, permeabilized with BD Perm/Wash ™ buffer, then directly stained with anti-mouse EBI3 mAb (R&D Systems or LifeSpan BioSciences, Inc.) labeled with Alexa Fluor 488 using Alexa Fluor® 488 Microscale Protein Labeling Kit (Molecular Probes ™, Invitrogen) or its isotype control labeled with Alexa Fluor® 488 (R&D Systems), and with anti-mouse p35-Alexa Fluor 647 mAb (eBioscience), anti-IL-27p28-PE mAb (BioLegend, San Diego, CA), or with their isotype controls labeled with Fluor® 647 or PE (eBioscience).

Adoptive transfer of CD39⁺CD4⁺ T cells

CIA was induced in EBI3^−/− mice on day 0. Naive wild-type and EBI3^−/− spleen and combined peripheral LN CD4⁺ T cells were enriched by negative selection using Dynabeads^® (Invitrogen), then, after staining with anti-CD4-FITC (eBioscience) and anti-CD39-PE (BioLegend), CD39⁺CD4⁺ T cells were further cell-sorted using BD FACS Aria II (>95% purity). On day 14, 2×10⁵ wild-type or a mixture of wild-type and EBI3^−/− (10⁵:10⁵) CD39⁺CD4⁺ T cells were adoptively transferred to CII- challenged EBI3^−/− recipients followed by oral CFA/I fimbriae treatment. On days 21–25, recombinant mouse IL-35 (1.0 µg/dose; 23) was given i.p. daily.

Statistics

Mann-Whitney U test was applied to analyze statistically clinical scores, histology scores, and level of cartilage loss. Difference in arthritis incidence between experimental groups was checked with Fisher’s exact probability test. One-way ANOVA was performed to analyze ELISA and flow cytometry results. Data were considered statistically significant, if p-value was <0.05.

Results

Soluble CFA/I fimbriae can ameliorate CIA

It was previously shown that E. coli CFA/I fimbriae, when expressed by an attenuated Salmonella, resulted in suppression of CIA (41). To ascertain if purified, soluble CFA/I fimbriae are effective against CIA, C57BL/6 mice were challenged with CII on day 0 and, on day 14, orally treated with a single 80 µg dose of purified endotoxin-free CFA/I protein. Control mice were given sterile PBS. By day 31, all PBS-treated mice showed clinical symptoms of arthritis. In CFA/I fimbriae-treated group, 70% of mice remained protected over the course of the experiment - 40 days (Fig. 1A). The average clinical score for the CFA/I fimbriae-treated group was significantly less than PBS-treated mice (Fig. 1A). Histopathological analysis of H&E-stained joint sections revealed significantly less inflammation and joint destruction confirming the clinical findings. CFA/I fimbriae-treated mice also had less chondrocyte and proteoglycan matrix loss as evident from the toluidine blue-stained sections (Fig. 1B, C).

FIGURE 1.

Oral CFA/I fimbriae after collagen II (CII) challenge protects against collagen-induced arthritis (CIA). CIA was induced in C57BL/6 mice (10 per group) on day 0 upon s.c. injection of emulsified chick CII. On day 14 post-CII challenge, mice were orally dosed with sterile PBS or 80 µg soluble CFA/I fimbriae. A, average clinical score per group and incidence of arthritis (percent of mice within a group with clinical symptoms); *p < 0.005, ^□p<0.05 compared to PBS-treated mice. Data are representative of 3 experiments. B, Histology score and cartilage loss were estimated for each limb and knee sections. Total maximum score of 18 possible per mouse; *p < 0.05 compared to PBS-treated mice. C, H&E - (top panels) and toluidine blue-stained (bottom panels) knee sections upon termination of study (median for each group). D, CII-specific CD4⁺ T cell cytokine production. Upon termination of the studies, purified CD4⁺ T cells (10⁶ cells/ml) from peripheral lymph nodes (LNs) were CII restimulated in the presence of irradiated syngenic APCs for 4 days. Mean cytokine concentrations from triplicate cultures ± SEM are shown; *p < 0.001 compared to PBS-treated mice.

Upon conclusion of the study, CII-specific CD4⁺ T cells cytokine responses were measured using collected supernatants from Ag-restimulated popliteal, axillary, inguinal, and iliac LN CD4⁺ T cells. Soluble CFA/I fimbriae treatment markedly inhibited Th1 and Th17 cell responses along with the stimulation of IL-10- and TGF-β-producing CD4⁺ T cells (Fig. 1D). Cytokine responses by CD4⁺ T cells were also evaluated one week after CFA/I fimbriae treatment, and similar cytokine profiles were observed (data not shown). The results of these studies show that orally administered CFA/I fimbriae alone can mitigate CIA by suppressing Th1 and Th17 cell responses.

Soluble CFA/I fimbriae stimulate CD39⁺CD4⁺ T cells with regulatory phenotypes

Protection to CIA by Salmonella-CFA/I was found associated with induced CD39⁺CD4⁺ T cells (41). The frequency of CD39⁺ Tregs in spleens, MLNs, and LNs from CFA/I fimbriae-treated CIA mice was evaluated. By flow cytometry, all three tissues showed significantly elevated percentages of CD39⁺CD4⁺ T cells (Fig. 2A). The MLN and LN CD39⁺CD4⁺ T cells from CFA/I fimbriae-treated mice also showed a significantly greater frequency of Foxp3 expressing CD39⁺CD4⁺ T cells (Fig. 2B). Concordant with our previous findings for Salmonella-CFA/I (41), TGF-β mostly associated with Foxp3⁻ CD39⁺CD4⁺ T cells and IL-10 with Foxp3⁺CD39⁺CD4⁺ T cells (Fig. 2C). The percentage of LN TGF-β⁺ Foxp3⁻CD39⁺CD4⁺ T cells for the CFA/I fimbriae-treated mice was enhanced nearly two-fold compared to PBS-treated mice (Fig. 2C). The frequency of IL-10-expressing Foxp3⁺CD39⁺CD4⁺ T cells was also significantly increased after treatment with soluble fimbriae (Fig. 2 C).

FIGURE 2.

Soluble CFA/I fimbriae are potent in stimulating TGF-β- and IL-10-producing and Foxp3-expressing CD39⁺CD4⁺ T cells. CIA was induced and mice were treated with CFA/I fimbriae as in Figure 1. Flow cytometry analysis on stained lymphocyte populations was performed upon termination of clinical study. A, Frequencies of CD39⁺CD4⁺ T cells in lymphoid tissues; *p < 0.01, **p < 0.05 versus PBS-treated group. B, Frequencies of Foxp3-expressing CD39⁺CD4⁺ T cells in lymphoid tissues upon termination of clinical study; *p < 0.05 versus PBS control group. C, Percentage of TGF-β-producing Foxp3⁻CD39⁺CD4⁺ T cells and IL-10-producing Foxp3⁺CD39⁺CD4⁺ T cells in draining LNs; *p < 0.001, **p < 0.01 as compared to PBS-treated mice.

Soluble CFA/I fimbriae stimulate IL-35 production by Foxp3⁺CD39⁺CD4⁺ T cells and suppresses IL-27 production by CD11c⁺ cells

Exogenously administered recombinant IL-35 was shown to mitigate CIA by stimulating protective CD39⁺CD4⁺ T cells (23). Thus, we queried whether CFA/I fimbriae can stimulate endogenous IL-35 by these induced Tregs. At the conclusion of the study, draining LN lymphocytes were isolated and gated on CD39⁺CD4⁺ T cells to measure by flow cytometry the extent of EBI3 and p35 expression. In protected CFA/I fimbriae-treated mice, the percentage of CD39⁺CD4⁺ T cells simultaneously expressing EBI3 and p35 subunits was 4 times greater than PBS-treated mice and nearly 15 times greater than in naive mice (Fig. 3A, C). Moreover, the EBI3- and p35-specific mean fluorescence intensities (MFIs) were correspondingly increased in CFA/I fimbriae-treated mice (Fig. 3B). Soluble IL-35 production by CD4⁺ T cells correlated with the flow cytometry analysis for IL-35 (Fig. 3D). CD4⁺ T cells from CFA/I fimbriae-treated mice showed a 4-fold increase in IL-35 production following CII restimulation. Subsequently, gating on Foxp3⁻ and Foxp3⁺ from CD39⁺CD4⁺ T cells, IL-35 predominantly associated with the Foxp3⁺CD39⁺CD4⁺ T cells (Fig. 3E). These results show that CFA/I fimbriae stimulate endogenous IL-35 in mice protected from CIA.

FIGURE 3.

Oral CFA/I fimbriae stimulate IL-35 expression by Foxp3⁺ CD39⁺CD4⁺ T cells. C57BL/6 mice were challenged with CII and treated with CFA/I fimbriae as described, and on day 40, flow cytometry analysis of CD39⁺CD4⁺ T cells stained for EBI3 and p35 subunits was performed. A, Representative plots of gated LN CD39⁺CD4⁺ T cells. B, Representative histograms of EBI3 and p35 expression by LN CD39⁺CD4⁺ T cells. Numbers indicate specific mean fluorescence intensity (MFI). C, Percentage of CD39⁺CD4⁺ T cells simultaneously expressing EBI3 and p35 subunits of IL-35; *p < 0.001, **p < 0.005 as compared to PBS-treated mice; ***p < 0.05, ^□p < 0.001 as compared to naive mice. D, IL-35 concentrations in supernatants of CII-restimulated LN CD4⁺ T cells; *p < 0.05. E, EBI3 and p35 expression by LN Foxp3⁻ and Foxp3⁺ CD39⁺CD4⁺ T cells. Left panels show gates for Foxp3⁻ and Foxp3⁺ subsets for the gated CD39⁺CD4⁺ T cells One of 4 individual CFA/I fimbriae-protected mice is depicted.

Since IL-27 and IL-35 share EBI3 subunit, we queried whether soluble CFA/I fimbriae can also stimulate IL-27. IL-27 is produced by DCs and has been shown to have immunoregulatory properties upon adjuvant-induced arthritis (27) and EAE (28, 30). To assess its expression, flow cytometry was used to analyze splenic and draining LN cells from PBS- and CFA/I fimbriae-treated mice at the termination of the study - day 40. Relatively very few CD4⁺ T cells expressed both EBI3 and IL-27p28 subunits simultaneously (Fig.4A, C). Gated LN CD11c⁺ cells from CFA/I fimbriae-protected mice showed significantly less EBI3 and p28 co-expression relative to those cells from PBS control mice (Fig. 4A, C). Notably, the MFI for p28 was suppressed by CFA/I fimbriae (Fig. 4 B). In contrast, the MFI for EBI3 was slightly enhanced (Fig. 4 B). To confirm these results, an IL-27 ELISA was performed for CII-restimulated LN cells, and collected supernatants were analyzed revealing nearly 3-fold less IL-27 secretion by lymphocytes from CFA/I fimbriae-protected mice than PBS control mice (Fig. 4 D). Thus, these data show IL-27 is not being augmented by CFA/I fimbriae, rather IL-27 is being suppressed.

FIGURE 4.

Oral CFA/I fimbriae do not stimulate, rather suppress IL-27 expression by CD4⁺ and CD11c⁺ cells. CIA was induced, and mice were treated with CFA/I fimbriae as described above. Flow cytometry was performed on day 40 post-challenge. A, Representative FACS plots of gated CD4⁺ T cells and CD11c⁺ cells from LNs of CFA/I fimbriae- or PBS-treated mice with induced CIA. B, Representative histograms of EBI3 and p28 expression by LN CD11c⁺ cells. Numbers indicate EBI3- and p28-specific MFI. C, Frequencies of EBI3 and p28 expressing CD4⁺ T cells and CD11c⁺ cells; *p < 0.001, **p < 0.05 between groups. D, IL-27 concentrations in supernatants of CII-restimulated LN lymphocytes from PBS- and CFA/I fimbriae-treated mice. One of three experiments is depicted. Mean IL-27 concentration of triplicate cultures ± SEM is shown; *p=0.005.

Protection elicited by soluble CFA/I fimbriae is IL-35-, not IL-27-dependent

Since TGF-β and IL-10 are important for the protection conferred by CFA/I fimbriae, we queried the role of endogenous IL-35 subsequent fimbriae treatment. Mice deficient of IL-35 (EBI3^−/−) were induced with CIA, and treated with CFA/I fimbriae on day 14. Interestingly, the disease severity and incidence in EBI3^−/− mice were not significantly different from B6 mice; however, protection by CFA/I fimbriae was completely abated (Fig. 5A). To ascertain the contribution of endogenous IL-27 in protection to CIA, subsequent studies tested the therapeutic effect of CFA/I fimbriae in WSX-1^−/− mice. As with EBI3^−/− mice, WSX-1^−/− mice were induced with CIA and treated on day 14 with CFA/I fimbriae. Although PBS-treated mice developed arthritis with slightly delayed onset and essentially lower average clinical score, CFA/I fimbriae treatment was as effective as in B6 mice ameliorating CIA (Fig. 5A) suggesting that endogenously induced IL-27 is not important for protection as conferred by CFA/I fimbriae. These data implicate the relevance of endogenously induced IL-35 for protection as mediated by CFA/I fimbriae since EBI3^−/− mice were unable to resolve CIA. While EBI3^−/− mice are deficient in both IL-27 and IL-35, the failure of IL-27 signaling in WSX-1^−/− mice suggests IL-27 has no or a lesser role for CFA/I fimbriae-conferred protection.

FIGURE 5.

IL-27 signaling is not essential for protection against autoimmune arthritis by CFA/I fimbriae, whereas the lack of the shared EBI3 subunit results in loss of protection. A, On day 14 after CII challenge, EBI3^−/− or WSX-1^−/− mice (5/group) were treated with CFA/I fimbriae, and clinical symptoms were monitored; *p < 0.05 compared to PBS-treated mice. One of 3 experiments is depicted. B, CFA/I fimbriae-specific IgG production is reduced in the absence of IL-27 signaling, but not affected in EBI3-deficient mice. Sera samples were analyzed by ELISA after clinical study; *p < 0.001 as compared to EBI3^−/− mice; ^□p < 0.005 as compared to wild-type (WT) C57BL/6 mice. C, CII-specific IgG responses correlate with clinical responses in WT, EBI3^−/−, and WSX-1^−/− mice; *p < 0.001, **p < 0.005, ^□p < 0.05 as compared to corresponding PBS control mice.

To determine whether the immunodeficient mice could recognize CFA/I fimbriae, endpoint serum IgG anti-CFA/I fimbriae Ab titers were measured. Both B6 and EBI3^−/− mice showed very similar IgG Ab titers unlike WSX-1^−/− mice exhibited significantly reduced CFA/I fimbriae-specific IgG1, IgG2a, and IgG2b serum titers than B6 mice (Fig. 5B). The absence of IL-27 signaling correlated with less recognition of the fimbriae, whereas Ab production by EBI3^−/− mice was not significantly different from B6 mice.

CII-specific IgG Ab responses were also tested amongst the three mouse strains on day 35 post-CII challenge. Correlating with clinical and histological findings, significantly less IgG1, IgG2a, and IgG2b anti-CII Ab titers were found in B6 mice treated with CFA/I fimbriae compared with PBS-treated mice (Fig. 5C). In WSX-1^−/− mice, only IgG2a titer was significantly less than PBS-treated controls (Fig. 5C). In EBI3^−/− mice, CFA/I fimbriae treatment failed to reduce CII-specific IgG subclass Ab titers. Relative to B6 or WSX-1^−/− mice, CFA/I fimbriae-treated EBI3^−/− mice had elevated levels of CII-specific IgG1, IgG2a, and IgG2b Abs (Fig. 5C). Thus, these data show that strength of CII-specific IgG Ab responses as well as average arthritis scores were muted upon CFA/I fimbriae treatment in mice deficient of IL-27 signaling.

CFA/I fimbriae suppress Th1 and Th17 cell responses in B6 and WSX-1^−/− mice while sustaining Treg responses

To assess what impact on Th cell responses CFA/I fimbriae has as a consequence of the loss of IL-27 and IL-35 function, Ag-specific LN CD4⁺ T cells from B6, EBI3^−/−, and WSX-1^−/− mice were isolated and CII-restimulated in vitro at day 40 post-CII challenge. Supernatants were subsequently evaluated for proinflammatory and regulatory cytokine production. CFA/I fimbriae-treated B6 mice showed 3- to 4-fold reductions in Th1 and Th17 cell responses relative to PBS-treated mice (Fig. 6A). In contrast, CFA/I fimbriae-treated EBI3^−/− mice failed to reduce IFN-γ and IL-17 production relative to PBS-treated mice, although IL-6 was partially diminished by 1.6-fold (Fig. 6A). CFA/I fimbriae-treated WSX-1^−/− mice also showed significant reductions in IFN-γ and IL-6, but not for IL-17 relative to PBS-treated WSX-1^−/− mice (Fig. 6A). Thus, in the absence of EBI3, proinflammatory cytokine responses go unabated upon CFA/I fimbriae treatment, and in the absence of IL-27 signaling, IFN-γ and IL-6 still are repressed by CFA/I fimbriae.

FIGURE 6.

A, CII-specific CD4⁺ T cells cytokine responses in C57BL/6, EBI3^−/− and WSX-1^−/− mice upon termination of clinical studies; bars indicate differences within species tested; *p < 0.001, **p < 0.005 as compared to similarly treated WT and WSX-1^−/− mice; ***p ≤ 0.01as compared to similarly treated WT and EBI3^−/− mice; ^□p < 0.05 as compared to WT mice; ^□p=0.005 as compared to CFA/I fimbriae-treated WT mice. B, LN CD39⁺CD4⁺ T cells are induced by CFA/I fimbriae in WT and WSX-1^−/− mice. Animals were treated with CFA/I fimbriae on day 14 post-CII challenge as described above. The frequencies of CD39⁺CD4⁺ T cells from individual mice were analyzed by flow cytometry on day 40; bars indicate differences within species tested; *p < 0.001 as compared to CFA/I fimbriae-treated EBI3^−/− and WT mice; **p < 0.05 as compared to PBS-treated EBI3^−/− and WT mice; ^□p < 0.005 as compared with CFA/I fimbriae-treated EBI3^−/− mice. C, Foxp3-expressing CD39⁺CD4⁺ T cells are induced by CFA/I fimbriae in LNs of WT and WSX-1^−/− mice; bar indicates differences within WT mice; *p < 0.001 as compared to CFA/I fimbriae-treated WT and WSX-1^−/− mice; **p < 0.005 as compared to WT and EBI3^−/− PBS-treated mice.

To assess their magnitude as the result of CFA/I fimbriae treatment in B6, EBI3^−/−, and WSX-1^−/− mice, IL-10 and TGF-β were measured. IL-10 levels were significantly enhanced by 12.3-fold and 5.2-fold in B6 and WSX-1^−/− mice relative to their respective PBS control group, unlike EBI3^−/− mice showing no significant change upon treatment (Fig. 6A). Baseline TGF-β levels were present for all three species treated with PBS with modest increases by 4.7- and 2.1-fold by B6 and EBI3^−/− mice, respectively, upon CFA/I fimbriae treatment (Fig. 6A). Notably, WSX-1^−/− mice exhibited a large increase in TGF-β production by 12.7-fold (Fig. 6A). Thus, as with B6 mice, WSX-1^−/− mice are responsive to CFA/I fimbriae treatment evident by the enhanced IL-10 and TGF-β stimulation with concomitant reductions in IFN-γ and IL-6 which correlate with the reduced disease severity in these mice.

It was previously demonstrated that TGF-β contributes to the upregulation of CD39 since neutralization of TGF-β ultimately dampens the phosphorylation of CREB lessening CD39 apyrase expression by CD4⁺ T cells (41). To assess the impact on CD39 expression by soluble CFA/I fimbriae, CD39⁺CD4⁺ T cell frequencies were measured in PBS- and CFA/I fimbriae-treated B6, EBI3^−/−, and WSX-1^−/− mice. The elevated TGF-β in B6 and WSX-1^−/− mice treated with CFA/I fimbriae correlated with a significant increase in the percentages of CD39⁺CD4⁺ T cells present in draining LNs (Fig. 6B). In contrast, CFA/I fimbriae-treated EBI3^−/− mice failed to show modulation of CD39 expression by their CD4⁺ T cells (Fig. 6B). Interestingly, LN CD4⁺ T cells showed enhanced expression of CD39 in the PBS-treated WSX-1^−/− relative to B6 mice (Fig. 6 B) implicating that these mice may be more resistant to CIA. As it was observed, PBS-treated WSX-1^−/− mice exhibited lessened disease severity, and this correlated with the enhanced frequency of Foxp3⁺ CD39⁺CD4⁺ T cells in draining LNs relative to similarly treated B6 mice (Fig. 6 C). CFA/I fimbriae treatment enhanced Foxp3 expression in B6 mice, but not in WSX-1^−/− mice since these mice already had enhanced Foxp3 expression (Fig. 6 C). In contrast, the percentage of Foxp3⁺CD39⁺CD4⁺ T cells in EBI3^−/− mice remained unchanged from PBS-treated controls, and was reduced by 30% relative to B6 and WSX-1^−/− mice (Fig. 6 C). Thus, these data show that protection to CIA by CFA/I fimbriae is not IL-27-dependent since IL-10 and TGF-β remained to be stimulated; increases in CD39 expression were evident in WSX-1^−/− mice, and in fact, augmented by their CD4⁺ T cells. A lack of protection to CIA correlated with the loss EBI3 expression abolishing CFA/I fimbriae-induced regulatory cytokine and CD39⁺CD4⁺ T cells.

Endogenous IL-35 is required for protection against CIA since EBI3^−/− mice are unable to sustain protection without exogenous IL-35 intervention

Since IL-35 cannot be produced by EBI3^−/− mice, we queried whether exogenous IL-35 could substitute for protection. CIA was induced in EBI3^−/− mice, and IL-35 treatments were initiated at disease onset on day 21 for 5 consecutive days similar to that described with wild-type mice (23). While IL-35 treatments surely reduced clinical disease and incidence of disease (Fig. 7A), shortly after cessation of treatment, mice resumed disease course suggesting that an endogenous source of IL-35 is required for continuing protection to CIA. Thus, to assess the capacity of Treg-derived IL-35, adoptive transfer studies were performed in EBI3^−/− recipients. Groups of EBI3^−/− mice were induced with CIA as described above on day 0 followed by adoptive transfer on day 14 with naive wild-type or 1:1 mix of wild-type and EBI3^−/− CD39⁺CD4⁺ T cells to demonstrate significance of this subset’s capacity to generate IL-35 subsequent in vivo CFA/I fimbrial stimulation. On the same day following adoptive transfer, recipients were given a single, oral dose of CFA/I fimbriae. One week later at the time of clinical onset, five consecutive daily treatments with 1 µg of recombinant IL-35 were given similar to that previously described (23) (Fig. 7B). Clinical improvement was evident in EBI3^−/− recipients treated with IL-35 or CFA/I fimbriae plus IL-35 over the course of the disease observation (maximum average clinical score 2.8 and 3.2 respectively versus 8 in PBS-treated group; Fig. 7C). Interestingly, over the course of IL-35 treatment and through day 29, 60 – 80% of the B6 CD39⁺CD4⁺ T cell recipients (not treated with CFA/I fimbriae) did not produce clinical symptoms of arthritis, though after day 35, disease incidence reached 80% (Fig. 7C). Over the same time frame, CFA/I fimbriae plus IL-35-treated recipients showed 60% protection early and dropped to 40% over the course of the disease (Fig. 7C). Notably, in the EBI3^−/− recipients adoptively transferred with half the amount of wild-type CD39⁺CD4⁺ T cells and substituted with EBI3^−/− CD39⁺CD4⁺ T cells, the clinical symptoms were not significantly reduced by CFA/I fimbriae plus IL-35 treatment, and all mice developed CIA (Fig. 7C). Thus, an effective regulatory response could be achieved upon sufficient presence of IL-35-producing CD39⁺CD4⁺ T cells. These data show that reconstituted EBI3^−/− mice with B6 CD39⁺CD4⁺ T cells require a continuous intervention with IL-35 to abate the increased incidence of disease. Furthermore, CD39⁺CD4⁺ T cells represent an essential source of endogenous IL-35 induced upon treatment with CFA/I fimbriae for sustaining protection to CIA. It appears that in the absence of sustained or intermittent IL-35 signaling, multiple supportive doses of CFA/I fimbriae may be required for treating these reconstituted EBI3^−/− mice.

FIGURE 7.

Endogenous IL-35 from CD39⁺CD4⁺ T cells is required for protection against arthritis when treated with CFA/I fimbriae. A, Severity and incidence of CIA in EBI3^−/− mice treated with 5 doses of recombinant IL-35 from day 21 to day 25 post-challenge (arrowheads). B, Scheme of adoptive transfer of donor Tregs into EBI3^−/− recipients. CIA was induced in EBI3^−/− mice on day 0, and on day 14, challenged EBI3^−/− mice received naive wild-type (WT) or an equal mix of WT and EBI3^−/− CD39⁺CD4⁺ T cells. On the same day, EBI3^−/− recipients were orally treated with CFA/I fimbriae. Treatments with recombinant IL-35 were performed upon disease onset as described in A. C, Severity and incidence of CIA in recipients treatment groups: *p < 0.005, ^□p < 0.05, as compared to PBS control group; D, Inflammatory and regulatory cytokine production by CII-specific LN CD4⁺ T cells after following the disease course: *p < 0.001 versus each experimental group; **p < 0.005, ^□p < 0.001 as compared to treated EBI3^−/− mice given only donor WT CD39⁺CD4⁺ T cells; ***p < 0.005, ^□p < 0.001 as compared to PBS-treated EBI3^−/− mice. E, The frequencies of surface TGF-β⁺ and intracellular Foxp3⁺ CD39⁺CD4⁺ T cells in treatment groups: *p < 0.005 as compared to treated EBI3^−/− recipients given WT CD39⁺CD4⁺ T cells; **p < 0.01 as compared to EBI3^−/− recipients given a mix of WT:EBI3^−/− (1:1) CD39⁺CD4⁺ T cells; ^□p < 0.05 versus each experimental group.

Subsequent analysis aimed to evaluate the cytokine profiles following the intervention of these EBI3^−/− recipients. Exogenous IL-35 was responsible for significantly suppressing IFN-γ and IL-17 by CII-restimulated LN CD4⁺ T cells (Fig. 7D), and this was significantly augmented upon in vivo co-treatment with CFA/I fimbriae showing even greater reduction in IFN-γ production; both groups treated with either IL-35 or CFA/I fimbriae plus IL-35 showed similar reduction in IL-17 (Fig. 7D). The EBI3^−/− recipients given the wild-type plus EBI3^−/− mixture showed no change in IFN-γ or IL-17 production relative to untreated mice (Fig. 7D). Improvement of arthritis was associated with significant elevation of IL-10 in wild-type Treg recipients treated with either IL-35 or CFA/I fimbriae plus IL-35, whereas TGF-β was greatly augmented in wild-type Treg recipients treated with CFA/I fimbriae plus IL-35, more so than recipients treated only with IL-35 (Fig. 7D). In support of this cytokine secretion analysis, the number of TGF-β⁺ Tregs as analyzed by flow cytometry was significantly elevated for all EBI3^−/− recipients given the wild-type Tregs and treated with CFA/I fimbriae plus IL-35 (Fig. 7E). Likewise, all intervention groups showed an increase in Foxp3⁺ Tregs (Fig. 7E). Thus, these data show CFA/I fimbriae-induced IL-35 is required for protection to CIA, and that an endogenous source of IL-35 is required to sustain this protection since cessation of IL-35 treatment or dilution of CD39⁺CD4⁺ Tregs results in the continued susceptibility to CIA.

Discussion

The pathogenesis of autoimmune arthritis involves autoreactive Th1 and Th17 cells mediating inflammation of the joints resulting in cartilage and bone destruction. In addition to autoreactive T cells, autoreactive B cells and the recruitment of innate cells into the joints further perpetuate tissue destruction as well as their contribution of soluble mediators including TNF-α, IL-1β, and IL-6 (42, 43). Current treatments rely mostly on treating the symptoms and not the cause of the disease, and arthritis is in large, plagued by the lack of efforts focusing on the development of Ag-specific interventions (44). Thus, drug-driven induction of Tregs and/or DCs may offer a viable alternative in treating arthritis. In furthering such efforts, CIA is used to test novel therapeutics since it shares features with human rheumatoid arthritis both for joint pathology and T cell aspects of this disease (45, 46). Exploiting this model, we have previously shown the potency of CFA/I fimbriae, when delivered by an attenuated Salmonella vector, to stimulate elevated immune responses to CFA/I fimbriae. When applied to mice with CIA, the fimbriae could also stimulate CII-specific Tregs suppressing Th1 and Th17 cells (40, 41). Interestingly, the induced Tregs showed a CD39⁺CD4⁺ phenotype. Further analysis revealed these Tregs could be subdivided into two additional subsets based on whether they expressed Foxp3: TGF-β-producing Foxp3⁻CD39⁺CD4⁺ and IL-10-producing Foxp3⁺CD39⁺CD4⁺ T cells (41). Although each subset could confer protection upon adoptive transfer, each provided suboptimal protection when compared to recipients given the total CD39⁺CD4⁺ T cell population; CD39⁻CD4⁺ T cells were unable to confer any protection (41).

Given these past findings, the objectives for these studies sought to determine whether CFA/I fimbriae could be administered independent of Salmonella, and further understand CFA/I fimbriae’s mechanisms of protection. As depicted, a single oral dose was sufficient to confer protection against CIA. Consistent with the Salmonella studies (41), soluble CFA/I fimbriae protected against CIA when given on day 14 post-CII challenge, the time when immune activation in the joints occurs. CFA/I fimbriae effectively offset the development of inflammation in the joints of 70% of the treated mice by suppressing Th1 and Th17 cells with the concomitant stimulation of IL-10- and TGF-β-producing CD4⁺ T cells. The latter is important since we have previously shown that TGF-β is responsible for promoting CD39 expression (41). Moreover, soluble CFA/I fimbriae stimulated Foxp3 expression by a portion of these CD39⁺CD4⁺ T cells. Although Salmonella-CFA/I was previously shown to activate Foxp3⁺ CD25⁺CD4⁺ T cells during EAE, our results with CIA consistently show the induction of CD39⁺CD4⁺ Tregs instead. This is believed to be a consequence of the disease, perhaps because of the increased apoptosis associated with CIA releasing ATP (47), which has been shown to activate CD39 cells (48, 49). Expression of CD39, ectonucleoside triphosphate diphosphohydrolase-1, on CD4⁺ T cells plays an important role in immune suppression by induced Tregs (48, 49) and Langerhans cells (50) by hydrolyzing ATP into AMP. In turn, AMP is rendered into adenosine by CD73 (49). CD39 is transcriptionally regulated through cAMP/CREB (51). CFA/I fimbriae-stimulated TGF-β has been shown to be involved in activation of CREB upon its S133 phosphorylation and, thus, promoting CD39 expression (41). Along these lines, when recombinant IL-35 is exogenously administered to mice with CIA, protection correlates with the CD39⁺CD4⁺ T cell subset as well, and this property is lost in IL-10^−/− mice further implicating the importance of IL-10 in the IL-35 pathway against CIA (23). Because of this similarity in stimulating CD39⁺CD4⁺ T cells during CIA, we queried if CFA/I fimbriae can also stimulate endogenous IL-35 since previous studies have shown that IL-35 is produced by Tregs (18, 21). Upon examination of CD39⁺CD4⁺ T cells from CIA mice treated with soluble CFA/I fimbriae, flow cytometry analysis revealed the simultaneous expression of EBI3 and p35 subunits. Consistent with what others have found (21), IL-35 was predominantly derived from Foxp3⁺ CD4⁺ T cells, but being CD39⁺. In the context of the two Treg subsets induced by CFA/I fimbriae, IL-35 associated with the Foxp3⁺ CD39⁺CD4⁺ T cells again suggesting that IL-35 may be linked with the IL-10-producing Tregs and not a separate entity as suggested by others (52).

IL-35 shares the EBI3 subunit with IL-27 which can also have immunoregulatory properties (19, 30–33); thus, it is important to assess IL-27’s role in these studies. What aids in differentiating their role is that IL-27 and IL-35 are produced by different cells: IL-27 is mostly secreted by APCs (rev. in 53), and IL-35 mostly by Tregs (18, 21). Flow cytometry analysis revealed that IL-27 was derived from CD11c⁺ cells from diseased mice and, not from Tregs. In contrast, IL-27 levels were reduced in CFA/I fimbriae-treated CIA mice as measured by flow cytometry and in Ag restimulation assays. Finally, CFA/I fimbriae could confer protection in mice compromised of their IL-27 signaling further implicating that the observed protection in B6 mice is independent of IL-27, but dependent on IL-35. In this respect, CFA/I-driven increase in IL-35 was associated with B6 Foxp3⁺ CD39⁺CD4⁺ Tregs, and not derived from CD11c⁺ cells, although CD11c⁺ cells from protected mice did significantly express more EBI3, but not simultaneously with p35 (data not shown). Additional FACS analysis failed to reveal B cells expressing IL-35 (data not shown) unlike that shown with EBV-transformed human cells (20), but our analyses do not exclude a potential nonlymphoid source of IL-35 (20). Nonetheless, the IL-35 secretion analysis performed with Ag-restimulated, purified CD4⁺ T cells obtained from CFA/I fimbriae-treated CIA mice (Fig. 3D) confirm production from CD4⁺ T cells. In the absence of IL-35 (EBI3^−/− mice), mice lost the capacity to induce elevated quantities of IL-10 and TGF-β subsequent CFA/I fimbriae treatment. In fact, the disease progressed unabated with enhanced IFN-γ and IL-17 production.

IL-27’s mode of action is believed to be attributed to its ability to inhibit RORγt (31) and control Th17 cell development at the priming stage by inducing the expression of PD-L1 on CD4⁺ T cells (19). IL-27 has potent effects in controlling EAE (19, 30,31), and this protective response is lost in EBI3^−/− mice (32). In fact, EAE was exacerbated in WSX-1^−/− mice (33). Similar to EAE, exogenously administered IL-27 was found to be therapeutic for CIA (53, 54); however, few studies have examined the role of endogenous IL-27 (53, 55) or IL-35 (18) in arthritis. Of particular interest in the present studies is the observation that CFA/I fimbriae induced IL-35 production by CD39⁺CD4⁺ T cells while concomitantly suppressing IL-27 by CD11c⁺ cells. CFA/I fimbriae lost their protective capacity in EBI3^−/− mice, but not in WSX-1^−/− mice implicating the importance of endogenous IL-35 to mediate CFA/I fimbriae’s protection. Interestingly, the EBI3 deficiency did not affect disease severity or incidence of CIA, and disease onset and clinical scores were similar in both B6 and EBI3^−/− mice supporting the notion that unprotected diseased mice produce minimal to no IL-35. This was further substantiated by the analysis of CD39⁺CD4⁺ T cells showing minimal IL-35 production in untreated CIA mice. In contrast, WSX-1^−/− mice exhibited less severe disease compared to B6 mice perhaps implicating IL-27’s importance for effector T cell survival as recently suggested by the absence of colitis in Rag^−/− mice adoptively transferred with WSX-1^−/− CD45RB^high CD4⁺ T cells (56). As in WSX-1^−/− mice, in vivo neutralization of IL-27p28 in B6 mice had no impact upon CFA/I fimbriae’s capacity to protect against CIA (data not shown). Thus, these collective findings further substantiate IL-35’s importance for CFA/I-mediated protection rather than IL-27. Although it remains unclear as to how CFA/I fimbriae ultimately impact the stimulation ofCD39⁺CD4⁺ T cells, past studies with macrophages showing CFA/I fimbriae to dampen proinflammatory cytokines, IL-1, IL-6, and TNF-α (38) implicate the fimbriae interact with APCs first, and these in turn, stimulate IL-35 production by CD39⁺CD4⁺ T cells. Such possibility is currently being explored.

For the most part, the observed Th1 and Th17 cell responses corresponded to the clinical findings in B6, EBI3^−/−, and WSX-1^−/− mice. Unprotected B6 and EBI3^−/− mice showed increased production of IFN-γ, IL-6, and IL-17 and reduced regulatory cytokines, IL-10 and TGF-β. Subsequent CFA/I fimbriae treatment, B6 and WSX-1^−/−, not EBI3^−/−, mice showed corresponding reductions in IFN-γ, IL-6, and IL-17. On the other hand, WSX-1^−/− mice showed marked elevation in TGF-β when compared to similarly treated B6 mice. This resistance to CIA may be in part attributed to the noted increases in CD39⁺ Tregs as these were increased in both PBS- and CFA/I fimbriae-treated WSX-1^−/− mice. TGF-β has been previously shown to augment CD39 expression (41), and the increased presence of Tregs and/or lessen activation of effector T cells (56) could account for these findings. In contrast, CFA/I fimbriae-treated EBI3^−/− mice showed elevated IFN-γ and IL-17 relative to PBS-treated CIA mice and IL-10 and TGF-β remained unchanged despite treatment.

The importance of endogenous IL-35 in CFA/I fimbriae-mediated protection to arthritis became evident in EBI3^−/− mice being unresponsive to CFA/I fimbriae treatment. Such finding compelled testing whether exogenous IL-35 could substitute for CFA/I fimbriae in the absence of endogenous IL-35, and the data showed that such treatment subdued disease as long as IL-35 treatments were maintained, and once interventions ceased, so did protection. These results beckoned then whether wild-type Tregs responding to CFA/I fimbriae treatment could restore protection in EBI3^−/− mice. It was previously shown that intervention with recombinant IL-35 at the onset of CIA potently protected B6 mice during the entire disease course (23 and unpublished observations). In this current study, EBI3^−/− mice required continuous administration of IL-35 since upon cessation of treatment, disease quickly developed. Likewise, soluble CFA/I fimbriae protected immunocompetent mice against CIA, or as long mice have an endogenous source of IL-35. To restore protection against CIA in EBI3^−/− mice, wild-type CD39⁺CD4⁺ T cells were adoptively transferred as a source of endogenous IL-35 when induced by CFA/I fimbriae. Since naive Tregs were used in the adoptive transfer, exogenous IL-35 was also administered and compared to EBI3^−/− recipients treated only with IL-35. EBI3^−/− recipients treated with CFA/I fimbriae plus IL-35 or IL-35 alone showed similar clinical disease, but mice treated with IL-35 alone showed greater disease incidence. These data point to the fact that an additional group of EBI3^−/− recipients treated with CFA/I fimbriae alone would not be sufficient for sustaining IL-35 signaling to thwart further disease, and possibly implicating another cell source of IL-35. Such consideration is currently being investigated. Collectively, these data further implicate the importance of endogenous IL-35 whether solely derived from Tregs and/or other unknown sources. This was more prominent in EBI3^−/− recipients given equal amounts of wild-type and EBI3^−/− CD39⁺CD4⁺ T cells diluting the wild-type Treg effect where no protection was observed even upon optimal intervention by CFA/I fimbriae plus IL-35 combination. Minimally, these findings point to a quantitative threshold of a sufficient source of endogenous IL-35 must be present to attain a qualitative therapeutic effect. This loss of protection correlated with enhanced IFN-γ and IL-17 production whereas protected EBI3^−/− recipients treated with CFA/I fimbriae plus IL-35 showed marked reductions in proinflammatory cytokines and augmented TGF-β and IL-10 production with concomitant increases in Tregs. These findings are consistent with that previously shown for CFA/I fimbriae stimulating TGF-β and IL-10 by CD39⁺ Tregs for protection to CIA (23,41). Coupled here now is the additional requirement for IL-35 for guarding against arthritis, and what remains to be determined is the sequence of each’s release during protection. Clearly, in the absence of IL-35, TGF-β and IL-10 are markedly reduced, and conversely, in the absence of IL-10, IL-35 cannot render protection to CIA (23). It remains to be determined how these three cytokines interact, and whether IL-35 serves as an anti-inflammatory amplifier (adjuvant) to augment, and possibly sustain TGF-β and IL-10 production.

In conclusion, these studies demonstrate that soluble CFA/I fimbriae have potent immuno-modulatory activity disallowing the requirement for a live vector. Importantly, CFA/I fimbriae conferred protection against CIA with a single, low oral dose and prior knowledge of the disease Ag-specificity is not required. Another salient feature of this immunotherapeutic is the capacity of CFA/I fimbriae to stimulate Tregs to produce IL-35. Such IL-35 production complements the already induced IL-10 by Foxp3⁺ Tregs and TGF-β by Foxp3⁻ Tregs. CFA/I fimbriae also act upon dendritic cells to diminish IL-27 production. Protection by CFA/I fimbriae was indeed IL-35-dependent as noted by the loss of protection in EBI3^−/− mice and the failure to induce sufficient IL-10⁺ and TGF-β⁺ CD39⁺ Tregs. In the absence of IL-27 signaling, CFA/I fimbriae retained the capacity to confer protection against CIA, further implicating that the lack of protection in EBI3^−/− mice is attributed to the loss of IL-35’s, not IL-27’s activity. Further evidence showed that sustained or intermittent IL-35 signaling is required for CFA/I fimbriae to maintain their protective effect against CIA. Diluting the presence of wild-type CD39⁺CD4⁺ T cells as a source of IL-35 generation in diseased EBI3^−/− recipients resulted in the loss of CFA/I fimbriae-mediated protection against arthritis.

Abbreviations used in this article

CFA/I

colonization factor antigen I

CIA

collagen-induced arthritis

CII

collagen II

CREB

cAMP-response element-binding protein

EAE

experimental autoimmune encephalomyelitis

EBI3

Epstein-Barr virus-induced gene 3

Foxp3

forkhead box P3

LNs

lymph nodes

MFI

mean fluorescence intensity

MLNs

mesenteric LNs

Tregs

regulatory T cells