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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

To examine the role of interleukin-23 (IL-23) in subgroup polarization of IL-17A–positive and/or interferon-γ (IFNγ)–positive T cells in autoimmune disease–prone DBA/1 mice with and without collagen-induced arthritis.

Methods

A magnetic-activated cell sorting system was used to isolate CD4+ T cells from the spleen of naive and type II collagen (CII)–immunized DBA/1 mice. These CD4+ T cells were stimulated in vitro under Th0, Th1, or different Th17 culture conditions. Intracellular staining for IL-17A and IFNγ was evaluated by flow cytometry. In addition, Th17 cytokines and T helper–specific transcription factors were analyzed by enzyme-linked immunosorbent assay and/or quantitative polymerase chain reaction.

Results

In CD4+ T cells from naive DBA/1 mice, IL-23 alone hardly induced retinoic acid–related orphan receptor γt (RORγt), Th17 polarization, and Th17 cytokines, but it inhibited T-bet expression. In contrast, transforming growth factor β1 (TGFβ1)/IL-6 was a potent inducer of RORγt, RORα, IL-17A, IL-17F, IL-21, and FoxP3 in these cells. In contrast to TGFβ1/IL-6, IL-23 was critical for the induction of IL-22 in CD4+ T cells from both naive and CII-immunized DBA/1 mice. Consistent with these findings, IL-23 showed a more pronounced induction of the IL-17A+IFNγ– subset in CD4+ T cells from CII-immunized mice. However, in CD4+ T cells from naive mice, IL-23 significantly increased the TGFβ1/IL-6–induced Th17 polarization, including elevated levels of IL-17A and IL-17F and decreased expression of T-bet and FoxP3. Of note, the IL-23–induced increase in IL-17A and IL-17F levels was prevented in T-bet–deficient mice.

Conclusion

IL-23 promotes Th17 differentiation by inhibiting T-bet and FoxP3 and is required for elevation of IL-22, but not IL-21, levels in autoimmune arthritis. These data indicate different mechanisms for IL-23 and TGFβ1/IL-6 at the transcription factor level during Th17 differentiation in autoimmune experimental arthritis.

A novel pathogenic T cell population, Th17 cells, has been identified that induces autoimmune inflammation in mice (1). These Th17 cells are far more efficient at inducing Th1-mediated autoimmune inflammation in mice than are classic Th1 cells (interferon-γ [IFNγ]), although the pathogenic potential of Th17 cells, as well as Th1 cells, has been demonstrated (2, 3). It was shown that Th17 cells were induced by interleukin-23 (IL-23; p19/p40), a novel member of the IL-12 (p35/p40) family.

Dendritic cells secrete cytokines of the IL-12 family, such as IL-12 and IL-27 (p28/EBI3), and play a role in Th1 cell proliferation. Their activation results in the production of IFNγ. IL-23 induces the differentiation of naive T cells into Th17 cells through a mechanism distinct from the signals that drive the development of Th1 cells and Th2 cells. Neutralization of both IFNγ and IL-4 has been found to favor IL-23–induced IL-17–producing cells (4, 5). However, despite a requirement for IL-23 in vivo in the development of Th17 cell–mediated diseases such as experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA) (1, 6, 7), IL-23 cannot drive Th17 cell differentiation in naive CD4+ T cells (8) in vitro. This in vitro Th17 cell differentiation is driven by the combination of IL-6 and transforming growth factor β1 (TGFβ11) (8–10). During Th17 development induced by IL-6 and TGFβ1, Th17 cells become responsive to IL-23, which subsequently serves as a survival factor for committed Th17 cells (8). More recently, it was shown that IL-21 is an autocrine cytokine that is sufficient and necessary for Th17 differentiation (11, 12)

Th17 cells are characterized by the expression of IL-17 (IL-17A), and they also reportedly express IL-17F, IL-6, tumor necrosis factor (TNF), granulocyte–macrophage colony-stimulating factor (GM-CSF), IL-21, and IL-22, but not IFNγ or IL-4 (1, 4, 5, 11–16). In mice, Th17 cells have been established as a separate lineage of T helper cells that are distinct from conventional Th1 and Th2 cells (4, 5). Transcription factors and signaling molecules that are important for the differentiation of Th1 or Th2 cells, including STAT-1, STAT-4, and STAT-6, which are signal transducers and activators of transcription, and T-bet, are dispensable for the development of Th17 cells (4, 5). Recently, retinoic acid–related orphan receptor γt (RORγt) and RORα were found to be novel Th17 transcription factors (17, 18). Moreover, Th1 cytokines (IFNγ and IL-12) and Th2 cytokines (IL-4 and IL-13) repress Th17 cell development (4, 5, 19).

IL-23 is required for the development of IL-17–producing T cells in C57BL/6 mice with CIA, since it was shown that IL-23p19–deficient C57BL/6 mice lacked these cells (7). However, the role of IL-23 in subgroup polarization of IL-17A+IFNγ– (Th17), IL-17A+IFNγ+ (double-positive), and IL-17A–IFNγ+ (Th1) cells in the autoimmune disease–prone DBA/1 mouse with or without CIA is still unknown.

In the present study, we demonstrated that IL-23 promotes Th17 differentiation by inhibiting T-bet and FoxP3 expression. Furthermore, IL-23 is required for IL-22, but not IL-21, in autoimmune experimental arthritis. These data indicate different mechanisms for IL-23 and TGFβ1/IL-6 at the transcription factor level during Th17 differentiation in autoimmune experimental arthritis.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Mice.

DBA/1 mice were purchased from Harlan (Horst, The Netherlands). B6.129S6-Tbx21tm1Glm/J (T-bet–deficient) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained under specific pathogen–free conditions and were provided with food and water ad libitum. Mice were used in the experiments at ages 8–12 weeks. All experiments were approved by the Dutch Animal Ethics Committee.

CIA induction.

CIA was induced by an intradermal injection of bovine type II collagen (CII; Chondrex, Redmond, WA) emulsified in Freund's complete adjuvant (Difco, Detroit, MI), as described previously (20). The animals were killed 10 days after immunization.

Purification and in vitro culture of T cells.

CD4+ T cells were purified from spleens obtained from naive and CII-immunized DBA/1 mice and from naive T-bet–deficient mice by negative isolation using a magnetic-activated cell sorter. The purity of the obtained fractions was typically >98%. CD4+ T cell fractions were cultured at concentrations of 1 × 106 cells/ml in Iscove's modified Dulbecco's medium (BioWhittaker, Walkersville, MD), containing 10% heat-inactivated fetal calf serum (Sigma, St. Louis, MO) and 5 × 10–5M β-mercaptoethanol (Merck, Darmstadt, Germany) supplemented with various cytokines (R&D Systems, Minneapolis, MN), as described below. Plates were coated overnight at 4°C with anti-CD3 and anti-CD28 (145-2C11 and 37.51, respectively; BD Biosciences, Heidelberg, Germany) at a concentration of 10 μg/ml each in phosphate buffered saline.

For Th1 polarizing conditions, IL-12 (10 ng/ml) and anti–IL-4 (10 μg/ml) (11B11; Bioceros, Utrecht, The Netherlands) were added. For Th17 polarizing conditions, anti–IL-4 (10 μg/ml) and anti-IFNγ (5 μg/ml) (XMG1.2; Bioceros) were added, along with TGFβ1 (3 ng/ml), IL-6 (20 ng/ml), and/or IL-23 (50 ng/ml). On day 3, anti-CD3/CD28 activation was stopped. T cell cultures were expanded in the presence of the indicated cytokines supplemented with IL-2 (5 ng/ml) for up to 7 days. Then, supernatants were collected, cells were collected for RNA isolation, and the expanded cells were stained for detection of intracellular cytokine.

Flow cytometric analyses.

Single-cell suspensions from the spleen were prepared and incubated with monoclonal antibodies (mAb) for 4-color flow cytometry as previously described (21). Monoclonal antibodies were purchased from BD Biosciences. For intracellular detection of cytokines, cells were stimulated for 4 hours with phorbol myristate acetate (50 ng/ml) and ionomycin (500 ng/ml) in the presence of GolgiStop (BD Biosciences). Cells were harvested, extracellularly stained with anti-CD4 mAb, and then intracellularly stained (IL-17A and IFNγ; BD Biosciences) with 2% paraformaldehyde and 0.5% saponin. Data were acquired on a FACSCalibur flow cytometer and were analyzed using CellQuest (Becton Dickinson, Sunnyvale, CA) and FlowJo (Tree Star, Ashland, CA) software. Live events were collected based on forward and side scatter patterns.

Enzyme-linked immunosorbent assay (ELISA).

Cytokines in culture supernatants were measured by ELISA for IL-17A and IL-21 (both from R&D Systems), for IFNγ (OptEIA; BD Biosciences), and for IL-22 (Antigenix, Huntington Station, NY) according to the manufacturer's instructions. The IL-17F ELISA was performed using the protocol recommended by R&D Systems, using recombinant mouse IL-17F and coating IL-17F antibodies, which were kindly provided by Dr. J. Wright (Wyeth, Cambridge, MA).

Quantitative polymerase chain reaction (PCR) analyses.

Total RNA was extracted using a GenElute Mammalian Total RNA Miniprep system (Sigma), and 1 μg was used as a template for complementary DNA synthesis, using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) and random hexamer primers. PCR primers spanning at least 1 intron–exon junction were designed manually or by using ProbeFinder software (Roche Applied Science, Indianapolis, IN). Probes were chosen from the universal probe library (Roche Applied Science) or were designed manually and purchased from Eurogentec (Seraing, Belgium). Quantitative real-time PCR was performed using an ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA). To confirm the specificity of the amplification products, samples were analyzed by standard agarose gel electrophoresis. Threshold levels were set and further analysis was performed using the SDS version 1.9 software (Applied Biosystems). The obtained Ct values were normalized to those of GAPDH.

Statistical analysis.

Student's paired t-test was used to test differences between 2 groups within the naive or the collagen mouse population. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

IL-23 induces more prominent expression of the IL-17+IFNγ– subset in CD4+ T cells from CII-immunized DBA/1 mice than those from naive DBA/1 mice.

IL-23 did not markedly induce the IL-17+IFNγ– subset (Th17) in vitro in CD4+ T cells isolated from the spleen of naive DBA/1 mice. This is in contrast to a marked induction of the IL-17+IFNγ– subset in CD4+ T cells from CII-immunized DBA/1 mice (Figure 1). TGFβ1/IL-6, on the other hand, induced a relatively high percentage of the IL-17+IFNγ– subset of CD4+ T cells from naive animals; this was more prominent in CII-immunized mice (Figure 1). In CII-immunized mice, a lower percentage of cells in the IL-17–IFNγ+ (Th1) subset was induced by TGFβ1/IL-6 as compared with IL-23 alone (Figure 1). Furthermore, the percentage of IL-17+IFNγ+ double-positive cells induced by TGFβ1/IL-6 was higher than that induced by IL-23 alone. Adding IL-23 to TGFβ1/IL-6–stimulated CD4+ T cells markedly increase the percentage of Th17, but not Th1 cells or double-positive cells, in naive mice as compared with TGFβ1/IL-6 alone, and this increase was less prominent in CII-immunized mice (Figure 1).

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Figure 1. Interleukin-23 (IL-23) markedly increases the transforming growth factor β1 (TGFβ1)/IL-6–induced IL-17+IFNγ– cell subset in CD4+ T cells from naive DBA/1 mice. Flow cytometric analysis of intracellular IL-17A and interferon-γ (IFNγ) expression in CD4+ T cells isolated from the spleen of naive DBA/1 mice and DBA/1 mice immunized with type II collagen (CII) was performed 10 days after immunization. CD4+ T cells were activated with anti-CD3/anti-CD28 and cultured for 7 days under Th0, Th1 (IL-12/anti–IL-4 antibody), or different Th17 (IL-23, TGFβ1/IL-6, or IL-23 plus TGFβ1/IL-6) culture conditions. Numbers in each compartment are the percentage of cells. Results are representatives of 3 separate experiments using CD4+ T cells from a total of 5 mice per group.

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IL-23 decreases the expression of the Th1-specific transcription factor T-bet in CD4+ T cells.

The expression of the Th1-specific T-bet transcription factor was induced under Th1 culture conditions as expected (P < 0.05) (Figure 2A). IL-23 suppressed T-bet expression in CD4+ T cells isolated from the spleen of naive mice as compared with Th0 and Th1 culture conditions (P < 0.01 for both comparisons). Adding IL-23 to TGFβ1/IL-6 resulted in a reduction of T-bet expression in CD4+ T cells from naive DBA/1 mice as compared with TGFβ1/IL-6 alone, although the difference did not reach statistical significance (P = 0.07) (Figure 2A). Of note, T-bet expression was significantly lower in TGFβ1/IL-6–stimulated CD4+ T cells as compared with Th0 culture conditions in CII-immunized DBA/1 mice (P < 0.05) (Figure 2A). In contrast to the findings in naive mice, IL-23 alone did not significantly suppress T-bet expression in CD4+ T cells from CII-immunized DBA/1 mice as compared with the CII/Th0 culture conditions. However, compared with the CII/Th1 culture conditions, T-bet expression was significantly suppressed under all 3 Th17 culture conditions (P < 0.05 for all 3 comparisons) (Figure 2A). These data indicate that IL-23 directly or indirectly decreased the expression of T-bet in CD4+ T cells from both naive and CII-immunized DBA/1 mice.

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Figure 2. Interleukin-23 (IL-23) suppresses T-bet expression, and transforming growth factor β1 (TGFβ1)/IL-6 induces FoxP3 expression. Quantitative reverse transcription–polymerase chain reaction analysis of A, T-bet, B, retinoic acid–related orphan receptor γt (RORγt), C, RORα, and D, FoxP3 expression in activated CD4+ T cells from naive DBA/1 mice and DBA/1 mice immunized with type II collagen under Th0, Th1 (IL-12/anti–IL-4 antibody), or different Th17 (IL-23, TGFβ1/IL-6, or IL-23 plus TGFβ1/IL-6) culture conditions was performed after 7 days of culture. Expression levels were normalized to GAPDH; results for activated CD4+ T cells from naive DBA/1 mice were set at 1. Values are the mean and SEM of 3 separate experiments using CD4+ T cells from a total of 5 mice per group. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, by Student's paired t-test.

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IL-23 induces the expression of the Th17 transcription factors RORγt and RORα in CD4+ T cells from CII-immunized, but not naive, DBA/1 mice.

IL-23 barely induced RORγt and RORα expression in CD4+ T cells from naive DBA/1 mice as compared with induction by TGFβ1/IL-6 (Figures 2B and C). Adding IL-23 to TGFβ1/IL-6 further increased RORγt (P = 0.05) and RORα expression in CD4+ T cells from naive DBA/1 mice, although it did not reach statistical significance (Figures 2B and C). Moreover, compared with naive mice, IL-23 significantly increased RORγt and RORα expression in CD4+ T cells from CII-immunized DBA/1 mice (P < 0.001 and P < 0.05, respectively) (Figures 2B and C). These data suggest that IL-23 influenced RORγt and RORα expression and that the increase in IL-17+IFNγ– subset of CD4+ T cells from naive DBA/1 mice was accompanied by lower T-bet and higher RORγt expression after IL-23 was added to TGFβ1/IL-6 (Figure 1).

TGFβ1/IL-6–induced FoxP3 expression in CD4+ T cells from naive DBA/1 mice is inhibited by IL-23.

TGFβ1/IL-6, but not IL-23, increased the Treg cell transcription factor FoxP3 in CD4+ T cells from naive DBA/1 mice as compared with Th0 culture conditions (P < 0.05) (Figure 2D). Interestingly, the presence of IL-23 significantly suppressed the TGFβ1/IL-6–induced expression of FoxP3 in these cells (P < 0.05) (Figure 2D). In contrast to CD4+ T cells from naive mice, FoxP3 expression in CD4+ T cells from CII-immunized DBA/1 mice was low under the TGFβ1/IL-6 culture conditions tested (Figure 2D).

Expression of the Th2 transcription factor GATA-3 was significantly suppressed under all Th17 culture conditions in CD4+ T cells from naive and CII-immunized DBA/1 mice as compared with the Th0 culture conditions (P < 0.05) (data not shown).

These data indicate that in addition to the induction of RORγt and RORα, TGFβ1/IL-6 also induces the Treg transcription factor FoxP3 and that the expression of this transcription factor is suppressed by IL-23.

IL-23 has an additive effect on the IL-17A and IL-17F levels induced by TGFβ1/IL-6.

IL-23 alone is less potent in inducing IL-17A and IL-17F in CD4+ T cells isolated from naive DBA/1 mice as compared with induction by TGFβ1/IL-6 (Figures 3A and B). Significantly higher levels of IL-17A and IL-17F were detected in the presence of TGFβ1/IL-6 as compared with the presence of IL-23 alone (P < 0.01 for both comparisons). Although IL-23 alone induced a significant increase in IL-17A and IL-17F in CD4+ T cells from naive DBA/1 mice (P < 0.05 for IL-17A and P < 0.01 for IL-17F), the IL-23–induced IL-17A and IL-17F levels were more pronounced in CD4+ T cells isolated from CII-immunized DBA/1 mice (P < 0.01 for both comparisons) (Figures 3A and B). In addition, when CD4+ T cells from naive and CII-immunized DBA/1 mice were incubated with the combination of IL-23 plus TGFβ1/IL-6, a significant increase in the levels of IL-17A (P < 0.01 for both groups of mice) and IL-17F (P < 0.01 for naive mice and P < 0.05 for CII-immunized mice) was noted as compared with incubation with TGFβ1/IL-6 alone (Figures 3A and B).

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Figure 3. Interleukin-23 (IL-23) increases the levels of IL-17A and IL-17F induced by transforming growth factor β1 (TGFβ1)/IL-6 in CD4+ T cells from DBA/1 mice immunized with type II collagen (CII). Enzyme-linked immunosorbent assay for levels of IL-17A (A and C) and IL-17F (B and D) was performed on culture supernatants of activated CD4+ T cells from naive DBA/1 mice and DBA/1 mice immunized with CII (A and B) and from T-bet–deficient mice (C and D) under Th0, Th1 (IL-12/anti–IL-4 antibody), or different Th17 (IL-23, TGFβ1/IL-6, or IL-23 plus TGFβ1/IL-6) culture conditions after 7 days of culture. Values in A and B are the mean and SEM of 3 separate experiments using CD4+ T cells from a total of 5 mice per group. Values in C and D are the mean and SEM of 3 separate experiments using CD4+ T cells from a total of 4 mice per group. ∗ = P < 0.05; ∗∗ = P < 0.01, by Student's paired t-test.

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To further examine the importance of suppressing the IL-23–induced T-bet expression for the increase in IL-17A and IL-17F levels by treatment with the combination of IL-23 and TGFβ1/IL-6 as compared with TGFβ1/IL-6 alone, we studied these effects in T-bet–deficient mice. Interestingly, the lack of T-bet expression prevented the IL-23–induced increase in IL-17A and IL-17F levels in cultures treated with the triple combination (Figures 3C and D). This finding underscores the functional link between IL-23 and T-bet under the Th17 culture conditions used.

TGFβ1/IL-6, but not IL-23, is critical for the induction of IL-21.

Incubation of CD4+ T cells isolated from naive DBA/1 mice with TGFβ1/IL-6 resulted in a significant increase in the IL-21 level as compared with the Th0 culture conditions (P < 0.05) (Figure 4). In contrast, incubation of CD4+ cells from naive mice with IL-23 showed a significantly lower level of IL-21 as compared with the Th0 culture conditions (P < 0.01) (Figure 4). These data show that IL-23 alone is not a potent inducer of IL-21. No significant increase in the IL-21 level was found after incubation of CD4+ T cells from naive or CII-immunized DBA/1 mice with the combination IL-23/TGFβ1/IL-6 as compared with TGFβ1/IL-6 alone (Figure 4). These data indicate that IL-23 is not critical for the increase in IL-21 levels in mice with autoimmune arthritis.

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Figure 4. Transforming growth factor β (TGFβ1)/interleukin-6 (IL-6), but not IL-23, is critical for the induction of IL-21. Enzyme-linked immunosorbent assay for levels of IL-21 was performed on culture supernatants of activated CD4+ T cells from naive DBA/1 mice and DBA/1 mice immunized with type II collagen under Th0, Th1 (IL-12/anti–IL-4 antibody), or different Th17 (IL-23, TGFβ1/IL-6, or IL-23 plus TGFβ1/IL-6) culture conditions after 7 days of culture. Values are the mean and SEM of 3 separate experiments using CD4+ T cells from a total of 5 mice per group. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, by Student's paired t-test.

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IL-23 is critical for the induction of IL-22.

Th1 cells and Th17 cells can produce IL-22. Therefore, we examined the expression of IL-22 under both culture conditions using CD4+ T cells from naive and CII-immunized DBA/1 mice. IL-23 was a potent inducer of IL-22, and significantly higher levels of this cytokine were found under Th17/IL-23 culture conditions as compared with the Th0 and the Th1 culture conditions in CD4+ T cells from both naive and CII-immunized DBA/1 mice (P < 0.05 for all comparisons) (Figure 5). Incubation with TGFβ1/IL-6 alone did not result in a significant increase in IL-22 as compared with the Th0 and Th1 culture conditions, both in CD4+ T cells isolated from naive DBA/1 mice and from CII-immunized DBA/1 mice (Figure 5). IL-23 was required to significantly increase the levels of IL-22 under TGFβ1/IL-6 culture conditions (P < 0.05 for both groups of mice) (Figure 5). These data indicate the requirement of IL-23 for the induction of elevated IL-22 levels in mice with autoimmune arthritis.

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Figure 5. Interleukin-23 (IL-23), but not transforming growth factor β1 (TGFβ1)/IL-6, is critical for the induction of IL-22. Enzyme-linked immunosorbent assay analysis for levels of IL-22 was performed on culture supernatants of activated CD4+ T cells from naive DBA/1 mice and DBA/1 mice immunized with type II collagen under Th0, Th1 (IL-12/anti–IL-4 antibody), or different Th17 (IL-23, TGFβ1/IL-6, or IL-23 plus TGFβ1/IL-6) culture conditions after 7 days of culture. Values are the mean and SEM of 3 separate experiments using CD4+ T cells from a total of 5 mice per group. ∗ = P < 0.05, by Student's paired t-test.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

In the present study, we analyzed the effects of IL-23 and/or TGFβ1/IL-6 on the subgroup polarization of IL-17+IFNγ– (Th17), IL-17+IFNγ+ (double-positive), and IL-17–IFNγ+ (Th1) cell subsets in CD4+ T cells from autoimmune disease–prone DBA/1 mice with and without CIA. We demonstrated that IL-23 and TGFβ1/IL-6 differentially contributed to the induction of Th17 cytokines in the autoimmune CIA model by regulating the expression of T helper transcription factors in differing ways. Under Th17 polarizing conditions, we detected a higher percentage of Th17 cells among CD4+ T cells from CII-immunized DBA/1 mice as compared with naive DBA/1 mice. Splenic CD4+ T cells isolated from CII-immunized DBA/1 mice 10 days after immunization and stimulated for 4 hours also showed an increase in the percentage of IL-17+IFNγ– (Th17) CD4+ T cells as compared with splenic CD4+ T cells from naive DBA/1 mice (data not shown). IL-23 induced a more pronounced expression of IL-17+IFNγ– (Th17) cells in CII-immunized mice. Furthermore, we demonstrated that IL-23 promoted Th17 differentiation by inhibiting T-bet and FoxP3 expression. We also demonstrated that IL-23 was required for IL-22, but not IL-21, expression in autoimmune experimental arthritis. Whether an environment with IL-23 or TGFβ1/IL-6 or an environment containing the combination of IL-23 and TGFβ1/IL-6 will influence the pathogenic potential of these Th17 cells in experimental arthritis is currently under investigation.

It has been shown that IL-23 is essential for IL-17–producing CD4+ T cell function in CIA (7). IL-23 is considered to be a survival factor for Th17 cells, and it could also be involved in Th17 proliferation, but it is not essential for early Th17 polarization, since naive T cells do not express the IL-23 receptor (8–10, 22). Of note, T-bet expression was reduced in CD4+ T cells from naive DBA/1 mice under IL-23 culture conditions. However, these cells were not triggered by IL-23 to significantly increase their RORγt and RORα expression, although a small, but significant, increase in IL-17A and IL-17F levels was noted. Consistent with the idea that IL-23 is not essential for early Th17 polarization, we found that IL-23 showed a more pronounced induction of Th17 cells in CII-immunized mice. On the other hand, TGFβ1, IL-6, and recently, IL-21 have been recognized as key cytokines for the differentiation of naive T helper cells into Th17 cells (11, 12). Here, we showed that the combination TGFβ1/IL-6, but not IL-23, was critical for IL-21, a finding consistent with the idea that TGFβ1/IL-6 is important in early Th17 differentiation.

TGFβ1 is a potent inducer of the Treg cell transcription factor FoxP3. In the present study, we found that TGFβ1, together with IL-6, significantly induced FoxP3 expression as well as RORγt. It has been reported that the transcription factor FoxP3 can bind to RORγt and therefore that FoxP3 may directly regulate RORγt activity (23). Our data reveal that IL-23 markedly influenced the capacity of TGFβ1/IL-6 to induce FoxP3 expression. In fact, TGFβ1/IL-6–induced FoxP3 expression was significantly suppressed by IL-23, whereas RORγt expression was increased. These data suggest that IL-23 promotes Th17 polarization and activity by influencing the balance between FoxP3 and RORγt expression in CD4+ T cells. Moreover, this change in balance may also be important for the pathogenic potential of these Th17 cells and for plasticity between Treg cells and Th17 cells in arthritis, and this is presently under investigation (24).

IL-23 plays an important role in regulating IL-22 expression (16, 19). Herein, we showed that IL-23 and not TGFβ1/IL-6 was critical for IL-22 expression in CII-immunized DBA/1 mice. IL-22 is a member of the IL-10 family and can function as a proinflammatory cytokine. It has been shown that IL-22 mediates IL-23–induced acanthosis and dermal inflammation (19). Of interest, IL-22 together with IL-17A and/or IL-17F synergistically regulates the expression of β-defensin 2 and S100 calcium-binding protein A9 (S100A9) (16). However, it was shown that IL-22 deficiency did not protect mice against EAE (25), but decreased the severity of CIA, although the CIA incidence was enhanced (ref.26 and Lubberts E, et al: unpublished observations). Further studies may better elucidate the role of IL-22 in experimental arthritis, as well as the importance of the interaction and coexpression of IL-23 and IL-22 in particular.

Compared with TGFβ1/IL-6, IL-23 alone was not a strong inducer of RORγt, IL-17A, or IL-17F in CD4+ T cells from naive DBA/1 mice. Of note, IL-23 markedly increased the TGFβ1/IL-6–induced expression of IL-17A and IL-17F in CD4+ T cells from both naive and CII-immunized DBA/1 mice. A trend toward an increase in RORγt was also found. Whether IL-23 truly induced RORγt or just increased the percentage of cells expressing RORγt is unclear. We observed that the cell numbers were not very different between the groups, indicating that IL-23 stimulation does not result in marked T cell proliferation. This suggests that IL-23 may have truly increased RORγt expression in our CD4+ T cells, although we cannot exclude the possibility that IL-23 in particular increased the number of RORγt positive cells.

The role of IL-17A in experimental arthritis has been well described (27). Recently, it was shown that IL-17F is an important regulator of inflammatory responses that seems to function differently than IL-17A in immune responses and diseases (28, 29). It has been shown that in IL-1Ra–/– mice, the spontaneous develoment of arthritis was considerably, but only partially, suppressed after crossing these mice with IL-17F–deficient mice (IL-17F–/–IL-1Ra–/– mice) as compared with their littermate controls (29). However, CIA developed normally in IL-17F–/– mice (29). Further studies may better elucidate the role of IL-17F in experimental arthritis as well as the interaction of IL-17F with other Th17 cytokines.

IL-23 was able to suppress T-bet expression in Th17 cells. However, IL-23 did not repress T-bet expression in Th0 or Th1 cells (data not shown). Interestingly, IL-23 was not able to increase IL-17A and IL-17F levels in TGFβ1/IL-6–treated CD4+ T cells from T-bet–deficient mice. To our knowledge, these data are the first to show a functional link between IL-23 and the Th1 transcription factor T-bet under Th17 culture conditions.

In conclusion, this study revealed that IL-23 promotes Th17 differentiation by inhibiting T-bet and FoxP3 and that IL-23 is required for elevation of IL-22, but not IL-21, in autoimmune arthritis. These data indicate different mechanisms for IL-23 and TGFβ1/IL-6 at the transcription factor level during Th17 differentiation in autoimmune experimental arthritis. Further studies are needed to unravel the pathogenic potential of IL-23 or TGFβ1/IL-6, or the combination of IL-23 and TGFβ1/IL-6, in the induction of Th17 cells in experimental arthritis.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Lubberts had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Mus, Cornelissen, Asmawidjaja, van Hamburg, Hendriks, Lubberts.

Acquisition of data. Mus, Cornelissen, Asmawidjaja.

Analysis and interpretation of data. Mus, Cornelissen, Asmawidjaja, van Hamburg, Boon, Hendriks, Lubberts.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We acknowledge the staff of the EDC Animal Facility (Erasmus MC, Rotterdam, The Netherlands) for good animal care. We highly appreciate the kind gift of recombinant IL-17F and IL-17F antibody from Dr. J. Wright (Wyeth, Cambridge, MA).

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES