Interleukins 1β and 6 but not transforming growth factor-β are essential for the differentiation of interleukin 17–producing human T helper cells

Abstract

Interleukin 17 (IL-17)–producing CD4+ helper T cells (TH-17 cells) have been linked to host defense and autoimmune diseases. In mice, the differentiation of TH-17 cells requires transforming growth factor-β and IL-6 and the transcription factor RORγt. We report here that for human naive CD4+ T cells, RORγt expression and TH-17 polarization were induced by IL-1β and enhanced by IL-6 but were suppressed by transforming growth factor-β and IL-12. Monocytes and conventional dendritic cells, but not monocyte-derived dendritic cells activated by microbial stimuli, efficiently induced TH-17 priming, and this function correlated with antigen-presenting cell production of IL-1β and IL-6 but not IL-12. Our results identify cytokines, antigen-presenting cells and microbial products that promote the polarization of human TH-17 cells and emphasize an important difference in the requirements for the differentiation of TH-17 cells in humans and mice.

Main

The TH-17 lineage of effector CD4+ T helper lymphocytes produces interleukin 17 (IL-17)1,2, a cytokine that induces the production of chemokines and antimicrobial peptides by tissue cells, leading to the recruitment of neutrophils and inflammation3. Beyond their possible involvement in host defense against microbes, TH-17 cells have been linked to the pathogenesis of several inflammatory and autoimmune diseases. In experimental animal models, IL-17-producing T cells are responsible for autoimmune encephalitis, collagen-induced arthritis, colitis and psoriasis4,5,6. In addition, mice deficient in the IL-17 receptor are more susceptible to certain pathogens and are resistant to chemical-induced colitis7,8. In humans, several studies have demonstrated a correlation between the severity of inflammatory disease and IL-17 concentrations in serum or tissues9,10,11.

Since the identification of TH-17 cells, ongoing efforts have been made to characterize TH-17 cells and the factors regulating their differentiation and function. Early reports indicated a requirement for IL-23 in TH-17 differentiation4,12. However, it was subsequently shown that IL-23 is required for IL-17-mediated effector function13 and the survival but not differentiation of TH-17 cells14. Studies in mice have identified transforming growth factor-β (TGF-β) and IL-6 as the critical cytokines driving the TH-17 differentiation of naive T cells14,15,16. In addition to TGF-β and IL-6, proinflammatory cytokines such as IL-1β and tumor necrosis factor can increase the efficiency of TH-17 differentiation17. Several cytokines, including type I and type II interferon, IL-4, IL-12, IL-27 and IL-2, inhibit TH-17 differentiation1,2,4,18,19,20.

The orphan nuclear receptor RORγt has been identified as the key transcription factor needed to orchestrate the differentiation of mouse TH-17 cells21. Expression of RORγt mRNA is induced by stimulation with TGF-β and IL-6 and is upregulated by IL-6- and IL-23-induced activation of the STAT3 transcription factor22. In contrast, IL-2-induced activation of STAT5 suppresses the differentiation of mouse TH-17 cells in vitro and in vivo20. The T helper type 1 (TH1) and TH2 lineage-specifying transcription factors T-bet and GATA-3, respectively, are not expressed by human memory TH-17 cells23 and can, at least in the case of T-bet, suppress the differentiation of mouse TH-17 cells24.

At present, understanding of TH-17 differentiation is limited to the mouse system and it remains to be established whether the factors that promote or inhibit mouse TH-17 differentiation exert similar effects in humans. A related issue that still must be addressed in both mice and humans is the nature of the antigen-presenting cells (APCs) capable of inducing TH-17 responses. It is well appreciated that TH1 differentiation can be promoted by dendritic cells (DCs) that are stimulated by particular microbial stimuli and by CD40 ligand (CD40L) to release large amounts of IL-12 (ref. 25). Whether DCs are the main APCs mediating TH-17 responses and what microbial stimuli optimally induce TH-17 differentiation remain to be determined. Understanding the mechanisms driving the differentiation of human TH-17 cells is of relevance to both immunopathology and vaccination.

Here we report that in human naive CD4+ T cells, RORγt expression and TH-17 polarization were induced by IL-1β and IL-6 and were inhibited by TGF-β and IL-12. Monocytes and circulating conventional DCs (cDCs) activated by lipopolysaccharide (LPS) and peptidoglycan, which produce large amounts of IL-1β and IL-6 but little IL-12, were the most efficient APCs for TH-17 differentiation, whereas monocyte-derived DCs that produced IL-12 but not IL-1β in response to LPS or peptidoglycan failed to promote TH-17 differentiation. Monocyte-induced TH-17 differentiation was inhibited by antibodies that neutralized IL-1β and IL-6 and by inhibitors of caspase-1, an enzyme required for the release of IL-1β. Our results indicate that cytokines needed for the priming of human TH-17 cells differ from those needed to prime mouse TH-17 cells; they also demonstrate an essential function for caspase-1-mediated release of IL-1β in the induction of human TH-17 cells.

Results

Cytokines inducing the differentiation of human TH-17 cells

To identify conditions permissive for the differentiation of human TH-17 cells, we stimulated highly purified CD4+CD45RA+CCR7+CD25 naive T cells for 5 d with beads coated with antibody to CD3 (anti-CD3) and anti-CD28 in the presence of antibodies neutralizing IL-4 and interferon-γ (IFN-γ). We allowed the cells to proliferate for additional 7 d in low doses of IL-2 and analyzed their capacity to produce IL-17 and IFN-γ by intracellular cytokine staining and by enzyme-linked immunosorbent assay (ELISA; Fig. 1a–d). In the absence of exogenous cytokines, a small percentage of primed T cells acquired the ability to produce IL-17, alone or in combination with IFN-γ. IL-6 did not enhance this apparently spontaneous TH-17 differentiation, whereas TGF-β suppressed the production of both IL-17 and IFN-γ. TGF-β and IL-6, a combination that promotes the differentiation of mouse TH-17 cells, failed to induce the differentiation of human TH-17 cells and actually reduced the spontaneous TH-17 differentiation. In contrast, IL-1β strongly induced the differentiation of IL-17-producing cells, most of which also produced IFN-γ (Fig. 1a–d). Furthermore, the combination of IL-1β and IL-6 led to a significant increase in the overall percentage of IL-17-producing cells; this 'boost' mainly affected cells producing IL-17 but not IFN-γ (Fig. 1c). Unexpectedly, the addition of TGF-β to cultures stimulated with IL-1β and IL-6 significantly decreased TH-17 differentiation. In the same culture conditions, IL-12 inhibited TH-17 differentiation but promoted TH1 differentiation (Fig. 1a,b).

Figure 1: Polarization of human TH-17 cells is induced by IL-1β and IL-6 but is inhibited by TGF-β and IL-12.
figure1

(a) Intracellular cytokine staining for IL-17 and IFN-γ in naive CD4+ T cells primed for 5 d with beads coated with anti-CD3 and anti-CD28, in the presence of various combinations of cytokines (above plots) plus neutralizing anti-IFN-γ and anti-IL-4, then incubated for 7 d more in IL-2 and stimulated for 5 h with PMA and ionomycin. (b) ELISA of IL-17 and IFN-γ in 36-hour culture supernatants of cell populations primed and expanded as described in a and then stimulated with anti-CD3 and PdBu. (c) Flow cytometry to determine the percentage of cells producing IL-17 and IFN-γ among cells from six different donors, primed and activated as described in a. Each symbol represents a single donor; small horizontal bars indicate the mean. NS, not significant. (d) ELISA of IL-17 and IFN-γ in 36-hour culture supernatants of cells from six donors, primed and activated as described in b. (e) Flow cytometry of the production of IL-17, tumor necrosis factor (TNF), IL-22 and IL-4 by naive CD4+ T cell populations primed, expanded and activated as described in a. (f) Flow cytometry and ELISA of naive CD4+ T cells primed as described in a. Below, concentrations of IL-17 and IFN-γ in 36-hour culture supernatants of cells sorted according to CCR6 expression (gating, above) and then stimulated with anti-CD3 and PdBu. SSC, side scatter. Numbers above outlined areas (f) or in quadrants (a,e) indicate percent cells in each (right (a,e): top number, top right quadrant; bottom number, bottom right quadrant). Data are representative of six (ad) or three (e,f) separate experiments (mean + s.d. of duplicate cultures (b,f) or six donors (d)).

To further characterize the human TH-17 cells differentiated in vitro, we measured the production of additional cytokines. All IL-17-producing T cells produced large amounts of tumor necrosis factor, and more than half produced IL-22, a TH-17-related cytokine; in contrast, almost no IL-17-producing T cells produced IL-4 (Fig. 1e). In addition, TH-17 cells differentiated in vitro expressed CCR6 (Fig. 1f), a chemokine receptor that has been identified as a marker of human memory TH-17 cells23.

To determine whether TGF-β exerts dose-dependent effects on the differentiation of human TH-17 cells, we primed naive CD4+ T cells in the presence of IL-1β, IL-6 or a combination of IL-1β and IL-6, together with 'graded' concentrations of TGF-β. In all conditions, we noted a TGF-β dose–dependent inhibition of the IL-17- and IFN-γ-producing cells (Supplementary Fig. 1 online). Notably, T cells producing only IL-17 seemed to be more resistant to TGF-β-mediated inhibition than were T cells producing only IFN-γ or both IL-17 and IFN-γ.

To investigate the kinetics of the differentiation of human TH-17 cells and the effects of other cytokines on the development of human TH-17 cells, we primed naive human CD4+ T cells with IL-1β and IL-6 in the presence or absence of various doses of IL-2 and analyzed the cells 5 d or 12 d after incubation in IL-2 (Fig. 2a). We noted TH-17 differentiation as early as day 5 in cultures incubated in the presence or absence of low doses of IL-2; this was partially inhibited by high doses of IL-2 added at the initiation of culture. The addition of IL-2 at 3 d after priming did not substantially alter the percentage of IL-17-producing T cells (Fig. 2a). Notably, in cultures stimulated with IL-1β alone or with IL-1β plus IL-6, the addition of IL-23 on day 3 increased the percentage of IL-17-producing T cells recovered on day 12; this increase was more evident in the population of cells producing only IL-17 and in cultures stimulated with IL-1β alone (Fig. 2b).

Figure 2: Effects of IL-2 and IL-23 on TH-17 differentiation.
figure2

(a) Intracellular cytokine staining of IL-17 and IFN-γ in naive CD4+ T cells primed with beads coated with anti-CD3 and anti-CD28, in the presence of IL-1β, IL-6 and neutralizing anti-IFN-γ and anti-IL-4 and in the absence or presence of IL-2 (20 or 200 IU/ml) added on day 0 or day 3 after priming, then stimulated for 5 h with PMA and ionomycin on day 5 or day 12. (b) Intracellular cytokine staining of IL-17 and IFN-γ in naive CD4+ T cells primed for 5 d with beads coated with anti-CD3 and anti-CD28, in the presence of IL-1β alone or with IL-6 (left margin), plus neutralizing anti-IFN-γ and anti-IL-4, with IL-2 (left) or IL-2 plus IL-23 (right) present from day 3 to day 12, and then stimulated for 5 h with PMA and ionomycin. Numbers in quadrants indicate percent cells in each (as described in Fig. 1). Data are representative of three separate experiments.

These results collectively indicate that IL-1β and IL-6 enhance, whereas TGF-β and IL-12 inhibit, the TH-17 differentiation of human naive T cells. Our findings also indicate that the TH-17 phenotype is acquired rapidly, is maintained even after incubation in IL-2 and is enhanced by IL-23.

Regulation of lineage-specifying transcription factors

To investigate early events in the differentiation of human TH-17 cells, we measured the kinetics of expression of mRNA encoding the human orthologs of mouse RORγt (RORC variant 2) and T-bet (TBX21), as well as mRNA encoding the cytokines IL-17 (IL17A) and IFN-γ (IFNG). RORC and IL17A were induced by IL-1β alone and, in a more sustained way, by the combination of IL-1β and IL-6 (Fig. 3a). In contrast, IL-12, IL-6 and TGF-β, either alone or in combination, failed to induce or induced low expression of RORC, consistent with their inability to induce the differentiation of human TH-17 cells. Furthermore, TBX21 was induced slightly in the presence of IL-1β and IL-6 but was inhibited in the presence of TGF-β. As expected, TBX21 mRNA and IFNG mRNA were strongly induced by IL-12 (Fig. 3a). In T cells exposed to IL-1β and IL-6, RORC mRNA and IL17A mRNA peaked on days 2–5 and decreased on day 12, but could be upregulated within 4 h of restimulation (Fig. 3b). These results collectively indicate that IL-1β and IL-6 induce sustained expression of RORC in human CD4+ T cells, which is consistent with their ability to promote TH-17 differentiation. In contrast, TGF-β and IL-6, a combination capable of inducing RORγt in mouse naive T cells, modestly stimulated human RORC expression and failed to induce IL17A.

Figure 3: IL-1β and IL-6 induce RORγt expression in differentiating TH-17 cells.
figure3

(a) RT-PCR of the expression of RORC, IL17A, TBX21 and IFNG in naive CD4+ T cells primed for various times (horizontal axes) with beads coated with anti-CD3 and anti-CD28, in the presence of various cytokines (key) plus neutralizing anti-IFN-γ and anti-IL-4. *, P < 0.05, IL-1β versus IL-1β and IL-6; **, P < 0.05, IL-12 versus medium (two-tailed t-test). (b) RT-PCR of the expression of RORC and IL17A in cells primed with IL-1β and IL-6 as described in a and analyzed on day 0, 2 or 5 of priming, or after an additional 7 d in IL-2 (12), or after 4 h of stimulation with anti-CD3 and PdBu on day 12 (12+). AU, arbitrary units. Data represent the mean ± s.d. of four donors (a) and are representative of four (a) or three (b) separate experiments.

APCs and innate stimuli that trigger human TH-17 differentiation

Having established in an APC-free system the cytokines that elicit or inhibit the differentiation of human TH-17 cells, we next set out to elucidate the nature of the APCs and innate stimuli that trigger TH-17 differentiation. We stimulated freshly isolated monocytes and cDCs, as well as cultured monocyte-derived DC populations, with various microbial products alone or in combination with CD40L and measured the upregulation of costimulatory molecules and cytokine after 24 h (Fig. 4 and Supplementary Fig. 2 online). All stimuli induced APC activation, as assessed by the upregulation of CD80 and CD86 (data not shown). In contrast, cytokine production depended both on the type of APCs and on the nature and combination of the stimuli. In particular, monocytes produced large amounts of IL-1β and IL-6 in response to LPS (a Toll-like receptor 4 (TLR4) agonist), peptidoglycan (PGN; a TLR2 agonist), Pam3CSK4 (a synthetic bacterial lipoprotein that triggers TLR1 and TLR2), and a combination of LPS and R848 (imidazoquinoline resiquimod, a TLR8 agonist). We obtained optimal production of IL-1β and IL-6 by monocytes in most cases even in the absence of CD40L stimulation and, notably, this was not associated with IL-12 production. Circulating cDCs were also able to produce IL-1β and IL-6 and no IL-12, but did so only in response to some stimuli (PGN and Pam3CSK4); in other cases, they either failed to produce IL-1β or concomitantly produced IL-12 and IL-23. In contrast, DCs derived from the culture of monocytes in the presence of granulocyte-macrophage colony-stimulating factor plus IL-4, TGF-β or IFN-α were either unable to release IL-1β or produced small amounts of IL-1β in response to three synergizing stimuli (LPS, R848 and CD40L). In addition, these cells had substantial production of IL-12 and IL-23 (Fig. 4 and Supplementary Fig. 2 online).

Figure 4: APCs vary in their ability to secrete polarizing cytokines after activation.
figure4

Cytometric bead assay of IL-1β and IL-6 (left) and ELISA of IL-12p70 and IL-23 (right) in peripheral CD14+ monocytes, circulating CD1c+ cDCs, and DCs derived from the culture of monocytes with granulocyte-macrophage colony-stimulating factor and IL-4 (IL-4–DCs), activated for 24 h with LPS, PGN, Pam3CSK4 (PamCSK), or LPS plus R848 in the presence (black bars) or absence (gray bars) of CD40L. Data are the mean + s.d. of triplicates and are representative of six separate experiments.

We next compared the ability of various APCs and microbial stimuli to prime TH-17 responses. We did these experiments in the absence of IL-4- and IFN-γ-neutralizing antibodies and exogenous cytokines. We labeled naive CD4+ T cells with CFSE (carboxyfluorescein diacetate succinimidyl diester) and stimulated them for 5 d with allogeneic monocytes, cDCs or monocyte-derived DCs stimulated with LPS or PGN, in the absence or presence of CD3-crosslinking antibodies. We measured T cell proliferation on days 5 and 12 after incubation in IL-2 and measured the production of IL-17, IFN-γ and IL-4 on day 12 (Fig. 5 and Supplementary Fig. 3 online). Both monocytes and DCs induced T cell proliferation, although on a per-cell basis, monocytes and cDCs were less efficient than monocyte-derived DCs and nonactivated APCs were much less efficient than APCs activated by microbial products (data not shown and Supplementary Fig. 3). Monocytes activated by LPS and especially those activated by PGN were the APCs most efficient in triggering the differentiation of TH-17 cells (Fig. 5a,b). Notably, the proportion of cells producing IL-17 but not those producing IFN-γ was higher after priming of naive T cells by APCs than after priming by anti-CD3 and anti-CD28 (Fig. 1a). Circulating cDCs were also capable of inducing TH-17 differentiation, especially when stimulated with PGN (Fig. 5a,b). In contrast, monocyte-derived DCs failed to induce TH-17 differentiation but elicited varying degrees of TH1 differentiation. When T cells were primed by alloantigens (in the absence of anti-CD3), only monocytes and cDCs, not DCs derived from the culture of monocytes in the presence of IL-4, primed IL-17-producing T cells, as assessed by intracellular cytokine staining and ELISA (Fig. 5c,d). The extent of TH-17 differentiation in these cultures was lower than that in cultures stimulated by monocytes coated with anti-CD3 (Fig. 5a,b). These results collectively delineate a combination of APCs and microbial stimuli optimal for the induction of human TH-17 responses in vitro. In particular, among the APCs and stimuli analyzed, monocytes and PGN seemed to be most effective for the induction of human TH-17 cells.

Figure 5: Circulating CD14+ monocytes and CD1c+ cDCs stimulated with PGN or LPS efficiently trigger the differentiation of human IL-17-secreting T cells.
figure5

(a) Intracellular cytokine staining of IL-17 and IFN-γ in naive CD4+ T cells primed for 5 d with allogeneic APCs activated with LPS or PGN in the presence of anti-CD3, then incubated for 7 d in IL-2 and stimulated for 5 h with PMA and ionomycin. (b) Intracellular cytokine staining to determine the percentage of cells secreting IL-17 and IFN-γ among cells from various donors, treated as described in a. Each symbol represents a single donor; small horizontal bars indicate the mean. *, P < 0.05 (t-test with Welch's correction). (c) Intracellular cytokine staining of IL-17 and IFN-γ in naive CD4+ T cells primed by allogeneic APCs in the absence of anti-CD3, incubated for 7 d in IL-2 and then stimulated for 5 h with PMA and ionomycin. (d) ELISA of IL-17 and IFN-γ in 36-hour culture supernatants of cells treated as described in c and then stimulated with anti-CD3 and PdBu. Numbers in quadrants (a,c) indicate percent cells in each (as described in Fig. 1). IL-4–DCs, TGF-β–DCs and IFN-α–DCs, DCs derived from monocytes cultured in the presence of granulocyte macrophage-colony stimulating factor plus IL-4, TGF-β or IFN-α, respectively. Data are representative of ten (a,b) or six (c,d) separate experiments (d, mean + s.d. of duplicate cultures).

IL-1β and IL-6 in TH-17 priming by monocytes

To further document the function of IL-1β and IL-6 in the ability of LPS- or PGN-activated monocytes to induce TH-17 responses, we evaluated the effects of antibodies neutralizing IL-1β, IL-18 or the IL-6 receptor (IL-6R), and of YVAD, a cell-permeable caspase-1 inhibitor that blocks the release of IL-1β and IL-18 (ref. 26). Treatment with antibodies neutralizing IL-1β or both IL-1β and IL-6R completely abolished the TH-17 differentiation induced by LPS-activated monocytes, whereas neutralization of IL-18 did not affect TH-17 development (Fig. 6a). The addition of YVAD to cultures of monocytes stimulated with LPS or PGN nearly completely abolished the release of IL-1β (Supplementary Fig. 4 online). Consistent with that finding, treatment with YVAD abolished the ability of monocytes to induce TH-17 differentiation, which was partially reconstituted by the addition of exogenous IL-1β but not by the addition of IL-18 (Fig. 6b). Antibody-mediated neutralization of IL-12, IL-23 or TGF-β did not affect TH-17 differentiation induced by monocytes stimulated with LPS or PGN (Fig. 6c). These results collectively demonstrate that the ability of monocytes to induce TH-17 differentiation is critically dependent on their ability to produce IL-1β but occurs independently of IL-18, IL-23 or TGF-β.

Figure 6: Monocyte-derived IL-1β and IL-6 are required for effective TH-17 polarization.
figure6

Flow cytometry of the production of IL-17 and IFN-γ by naive CD4+ T cells primed for 5 d with allogeneic monocytes activated with LPS or PGN, in the presence of anti-CD3 plus antibodies neutralizing IL-1β, IL-6R, both IL-1β and IL-6R, or IL-18 (a), the caspase-1 inhibitor YVAD alone or in combination with IL-1β or IL-18 (b), or antibodies neutralizing IL-12p35, IL-23 or TGF-β (c), then incubated for 7 d in IL-2 and stimulated for 5 h with PMA and ionomycin. Numbers in quadrants indicate percent cells in each (as described in Fig. 1). Ab, antibody. Results are representative of six (a,c) or four (b) separate experiments.

Discussion

Using two complementary experimental approaches, we have shown here that the differentiation of human naive CD4+ T cells into TH-17 cells is promoted by IL-1β and IL-6. In particular, IL-1β was sufficient to induce the expression of RORγt and production of both IL-17 and IFN-γ. IL-6, when added to IL-1β, sustained the expression of RORγt and promoted the differentiation of T cells producing IL-17 but not IFN-γ. The addition of TGF-β, which in the mouse has been identified as a cytokine essential for the development of TH-17 cells14,15,16, did not induce and actually suppressed the TH-17 differentiation of human CD4+ T cells. Thus, in humans, TGF-β seems to inhibit the three main pathways (TH1, TH2 and TH-17) of effector T cell differentiation. The fact that T cells producing only IL-17 were more resistant to TGF-β-mediated inhibition than were T cells producing only IFN-γ or both IL-17 and IFN-γ would be consistent with variable sensitivity of TH1 and TH-17 differentiation to the inhibitory effect of TGF-β. In addition, whereas in mice IL-6 is required for TH-17 differentiation and IL-1β exerts an enhancing effect, in humans it seems that IL-1β has a chief function and IL-6 enhances IL-1β-induced TH-17 differentiation.

The differential requirements for TH-17 differentiation in mice and humans are unexpected, given that no differences have been reported in mice versus humans for the signaling pathways triggered by TGF-β, IL-1β and IL-6. However, in some cases analogous cytokine receptors have been shown to couple different signaling pathways in mice and humans27. A possible explanation for the divergent requirements, at least in vitro, for the differentiation of human versus mouse TH-17 cells may be related to the source of naive T cells or to their developmental stage. Another possibility is that the relative contributions of TGF-β, IL-1β and IL-6 depend on the stimulatory conditions, both in vitro and in vivo. For example, involvement of IL-1 in the generation of IL-17-producing cells has been documented in mouse models of arthritis, encephalomyelitis and vaccination against bacterial infections28,29,30.

IL-2 and IL-23 seem to modulate the differentiation of human and mouse TH-17 cells in a similar way14,20. Indeed, the differentiation of human TH-17 cells was inhibited by large doses of IL-2 and was enhanced by IL-23. Notably, it has been reported that APCs activated by the hyphal form of Candida albicans or by the dectin-1 ligand curdlan selectively trigger the production of IL-23 but not IL-12 and induce TH-17 differentiation in vitro and in vivo23,31.

Another unexpected finding here was that TH-17 differentiation was efficiently elicited by activated monocytes and to a lesser extent by circulating cDCs but not by monocyte-derived DCs, which are the APCs most potent in triggering TH1 responses. This difference in T cell–polarizing capacity correlated with the pattern of cytokines produced by the various APCs when activated by microbial stimuli and T cell help. Indeed, when activated by LPS or PGN, monocytes produced IL-1β and IL-6 but no IL-12, even when costimulated by CD40L. In contrast, different types of monocyte-derived DCs produced little IL-1β and large amounts of IL-12, which has a strong inhibitory effect on TH-17 differentiation. Monocytes are increasingly appreciated as a heterogeneous pool of DC precursors that can be mobilized in immune responses to incoming pathogens and can rapidly differentiate into DCs in response to microbial products or after transmigrating across endothelial cells32,33,34,35,36. It remains to be established which APCs and maturation stimuli other than fungal products induce TH-17 responses in vivo in physiological and pathological conditions. Our results also indicate that the various types of monocyte-derived DCs now in use for therapeutic vaccination against cancer may not be suitable for the induction of TH-17 responses.

This realization of the central function of IL-1β in human TH-17 polarization has obvious therapeutic implications. Microbial and endogenous stimuli that trigger the synthesis and release of IL-1β are well known and could be used as adjuvants to promote TH-17 responses, with the possibility of eliciting strong tissue-specific inflammatory responses whenever this may be desirable. IL-1 and IL-6 have been identified in the pathogenesis of several autoimmune and chronic inflammatory diseases as effector cytokines that mediate damage in peripheral tissues37,38. The finding that IL-1β and IL-6 are also critical inducers of TH-17 responses provides an additional rationale for targeting these cytokines in autoimmune disease to reduce not only inflammation but also the priming of inflammatory TH-17 cells39. Thus, neutralization of IL-1β with specific antibodies or IL-1 receptor antagonists, which are now used to block the systemic effects of IL-1β40, may exert a beneficial effect by inhibiting TH-17 responses as well. Finally, the finding that in humans, TGF-β inhibits TH-17 as well as TH1 and TH2 differentiation suggests particular caution should be taken in the use of TGF-β-blocking antibodies in autoimmune diseases, as they may unleash powerful pathogenic T cells.

Methods

Cell purification and sorting.

Blood samples for transfusion were obtained from the Basel Swiss Blood Center. Permission for experiments with human primary cells was obtained from the Federal Office of Public Health (A000197/2 to F.S.). Peripheral blood CD14+ monocytes and CD4+ T cells were isolated by positive selection with CD14- and CD4-specific microbeads, respectively (Miltenyi Biotec). Circulating CD1c+ myeloid DCs were isolated by positive selection with specific fluorescein isothiocyanate–labeled anti-CD1c (AD5-8E7; Miltenyi Biotec) and microbeads (Miltenyi Biotec), followed by cell sorting for exclusion of contaminating monocytes and B cells. DCs were obtained from monocytes after culture with granulocyte-macrophage colony-stimulating factor and IL-4, TGF-β or IFN-α as reported before41,42,43. Naive T cells were isolated to over 99% purity as CD45RA+CCR7+CD25 after being stained with fluorescein isothiocyanate–conjugated anti-CD25 (B1.49.9; Immunotech), phycoerythrin-conjugated anti-CD45RA (ALB11; Immunotech) and anti-CCR7 (150503; R&D Systems), followed staining with biotinylated anti–immunoglobulin G2a and streptavidin–Pacific blue (Molecular Probes; Invitrogen) and cell sorting with a FACSAria (BD Bioscience). Where indicated, primed cells were sorted by CCR6 expression with phycoerythrin-conjugated anti-CCR6 (11A9; BD Biosciences).

Cell culture.

Cells were cultured in RPMI 1640 medium supplemented with 2 mM glutamine, 1% (vol/vol) nonessential amino acids, 1% (vol/vol) sodium pyruvate, kanamycin (50 μg/ml), penicillin (50 U/ml) and streptomycin (50 μg/ml) and containing 10% (vol/vol) FCS (Gibco BRL). Naive T cells were primed with beads coated with anti-CD3 and anti-CD28 (Miltenyi Biotec) according to the manufacturer's instructions. Naive CD4 cells (5 × 104) were cultured with beads (2.5 × 104) in U-bottomed 96-well plates. After 3 d of priming, cells were gently pipetted to break up clumps and were supplemented with recombinant IL-2 (20 IU/ml; or with IL-2 and IL-23 (20 ng/ml) for Fig. 2b). Where indicated, TGF-β (5 ng/ml), IL-1β (10 ng/ml), IL-12 (10 ng/ml; R&D Systems), IL-6 (50 ng/ml; BD Biosciences) or neutralizing anti-IL-4 (10 μg/ml; 34019.111; R&D Systems) and anti-IFN-γ (10 μg/ml; B27; BD Biosciences) were added to the cultures. Alternatively, T cells were primed with various APCs activated with Escherichia coli (100 ng/ml; 0111:B4 LPS Ultra-Pure), PGN from Staphylococcus. aureus (5 μg/ml), Pam3CSK4 (1 μg/ml; Invivogen) or LPS plus R848 (2.5 μg/ml; GLSynthesis) in the presence of the CD3-specific antibody OKT3 (produced 'in-house' from a B cell hybridoma), except when alloantigen-specific T cell responses were evaluated. Neutralizing anti-IL-4 and anti-IFN-γ were omitted in the T cell–priming experiments with APCs, and in some, neutralizing antibodies binding the following were used: IL-1β (8516.311), IL-6R (17506), IL-23 (253810), TGF-β (9016), IL-18 (52713.11) and IL-12p35 (29910.1; all at a concentration of 10 μg/ml; all from R&D Systems). For caspase-1 inhibition, 50 μM YVAD (Alexis Biochemicals) was added to the cultures 30 min before stimulation with LPS or PGN. Naive CD4+ T cells were labeled with CFSE according to standard protocols. After 5 d of priming, primed cells were washed, beads or APCs were removed by magnetic depletion or transfer to new plates, and primed cells were incubated with recombinant IL-2 (20 IU/ml; or with IL-2 and IL-23 (20 ng/ml) for Fig. 2b).

ELISA and intracellular cytokine staining.

The cytokine-producing capacity of primed T cells was assessed by stimulation of cells (1 × 106 per ml) for 36 h with soluble anti-CD3 (1 μg/ml) and phorbol-12-13-dibutyrate (PdBu; 50 nM). Cytokine secretion by APCs was assessed after stimulation of cells (0.5 × 106 per ml) for 24 h with various stimuli. Cytokines in culture supernatants were measured by ELISA according to a standard protocol and data were analyzed with the Softmax program or by cytometric bead assay (BD Bioscience) according to the manufacturer's instructions. Intracellular staining for IFN-γ, IL-17, IL-4, IL-22 and tumor necrosis factor was done on T cells stimulated for 5 h with PMA and ionomycin in the presence of GolgiStop (BD Bioscience) for the final 3 h of culture. Cells were fixed and made permeable with BD Cytofix/Cytoperm Plus (BD Bioscience) according to the manufacturer's instructions. Cells were incubated with fluorescein isothiocyanate–labeled anti-IFN-γ (B27; BD Biosciences), phycoerythrin-labeled anti-IL-4 (8D4-8; BD Biosciences), phycoerythrin-labeled anti-IL-22 (142928; R&D Systems) and allophycocyanin-labeled anti-IL-17 (eBIO64DEC17; eBioscience), then cells were washed and data were acquired on a FACSCanto or FACSCalibur (BD Biosciences) and analyzed with FlowJo software (Tree Star).

Real-time quantitative RT-PCR.

Total RNA was extracted with the ABI PRISM 6100 Nucleic Acid PrepStation (Perkin-Elmer Applied Biosystems) according to the manufacturer's instructions. Random hexamers and an MMLV Reverse Transcriptase kit (Stratagene) were used for cDNA synthesis. Transcripts were quantified by real-time quantitative PCR on an ABI PRISM 7700 Sequence Detector (Perkin-Elmer Applied Biosystems) with Applied Biosystems predesigned TaqMan Gene Expression Assays and reagents according to the manufacturer's instructions. The following probes were used (identified by Applied Biosystems assay identification number): TBX21, Hs00203436_m1; IL17A, Hs99999082_m1; IFNG, Hs99999041_m1. Because a probe specific for RORC variant 2 was not available and the RORC variant 2 (Applied Biosystems assay identification number Hs00172858_m1) was not detected in any of the condition tested, RORC variant 2 was assessed with a probe that recognizes both forms of RORC (Applied Biosystems assay identification number Hs01076112_m1 probe). For each sample, mRNA abundance was normalized to the amount of 18S rRNA and is expressed as arbitrary units.

Statistics.

A standard two-tailed t-test or a t-test with Welch's correction was used for statistical analysis; P values of 0.05 or less were considered significant.

Note: Supplementary information is available on the Nature Immunology website.

References

  1. 1

    Park, H. et al. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat. Immunol. 6, 1133–1141 (2005).

    CAS  Article  Google Scholar 

  2. 2

    Harrington, L.E. et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat. Immunol. 6, 1123–1132 (2005).

    CAS  Article  Google Scholar 

  3. 3

    Weaver, C.T., Hatton, R.D., Mangan, P.R. & Harrington, L.E. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu. Rev. Immunol. 25, 821–852 (2007).

    CAS  Article  Google Scholar 

  4. 4

    Murphy, C.A. et al. Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. J. Exp. Med. 198, 1951–1957 (2003).

    CAS  Article  Google Scholar 

  5. 5

    Yen, D. et al. IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. J. Clin. Invest. 116, 1310–1316 (2006).

    CAS  Article  Google Scholar 

  6. 6

    Zheng, Y. et al. Interleukin-22, a TH17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis. Nature 445, 648–651 (2007).

    CAS  Article  Google Scholar 

  7. 7

    Ye, P. et al. Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense. J. Exp. Med. 194, 519–527 (2001).

    CAS  Article  Google Scholar 

  8. 8

    Zhang, Z., Zheng, M., Bindas, J., Schwarzenberger, P. & Kolls, J.K. Critical role of IL-17 receptor signaling in acute TNBS-induced colitis. Inflamm. Bowel Dis. 12, 382–388 (2006).

    Article  Google Scholar 

  9. 9

    Chabaud, M. et al. Human interleukin-17: A T cell-derived proinflammatory cytokine produced by the rheumatoid synovium. Arthritis Rheum. 42, 963–970 (1999).

    CAS  Article  Google Scholar 

  10. 10

    Lock, C. et al. Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis. Nat. Med. 8, 500–508 (2002).

    CAS  Article  Google Scholar 

  11. 11

    Fujino, S. et al. Increased expression of interleukin 17 in inflammatory bowel disease. Gut 52, 65–70 (2003).

    CAS  Article  Google Scholar 

  12. 12

    Aggarwal, S., Ghilardi, N., Xie, M.H., de Sauvage, F.J. & Gurney, A.L. Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17. J. Biol. Chem. 278, 1910–1914 (2003).

    CAS  Article  Google Scholar 

  13. 13

    Veldhoen, M., Hocking, R.J., Flavell, R.A. & Stockinger, B. Signals mediated by transforming growth factor-β initiate autoimmune encephalomyelitis, but chronic inflammation is needed to sustain disease. Nat. Immunol. 7, 1151–1156 (2006).

    CAS  Article  Google Scholar 

  14. 14

    Veldhoen, M., Hocking, R.J., Atkins, C.J., Locksley, R.M. & Stockinger, B. TGFβ in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 24, 179–189 (2006).

    CAS  Article  Google Scholar 

  15. 15

    Mangan, P.R. et al. Transforming growth factor-β induces development of the TH17 lineage. Nature 441, 231–234 (2006).

    CAS  Article  Google Scholar 

  16. 16

    Bettelli, E. et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441, 235–238 (2006).

    CAS  Article  Google Scholar 

  17. 17

    Cho, M.L. et al. STAT3 and NF-κB signal pathway is required for IL-23-mediated IL-17 production in spontaneous arthritis animal model IL-1 receptor antagonist-deficient mice. J. Immunol. 176, 5652–5661 (2006).

    CAS  Article  Google Scholar 

  18. 18

    Hoeve, M.A. et al. Divergent effects of IL-12 and IL-23 on the production of IL-17 by human T cells. Eur. J. Immunol. 36, 661–670 (2006).

    CAS  Article  Google Scholar 

  19. 19

    Batten, M. et al. Interleukin 27 limits autoimmune encephalomyelitis by suppressing the development of interleukin 17-producing T cells. Nat. Immunol. 7, 929–936 (2006).

    CAS  Article  Google Scholar 

  20. 20

    Laurence, A. et al. Interleukin-2 signaling via STAT5 constrains T helper 17 cell generation. Immunity 26, 371–381 (2007).

    CAS  Article  Google Scholar 

  21. 21

    Ivanov, I.I. et al. The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126, 1121–1133 (2006).

    CAS  Article  Google Scholar 

  22. 22

    Yang, X.O. et al. STAT3 regulates cytokine-mediated generation of inflammatory helper T cells. J. Biol. Chem. 282, 9358–9363 (2007).

    CAS  Article  Google Scholar 

  23. 23

    Acosta-Rodriguez, E.V. et al. Surface phenotype and antigenic specificity of human interleukin 17–producing T helper memory cells. Nat. Immunol. 8, 639–646 (2007).

    CAS  Article  Google Scholar 

  24. 24

    Mathur, A.N. et al. T-bet is a critical determinant in the instability of the IL-17-secreting T-helper phenotype. Blood 108, 1595–1601 (2006).

    CAS  Article  Google Scholar 

  25. 25

    Reis e Sousa, C. Activation of dendritic cells: translating innate into adaptive immunity. Curr. Opin. Immunol. 16, 21–25 (2004).

    CAS  Article  Google Scholar 

  26. 26

    Thornberry, N.A. et al. Inactivation of interleukin-1β converting enzyme by peptide (acyloxy)methyl ketones. Biochemistry 33, 3934–3940 (1994).

    CAS  Article  Google Scholar 

  27. 27

    Yang, J., Zhu, H., Murphy, T.L., Ouyang, W. & Murphy, K.M. IL-18-stimulated GADD45β required in cytokine-induced, but not TCR-induced, IFN-γ production. Nat. Immunol. 2, 157–164 (2001).

    CAS  Article  Google Scholar 

  28. 28

    Nakae, S. et al. IL-17 production from activated T cells is required for the spontaneous development of destructive arthritis in mice deficient in IL-1 receptor antagonist. Proc. Natl. Acad. Sci. USA 100, 5986–5990 (2003).

    CAS  Article  Google Scholar 

  29. 29

    Sutton, C., Brereton, C., Keogh, B., Mills, K.H. & Lavelle, E.C. A crucial role for interleukin (IL)-1 in the induction of IL-17-producing T cells that mediate autoimmune encephalomyelitis. J. Exp. Med. 203, 1685–1691 (2006).

    CAS  Article  Google Scholar 

  30. 30

    Higgins, S.C., Jarnicki, A.G., Lavelle, E.C. & Mills, K.H. TLR4 mediates vaccine-induced protective cellular immunity to Bordetella pertussis: role of IL-17-producing T cells. J. Immunol. 177, 7980–7989 (2006).

    CAS  Article  Google Scholar 

  31. 31

    Leibundgut-Landmann, S. et al. Syk- and CARD9-dependent coupling of innate immunity to the induction of T helper cells that produce interleukin 17. Nat. Immunol. 8, 630–638 (2007).

    CAS  Article  Google Scholar 

  32. 32

    Krutzik, S.R. et al. TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells. Nat. Med. 11, 653–660 (2005).

    CAS  Article  Google Scholar 

  33. 33

    Randolph, G.J., Inaba, K., Robbiani, D.F., Steinman, R.M. & Muller, W.A. Differentiation of phagocytic monocytes into lymph node dendritic cells in vivo. Immunity 11, 753–761 (1999).

    CAS  Article  Google Scholar 

  34. 34

    Ginhoux, F. et al. Langerhans cells arise from monocytes in vivo. Nat. Immunol. 7, 265–273 (2006).

    CAS  Article  Google Scholar 

  35. 35

    Palframan, R.T. et al. Inflammatory chemokine transport and presentation in HEV: a remote control mechanism for monocyte recruitment to lymph nodes in inflamed tissues. J. Exp. Med. 194, 1361–1373 (2001).

    CAS  Article  Google Scholar 

  36. 36

    Janatpour, M.J., Hudak, S., Sathe, M., Sedgwick, J.D. & McEvoy, L.M. Tumor necrosis factor-dependent segmental control of MIG expression by high endothelial venules in inflamed lymph nodes regulates monocyte recruitment. J. Exp. Med. 194, 1375–1384 (2001).

    CAS  Article  Google Scholar 

  37. 37

    Chabaud, M., Fossiez, F., Taupin, J.L. & Miossec, P. Enhancing effect of IL-17 on IL-1-induced IL-6 and leukemia inhibitory factor production by rheumatoid arthritis synoviocytes and its regulation by Th2 cytokines. J. Immunol. 161, 409–414 (1998).

    CAS  PubMed  Google Scholar 

  38. 38

    Koenders, M.I. et al. Interleukin-17 receptor deficiency results in impaired synovial expression of interleukin-1 and matrix metalloproteinases 3, 9, and 13 and prevents cartilage destruction during chronic reactivated streptococcal cell wall-induced arthritis. Arthritis Rheum. 52, 3239–3247 (2005).

    CAS  Article  Google Scholar 

  39. 39

    McInnes, I.B. & Liew, F.Y. Cytokine networks–towards new therapies for rheumatoid arthritis. Nat. Clin. Pract. Rheumatol. 1, 31–39 (2005).

    CAS  Article  Google Scholar 

  40. 40

    Martinon, F. & Tschopp, J. Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases. Cell 117, 561–574 (2004).

    CAS  Article  Google Scholar 

  41. 41

    Sallusto, F. & Lanzavecchia, A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J. Exp. Med. 179, 1109–1118 (1994).

    CAS  Article  Google Scholar 

  42. 42

    Geissmann, F. et al. Transforming growth factor β1, in the presence of granulocyte/macrophage colony-stimulating factor and interleukin 4, induces differentiation of human peripheral blood monocytes into dendritic Langerhans cells. J. Exp. Med. 187, 961–966 (1998).

    CAS  Article  Google Scholar 

  43. 43

    Santini, S.M. et al. Type I interferon as a powerful adjuvant for monocyte-derived dendritic cell development and activity in vitro and in Hu-PBL-SCID mice. J. Exp. Med. 191, 1777–1788 (2000).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank D. Jarrossay for cell sorting and discussions. Supported by the Swiss National Science Foundation (31-101962 to F.S.), the US National Institutes of Health (U19 AI057266-01) and the Helmut Horten Foundation (for The Institute for Research in Biomedicine).

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Eva V Acosta-Rodriguez or Federica Sallusto.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 145 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Acosta-Rodriguez, E., Napolitani, G., Lanzavecchia, A. et al. Interleukins 1β and 6 but not transforming growth factor-β are essential for the differentiation of interleukin 17–producing human T helper cells. Nat Immunol 8, 942–949 (2007). https://doi.org/10.1038/ni1496

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing