Immunotherapy Delay Type 1 Diabetes Mice Following Mycobacterial ...

The Journal of Immunology

Th17 Polarized Cells from Nonobese Diabetic Mice Following Mycobacterial Adjuvant Immunotherapy Delay Type 1 Diabetes Enayat Nikoopour,* Jordan A. Schwartz,* Katrina Huszarik,* Christian Sandrock,* Olga Krougly,* Edwin Lee-Chan,* and Bhagirath Singh*,†,‡ IL-17–producing T cells are regarded as potential pathogenic T cells in the induction of autoimmune diseases. Previously, we have shown that injection of adjuvants containing Mycobacterium, such as CFA or bacillus Calmette-Gue´rin, can prevent type 1 diabetes in NOD mice. We injected NOD mice with mycobacterial products s.c. and analyzed the IL-17–producing cells from the draining lymph nodes and spleen by restimulating whole-cell populations or CD4+ T cells in vitro with or without IL-17–polarizing cytokines. Mice receiving CFA had a concomitant rise in the level of IL-17, IL-22, IL-10, and IFN-g in the draining lymph node and spleen. Adoptive transfer of splenocytes from CFA-injected NOD mice polarized with TGF-b plus IL-6 or IL-23 delayed the development of diabetes in recipient mice. IL-17–producing cells induced by CFA maintained their IL-17–producing ability in the recipient mice. Injection of CFA also changed the cytokine profile of cells in pancreatic tissue by increasing IL-17, IL-10, and IFN-g cytokine gene expression. We suggest that the rise in the level of IL-17 after adjuvant therapy in NOD mice has a protective effect on type 1 diabetes development. The Journal of Immunology, 2010, 184: 4779–4788.

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mmunization with mycobacterial preparations, such as bacillus Calmette-Gue´rin (BCG) or CFA, prevents the onset and recurrence of type 1 diabetes in NOD mice (1–5) and biobreeding rats (6, 7). We have previously reported that a single injection of CFA at an early age confers protection from developing diabetes in NOD mice (4). Several mechanisms have been proposed to explain the prevention of spontaneous diabetes by mycobacterial preparations (CFA or BCG). The protective effect of CFA could be due to the induction of regulatory T cells (8– 11), NK cells (12, 13), T cell apoptosis (14–17), b cell regeneration (10), or the modulation of APC function (8, 10). CFA has been shown to prevent the onset of diabetes by inducing CD4+CD25+ forkhead box P3 (FoxP3+) regulatory T cells (Tregs) in the spleen and the pancreatic lymph node (LN) (10). We found that a CD4+CD8+ CD25+ Treg clone derived from the LN cells of NOD mice immunized with CFA is capable of suppressing the immune response by secreting Granzyme B/Perforin in a cell contact-independent manner (9). Apoptosis of diabetogenic T cells has been suggested as another mechanism for the prevention of the recurrence of diabetes in islet-transplanted NOD mice (17, 18). BCG downregulates destructive autoimmunity by TNF-a/IFN-g–induced apoptosis of diabetogenic T cells through both Fas and TNF pathways (17). After BCG administration, there is an IFN-g– dependent upregulation of many apoptosis-related genes in effector *Department of Microbiology and Immunology, †Centre for Human Immunology, and ‡Robarts Research Institute, University of Western Ontario, London, Ontario, Canada Received for publication August 27, 2009. Accepted for publication March 4, 2010. This work was supported by grants from the Canadian Institutes of Health Research. Address correspondence and reprint requests to Dr. Bhagirath Singh, Department of Microbiology and Immunology, University of Western Ontario, Dental Sciences Building, London, Ontario N6A 5C1, Canada. E-mail address: [email protected] Abbreviations used in this paper: BCG, bacillus Calmette-Gue´rin; EAE, experimental autoimmune encephalomyelitis; FoxP3, forkhead box P3; ICS, intracellular staining; LN, lymph node; Treg, regulatory T cell. Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902822

CD4+ T cells (14–16). Also, NK cells mediate the protective effect of CFA through secretion of IFN-g (12, 13). These NK cells prevent the accumulation of b cell-specific CTLs in pancreatic islets (12). The Th17 subset of CD4+ T cells secretes IL-17, IL-22, and IL-21 and is differentiated from naive precursor cells in the presence of TGFb and IL-6 (19, 20). With the growing evidence for the role of Th17 cells in the pathogenesis of many autoimmune diseases, these cells are considered a contributing factor in the pathogenic process of type 1 diabetes. The transfer of highly purified Th17 cells from BDC2.5 transgenic mice caused diabetes in NOD/SCID recipients (21, 22). However, BDC2.5+ cells recovered from diabetic NOD/SCID mice showed predominant IFN-g expression over IL-17 expression, indicating conversion of donor cells into Th1 cells (21, 22). Although islet-reactive Th17 cells promote pancreatic inflammation, conversion of these cells into IFN-g producers contributes to diabetes pathogenesis (22). Th17 cells differentiated in the presence of TGF-b plus IL-6 produce both IL-17 and IL-10 and have suppressive activity in contrast to IL-23–expanded Th17 cells (23). Likewise, IL-17–producing CD8+ T cells differentiated from TGF-b1 plus IL-6 are not diabetogenic, whereas IL-23–treated cells potently induce the disease (24). In this study, we elucidated the immune response mediated by Th17 cells after CFA administration and their role in the protective effect of CFA. We found that CFA injection raised the IL-17 levels in the culture supernatants of the splenocytes or draining LN cells from NOD mice. However, the change in the cytokine profile following CFA treatment is not restricted to IL-17 as other cytokines, such as IFN-g and IL-10, were also increased. We found that adoptive transfer of Th17 polarized cells from CFA-injected NOD mice can delay diabetes development, suggesting that CFA induces a regulatory Th17 subset.

Materials and Methods Mice and immunizations Female NOD/Lt and NOD/SCID mice were bred in the animal facility at the Robarts Research Institute at the University of Western Ontario (London, Ontario, Canada). Mice were maintained in the specific pathogen-free facility at the University of Western Ontario. All of the experiments were

4780 performed in accordance with institutional guidelines for animal care. Mice were used at 4–8 wk of age, unless otherwise stated. Mice were monitored for the development of diabetes by measuring urine glucose with Diastix strips (Bayer, Mishawaka, IN) twice a week. Mice were recorded as diabetic based on two consecutive positive (.11.5 mmol) glucose urine tests. For immunization of mice, CFA, IFA, and zymosan A were purchased from Sigma-Aldrich (St. Louis, MO). Mice were immunized with a 1:1 emulsion of CFA or IFA in saline or PBS. Zymosan (250 mg/ml in saline) was emulsified 1:1 with IFA. BCG was purchased from Sanofi Pasteur (Toronto, Ontario, Canada). Mice were immunized with 100 mg BCG in PBS.

Cell culture Splenocytes or draining LN cell suspensions were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 0.5% HEPES, 5 mg/ml penicillin, 100 U/ml streptomycin (Life Technologies, Grand Island, NY), and 10% (v/v) FCS (HyClone Laboratories, Logan, UT). Murine cytokines IL-6, IL-17, IL-23, and recombinant human TGF-b1 were purchased from Cedarlane Laboratories (Hornby, ON, Canada).

Abs Anti-CD3ε (clone no. CL7202NA) activating Ab was purchased from Cedarlane Laboratories, and 5 mg/ml Ab was used to coat plates overnight at 4˚C. Purified and biotin-labeled anti–IL-17 were both purchased from BD Biosciences (San Jose, CA). The following fluorescent Abs labeled with PE, FITC, or allophycocyanin were used: anti-CD4 allophycocyanin, anti-CD8 allophycocyanin (eBiosciences, San Diego, CA), anti–IL-17 FITC or PE, anti-CD11b FITC, anti-CD11c FITC, or anti-B220 FITC and anti–IFN-g FITC. All of the Abs were purchased from BD Biosciences unless otherwise stated. For neutralization of IL-17, anti-mouse IL-17A was purchased from eBiosciences.

In vitro stimulation of spleen cells

ADJUVANT THERAPY PREVENTS DIABETES DEVELOPMENT sample were used in first-strand synthesis reactions using oligo(dT)12–18 primer from SuperScript III First-Strand Synthesis SuperMix for quantitative real-time PCR (Invitrogen). Resultant cDNA was diluted to a concentration of 225 ng/ml in diethyl pyrocarbonate water and amplified by PCR using QuantiFast CYBR Green PCR Kit purchased from Qiagen following the manufacturer’s protocols. Primers of the following sequences were used at a concentration of 1.25 mΜ: IL-17, forward, 59-GCTCCAGAAGGCCCTCAGA-39, reverse, 59-AGCTTTCCCTCCGCATTGA-39; IFN-g, forward, 59-TCAAGTGGCATAGATGTGGAA-39, reverse, 59-TGGCTCTGCAGGATTTTCATG-39; IL-10, forward, 59-GTTGCCAACTATGGA-39, reverse, 59-ACCTGCTCCACTGCCTTGCT-39; b-actin, forward, 59-ATCCGAAAACCTCTATGC-39, reverse, 59-AACGCAGCTCAGTAACSGTC-39. PCR reactions were performed in a Rotor-Gene 6000 real-time PCR analyzer (Corbett Life Sciences, San Francisco, CA) with an initial denaturing at 95˚C for 5 min followed by 40 cycles at 95˚C for 10 s and 60˚C for 30 s. Melting was performed at 60–95˚C. Results are normalized to b-actin expression levels in the pancreatic tissue.

Adoptive transfers NOD mice were injected with 50 ml of saline-emulsified CFA (1:1), and 2 wk later popliteal LNs and spleens were harvested and single-cell suspensions made. Splenocytes or cells from LNs (4 3 106 cells per well) were added to plates that were precoated with anti-CD3 overnight. The IL-17–polarizing cytokines TGF-b plus IL-6 or IL-23 alone (as indicated) were added, and cells were incubated for 4 d. Splenocytes were washed with PBS to remove any excess cytokine and suspended in PBS at a concentration of 10 3 107 cells per milliliter. Cells were mixed with diabetic splenocytes (10 3 106) and injected i.v. through the tail vein into NOD/SCID mice. Mice were housed in a specific pathogen-free exclusion barrier, where they were monitored for diabetes development by measuring urine glucose using Diastix strips every 3 d. A positive test for high glucose was confirmed by blood glucose measurements using a glucometer (Bayer).

Spleen cell or draining LN cell suspensions were cultured in 24-well plates precoated with anti-CD3 (5 mg/ml) at 4 3 106 cells per well in the presence of IL-23 (10 ng/ml), IL-6 (10 ng/ml), and TGF-b (10 ng/ml). Cells were cultured for 4 d followed by restimulation with phorbol-4,5-myristate (50 ng/ml) and ionomycin (500 ng/ml) and brefeldin A (10 mg/ml) for 4 h. Supernatants were collected before PMA and ionomycin stimulation and stored at 220˚C for cytokine analysis by ELISA. After restimulation and brefeldin A treatment, cells were prepared for FACS and intracellular staining (ICS) as described below.

Statistical analysis

Flow cytometry and ICS

CFA administration promotes IL-17 production in lymphoid organs

Cells were harvested from culture and washed with PBS followed by surface staining in 1% BSA in PBS buffer with FITC-, PE-, and allophycocyaninconjugated Abs. After surface staining, cells were washed and fixed with 2% formaldehyde and 1% BSA in PBS buffer for 10 min followed by washing with PBS. Cells were permeabilized for ICS with 0.5% saponin (SigmaAldrich) in PBS buffer containing 2% BSA with anti–IL-17 FITC, anti– IFN-g FITC, and anti–IL-10 FITC. Cells were washed with PBS and captured on a FACScalibur (BD Biosciences). FACS analysis was performed on FlowJo (Tree Star, Ashland, OR).

ELISA DuoSet ELISA kits from R&D Systems (Minneapolis, MN) were used to determine the concentrations of IL-22, IFN-g, and IL-10 in cell culture supernatants using the kit protocol. Quantification of IL-17 in culture supernatants was performed using a protocol adapted from a BD OptEIA kit (BD Biosciences). ELISA plates (Costar 96 Well Easy Wash; Lowell, MA) were coated with purified anti–IL-17 in PBS buffer with an Ab concentration of 1 mg/ml. Plates were washed as recommended and blocked in reagent diluent (10% FCS in PBS) for an hour. Recombinant IL-17 was used as a standard, and samples were diluted in reagent diluent and incubated for 2 h. Biotinlabeled anti–IL-17 (0.5 mg/ml) was diluted in reagent diluent and incubated for an hour, followed by 1:1000 streptavidin–alkaline phosphatase (BD Biosciences) for 30 min. Plates were washed thoroughly and activated with phosphatase substrate (Sigma-Aldrich) in diethanolamine (Sigma-Aldrich). Optical absorbance was read at a wavelength of 405 nm on a Benchmark Microplate Reader (Bio-Rad, Hercules, CA) and analyzed using Microplate Manager, version 4.0 (Bio-Rad).

Real-time PCR Total RNA was extracted from the pancreas by RNeasy Midi-prep (Qiagen, Mississauga, Ontario, Canada). RNA was decontaminated with a DNA-free kit (Ambion, Austin, TX). Equal concentrations of RNA (5 mg) from each

All of the experiments were repeated at least three times with reproducible results. Figures show data from representative experiments. Fisher exact probability test is used to analyze adoptive transfer data for diabetes development, and a p value of ,0.05 was considered statistically significant.

Results

NOD mice were injected in the footpad with 50 ml saline-emulsified CFA (1:1). Ten days later, draining popliteal LNs and spleens were harvested, and single-cell suspensions were made. Splenocytes or LN cells (3 3 106 cells per well) were added to plates that were precoated overnight with anti-CD3 Ab. TGF-b, IL-6, or IL-23 cytokines were added as indicated, and cells were incubated for 4 d. Supernatants of splenocytes or LN cell cultures were collected after 4 d of incubation with Th17 polarizing cytokines and analyzed for the levels of IL-17, IL-22, IL-21, IFN-g, and IL-10. Injection of the mycobacterial adjuvant increased the IL-17 level in culture supernatants compared with that in IFA-injected controls (Fig. 1). Splenocytes or draining LN cells from CFA-treated mice that were grown in IL-23 also showed an increase in IL-17 levels within culture supernatants compared with those of cultures from mice injected with IFA control. Indeed, IL-23 in culture is able to effectively maintain the CFA-stimulated Th17 memory populations, and it magnified the difference in IL-17 levels between cultures from the CFA and control groups. Analysis of the cytokine profile of cells from CFA-treated animals also revealed an increase in IFN-g and IL-10 levels (Fig. 1). In vitro IL-23 treatment of cells from mice injected with CFA did not suppress IFN-g and IL-10 cytokine levels but rather supported the development of cells secreting IFN-g, IL-10, and IL-17. The measurement of cytokines related to IL-17, such as IL-22 and IL-21, revealed a modest increase in the level of IL-22 in culture supernatants of all in vitro treatment groups following injection of


4781 mycobacterial adjuvant (Fig. 1), although the level of IL-21 was not in the detectable range (data not shown). The specific cytokine profile induced after administration of mycobacterial adjuvant was not limited to NOD mice because a similar pattern was seen in other mouse strains, such as C57BL/6 and BALB/c (data not shown). Because we were interested to know the cellular source of IL-17 production in cell culture supernatants, we analyzed cells gated for intracellular IL-17 for the expression of specific cell markers, such as TCR-b and DX5 (NKT cells), gd TCR (gd T cells), CD4, CD8, FoxP3, and RORgt (Fig. 1B). The percentage of NKT cells was not different between CFA-injected NOD mice and a control group. There was an increase in the percentage of gd T cells (6.3% in the CFA group versus 2.9% in the control group) among IL-17+ cells in mice treated with adjuvant. We found an increase in IL17+FoxP3+ cells (65.4% in CFA group versus 12.2% in control group). CFA could induce IL-17 in both CD4+ and CD8+ T cells, with the CD4+ T cells as a major cell type for IL-17 production. The ratio of the IL-17–producing CD4+ to CD8+ T cells remained the same in both experimental and control groups. CFA also caused more expression of the transcription factor RORgt. Kinetics of cytokine response after adjuvant therapy We monitored the levels of IL-17, IL-22, IL-10, and IFN-g on days 1, 3, 6, 12, 24, 32, and 40 after CFA injection of NOD mice and subsequent in vitro restimulation of cells from popliteal LNs in anti-CD3–precoated plates with IL-17 polarizing cytokine mixture. Analysis of cytokines in supernatants of cells from LN cells treated with TGF-b plus IL-6 (Fig. 2A) showed an increase in IL-17, IL-22, IL-10, and IFN-g cytokine levels over the course of 40 d, with the highest level between days 6 and 24. Analysis of kinetics of cytokine production in supernatants of cells from LN cells not polarized with cytokines showed higher levels of IL-17, IL-22, and IFN-g in the CFA treatment group (Fig. 2B). Comparison of the kinetics of IL-10 secretion in TGF-b plus IL-6–treated and nontreated cells from CFA-injected mice showed that IL-10 production is much higher in the TGF-b plus IL-6–treated group. CD4+ T cells are the source of the specific cytokine profile observed following adjuvant therapy We purified CD4+ cells from pooled LN cells of NOD mice injected with CFA and restimulated them with TGF-b plus IL-6 or IL-23 in plates precoated overnight with anti-CD3 and anti-CD28 (Fig. 3A). We observed the same pattern of the rise in the level of IL-17, IL-22, IL-10, and IFN-g cytokines, showing that CD4+ T cells are the main source of the secreted cytokines. Also, we did ICS on purified CD4+ T cells from CFA-injected mice after 4 d stimulation with TGF-b plus IL-6 in plates coated with anti-CD3 and anti-CD28 (Fig. 3B). CD4+ T cells from CFA-injected mice FIGURE 1. Polarized Th17 cells from CFA-immunized NOD mice produce IL-17, IL-22, IFN-g, and IL-10. NOD mice were injected with 50 ml of either saline–CFA (1:1) emulsion or saline–IFA (1:1) emulsion in footpad. After 10 d, mice were sacrificed, and a single-cell suspension of popliteal LN and spleen was made. Cells (3 3 106) were incubated for 4 d in plates precoated overnight with anti-CD3 Ab (5 mg/ml) with or without Th17 polarizing cytokines TGF-b (3 ng/ml), IL-6 (10 ng/ml), and IL-23 (10 ng/ml). A, Culture supernatants were collected on day 4 analyzed for IL-17, IL-22, IL-21, IFN-g, and IL-10 by ELISA as described in Materials and Methods. Data are representative results of three experiments. B, Cells were stained intracellularly for IL-17, and after gating on IL-17+, they were analyzed for expression of surface markers TCR-b, DX5, gd TCR, CD4, CD8, and transcription factors FoxP3 and RORgt for analysis of IL17–producing cell subpopulations. Data are representative of three reproducible experiments.

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ADJUVANT THERAPY PREVENTS DIABETES DEVELOPMENT

FIGURE 2. Kinetics of Th17 cytokine production by polarized lymphoid cells from CFAimmunized NOD mice. A and B, NOD mice were injected with 50 ml of either saline–CFA (1:1) emulsion or saline–IFA (1:1) emulsion in footpad. After 10 d, mice were sacrificed, and a single-cell suspension from popliteal LN was made. Cells (3 3 106) were incubated for 5 d in plates precoated overnight with anti-CD3 Ab (5 mg/ml) (A) with or (B) without Th17 polarizing cytokines TGF-b (5 ng/ml) and IL-6 (20 ng/ml). Culture supernatants are collected on day 5 for the cytokine analysis of IL-17, IL-22, IFN-g, and IL-10 by ELISA as described in Materials and Methods. Time course of the observed cytokine profile in LN cells of CFAor IFA-immunized NOD mice after 1, 3, 6, 12, 24, 32, and 40 d of injection is shown.

showed a concomitant increase in IL-17, IL-10, and IFN-g compared with that of the IFA-injected controls. Induction of IL-17–producing cells in the pancreas by mycobacterial adjuvant and zymosan The cytokine profile observed in the peripheral lymphoid organs prompted us to analyze the cytokine profile in the target organ in NOD mice. We analyzed gene expression of IL-17 and other cytokines in the pancreas to see changes in the cytokine milieu after injection of the mycobacterial product, BCG. We also included another adjuvant in our experiment called zymosan that has been shown previously to induce an IL-17 response (25). Three groups of NOD mice were injected i.p. with BCG, zymosan, and saline and sacrificed after 10 d to harvest the pancreas for RNA extraction and cDNA synthesis. Real-time PCR analysis of pancreatic samples showed an increase in IL-17 gene expression (normalized to b-actin gene expression) in BCG-treated animals compared with that in the saline group (Fig. 4). In line with higher secretion of IL-17, IFN-g, and IL-10 in the culture supernatants of the lymphoid organs of NOD mice injected with mycobacterial adjuvant, real-time PCR analysis of pancreatic samples also showed a significant upregulation of IFN-g gene expression and

a modest increase in IL-10 gene expression in the target organ. There was no IL-17 in the pancreatic tissue of mice treated with zymosan. However, both BCG and zymosan increased IL-21 gene expression. Mycobacterial adjuvants also led to higher expression of TGF-b and IL-6 genes in the pancreas. These cytokines are involved in the early differentiation of Th17 cells. This indicates that mycobacterial adjuvants provide a cytokine microenvironment in the pancreatic islets conducive to the development of Th17 cells. BCG induced a higher expression of the FoxP3 gene along with increased expression of TGF-b and IL-2 genes in pancreas compared with that in the saline control. Likewise, BCG increased expression of IL-12, a cytokine conducive to the production of IFN-g in pancreatic tissue. Adoptive transfer of IL-17–producing cells from CFA-treated NOD mice into NOD/SCID mice delayed diabetes development We sought to determine whether the cytokine profile observed after administration of mycobacterial adjuvants has any functional effect. For this purpose, we cultured splenocytes from mice injected with CFA or IFA (as a control) in TGF-b and IL-6 with plate-bound anti-CD3 stimulation for 4 d. These IL-17–producing cells (10 3 106) were mixed with splenocytes (10 3 106) from diabetic mice


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FIGURE 3. CD4+ T cells are the main source of the observed cytokine profile in polarized Th17 cells from CFA-immunized NOD mice. NOD mice were injected with 50 ml of either saline–CFA (1:1) emulsion or saline–IFA (1:1) emulsion in footpad. After 1 wk, mice were sacrificed, and CD4+ T cells were purified from the pooled popliteal LNs of four NOD mice by magnetic beads as described in Materials and Methods. Cells (3 3 106) incubated for 4 d in plates precoated overnight with anti-CD3 (5 mg/ml) and anti-CD28 (1 mg/ml) Abs with or without Th17 polarizing cytokines TGF-b (3 ng/ml), IL-6 (10 ng/ml), and IL-23 (10 ng/ml). A, Culture supernatants were collected on day 4 for the analysis of IL-17, IL-22, IFN-g, and IL-10 by ELISA as described in Materials and Methods. B, PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (10 mg/ml) were added to the cells for the last 6 h, and ICS for IL-17, IFN-g, and IL-10 on purified CD4+ T cells was done as described in Materials and Methods.

and adoptively transferred into NOD/SCID recipient mice (n = 5). NOD/SCID mice were monitored for diabetes development by measuring urine or blood glucose levels. As shown in Fig. 5A, cotransfer of polarized splenocytes from CFA-injected NOD mice with splenocytes from diabetic NOD mice significantly delayed the development of diabetes in NOD/SCID mice (Fig. 5A) compared with cotransfer of splenocytes from IFA-injected control NOD mice (p # 0.01). This indicated that the induced cytokine profile following CFA administration had a suppressive effect on diabetogenic T cells. We neutralized IL-17 in cultured cells to assess the contribution of IL-17 to the overall protective effect of the induced cytokine profile (Fig. 5B). Adoptive transfer of IL-17– neutralized cells compared with untreated Th17 polarized cells showed a reduction in diabetes protection, suggesting a role for IL-17 in mycobacterial adjuvant therapy (Fig. 5C). Although adoptive transfer of Th17 polarized cells from CFA-injected mice reduced diabetes incidence significantly (p # 0.05), disease protection from IL-17–neutralized cells was not significantly different from splenocytes from diabetic NOD mice. We observed the same

phenomenon for the splenocytes from CFA-injected NOD mice after in vitro treatment with IL-23 cytokine in culture. Cotransfer of these IL-23–treated cells with splenocytes from diabetic mice also delayed the development of diabetes (p # 0.02) in the recipient NOD/SCID mice (Fig. 6B). The suppressive effect of splenocytes from CFA-injected NOD mice on diabetogenic cells was effective irrespective of culturing these cells in media containing TGF-b plus IL-6 or IL-23 cytokine. This further supported the idea that these cells are functional and could exert an inhibitory effect on diabetogenic splenocytes. Adoptive transfer of Th17 cells from zymosan-treated NOD mice into NOD/SCID mice did not delay diabetes development Because zymosan administration in B6 mice has been shown to increase the level of IL-17 cytokine (25), we injected zymosan as a control in NOD mice. For this purpose, splenocytes and draining LN cells were cultured in IL-23 for 4 d, and culture supernatants were analyzed for IL-17, IL-22, IL-10, and IFN-g (Fig. 6A). Supernatants from cultured draining popliteal LN cells of mice

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FIGURE 4. Adjuvant therapy induces expression of the IL-17 cytokine gene in pancreas. NOD mice were injected with BCG (100 mg in PBS) (n = 10), zymosan (250 mg in IFA) (n = 10), or saline (n = 10). After 10 d, pancreases were harvested and homogenized, and mRNA was extracted. Real-time PCR analysis of mRNA transcripts encoding IL-17, IL-10, IFNg, TGF-b, IL-6, IL-27 p28, GM-CSF, IL-21, IL-2, IL-12, TNF-a, and FoxP3 genes was performed as described in Materials and Methods. The expression of various genes was normalized with respect to b-actin gene, and increases are represented as fold changes. Data are representative results of three experiments.

injected with zymosan in IFA showed an increase in IL-17, IL-22, IL-10, and IFN-g cytokine levels compared with those of the NOD mice injected with IFA as a vehicle. In fact, zymosan injection induced the same cytokine secretion pattern as was seen in CFAinjected NOD mice. Therefore, it was interesting to determine whether splenocytes from zymosan-treated NOD mice could induce the same delay in diabetes progression that was observed following adoptive transfer of splenocytes from CFA-injected NOD mice. For this purpose, splenocytes and draining LN cells of mice injected with CFA or zymosan were cultured in IL-23 for 4 d and mixed with diabetogenic splenocytes and were used for adoptive transfer into NOD/SCID recipients (Fig. 6B). NOD/SCID

mice were monitored for diabetes development by measuring urine or blood glucose levels. Although the use of mycobacterial adjuvants helped to delay the onset of diabetes in NOD mice (p # 0.02), injection of another adjuvant, namely, zymosan, that induced a similar cytokine profile as mycobacterial adjuvants did not delay the development of diabetes. IL-17–producing cells maintained IL-17 production potential after adoptive transfer into immunodeficient mice We next followed the fate of IL-17–producing donor cells adoptively transferred into NOD/SCID recipients. For this purpose, splenocytes were isolated and stimulated in vitro with anti-CD3


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FIGURE 5. Adoptive transfer of IL-17–producing cells from CFA-treated NOD mice into NOD/SCID mice delayed diabetes development, and the effect was reversed by anti-IL-17 Ab. A, NOD mice (n = 5 per group) were injected with 50 ml of either saline–CFA (1:1) emulsion or saline–IFA (1:1) emulsion in footpad. After 10 d, splenocytes and LN cells were harvested and incubated in anti-CD3 (5 mg/ml) and anti-CD28 (1 mg/ml) Ab-coated plates with or without Th17 polarizing cytokines TGF-b (10 ng/ml) and IL-6 (10 ng/ml). After 4 d, cells (10 3 106) were mixed with splenocytes (10 3 106) from diabetic mice and injected into tail vein of NOD/SCID mice. Mice were monitored for the development of diabetes for 48 d after adoptive transfer, and the CFA-treated group showed strong protective response (p # 0.01). B, IL-17 production from polarized cells from CFA-immunized NOD mice was blocked by anti- IL-17 Ab when added at the beginning of culture to neutralize IL-17 production. C, After 4 d, cells (10 3 106) from B were mixed with splenocytes (10 3 106) from diabetic mice and injected into tail vein of NOD/SCID mice. Mice were monitored for the development of diabetes. Anti–IL-17 treatment reduced the protective effect of CFA on adoptive transfer of diabetes (p = NS).

and treated with IL-23 or left untreated at day 4 following the adoptive transfer. Analysis of IL-17 levels in culture supernatants showed that mice with donor cells from BCG-injected NOD mice still released more IL-17 compared with that from NOD/SCID mice that received donor cells from saline-injected mice (Fig. 7). There was no significant difference in the IFN-g levels between the two study groups. These results indicated that IL-17–producing cells maintained their potential for IL-17 production after adoptive transfer into immunodeficient mice. To exclude the possibility that cells from NOD/SCID mice could provide a source of IL-17, we directly injected NOD/SCID mice with BCG and saline i.p. Restimulation of splenocytes from both groups with anti-CD3 and anti-CD28 did not yield any IL-17 or IFN-g production.

Discussion In this study, we found that administration of mycobacterial products into NOD mice could give rise to a new balance in the level of IL-17, IL-10, and IFN-g in the peripheral lymphoid organs and the pancreas of mice. The adjuvant-induced cytokine profile helped to decrease the diabetogenic potential of cells from diabetic NOD mice upon transfer into immunodeficient NOD/SCID mice. Since the discovery of adjuvant immunotherapy for preventing diabetes in NOD mice (1), various mechanisms of action have been proposed to explain this phenomenon. In the era of Th1/Th2 paradigm governance, the traditional view on the mechanisms involved in this process mainly focused on the activation of a Th2 subset of T cells that could produce the cytokines IL-4 and IL-10 and consequently downregulate the Th1 cell-mediated autoimmune response (26). Later findings by Serreze et al. (27) showed that the protective effect of CFA or BCG in NOD mice can occur even in the absence of the IL-10 or IL-4 genes, whereas the IFN-g gene is necessary for this effect. Therefore, contrary to its presumed role in the pathogenesis of diabetes, IFN-g was found to be involved in the mechanism of diabetes inhibition following adjuvant therapy (27). These findings ruled out the possibility that a Th1 to Th2 immune deviation is involved in the mechanism of protection elicited by mycobacterial adjuvants. In light of recent advances in the IL-17 family of cytokines, we attempted to re-evaluate the cytokine profile in NOD mice with respect to the raised IL-17 level in the course of adjuvant therapy.

We found an increase in IL-17, IL-22, IFN-g, and IL-10 in supernatants of splenocytes and draining LN cells of NOD mice treated with CFA (Fig. 1). Scriba et al. (28) also showed that stimulation of human CD4+ T cells with mycobacterial products, such as BCG or purified protein derivative, caused elevated levels of IL-17 and IL-22 in CD4+ T cells. In our previous findings, treatment of NOD splenocytes with Th17 polarizing cytokines did not increase their diabetogenic potential (29). In this study, we found that mixing diabetogenic splenocytes from diabetic NOD mice with Th17 polarized splenocytes from NOD mice injected with CFA can reduce the pathogenic ability of the diabetogenic splenocytes (Fig. 5). McGeachy et al. (23) showed that TGF-b plus IL-6 could lead to production of IL-17 and IL-10 in Th17 cells and that these cells could exert a regulatory effect by preventing autoimmunity. We found that CFA administration into NOD mice could magnify the production of IL-17 and IL-10 in CD4+ T cells following treatment of the draining LN cells with a mixture of TGF-b and IL-6. Furthermore, CFA administration helped to preserve the increased level of IL-17 and IL-10 in draining LN cells cultured with IL-23. In our experiments, we found that splenocytes from CFA-injected mice were more responsive to IL-23 treatment in terms of increased cytokine production, which could result from the upregulation of the IL-23R in the lymphoid organs following CFA treatment. Lymphoid cells obtained from CFA-injected mice that were cultured with either TGF-b plus IL-6 or with IL-23 alone could suppress the pathogenic ability of splenocytes from diabetic NOD mice upon adoptive transfer into NOD/SCID recipients (Figs. 5, 6B). However, because mycobacterial adjuvants are able to suppress autoimmunity in IL-10–deficient mice (27), the increased IL-10 level cannot be interpreted as the main factor mediating the protective effect of CFA. Because IFN-g is required for this protective effect and administration of CFA into IFN-gnull mice is not protective (30), we speculate that a new balance between IFN-g and IL-17 cytokine levels might also play a role in CFA-mediated autoimmune protection. Although, Th1 cells were traditionally considered to be pathogenic in diabetes development, there are some reports on the beneficial effect of IFN-g in preventing autoimmunity in NOD mice. For example, injection of rIFN-g into NOD

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FIGURE 7. Polarized IL-17–producing cells from BCG-immunized NOD mice maintained IL-17 production after adoptive transfer into NOD/ SCID mice. NOD mice were injected with BCG (100 mg in PBS) or saline as a control. After 10 d, splenocytes were harvested and stimulated in the presence of IL-23 (10 ng/ml) in plates precoated with anti-CD3 (5 mg/ml) and anti-CD28 (1 mg/ml) Ab. After 4 d, cells were adoptively transferred into NOD/SCID mice. Two groups of NOD/SCID mice injected with BCG or saline were used as controls. After 4 d, mice were sacrificed, and a single-cell suspension was made from splenocytes. Cells were incubated with or without IL-23 (10 ng/ml) in plates precoated with anti-CD3 (5 mg/ ml) and anti-CD28 (1 mg/ml) Abs. After 4 d, culture supernatants were collected and analyzed for the IL-17 and IFN-g cytokines by ELISA as described in Materials and Methods. Data are representative results of three experiments.

mice has been shown to reduce the chance of diabetes development (31). Also, Lee et al. (12, 13) found that NK cells mediate the protective effects of CFA through secretion of IFN-g. In addition, O’Connor et al. (16) found that apoptosis of activated CD4+ T cells following BCG administration required IFN-g production and IFN-g receptor expression by the CD4+ T cells (14). Therefore, although Th1 cells are able to induce diabetes, a concomitant increase in IL-17 and IFN-g appears to have a beneficial effect on the disease. O’Connor et al. (32) showed that IL-17 can suppress Th1 differentiation by suppressing T-bet, the master FIGURE 6. Adoptive transfer of Th17 cells from zymosan-treated NOD mice into NOD/SCID mice did not delay diabetes development. NOD mice (n = 10 per group) were injected with 50 ml of saline–CFA (1:1) emulsion, saline–IFA (1:1) emulsion, or 250 mg zymosan in saline–IFA (1:1) emulsion in footpad. After 20 d, mice were sacrificed, and a single-cell suspension of popliteal LN cells (3 3 106) was incubated with IL-23 (10 ng/ml) in plates precoated overnight with anti-CD3 (5 mg/ml) and antiCD28 (1 mg/ml) Abs. Cells were incubated for 5 d at 37˚C and in 5% CO2. A, Culture supernatants are collected on day 5 for the cytokine analysis of

IL-17, IL-22, IFN-g, and IL-10 by ELISA as described in Materials and Methods. Data are representative results of three experiments. B, Splenocytes (10 3 106) from various groups in A were mixed with an equal number of diabetic splenocytes (10 3 106) and adoptively transferred into NOD/SCID mice. Mice were monitored for the development of diabetes for 80 d after adoptive transfer, and the IL-23–treated CFA group showed a protective response (p # 0.02).

The Journal of Immunology regulator of Th1 differentiation. However, Harrington et al. (33) reported that IFN-g suppressed Th17 development from naive precursors. These studies showed that although IFN-g and IL-17 cytokines can prevent the early differentiation programs of naive T cells into Th17 and Th1 cells, respectively, they are not able to suppress mature Th17 or Th1 cells. However, each of these two Th cells can affect the function and activity of other cell type by cross-regulation. For example, Goverman et al. (34, 35) found that different ratios of Th1 and Th17 cells in the CNS affected the pattern of T cell infiltration and inflammation in the brain parenchyma. Infiltration of the brain parenchyma and the induction of brain inflammation occurred only when Th17 cells outnumbered Th1 cells (34). On the basis of these findings, we suggest that the interaction between Th17 and Th1 cells and the induction of IL-10 production can contribute to the mechanism of diabetes protection induced by mycobacterial adjuvants. Previous studies have been shown that IL-23 alone is pathogenic, whereas cells polarized with TGF-b plus IL-6 in combination with or without IL-23 can have a suppressive effect (23). As shown in Figs. 1A and 3A, IL-23 polarized cells from CFA-immunized NOD mice become regulatory Th17. Thus, mycobacterial adjuvants can induce TGF-b and IL-6. We indeed found an increased level of IL-6 and TGF-b after growing bone marrow dendritic cells with BCG (data not shown). One possibility is that injections of mycobacterial adjuvants induce dendritic cells to secrete cytokines that favor polarization of regulatory Th17 cells. In our experiments, we did not use naive CD4+ cells because IL-23 worked on the memory population of CD4 cells from CFA-injected mice. It should be noted that gd T cells also contributed to the production of IL-17 in CFA-injected mice (Fig. 1B). IL-23 can expand IL-17– producing gd T cells in NOD mice that are protective in adoptive transfer to NOD/SCID mice (36). We found a similar pattern of IL-17, IFN-g, and IL-10 cytokine levels in culture supernatants of splenocytes from mice injected with an adjuvant called zymosan as was observed following mycobacterial adjuvant injection (Fig. 6A). Although zymosan induced a high level of these cytokines, the adoptive transfer of splenocytes from zymosan-injected NOD mice along with diabetogenic cells had the same kinetic effect on diabetes development in NOD/SCID mice as did transfer of diabetogenic cells alone. This indicates that cells from the zymosan-treated mice did not have a protective effect on diabetes development (Fig. 6B). Therefore, the mechanism of CFA-induced protection is more complex than simply the induction of high levels of IL-17, IFN-g, and IL-10 cytokines. We found that the pattern of gene upregulation in the pancreas of mice injected with zymosan was different from that in mice injected with CFA (Fig. 4). Although zymosan induced a high level of IL-17 in splenocyte culture supernatants, it did not change the production of IL-17 in the target organ. This indicates that the infiltration of T cells into pancreatic tissue is different following CFA treatment compared with that following zymosan treatment. Contrary to our findings on the role of adjuvants in autoimmune diseases, in the experimental autoimmune encephalomyelitis (EAE) mouse model Veldhoen et al. (25) showed that injection of zymosan could lead to the reversal of symptoms after the acute phase of inflammation. Zymosan elicited Th17 cell differentiation and supported the initiation of myelin oligodendrocyte glycoprotein-induced EAE. However, in contrast to mycobacterial adjuvant (CFA), zymosan did not sustain chronic inflammation or the production of IL-23. As a consequence, mice with EAE induced by zymosan and myelin oligodendrocyte glycoprotein had a notable reversal of symptoms after the acute phase of the disease (25). It should be noted that we used adjuvants without autoantigens in our protection model. It has been shown that multiple injections of

4787 zymosan, three times in the first week and injection in weeks 3, 5, 7, 9, and 11, could prevent diabetes development (37). We just used a single injection of zymosan. Because Th17 cells induced by a single injection of zymosan are quite unstable (25), repeated injection of zymosan might make the induced Th17 cells to persist in vivo, and that could be a reason for the protection seen in mice with multiple injections. We found that IL-17–producing cells maintained their ability to produce IL-17 after adoptive transfer of IL-17 polarized cells from CFA-injected NOD mice into NOD/SCID mice (Fig. 7). Nurieva et al. (38) showed that in vitro generated Th17 cells from naive T cells of IL-17F–red fluorescent protein reporter mice rapidly lost their IL-17 expression and were converted to Th1 cells upon adoptive transfer into lymphopenic hosts. Also in these experiments, Th17 cells were able to maintain IL-17 production in normal mice, and antigenic peptide plus CFA injection in the recipient mice helped to further expand and stabilize Th17 cells (38). There might be a role for CFA in potentiating receptor–ligand interactions on Th17 cells by making them more responsive to IL-23 or upregulating IL-23R because treatment of cells from CFA-injected NOD mice with IL-23 increased IL-17 level in culture (Fig. 1). McGeachy et al. (39) found that in the absence of IL-23 Th17 development was stalled at the early activation stage and failed to maintain IL-17 production. The administration of CFA in donor mice may stabilize Th17 donor cells upon adoptive transfer, or CFA administration in the recipient mice after receiving Th17 donor cells can help to maintain the Th17 phenotype. Because IL-17 polarized T cells of BDC2.5 TCR NOD transgenic mice lost IL-17 production after adoptive transfer into NOD/SCID mice (21), CFA injection of NOD/ SCID mice after receiving IL-17 polarized donor cells might be helpful in maintaining the Th17 phenotype in the lymphopenic host. We previously showed that T cells from CFA-injected NOD mice were suppressive upon adoptive transfer into NOD/SCID mice. The increase in number and function of the regulatory T cells have been suggested as a mechanism for the CFA suppressive effect (9, 10). In this study, we showed that CFA-induced suppressive function of T cells persists even in the presence of Th17 polarizing conditions. This means that the increase in IL-17 is at least not detrimental to the suppressive effect of the adjuvant therapy and the overall cytokine profile induced following CFA injection contributes to the this effect. We suggest that the suppressive effect could be the result of cross-regulation among Treg, Th17, and Th1 cell subsets following adjuvant therapy. In conclusion, the overall cytokine profile induced in the peripheral lymphoid organs and pancreas following adjuvant therapy in NOD mice indicates that a rise in IL-17 levels does not increase the susceptibility for the development of autoimmune diseases and it may have a protective role in preventing diabetes in NOD mice.

Disclosures The authors have no financial conflicts of interest.

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