Research ArticleT CELL DIFFERENTIATION

TH17 cells require ongoing classic IL-6 receptor signaling to retain transcriptional and functional identity

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Science Immunology  17 Jul 2020:
Vol. 5, Issue 49, eaaw2262
DOI: 10.1126/sciimmunol.aaw2262

TH17 sustenance via IL-6

The pathogenic role of the TH17 subset of CD4+ T cells in multiple immune-mediated diseases has prompted close scrutiny of the cytokine signals that promote differentiation and maintenance of TH17 cells. IL-6 and TGF-β are key cytokines for TH17 commitment by naïve T cells whereas IL-23 supports maintenance of a TH17 identity. Harbour et al. investigated whether persistent IL-6 signaling is also needed to sustain TH17 cell functions. IL-6Rα–deficient mouse T cells could not maintain a TH17 phenotype and were attenuated in their ability to elicit colitis in an in vivo cell transfer model. These studies provide deeper insights into the set of signals required for TH17 cell maintenance and suggest additional molecular targets for pharmacological interventions aimed at antagonizing pathogenic TH17 immunity.

Abstract

Acting in concert with TGF-β, interleukin-6 (IL-6) signaling induces T helper 17 (TH17) cell development by programming TH17-related genes via signal transducers and activators of transcription 3 (STAT3). A role for IL-6 signaling beyond the inductive phase of TH17 cell development has not been defined because IL-23 signaling downstream of TH17 cell induction also activates STAT3 and is thought responsible for TH17 cell maintenance. Here, we find that IL-6 signaling is required for both induction and maintenance of mouse TH17 cells; IL-6Rα–deficient TH17 cells rapidly lost their TH17 phenotype and did not cause disease in two models of colitis. Cotransfer of wild-type TH17 cells with IL-6Rα–deficient TH17 cells induced colitis but was unable to rescue phenotype loss of the latter. High IL-6 expression in the colon promoted classic, or cis, rather than transreceptor signaling that was required for maintenance of TH17 cells. Thus, ongoing classic IL-6 signaling underpins the TH17 program and is required for TH17 cell maintenance and function.

INTRODUCTION

Distinct subsets of effector CD4+ T cells differentiate from multipotent naïve precursors under control of antigen- and cytokine-induced signals. T helper 17 (TH17) cells arise from antigen-activated naïve CD4+ T cells in response to transforming growth factor–β (TGF-β) and interleukin-6 (IL-6) (14), the latter inducing a signaling cascade that recruits Janus kinases JAK1, JAK2, and TYK2. These, in turn, induce tyrosine phosphorylation of both signal transducers and activators of transcription 3 (STAT3) and STAT1 (5). STAT3 induces the expression of TH17-related genes including Il17a, Il17f, Il22, Il23r, and the master transcription factor Rorc (68), which itself is a positive regulator of key TH17 genes and a negative regulator of alternative lineage fates (912). Expression of Il23r and other TH17-specific genes is required for several TH17-mediated diseases, including inflammatory bowel disease (IBD) (1315), psoriasis (16), and ankylosing spondylitis (17), and exposure of TH17 cells to IL-23 is critical for TH17 maintenance and pathogenesis (15, 1822).

TH17 cells are highly plastic (19, 23). Under the influence of IL-12, most TH17 cells that rapidly extinguish retinoic acid receptor–related orphan receptor γt (RORγt) expression up-regulate T-bet and a TH1-like program (19, 24, 25). Although IL-23 has been shown to be important for TH17 cell maintenance due to its activation of STAT3, reiterative IL-23 signaling also induces TH17 cells to extinguish Il17a and Rorc in favor of TH1-related genes (19, 24, 25). This transdifferentiation of TH17 cells is dependent on both STAT4 and T-bet (19, 23, 26, 27). Thus, the balance of cytokines in the local environment plays a critical role in TH17 maintenance and function. Although the role of IL-6 in TH17 cell induction is well characterized (2, 4), it is unknown whether ongoing IL-6 signaling contributes to TH17 maintenance or whether there are additional factors that antagonize lineage-destabilizing signals such as IL-12– and IL-23–induced STAT4 to preserve the TH17 phenotype.

IL-6 is produced by a wide range of cells, including hematopoietic-derived monocytes, macrophages, and dendritic cells, and nonhematopoietic stromal cells such as fibroblasts and endothelial cells (28). Various inflammatory cytokines [e.g., IL-1 and tumor necrosis factor–α (TNFα)] and innate sensing mechanisms contribute to the generation of IL-6, which elicits a broad range of biological functions on target cells (29). Effects range from control of organ development, the regulation of the acute-phase response, and immune-related functions such as cell survival, differentiation, proliferation, and apoptosis (30). The pleiotropic effects of IL-6 are transmitted through a tightly controlled variable mode of signaling (31). Classic, or cis, IL-6 receptor signaling occurs in cells that express both membrane-bound IL-6Rα and the signal transducing component, gp130, which bind IL-6 in a hexameric complex where IL-6, IL-6Rα, and gp130 exist in a 2:2:2 stoichiometry (32). Whereas gp130 is ubiquitously expressed (33), IL-6Rα expression is restricted to hepatocytes (34) and subsets of leukocytes, including naïve T cells (35). In activated cells that express IL-6Rα, particularly activated neutrophils, monocytes, and T cells (36), the extracellular domain can be shed by proteolysis to liberate a functional soluble IL-6Rα (sIL-6Rα) (37, 38). sIL-6Rα binds IL-6 to form an agonistic dimer that can bind gp130 to induce IL-6 signaling through a mechanism termed IL-6 trans-signaling. A soluble isoform of gp130 (sgp130) buffers the activity of IL-6 trans-signaling and restricts the bioavailability of IL-6–sIL-6Rα complexes for signaling through membrane-bound gp130 (39). Given that gp130 is ubiquitously expressed, the local balance between IL-6–, sIL-6Rα–, and sgp130 IL-6–responsive target cells thus determines the prominent mode of IL-6 signaling acting on any cell type (40). An additional form of IL-6 signaling, referred to as transpresentation, involves the binding of the cell surface–associated IL-6–sIL-6Rα complexes on a subset of dendritic cells to gp130 expressed on naïve T cells and has been proposed as a mechanism that promotes the development of pathogenic TH17 cells (41).

An excess of IL-6 relative to sIL-6Rα in acute injury leads to classic signaling, which is believed to have more homeostatic effects such as the acute phase response (42). An excess of sIL-6Rα, however, is a marker of active inflammation (43) and allows for trans-signaling to a wide variety of cell targets and amplifies inflammatory responses through the recruitment and activation of mononuclear cells (44, 45). IL-6 trans-signaling has been shown to affect the recruitment of TH17 cells in model of peritoneal inflammation (46), although it is unknown whether these effects were direct or indirect.

Here, we have used a mouse model of TH17 cell–induced colitis (19) to examine a possible contribution of IL-6 to TH17 biology beyond the early phase of TH17 induction. We find that expression of IL-6Rα by mature TH17 cells is indispensable for maintenance of the TH17 phenotype and pathogenic response. Classic IL-6 receptor signaling was dominant over IL-6 trans-signaling in the colon because of the high levels of IL-6 produced there. Our findings establish that ongoing IL-6 is required for maintenance of TH17 cells and suggest that a major contributor to TH17 plasticity is the loss of IL-6 signaling.

RESULTS

Naïve CD4+ T cells require IL-6 signaling for development of colitis

The TH17 pathway is critical for IBD pathogenesis. In view of the requirement for IL-6 in TH17 cell development, we examined whether naïve CD4+ T cells deficient in IL-6Rα were able to develop into TH17 cells and drive intestinal inflammation in vivo. Wild-type (WT) and Il6ra−/− naïve T cells were used as donors in the CD4+ CD45RBHi T cell transfer colitis model (47). Eight weeks after transfer into Rag1−/− recipients, T cells from WT mice were found to induce substantial disease. This was reflected by a significant loss of weight (Fig. 1A) and an increase in CD4+ cells recovered from the colonic lamina propria (CLP) of colitic mice (Fig. 1B). Although the transfer of WT CD4+ cells resulted in pronounced pathology (Fig. 1, C and D), the transfer of Il6ra−/− CD4+ cells failed to induce weight loss or significant colonic inflammation.

Fig. 1 Naïve CD4+ T cells require IL-6 for development and colitis pathogenesis.

FACS-sorted CD4+ CD45RBHi T cells isolated from WT and Il6ra−/− mice were transferred into Rag1−/− recipients (4 × 105 per mouse) (n = 5 per group) and analyzed for colitis 8 weeks after transfer. (A) Weight loss expressed as a percentage of starting weight (means ± SEM). *P < 0.05 and **P < 0.01. (B) Total numbers of CD4+ T cells recovered from the CLP of colitic mice (means + SEM). **P = 0.0044. (C) Histological scores (means + SEM). **P = 0.005 (D) Representative hematoxylin and eosin (H&E)–stained sections of colon from recipients of WT (top) or Il6ra−/− CD4+ T cells. H&E stain; original magnification, ×10. Scale bars, 100 μm. (E) Representative FACS plots showing Foxp3 expression in CLP CD4+ T cells ex vivo from WT or Il6ra−/− recipients after development of colitis. (F) Relative frequencies (top) and total numbers (bottom) of Foxp3+ CD4+ T cells from the CLP (means ± SEM). P = 0.1724 (top) and P = 0.054 (bottom). (G) Representative FACS plots showing IL-17A and IFN-γ in CLP CD4+ T cells ex vivo after development of colitis. (H) Relative frequencies (top) and total numbers (bottom) of IL-17A+, IL-17A+IFN-γ +, and IFN-γ + CD4+ T cells in the CLP of colitic mice (means ± SEM). *P = 0.0268, *P = 0.0121, and P = 0.0803 [(top) IL-17A+, IL-17A+IFN-γ +, and IFN-γ + CD4+ T cells]. **P = 0.0023, ***P = 0.0008, and *P = 0.0191 [(bottom) IL-17A+, IL-17A+IFN-γ +, and IFN-γ + CD4+ T cells]. Data are representative of at least three independent experiments (A and D to H; n = 5 per group) or pooled from two independent experiments (B and C; n = 10 per group). P values, two-way ANOVA with Bonferroni posttest (A) or unpaired two-tailed Student’s t test (B, C, F, and H).

Consistent with previous reports, flow cytometric analysis of T cells recovered from the inflamed CLP and mesenteric lymph node (MLN) identified four populations of IL-17A– and interferon-γ (IFN-γ)–expressing cells in WT recipients (Fig. 1G and fig. S1) (19). In contrast, there was a significant reduction in the percentage and number of IL-17A+ and IL-17A+IFN-γ+ cells in the CLP (Fig. 1, G and H) and MLN (fig. S1) of the recipients of Il6ra−/− T cells. The relative percentage of IFN-γ–producing cells in Il6ra−/− recipient T cells was unchanged in the CLP (Fig. 1H) and increased in the MLN (fig. S1), but their numbers were not increased. Thus, IL-6 receptor signaling in these cells is not required for development of an intact TH1 pathway but is essential for control of pathogenic T cells displaying a TH17 program (15, 48). The numbers of CD4+ Foxp3+ cells in the CLP (Fig. 1, E and F) and MLN (fig. S1) remained unchanged between recipients of WT and Il6ra−/− cells, suggesting that reduction in disease in Il6ra−/− recipients was not because of an increase in regulatory T (Treg) cells and suppression of an inflammatory response in the absence of IL-6. Together, these results indicate that IL-6 signaling in naïve CD4+ T cells is required for the pathogenesis of IBD and that trans-signaling in the absence of membrane-bound IL-6Rα is not sufficient to induce development of a pathogenic TH17 response. These findings are in agreement with other studies that showed defective TH17 responses in the absence of IL-6Rα (49).

Maintenance and pathogenesis of TH17 cells are dependent on ongoing IL-6 signaling

Previous results using IL-6–deficient animals or blocking IL-6 interventions suggest that IL-6 is required to promote TH17-induced disease (12, 50, 51). However, it is currently unknown whether ongoing IL-6 signaling is required to maintain TH17 responses. To investigate this, we used a TH17 transfer model of colitis (19), wherein TH17-polarized cells are cultured ex vivo from naïve CD4+ T cells of Il17fThy1.1 reporter mice (Fig. 2A) and sorted on the basis of Thy1.1 (IL-17F) expression (Fig. 2B). Transfer of Thy1.1+ (IL-17F+) TH17 cells into Rag1−/− mice induces a rapid onset of disease that is equivalent in severity to that caused by transfer of CD4+ CD45RBHi T cells (19).

Fig. 2 Maintenance and pathogenesis of TH17 cells is dependent on persistent IL-6 signaling.

CD4+ T cells from Il17fThy1.1 mice were cultured with splenic feeder cells under TH17-polarizing conditions [anti-CD3 (2.5 μg/ml), TGF-β (2.5 ng/ml), IL-6 (10 ng/ml), anti–IFN-γ (10 μg/ml), anti–IL-4 (10 μg/ml)] for 7 days. (A) Expression of IL-17A, IFN-γ, and Thy1.1 in a fraction of in vitro cultured CD4+ T cells after PMA/ionomycin stimulation for 5 hours in the presence of monensin. (B) Thy1.1 expression in CD4+ T cells after purification of Thy1.1+ (IL-17F+) TH17 cells. TH17 cells (4 × 105 per mouse) were transferred into Rag1−/− (n = 4) or Il6−/−.Rag1−/− (n = 8) mice. Four weeks after transfer, mice were analyzed for development of colitis. (C) Histological scores (means + SEM). *P = 0.0264. (D) Representative H&E-stained sections of colon from Rag1−/− (left) or Il6−/−.Rag1−/− (right) recipients of WT TH17 cells. H&E stain; original magnification, ×10. Scale bars, 100 μm. (E) Representative FACS plots showing IL-17A and IFN-γ in CLP CD4+ T cells ex vivo. (F) Relative frequencies (top) and total numbers (bottom) of IL-17A+, IL-17A+ IFN-γ+, and IFN-γ+ CD4+ T cells in the CLP of colitic mice (means ± SEM). **P = 0.0099 and *P = 0.0302. Data are representative of at least two independent experiments. P values, unpaired two-tailed Mann-Whitney test (C) or unpaired two-tailed Student’s t test (F).

Isolated TH17 cells were transferred into groups of Rag1−/− or Il6−/−.Rag1−/− mice. When compared with Il6−/−.Rag1−/− recipients, Rag1−/− recipients showed significantly elevated disease activity (Fig. 2, C and D). In this regard, the absence of IL-6 led to a reduction in the percentage and number of IL-17A+ CD4+ cells recovered from the CLP of colitic mice (Fig. 2, E and F). However, the percentage and numbers of double IL-17A+IFN-γ+ and single IFN-γ+ cells in Il6−/−.Rag1−/− recipients were comparable with that seen in Rag1−/− recipients. The percentage of CD4+ Foxp3+ cells in the CLP of recipient mice was also unchanged (fig. S2), suggesting that disease disparity was not due to elevated Treg numbers in the absence of IL-6. Whereas IL-6 signaling is known to promote anti-apoptotic mechanisms (44), annexin V staining of CD4+ cells recovered from the CLP of colitic Rag1−/− and Il6−/−.Rag1−/− recipients showed no difference in the percentage of apoptotic (annexin V+ propidium iodide+) cells (fig. S2), suggesting that the differences observed was not due to a loss of IL-6–mediated T cell survival at this stage of disease. Although IL-6–deficient mice are known to have other defects that may inhibit an immune response, particularly in the innate immune compartment, these findings support a role for IL-6 in the maintenance of TH17 responses required for chronic disease progression. We therefore considered the mode of IL-6 receptor signaling responsible for this outcome.

Classic IL-6 signaling is required for maintenance of pathogenic TH17 cells

The previous experiments suggested that ongoing IL-6 signaling is required to maintain a pathogenic TH17 response but did not distinguish whether IL-6 was acting via classic versus IL-6 trans-signaling to maintain TH17 cells. Because naïve CD4+ T cells from Il6ra−/− mice are not responsive to direct IL-6 activation (Fig. 3A), we used TH17 cells from Il6ra−/− mice as donors in the TH17 transfer colitis model to interrogate the requirement for classic IL-6 receptor signaling. Consistent with our previous work (46), under TH17-polarizing conditions, Il6ra−/− CD4+ T cells could not produce IL-17A in response to TGF-β and IL-6. However, this lack of IL-6–induced TH17 differentiation was restored by using a recombinant IL-6–sIL-6Ra fusion protein [hyper-DS-sIL-6R (HDS)], which acts exclusively via IL-6 trans-signaling (Fig. 3B). Both IL-6 and HDS generated a similar proportion of TH17 cells when combined with TGF-β (Fig. 3B and fig. S3). There was no difference in the proliferative capacity of WT and Il6ra−/− TH17 cells in vitro as measured by vital dye dilution, and RNA sequencing (RNA-seq) analysis of WT (IL-6), WT (HDS), and Il6ra−/− TH17 precursors showed a very high degree of similarity by principal components analysis of gene expression, with only three genes being >1.5-fold differentially expressed with a false discovery rate (FDR) of <0.05 (Serpine2, Kit, and Nav1) (fig. S3). In addition, pSTAT3 levels were equivalent between TH17 cells restimulated with IL-6 or HDS in vitro (fig. S3). Thus, naïve CD4+ T cells display a comparable capacity to differentiate into TH17 cells in response to either classic or trans-signaling by the IL-6 receptor.

Fig. 3 Classic IL-6 signaling is required for maintenance of pathogenic TH17 cells.

(A) Model of classic versus IL-6 trans-signaling in TH17 development. Early TH17 development is mediated by classic IL-6 signaling involving membrane IL-6Rα associating with gp130 (left). After activation, IL-6Rα is cleaved by ADAM proteases (middle), and late development is believed to be mediated by trans-signaling (right), where shed sIL-6Rα complexes with IL-6 and binds gp130 to induce signaling. (B) CD4+ T cells from Il17fThy1.1 (WT) and Il6ra−/−.Il17fThy1.1 (Il6ra−/−) were cultured with irradiated splenic feeder cells under TH17-polarizing conditions [anti-CD3 (2.5 μg/ml), TGF-β (2.5 ng/ml), anti–IFN-γ (10 μg/ml), and anti–IL-4 (10 μg/ml)] for 7 days with IL-6 (10 ng/ml) or recombinant IL-6–sIL-6Ra (10 ng/ml) (HDS). A fraction of cells was analyzed for expression of CD4 and intracellular IL-17A and IFN-γ after PMA/ionomycin stimulation for 5 hours in the presence of monensin. FACS plots are gated on CD4+ cells. (C) WT (IL-6), WT (HDS), and Il6ra−/− Thy1.1+ TH17 cells obtained in (B) were transferred into Rag1−/− mice (4 × 105 per mouse). Histological scores were analyzed 4 weeks after transfer (means + SEM) [WT (IL-6), n = 8; WT (HDS), n = 13; and Il6ra−/−, n = 12]. *P = 0.0305. (D) Serum sIL-6Rα levels from naïve Rag1−/− mice and colitic recipients of WT (hIL-6) and Il6ra−/− TH17 cells at 4 weeks after transfer (n = 3 per group) were quantified by MILLIPLEX assay (means + SEM). *P < 0.05. (E) Representative FACS plots showing IL-17A and IFN-γ in CLP CD4+ T cells ex vivo. (F) Representative FACS plots showing Foxp3 in CLP CD4+ T cells ex vivo. (G) Relative frequencies (top) and total numbers (bottom) of IL-17A+, IL-17A+ IFN-γ+, and IFN-γ+ CD4+ T cells in the CLP of colitic mice (means ± SEM) (n = 5 per group). (H) Relative frequencies and (I) total numbers of CD4+ Foxp3+ T cells in the CLP of colitic mice (means ± SEM). Data are pooled from (C) or representative of (E to I) three independent experiments. P values, unpaired two-tailed Mann-Whitney test (C), one-way ANOVA with Bonferroni posttest (D, G to I). **P < 0.01, ***P < 0.001

Exploiting this dual form of TH17 cell regulation, we next considered the relative importance of these IL-6 receptor signaling mechanisms in maintaining local TH17 responses. Thy1.1+ TH17 precursors from WT (IL-6 polarized), WT (HDS polarized), and Il6ra−/− (HDS polarized) CD4+ T cells were transferred into Rag1−/− mice. Disease activity and T cell phenotype were assessed 4 weeks after transfer. Whereas TH17 cells from WT mice caused severe disease in Rag1−/− recipients, TH17 cells from Il6ra−/− mice significantly induced less pathology (Fig. 3C and fig. S3). There was no difference in the ability of WT (IL-6) versus WT (HDS) TH17 cells to induce disease, suggesting that these cells are functionally comparable. The reduced disease observed in recipients of Il6ra−/− TH17 cells was associated with a significant loss of IL-17A+ and IL-17A+IFN-γ+ T cells recovered from the CLP, with a concomitant increase in the percentage of IFN-γ+ T cells when compared with recipients of WT TH17 cells (Fig. 3, E and G). As with disease scores, there were no detectable differences between phenotypes of recovered CD4+ cells from WT (IL-6) and WT (HDS) recipients. Thus, in the absence of IL-6–sustained signaling, TH17 cells were unstable, indicating that ongoing, classic IL-6 signal is required to maintain a TH17 response.

In addition, there were no differences in the percentage or number of CD4+ Foxp3+ cells in the CLP of Il6ra−/− versus WT recipients (Fig. 3, F, H, and I), suggesting that the loss of TH17 phenotype is not due to the conversion to Treg in the absence of IL-6 signaling. Serum sIL-6Rα, which is elevated during inflammation due to shedding from membrane-bound IL-6Rα–expressing cells, was significantly increased in recipients of WT TH17 cells. This was in contrast to recipients of Il6ra−/− TH17 cells (Fig. 3D) in which serum sIL-6Rα levels were equivalent to untreated Rag1−/− mice, suggesting that sIL-6Rα is a marker of disease activity in this model. Colon explant cultures showed a significant reduction in IL-17A in Il6ra−/− TH17 cell recipients, despite showing an increase in IL-23 and IL-21 levels (fig. S3), suggesting that these STAT3-inducing cytokines were present but unable to compensate for the loss of IL-6 responsiveness in CD4+ cells. Together, these results suggest that continued classic IL-6 receptor signaling, rather than IL-6 trans-signaling, is required to maintain the pathogenic phenotype of TH17 cells in vivo and that the TH17 phenotype is unstable in the absence of classic signaling.

Loss of TH17 phenotype in Il6ra−/− mice cannot be rescued by cotransfer of WT TH17 cells

Coordinate increases of sIL-6Rα and IL-6 potentiate IL-6 trans-signaling. To rule out the possibility that reduced disease in recipients of Il6ra−/− TH17 cells (Fig. 3C) was due to lack of availability of sIL-6Rα, cotransfer experiments were performed to assess the ability of Il6ra−/− TH17 cells to maintain their phenotype in the presence of inflammation induced by WT TH17 cells. TH17 precursors from congenically marked WT and Il6ra−/− mice were transferred into Rag1−/− recipients either individually or together (WT + Il6ra−/−; 4 weeks), with a fourth group receiving Il6ra−/− TH17 cells 2 weeks after WT TH17 cell transfer (WT + Il6ra−/−; 2 weeks) (Fig. 4A).

Fig. 4 Loss of TH17 phenotype in IL-6Rα–deficient TH17 cells is not rescued by cotransfer of WT TH17 cells.

(A) Experimental schematic. CD4+ T cells from Il17fThy1.1CD45.1 (WT) and Il6ra−/−.Il17fThy1.1 (Il6ra−/−) were cultured with irradiated splenic feeder cells under TH17-polarizing conditions for 7 days with HDS (10 ng/ml). Thy1.1+ TH17 cells were transferred separately (4 × 105 per mouse) or together (2 × 105 each per mouse) into Rag1−/− recipients [WT, n = 4; Il6ra−/−, n = 4; WT and Il6ra−/−, n = 5; and WT and Il6ra−/− (2 weeks), n = 5]. (B) Histological scores were analyzed 4 weeks after transfer (means + SEM). *P < 0.05. (C) Relative frequencies (top) and total numbers (bottom) of IL-17A+, IL-17A+IFN-γ+, and IFN-γ+ CD4+ T cells in the CLP of colitic mice (means ± SEM). *P < 0.05, **P < 0.01, and ***P < 0.001. (D) Representative FACS plots showing IL-17A and IFN-γ in CLP CD4+ T cells from recipients of single or cotransfers ex vivo after development of colitis. (E and F) Levels of sIL-6Rα, sgp130, and IL-6 in colon explants and serum of naïve Rag1−/− mice and colitic recipients of single or cotransfer TH17 cells were quantified by MILLIPLEX assay. Molar ratio of IL-6–IL-6Rα (E) and IL-6Rα–sgp130 (F) in colon explants (squares) and serum (circles) from colitic mice are shown. (G) Colon tissue from WT and Il6ra−/− TH17 cotransfers were stained with anti-EpCAM1 (blue), anti-CD45.1 (green), and anti-CD3 (red). (Left) Anti-CD45.1 (WT), (middle) anti-CD3 (total T cells), and (right) merged image (red arrows, Il6ra−/− CD4+ T cells; green arrows, WT CD4+ T cells). Scale bar, 20 μm. (H) Quantification of WT and Il6ra−/− CD4+ T cells from (G). Data are representative of three independent experiments. P values, nonparametric Kruskal-Wallis with Dunn’s post comparison test (B) and one-way ANOVA with Bonferroni posttest (C).

Four weeks after initial transfer, WT and Il6ra−/− TH17 cells in recipients of both (WT + Il6ra−/−; 4 weeks) had repopulated the colon in equivalent numbers (fig. S4), suggesting that there was no survival advantage by population. Disease severity trended with the total number of recovered CLP CD4+ T cells (fig. S4). For example, disease was significantly ameliorated in Rag1−/− mice that received single Il6ra−/− TH17 cell transfers when compared with recipients of WT TH17 cells (Fig. 4B and fig. S5). Both cotransfer groups developed a disease that was equivalent to WT recipients, reflecting the increased total number of pathogenic CD4+ T cells in these mice. CD4+ T cells recovered from the CLP of recipient mice showed that regardless of whether they were transferred individually or with WT cells, Il6ra−/− TH17 cells failed to retain their TH17 identity, as evidenced by a significant reduction in the percentage of IL-17A+ and IL-17A+IFN-γ+, as well as IL-17A+TNF-α+ and IL-17A+GM-CSF+ CD4+ T cells compared with recipients of WT cells (Fig. 4, C and D, and fig. S4). This correlated with a significant reduction in the numbers of IL-17A+ and IL-17A+IFN-γ+ T cells recovered from most of the Il6ra−/− transfer recipients (Fig. 4C).

Loss of the TH17 phenotype by Il6ra−/− cells occurred as early as 2 weeks after transfer (Fig. 4D, bottom, and fig. S4). In addition, although the percentage of IFN-γ+ cells was increased (and IL-17A+ cells lost) in the CLP of Il6ra−/− TH17 cell recipients in both cotransfer groups, the number of IFN-γ+ cells was significantly decreased compared with WT cells in all groups except that of WT and Il6ra−/− cell recipients (4 weeks).

The balance between classic IL-6 receptor signaling and IL-6 trans-signaling is tightly controlled by the bioavailability of IL-6, sIL-6Rα, and sgp130. These were therefore quantified in serum and colon explant cultures from recipient mice (fig. S4). Significant differences were found: Molar ratios of each analyte showed that systemic sIL-6Rα was in excess of both IL-6 and sgp130 in all recipient groups (Fig. 4, E and F)—a stoichiometry that favors IL-6 trans-signaling. Conversely, colon explant cultures showed that IL-6 was in molar excess relative to sIL-6Rα in all groups (Fig. 4E), whereas sIL-6Rα was again in excess of sgp130 (Fig. 4F), enabling both classic and trans-signaling in the colon microenvironment.

IL-6 signaling promotes T cell proliferation and inhibits apoptosis via its actions to increase Bcl-2 (52). We therefore measured cell cycling in WT and Il6ra−/− TH17 cells after transfer. There were no differences in either 5-bromo-2′-deoxyuridine–positive (BrdU+) or Ki67+ T cells isolated from the spleens and MLNs (fig. S5) of recipient Rag1−/− mice 7 days after transfers of WT, Il6ra−/−, or cotransferred WT and Il6ra−/− TH17 cells. From this, we inferred that there was no difference in the proliferative capacity of Il6ra−/− TH17 cells, whether transferred individually or cotransferred with WT TH17 cells. Similarly, there were equivalent percentages of CD4+ T cells from the CLP and MLN (fig. S5) that were BrdU+ and Ki67+ 4 weeks after transfer, suggestive of no defect in the proliferative capacity of Il6ra−/− TH17 cells in active colitis. Nearly 70% of CLP T cells from Il6ra−/− TH17 recipients were BrdU+ 4 weeks after transfer, although these cells were not pathogenic. Immunofluorescent staining of colon sections from WT and Il6ra−/− recipients showed that WT and Il6ra−/− TH17 cells repopulate the colon equivalently at 4 weeks after transfer (Fig. 4, G and H), indicating no defect in the ability of Il6ra−/− TH17 cells to traffic to the colon.

Together, these findings establish that persistent IL-6 signaling is required to maintain the TH17 phenotype in vivo and that classic rather than IL-6 receptor trans-signaling is favored for maintenance of the TH17 program in the colon microenvironment; the requirement for IL-6 signaling is not related to effects on cell cycle or cell survival but, instead, appears to be intrinsic to TH17 lineage maintenance.

IL-6 trans-signaling activates STAT1 and STAT3 in IL-6Rα–deficient TH17 cells ex vivo

IL-6 signaling activates the transcription factors STAT3 and STAT1 in naïve CD4+ T cells, and studies have shown that CD4+ T cell activation status can alter the balance of STAT3/STAT1 activation downstream of IL-6 signaling (53, 54). Il6ra−/− TH17 cells appeared to be either unresponsive or unable to receive IL-6 trans-signaling in vivo (Fig. 4). To determine whether this might be due to a lack of inflammation-driven availability of the IL-6–IL-6Rα complex or unresponsiveness of TH17 cells to this complex, we cotransferred congenically marked Thy1.1+ TH17 cells from WT and Il6ra−/− mice into Rag1−/− recipients. Four weeks after transfer, we examined the responsiveness of recovered MLN T cells to IL-6 or HDS ex vivo.

Overall, responsiveness to IL-6 signaling was lower in ex vivo T cells compared with naïve T cells (Fig. 5, A and B) due, at least in part, to reduced expression of IL-6Rα and gp130 on recovered cells (fig. S6). IL-6 trans-signaling mediated by HDS induced both pY-STAT3 (Fig. 5, A and B) and pY-STAT1 (Fig. 5, C and D) in a small but significant fraction of both WT and Il6ra−/− T cells stimulated ex vivo, although the percentage of cells positive for pY-STAT3 was substantially higher than those positive for pY-STAT1. In contrast, only WT CD4+ T cells were responsive to classic IL-6 signaling ex vivo. IL-6 stimulation induced pY-STAT3 in a significant fraction of WT cells but, interestingly, did not induce pY-STAT1 in the same population, indicating that, unlike naïve precursors, coupling of the IL-6 receptor to STAT1 via classic signaling is lost in TH17 cells.

Fig. 5 IL-6Rα–deficient TH17 cells are responsive to trans-signaling ex vivo.

Thy1.1+ TH17 cells from Il17fThy1.1CD45.1 (WT) and Il6ra−/−.Il17fThy1.1 (Il6ra−/−) were adoptively transferred together (2 × 105 each per mouse) into Rag1−/− recipients (n = 4 mice). Four weeks after transfer, CD4+ cells from the MLN of recipient mice or naïve CD4+ cells from the MLN of WT mice were stimulated with equimolar concentrations of IL-6 or HDS (0.95 nM) for 15 min. (A) Representative FACS plots showing pSTAT3 (Y705) in MLN CD4+ T cells from naïve or colitic mice. (B) Representative FACS plots showing pSTAT1 (Y701) in MLN CD4+ T cells from naïve or colitic mice. (C). Relative frequencies of pSTAT3+ (top) and pSTAT1+ CD4+ T cells (bottom) in the MLN of colitic mice (means + SEM). *P < 0.05 and **P < 0.01. Data are representative of two independent experiments. P values, two-way ANOVA with Bonferroni posttest (C).

Collectively, these data show that both WT and Il6ra−/− CD4+ T cells are responsive to trans-signaling ex vivo, but this is limited by diminished gp130 expression, consistent with activation-induced down-regulation of gp130 in CD4+ T cells (53, 55, 56). Moreover, classic IL-6 receptor signaling in WT cells, which promotes retention of the TH17 phenotype, preferentially induces pY-STAT3 without pY-STAT1. Therefore, ongoing classic IL-6 signaling in TH17 cells is required to maintain tyrosine phosphorylation of STAT3, and thus, IL-6–induced STAT3 activation is critical for both the development and maintenance of TH17 cells.

Maintenance of the TH17 transcriptional program is dependent on ongoing IL-6 receptor signaling

To further characterize the phenotypic divergence of Il6ra−/− TH17 cells, we performed RNA-seq analysis of T cells that were isolated from the CLP of Rag1−/− mice that received cotransfers of congenically marked WT and Il6ra−/− TH17 cells (Fig. 6A). Recipient mice were repopulated with equivalent percentages (Fig. 6B) and numbers (Fig. 6C) of TH17 cells in the CLP 4 weeks after transfer. Transcriptome analysis revealed a number of differences in gene expression in recovered WT and Il6ra−/− CD4+ T cells ex vivo (Fig. 6D). Gene set enrichment analysis (GSEA) using a previously defined TH17 to TH1 gene set (57) identified significant enrichment of TH1-related genes in Il6ra−/− T cells ex vivo (Fig. 6E, right, and fig. S7). Conversely, and in accord with our previous findings, the transcriptome of WT T cells was enriched for a subset of genes related to TH17 cells ex vivo (Fig. 6E, left, and fig. S7). Signature TH17-related genes that were retained in WT but not in Il6ra−/− T cells ex vivo included Il17a, Il17f, Il21, Il22, Il23r, Rorc, and Ccr6 (Fig. 6F). A number of TH1-related genes were enriched in Il6ra−/− T cells, including Ifng, Tbx21, and Il12rb2, indicative of a molecular program more similar to TH1 cells than the TH17 precursors. GSEA using a dataset derived from colonic CD4+ T cells (GSE58147) (58) showed that the expression pattern of WT and Il6ra−/− CD4+ T cells was significantly related to that of WT and Il23r−/− CD4+ T cells, respectively (fig. S7). Likewise, a subset of genes induced by Tbx21 was significantly associated with Il6ra−/− T cells (fig. S7), consistent with the transdifferentiation of these cells to a TH1-like program.

Using a previously published dataset defining the transcriptional program of TH17 cells (9), significant enrichment of Stat3- and Rorc-induced genes was found in WT T cells ex vivo, as expected, whereas Stat3- and Rorc-repressed genes were significantly enriched in Il6ra−/− T cells (Fig. 6, G to J). In addition, expression of genes regulated by other transcription factors associated with the TH17 lineage, including Batf, Irf4, Fosl2, and Hif1a, was significantly reversed in Il6ra−/− T cells ex vivo (fig. S7) in fitting with the loss of TH17 phenotype in these cells. As expected, the Stat3- and Rorc-induced genes enriched in WT cells contained some overlap, including Il17a, Il17f, Il23r, Il1r1, and Lif (Fig. 6, H and J). There was little overlap in genes repressed by Stat3 and Rorc in Il6ra−/− CLP T cells. Targets of Rorc included Ahr, Ccr5, and Il12rb2 (Fig. 6H), whereas Stat3-repressed genes included Fasl and the TH1-related transcription factors Tbx21 and Irf1 (Fig. 6J). This is consistent with data suggesting that Stat3 is a key transcriptional activator in TH17 cells, whereas Rorc is thought to be an expression modulator (9).

Our previous work has shown that TH17 cells develop divergent gene expression profiles under the influence of IL-23 signaling (19) such that an increase in the expression of Tbx21, Ifng, Ccr5, and Fasl is correlated with a significant portion of IFN-γ–producing ex-TH17 cells, whereas a decrease in the expression of Il17a, Il17f, Ccr6, and Ccl20 is correlated with a reduction in IL-17A+ TH17 cells. We find here that Il6ra−/− CLP CD4+ T cells were enriched for the aforementioned TH1 genes. Conversely, expression of TH17-related genes was associated with WT, rather than Il6ra−/− CLP CD4+ T cells, further indicating that ongoing IL-6 signaling is required to maintain TH17 program stability. Together, these data confirmed our finding that Il6ra−/− TH17 cells rapidly lose their TH17 phenotype in vivo and extended them to show that, without classic ongoing IL-6 signaling, the entire TH17 gene expression program is down-regulated, favoring the transition of many cells to a TH1-like program.

Fig. 6 IL-6 is required to maintain a TH17 gene program in vivo.

(A) Experimental schematic. Thy1.1+ TH17 cells from Il17fThy1.1CD45.1 (WT) and Il6ra−/−.Il17fThy1.1 (Il6ra−/−) were adoptively transferred together (2 × 105 each per mouse) into Rag1−/− recipients (n = 4). Four weeks after transfer, congenically marked CD4+ cells from the CLP of recipient mice were isolated, and gene expression was assessed by RNA-seq. (B) Recovery of CLP CD4+ cells from Rag1−/− recipients of WT and Il6ra−/− TH17 cells (means ± SEM). (C) Total number of CLP CD4+ T cells recovered from Rag1−/− recipients (means + SEM). (D) MA plot displaying differentially expressed genes in WT (bottom) and Il6ra−/− (top) CD4+ T cells ex vivo. Red circles indicate genes with FDR of <0.05. (E) Barcode plots of GSE results obtained using a set of genes previously reported to be differentially expressed between TH17 cells (left) or TH1 cells (right) (data from GSE 14308) (NES). (F) Expression of representative TH1 and TH17 cell–associated genes identified by GSE in (E) are depicted by heat map with z-scored expression values. (G) Barcode plots of GSE results obtained using a set of genes previously reported to be regulated by Rorc in TH17 cells (GSE 40918). (H) Expression of representative genes identified by GSE in (G) is depicted by heat map with z-scored expression values. (I) Barcode plots of GSE results obtained using a set of genes previously reported to be regulated by Stat3 in TH17 cells (GSE 40918) (J). Expression of representative genes identified by GSE in (I) is depicted by heat map with z-scored expression values. Data are from two independent experiments.

Enforced RORγt expression in IL-6R–deficient TH17 cells supports the maintenance of the TH17 program but does not completely prevent TH1 transdifferentiation

A critical gene target of IL-6 signaling in TH17 cells is Rorc, which underpins the TH17 gene expression program while inhibiting alternative programs. Important functions of IL-6/STAT3–induced RORγt include inhibition of TH1 pathway signaling via suppression of T-bet (59), although RORγt also sustains expression of Il23r, which has been shown to be central to the transdifferentiation of TH17 cells into TH1-like cells in vivo via STAT4 signaling (19, 26, 60). Our gene expression analysis showed that in IL-6Rα–deficient TH17 cells, Rorc and Il23r expression were rapidly lost along with other genes of the TH17 program after adoptive transfers (Fig. 6F). To determine whether loss of RORγt was responsible for loss of the TH17 phenotype, we polarized congenically marked naïve WT and Il6ra−/−.Il17fThy1.1 T cells under TH17 conditions with HDS and transduced cells with retroviruses that expressed green fluorescent protein (GFP) alone or GFP and RORγt at 24 hours of a 5-day culture (Fig. 7A). As expected, TH17 precursors from both WT and Il6ra−/− mice expressed a higher percentage of IL-17A after enforced RORγt expression (Fig. 7B, right). GFP+Thy1.1+ TH17 cells were sorted 4 days after transduction, and WT TH17 cells expressing GFP alone (WT pMIG) were transferred together with either Il6ra−/− cells expressing GFP alone (Il6ra−/− pMIG) or Il6ra−/− cells expressing both GFP and RORγt (Il6ra−/− pMIG-RORγt) into Rag1−/− recipients.

Fig. 7 Enforced RORγt expression in IL-6Ra–deficient TH17 precursors rescues the TH17 phenotype and reduces TH1-like cell transdifferentiation in vivo.

(A) CD4+ T cells from Il17fThy1.1 CD45.1 (WT) and Il6ra−/−.Il17fThy1.1 (Il6ra−/−) were cultured under TH17-polarizing conditions [TGF-β (2.5 ng/ml), anti–IFN-γ (10 μg/ml), anti–IL-4 (10 μg/ml), and HDS (10 ng/ml)] with CD3/CD28 expander beads (Thermo Fisher Scientific) and transduced with retroviruses encoding GFP alone (pMIG) or GFP with RORγt (pMIG-RORγt). FACS plots show GFP and Thy1.1 expression on day 6 and are gated on CD4+ T cells. (B) TH17 precursors were stained intracellularly for IL-17A and IFN-γ after PMA/ionomycin activation for 5 hours in the presence of monensin. FACS plots are gated on CD4+ GFP+Thy1.1+ cells. (C) GFP+Thy1.1+ TH17 cells from (A) were FACS purified and transferred together (2 × 105 each per mouse) into Rag1−/− recipients (WT pMIG and Il6ra−/− pMIG, n = 7; WT pMIG and Il6ra−/− pMIG-RORγt, n = 7). Histological scores were analyzed 3 weeks after transfer (means + SEM). (D) Representative FACS plots showing IL-17A and IFN-γ in CLP CD4+ T cells from recipients of cotransfers ex vivo after development of colitis. (E) Relative frequencies of IL-17A+, IL-17A+ IFN-γ+, and IFN-γ+ CD4+ T cells in the CLP of colitic mice (means ± SEM). *P < 0.05, **P < 0.01, and ***P < 0.001. (F) Representative H&E-stained sections of colon from recipients of WT pMIG and Il6ra−/− pMIG or WT pMIG and Il6ra−/− pMIG-RORγt CD4+ T cells. H&E stain; original magnification, ×10. Scale bars, 100 μm. (G) RNA isolated from FACS purified WT and Il6ra−/− CLP CD4+ T cells from cotransfer recipients was analyzed ex vivo by quantitative PCR for expression of Il17a, Il17f, Rorc, Tbx21, Il12rb2, and Il23r (means + SEM of three biological replicates). *P < 0.05 and **P < 0.01. Data are representative of three experiments. P values, one-way ANOVA with Tukey’s multiple comparison test (G) or one-way ANOVA with Bonferroni posttest (E). ns, not significant.

Three weeks after transfer, both cotransfer groups had developed comparable disease (Fig. 7, C and F). CD4+ T cells recovered from the CLP showed that, consistent with previous results (Fig. 4), TH17 cells transfected with empty vector (Il6ra−/− pMIG) rapidly lost their TH17 phenotype as shown by a significant reduction in IL-17A+ cells compared with cotransferred WT (WT pMIG) TH17 cell controls (Fig. 7, D and E), with a concomitant increase in the relative frequency of IFN-γ+ cells. In contrast, Il6ra−/− TH17 cells transfected with RORγt (Il6ra−/− pMIG-RORγt) had a much greater propensity to retain their TH17 phenotype even in the absence of classical IL-6 signaling. Thus, the frequencies of both IL-17A+ and IL-17A+ IFN-γ+ cells from recipients of RORγt-transfected Il6ra−/− TH17 cells were markedly increased in comparison with those transduced with vector alone (Fig. 7, D and E). Moreover, RORγt-transfected TH17 cells had significantly greater retention of IL-17 expression compared with WT controls, reflecting the role of RORγt in promoting TH17 cytokine production (12). Although RORγt-transfected Il6ra−/− TH17 cells had significantly decreased expression of IFN-γ compared with Il6ra−/− TH17 cells transfected with empty vector, they showed a modest but significant increase in the fraction of IFN-γ+ cells compared with WT TH17 cells. This is consistent with the development of TH1-like cells from the larger frequency of TH17 precursors maintained by exogenous RORγt (19) and suggested that enforced RORγt expression did not entirely repress transdifferentiation of TH17 cells into TH1-like cells.

In accord with the phenotypic data, gene expression analysis of CD4+ T cells recovered from CLP showed that RORγt-transfected Il6ra−/− TH17 cells had a significant increase in expression of genes of the TH17 program compared with empty vector Il6ra−/− TH17 cells, including Il17a, Rorc, Il17f, and Il23r (Fig. 7G). Furthermore, there was decreased Il12rb2 but not Tbx21, suggesting differential sensitivities of these two genes to RORγt-mediated repression. Expression of Rorc was not significantly increased over that of recovered WT controls, indicating that the levels of RORγt driven by retroviral transduction were physiologic. As anticipated, Il23r was significantly elevated in RORγt-transfected Il6ra−/− TH17 cells compared with both empty vector Il6ra−/− cells and cotransferred WT control cells, consistent with a central role for IL-6–induced RORγt in the maintenance of Il23r expression. Because IL-23 signaling via STAT4 is known to be a principal pathway mediating the transdifferentiation to TH1-like cells in vivo (19, 26), it is likely that the elevated expression of IL-23R by RORγt-transfected Il6ra−/− TH17 cells contributed to the modest increase in expression of IFN-γ by these cells compared with WT controls, despite ongoing RORγt expression. Together, these results suggest that ongoing classical IL-6 signaling supports the TH17 program by sustaining RORγt expression that contributes to the maintenance of TH17 cells, which provide a reservoir of cells that can transdifferentiate into pathogenic TH1-like cells that are essential to colitogenesis.

DISCUSSION

IL-6 is required for lineage specification of TH17 cells through its activation of STAT3, which antagonizes the IL-2/STAT5–driven specification of induced Treg cells with which TH17 cells share TGF-β–dependent developmental programming (6163). Here, we report that in addition to its key role in TH17 cell induction (3, 4), there is a requirement for persistent IL-6 signaling to maintain the TH17 genetic program and, thus, TH17 cells. Although it has been reported that IL-23 signaling is important for the maintenance of TH17 cells (18, 19, 21), we find that ongoing IL-6 signaling is indispensable and foundational, acting upstream of IL-23 via its unique function to sustain RORγt expression and thereby IL-23R expression. Although TH17 cells were fully competent to receive IL-6 trans-signaling, classic IL-6 signaling proved to be necessary for TH17 maintenance in the uninflamed and inflamed intestines. This appears to be due to the combined effects of high local production of IL-6, which was incompletely buffered by soluble IL-6Rα, and the abundance of gp130-expressing non–T cells (e.g., intestinal epithelial cells), which act as a sink for trans-signaling. Possible differences between the classic and trans-signaling modes may also be contributory and deserve further study.

In contrast to TH1 and TH2 cells, TH17 cells show substantially greater plasticity (19, 23, 64). This reflects intrinsic instability in the transcriptional circuit upon which TH17 cell development is based, reflected in the propensity of several cytokines including IL-23, IL-12, and both type I and II interferons to promote transdifferentiation of TH17 into TH1-like cells. Whereas the TH1 and TH2 programs are reinforced by cell-extrinsic and cell-intrinsic mechanisms that enhance expression of T-bet and GATA-3, respectively (66), mechanisms by which RORγt expression is sustained and the TH17 program maintained have been unclear. IL-23 has been reported to be a trophic factor for TH17 cells (19, 23, 64), but IL-23 signaling also promotes TH17 to TH1-like transdifferentiation (19, 26), thereby contributing to the instability of TH17 cells. Despite its dominant activation of STAT3, IL-23 signaling also activates STAT4, leading to expression of T-bet, which represses RORγt via direct binding to the Rorc locus (26, 59). Our finding that TH17 cells require tonic or intermittent classic IL-6 signaling, which shifts from a STAT3-STAT1 output during early development to a pure STAT3 output in mature cells, indicates that this pathway of STAT3 activation may be essential to counterbalance the STAT4 output of the IL-23 (or IL-12) receptor to sustain RORγt and the TH17 program. However, it also appears that the IL-6–dependent maintenance of a pool of TH17 cells provides precursors from which pathogenic TH1-like cells can arise. In view of the findings herein, it would appear that stable expression of RORγt does not completely block TH1 reprogramming, consistent with a dominant role of T-bet in overriding the TH17 program.

In view of the role for STAT1 in modulating the STAT3-dominant output of the IL-6 receptor via formation of STAT3-STAT1 heterodimers (66), results herein suggest that the maintenance of mature TH17 cells via classic signaling may be strictly dependent on STAT3 homodimers. Moreover, because STAT1 signaling impairs TH17 programming (67), the loss of STAT1 activation in TH17 cells receiving classic IL-6 signaling may further reinforce maintenance of the TH17 program. Because IL-6 trans-signaling in TH17 cells retained a STAT1 output, which can also induce T-bet expression, it is possible that TH17 cells exposed to this mode of IL-6 signaling may be less resistant to transdifferentiation mediated by TH1-promoting cytokines that preferentially activate STAT1 (e.g., IFNs) or STAT4 (e.g., IL-12). This will require further study. Despite its elevated expression in the colons of recipients of IL-6Rα–deficient TH17 cells, IL-21, which also activates STAT3 and has been implicated as an autocrine factor for TH17 cells (8, 68), was also unable to compensate for deficiency of IL-6 signaling in maintenance of the TH17 program. The basis for the developmentally linked shift in STAT activation by the IL-6 receptor and insufficiency of autocrine IL-21 to substitute for IL-6 signaling will also require further study. In any case, collectively, these findings suggest that the local balance of IL-6 and IL-23 is critical in sustaining or destabilizing the TH17 program, resulting in a protective versus pathogenic TH17 response.

The finding of a critical requirement for paracrine IL-6 in both the induction and maintenance of TH17 cells draws a further parallel between TH17 cells and Treg cells (61). Treg cells require IL-2 for both their development and maintenance (69) but are unable to produce IL-2 themselves. Instead, they are thought to rely on production of IL-2 by activated naïve or other effector T cells (69), akin to the reliance of TH17 cells on IL-6 produced by innate immune cells. For both lineages, the dependence on a single cytokine as both developmental inducer and maintenance factor renders them particularly susceptible to lineage instability. Moreover, because these two cytokines are cross-inhibitory, IL-6 promotes the development of TH17 cells and inhibits Tregs, whereas IL-2 promotes the development of Tregs and inhibits TH17 cells; this suggests that competition for their respective inductive/maintenance cytokine is a major contributor to TH17 versus Treg pool size both at homeostasis and during the emergence and subsidence of an antigen-driven inflammatory response. This is of particular importance in intestinal tissues, which are enriched in both TH17 and Treg cells and are subject to ongoing antigenic stimulation from the intestinal microbiome (70). In view of the current data that implicate the importance of the balance of these cytokines in controlling TH17-Treg cell balance during both evolving and waning antigenic response, it will be important to better define the precise sources and dynamics of IL-2 and IL-6 production.

The relative contribution of TH17 and TH1 cells to immune-mediated diseases has been an area of considerable interest. Early seminal studies identified a definitive link between TH1 cells and colitis (47, 71), whereas subsequent studies established an essential role for the IL-23–TH17 axis in disease pathogenesis (15, 72). A recent study from our group reconciled these findings by demonstrating that TH1-like cells, which derived from TH17 precursors contingent on intact IL-23–induced STAT4 activation and T-bet expression, were critical for colitis development (60). A notable feature of our current findings is that IL-6Rα–deficient TH17 cells more readily transitioned to IFN-γ–producing TH1 cells in vitro yet failed to cause colitis in vivo. Although this appears to contradict a requirement for TH1 cells in the pathogenesis of IBD, our previous studies showed that TH17 cells assisted the development and/or survival of pathogenic TH1 cells that were unable to drive disease on their own (60). Although the basis for TH17-mediated support for de novo TH1 development remains to be defined, a trivial explanation for the observed lack of disease may be that the rapid loss of TH17 cells unable to receive IL-6 signaling resulted in an inadequate number of TH17 precursors to give rise to sufficient TH1-like cells or promote their de novo development.

In light of a critical role for ongoing classic IL-6 signaling in TH17 maintenance, a more detailed understanding of the dynamics of IL-6Rα and gp130 expression by TH17 cell populations is needed, as is a better understanding of factors regulating the local concentrations of sIL-6Rα, IL-6, and sgp130 in specific tissue microenvironments such as the intestines. Although we and others have found that IL-6Rα and gp130 are down-regulated upon T cell activation (31), the mechanisms are different. IL-6Rα appears to be down-regulated primarily by cleavage of the extracellular domain by the metalloproteinases ADAM10 and ADAM17 in humans (73) or only ADAM 10 in mice (74), which would appear to favor a shift from classic to IL-6 trans-signaling in developing TH17 cells. Surface expression of gp130 is down-regulated by internalization, alternative splicing of the encoding mRNA to favor the secreted form and/or other unidentified mechanisms (31). Although IL-2 signaling has been shown to inhibit expression of Il6ra and Il6st (gp130) (75), little is known about conditions that promote retention or reexpression of IL-6Rα and gp130 by T cells, which now appear to be a key in determining which cells are competent to retain in the TH17 program and by what mode of IL-6R signaling. Although IL-6 trans-signaling was intact and comparable in WT and IL-6Rα–deficient T cells recovered from colitic mice, it was substantially diminished, reflecting the diminished gp130 expression and/or signaling competency. This affected classic signaling in WT cells as well and suggests that mechanisms controlling the expression of gp130 by effector T cells are limiting for TH17 maintenance, irrespective of the expression of IL-6Rα and the local balance of free versus sIL-6Rα–bound IL-6.

Although IL-6 trans-signaling was fully sufficient to differentiate TH17 cells in vitro, it was insufficient for either TH17 development or maintenance in vivo, despite elevated sIL-6Rα. The reasons for this are unclear but are unlikely to involve complete buffering by sgp130 because sIL-6Rα was in substantial excess in colitic mice. Rather, competition from intestinal innate, epithelial, and stromal cells, all of which can express gp130 and can signal in trans, appeared to prevent IL-6 trans-signaling in TH17 cells. At least in the colon, a high IL-6–sIL-6Rα ratio due to high local production of IL-6 favored sufficiently high concentrations of free IL-6 to enable classic signaling to the relatively small percentage of T cells coexpressing membrane-bound IL-6Rα and gp130. The stoichiometry of IL-6–IL-6Rα was comparable in both the homeostatic and inflamed intestines, consistent with a dominant role for classic signaling in TH17 maintenance in this tissue both at the baseline and in the diseased state. Whether this is a common feature of mucosal and nonmucosal barrier tissues is unknown.

An association between polymorphisms in the IL6RA gene and susceptibility to both rheumatoid arthritis (RA) and IBD has been found (76, 77). The IL6RA variant rs2228145, which leads to increased proteolytic cleavage of IL-6Rα and, thus, a reduction in membrane-bound IL-6Rα in favor of increased serum levels (78), is associated with reduced risk of both RA (76) and IBD (77). Although the mechanism of protection remains to be characterized, it is possible that it involves both increased buffering of free IL-6 and reduced expression of cell-surface IL-6Rα such that classic signaling required for TH17 development, and thus disease induction, is impaired. Given the findings herein, it is possible that this variant also compromises the maintenance of TH17 cells.

Our finding that ongoing IL-6 signaling is required for TH17 maintenance has implications for therapeutics that target TH17-mediated diseases. In systemic TH17-related autoimmune diseases such as RA, juvenile idiopathic arthritis, and systemic sclerosis, treatment with biologics that inhibit IL-6 is an effective or promising therapy (7981). This is in contrast to tissue-specific immune-mediated diseases, including IBD, where therapies that inhibit IL-6 signaling have not gained clinical use despite evidence that antibodies targeting IL-6 or IL-6Rα may have efficacy (82). Given that IL-17A– and IL-22–producing TH17 cells appear to be protective rather than pathogenic in the intestines (83), persistent classic IL-6 receptor signaling that sustains intestinal TH17 responses may enhance barrier function to restrain disease. This is in line with the view that classic and trans-signaling promote anti- and proinflammatory responses, respectively (84), and suggests that inhibiting classic signaling and, thus, the protective effects of IL-6 may exacerbate disease. Given that IL-6 can amplify inflammation-induced injury to a variety of cells and tissues, it remains a valid therapeutic target (31), although it may be important to selectively target the proinflammatory effects of IL-6 while sparing its protective effects. The development of inhibitors that specifically target trans-signaling by inhibiting the docking of the IL-6–IL-6Rα complex to gp130 may offer a clinical advantage in selectively targeting the deleterious effects of IL-6 without affecting the more beneficial effects, including the maintenance of protective TH17 cells.

MATERIALS AND METHODS

Study design

This study was designed to investigate whether ongoing IL-6 is required for TH17 function and whether classical or trans-signaling was more important for maintenance of TH17 cells. Experiments consisted of analyzing T cell populations in the inflamed intestines of immunodeficient recipients as part of two established T cell transfer models of colitis. All animals were bred and maintained in accordance with the Institutional Animal Care and Use Committee regulations. Littermate comparisons were used for all experiments where possible. Gender- and age-matched male and female mice were used, and animals were randomly assigned into different groups. Group sizes were the minimum requirement to detect statistically significant differences based on previous experience. Experiments were not blinded to investigators, but samples for histopathological analyses were blinded before scoring. All experiments were replicated as presented in the figure legends.

Mice

The following mice were purchased from the Jackson laboratories: C57BL/6.CD45.1, Il6−/−, B6.FVB-Tg(EIIaCre), and Rag1−/−. The generation of Il17fThy1.1/Thy1.1 reporter mice has been described previously (19). Il17fThy1.1 mice were crossed with B6.CD45.1 to create Il17fThy1.1(CD45.1). Il6rafl/fl mice were a gift from A. Drew (University of Cincinnati) and were crossed with EIIaCre mice to generate Il6ra−/−. Where indicated, crosses between Il17fThy1.1/Thy1.1 reporter mice and individual knockout mice were made in our facility to generate mice homozygous for Il17fThy1.1 and respective gene deletion alleles (e.g., Il6ra−/−). All mice were on a C57BL/6 background.

CD4 T cell preparation and culture

CD4+ T cells were purified from pooled spleen and lymph nodes by Dynabeads mouse CD4, followed by DETACHaBEAD mouse CD4, according to the manufacturer’s instructions (Thermo Fisher Scientific). Naïve CD4+ CD62L+ CD25 T cells were obtained by fluorescence-activated cell sorting (FACS). CD4+ T cells were cultured at a ratio of 1:5 with irradiated splenic feeder cells [or, where noted, Dynabeads T-activator CD3/CD28 beads (Thermo Fisher Scientific)] for 7 days in RPMI containing 10% fetal bovine serum (FBS), penicillin (100 IU/ml), streptomycin (100 μg/ml), 1 μM sodium pyruvate, 1× nonessential amino acids, 2.5 μM β-mercaptoethanol, and 2 μM l-glutamine (R10 medium). Cells were stimulated with anti-CD3 (2.5 μg/ml) (clone 145-11), and for TH17, polarizations were supplemented with rhTGF-β1 (2.5 ng/ml) (R&D Systems), rmIL-6 (10 ng/ml) (R&D Systems) or hyper–IL-6 (10 ng/ml) (S. Jones, Cardiff University), anti–IFN-γ (10 μg/ml) (clone XMG1.2), and anti–IL-4 (10 μg/ml) (clone 11B11). In restimulation cultures, viable T cells were recovered on day 7 of initial cultures and activated with fresh splenic feeders or CD3/CD28 activator beads, cytokines, and antibody mixtures as indicated. Unless otherwise noted, rmIL-23 and rmIL-12 (R&D Systems) were added at 20 and 2.5 ng/ml, respectively.

CD4 adoptive transfer model of colitis

CD4 cells were cultured under TH17-polarizing conditions for 7 days. Viable cells were recovered (Ficoll), and Thy1.1+ cells were isolated by magnetic sorting (Miltenyi Biotec). A total of 4 × 105 Thy1.1+ TH17 cells were injected intraperitoneally into age- and sex-matched Rag1−/− recipients. For cotransfer experiments, 2 × 105 each of WT and Il6ra−/− TH17 cells were transferred. For the naïve CD4 transfer model, CD4+ CD45RBHi cells were FACS sorted from pooled spleen and lymph nodes, and a total of 4 × 105 cells were injected into age-matched Rag1−/− recipients. Mice were monitored regularly for signs of disease and weighed weekly. At 4 or 8 weeks after transfer, mice were sacrificed, and MLNs and colons were recovered. Colons were cut longitudinally, and small lengths of tissue were obtained from the proximal, middle, and distal portions of the colon, fixed in 10% formalin, and processed for histopathological analyses. Samples were scored by a pathologist in a blinded fashion as previously described (85).

Isolation of colonic lymphocytes

Lamina propria lymphocytes from the colon (CLP) were isolated as described previously (85). Briefly, colons were cut longitudinally and were cut into 1-cm-long strips. Tissues were washed in cold 1× Hanks’ balanced salt solution containing 2% (v/v) FBS and penicillin (100 IU/ml) and streptomycin (100 μg/ml). After being washed, tissues were digested at 37 C for 45 min with gentle stirring in R10 medium containing collagenase D (100 U/ml) and deoxyribonuclease I (20 mg/ml) (Sigma-Aldrich). Lamina propria lymphocytes were purified on a 40/75% (v/v) Percoll gradient by centrifugation for 20 min at 25°C.

Flow cytometric analysis

Where indicated, CD4+ cells were stimulated with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml; Sigma-Aldrich) and ionomycin (750 ng/ml; EMD Millipore) for 5 hours in the presence of GolgiPlug (BD Biosciences). Intracellular staining was performed as previously described (85). LIVE/DEAD Fixable near-IR Dead Cell Stain (Invitrogen) was used to exclude dead cells. Staining of CD4+ T cells was performed with antibodies to CD4 (RM4-5), IL-6Rα (D7715A7), IL-17A (TC11-18H10), T cell receptor–β (TCR-β) (H57-597), and Foxp3 (FJK-16s) from BD Biosciences; IFN-γ (XMG1.2), TCR-β (H57-597), gp130 (KGP130), and Thy1.1 (His51) from eBioscience; and Thy1.1 (OX-7) from BioLegend. For apoptosis analysis, annexin V and propidium iodide staining were performed using an annexin V apoptosis detection kit (eBioscience) according to the manufacturer’s instructions. Samples were acquired on an LSR II or Attune NxT flow cytometer, and data were analyzed with FlowJo software (Tree Star).

Phospho-STAT staining

CD4+ T cells from in vitro cultures or MLN cells from recipient Rag1−/− mice were rested for 4 hours at 37°C in RPMI and then stimulated for 15 min with 0.95 nM IL-6 or HDS. Cells were fixed for 15 min at 37°C with 3% paraformaldehyde (PFA), washed, and permeabilized for 30 min at 4°C with 90% methanol. After two washes, cells were stained for 1 hour at room temperature with antibodies against CD4 (clone RM4-5), pSTAT3 (Y705; clone 4/P-STAT3), and pSTAT1 (Y701; clone 4a), all from BD Biosciences.

BrdU staining

Where noted, CD4+ T cell–recipient Rag1−/− mice were fed with BrdU (1 mg/ml; Sigma) in tap water with 1% sucrose (w/v) for 7 days before sacrifice. At 1 and 4 weeks after transfer, CD4+ cells from the spleen, MLN, and CLP were harvested, and BrdU was detected using the BrdU Flow Kit (BD Biosciences) according to the manufacturer’s instructions. CD4+ cells were costained with CD4 (RM4-5), LIVE/DEAD Fixable near-IR Dead Cell Stain, TCR-β (H57-597), and Ki67 (eBioscience, SolA15).

Measurement of IL-6, sIL6Rα, sgp130, and other analytes

Blood was collected from mice upon euthanasia, and serum was obtained by centrifugation. For colon explant cultures, colon tissue was flushed, cut into three equal sections, and then opened longitudinally. Individual tissue segments were minced and placed in 0.4 ml of R10 medium for 2 days, and then supernatant was collected by centrifugation. Cytokines and other analytes were measured by MILLIPLEX assay (Millipore) following the manufacturer’s instructions.

Immunohistology

For immunostaining experiments, colons were fixed in 2% (v/v) PFA for 2 hours at room temperature, washed in phosphate-buffered saline (PBS), and then set overnight at 4°C in 20% (w/v) sucrose solution. Tissue was then rinsed in PBS, cut into equal segments, embedded in O.C.T. (Tissue-Tek), and snapped frozen. Tissue sections (6 to 8 μm) were stained with biotinylated anti-CD45.1 (clone A20, eBioscience) followed by streptavidin–Alexa Fluor 488 (Invitrogen), allophycocyanin–conjugated anti-EpCAM1 (G8.8, eBioscience), and Alexa Fluor 594–conjugated anti-CD3 (17A2, eBioscience), followed by donkey anti-goat immunoglobulin G Alexa Fluor 594 to amplify the signal. Images were obtained using a Nikon A1R confocal microscope and processed with the Nikon Elements software.

RNA-seq analysis

Adaptors were trimmed and aberrant reads were removed using Trim Galore (version 0.4.5). The quality-controlled reads were mapped onto the mouse genome build GRCm38 (ENSEMBL.mus_musculus.release-75) (86) using STAR (version 2.5.3) (87). BAM files were sorted using SAMtools (version 0.1.18) (88), and reads were counted for each gene using HTSeq (version 0.7.2) (89). Differential expression analysis was performed using DESeq2 (version 1.18.1) (90) using R (version 3.4.3). Dispersion shrinkage of fold changes was performed with the adaptive shrinkage R algorithm (91). To prepare data for GSEA, results of differential gene expression analysis were ranked by signed P value. Gene lists were ranked by the P value of Wald test multiplied by a sign of fold change and analyzed for enrichment of curated query gene sets using the fast gene set enrichment analysis (version 1.4.0) with 1 million permutations. Gene set enrichment (GSE) P values of normalized enrichment scores (NES) were corrected with the Benjamini-Hochberg procedure. When available, gene sets were made from reported results of transcriptome analysis. However, for many published datasets, only raw data were available, and gene sets were created using in-house scripts. Query gene sets were created from publicly available data. Raw data was obtained from the Gene Expression Omnibus (GEO) (92). Bulk RNA-seq data were analyzed as described above, and gene sets were made by selecting the top 250 up- and down-regulated genes by P value with a fold change of at least 1.5. Microarray data were analyzed using the limma R package (93). The functions model.matrix and lmFit were used to create and fit linear models, respectively. Because of the increased noise in microarray data, the top 125 up- and down-regulated genes were selected by P value with a fold change of at least 1.5 to create gene sets. All query gene sets used are available in table S1.

The Jaccard indexes were calculated for each leading edge gene set versus all others by dividing the number of genes in the intersection by the number of genes in the union of each comparison. The resulting matrix underwent dimension reduction by using t-stochastic neighbor embedding (t-SNE) as implemented in the R package Rtsne (version 0.13; with settings dim = 2, perplexity = 5, and max_iter = 50,000).

Gene set enrichment analysis

GSEA was performed using software from the Broad Institute (94). A preranked analysis was performed on a gene list ordered by signed P value using classic enrichment statistic, 10,000 permutations, and meandiv normalization mode. TH17- and TH1-associated gene sets were created from publicly available microarrays (GEO GSE14308)(57). Limma (3.32.8) was used to calculate differential expression by comparing TH1 samples with TH17 samples. The top 250 genes by most significant adjusted P value were separated by fold change. Those with a log2-transformed fold change greater than zero generated the TH1 gene set, those with fold changes less than zero generated the TH17 gene set. Hallmark gene sets were obtained from the MSigDB (95). Heat maps were created using R 3.0.1 and the function heatmap.2 from the ggplot2 package (96).

Retrovirus production and CD4+ T cell transduction

GFP and RORγt GFP retroviruses were produced by transfecting Platinum-E packaging cells (Cell Biolabs) with pMIG (Addgene no. 9044) or MIGR-RORgt (Addgene no. 24069) retroviral plasmids using Lipofectamine 2000 (Thermo Fisher Scientific) and collecting supernatant 24 and 48 hours later. To transduce TH17 cells, FACS-sorted naïve CD4+ T cells from Il17fThy1.1 mice were cultured in R10 medium with Dynabeads mouse T-activator CD3/CD28 beads (Thermo Fisher Scientific) at a ratio of five beads per CD4+ cell. After 24 hours, cells were spin infected with retrovirus-containing media for 90 min (2500 rpm, 30°C). Cells were then washed with R10 medium and cultured for a further 4 days under TH17-polarizing conditions. For transfers, CD4+ GFP+Thy1.1+ cells were FACS sorted and injected intraperitoneally into age- and sex-matched Rag1−/− recipients.

Real-time PCR

Congenically marked CD4+ TCR-β+ cells were sorted from the CLP of Rag1−/− recipients. Total RNA was isolated using an miRNeasy isolation kit (Qiagen) according to the manufacturer’s instructions. complementary DNA was synthesized with the iScript Reverse Transcription Supermix (Bio-Rad), and real-time polymerase chain reaction (PCR) was performed with SsoAdvanced Universal Probes Supermix or SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) for 35 to 40 cycles. Reactions were run in duplicate and normalized to B2m.

Statistical analysis

Statistical significance was calculated using the Prism software (GraphPad). The nonparametric two-tailed Mann-Whitney test or Kruskal-Wallis test was used to determine the significance for pathology data; all other data was analyzed using unpaired two-tailed Student’s t test or one-way analysis of variance (ANOVA) as appropriate. All P values of ≤0.05 were considered significant.

Supplementary Materials

immunology.sciencemag.org/cgi/content/full/5/49/eaaw2262/DC1

Fig. S1. Classical IL-6 signaling is required for development of colitis.

Fig. S2. Innate-derived IL-6 is required for TH17 pathogenicity and maintenance.

Fig. S3. Classical IL-6 signaling is required for maintenance of TH17 cells.

Fig. S4. Cotransfer of Il6ra−/− with WT TH17 cells is unable to rescue their phenotype.

Fig. S5. Cotransfer of Il6ra−/− with WT TH17 cells is unable to rescue their TH17 phenotype.

Fig. S6. IL-6Ra and gp130 expression on TH17 cells ex vivo.

Fig. S7. Il6ra−/− TH17 cells lose their TH17 gene expression signature in vivo.

Table S1. Full gene sets used for GSEA analysis (Excel spreadsheet).

Table S2. Raw data (Excel spreadsheet).

REFERENCES AND NOTES

Acknowledgments: We are grateful to members of the Weaver laboratory for helpful comments and suggestions. We thank D. O’Quinn, D.J. Wright, and H. Turner for technical assistance. We also thank A. Drew (University of Cincinnati) for the provision of Il6rafl/fl mice. We acknowledge the University of Alabama at Birmingham (UAB) Comparative Pathology laboratory for histology, the UAB High Resolution Imaging Facility (NIH P30 AR048311) for confocal microscopy images, the UAB Epitope Recognition and Immunoreagent Core Facility (NIH P30 AR048311) for antibody preparations, and the Center for AIDS Research Basic and Translational Sciences Core Facility (NIH P30 AI27667) for FACS. Funding: This work was supported by grants from NIH (R01 DK093015 and R01 DK103744), the Crohn’s and Colitis Foundation of America, and UAB institutional funds (to C.T.W.). Additional support was provided by a Career Development Fellowship from Versus Arthritis (reference no. 20305) to G.W.J. and grants from Versus Arthritis (reference nos. 20770 and 19796) and Wellcome Trust (reference no. 079044) to G.W.J. and S.A.J. Author contributions: S.N.H. and C.T.W. designed the research strategy and wrote the manuscript. S.N.H., DDT, C.L.Z., and R.D.H. performed experiments. S.N.H., S.J.W., T.R.S., M.G., and C.T.W. analyzed experiments. S.A.J. and G.W.J. provided key reagents and made helpful suggestions. Competing interests: S.A.J. has acted as an advisory consultant for pharmaceutical companies engaged in the development of biological drugs against the IL-6 cytokine system (Roche, Chugai Pharmaceuticals, Sesen Bio, NovImmune AG, Regeneron, Sanofi, EUSA Pharma, Janssen, Genentech) and received related grant funding from Roche, GSK, Janssen, NovImmune AG, Ferring Pharmaceuticals and Mestag Therapeutics. The other authors declare that they have no competing interests. Data and materials availability: Gene expression data obtained by RNA-seq have been deposited in the NCBI Gene Expression Omnibus with the accession number GSE 115141. All other data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.

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