Articles

Interferon and IL-27 antagonize the function of group 2 innate lymphoid cells and type 2 innate immune responses © 2015 Nature America, Inc. All rights reserved.

Kazuyo Moro1–4,9, Hiroki Kabata1,2,5,9, Masanobu Tanabe6, Satoshi Koga1,2,4, Natsuki Takeno1,2, Miho Mochizuki1,2, Koichi Fukunaga1,2,5, Koichiro Asano5,7, Tomoko Betsuyaku5 & Shigeo Koyasu1,8 Group 2 innate lymphoid cells (ILC2 cells) are type 2 cytokine–producing cells of the innate immune system with important roles in helminth infection and allergic inflammation. Here we found that tissue-resident ILC2 cells proliferated in situ without migrating during inflammatory responses. Both type I and type II interferons and interleukin 27 (IL-27) suppressed ILC2 function in a manner dependent on the transcription factor STAT1. ILC2-mediated lung inflammation was enhanced in the absence of the interferon- (IFN-) receptor on ILC2 cells in vivo. IFN- effectively suppressed the function of tissue-resident ILC2 cells but not that of inflammatory ILC2 cells, and IL-27 suppressed tissue-resident ILC2 cells but not tissue-resident T H2 cells during lung inflammation induced by Alternaria alternata. Our results demonstrate that suppression mediated by interferon and IL-27 is a negative feedback mechanism for ILC2 function in vivo. Impaired termination of immune responses is detrimental and often results in chronic inflammation and disease. Most activated T cells and B cells die after antigen clearance, and only a small fraction of cells survive as memory cells. In addition, the effector functions of T cells are counteracted by various cytokines, which contributes to the proper regulation of immune responses and homeostasis. For example, the balance of cytokines from the TH1 subset of helper T cells and those from TH2 cells in acquired immunity is regulated by interferon-γ (IFN-γ) and interleukin 4 (IL-4) via suppression of the activity of TH2 cells and that of TH1 cells, respectively1,2. Such regulatory mechanisms are believed to be important in the prevention of excessive inflammatory reactions. In contrast, innate immune responses are usually terminated by the death of effector cells. The lifespan of myeloid cells is generally 1 day to a few days, and once activated during immune responses, most will die immediately in situ3–5. Studies have revealed additional types of lymphocytes that function in innate immune responses, collectively called ‘innate lymphoid cells’ (ILCs). ILCs are classified into three groups (group 1, group 2 and group 3) on the basis of their ability to produce distinct sets of cytokines6–8. Unlike T lymphocytes and B lymphocytes, ILCs lack antigen-specific receptors dependent on the RAG recombinase and are activated by cytokines produced by other cells of the innate immune system or epithelial cells. Because ILCs have a much longer

lifespan than that of myeloid cells, an appropriate mechanism(s) is (are) needed to terminate responses mediated by ILCs. Among ILCs, group 2 ILCs (ILC2 cells), originally identified as natural helper cells in fat-associated lymphoid clusters (FALCs) in visceral adipose tissue9, are activated by epithelial cell–derived cytokines, including TSLP, IL-25 and IL-33, and have important roles in helminth infection and allergic diseases10–13. ILC2 cells are unique in that they constitutively produce type 2 cytokines even in the absence of external stimulation and are involved in the self-renewal of B-1 cells and the production of immunoglobulin A9. IL-33 and IL-25 strongly activate ILC2 cells to produce large amounts of IL-5 and IL-13, which induce eosinophilia and goblet cell hyperplasia, respectively, in the lungs and intestine upon infection with Nippostrongylus brasiliensis9. Eosinophilia induced by IL-5 and eotaxin is an important effector mechanism in both the lungs and intestine, and IL-13-dependent production of mucin by goblet cells is critical for anti-helminth immunity in the intestine14. Both ILC2 cells and TH2 cells provide IL-5 and IL-13 during the early phase of infection and late phase of infection, respectively, and cooperatively eradicate helminths. However, little is known about the timing and mechanism of the replacement of these two cell types during inflammation. In allergic inflammatory responses such as asthma 15,16, allergic dermatitis17,18 and allergic lung inflammation induced by fungal

1Laboratory

for Immune Cell Systems, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan. 2Laboratory for Innate Immune Systems, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan. 3Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Tokyo, Japan. 4Division of Immunobiology, Department of Medical Life Science, Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan. 5Division of Pulmonary Medicine, Department of Medicine, Keio University School of Medicine, Tokyo, Japan. 6Department of Infectious Diseases, Keio University School of Medicine, Tokyo, Japan. 7Division of Pulmonary Medicine, Department of Medicine, Tokai University School of Medicine, Kanagawa, Japan. 8Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan. 9These authors contributed equally to this work. Correspondence should be addressed to K.M. ([email protected]). Received 29 June; accepted 28 September; published online 23 November 2015; doi:10.1038/ni.3309

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Articles components such as those of the Alternaria alternata15, IL-25- and IL-33-mediated activation of ILC2 cells exacerbates these reactions. TSLP alone does not induce the production of type 2 cytokines by ILC2 cells but is involved in the resistance of ILC2 cells to corticosteroids during asthma19. The relationship between ILC2 cells and epithelial cells is not one sided. Amphiregulin, a cytokine that binds to the epidermal growth factor receptor on epithelial cells, is known to be produced by ILC2 cells and is involved in the maintenance of epithelial integrity and tissue repair20,21. Thus, ILC2 cells and epithelial cells establish a reciprocal relationship and provide protection at the ‘front line’ of the body’s defenses. Since ILC2 cells survive much longer than myeloid cells and persist for an extended period of time following activation by IL-25 or IL-33, it is possible that once ILC2 populations expand under IL-25- or IL-33-mediated immune responses, systemic production of type 2 cytokines results in chronic type 2 diseases. However, although helminth infection strongly activates ILC2 cells and triggers acute type 2 innate immune responses, chronic type 2 diseases are not generally induced after the eradication of helminths22, which would indicate the existence of an effective mechanism(s) for the suppression of ILC2 function. Here we report the migratory ability of ILC2 cells and interferon- and IL-27-dependent mechanisms for the suppression of ILC2 cells during IL-33-mediated lung inflammation. These cytokine-mediated counterbalance mechanisms might be critical for the conversion from an innate to an acquired type 2 immune response during helminth or fungus infection and allergic inflammation. RESULTS ILC2 cells reside in tissues and do not migrate to other organs To study the dynamic activity of ILC2 cells in vivo, we used a cytokineinduced lung-inflammation model that consisted of the intratracheal administration of IL-33 alone or a combination of IL-2 plus IL-25. We confirmed that both IL-33 alone and the combination of IL-2 plus IL-25 induced strong eosinophilic inflammation in the lungs of both wild-type mice and mice deficient in the recombinase component RAG-2 (Rag2−/− mice), which was associated with the appearance of ILC2 cells in the bronchoalveolar lavage fluid (BALF) (Fig. 1a). Next, to determine whether FALC-derived ILC2 cells were able to migrate to the lungs, we isolated ILC2 cells from FALCs and transferred the cells intravenously into Il2rg−/−Rag2−/− mice, which lack all lymphocytes, including ILCs, and subsequently administered cytokines intratracheally. FALC-derived ILC2 cells appeared in the BALF after treatment with IL-33 alone or the combination of IL-2 plus IL-25 (Fig. 1a), which demonstrated the ability of the transferred FALCderived ILC2 cells to migrate to the bronchioles and alveoli under the conditions of cytokine stimulation and/or environmental factors. We observed transferred ILC2 cells in the mesentery and spleen, but not in the lymph nodes, with or without cytokine administration (data not shown), consistent with the observation that FALC ILC2 cells did not express the chemokine receptors CCR7 and CXCR5, which are critical for the migration of lymphocytes through high endothelial venules into lymph nodes23,24 (Supplementary Fig. 1). The substantial eosinophilia in the BALF was probably due to IL-5 production by the transferred ILC2 cells in situ, as IL-5 was readily detectable in the BALF of cytokine-treated mice but not that of PBS-treated mice (Fig. 1b). Consistent with a published report9, we found that ILC2 cells constitutively produced small but physiologically relevant amounts of IL-5 and IL-13 and rapidly produced large amounts of IL-5, IL-6 and IL-13 following stimulation with IL-33, as shown by intracellular staining (Fig. 1c). In addition, stimulation with IL-33 alone or with IL-2 plus IL-25 induced ILC2 cells to produce eotaxin 

(Fig. 1d), which has a major role in the recruitment of eosinophils to the lungs25. These results indicated that ILC2 cells were necessary and sufficient for the induction of eosinophilia in the inflamed lungs through the production of not only IL-5 but also eotaxin. When we compared the phenotypes of surface markers on lung and BALF ILC2 cells with those of FALC ILC2 cells, BALF ILC2 cells induced by cytokines were more similar to FALC ILC2 cells than lung ILC2 cells (Supplementary Fig. 2). The migration of transferred FALC ILC2 cells to the bronchioles and alveoli upon the administration of cytokines following adoptive transfer (Fig. 1a) suggested that ILC2 cells might circulate among different tissues and organs. To assess this possibility, we performed parabiosis analysis whereby we joined a C57BL/6 (CD45.2+) mouse with a congenic B6.SJL (CD45.1+) mouse via skin. After 1 month, we administered IL-33 intratracheally into the lungs of each mouse and assessed the frequency of CD45.1 + and CD45.2+ T cells, ILC2 cells and eosinophils in each mouse. As expected, T cells and, to a lesser extent, eosinophils were mixed by parabiosis, but ILC2 cells in the lungs, BALF and FALCs were rarely mixed between the mice (Fig. 1e); this indicated that the ILC2 cells did not circulate in either steady-state conditions or inflammatory conditions, even though they were able to migrate to the lungs after intravenous transfer. It is likely that the accumulation of ILC2 cells in BALF obtained after inflammation resulted from the proliferation of a small number of ILC2 cells present in the bronchioles and alveoli of naive mice or the exudation of proliferated ILC2 cells in the lung parenchyma but not via the bloodstream. The results reported above suggested that the ILC2 cells proliferated in tissues locally. ILC2 cells proliferate slowly9, and we found that they survived for more than half a year in the presence of IL-2 in vitro (data not shown). We also observed rapid proliferation and substantial survival of ILC2 cells in the presence of IL-33 alone or both IL-2 plus IL-25, whereas IL-25 alone did not support their survival or proliferation (Fig. 1f). An ILC2 population called ‘inflammatory ILC2’ (iILC2)26, induced by the in vivo administration of IL-25, has characteristics distinct from those of tissue-resident ILC2 cells such as FALC ILC2 cells that are present in naive mice. To compare the responsiveness of tissue-resident ILC2 cells and iILC2 cells to cytokines, we isolated iILC2 cells from the mesenteric lymph nodes of Rag2−/− mice after intraperitoneal administration of IL-25 and assessed their response to cytokines in vitro. The maximum duration of iILC2 population expansion in vitro was much shorter than that of tissue-resident ILC2 cells from naive mice (Fig. 1g). When naive FALC ILC2 cells and iILC2 cells were cultured, iILC2 cells did not proliferate after 9 d of stimulation with a variety of cytokines, whereas naive FALC ILC2 cells continued to proliferate under stimulation with IL-2, IL-33, IL-2 plus IL-25, or IL-7 plus IL-33 (Fig. 1g). IL-33 alone was unable to induce the proliferation of iILC2 cells (Fig. 1g). While IL-7 supported the survival of FALC ILC2 cells for a long period of time, iILC2 cells started to die after 9 d (Fig. 1g and data not shown). These results suggested that once the tissue-resident ILC2 cells proliferated and were activated after helminth or fungal infection, they tended to stay at the inflammatory site and continue to produce type 2 cytokines constitutively for a prolonged period, which would place the host at risk for developing allergic inflammation. We thus hypothesized that there must be negative regulatory mechanisms that terminate ILC2 function. Interferons suppress ILC2 proliferation and cytokine production Cytokines and their receptors are involved in the regulation of cellular activities. To address the question of how ILC2-dependent type 2 innate immune responses are terminated, we assessed the expression aDVANCE ONLINE PUBLICATION  nature immunology

Articles patterns of cytokine receptors on ILC2 cells before and after stimulation. Consistent with the observation that ILC2 cells respond to IL-2, IL-4, IL-7, IL-25 and IL-33 (refs. 9,27), naive ILC2 cells expressed receptors for those cytokines (Fig. 2). In addition, naive ILC2 cells expressed receptors for IL-10 and IL-12 but not receptors for IL-1, IL-5 or IL-6 (Fig. 2). ILC2 cells expressed IL-3Rα but not IL-3Rβ (βc),

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Figure 1  Characteristics of tissue-resident ILC2 cells. (a) Flow cytometry of BALF cells from wild-type (WT), Rag2−/− or Il2rg−/−Rag2−/− host mice given intravenous transfer of IL-2-cultured ILC2 cells (2.1 × 10 6 per mouse) 3 h before the first administration of PBS, IL-2 plus IL-25, or IL-33 (above plots), injected intratracheally into the host mice on days 0 and 3, followed by analysis on day 6. Outlined areas indicate ILC2 cells (CD25 +IL-1RL1+ cells among cells gated as FSCloSSCloPI−CD45+) (top row) or eosinophils (PI−SSChiSiglec-Fhi) (bottom row). SSC, side scatter. (b) Concentration of IL-5 in BALF obtained from wild-type mice (n = 3–4) on day 3 after intratracheal administration of PBS, IL-2 plus IL-25, or IL-33. (c) Intracellular expression of IL-5, IL-6 and IL-13 by FALC ILC2 cells cultured for 0, 6, 24 or 48 h (above plots) with IL-33, assessed by flow cytometry with cyokinespecific antibody (Specific Ab) or isotype-matched control monoclonal antibody (Isotype). (d) Enzyme-linked immunosorbent assay (ELISA) of eotaxin in supernatants of FALC ILC2 cells (5 × 103 per well; n = 3 wells per condition) cultured for 6 d with medium alone (–) or various cytokines (horizontal axis). (e) Frequency of CD45.1+ (C57BL/6) cells or CD45.2+ (B6.SJL) cells (numbers in bars) among T cells (FSC loSSCloPI−CD3+), ILC2 cells (FSCloSSCloPI−Lin−c-Kit+Thy-1+) and eosinophils (FSCloSSChiPI−CD11c−CD19−F4/80−NK1.1−Mac-1−TER119−) in FALCs, lungs and BALF of C57BL/6 (CD45.2+) mice (B6) and B6.SJL (CD45.1+) mice (SJL) in parabiotic pairs given IL-33 intratracheally on days 34 and 36, followed by flow cytometry after 39 d of parabiosis. (f) Quantification of FALC ILC2 cells (3 × 103 cells; duplicate wells) cultured for 15 d with various cytokines (key), with culture medium changed and samples assessed every 3 d during culture. (g) Quantification of ILC2 cells isolated from the mesentery of untreated Rag2−/− mice or iILC2 cells isolated from the mesenteric lymph nodes of IL-25-treated Rag2−/− mice (key), cultured (2 × 103 cells per well) with various cytokines and assessed every 3 d. ND, not detected. Each symbol (b,d) represents an individual mouse (b) or well (d); small horizontal lines indicate the mean. Data are representative of two to four independent experiments with similar results.

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Articles Specific Ab Naive

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© 2015 Nature America, Inc. All rights reserved.

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Figure 2  Cytokine receptors on ILC2 cells. Quantification of cytokine receptors expressed on FALC ILC2 cells freshly sorted (Naive) or sorted and then cultured for 6 d with IL-2 alone, IL-2 plus IL-25, or IL-33 alone (above plots), assessed by flow cytometry. Data are representative of two independent experiments with similar results.

with IL-2 plus IL-25 (Fig. 2). We also assessed receptors for interferons and found that a small percentage of ILC2 cells expressed the IFN-γ receptor IFNGR1 but not IFN-α and IFN-β receptor IFNAR1, and stimulation with cytokines, particularly IL-2, induced both IFNAR1 and IFNGR1 on ILC2 cells (Fig. 2). Because ILC2 cells expressed receptors for the anti-inflammatory cytokine IL-10 as well as receptors for type 1 cytokines, including interferons that suppress type 2 responses in T cells (Fig. 2), we next investigated whether these cytokines had suppressive effects on ILC2 cells in vitro. We cultured ILC2 cells with IFN-β, IFN-γ, IL-10 or IL-12 under conditions of stimulation with IL-2 alone, IL-2 plus IL-25, or IL-33 alone. Whereas IL-10 did not suppress the proliferation (Fig. 3a) or cytokine production (Fig. 3b) of ILC2 cells despite expression of the receptor for this cytokine (Fig. 2), IL-12 slightly suppressed the production of type 2 cytokines but weakly suppressed the proliferation of ILC2 cells, and IFN-β and IFN-γ strongly suppressed the proliferation of ILC2 cells (Fig. 3a). This suppression by IFN-β and IFN-γ was not due to the induction of early apoptosis (annexin V–positive and propidium iodide–negative cells) or late apoptosis and/or necrotic cell death (annexin V–and propidium iodide–positive cells)28 during the cultivation of ILC2 cells in conditions of stimulation with IL-2 alone, IL-2 plus IL-25, or IL-33 alone (Supplementary Fig. 3). Instead, interferons protected ILC2 cells from death (Supplementary Fig. 3). Such a protective role for interferons in T cells has also been reported29. Moreover, IFN-β and IFN-γ strongly suppressed the production of IL-5 and IL-13 by ILC2 cells mediated by IL-2 plus IL-25 or by IL-33 (Fig. 3b). Intracellular cytokine staining also demonstrated that IFN-γ suppressed not only cytokine secretion from but also cytokine expression in ILC2 cells (Fig. 3c) in an IFNGR1-dependent manner (Fig. 3d). Collectively, these results indicated that both type I interferons and type II interferons suppressed ILC2 function. IFN- suppresses ILC2-mediated type 2 responses in vivo To demonstrate the suppressive effect of IFN-γ in vivo, we first administered IL-33 alone or the combination of IL-2 plus IL-25 

intratracheally into the lungs of wild-type mice, with or without IFN-γ. The number of ILC2 cells and eosinophils in the BALF was significantly decreased by IFN-γ (Fig. 4a,b). Furthermore, the amount of both IL-5 and IL-13 in the BALF was strongly suppressed by IFN-γ (Fig. 4c). To assess whether IFN-γ suppressed not only the induction of ILC2-mediated inflammation but also ongoing inflammation, we injected IFN-γ into mice 3 d after the administration of IL-33. The number of ILC2 cells in the BALF was significantly decreased by treatment with IFN-γ during ongoing inflammation (Fig. 4d), which suggested that IFN-γ also had a suppressive effect on activated ILC2 cells in vivo. Moreover, IFN-γ suppressed the production of eotaxin by ILC2 cells under stimulation with IL-33 (Fig. 4e). These results collectively indicated that IFN-γ suppressed both the proliferation and the cytokine production of ILC2 cells even after inflammation was induced and, accordingly, terminated type 2 immune responses. Consistent with the findings reported above, IL-33-induced airway hyper-reactivity was ameliorated by the injection of IFN-γ (Fig. 4f). Furthermore, cellular infiltration, caused by mainly IL-5- and eotaxindependent eosinophilia, and IL-13-dependent goblet cell hyperplasia were strongly suppressed by IFN-γ (Fig. 4g). Next we assessed the effect of IFN-γ on immune reactions induced by helminth infection. We first infected Rag2−/− mice and Il2rg−/− Rag2−/− mice with N. brasiliensis with or without adoptive transfer of ILC2 cells to confirm that the eosinophilia that developed during infection with N. brasiliensis was dependent on ILC2 cells. Both Rag2−/− mice and Il2rg−/−Rag2−/− mice given transfer of ILC2 cells showed severe eosinophilia in the BALF, whereas Il2rg−/−Rag2−/− mice not given ILC2 cells did not show eosinophilia (Fig. 4h). N. brasiliensis is known to invade the host skin and migrate to the lungs via the bloodstream 24–48 h after infection. It is then coughed up and swallowed and moves to the intestine on day 3 or 4. Adult worms begin to produce eggs 5–6 d after infection14. To assess the effect of IFN-γ on immune reactions during infection with N. brasiliensis, we injected IFN-γ intratracheally into mice 3 h after infection with N. brasiliensis and collected BALF on day 6, when the aDVANCE ONLINE PUBLICATION  nature immunology

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© 2015 Nature America, Inc. All rights reserved.

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Figure 3  Interferons suppress the proliferation and cytokine production of ILC2 cells. (a) Quantification of sorted FALC ILC2 cells cultured (5 × 103 cells per well) for 6 d with medium alone (Med) or stimulated with IL-2 alone, IL-2 plus IL-25, or IL-33 alone (horizontal axis) in the presence of no additional cytokines (–), IFN-β, IFN-γ, IL-10 or IL-12 (key), assessed by trypan blue staining. (b) ELISA of IL-5, IL-6 and IL-13 in supernatants of ILC2 cells cultured as in a. (c) Intracellular staining of IL-5 and IL-13 in ILC2 cells cultured for 7 d with IL-2 plus IL-25 or with IL-33 (above plots), in the presence (+IFN-γ) or absence (−IFN-γ) of IFN-γ (key). (d) Intracellular staining of IL-5 and IL-13 in ILC2 cells obtained from the lungs of wild-type or Ifngr1−/−Rag2−/− mice (above plots) and treated with IL-2 plus IL-7 for population expansion, then cultured for 2 d with IL-2 plus IL-25, with or without IFN-γ (key). NS, not significant (P > 0.05); *P < 0.05 (Mann-Whitney U-test). Data are representative of two to three independent experiments with similar results (mean and s.e.m. of n = 4 wells in a,b).

helminths had already passed through the lungs and reached the small intestine. Similar to the results obtained in the cytokine-injection experiments, IFN-γ suppressed the accumulation of ILC2 cells and eosinophils in BALF during N. brasiliensis infection (Fig. 4i). It is known that IL-5-induced eosinophilia and IL-13-induced secretion of mucin from goblet cells accelerate the expulsion of N. brasiliensis. Accordingly, the suppression of ILC2 cells in BALF by injection of IFN-γ resulted in an increased number of adult worms in the small intestine and eggs in the feces on day 7, a time point when hosts began expelling worms (Fig. 4j). Furthermore, worms isolated from mice treated with IFN-γ were larger and healthier than those from untreated (control) mice (data not shown), consistent with the current understanding that the immunological attack by eosinophils in the lungs, in addition to mucin production via goblet-cell hyperplasia in the intestine, has an important role in the defense against helminth infection. These results demonstrated that IFN-γ suppressed type 2 innate immune responses in vivo. Defective IFN- action enhances type 2 responses in vivo As shown above, exogenous treatment with IFN-γ strongly suppressed ILC2 effector function. We next assessed the effect of endogenous IFN-γ on anti-helminth immune responses. First, to determine the cell types that produce IFN-γ, we studied mice with cDNA encoding the fluorescent protein Venus inserted into the Ifng locus. We infected these mice with N. brasiliensis and assessed the expression of Venus as a ‘readout’ of IFN-γ expression in various cell types on day 8. Natural killer (NK) cells, NKT cells and a portion of dendritic cells were Venus+ in the lungs, even under steady-state conditions (Fig. 5a). Furthermore, NKT cells increased their expression of Venus and a fraction of T cells became Venus+ after infection (Fig. 5a), which suggested that acquired cells of the immune system, such as NKT cells and T cells, produced IFN-γ during helminth infection. Next, we infected Ifngr1−/−Rag2−/− mice with N. brasiliensis to assess whether the absence of IFN-γ signaling would prolong type 2 immune responses during helminth infection. The number of ILC2

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cells in BALF was significantly higher in Ifngr1−/−Rag2−/− mice than in Ifngr1+/−Rag2−/− mice after infection (Fig. 5b), whereas lung ILC2 cells did not significantly increase in Ifngr1−/−Rag2−/− mice after infection, although both BALF ILC2 cells and lung ILC2 cells had similar amounts of IFN-γ receptors (Fig. 5c). Eosinophilia in both BALF and lung parenchyma was more severe in Ifngr1−/−Rag2−/− mice than in Ifngr1+/−Rag2−/− mice (Fig. 5b). These data supported the hypothesis that endogenous IFN-γ was involved in the suppression or termination of ILC2-dependent inflammation after helminths had exited from the lungs. The enhanced responses of ILC2 cells in the absence of IFN-γ signaling suggested that IFN-γ was produced at least locally during the helminth infection. To confirm the proposal that IFN-γ acted directly on ILC2 cells in vivo and suppressed type 2 immune responses, we performed adoptive-transfer experiments. We isolated ILC2 cells from the lungs of wild-type or Ifngr1−/−Rag2−/− mice, cultured the cells under stimulation with IL-2 plus IL-7 in vitro to elicit proliferation, then transferred the cells into Il2rg−/−Rag2−/− mice. We injected IL-33 together with IFN-γ intratracheally into mice on days 0 and 3, and isolated BALF and lungs on day 6. The number of ILC2 cells and eosinophils in both BALF and lungs was much higher in mice given transfer of Ifngr1−/−Rag2−/− ILC2 cells than in those of mice given transfer of wild-type ILC2 cells (Fig. 5d,e). Moreover, titers of type 2 cytokines in the BALF were also significantly higher in mice given transfer of Ifngr1−/−Rag2−/− ILC2 cells than in mice given transfer of wild-type ILC2 cells (Fig. 5f); this demonstrated that IFN-γ directly suppressed the ILC2 cells, which resulted in the termination of type 2 inflammation. These data collectively suggested that acquired cells of the immune system produced IFN-γ to terminate type 2 immune responses by ILC2 cells, which were acute and broad but not antigen specific, and replaced ILC2-mediated innate responses with antigenspecific responses mediated by acquired cells of the immune system, such as TH2 cells. Such a mechanism would probably be important for the prevention of excess type 2 immune responses that can lead to the induction of chronic allergic inflammation.



Articles receptors30. We therefore assessed the role of STAT1 by isolating ILC2 cells from Stat1−/− mice and incubating them with or without IFN-γ under the condition of stimulation with IL-33. The effects of IL-33

Suppressive effects of interferon on ILC2 cells mediated by STAT1 STAT1 is a major transcription factor and transducer for signaling through both type I interferon receptors and type II interferon

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© 2015 Nature America, Inc. All rights reserved.

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Figure 4  IFN-γ suppresses type 2 immune responses mediated by ILC2 cells. (a) Flow cytometry of BALF cells collected from C57BL/6 mice (n = 3 per condition) on day 6 after intratracheal injection (on days 0 and 3) of PBS, IL-2 plus IL-25, or IL-33 (top), with (+) or without (–) IFN-γ (1 µg per mouse) (above plots). Numbers adjacent to outlined areas indicate percent ILC2 cells (FSC loSSCloLin−CD25+IL-1RL1+) (top row) or percent eosinophils (SSChiSiglec-Fhi) (bottom row). (b) Quantification of ILC2 cells and eosinophils in BALF of mice as in a. (c) ELISA of IL-5 and IL-13 in BALF of mice as in a. (d) Quantification of ILC2 cells in mice given intratracheal injection of IL-33 on day 0, alone (n = 4 mice) or with additional injection of IFN-γ on day 3 (n = 4 mice), assessed on day 6. (e) Eotaxin secreted by ILC2 cells sorted from the mesentery and cultured (5 × 10 3 cells per well; n = 5 wells per group) for 5 d with IL-33 alone or with IL-33 plus IFN-γ (horizontal axis). (f) Airway hyper-reactivity in mice (n = 5 per group) given intratracheal injection of PBS, IL-33 alone, or IL-33 plus IFN-γ (key) on days 0 and 3, assessed as airway resistance (R) on day 6 after challenge with various concentrations of aerosolized methacholine (horizontal axis). (g) Histological analysis of lungs from mice as in f, stained with hematoxylin and eosin (H&E) or periodic acid–Schiff reagent (PAS). Outlined areas in top row indicate areas enlarged below. Scale bars, 400 µm (top row of each group) or 100 µm (bottom row of each group). (h) Flow cytometry of BALF cells from Rag2−/− mice given no ILC2 cells (−ILC2) or from Il2rg−/−Rag2−/− mice given no ILC2 cells (−ILC2) or given intravenous transfer of IL-2-cultured FALC ILC2 cells (7 × 10 6 per mouse) (+ILC2), then, 4 h later, left uninfected (UI) or infected by subcutaneous injection of 500 viable third-stage N. brasiliensis larvae (NB), followed by analysis 5 d after infection. Numbers adjacent to outlined areas indicate percent ILC2 (FSCloLin−MHCII−CD25+ IL-1RL1+) cells (top row) or eosinophils (FCSloT IL-1RL1−SSChiSiglec-Fhi) (bottom row). (i) Quantification of ILC2 cells (FSCloSSCloCD45+Lin−KLRG-1+Thy-1+ IL-1RL1+CD25+) and eosinophils (FCSloSSChiCD45+Siglec-FhiF4/80int) in BALF from wild-type mice (n = 6–7 per group) left uninfected or infected as in h, then given intratracheal injection of PBS or IFN-γ (1 µg per mouse) 3 h after infection and on days 2 and 4, followed by analysis on day 7. (j) Quantification of adult worms in the small intestine and eggs in the feces of infected mice as in i, assessed on day 7. Each symbol (b–e,i,j) represents an individual mouse (b,c,d,i,j) or well (e); small horizontal lines indicate the mean (b,c,d,i,j) or average (e). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (one-way analysis of variance (ANOVA) followed by Dunnett’s test (b,c,f), or Mann-Whitney U-test (d,e,i,j)). Data are representative of two to five independent experiments (mean ± s.e.m. in f).



aDVANCE ONLINE PUBLICATION  nature immunology

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on ILC2 cells, such as proliferation and cytokine production, were not suppressed by IFN-γ in the absence of STAT1 (Fig. 6a), which indicated that the suppressive effects of IFN-γ on IL-33-triggered proliferation and type 2 cytokine production by ILC2 cells were mediated by STAT1. It has been shown that IL-33-induced proliferation of ILC2 cells is inhibited by IL-27 (ref. 19), a cytokine whose signaling pathway also requires STAT1 in T cells31. Indeed, IL-27 suppressed both the IL-33-induced proliferation and the cytokine production of ILC2 cells similarly to IFN-γ (Supplementary Fig. 4). Therefore, we assessed the effect of IL-27 on ILC2 cells from wild-type and Stat1−/− mice. We found that IL-27 also suppressed the IL-33-mediated proliferation and type 2 cytokine production of ILC2 cells in a STAT1-dependent manner (Fig. 6a). Gene-expression analysis by RNA sequencing showed that both IL-27 and IFN-γ upregulated the expression of Stat1 mRNA and Irf1 mRNA (which encodes the transcription factor IRF1), both of which are regulated downstream of STAT1-mediated signaling, but produced no difference in the expression of Gata3, which encodes a transcription factor critical for ILC2 function32 (Fig. 6b and Supplementary Fig. 5). Similar to the effects of interferons

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Figure 5  A defect in the action of IFN-γ exacerbates type 2 immune responses. (a) Expression of Venus (reporting IFN-γ) by cells from the lungs of an uninfected wildtype mouse (UI WT (lung)) or IfngVenus/+ mouse (UI (lung)) and from BALF and lungs of IfngVenus/+ mice on day 8 after infection with N. brasiliensis (as in Fig. 4h), assessed by flow cytometry of cells gated as CD11c−F4/80+Gr-1+ (macrophages), CD11c+MHCII+ (DCs), NK1.1+CD3− (NK cells), NK1.1+CD3+ (NKT cells) or NK1.1−CD3+ (T cells). (b) Quantification of ILC2 cells (FSCloSSCloPI−Lin−Thy-1+KLRG-1+ IL-1RL1+) and eosinophils (FCSloSSChiKLRG-1−Siglec-F+) in the BALF and lungs of Ifngr1+/−Rag2−/− or Ifngr1−/−Rag2−/− mice (n = 7–8) on day 6 after infection as in a, analyzed by flow cytometry. (c) IFNGR1 expression on ILC2 cells (FSCloSSCloLin−CD45+KLRG1+Sca-1+) of BALF and lungs from C57BL/6 mice given intranasal injection of IL-2 plus IL-25 or of IL-33 on days 0 and 2, followed by analysis on day 4 with monoclonal antibody (mAb) to IFNGR1 or istoype-matched control antibody. (d) Flow cytometry of ILC2 cells (FSCloSSCloPI−Lin−C D45+KLRG1+Thy-1+ IL-1RL1+CD25+) from BALF and lungs of Il2rg−/−Rag2−/− host mice (n = 4) given intravenous injection, on day 0, of wild-type (WT) or Ifngr1−/−Rag2−/− (KO) lung ILC2 cells (8.2 × 105 cells per host mouse) cultured with IL-2 plus IL-7 (10 ng/ml each), along with intratracheal injection of IL-33 plus IFN-γ (500 ng per mouse) into the host mice on days 0 and 3, followed by analysis on day 6. (e) Quantification of ILC2 cells (top row) and eosinophils (FSCloSSChiPI−CD45+SiglecFhiF4/80int) (bottom row) in the BALF and lungs of mice as in d. (f) ELISA of IL5 and IL-13 in BALF of mice as in d. Each symbol (b,e,f) represents an individual mouse; small horizontal lines indicate the average. *P < 0.05, **P < 0.01 and ***P < 0.001 (Mann-Whitney U-test). Data are representative of two to three independent experiments with similar results.

IL-5 (pg/ml)

© 2015 Nature America, Inc. All rights reserved.

Articles

KO

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described above, IL-27 also suppressed lung inflammation induced by the administration of IL-33 in vivo (data not shown). The administration of IL-25 induces iILC2 cells in the lungs and mesenteric lymph nodes26. We next assessed the responsiveness of iILC2 cells to IFN-γ and IL-27. We isolated iILC2 cells from mesenteric lymph nodes of Rag2−/− mice that were given administration of IL-25, then stimulated the cells with IL-33 in the presence of IFN-γ or IL-27. Because iILC2 cells did not respond to IL-33 alone, and IL-7 was required for their activation33 (Fig. 1g), we added IL-7 to the cultures in this experiment. The proliferation and cytokine production of FALC ILC2 cells and lung ILC2 cells from naive mice were similarly suppressed by either IFN-γ or IL-27 with or without IL-7 (Fig. 6c and Supplementary Fig. 6). Notably, iILC2 cells were not suppressed by IFN-γ in the presence of IL-7, and the effect of IL-27 was weaker on iILC2 cells than on FALC ILC2 cells (Fig. 6c). Furthermore, IFN-γ had no effect on the generation or proliferation of iILC2 cells in mesenteric lymph nodes after the administration of IL-25 (Fig. 6d). These results demonstrated a difference in the sensitivity of iILC2 cells to IFN-γ and IL-27 and that of tissue-resident 

Articles a

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Figure 6  STAT1-mediated suppression of ILC2 NS 10 200 600 NS NS * function by IFN-γ and IL-27. (a) Quantification *** of ILC2 cells sorted from the mesentery of 7.5 150 * 400 IL-33 wild-type and Stat1−/− mice and cultured 5.0 100 IL-33 + IFN-γ (1,650 cells per well; n = 3 wells per group) IL-33 + IL-27 200 2.5 50 **** for 6 d in the presence of IL-33 alone, IL-33 *** **** ND **** **** plus IFN-γ, or IL-33 plus IL-27 (key), assessed 0 0 0 –/– –/– WT Stat1–/– WT Stat1 WT Stat1 by flow cytometry with propidium iodide staining (far left), and ELISA of IL-5 (middle) 10 10 10 IL-33 IL-33 ***** NS + and IL-13 (right) in the supernatants of IL-33 + ******* NS IFN-γ IL-27 8 8 8 those cells. (b) Expression of Stat1, Irf1 IL-33 Stat1 6 6 6 IL-33 + IFN-γ and Gata3 by ILC2 cells from the mesentery Irf1 IL-33 + IL-27 4 4 4 Gata3 48 h after stimulation as in a (left), 2 2 2 presented as log2 FPKM values (fragments LOW High 0 0 0 Expression per kilobase of transcript per million mapped IL-33 ILC2 cells ILC2 cells reads) at right. (c) Quantification (as in a) IL-33 + IFN-γ 25 500 1,500 IL-33 + IL-27 of ILC2 cells sorted from the mesentery 400 20 of naive wild-type mice and of iILC2 cells 1,000 300 15 sorted from mesenteric lymph nodes **** −/− **** 200 10 of Rag2 mice on day 4 after intraperitoneal **** *** 500 **** **** **** **** **** 100 5 injection of IL-25 (0.4 µg per mouse, on **** **** **** days 0, 1, 2 and 3), then cultured (3,800 0 0 0 –IL-7 +IL-7 –IL-7 +IL-7 –IL-7 +IL-7 cells per well) as in a (key) in the presence iILC2 cells iILC2 cells iILC2 cells or absence of IL-7 (horizontal axes) (left), and 30 400 2,500 NS ELISA of IL-5 (middle) and IL-13 (right) NS NS 2,000 ** 300 20 in the supernatants of those cells on day 5. 1,500 ** 200 (d) Flow cytometry of cells collected from 1,000 ** 10 mesenteric lymph nodes of Rag2−/− mice 100 ** ** 500 on day 4 after intraperitoneal injection of ** ** * * 0 0 0 IL-25 (0.5 µg per mouse) or IL-25 plus +IL-7 –IL-7 +IL-7 –IL-7 +IL-7 –IL-7 IFN-γ (0.5 µg per mouse) on days IL-25 + IFN-γ IL-25 4 10 0, 1 and 2. Numbers adjacent to outlined areas indicate percent iILC2 cells (Lin −KLRG-1+ICOShi) in the 3 + 10 CD45 fraction of the lymphocyte gate, determined by forward and side scatter. Each symbol (a,c) represents 95 93 2 an individual well; small horizontal lines indicate the average. *P < 0.05, **P < 0.01, ***P < 0.001 and 10 1 ****P < 0.0001, versus open symbols or bars (one-way ANOVA followed by Dunnett’s test). Data are 10 representative of two independent experiments with similar results (a,c,d) or are representative of three 100 100 101 102 103 104 independent experiments (b; mean and s.e.m.). Gata3 (log2 FPKM)

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© 2015 Nature America, Inc. All rights reserved.

3

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ILC2 cells. We speculate that a strong suppression mechanism would be necessary for tissue-resident ILC2 cells but not for iILC2 cells, because tissue-resident ILC2 cells survived for a long period of time after activation (Fig. 1g). IL-27 suppresses type 2 immune responses of ILC2 cells in vivo A. alternata is a major fungus associated with ILC2-mediated asthma34,35. Intranasal injection of an extract of A. alternata into wild-type mice increased the amount of IL-33 in the BALF, which was not suppressed by IFN-γ or IL-27 (Fig. 7a and data not shown). To assess the ability of ILC2 cells and CD4+ T cells to produce IL-5 and IL-13, we isolated both types of cells from the lungs 8 d after injection of A. alternata extract and stimulated the cells with the phorbol ester PMA plus ionomycin. Both antigen-specific acquired immunity and epithelial cell–derived cytokine-mediated innate immunity were involved in this model (Fig. 7b). To investigate whether IFN-γ and IL-27 suppressed A. alternata–induced asthmatic symptoms, we injected A. alternata extract intranasally into wild-type mice with or without IL-27 or IFN-γ. IFN-γ and IL-27 significantly suppressed eosinophilia and the production of type 2 cytokines in the lungs (Fig. 7c and data not shown), and an in vitro experiment showed that IL-27 also strongly suppressed eotaxin production by ILC2 cells (Supplementary Fig. 7). Although the number of ILC2 cells in both BALF and lung was decreased by IL-27, the number of CD4+ T cells was decreased only in BALF, not in the lungs (Fig. 7d). Intracellular cytokine staining also showed that IL-27 suppressed the expression of type 2 cytokines by ILC2 cells but not by TH2 cells (Fig. 7e,f). These data indicated that IL-27 shut down the innate immune response of 

ILC2 cells but moderately affected acquired immune responses governed by T cells. Inflammation, as measured by cellular infiltration and goblet-cell hyperplasia, became milder via injection of IL-27 in the A. alternata model (Fig. 7g), but this moderate effect was probably due to the remaining T cell responses (Fig. 7f). Expression of the IL-27 receptor components WSX-1 and gp130 was much higher in ILC2 cells than in CD4+ T cells (Fig. 7h), which suggested that the differential effects of IL-27 on T cells and ILC2 cells resulted from differences in expression of the IL-27 receptor. These data collectively suggested that the functions of ILC2 cells were terminated by type 1 cytokines, IFN-γ and IL-27. DISCUSSION Many laboratories have reported a variety of ILC2 populations with different phenotypes in various tissues and organs33,36,37. Although the functions, mechanism of differentiation and signaling pathways of ILC2 cells are well studied, it has remained unclear whether ILC2 cells in different tissues and organs with different phenotypes have distinct differentiation pathways and functions. It is also unknown whether ILC2 cells migrate and circulate in the body or are stably localized in tissues and organs. Our parabiosis analysis demonstrated that tissue-resident ILC2 cells did not migrate to other tissues in vivo, which indicated that tissue-resident ILC2 cells were activated in situ during inflammation but were able to migrate when artificially transferred into the circulation. It was unexpected that BALF ILC2 cells were not mixed in parabiotic mouse pairs, even though ILC2 cells are detectable in the peripheral blood38. It is likely that the BALF ILC2 cells were not derived from ILC2 cells circulating in the peripheral aDVANCE ONLINE PUBLICATION  nature immunology

Articles

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© 2015 Nature America, Inc. All rights reserved.

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Figure 7  IL-27 suppresses ILC2 cells in vitro and in vivo. (a) ELISA of IL-33 in the BALF of wild-type mice (n = 4 per group) 1 h after intranasal administration of PBS or A. alternata extract (AA) (10 µg per mouse). (b) Intracellular staining of IL-5 and IL-13 in T cells (FSC loSSCloLin+CD4+) and ILC2 cells (FSCloSSCloLin−Thy-1.2+ doublet-negative) in the lungs of mice on day 8 after intranasal administration of PBS or A. alternata extract (10 µg per mouse) on days 0, 3 and 6, assessed by flow cytometry. (c) Quantification of eosinophils in the BALF (left) (n = 3 mice) and ELISA of IL-5 (middle) and IL-13 (right) on day 8 after intranasal administration, on days 0, 3 and 6, of PBS or A. alternata extract (10 µg per mouse; n = 3–7 mice per group) with (AA + IL-27) or without (AA) IL-27 (0.1 µg per mouse). (d) Quantification of ILC2 cells (FSCloSSCloLin−Thy-1.2+) and CD4+ T cells (FSCloSSCloCD4+) in BALF and one half of the lungs of mice (n = 4) on day 8 after administration of A. alternata extract and IL-27 as in c, assessed by flow cytometry. (e) Intracellular staining of IL-5 and IL-13 in lung cells of mice as in c. Numbers adjacent to outlined areas indicate percent T H2 cells (FSCloSSCloLin+) (black gate) or ILC2 cells (FSCloSSCloLin−) (red gate). (f) Quantification of IL-5+ or IL-13+ ILC2 cells and T cells (n = 4 mice) gated as in d, assessed by flow cytometry. (g) Histological analysis of lungs from mice treated as in c. Outlined areas at left enlarged at right. Scale bars, 400 µm (left column of each pair) or 100 µm (right column of each pair). (h) Expression of mRNA encoding WSX-1 (Il27ra) or gp130 (Il6st) in ILC2 cells from the mesentery and CD4+ T cells from the spleen of untreated wild-type mice (n = 6); results are normalized to those of Gapdh mRNA. Each symbol represents an individual mouse (a,c,d,f) or sample (h); small horizontal lines indicate the average. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, versus IL-27 at 0 ng/ml for d,f (Mann-Whitney U-test (a,h) or one-way ANOVA followed by Dunnett’s test (c,d,f)). Data are representative of two to three independent experiments with similar results.

blood but were derived from tissue-resident ILC2 cells present in the lungs, bronchioles or alveoli. The inability of ILC2 cells to migrate between tissues and organs might be the cause of reported differences in the phenotypes of ILC2 cells in the various tissues and organs from which they are isolated. ILC2 cells are important in anti-helminth and anti-fungal immunity and, at the same time, exacerbate allergic inflammation. In fact, Löffler’s syndrome, induced by helminth infection, results in substantial eosinophilia in the lungs, similar to the eosinophilic inflammation observed in eosinophilic pseumonia39. Here we addressed the question of how such a strong inflammatory response is terminated. nature immunology  aDVANCE ONLINE PUBLICATION

We found that IFN-β, IFN-γ and IL-27 strongly suppressed the proliferation of ILC2 cells and expression of type 2 cytokines by ILC2 cells through a STAT1-dependent pathway. These data demonstrated that these cytokines mediated the suppression of type 2 responses as a negative feedback regulatory mechanism for both innate immunity and adaptive immunity in vivo. NK cells, NKT cells and CD11c+MHCII+ dendritic cells constitutively produced IFN-γ, and T cells were induced to produce IFN-γ several days after helminth infection, which suggested that acquired cells of the immune system suppressed ILC2 function to switch the immune response from an antigenindependent phase to an antigen-dependent phase. The finding 

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Articles that ILC2 cells expressed type I interferon receptors indicated that ILC2-mediated inflammatory responses would probably be suppressed during viral infection. The accompanying paper demonstrates this is indeed the case40. Myeloid cells such as dendritic cells produce IL-27 during inflammation41,42, which might be the source of IL-27 in vivo. STAT1 has a critical role in receptor signaling for both IFN-γ30 and IL-27 (ref. 31). In fact, our results demonstrated that STAT1 was critical for the suppressive functions of both of these cytokines. Tissue-resident naive ILC2 cells or natural helper cells express IL-1RL1 and vigorously respond to IL-33 but do not respond to IL-25 alone9. It is reported that in reporter mice expressing green fluorescent protein (GFP)-tagged IL-13, helminth infection as well as administration of IL-25 or IL-33 induces GFP+ cells (a cell type called ‘nuocytes’ by these authors)33. The administration of IL-25 induces GFP+ nuocytes, including IL-1RL1+ and IL-1RL1− populations, in lymph nodes33. These nuocytes are induced to express uniformly large amounts of IL-1RL1 after cultivation with IL-7 and IL-33. Another study has identified a similar cell population in the lungs and mesenteric lymph nodes induced by the administration of IL-25; these IL-1RL1− cells have been called ‘iILC2 cells’26. iILC2 cells acquire expression of IL-1RL1 and responsiveness to IL-33 after culture in vitro or adoptive transfer in vivo. Stimulation of iILC2 cells by IL-33 after cultivation with IL-7 results in the production of IL-5 and IL-13, but stimulation with IL-25 after such culture does not 26. Because helminth infection resulted in the induction of both IL-25 and IL-33, it is likely that the GFP + nuocyte populations induced by helminth infection33 include both IL-33-activated tissue-resident ILC2 cells or natural helper cells9 and IL-25-activated iILC2 cells26. We found here that ILC2 cells and iILC2 cells showed different sensitivity to IFN-γ. Contrary to tissue-resident ILC2 cells, iILC2 cells isolated from the mesenteric lymph nodes of IL-25-treated Rag2−/− mice were resistant to IFN-γ in the presence of IL-7. In addition, the induction of iILC2 cells in the mesenteric lymph nodes was not blocked by IFN-γ. The effects of IFN-γ on the number and cytokine production of ILC2 cells in the lungs during helminth infection were not as strong as those in lung inflammation induced by cytokine administration. This was probably because ILC2 cells in BALF during helminth infection included activated lung-resident ILC2 cells and iILC2 cells, and iILC2 cells are not suppressed by IFN-γ in the presence of IL-7, a cytokine that is constitutively produced in vivo. When anti-helminth adaptive immunity is established and activated T cells produce IFN-γ, its signaling would suppress TH2 cells and tissue-resident ILC2 function but not iILC2 cells. IL-27 seemed to be more important for the suppression of iILC2 cells than that of tissueresident ILC2 cells. Functional cooperation between ILC2 cells and regulatory T cells during metabolic homeostasis has been reported43. That study showed a suppressive effect of IFN-γ on ILC2 cells similar to that observed in our study here. We further showed that IFN-γ acted directly on ILC2 cells. Contrary to our results, however, that earlier study observed no effect of IL-27 on ILC2 cells43. The reason for this discrepancy is currently unknown. Our study has demonstrated negative feedback mechanisms for type 2 innate inflammatory responses by type 1 cytokines, as observed in TH2 responses. Future studies should investigate whether similar negative feedback mechanisms are involved in the regulation of other ILCs, including group 1 ILCs and group 3 ILCs. Methods Methods and any associated references are available in the online version of the paper. 10

Accession codes. GEO: RNA sequence data, GSE73272. Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments We thank M. Kubo (Tokyo University of Science) for IfngVenus/+ mice; M. Miyasaka (Osaka University) for antibody to mouse IL-2Rβ (TM-β1); S. Wada, T. Yamamoto, T. Shitamichi, U. Tran and S. Tada for animal care; M. Yamamoto, S. Kagawa, T. Fukushima and J. Furusawa for help in some experiments; Kafi N. Ealey for critical reading of this manuscript; and members of the Laboratory for Immune Cell Systems at RIKEN Integrative Medical Sciences for discussion. Supported by Precursory Research for Embryonic Science and Technology from Japan Science and Technology Agency Japan Society for the Promotion of Science (Grant-in Aid for Scientific Research (B) 26293110; Grant-in-Aid for Challenging Exploratory Research 24659373 to K.M.; Grant-in-Aid for Scientific Research (S) 22229004 to S.K.) and the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Scientific Research on Innovative Areas 15H01166 to K.M.). AUTHOR CONTRIBUTIONS K.M. designed and interpreted the experiments and wrote the manuscript; H.K. designed and performed IL-27 experiments and wrote the manuscript; M.T. maintained N. brasiliensis and supervised helminth infection experiments; Sa.K. assessed chemokine receptor expression and performed helminth infection experiments; N.T. and M.M. performed experiments under the supervision of K.M.; K.F., K.A. and T.B. discussed research with H.K. and provided insight into the study design; and Sh.K. interpreted the experiments and wrote the manuscript. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests: details are available in the online version of the paper. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. Zhao, G., Zhou, S., Davie, A. & Su, Q. Effects of moderate and high intensity exercise on T1/T2 balance. Exerc. Immunol. Rev. 18, 98–114 (2012). 2. Matsuzaki, J., Tsuji, T., Imazeki, I., Ikeda, H. & Nishimura, T. Immunosteroid as a regulator for Th1/Th2 balance: its possible role in autoimmune diseases. Autoimmunity 38, 369–375 (2005). 3. Greenberg, S. & Grinstein, S. Phagocytosis and innate immunity. Curr. Opin. Immunol. 14, 136–145 (2002). 4. Elbim, C. & Estaquier, J. Cytokines modulate neutrophil death. Eur. Cytokine Netw. 21, 1–6 (2010). 5. Lawrence, T. & Fong, C. The resolution of inflammation: anti-inflammatory roles for NF-kappaB. Int. J. Biochem. Cell Biol. 42, 519–523 (2010). 6. Spits, H. & Di Santo, J.P. The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodeling. Nat. Immunol. 12, 21–27 (2011). 7. Eberl, G., Di Santo, J.P. & Vivier, E. The brave new world of innate lymphoid cells. Nat. Immunol. 16, 1–5 (2015). 8. Cortez, V.S., Robinette, M.L. & Colonna, M. Innate lymphoid cells: new insights into function and development. Curr. Opin. Immunol. 32, 71–77 (2015). 9. Moro, K. et al. Innate production of TH2 cytokines by adipose tissue-associated c-Kit+Sca-1+ lymphoid cells. Nature 463, 540–544 (2010). 10. Liu, Y.J. Thymic stromal lymphopoietin: master switch for allergic inflammation. J. Exp. Med. 203, 269–273 (2006). 11. Zaph, C. et al. Epithelial-cell-intrinsic IKK-β expression regulates intestinal immune homeostasis. Nature 446, 552–556 (2007). 12. Bulek, K., Swaidani, S., Aronica, M. & Li, X. Epithelium: the interplay between innate and Th2 immunity. Immunol. Cell Biol. 88, 257–268 (2010). 13. Ziegler, S.F. & Artis, D. Sensing the outside world: TSLP regulates barrier immunity. Nat. Immunol. 11, 289–293 (2010). 14. Koyasu, S., Moro, K., Tanabe, M. & Takeuchi, T. Natural helper cells: a new player in the innate immune response against helminth infection. Adv. Immunol. 108, 21–44 (2010). 15. Bartemes, K.R. et al. IL-33-responsive lineage− CD25+ CD44hi lymphoid cells mediate innate type 2 immunity and allergic inflammation in the lungs. J. Immunol. 188, 1503–1513 (2012). 16. Halim, T.Y., Krauss, R.H., Sun, A.C. & Takei, F. Lung natural helper cells are a critical source of Th2 cell-type cytokines in protease allergen-induced airway inflammation. Immunity 36, 451–463 (2012). 17. Kim, B.S. et al. TSLP elicits IL-33-independent innate lymphoid cell responses to promote skin inflammation. Sci. Transl. Med. 5, 170ra116 (2013). 18. Salimi, M. et al. A role for IL-25 and IL-33-driven type-2 innate lymphoid cells in atopic dermatitis. J. Exp. Med. 210, 2939–2950 (2013). 19. Kabata, H. et al. Thymic stromal lymphopoietin induces corticosteroid resistance in natural helper cells during airway inflammation. Nat. Commun. 4, 2675 (2013).

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Articles 20. Monticelli, L.A. et al. Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus. Nat. Immunol. 12, 1045–1054 (2011). 21. Zaiss, D.M., Gause, W.C., Osborne, L.C. & Artis, D. Emerging functions of amphiregulin in orchestrating immunity, inflammation, and tissue repair. Immunity 42, 216–226 (2015). 22. Chen, F. et al. An essential role for TH2-type responses in limiting acute tissue damage during experimental helminth infection. Nat. Med. 18, 260–266 (2012). 23. Kehrl, J.H., Hwang, I.Y. & Park, C. Chemoattract receptor signaling and its role in lymphocyte motility and trafficking. Curr. Top. Microbiol. Immunol. 334, 107–127 (2009). 24. Chiba, K., Matsuyuki, H., Maeda, Y. & Sugahara, K. Role of sphingosine 1-phosphate receptor type 1 in lymphocyte egress from secondary lymphoid tissues and thymus. Cell. Mol. Immunol. 3, 11–19 (2006). 25. Conroy, D.M. et al. The role of the eosinophil-selective chemokine, eotaxin, in allergic and non-allergic airways inflammation. Mem. Inst. Oswaldo Cruz 92 (suppl. 2), 183–191 (1997). 26. Huang, Y. et al. IL-25-responsive, lineage-negative KLRG1hi cells are multipotential ‘inflammatory’ type 2 innate lymphoid cells. Nat. Immunol. 16, 161–169 (2015). 27. Motomura, Y. et al. Basophil-derived interleukin-4 controls the function of natural helper cells, a member of ILC2s, in lung inflammation. Immunity 40, 758–771 (2014). 28. Suzuki, J., Denning, D.P., Imanishi, E., Horvitz, H.R. & Nagata, S. Xk-related protein 8 and CED-8 promote phosphatidylserine exposure in apoptotic cells. Science 341, 403–406 (2013). 29. Moro, H., Otero, D.C., Tanabe, Y. & David, M. T cell-intrinsic and -extrinsic contributions of the IFNAR/STAT1-axis to thymocyte survival. PLoS ONE 6, e24972 (2011). 30. Jahnke, A. & Johnson, J.P. Synergistic activation of intercellular adhesion molecule 1 (ICAM-1) by TNF-α and IFN-γ is mediated by p65/p50 and p65/c-Rel and interferon-responsive factor Stat1 alpha (p91) that can be activated by both IFN-γ and IFN-α. FEBS Lett. 354, 220–226 (1994). 31. Takeda, A. et al. Cutting edge: role of IL-27/WSX-1 signaling for induction of T-bet through activation of STAT1 during initial Th1 commitment. J. Immunol. 170, 4886–4890 (2003).

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32. Furusawa, J. et al. Critical role of p38 and GATA3 in natural helper cell function. J. Immunol. 191, 1818–1826 (2013). 33. Neill, D.R. et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 464, 1367–1370 (2010). 34. Kim, H.K. et al. Innate type 2 response to Alternaria extract enhances ryegrassinduced lung inflammation. Int. Arch. Allergy Immunol. 163, 92–105 (2014). 35. McSorley, H.J., Blair, N.F., Smith, K.A., McKenzie, A.N. & Maizels, R.M. Blockade of IL-33 release and suppression of type 2 innate lymphoid cell responses by helminth secreted products in airway allergy. Mucosal Immunol. 7, 1068–1078 (2014). 36. Chang, Y.J. et al. Innate lymphoid cells mediate influenza-induced airway hyperreactivity independently of adaptive immunity. Nat. Immunol. 12, 631–638 (2011). 37. Price, A.E. et al. Systemically dispersed innate IL-13-expressing cells in type 2 immunity. Proc. Natl. Acad. Sci. USA 107, 11489–11494 (2010). 38. Bartemes, K.R., Kephart, G.M., Fox, S.J. & Kita, H. Enhanced innate type 2 immune response in peripheral blood from patients with asthma. J. Allergy Clin. Immunol. 134, 671–678 (2014). 39. Culley, F.J., Brown, A., Girod, N., Pritchard, D.I. & Williams, T.J. Innate and cognate mechanisms of pulmonary eosinophilia in helminth infection. Eur. J. Immunol. 32, 1376–1385 (2002). 40. Duerr, C.U. et al. Type I interferon restricts type 2 immunopathology through the regulation of group 2 innate lymphoid cells. Nat. Immunol. doi: 10.1038/ni.3309 (23 November 2015). 41. Siegemund, S. et al. Production of IL-12, IL-23 and IL-27p28 by bone marrowderived conventional dendritic cells rather than macrophages after LPS/TLR4dependent induction by Salmonella Enteritidis. Immunobiology 212, 739–750 (2007). 42. Smits, H.H. et al. Commensal Gram-negative bacteria prime human dendritic cells for enhanced IL-23 and IL-27 expression and enhanced Th1 development. Eur. J. Immunol. 34, 1371–1380 (2004). 43. Molofsky, A.B. et al. Interleukin-33 and Interferon-gamma counter-regulate group 2 innate lymphoid cell activation during immune perturbation. Immunity 43, 161–174 (2015).

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ONLINE METHODS

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Mice. Mice used in this study were on a C57BL/6 background and were maintained in the RIKEN Center for Integrative Medical Sciences or Keio University School of Medicine under specific pathogen–free conditions. Wild-type C57BL/6 mice were purchased from Charles River Japan, Japan SLC or CLEA Japan. B6-Rag2−/− mice (RAGN12), B6.SJL-Rag2−/− mice (461), Il2rg−/−Rag2−/− mice (4111) and B6.SJL mice (4007) were purchased from Taconic. Ifngr1−/− mice (003288) were purchased from Jackson Laboratory and were crossed with Rag2−/− mice to generate Ifngr1−/−Rag2−/− mice. Stat1−/− mice44 were provided by A. Yoshimura (Keio University School of Medicine), and IfngVenus/+ mice (provided by M. Kubo (Tokyo University of Science)) were established. ILC2 cells were usually isolated from retired female breeder mice unless stated otherwise. All experiments were approved by the Animal Care and Use Committee of RIKEN or Keio University and were performed in accordance with institutional guidelines. Antibodies and reagents. Monoclonal antibodies (mAbs) specific for mouse c-Kit (2B8), IFNGR1α (GR20), CD25 (IL-2Rα) (PC61), IL-3Rβ (JORO50), IL-4Rα (mIL4R-M1), IL-5Rα (T21), IL-12Rβ1 (114), CXCR4 (2B11/CXCR4), CDCR5 (2G8), FcγR (2.4G2), CD3ε (145-2C11), CD4 (GK1.5), CD8α (53-6.7), CD5 (53-7.3), Gr-1 (RB6-8C5), erythroid cell marker (TER119), CD19 (1D3), CD11c (HL3), Mac-1 (M1/70), NK1.1 (PK136), Sca-1 (D7), CD45.1 (A20-1.7), CD45.2 (104), Siglec-F (E50-2440), MHC class II (M5/114.15.2), KLRG1(2F1) and annexin V were purchased from BD Pharmingen; mAbs to mouse IL-5 (TRFK5), IL-7Rα (A7R34), FcεRIα (MAR-1), CCR7 (4B12) and IL-13 (eBio13A) from eBioscience; mAbs to mouse CCR2 (475301) and CXCR6 (221002) were from R&D Systems; and mAb to mouse IL-1RL1 (DJ8) was from MD Biosciences. mAbs to mouse F4/80 (CI:A3-1), IL-17RB (9B10), IFNAR1 (MAR1-5A3), ICOS (C398.4A), IL-1R1 (JAMA-147), IL-3Rα (5B11), IL-6Rα (D7715A7), IL-10R (1B1.3a), CCR3 (TG14/CCR3), CCR4 (2G12), CCR5 (HM-CCR5), CCR6 (29-2L17), CCR9 (CW-1.2), CXCR2 (TG11/CXCR2), CXCR3 (CXCR3-173) and CXCR7 (8F11-M16) were purchased from BioLegend. Allophycocyanin-conjugated streptavidin (BD), phycoerythrin (PE)–indotricarbocyanine (Cy7)–conjugated streptavidin (BD), peridinin chlorophyll protein (PerCP)–cyanine 5.5–conjugated streptavidin (BD) or allophycocyanin-Cy7-conjugated streptavidin (BD) was used in conjunction with staining with biotinylated mAbs. Dead cells were gated out through the use of propidium iodide. Antibody to mouse IL-2Rβ (TM-β1) was provided by M. Miyasaka (Osaka University). Four-, eight-, nine- and eleven-color flow cytometry was performed on a FACSCalibur, FACSCantoII, FACSAriaIIu and FACSAriaIII (all from BD Bioscience). Data were analyzed with FlowJo Software (TreeStar). Mouse IL-10, mouse IL-12 and mouse IFN-γ were from PeproTech; mouse IFN-β was from PBL InterferonSource; and mouse IL-2, mouse IL-7, mouse IL-25, mouse IL-27 and mouse IL-33 were from R&D Systems. The extract of A. alternata was obtained from Greer Laboratories. Preparation of cell suspensions. Naive ILC2 cells were isolated from the mesentery or lungs as described45 with anti-c-Kit and anti-Sca-1 (both identified above) for FALC ILC2 cells and with anti-KLRG1 and anti-Sca-1 (both identified above) for lung ILC2 cells. BALF cells were collected from the lung by gentle washing with Hank’s balanced-salt solution containing 10% FCS, with 18G plastic cannulae and 1-ml syringes45. After BALF cells were washes, lung cells were isolated by a published method45. Inflammatory ILC2 cells were prepared from mesenteric lymph nodes of IL-25-treated Rag2−/− mice (0.4 µg per mouse on days 0, 1, 2 and 3) by a published method33. Measurement of cytokines. Sorted naive ILC2 cells were seeded into 96-well round-bottomed tissue culture plates in RPMI-1640 medium (Sigma) containing 10% FCS, 50 µM 2-mercaptoethanol (Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco), 1× non-essential amino acids (Sigma), 10 mM HEPES (Sigma) and 1 mM sodium pyruvate (Gibco), plus various stimulants, including IL-2 (10 ng/ml), IL-7 (10 ng/ml), IL-10 (10ng/ml), IL-12 (10 ng/ml), IL-25 (10 ng/ml), IL-33 (10 ng/ml), IL-27 (10 ng/ml), IFN-β (10 ng/ml) and IFN-γ (10 ng/ml). The concentration of cytokines in culture supernatants were determined by ELISA with Quantikine kits (R&D Systems)

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according to the manufacturer’s protocols, or by a multiplex system with Bio-Plex Pro Mouse cytokine 23-plex assay kits (Bio-Rad). Flow cytometry. For analysis of the expression of cytokine and chemokine receptors, ILC2 cells were left untreated or were cultured for 5–7 d with IL-2, a combination of IL-2 and IL-25, or IL-33. Freshly sorted ILC2 cells are called ‘naive ILC2 cells’ here. For intracellular staining of cytokines in in vitro experiments, ILC2 cells from the mesentery or lungs were stimulated with IL-2 plus IL-25 or with IL-33 (10 ng/ml each). For the final 3 h of culture, ILC2 cells were treated with brefeldin A at the recommended concentrations (eBioscience). For analysis of the cytokine-producing ability of ILC2 cells and T cells in vivo, isolated lung cells were stimulated for 3 h with PMA (phorbol 12-myristate 13-acetate; 30 ng/ml; Sigma), ionomycin (500 ng/ml; Calbiochem) and brefeldin A. Cells were fixed and permeabilized with IntraPrep (Beckman Coulter) and then stained intracellularly with phycoerythrin (PE)-conjugated anti-IL-5 and eFluor 660–conjugated anti-IL-13 (both identified above). In this study, mAbs to CD3ε, CD4, CD5, CD8α, CD11c, F4/80 or Mac-1, CD19, NK1.1, Gr-1, TER119 and FcεRIα (all identified above) were used for detection of lineage markers. Gating strategies for flow cytometry of ILC2 cells and eosinophils. ILC2 cells can be identified as Lin−CD25+IL-1RL1+Thy-1+Sca-1+KLRG-1+ cells44, but there is no marker that can detect ILC2 cells by single staining. Therefore, in this study, we detected ILC2 cells with several combinations of mAbs in flow cytometry. Because the optimal combination of antibodies depends on the tissues, treatments and knockout mouse strains, antibodies used in various experiments are different. However, all gating strategies for ILC2 cells here accurately gated GATA3+ ILC2 cells (gating strategies for FALC ILC2 cells, lung ILC2 cells and BALF ILC2 cells , Supplementary Fig. 8). SSChi cells are all Siglec-Fhi cells, which are eosinophils (Supplementary Fig. 8). We thus present eosinophils as SSChiSiglec-Fhi cells. For both the helminth-infection experiments and intracellular staining of cytokines, cells could not be stained with propidium iodide because cells were fixed to prevent bacterial contamination of the flow cytometer and were also fixed and permeabilized to facilitate intracellular staining. However, we gated out the dead cells as much as possible by gating on FSC-W versus FSC-H and SSC-W versus SSC-H. Cytokine administration. Mice were treated with IL-2 (0.5 µg per mouse), IL-25 (0.5 or 1.0 µg per mouse), IL-33 (0.5 µg per mouse), IFN-γ (0.5 or 1.0 µg per mouse) and IL-27 (0.02 or 0.5 µg per mouse) by intranasal or intratracheal injection. For intranasal injection, 40 µl of cytokines in PBS was administered intranasally to mice anesthetized with ketamine or isoflurane. For intratracheal injection, mice were anesthetized with nembutal or isoflurane, and 30–40µl of cytokines in PBS was injected with 400 µl of air directly into the bronchial tube via a 22G plastic cannulae. We used a small animal laryngoscope for visualization of the epiglottis and glottis. Administration of helminths and A. alternata extract. Mice were given subcutaneous inoculation of 500 viable third-stage N. brasiliensis larvae in 500 µl PBS and were killed 5–8 d after infection. BALF was collected for ELISA,and BALF cells and lung cells were collected for flow cytometry. For administration of A. alternata, A. alternata extract (10 µg per mouse) in 40 µl PBS was administered intranasally three times (days 0, 3 and 6) to mice anesthetized with ketamine, then mice were killed on day 8. BALF was collected for ELISA, and BALF cells and one half of the lung cells were collected for flow cytometry and cell counts. Eosinophils in the BALF were identified with a hemocytometer, and differential cell counts were performed on cytospin preparations stained with Diff-Quick (Symex); >200 cells were analyzed by conventional morphological criteria. Adoptive-transfer experiments. For helminth infection, ILC2 cells from the mesentery of wild-type mice were cultured for 4 weeks with 10 ng/ml IL-2 and were transferred intravenously into Il2rg−/−Rag2−/− host mice 4 h before infection with N. brasiliensis. For IL-33-mediated inflammatory responses, ILC2 cells from the lungs of wild-type mice or Ifngr1−/−Rag2−/− mice were cultured for 4 weeks with 10 ng/ml IL-2 plus 10 ng/ml IL-7 and were transferred

doi:10.1038/ni.3309

intravenously into Il2rg−/−Rag2−/− host mice just before the first administration of cytokines. Both transfers were done without irradiation. Histological analysis. Lungs were fixed for at least 24 h with 10% formalin and were embedded in paraffin. Sections (5 µm in thickness) were stained with hematoxylin (Sakura Fintek Japan) and eosin (Sakura Fintek Japan) or with alcian blue and periodic acid–Schiff (Polysciences).

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Parabiosis. Weight-matched C57BL/6 mice and B6.SJL mice were used for parabiosis. Opposite sides of the mice, which were under inhalational anesthesia, were ‘shaved’ with a depilatory agent before surgery. In mice under anesthesia, lateral skin in a ‘lens shape’ (5 cm × 3 cm) was removed, and the skin was joined with 9-mm wound clips (BD) after temporary suture with 4-0 dissolvable stitches (Ethicon). Interosseous ligation and intermuscular ligation were not performed in this experiment. RNA sequencing. ILC2 cells isolated from the mesentery of wild-type mice were cultured for 48 h with IL-33 (10 ng/ml), IL-33 plus IFN-γ (10 ng/ml), or IL-33 plus IL-27 (10 ng/ml each). RNA was isolated with an Allprep DNA/RNA Micro Kit (Qiagen), and cDNA libraries were prepared with a TruSeq RNA Sample Preparation kit v2 (Illumina) according to the manufacturer’s ‘low sample’ protocol. A HiSeq 1500 system (Illumina) was used for 50 singleend–base sequencing. Sequenced reads were trimmed for adaptor sequences and were masked for low-complexity or low-quality sequences, then were mapped to the reference genome (mm9 assembly of the mouse genome) with Bowtie2 software, version 2.1.0, and TopHat2 software, version 2.0.8. The abundance of transcripts was estimated as FPKM values (fragments

doi:10.1038/ni.3309 

per kilobase of exon million fragments mapped) with Cufflinks software, version2.1.1. Heat maps were produced by the microarray data-analysis tool MeV. Real-time PCR. The expression of WSX-1 (5′-AATATCTCCAGCCCCAAACC3′ and 5′-TGTGAAACTTCTGGCAAACG-3′) and GP130 (5′-TCATGTTCC TTCTATCGGGTC-3′ and 5′-CTGAGGGACCGGTGGTGT-3′) was measured by real-time quantitative PCR with SYBR Green Master Mix (Applied Biosystems) and an ABI7500 real-time PCR system (Applied Biosystems). RNA was extracted from splenic CD4+ T cells and mesenteric ILC2 cells cultured with IL-2 (10 ng/ml) with an RNeasy kit (Venlo) Statistical analysis. The sample size in each experiment was determined empirically for sufficient statistical power. No statistical methods were used to determine the sample size in the experiments. No samples were excluded from all experiments, and randomization methods and blinding were not used in the experiments. Data were analyzed with GraphPad Prism 6 software (GraphPad Software). A Mann-Whitney U-test (two-tailed) was used for analysis of the significance of differences between two groups, and one-way ANOVA followed by Dunnett’s test was used for analysis of the significance of differences among more than two groups. All P values reported were based on two-tailed tests unless stated otherwise. 44. Durbin, J.E., Hackenmiller, R., Simon, M.C. & Levy, D.E. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell 84, 443–450 (1996). 45. Moro, K., Ealey, K.N., Kabata, H. & Koyasu, S. Isolation and analysis of group 2 innate lymphoid cells in mice. Nat. Protoc. 10, 792–806 (2015).

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