Innate lymphoid cells drive IL-23 dependent innate intestinal pathology

The key role of IL-23 in the pathogenesis of autoimmune and chronic inflammatory disorders is supported by the identification of IL-23R susceptibility alleles associated with IBD, psoriasis and ankylosing spondylitis. IL-23 driven inflammation has primarily been linked to the actions of Th17 cells1. Somewhat overlooked, IL-23 also has inflammatory effects on innate immune cells2 and can drive T cellindependent colitis. However the downstream cellular and molecular pathways involved in this innate intestinal inflammatory response are poorly characterized. Here we show that bacteria-driven innate colitis is associated with increased IL-17 and IFN-γ production in the colon. Stimulation of colonic leukocytes with IL-23 induced IL-17 and IFN-γ production exclusively by innate lymphoid cells expressing Thy1, SCA-1, RORγt and IL-23R and these cells markedly accumulated in the inflamed colon. Importantly, IL-23 responsive innate intestinal cells are also a feature of T-cell dependent models of colitis. The transcription factor RORγt, which controls IL-23R expression, plays a functional role as Ror−/−Rag−/− mice failed to develop innate colitis. Lastly, depletion of Thy1+ innate lymphoid cells completely abrogated acute and chronic innate colitis. These results identify a novel IL-23 responsive innate lymphoid population that mediates intestinal immune pathology and may therefore represent a target in IBD. Th17 cells produce a variety of inflammatory cytokines including interleukin-17A (IL-17), IL-17F, IL-22, IL-6 and TNFα, and are implicated in both host defence against extracellular pathogens and the pathogenesis of several inflammatory disorders1. Recently Th17 cells have been shown to exhibit flexibility of function and acquisition of IFN-γ production has been linked to their pathogenicity in vivo3,4. Transforming growth factor-β (TGFβ) and IL-6 or IL-21 drive Th17 cell differentiation5 and this process is orchestrated by the transcription factor, retinoic acid related orphan receptor γt (RORγt) (ref 6). RORγt promotes IL-23R 5Corresponding Authors Contact Information Phone: -00-44-1865285489 Fax: -00-44-1865275591 fiona.powrie@path.ox.ac.uk or kevin.maloy@path.ox.ac.uk. Author contributions S.B and P.A designed and performed the experiments. H. U. performed histology. I.I.I and D.R.L provided Rag−/−Rorc−/− mice and were involved in the experiments using this strain. S.B wrote the manuscript with K.J.M and F.P and K.J.M and F.P directed the research. Author information Antibodies directed against IL-17 and the relevant isotype control were obtained from UCB Celltech. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. Supplementary information is linked to the online version of the paper at www.nature.com/nature. Europe PMC Funders Group Author Manuscript Nature. Author manuscript; available in PMC 2013 October 15. Published in final edited form as: Nature. 2010 April 29; 464(7293): 1371–1375. doi:10.1038/nature08949. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts expression, allowing IL-23 to control the expansion and maintenance of Th17 cells7. Interestingly, RORγt is also expressed by innate lymphoid cells such as intestinal cells that express NK markers, fetal LTi (lymphoid tissue inducer) cells and adult LTi-like cells8. Various innate tissue leukocytes, such as CD11c+ myeloid cells, LTi-like cells and mucosal NKp46+ cells, have been shown to produce IL-22 and/or IL-17 upon IL-23 stimulation, but the contribution of these tissue resident innate immune cells to pathology in the intestine is not known2, 9-11. We have shown that innate immune colitis in Rag−/− mice following infection with Helicobacter hepaticus is IL-23 dependent12. To identify the cellular and molecular pathways involved, we first analysed the expression of inflammatory cytokines in this model. Consistent with selective upregulation of IL-23 in the intestine12, we observed significant increases in the expression of Th17 and Th1 signature cytokines including IL-17, IL-22 and IFN-γ by colonic lamina propria cells (cLP) from H. hepaticus infected Rag−/− mice but not from spleen cells (Fig. 1a). IL-23 mediated pathology was not associated with an increase of IL-6 (Supplementary Fig. 1). To determine whether IL-23 acts directly on innate cells to induce Th1 and Th17 cytokines, cLP cells were isolated from healthy colons of Rag−/−mice and stimulated with IL-12 or IL-23. Addition of IL-23 induced secretion of IL-17, IL-22 and IFN-γ (Fig. 1b), whereas IL-12 induced IFN-γ only. To determine whether IL-17 and IFN-γ played a functional role in innate colitis, H. hepaticus infected Rag−/− mice were treated with neutralising α-IL-17 or α-IFN-γ mAbs. Blockade of either IL-17 or IFN-γ was sufficient to significantly reduce colitis (Fig. 1c and e), without affecting colonisation with H. hepaticus (Supplementary Fig. 2). Similarly, systemic immune activation, assessed by splenomegaly, was also abrogated by IL-17 or IFN-γ blockade (Fig. 1d). Collectively, these results indicate that H. hepaticus induced IL-23 regulates the innate expression of effector cytokines such as IL-17 and IFN-γ that play functional roles in the intestinal innate inflammatory response. To identify IL-23 responsive innate immune cells present in the inflamed intestine, we used a cell sorting approach. Using leukocyte lineage (Lin) markers CD11b, GR1 and B220, we found that cytokine expressing cells were CD45+Lin− and distinct from common innate cell populations (Supplementary Fig. 3). To identify these cells, we performed intracellular cytokine staining in combination with cell surface marker expression on IL-23 stimulated cLP cells from colitic mice. We found that IL-23 not only enhanced the frequency of IL-17+IFN-γ− cells but also increased the frequency of IL-17+IFN-γ+ cLP cells whereas the frequency of IL-17−IFN-γ+ cells did not increase (Fig. 2a). Analysis of surface markers showed that the vast majority of the cLP IL-17 secreting cells expressed high levels of Thy1 (Fig 2b). Lin−Thy1+ cells in Rag−/− mice include a population of cells required for secondary lymphoid organ (SLOs) organogenesis, termed LTi/LTi-like cells13. Similar to classical LTi-like cells, IL-17 expressing cells were found to be IL-7R+CD44+NKp46−CCR6+CD25+RORγ+ (Fig. 2c). They also expressed LTi related genes such as LTα and β, TRANCE and CXCR5, recently found to be important for the recruitment of LTi-like cells during inflammation (Supplementary Fig 4) (refs 13, 14). However, IL-17expressing innate lymphoid cells were also phenotypically distinct from LTi-like cells as they were CD4−c-kit− and also expressed SCA-1 (Fig. 2c and d) suggesting heterogeneity amongst Thy1+ innate lymphoid cells in the intestine. To further characterize Thy1hiSCA-1+ innate lymphoid cells, this population was sorted from the inflamed colon of H. hepaticus infected Rag−/− mice. Even in the absence of cytokine stimulation, the Thy1hiSCA-1+ population produced IL-17, IL-22 and IFN-γ ex vivo, in contrast to the majority of cLP cells (Fig. 2d). In addition, cytokine secretion was enhanced upon stimulation with IL-23 (Fig. 2d). Consistent with their production of Th17 signature cytokines and IFN-γ in response to IL-23 stimulation, Thy1hiSCA-1+ cells Buonocore et al. Page 2 Nature. Author manuscript; available in PMC 2013 October 15. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts expressed higher levels of IL-23R, RORγt and Tbx21 mRNA but not of AHR mRNA (Fig. 2e). Thy1hiSCA-1+ innate lymphoid cells were present at low frequency in the Rag−/− colon but increased significantly during intestinal inflammation (Fig. 3a). As there is around a 10-fold increase in leukocytes in inflamed colons compared to controls15 this represents about a 100-fold increase in total number of Thy1hiSCA-1+ cells. There was also a marked increase in the proportion of Thy1hiSCA-1+ cells that produced IL-17 de novo from H. hepaticus infected mice and following IL-23 stimulation suggesting not only accumulation, but also activation of this population in the inflamed intestine (Fig 3b). The presence of Thy1hiSCA-1+ innate lymphoid cells was not restricted to the colon, as these cells were also observed in the small intestine (Supplementary Fig. 5a). Furthermore, they were also present in other tissues where H. hepaticus has been shown to mediate immune pathology such as the liver15 where they also responded to IL-23 by secreting IL-22, IFN-γ and IL-17 (Supplementary Fig. 5b). A similar population of Lin−CD3ε−Thy1hi cells was also present in the colon of immunocompetent mice both at steady state and during intestinal inflammation induced by infection with H. hepaticus plus concomitant blockade of IL-10R (ref 16) (Supplementary Fig. 6a). Lin−CD3ε−Thy1+ cells again expressed IL-23R, RORγt, and Tbx21 (Supplementary Figure 6b) and secreted IL-17, IFN-γ and IL-22 in response to IL-23 stimulation (Supplementary Fig. 6c). Immunohistological analyses demonstrated that CD3ε−Thy1+ cells were mainly localized within leucocytic clusters and were often present in close association with CD3+ T cell infiltrates (Supplementary Fig. 7). However, some CD3ε−Thy1+ cells were also observed scattered throughout the lamina propria (Supplementary Fig. 7). To determine whether Thy1hiSCA-1+ innate lymphoid cells played a functional role in H. hepaticus innate immune driven typhlocolitis, we depleted these cells by injection of a αThy1 mAb during the course of infection (Supplementary Fig. 8). Efficient depletion of Thy1+ cells led to abrogation of both colitis and typhlitis (Fig 3c and e). IL-23 dependent systemic immune activation was also ablated as shown by the significantly reduced splenomegaly (Fig. 3d). Absence of intestinal inflammation following depletion of Thy1+SCA-1+ correlated with abrogation of both spontaneous and IL-23-induced IL-17 and IFN-γ production by cLP cells (Fig. 3f). We next assess