Shinichiro Sawa

Matthias Lochner

Naoko Satoh-Takayama

Sophie Dulauroy

Marion Bérard

Melanie Kleinschek

Daniel Cua

James P Di Santo

Gérard Eberl
email: gerard.eberl@pasteur.fr

RORγt + innate lymphoid cells regulate intestinal homeostasis by integrating negative signals from the symbiotic microbiota

RORγt + lymphoid cells are involved in containment of the large intestinal microbiota and defense against pathogens through the production of IL-17 and IL-22. They include adaptive T H 17 cells, as well as innate lymphoid cells (ILCs), such as lymphoid tissue inducer (LTi) cells and IL-22-producing NKp46 + cells. We find that, in contrast to T H 17 cells, both types of RORγt + ILCs constitutively produced most of the intestinal IL-22, and that symbiotic microbiota repressed this function through epithelial expression of IL-25. This function was increased in the absence of adaptive immunity and fully restored and required upon epithelial damage, demonstrating a central role for RORγt + ILCs in intestinal homeostasis. Our data reveal a finely tuned equilibrium between intestinal symbionts, adaptive immunity and RORγt + ILCs.

INTRODUCTION

The mammalian intestine hosts a large microbiota that includes an estimated 10 14 bacteria and 100 to 1000 individual species [START_REF] Backhed | Host-bacterial mutualism in the human intestine[END_REF] . In order to contain this microbiota and fight breaches by invading microbes, the intestinal immune system deploys a complex network of lymphoid tissues and cells [START_REF] Duerkop | Immune responses to the microbiota at the intestinal mucosal surface[END_REF][START_REF] Nagler-Anderson | Man the barrier! Strategic defences in the intestinal mucosa[END_REF] . Part of this system is programmed to develop during ontogeny, such as mesenteric lymph nodes and Peyer's patches, and part of it is induced by the microbiota, such as the formation of numerous isolated lymphoid follicles (ILFs) from pre-formed cryptopatches, and the recruitment of various subsets of intestinal lymphocytes [START_REF] Eberl | The development of intestinal lymphoid tissues at the interface of self and microbiota[END_REF] . An equilibrium is established between the microbiota and the immune system that is fundamental to intestinal homeostasis [START_REF] Round | The gut microbiota shapes intestinal immune responses during health and disease[END_REF][START_REF] Eberl | A new vision of immunity: homeostasis of the superorganism[END_REF] . Perturbation of homeostasis leads to inflammatory disease characterized by immune attack on microbial symbionts and tissue destruction, a scenario unfolding during inflammatory bowel disease (IBD), or on the contrary, to low reactivity of the intestinal immune system and increased susceptibility to pathogens [START_REF] Brandl | Vancomycin-resistant enterococci exploit antibiotic-induced innate immune deficits[END_REF] .

A number of cytokines play a key role in intestinal homeostasis. Whereas proinflammatory cytokines, such as interferon-γ (IFN-γ), IL-17 and tumor necrosis factor (TNF), promote the elimination of intruding microbes, the anti-inflammatory cytokines IL-10 and TGF-β limit the amplitude of inflammation and favor the production and secretion of IgA, essential to mucosal immunity [START_REF] Duerkop | Immune responses to the microbiota at the intestinal mucosal surface[END_REF][START_REF] Nagler-Anderson | Man the barrier! Strategic defences in the intestinal mucosa[END_REF] . Another member of the IL-10 cytokine family, IL-22, has been shown to directly induce epithelial defense through the expression of anti-microbial peptides, such as β-defensins [START_REF] Wolk | IL-22 increases the innate immunity of tissues[END_REF] , S100A7 (psoriasin), S100A8 (calgranulin A), S100A9 (calgranulin B) [START_REF] Wolk | IL-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis[END_REF] , RegIIIβ and RegIIIγ [START_REF] Zheng | Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens[END_REF] . IL-22 plays a critical role in defense against attaching and effacing bacterial pathogens in the intestine, such as Citrobacter rodentium [START_REF] Zheng | Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens[END_REF][START_REF] Sonnenberg | CD4(+) Lymphoid Tissue-Inducer Cells Promote Innate Immunity in the Gut[END_REF] , Gram-negative bacteria in the lung, such as Klebsiella pneumoniae [START_REF] Aujla | IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia[END_REF] and against the fungi Candida albicans [START_REF] De Luca | IL-22 defines a novel immune pathway of antifungal resistance[END_REF] . IL-22 also induces anti-apoptotic molecules, such as Bcl-2 family members, through the activation of STAT3 (ref. 14), and as a possible consequence, protects colon and hepatocytes from acute inflammation [START_REF] Zenewicz | Interleukin-22 but not interleukin-17 provides protection to hepatocytes during acute liver inflammation[END_REF][START_REF] Zenewicz | Innate and adaptive interleukin-22 protects mice from inflammatory bowel disease[END_REF] . IL-22 is efficiently induced by IL-23 [START_REF] Kastelein | Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation[END_REF] and produced at high amounts by CD4 + T H 17 cells [START_REF] Zheng | Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis[END_REF][START_REF] Kreymborg | IL-22 is expressed by Th17 cells in an IL-23-dependent fashion, but not required for the development of autoimmune encephalomyelitis[END_REF] , but also by other subsets of T helper cells [START_REF] Liang | Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides[END_REF] , CD8 + T cells, γδ T cells [START_REF] Zheng | Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis[END_REF] and NK cells [START_REF] Zenewicz | Innate and adaptive interleukin-22 protects mice from inflammatory bowel disease[END_REF][START_REF] Wolk | Cutting edge: immune cells as sources and targets of the IL-10 family members?[END_REF] . Recently, it was revealed that a subset of mucosal innate lymphoid cells (ILCs) expressing the pan-NK markers NKp46 in mouse and NKp44 in human, expressed IL-22 [START_REF] Spits | The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodeling[END_REF][START_REF] Satoh-Takayama | Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense[END_REF][START_REF] Cella | A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity[END_REF][START_REF] Sanos | RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells[END_REF][START_REF] Luci | Influence of the transcription factor RORgammat on the development of NKp46+ cell populations in gut and skin[END_REF][START_REF] Cupedo | Human fetal lymphoid tissue-inducer cells are interleukin 17-producing precursors to RORC+ CD127+ natural killer-like cells[END_REF] , and mice lacking these IL-22 + NKp46 + cells showed increased susceptibility to C. rodentium infection [START_REF] Satoh-Takayama | Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense[END_REF] . It was also shown that CD4 + lineage-negative (Lin -) cells in the spleen, a phenotype reminiscent of lymphoid tissue inducer (LTi) cells required for the development of secondary lymphoid tissues [START_REF] Mebius | Developing lymph nodes collect CD4 + CD3 -LTβ + cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells[END_REF] and isolated lymphoid follicles (ILFs) in the intestine [START_REF] Eberl | Thymic origin of intestinal αβ T cells revealed by fate mapping of RORγt + cells[END_REF] , express IL-22 [START_REF] Sonnenberg | CD4(+) Lymphoid Tissue-Inducer Cells Promote Innate Immunity in the Gut[END_REF][START_REF] Takatori | Lymphoid tissue inducer-like cells are an innate source of IL-17 and IL-22[END_REF][START_REF] Sawa | Lineage relationship analysis of RORγt+ innate lymphoid cells[END_REF] . Common to T H 17 cells 32 , IL-22 + NKp46 + or NKp44 + cells, and LTi cells [START_REF] Eberl | An essential function for the nuclear receptor RORγt in the generation of fetal lymphoid tissue inducer cells[END_REF] , is the expression of the nuclear hormone receptor RORγt, and the requirement for RORγt during their development. Common also to T H 17 cells [START_REF] Ivanov | Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine[END_REF] and a subset of IL-22 + NKp46 + cells, but not to LTi cells [START_REF] Sanos | RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells[END_REF][START_REF] Bouskra | Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis[END_REF] , is a development induced by intestinal symbiotic microbiota.

Using RORγt-EGFP reporter mice [START_REF] Lochner | In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORγt+ T cells[END_REF] , we now show that RORγt + ILCs, including IL-22 + NKp46 + cells and LTi cells, produced most of the intestinal IL-22. In contrast to T cells, this expression of IL-22 was constitutive, highest before weaning and independent of microbial colonization. However, microbiota repressed the production of IL-22 by RORγt + ILCs through the expression of IL-25 in epithelial cells, whereas the role of IL-23 was minimal. The IL-22-producing activity and numbers of RORγt + ILCs was increased in the absence of adaptive immunity and RORγt + T cells, presumably as a consequence of larger niche availability. Furthermore, upon epithelial damage and consequent intestinal inflammation, RORγt + ILCs regained full activity and were required for survival and recovery. Collectively, our data demonstrate that IL-22 production by RORγt + ILCs is programmed, and regulated by microbiota and adaptive immunity, presumably to maintain a necessary equilibrium between microbiota and the different forces of immunity.

RESULTS

RORγt + ILCs are the major source of intestinal IL-22

The nuclear hormone receptor RORγt is expressed by T cells [START_REF] Ivanov | The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17 + T helper cells[END_REF][START_REF] Lochner | In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORγt+ T cells[END_REF] and NKp46 + or NKp44 + cells that produce IL-22 [START_REF] Satoh-Takayama | Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense[END_REF][START_REF] Cella | A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity[END_REF][START_REF] Sanos | RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells[END_REF][START_REF] Luci | Influence of the transcription factor RORgammat on the development of NKp46+ cell populations in gut and skin[END_REF] . To determine the proportion of intestinal IL-22 + cells among RORγt + cells, we examined BAC-transgenic Rorc(γt)-Gfp TG mice [START_REF] Lochner | In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORγt+ T cells[END_REF] . We found that in fetuses (E15), pre-weaned (2 weeks) and adult mice (8 weeks), most IL-22 + cells expressed RORγt (Fig. 1a). The vast majority of IL-22 + cells (99%, 80% and 83%, respectively, at these ages) did not express CD3ε, and IL-22 + CD4 + T cells that include T H 17 cells represented only 11% of the total IL-22 + cells in the adult. RAG-deficient mice that lack T cells did not show a significant decrease in the expression of Il22 in the small intestine, whereas RORγt-or RAG/RORγt-double deficient mice, which lack RORγt + cells, showed no detectable expression of Il22 (Fig. 1b) and a consequent decreased expression of Reg3b and Reg3g (Fig. 1c). These results demonstrate that most intestinal IL-22 is produced by RORγt + ILCs.

In the fetus, RORγt + ILCs consist mainly of CD4 + LTi (LTi 4 ) cells [START_REF] Eberl | An essential function for the nuclear receptor RORγt in the generation of fetal lymphoid tissue inducer cells[END_REF] , whereas after birth, the pool of CD4 -RORγt + ILCs, including CD4 -LTi (LTi 0 ) cells [START_REF] Yoshida | IL-7 receptor α + CD3cells in the embryonic intestine induces the organizing center of Peyer's patches[END_REF] , expanded, and NKp46 + RORγt + ILCs were generated (Fig. 1d). RORγt + ILCs were found mostly in the intestinal lamina propria (Supplementary Fig. 1a) and did not include B cells, dendritic cells (DCs) or cells of the monocyte/macrophage lineage (Supplementary Fig. 1b). The expression of IL-22 by RORγt + ILCs was highest in fetal and pre-weaned animals, and substantially decreased thereafter (Fig. 2a). A similar expression profile was found in LTi 4 cells for IL-17 (Fig. 2b), co-expressed by a sizeable fraction of IL-22 + cells (Supplementary Fig. 2a andb), and to a lesser degree in all RORγt + ILCs for genes involved in lymphoid tissue development, such as Lta (coding for lymphotoxin α) (Fig. 2c) and Tnfsf11 (coding for Trance) [START_REF] Mebius | Organogenesis of lymphoid tissues[END_REF] (Supplementary Fig. 2c). The expression of Ifng transcripts, produced by the NK1.1 + subset of RORγt + NKp46 + cells [START_REF] Satoh-Takayama | Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense[END_REF] , was also decreased after weaning (Fig. 2c).

Finally, the remaining CD4 -NKp46 -RORγt + ILCs (Fig. 1d), which include c-kit hi LTi 0 cells and poorly characterized c-kit low RORγt + ILCs expressing IFNγ, also showed decreased expression of IL-22, IL-17, Lta and Ifng after weaning (Supplementary Fig. 3).

Microbiota represses IL-22 production by RORγt + ILCs

intestinal NKp46 + RORγt + ILCs [START_REF] Satoh-Takayama | Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense[END_REF][START_REF] Sanos | RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells[END_REF] . However, our data now show that LTi cells constitutively expressed IL-22 in the sterile environment of the fetus, and that expression of IL-22 by RORγt + ILCs was decreased after weaning. As microbial colonization of the intestine is vastly increased after weaning, we hypothesized that symbiotic microbiota is repressing the activity of RORγt + ILCs. First, and in contrast to RORγt + T cells, the number of RORγt + ILCs remained unchanged in germfree or antibiotic-treated Rorc(γt)-Gfp TG mice (Fig. 3a), in accordance with data obtained for LTi cells [START_REF] Bouskra | Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis[END_REF] . These observations were valid for both LTi cells and NKp46 + RORγt + ILCs, the latter including varying proportions of NK1.1 hi cells that are reported not to be affected by microbiota [START_REF] Satoh-Takayama | Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense[END_REF] . Furthermore, production of IL-22 by RORγt + ILCs was de-repressed in adult germfree and antibiotic-treated mice (Fig. 3b) to concentrations reached in fetal and pre-weaned mice (Fig. 2a). This microbiota-induced repression appeared specific for IL-22 production and not operative on IL-17 production (Fig. 3c).

Microbiota represses RORγt + ILCs through IL-25

It has been reported that the differentiation of T H 17 cells in the colon of BALB/c mice is inhibited by IL-25 induced by the microbiota [START_REF] Zaph | Commensal-dependent expression of IL-25 regulates the IL-23-IL-17 axis in the intestine[END_REF] . In accordance with these data, we found that the expression of Il25 in the ileal epithelium of germfree mice was markedly decreased as compared to SPF mice (Fig. 4a). In addition, in SPF mice, expression of Il25 by epithelial cells increased with age, presumably as a result of microbial colonization of the intestine. As this Il25 expression pattern inversely correlated with the production of IL-22 by RORγt + ILCs, we assessed the activity of RORγt + ILCs in IL-25-deficient mice. Whereas the number of RORγt + ILCs increased marginally in the absence of IL-25 (Fig. 4b), the production of IL-22 was significantly increased (Fig. 4c), but not to amounts reached in germfree or preweaned mice (Fig. 2a and3b). Administration of IL-25 to Rorc(γt)-Gfp TG mice efficiently repressed IL-22 production by RORγt + ILCs (Fig. 4d) and, as a probable consequence, inhibited the expression of Reg3b and Reg3g by epithelial cells (Fig. 4e).

The inhibitory effect of IL-25 on RORγt + ILCs was indirect as RORγt + ILCs did not express IL-25R (or IL-17BR) (Fig. 4f). In contrast, IL-17BR was expressed by RORγt -ILCs, recently reported to expand and produce IL-13 upon administration of IL-25 in mice and involved in early immunity against intestinal helminthes [START_REF] Neill | Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity[END_REF][START_REF] Moro | Innate production of T(H)2 cytokines by adipose tissue-associated c-Kit(+)Sca-1(+) lymphoid cells[END_REF] . IL-17RB was also expressed by CD11c + DCs in cryptopatches, which are clusters of LTi cells [START_REF] Eberl | Thymic origin of intestinal αβ T cells revealed by fate mapping of RORγt + cells[END_REF] . When IL-17BR + DCs were added to cultures of RORγt + ILCs, IL-25 efficiently repressed the production of IL-22 by RORγt + ILCs (Fig. 4g), demonstrating that IL-17RB + ILCs or DCs could mediate the inhibitory effect of IL-25 on the activity of RORγt + ILC. Transwell analysis showed that this inhibitory effect required contact between IL-17BR + DCs and RORγt + ILCs (Fig. 4h), in accordance with our recent data showing that in CCR6-deficient mice that develop abnormal cryptopatches, the expression of IL-22 by RORγt + ILC is increased [START_REF] Sonnenberg | CD4(+) Lymphoid Tissue-Inducer Cells Promote Innate Immunity in the Gut[END_REF][START_REF] Takatori | Lymphoid tissue inducer-like cells are an innate source of IL-17 and IL-22[END_REF][START_REF] Sawa | Lineage relationship analysis of RORγt+ innate lymphoid cells[END_REF] . IL-23 is a potent inducer of IL-22 [START_REF] Kastelein | Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation[END_REF][START_REF] Zheng | Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis[END_REF][START_REF] Kreymborg | IL-22 is expressed by Th17 cells in an IL-23-dependent fashion, but not required for the development of autoimmune encephalomyelitis[END_REF] , and may be modulated by microbiota and IL-25. We therefore assessed whether the increased IL-22 production by RORγt + ILCs in germfree mice was a consequence of increased IL-23 in intestinal tissues. However, as might be expected, the expression of Il23 was decreased in germfree mice (Supplementary Fig. 4a). Furthermore, the absence of IL-25 or administration of IL-25 had no effect on the expression of Il23 and IL-23 protein (Supplementary Fig. 4b), excluding that IL-25 regulated the activity of RORγt + ILCs through IL-23, and the expression of Il23r by RORγt + ILCs was not modulated by IL-25 (Supplementary Fig. 4c). Finally, in p19-deficient mice that lack IL-23 (Supplementary Fig. 4d), or in wild-type mice treated with neutralizing antibody specific for IL-23R (Supplementary Fig. 4e), the production of IL-22 by RORγt + ILCs and its repression by IL-25 were unaffected. Together, these data show that IL-23 is not necessary for or involved in the regulation of IL-22 production by RORγt + ILCs.

Adaptive immunity represses the activity of RORγt + ILCs

Both RORγt + T cells [START_REF] Zheng | Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis[END_REF][START_REF] Kreymborg | IL-22 is expressed by Th17 cells in an IL-23-dependent fashion, but not required for the development of autoimmune encephalomyelitis[END_REF] and RORγt + ILCs produce IL-22. These two cell types might therefore compete for regulating factors and thereby mutually regulate their activity. In accordance with this view, the production of IL-22 by RORγt + ILCs was markedly enhanced in RAG-2-deficient mice (Fig. 5a), and the absolute numbers of RORγt + ILCs was increased 4-fold (Fig. 5b). As pointed out earlier, de-repression of the IL-22 production by RORγt + ILCs was only partial in IL-25-deficient mice as compared to germfree mice (Fig. 4c and3b).

An additional level of repression is thus provided by adaptive immunity, possibly through

RORγt + T cells that compete for a cytokine niche also occupied by RORγt + ILCs. As microbiota induces the generation of RORγt + T H 17 cells [START_REF] Ivanov | Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine[END_REF] , our data indicate that microbiota represses IL-22 production by RORγt + ILCs both through IL-25 and adaptive immunity. Of note, adaptive immunity was not required for the IL-25-mediated repression of IL-22 production by RORγt + ILCs, as the administration of IL-25 efficiently repressed this activity in RAG-2-deficient mice (Fig. 5c).

Epithelial damage de-represses the activity of RORγt + ILCs

We next assessed the activity of RORγt + ILCs during intestinal inflammation. Epithelial damage was induced by oral exposure of Rorc(γt)-Gfp TG mice to dextran sodium sulfate (DSS), which affects the colon, and to a lesser extent, the small intestine [START_REF] Geier | Small-intestinal manifestations of dextran sulfate sodium consumption in rats and assessment of the effects of Lactobacillus fermentum BR11[END_REF] . After two cycles of DSS administration followed by water, the expression of Il22 was markedly increased in the intestine (Fig. 6a), as shown previously [START_REF] Zenewicz | Innate and adaptive interleukin-22 protects mice from inflammatory bowel disease[END_REF] , concomitant with an increase in Il23 and a sharp decrease in Il25 expression by epithelial cells. The IL-22 production by RORγt + ILCs was increased to concentrations found in germfree mice (Fig. 6b and3b) and RORγt + ILCs expanded 3 to 4-fold (Fig. 6c). Thus, whereas microbiota and adaptive immunity repress the activity of RORγt + ILCs during steady state, epithelial damage that induces intestinal inflammation fully de-represses this activity and leads to an expansion of RORγt + ILCs.

Remarkably, even in the context of intestinal inflammation, IL-25 repressed IL-22 production by RORγt + ILCs (Fig. 6d), demonstrating its dominant role in the regulation of RORγt + ILCs.

Finally, RORγt + ILCs were required for resistance to DSS-mediated colitis. Wild-type or RAG-2-deficient mice treated with IL-25 showed a significant increase in weight loss in response to DSS treatment, as well as a delayed recovery (Fig. 7a andb). In that context, IL-25 also decreased the number of RORγt + T cells and repressed their production of IL-22 (Supplementary Fig. 5). However, mice that lack RORγt + ILCs rapidly succumbed to DSSmediated colitis [START_REF] Lochner | Microbiota-induced tertiary lymphoid tissues aggravate inflammatory disease in the absence of RORγt and LTi cells[END_REF] , whereas RAG-2-deficient mice did not (Fig. 7b). Of note, IL-25 efficiently repressed expression of Reg3b and Reg3g (Fig. 4e), but only partially repressed IL-22 production by RORγt + ILCs (Fig. 4d, 6d, 7c and7d). In contrast, the absence of RORγt abolished expression of IL-22 (Fig. 1b) but not of Reg3b and Reg3g (Fig. 1c), indicating that the IL-22 producing activity of RORγt + ILCs is involved in protection and recovery from DSS-mediated epithelial damage and colitis.

DISCUSSION

Lymphoid cells derived from common lymphoid progenitors (CLP) [START_REF] Kondo | Biology of hematopoietic stem cells and progenitors: implications for clinical application[END_REF] include B and T cells, the hallmarks of adaptive immunity, and innate lymphoid cells (ILCs) including NK cells and LTi cells [START_REF] Eberl | Immunology: Close encounters of the second type[END_REF] , which lack antigen receptors generated by somatic recombination. LTi cells, which express the nuclear hormone receptor RORγt [START_REF] Eberl | An essential function for the nuclear receptor RORγt in the generation of fetal lymphoid tissue inducer cells[END_REF] , generate micro-inflammation at programmed sites in the fetus and adult intestine to induce the development of lymphoid tissues [START_REF] Eberl | An essential function for the nuclear receptor RORγt in the generation of fetal lymphoid tissue inducer cells[END_REF][START_REF] Bouskra | Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis[END_REF] . Recently, a new subset of RORγt + innate lymphoid cells (ILCs) has been described that resides mostly in the intestine. These cells express the pan-NK marker NKp46, but lack most other NK markers and are non-cytotoxic [START_REF] Satoh-Takayama | Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense[END_REF][START_REF] Cella | A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity[END_REF][START_REF] Sanos | RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells[END_REF][START_REF] Luci | Influence of the transcription factor RORgammat on the development of NKp46+ cell populations in gut and skin[END_REF][START_REF] Cupedo | Human fetal lymphoid tissue-inducer cells are interleukin 17-producing precursors to RORC+ CD127+ natural killer-like cells[END_REF] . We now show that RORγt + ILCs produce most of intestinal IL-22 at steady state and markedly increase this activity during inflammation. RORγt + ILCs therefore play a critical role in intestinal homeostasis and defense, as IL-22 induces the production of anti-bacterial peptides by epithelial cells, is required for intestinal defense against bacterial and fungal pathogens [START_REF] Wolk | IL-22 increases the innate immunity of tissues[END_REF][START_REF] Wolk | IL-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis[END_REF][START_REF] Zheng | Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens[END_REF][START_REF] Aujla | IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia[END_REF][START_REF] De Luca | IL-22 defines a novel immune pathway of antifungal resistance[END_REF] and is involved in protection of intestinal tissues from the effects of pathological inflammation, presumably through the induction of anti-apoptotic factors [START_REF] Zenewicz | Interleukin-22 but not interleukin-17 provides protection to hepatocytes during acute liver inflammation[END_REF][START_REF] Zenewicz | Innate and adaptive interleukin-22 protects mice from inflammatory bowel disease[END_REF] .

In marked contrast to RORγt + T cells [START_REF] Ivanov | Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine[END_REF] , absolute numbers of RORγt + ILCs are unaffected in germfree mice, showing that the development of RORγt + ILCs is programmed [START_REF] Sawa | Lineage relationship analysis of RORγt+ innate lymphoid cells[END_REF] . This result may seem counter-intuitive, as RORγt is induced in developing T H 17 cells through the activation of Stat3 [START_REF] Harris | Cutting edge: An in vivo requirement for STAT3 signaling in TH17 development and TH17-dependent autoimmunity[END_REF][START_REF] Mathur | Stat3 and Stat4 direct development of IL-17-secreting Th cells[END_REF] . T H 17 cells are enriched in the intestine [START_REF] Lochner | In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORγt+ T cells[END_REF] where activators of Stat3, such as IL-6, IL-21 and IL-23 [START_REF] Dong | TH17 cells in development: an updated view of their molecular identity and genetic programming[END_REF] abound, presumably in response to the large microbial community residing in the intestinal lumen. Microbiota also induces the generation of the NK1.1 lo/-subset of RORγt + NKp46 + cells [START_REF] Satoh-Takayama | Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense[END_REF][START_REF] Sanos | RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells[END_REF][START_REF] Luci | Influence of the transcription factor RORgammat on the development of NKp46+ cell populations in gut and skin[END_REF] . Nevertheless, RORγt appears to be programmed in thymic subsets of γδ T cells [START_REF] Jensen | Thymic selection determines gammadelta T cell effector fate: antigennaive cells make interleukin-17 and antigen-experienced cells make interferon gamma[END_REF] and iNKT cells [START_REF] Michel | Critical role of ROR-gammat in a new thymic pathway leading to IL-17-producing invariant NKT cell differentiation[END_REF] , thus presumably in the absence of germs, and in LTi cells generated in the sterile environment of the fetus [START_REF] Mebius | Developing lymph nodes collect CD4 + CD3 -LTβ + cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells[END_REF] .

In addition to a programmed development, RORγt + ILCs show constitutive expression of IL-22, as well as of IL-17 by LTi cells. A role of IL-22 and IL-17 produced by LTi cells in the fetus remains to be elucidated, but appears evolutionary conserved in human [START_REF] Cupedo | Human fetal lymphoid tissue-inducer cells are interleukin 17-producing precursors to RORC+ CD127+ natural killer-like cells[END_REF] . After birth, the key role of IL-22 in intestinal homeostasis and defense, and more generally in mucosal immunity, is well documented [START_REF] Zheng | Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens[END_REF][START_REF] Aujla | IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia[END_REF][START_REF] De Luca | IL-22 defines a novel immune pathway of antifungal resistance[END_REF] . It may therefore not come as a surprise that the immune system has evolved a constitutive pathway for the production of IL-22. However, as the size and structure of the intestinal symbiotic microbiota evolves with time, the amount of IL-22 produced might need constant re-adjustments. In accordance with this view, we show that microbiota regulates IL-22-production by RORγt + ILCs. As components of the symbiotic microbiota, such as Candidatus arthromitus, also termed Segmented Filamentous Bacteria (SFB), have been shown to induce T H 17 cells [START_REF] Gaboriau-Routhiau | The key role of segmented filamentous bacteria in the coordinated maturation of gut helper T cell responses[END_REF][START_REF] Ivanov | Induction of Intestinal Th17 Cells by Segmented Filamentous Bacteria[END_REF] , it will be important to assess whether SFB, and other species or phyla of symbiotic bacteria, regulate the activity of RORγt + ILCs.

We show that both microbiota and adaptive immunity repress the activity of RORγt + ILCs. It is possible that RORγt + T cells including T H 17 cells [START_REF] Lochner | In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORγt+ T cells[END_REF] repress RORγt + ILCs by competition for a common cytokine niche. As both type of cells express IL-17 and IL-22, they might be subject to similar regulation through a panel of cytokines that includes IL-6, IL-21, IL-23, IL-25, IL-27 [START_REF] Dong | TH17 cells in development: an updated view of their molecular identity and genetic programming[END_REF] . Our data demonstrate the major role of IL-25 in the repression IL-22 production by RORγt + ILCs. In contrast, IL-23, a major inducer of IL-22 [START_REF] Kastelein | Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation[END_REF] , does not regulate the production of IL-22 by RORγt + ILCs during homeostasis, and is not repressed by IL-25.

The role of other T H 17 regulators on RORγt + ILCs remains to be assessed.

Microbiota appears to be a positive regulator of IL-22 produced by T H 17 cells, but a negative regulator of IL-22 produced by innate RORγt + cells. In addition, adaptive immunity represses both the number and IL-22-production by RORγt + ILCs. Microbiota might therefore repress the activity of RORγt + ILCs through both IL-25 and adaptive immunity. From the point of view of the host, RORγt + ILCs preempt colonization of the intestine by symbiotic microbiota, whereas adaptive immunity reacts to this colonization and regulates the preemptive innate immunity. This complex regulatory network probably reflects the superposition of adaptive immunity over the more ancient innate preemptive system, and shows the subtle interplay between microbiota and the different forces of the vertebrate immune system to maintain intestinal homeostasis.

Finally, a rupture in intestinal homeostasis is induced by epithelial damage (achieved here by the administration of DSS) that provokes a strong inflammatory reaction to contain the breaching symbiotic microbiota [START_REF] Hans | The role of the resident intestinal flora in acute and chronic dextran sulfate sodium-induced colitis in mice[END_REF] . In that highly challenging context, both T H 17 [START_REF] Lochner | In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORγt+ T cells[END_REF] and RORγt + ILCs expand, and IL-22 production is highest. Thus, whereas during steady state, microbiota, T H 17 and RORγt + ILCs are controlled by a complex regulatory network, during tissue damage and consequent inflammation, both adaptive and innate RORγt + cells add up production of IL-22 to force a return to homeostasis. However, such situations are potentially dangerous to the host, as the pro-inflammatory activity of T H 17 cells and RORγt + ILCs may expand beyond control and induce inflammatory pathology [START_REF] Buonocore | Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology[END_REF][START_REF] Zenewicz | CD4 T-cell differentiation and inflammatory bowel disease[END_REF][START_REF] Sarra | IL-23/IL-17 axis in IBD[END_REF] . Slides were examined under an AxioImager M1 fluorescence microscope (Carl Zeiss, Inc.) equipped with a CCD camera, and images were processed with AxioVision software (Carl Zeiss, Inc.).

LEGENDS TO FIGURES

Statistical analysis

A two-tailed Student's t-test was used for all statistical analysis.

Figure 1

 1 Figure 1 RORγt + ILCs are the major producer of IL-22 in the intestine. (a) Small intestinal lamina propria leukocytes (SI-LPL) from E.15, 2 weeks and 8 weeks old Rorc(γt)-Gfp TG mice were analyzed by flow cytometry. The production of IL-22 was assessed by intracellular cytokine staining after 3 hours of stimulation with IL-23 ex vivo, and IL-22 + RORγt + cells were further analyzed for the expression of CD3ε and CD4. Numbers indicate percent cells per quadrant. The data are representative of three independent experiments. (b) Expression of transcripts for IL-22 in the terminal ileum of 8 weeks old wild type, RAG2-deficient, RORγtdeficient and RAG2/RORγt-double deficient mice. Data are mean of qPCR triplicates and n = 4 mice per group. NS, statistically not significant; unpaired t-test. (c) Expression of transcripts for RegIIIβ and RegIIIγ by epithelial cells from 8 weeks old wild type, RAG2-deficient and RORγt-deficient mice. Data are mean of qPCR triplicates and n = 4 mice per group. NS, statistically not significant; unpaired t-test. (d) RORγt + ILCs (GFP + CD3ε -cells) were analyzed for their surface expressions of CD4 and NKp46. Data are representative of at least n = 8 individual mice.

Figure 2

 2 Figure 2 The production of pro-inflammatory cytokines by RORγt + ILCs. SI-LPL from E15, 2 weeks and 8 weeks old Rorc(γt)-Gfp TG mice were analyzed by real-time qPCR for the expression of transcripts and by flow cytometry for the expression of protein for (a) IL-22, (b) IL-17, (c) LTα and IFNγ. qPCR data are mean of three independent experiments ± S.E.M.; * P < 0.05, NS, statistically not significant, unpaired t-test. ND, non-detected. The data are representative of five independent experiments. In histograms, numbers are mean of n = 10 mice ± S.E.M percentages of IL-22 + or IL-17 + cells compared to isotype controls (shown in blue). Cells were stimulated for 3 hours ex vivo with rIL-23 or PMA/ionomycin, respectively.

Figure 3

 3 Figure 3 Microbiota represses the production of IL-22 by RORγt + ILCs. (a) Absolute numbers of RORγt + ILCs and CD4 + T cells in SI-LPL from 10 weeks old germ free (GF) mice as compared with age matched specific pathogen free (SPF) mice (left panel), and in 6 weeks old antibiotic-treated mice as compared to non-treated controls (right panel). The histograms are compilations of data obtained from n = 4 mice per group. * P < 0.05, NS, statistically not significant, unpaired t-test. (b) RORγt + ILCs and CD4 + T cells from 8 weeks old SPF, antiobiotic (Abx)-treated or GF Rorc(γt)-Gfp TG mice were analyzed for the expression of

Figure 4

 4 Figure 4 Microbiota represses the activity of RORγt + ILCs through IL-25. (a) Epithelial cells were isolated by laser capture microdissection from the terminal ileum of 6 weeks old SPF or GF C57BL/6 mice, and from 2 weeks or 8 weeks old SPF C57BL/6 mice, and analyzed for the expression of transcripts for IL-25. Data are mean of qPCR triplicates and n = 2 mice per group. (b) Absolute numbers of RORγt + ILCs in SI-LPL of 8 weeks old wild-type and IL-25deficient (IL-25KO) mice. Data are the mean of qPCR triplicates and n = 3 mice per group. (c) Production of transcripts and protein for IL-22 by RORγt + ILCs isolated from 8 weeks old wild-type or IL-25-deficient mice. Protein expression was analyzed after 3 hr stimulation ex vivo with IL-23. FACS data are representative of two independent experiments and the numbers indicate mean ± S.E.M percentage of IL-22+ cells obtained from n = 4 mice. (d) IL-22 production by RORγt + ILCs in 6 weeks-old Rorc(γt)-Gfp TG mice treated with rIL-25 or PBS. Data are representative of three independent experiment and the numbers indicate the mean ± S.E.M percentage of IL-22 + cells obtained from n = 6 mice. (e) Expression of transcripts for RegIIIβ and RegIIIγ by epithelial cells from 6 weeks old mice treated with rIL-25 or PBS. (f) Expression of IL-17BR (IL-25R) by lineage -(CD5 -B220 -CD11b -Gr-1 - Ter119 -) cells from 6 weeks old Rorc(γt)-Gfp TG mice and CD3ε -CD19 -SI-LPL from wildtype mice treated with rIL-25 or PBS. Histology shows a cryptopatch in sections from the terminal ileum of 4 weeks-old Rorc(γt)-Gfp TG mice. Bar, 50µm. (g) RORγt + ILCs were isolated from 4 weeks-old Rorc(γt)-Gfp TG mice and cultured in the presence of rIL-25 with or without IL-17BR + CD11c + cells sorted from SI-LPL of 4 weeks-old wild type mice. (h) RORγt + ILCs were cultured in the presence of PBS (left) or rIL-25, with IL-17BR + CD11c + cells in the same well (middle) or separated by a transwell of 0.4 μm pore size (right). After 3-days, RORγt + ILCs were re-sorted and Il22 gene expression was analyzed by qPCR. Shown are mean of results obtained from triplicate wells. Data are compilations of two independent experiments. * P < 0.05, NS, statistically not significant, unpaired t-test.

Figure 5

 5 Figure 5 Adaptive immunity represses the activity of RORγt + ILCs. (a) SI-LPLs from 6 weeks old wild-type or RAG2-deficient Rorc(γt)-Gfp TG mice were analyzed for intracellular IL-22 expression after 3 hr stimulation ex vivo with IL-23. Data are representative of three independent experiments, and the numbers indicate mean ± S.E.M percentage of IL-22 + cells obtained from n = 6 mice. (b) Absolute numbers of RORγt + ILCs cells from 6 weeks old mice. Data shown are mean of n = 4 mice. (c) Expression of IL-22 by RORγt + ILCs from RAG2-deficient Rorc(γt)-Gfp TG mice treated with rIL-25 or PBS. Data are representative of three independent experiments. Numbers indicate mean ± S.E.M percentage of IL-22 + cells obtained from n = 6 mice. * P < 0.05, NS, unpaired t-test.

Figure 6

 6 Figure 6 Epithelial damage de-represses the activity of RORγt + ILCs. (a) Expression of transcripts for IL-22, IL-23 and IL-25 in total terminal ileum or microdissected epithelial cells from terminal ileum of mice treated with water or DSS. Data are mean of qPCR triplicates and n = 2 mice per group. (b) Expression of transcripts and protein for IL-22 by RORγt + ILCs isolated from 8 weeks old Rorc(γt)-Gfp TG mice treated with water or DSS. FACS data are representative of three independent experiments. Numbers indicate mean ± S.E.M percentage of IL-22 + cells obtained from n = 6 mice. (c) Absolute numbers of RORγt + ILCs in SI-LPL of 8 weeks old control and DSS-treated mice. Data are mean of n = 3 mice per group. (d) Expression of IL-22 by RORγt + ILCs in 6 weeks old Rorc(γt)-Gfp TG mice treated with DSS and injected daily with rIL-25 or PBS. FACS data are representative of two independent experiments. Numbers indicate mean ± S.E.M percentage of IL-22 + cells obtained from n = 6 mice. * P < 0.05, unpaired t-test. Data in histograms are representative of three independent experiments.

Figure 7

 7 Figure 7 The activity of RORγt + ILCs is required for protection and recovery from colitis. (a) Body weight of C57BL/6 mice injected with rIL-25 or PBS, or of (b) RAG2/RORγt-double deficient mice, or RAG2-deficient mice injected with rIL-25 or PBS, and treated with DSS. Data are the mean ± S.E.M of n = 5 mice per group. * P < 0.05, unpaired t-test. † indicates death of one IL-25-injected RAG-2-deficient mice at day 9. (c) Expression of IL-22 by RORγt + ILCs in 9 weeks old RAG-2-deficient mice injected with rIL-25 or PBS every two days, and treated with DSS. Cells were stimulated for 3 hours ex vivo with PMA/ionomycin. Numbers in histograms indicate mean ± S.E.M of n = 3 mice per group. (d) Absolute numbers of small intestinal lamina propria RORγt + ILCs of RAG2-deficient mice injected with rIL-25

ACKNOWLEDGEMENTS

We thank the members of the DTL lab for discussions and critical reading of the manuscript, Lucette Polomack for technical assistance, and José Perez and Eddie Maranghi for excellent work on germfree mice. We also thank B. Ryffel for providing us with p19-deficient mice.

This study was supported by the Institut Pasteur, grants from the Mairie de Paris, the Agence Nationale de la Recherche, and an Excellence Grant from the European Commission. M.L. was supported by the Deutsche Forschungsgemeinschaft and the Schlumberger Foundation.

The authors have no competing financial interests.

AUTHOR CONTRIBUTIONS

S.S. and G.E. designed the study and wrote the manuscript; S.S. did most of the experimental work; M.L. contributed to the analysis of T cells and DSS-mediated colitis; N.S.-T. contributed to the analysis of NKp46 + ILCs; S.D. performed laser capture microdissection; M.B. generated germfree mice; M.K. and D.C. generated and provided IL-25-deficient mice; J.P.D.S contributed to data analysis and manuscript writing.

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

METHODS

Mice

BAC-transgenic Rorc(γt)-Gfp TG mice [START_REF] Lochner | In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORγt+ T cells[END_REF] were kept in specific pathogen-free conditions. RAG2-deficient mice were further crossed with Rorc(γt)-Gfp TG mice. Germfree Rorc(γt)-Gfp TG mice were obtained by aseptic caesarean section followed by the adoption of the caesarean-derived pups by germ-free foster mothers. Health monitoring tests were performed to verify that the germ-free colony was also free of parasites and of mouse specific viruses. IL-23-or IL-25-deficient mice were described previously [START_REF] Cua | Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain[END_REF][START_REF] Kleinschek | IL-25 regulates Th17 function in autoimmune inflammation[END_REF] . All animal experiments were approved by the committee on animal experimentation of the Institute Pasteur and strictly followed French regulation on animal experimentation.

Mouse treatments

To eradicate the intestinal bacterial flora, Rorc(γt)-Gfp TG mice were treated with a cocktail of antibiotics containing 5g/L Streptomycin, 1g/L Colistin, 1g/L Ampicillin and 2.5% Sucrose (Sigma Aldrich) in the drinking water starting one day before birth, and until analysis at day 42. Antibiotic treatment was renewed every week. To induce gut inflammation, Dextran sulfate sodium (DSS) salt (M.W.= 36.000-50.000; MP Biomedicals, France) was dissolved in the drinking water at a concentration of 2,5% (m/v). 6 weeks old mice were exposed to DSS for 7 days followed by a recovery period of 10 days without DSS [START_REF] Lochner | In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORγt+ T cells[END_REF] . This cycle was repeated twice. The mice were analyzed at the age of 9 weeks. To assess the function of IL-25 in vivo, recombinant mouse IL-25 (R&D systems) was administrated (0.5μg in 100 μL PBS i.p) every other day for 21 days to adult Rorc(γt)-Gfp TG or RAG2-deficient Rorc(γt)-Gfp TG mice.

Recombinant mouse IL-23 (R&D systems) (0.5μg in 100 μL PBS i.p) and neutralizing polyclonal goat anti-IL-23p19 Ab (R&D Systems) was injected to RAG-2-deficient mice daily. Neutralizing anti-IL-23R Ab (clone 258010) (R&D Systems) (10μg in 100 μL PBS i.v) was injected daily to Rorc(γt)-Gfp TG mice.

Isolation of cells and flow cytometry

To isolate mononuclear cells from the small intestinal lamina propria (SI-LPL), Peyer's pathes were first removed, gut fragments were cut open and then incubated in PBS (Ca/Mg free) containing 30mM EDTA for 30 min at 4 o C. Tissues were then washed with PBS by vigorous shaking for 3 cycles. Washed gut pieces were subsequently cut into 1mm pieces and

Cell stimulation and intracellular staining

To assess intracellular IL-17A, IL-22 and IFNγ, cells were stimulated for 3hr in DMEM containing 50 ng/ml Phorbol 12-Myristate 13-Acetate (PMA) and 500 ng/ml ionomycin (Sigma-Aldrich). For the last 1.5 hr, 10μg brefeldin A (Sigma-Aldrich) was added to the culture medium. In another setting to measure IL-22 expression, cells were stimulated with 40 ng/ml mouse recombinant IL-23 (R&D systems) for 3 hr. For the last 1.5 hr, 10μg brefeldin A (Sigma-Aldrich) was added to the culture medium. After surface staining for c-kit (CD117), CD4, NKp46, IL-7Rα (CD127) and/or CD3ε, stimulated cells were fixed with 4% PFA (Sigma-Aldrich) and permeabilized with 1% saponin (Sigma-Aldrich) followed by intracellular cytokine staining. To detect intracellular RORγt, the Foxp3-staining buffer set (e-Bioscience) was used for fixation and permeabilization of the cells.

Cultures of RORγt + ILCs

Total RORγt (GFP) + ILCs were sorted by flow cytometry from SI-LPL of 4 weeks-old Rorc(γt)-Egfp TG mice. 2000 ILCs were re-suspended in culture medium and seeded into flat bottom 96-well plates coated with OP9 stroma cells. Culture medium was OPTI MEM containing 10% FCS, β2-Melcaptoethanol, ampicilin and streptomycin, in the presence of 20ng/ml mouse rIL-7, 20ng/ml mouse rSCF (PeproTec) and 20ng/mL mouse rIL-25 (R&D systems) in 24 well plates or in transwell plates with 12 mm diameter inserts (0.4 μm pore size) (Corning). After 3 days of culture, ILCs and OP9 stroma cells were dissociated with Cell Dissociation Buffer (GIBCO) and RORγt + ILCs were sorted by FACS ARIA. In another setting, IL-25R (IL17BR) + CD11c + cells were sorted from SI-LPL of 4 weeks-old wild type (littermate of Rorc(γt)-Egfp TG ) mice, and 2000 IL-25R + CD11c + cells were cultured with 2000 ILCs.

ELISA

Tissue fragments 1 cm in size were pepared from the terminal ileum of IL-25-treated or nontreated C57BL/6 wild type mice, washed with sterile PBS three times and cultured with 1 ml of serum free DMEM medium for 24hrs. Supernatants were centrifuged and assessed for IL-23a protein by ELISA using the Single Analyte ELISAssay TM Kit (SABiosciences).

RNA isolation and qPCR

To perform gene expression analysis, whole tissue from the middle and terminal part of the colon was immediately frozen in liquid nitrogen upon animal sacrifice. Tissue was homogenized using Ultra Turrax T8 (IKA-Werke, Germany) in TRIZOL regent and total RNA was purified according to the manufacture's protocol (Invitrogen). RNA was subjected to DNase I digestion and additional purification using RNeasy Mini kit (Quiagen). In the case of sorted cells and epithelial cells isolated by laser capture microdissection, mRNA was linearly amplified using the Message Booster kit for quantitative RT-PCR (Epicentre Biotechnologies). RNA was transcribed into cDNA using Superscipt III reverse transcriptase

Immunofluorescence histology

Tissues were washed and fixed overnight at 4°C in a fresh solution of 4% paraformaldehyde (Sigma-Aldrich) in PBS. The samples were then washed for 1 d in PBS, incubated in a solution of 30% sucrose (Sigma-Aldrich) in PBS until the samples sank, embedded in OCT compound 4583 (Sakura Finetek), frozen in a bath of isopentane cooled with liquid nitrogen, and stocked at -80°C. Frozen blocs were cut at 8-μm thickness, and sections were collected onto Superfrost/Plus slides (VWR). Slides were dried for 1 h and processed for staining or stocked at -80°C. For staining, slides were first hydrated in PBS-XG (PBS containing 0.1% Triton X-100 and 1% normal goat serum; Sigma-Aldrich) for 5 min and blocked with 10% bovine serum in PBS-XG for 1 h at 20 o C. Endogenous biotin was blocked with a biotin blocking kit (Vector Laboratories). Slides were then incubated with primary polyclonal


Danielle S Counotte

Natalia A Goriounova

Ka Wan Li

Maarten Loos

Roel C Van Der Schors

Dustin Schetters

Anton Nm Schoffelmeer

August B Smit

Huibert D Mansvelder

Tommy Pattij

Sabine Spijker
email: sabine.spijker@cncr.vu.nl

Lasting synaptic changes underlie attention deficits caused by nicotine exposure during adolescence

Tobacco smoking and nicotine exposure during adolescence interfere with prefrontal cortex (PFC) development and lead to cognitive impairments in later life. The molecular and cellular underpinning of these consequences remains elusive. We find that adolescent nicotine exposure induces lasting attentional disturbances and reduces mGluR2 protein and function on presynaptic terminals of PFC glutamatergic synapses. Restoring mGluR2 activity in vivo by local infusion of a group II mGluR agonist in adult rats that received adolescent nicotine rescued attentional disturbances.

Increased risk-taking and reckless behavior of adolescents has been linked to late development of brain areas involved in executive cognitive functioning such as the prefrontal cortex (PFC) [START_REF] Casey | Imaging the developing brain: what have we learned about cognitive development?[END_REF] , which shows dynamic changes in grey and white matter proceeding late into adolescence. Adolescence also marks a period of increased vulnerability to initiation and subsequent abuse of drugs including tobacco smoking [START_REF] Chassin | The natural history of cigarette smoking from adolescence to adulthood: demographic predictors of continuity and change[END_REF] . Nicotine exposure during adolescence interferes with PFC development [START_REF] Slotkin | Cholinergic systems in brain development and disruption by neurotoxicants: nicotine, environmental tobacco smoke, organophosphates[END_REF] and has long-lasting consequences for cognitive performance [START_REF] Jacobsen | Effects of smoking and smoking abstinence on cognition in adolescent tobacco smokers[END_REF][START_REF] Jacobsen | Gender-specific effects of prenatal and adolescent exposure to tobacco smoke on auditory and visual attention[END_REF][START_REF] Mathers | Consequences of youth tobacco use: a review of prospective behavioural studies[END_REF] . In line with human epidemiological data, nicotine exposure in adolescent rats has long-term cognitive consequences. Indeed, most adult smokers start their habit before the age of 19 years, and more than 70% of adolescents report to have tried a cigarette at least once [START_REF]WHO-Europe[END_REF] . Nicotine, acting on nicotinic acetylcholine receptors, hampers PFC function and may interfere with PFC maturation, altering structure and function that persist into adulthood. In adolescent smokers, PFC activity, working-memory and attention are reduced [START_REF] Jacobsen | Effects of smoking and smoking abstinence on cognition in adolescent tobacco smokers[END_REF][START_REF] Jacobsen | Gender-specific effects of prenatal and adolescent exposure to tobacco smoke on auditory and visual attention[END_REF] . In later life, behavioral disturbances and mental health problems are strongly correlated with adolescent nicotine use [START_REF] Mathers | Consequences of youth tobacco use: a review of prospective behavioural studies[END_REF] . What mechanisms underlie these long-term consequences of nicotine exposure during adolescence is unclear.

To uncover molecular and cellular mechanisms underlying long-term cognitive disturbances resulting from adolescent nicotine exposure, we exposed adolescent rats to nicotine and assessed visuospatial attention, protein expression and synaptic physiology in PFC during adulthood.

Between postnatal day (PND) 28 and 50, rats show typical adolescent-like behaviors, such as peerdirected and risk-taking behavior and altered sensitivity to drugs of abuse [START_REF] Spear | The adolescent brain and age-related behavioral manifestations[END_REF] . Similar to findings from human epidemiological data, adolescent nicotine exposure in rats has long-term effects on attentional processing [START_REF] Counotte | Long-lasting cognitive deficits resulting from adolescent nicotine exposure in rats[END_REF] . The 5-choice serial reaction time task (5-CSRTT) is the most widely employed translational paradigm that has tremendously contributed to our understanding of the neural correlates of divided and sustained attention and impulsive action [START_REF] Pattij | The neuropharmacology of impulsive behaviour[END_REF][START_REF] Robbins | The 5-choice serial reaction time task: behavioural pharmacology and functional neurochemistry[END_REF] . Nicotine treatment during adolescence (PND 34-43) increased impulsive behavior (p=0.042), and impaired measures of attention (7%, p=0.015) in adulthood following five weeks of abstinence [START_REF] Counotte | Long-lasting cognitive deficits resulting from adolescent nicotine exposure in rats[END_REF] (Fig. 1a). The latter is comparable to the decrease (3-4%) in visuospatial attention observed in male adolescent smokers [START_REF] Jacobsen | Effects of smoking and smoking abstinence on cognition in adolescent tobacco smokers[END_REF][START_REF] Jacobsen | Gender-specific effects of prenatal and adolescent exposure to tobacco smoke on auditory and visual attention[END_REF] . Increasing attentional load augmented the difference between these groups (Supplementary Fig. 1). In contrast, adult nicotine exposure (PND 60-69) did not have these long-term consequences.

Nicotine treatment had no effect on locomotor behavior, body weight, learning or motivation (Supplementary Figs. 2 and3). Thus, in rats nicotine exposure during adolescence has profound impact specifically on attention and impulsive behavior later in life.

Visuospatial attention in the 5-CSRTT strongly depends on the integrity of the medial PFC (mPFC) [START_REF] Dalley | Prefrontal executive and cognitive functions in rodents: neural and neurochemical substrates[END_REF] . Synaptic connectivity and dynamics of neuronal interactions in mPFC underlie attentional processing. To investigate whether long-term molecular changes were induced in synaptic connectivity in mPFC by adolescent nicotine exposure, we performed iTRAQ-based quantitative proteomics of synaptic membrane fractions of rat mPFC. Synaptic protein levels from animals exposed to either nicotine or saline during adolescence (PND 34-43) were quantified one day after nicotine exposure ended (PND 44), as well as after five weeks of abstinence (PND 78).

From 297 unique proteins reliably quantified at these two time points (Supplementary Fig. 4), nine synaptic proteins were significantly affected by age and pretreatment (Supplementary Table 1), indicating that adolescent nicotine exposure altered the developmental expression profile of these proteins. Post-hoc analyses revealed three significantly regulated proteins five weeks after nicotine pre-treatment in adolescents (PDN78) of which only mGluR2 could be confirmed by immunoblotting (p=0.048; Fig. 1b and Supplementary Fig. 4). In contrast, nicotine exposure during adulthood (PND 60-69) did not alter synaptic protein levels of mGluR2, neither after one day, nor after five weeks of abstinence. These findings show that in mPFC, synaptic metabotropic glutamate receptor protein levels are altered specifically by adolescent and not adult nicotine exposure. We next studied functional consequences of down-regulation of synaptic mGluR2 protein levels after adolescent nicotine exposure for mPFC glutamatergic synaptic function in adulthood. Activation of mGluR2s in developing somatosensory cortex suppresses glutamatergic transmission [START_REF] Mateo | Group II metabotropic glutamate receptors inhibit glutamate release at thalamocortical synapses in the developing somatosensory cortex[END_REF] . Similarly, in adult mPFC, activation of mGluRs by the group II agonist LY379268 (5 μM) strongly reduced extracellular evoked excitatory postsynaptic currents (eEPSCs) in wholecell recordings from mPFC layer V pyramidal neurons (Fig. 1c). These mGluR2 receptors were most likely localized on presynaptic glutamatergic terminals, since LY379268 increased pairedpulse ratio of eEPSCs and reduced miniature EPSC amplitudes recorded in the presence of TTX ( 1μM) (Supplementary Fig. 6). In pyramidal neurons from PND 78 animals, five weeks after adolescent nicotine exposure, mGluR2/3 activation reduced eEPSC amplitudes to a lesser extent than in those from saline-treated animals (p=0.013) (Fig. 1c,d). Baseline EPSCs were unaltered by nicotine exposure (Supplementary Fig. 7). The reduced mGluR-mediated synaptic inhibition in nicotine exposed animals (Fig. 1c-d; p=0.0006, Supplementary Fig. 7b) is in line with reduced synaptic mGluR2 protein expression in the mPFC of these animals (Fig. 1b). In adult nicotinetreated animals, eEPSC amplitude reduction by LY379268 was not different from their salinetreated controls, in line with unaltered synaptic mGluR2 protein levels.

Synaptic group II mGluRs have been implicated in short-term plasticity of glutamatergic synapses in cortical areas, and short-term depression and facilitation strongly shape information transfer in cortical networks. We tested whether reduction in synaptic mGluR2 levels following adolescent nicotine exposure affected short-term plasticity of glutamatergic synapses in adult mPFC (Fig. 2). Five weeks abstinence following adolescent nicotine exposure resulted in less depression of mPFC glutamatergic synapses compared with their saline-treated controls (Fig. 2a,b; p<0.001). This corroborated the observed reduced synaptic mGluR2 protein levels in adolescent nicotine-treated animals. Nicotine exposure during adulthood (PND 60-69) did not reduce synaptic depression (PND 104), but instead resulted in a slight increase of depression at short intervals (Fig. 2b; p=0.033). The group II mGluR antagonist MPPG (100-200 μM) reduced short-term depression ((Fig. 2c; p=0.012). The group II mGluR agonist LY379268 strongly reduced the amplitude of the first eEPSC in the train by 80% (p<0.001; Supplementary Fig. 8), confirming that mGluR2s are involved in synaptic depression at these synapses. Thus, adolescent nicotine exposure resulted in reduced synaptic mGluR2 signaling and short-term plasticity on mPFC output layer V pyramidal neurons after five weeks of abstinence, and may explain reduced mPFC functioning and disturbances in attention.

To test whether mGluR2 signaling in the mPFC is involved in attention, we infused the type II antagonist MPPG into the mPFC of control animals. Decreasing mGluR2 activity in these animals selectively reduced attentional performance (Fig. 3a; p=0.018; Supplementary Table 2), showing the importance of mPFC mGluR2 function in attention. Next, to determine whether reduced synaptic mGluR2 protein levels in the mPFC can indeed explain the observed decrements in attention after adolescent nicotine exposure, we increased mGluR2 activity in vivo by infusing LY379268 into the mPFC during behavioral testing (Fig. 3b,d). Adolescent nicotine exposure reduced attention (cf. Fig. 1, Supplementary Fig. 9, 10 and Supplementary Table 3), and LY379268 selectively improved attention in nicotine-treated animals in a dose-dependent manner (Fig. 3b; p=0.033). This indicates that augmenting mGluR2 activity in adult animals previously exposed to nicotine during adolescence restores attention performance. Infusion of LY379268 in mPFC did not ameliorate impulsivity in these animals (Fig. 3c, Supplementary Table 4). In saline-treated animals, LY379268 did not affect attention performance (Fig. 3c), but increased impulsivity (Fig. 3d; p=0.018) and MPPG did not affect impulsivity (Fig. 3c), indicating that mGluR2 activity affects attention and impulsivity differentially and emphasizing that these behavioral domains are under differential control by the mPFC [START_REF] Robbins | The 5-choice serial reaction time task: behavioural pharmacology and functional neurochemistry[END_REF][START_REF] Chudasama | Dissociable aspects of performance on the 5-choice serial reaction time task following lesions of the dorsal anterior cingulate, infralimbic and orbitofrontal cortex in the rat: differential effects on selectivity, impulsivity and compulsivity[END_REF] .

This study uncovers molecular and cellular changes induced by adolescent nicotine exposure that result in cognitive disturbances in adult life and reveals that a lasting down-regulation of mGluR2 on presynaptic terminals of glutamatergic synapses in PFC persists into adulthood causing disturbances in attention. Decreased mGluR2 functionality reduced short-term plasticity of glutamatergic inputs to PFC output layer V pyramidal neurons and thereby most likely alters information transfer in active networks underlying attention [START_REF] Zucker | Short-term synaptic plasticity[END_REF] . Restoring mGluR2 activity in vivo in the PFC of adult animals exposed to nicotine during adolescence remediated the attention deficit.

Our findings stress that nicotine impacts the brain in different ways during adolescence.

Although nicotine has acute behavioral effects on attention in adult rats [START_REF] Pattij | The neuropharmacology of impulsive behaviour[END_REF] , the adult mPFC does not seem to suffer from lasting consequences from nicotine exposure. Cognitive performance, synaptic mGluR2 protein levels and glutamatergic synaptic depression were all unaffected by nicotine exposure during adulthood. Thus, this clearly pinpoints adolescence as a period of increased vulnerability for the effects of nicotine. Not only from a behavioral, but also from a molecular point of view, the adolescent brain is more susceptible to consequences of nicotinic receptor activation. What sequence of molecular events ties nicotine exposure to altered synaptic mGluR2 protein levels, without increasing mGluR2 gene expression (data not shown), is as yet unknown.

Nicotine easily penetrates the blood-brain barrier at concentrations experienced by smokers and readily binds to nAChRs in PFC to enhance glutamatergic and GABAergic synaptic transmission.

Perhaps elevated mGluR2 levels following nicotine exposure at the end of adolescence compensate for nicotine's actions and inhibit neurotransmitter release. Regardless, altered glutamatergic synaptic transmission in mPFC plays a key role in attention and impulsivity and altered mGluR2 levels have been linked to several brain disorders. The sustained molecular and synaptic changes that result from nicotine exposure during adolescence and alter cognitive performance during adulthood may prompt us to reconsider our views on the aetiology of attention deficits.

experiments. DSC, TP analyzed behavioral experiments. DSC, HDM, TP, SS wrote the manuscript.

Figure 1

 1 Figure 1 Adolescent nicotine exposure affects measures of attentional performance, mGluR2 levels and function on the long term. (a) Visuospatial divided and sustained attention (accuracy; left) indicated by percentage of correct stimulus detections (average 5 baseline sessions, see methods), and impulsive behavior indicated by number of prematurely expressed responses before stimulus onset (right) measured 5 weeks after adolescent (n=11) or adult (n=11) nicotine exposure. (b) Quantification of immunoblot analysis of synaptic mGluR2 expression (n=8). (c) Time course of eEPSC amplitude reduction by LY379268 in adolescent nicotine (black, n=25) or saline exposed animals (gray, n=24), per cell each data point is an average of 7 eEPSCs. Insets: example eEPSC traces in control (a) and in presence of LY379268 (b). Right panel: average of last 10 responses in presence of LY379268. (d) mGluR2/3-dependent inhibition of eEPSC amplitudes is different due to adolescent nicotine treatment (saline, n=39; nicotine, n=47, F(3,128)=6.2; p=0.0006), with no difference in adult treated animals (saline, n=22; nicotine, n=26). Mean±SEM is presented. **p<0.01, *p<0.05. All experiments were approved (ethical committee VU University Amsterdam).

Figure 2

 2 Figure 2 Short-term depression in mPFC is reduced 5 weeks after nicotine exposure during adolescence. (a,d) Example of short-term plasticity recorded from a layer V pyramidal neuron after extracellular stimulation in layer II/III of adolescent nicotine-or saline-exposed animals. (b) Summary of short-term depression during 10 stimuli (25-200 ms intervals), measured in adolescent-treated (left panel, F (1,137) =21.6 p<0.001, saline n=10-26, nicotine n=10-23) and adulttreated animals (right panel F (1,255) =4.57 p=0.033; saline n=24, nicotine n=31). Average of last 3 responses in the train was normalized to the first one for analysis (c) mGluR group II/III antagonist MPPG reduces short-term plasticity in mPFC layer V pyramidal neurons (F (2,124) =7.03 p=0.012; control n=11, MPPG 100 µM n=10-11, MPPG 200 µM n=6). Mean±SEM is presented.

Figure 3

 3 Figure 3 Intra-mPFC infusion of mGluR2/3 agonist LY379268 reverses long-term attentional disturbances in adolescent nicotine-exposed animals. (a) Infusion of the group II antagonist MPPG decreased divided and sustained attention (accuracy) in control animals. (b) LY379268 normalized the nicotine-induced disturbances in divided and sustained attention (accuracy) in adolescent nicotine-exposed animals (dose F (1,10) =4.08, p=0.033), with no effect on adolescent saline-exposed animals (dose F (1,10) =1.71, n.s.). (c,d) Impulsive behavior was not affected in control animals by MPPG (c), nor in adolescent nicotine-exposed animals by LY379268, but was increased in salineexposed animals (dose F (1,10) =4.98, p=0.018). Mean±SEM is presented. *p<0.05.

Acknowledgements

Authors thank Yvar van Mourik, Mathijs Stegeman, Hans Lodder, Brendan Lodder, Christiaan de Kock and Rob Binnekade for excellent technical assistance. Funding was received from NWO (D.S.C., N.A.G., S.S., H.D.M., T.P., A.N.M.S., A.B.S.), CMSB (K.W.L, R.C.v.d.S.), and VU University (H.D.M.; Stg VU-ERC).

Supplementary Information is linked to the online version of the paper.

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Journal: Nature Neuroscience

Article Title:

Lasting synaptic changes underlie attention deficits caused by nicotine exposure during adolescence

Corresponding Author:

Sabine Spijker

Supplementary Item & Number

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Title or Caption

Supplementary Figure 1 Manipulation of task parameters in the 5-CSRTT following adult and adolescent nicotine exposure.

Supplementary Figure 2 Locomotor activity and bodyweight are not differentially affected by adolescent nicotine exposure.

Supplementary Figure 3

No learning defects in 5-CSRTT acquisition due to adolescent nicotine exposure.

Supplementary Figure 4 iTRAQ proteomics and immunoblotting for adolescent nicotine-induced changes.

Supplementary Figure 5

Adolescent nicotine exposure does not alter AMPA-R/NMDA-R current ratios.

Supplementary Figure 6 mGluR2 modulates EPSCs through a presynaptic action.

Supplementary Figure 7

Nicotine-exposure during adolescence does not alter baseline EPSCs in adult animals.

Supplementary Figure 8

Activation of mGluR2/3 by agonist LY379268 (5 µM) reduces short-term plasticity in pyramidal neurons in layer V of the mPFC.

Supplementary Figure 9

Performance in the 5-CSRTT over five baseline sessions before and after cannulae implantation.

Supplementary Figure 10 Performance in the 5-CSRTT before, during and after intracranial infusions of LY379268.

Supplementary Table 1

Protein changes following one day and five weeks of adolescent nicotine exposure.

Supplementary Table 2

Behavioral parameters in the 5-CSRTT after intracranial infusion of 0.5 and 2.0 nmol/side MPPG in the mPFC.

Supplementary Table 3

Behavioral parameters in the 5-CSRTT averaged over 5 baseline sessions.

Supplementary Table 4 Behavioral parameters in the 5-CSRTT after intracranial infusion of LY379268 in the mPFC.

Supplementary Results

& Discussion, Methods, and References