Final Report Summary - ILCHI (Innate lymphoid cells in intestinal homeostasis and immunity) Inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, impose a high burden on Public Health, as an estimated 1% of people are affected in industrialized nations. Current treatments include general immunosuppression and inhibition of TNF, antibiotics and surgery to remove affected parts of the digestive tract. However, more specific or less traumatic strategies are warranted. In addition, the prevalence of other inflammatory immunopathologies, such as type I diabetes, multiple sclerosis, arthritis, and obesity, is also on the rise. Therefore, a better understanding of the mechanisms leading to a loss of control of inflammation and immunopathological reactions is necessary to design new and more effective preventive and therapeutic strategies.The nuclear hormone receptor RORγt marks a family of pro-inflammatory lymphoid cells, which includes adaptive IL-17-producing helper T cells (Th17), as well as subsets of innate lymphoid cells (ILCs), such as lymphoid tissue inducer (LTi) cells and IL-22-producing NKp46+ cells. These cells are required for the development of lymphoid tissues, homeostasis with symbiotic microbiota and defense against pathogens. Whereas most T cells are induced to express RORγt by microbiota, the development and activity of RORγt+ ILCs, so-called ILC3, is programmed to pre-empt intestinal colonization by bacterial symbionts and in turn, symbionts regulate the activity of ILC3, ultimately leading to equilibrium between the host and its symbiotic microbiota. Type-17-cytokines are also involved autoimmune inflammatory pathologies. Thus, a finely balanced crosstalk between ILC3 and microbiota develops that is critical for intestinal homeostasis, a loss of which leads to inflammatory immunopathology. It appears obvious that RORγt possesses an important target to modulate inflammation. The objectives of this project aimed at understanding which and how symbiotic microbes induce and regulate ILC3, and, on the other hand, if and how ILC3 control microbial symbiota during homeostasis as well as resistance to pathology. The studies were carried out in the Lymphoid Tissue Development Unit at Institut Pasteur in Paris, France, and the Department of Infection Immunology at Twincore in Hannover, Germany.To determine novel mechanisms of in vivo ILC3 regulation, a panel of knockout mice was screened for activity and homeostasis of ILC3. For the identification of potential regulatory pathways, mice deficient for several innate immune pathways such as MyD88, NOD1, NOD2 or TLR5 were analyzed, but none of them showed altered regulation of ILC3 at the steady state. Moreover, cytokines such as TSLP as well as IL-7 showed no influence on ILC3 homeostasis or function, even though the latter has been described to be critically important for ILC3 generation, indicating a strong influence of housing conditions and therefore the microbial constitution on activation and homeostasis of ILC3. This was further corroborated by experiments where particular knockouts showed different phenotypes depending on whether they were compared to separately housed wild-type or to littermate controls. Preliminary experiments on correlation of microbial composition and activation status of ILC3 from different mutant mouse strains turned out to be of little promise due to high data fluctuation already at littermate level. This led to dismissal of this approach for identification of ILC3 modulating species. For identification of potentially ILC3 regulating symbionts, germ-free or conventionally raised mice were colonized with segmented filamentous bacteria (SFB), which have been shown before to regulate Th17 cells. However, SFB exerted no effect on ILC3 activation and numbers. To identify ILC3 regulating cells subsets, several lineage specific knockouts for CD3e, αβ or γδ T cells as well as for B cells or dendritic cells (DC) were analyzed. B cells and αβ Τ cells showed an activating effect on ILC3 activation, whereas depletion of Treg in the DEREG mouse model had no influence, neither under homeostatic conditions nor during Citrobacter rodentium infection. On the contrary, CD3e and αβ Τ cells as well as DC exerted an inhibitory function at the steady state. The latter goes in line with the previously reported indirect negative integration of IL-25 mediated microbial derived-signals via IL25R+ DC. It could be shown that among the DC population, particularly the CD11c+MHCII+CD103- subset, mainly comprising of the so-called CX3CR1+ intestinal macrophages, was exclusively expressing IL-25R. Even though B cells and ILC2 also expressed high IL-25R levels, they failed to regulate ILC3 in an in vitro modulation assay. Hence, the role of IL-25R expression on these cells and the consequence for ILC3 remains elusive.The complex interrelation of ILC3 with microbiota and innate as well as adaptive immune cells necessitates a novel ILC3 depletion system in order to concisely depicting their role in homeostasis and defense. In light of recently published data, an initially envisioned chimera-based ILC3 depletion system turned out to be suboptimal. Instead, bacterial artificial chromosome (BAC) technology was applied to generate a new transgenic mouse line that allows for the timely and specific ablation of ILC3 in vivo, and therefore, to establish the impact of RORγt+ ILC on the development of the immune system, on the activation of innate and adaptive responses, on the symbiotic microbiota and infection by pathogens, as well as on the development of inflammatory pathology. The results of this study will ultimately support a better understanding of the mechanisms leading to a loss of homeostasis and immunopathological reactions and will allow the design of new and more effective preventive and therapeutic strategies against intestinal immunopathologies that aim to re-establish and reinforce intestinal homeostasis through balanced activation, rather than blocking, of pro-inflammatory immunity. In addition, recent evidence indicate that loss of intestinal homeostasis induces a sub-inflammatory state of the host, which appears to affect tissues and organs distinct from the intestine, such as adipose tissues and the pancreas, and lead to physiologic deregulation and pathology, including obesity, diabetes and other autoimmune pathologies. Thus, regulation of intestinal homeostasis may open new avenues for the prevention and treatment of physiologic disturbances and autoimmune pathologies. Diagnostic tools may be developed based on the immune-status in the intestine and in the blood, as well as on the degree of microbial dysbiosis in the feces. Such assays may help predicting the risk of immunopathology in the intestine and beyond, and discriminate between different types of patients for the prognosis of distinct types of IBD and autoimmune disorders.