Periodic Reporting for period 1 - LIPID IMMUNITY (Regulation of lipid-mediated immunity in the intestine) Reporting period: 2016-03-01 to 2018-02-28 Summary of the context and overall objectives of the project The current project titled Regulation of lipid-mediated immunity in the intestine aimed to explore the mechanisms regulating lipid-mediated immunity in the intestine. This tissue, in mammals, has a unique environment colonized by a highly complex mixture of microorganisms that contribute to the establishment of tissue homeostasis. Both, the microbiota and the host, establish mutual relationships that comprise, among other contributors, the intestinal immune system. Several evidences propose that the dysregulation of the intestinal immune system or the alteration in the commensal bacteria populations can lead to inflammatory and autoimmune diseases development, such as inflammatory bowel disease (IBD). In the last decades, IBD has become a global disease with accelerating incidence in newly industrialised countries (1). Data reviewed in December 2017 by Kaplan et al. highlight the need for research into prevention of this complex and costly disease. In this context, understanding the immune mechanisms involved in the pathology of this disease is crucial for its prevention and for the development of efficient therapies. It is known that interactions between immune system and commensal bacteria are complex and involved several immune cell types. One of these cell types is the population called innate lymphoid cells (ILCs) which comprise several families of cells that can be classified into three groups: ILC type 1 (ILC1), ILC2 and ILC3. Given their functions and location, ILCs have been considered as orchestrators of immune defences at mucosal surfaces (2). Lately, it has been described a novel function for ILCs as antigen presenting cells (APCs) through MHC-II-dependent presentation of commensal-derived antigens (3-7). Both ILC2 and ILC3 populations are able to internalize and present peptide antigens on MHC-II and control T cell responses (3-7). Nonetheless, in addition of source of protein antigens, commensal bacteria are a source of lipids capable to activate the lipid-reactive cells named natural killer T (NKT) cells (8,9). NKT cells, through TCR recognition of self- or commensal-derived lipids presented by CD1d, contribute to establish immune homeostasis and to anti-microbial, anti-tumour and autoimmune responses (10,11). Despite increasing data during the last decades, how commensal lipids are handled by immune cells, the contribution of different APCs to lipid presentation, and their effects on mucosal NKT cells remain poorly understood. Taking this into consideration, this project has focussed in understanding the mechanisms controlling lipid-mediated immunity by examining the ILC-NKT cell crosstalk. Specific objectives include: the characterization of CD1d expression, intracellular trafficking and lipid presentation capacity of ILC3s (Aim 1); as well as the study of the role of ILC3s on lipid-mediated immunity (Aim 2). As a result of the progress of the project, it was found that NKT cell-APCs crosstalk is a key mechanism for the regulation of intestinal homeostasis. Specifically, a previously unknown role for ILC3s on CD1d-dependent immunity was discovered. 1. Ng et al. The Lancet, 2018; 2. Artis et al. Nature, 2015; 3. Hepworth et al. Nature, 2013; 4. Hepworth et al. Science, 2015; 5. Oliphant et al. Immunity, 2014; 6. von Burg et al. PNAS, 2014; 7. Mirchandani et al. J Immunol, 2014; 8. An et al. Cell, 2014; 9. Wieland Brown et al. PLoS Biol, 2013; 10. Brennan et al., Nat Rev Immunol, 2013; 11. Salio et al., Annu Rev Immunol, 2014. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far As mentioned above, the project aimed to investigate the mechanisms regulating lipid-mediated immunity in the intestinal mucosa by examining the crosstalk between ILCs and NKT cells. To achieve this, Aim 1 and 2 were divided into diverse specific objectives. Aim 1 included the characterization of CD1d expression, intracellular trafficking and lipid presentation capacity of ILC3s which were accomplished by combining in vitro and in vivo experiments. Flow cytometry and quantitative PCR analysis were preformed to determine the CD1d expression. Results showed that CD1d level on ILC3s is comparable of those levels found on classical CD1d+ cells, such as DCs and B cells (Attached Figure 1). After CD1d expression on ILC3s was confirmed, lipid loading and presentation capacity of ILC3s were analysed through both in vitro and in vivo experiments. Flow cytometry and ELISA analysis indicated that ILC3s are able to present Galcer through CD1d to NKT cells (Attached Figure 2). Finally, the effect of CD1d engagement on ILC3s was addressed. In vitro and in vivo experiments exhibited an increase in IL-22 levels after CD1d engagement (Attached Figure 3). The second part of the project aimed to analyse the effect of lipid-presentation by ILC3s on NKT cell responses (Aim 2). We test whether ILC3s could mediate CD1d-dependent lipid presentation in vivo and found that ILC3s able are to induce activation of NKT cells (Attached Figure 2). While most of this project has been focused on exploring the role of ILC3s in the regulation of lipid immunity, we have also extended our analyses to explore the role of other APCs in the regulation of mucosal NKT cells. We analysed the CD1d-mediated lipid presentation by CD11c+ APCs (macrophages and DCs) to intestinal NKT cells. Data unveiled a central role for CD11c+ cells in controlling lipid-dependent immunity in the intestinal compartment and revealed an NKT cell-DC crosstalk as a key mechanism for the regulation of gut homeostasis.Overall, our experiments demonstrate that ILC3s express CD1d, have the capacity to internalise lipid antigens and can mediate NKT cell activation in vitro and in vivo. These functions could be particularly relevant at mucosal sites, where has been proposed that ILC3s play a central role in the anatomical containment of commensals as well as that NKT cells and CD1d expression regulate mucosal homeostasis.Data obtained has resulted in 2 peer-reviewed publications:• CD1d-mediated activation of group 3 innate lymphoid cells drives IL-22 production. Jimeno et al., EMBO Rep, 2017 Jan,18:39-47.• CD11d-mediated lipid presentation by CD11c+ cells regulate intestinal homeostasis. Saez de Guinoa, Jimeno et al., EMBO J, 2018, pii:e97537.Moreover, data were presented in 2 research conferences:• British Society for Immunology/NVVI 2016 Congress (Liverpool, United Kingdom): December 2016 • CD1-MR1 2017 Congress (Napa, California): November 2017 Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Given the successful achievement of planned aims in the proposal, it can be considered that data generated in this project can positively impact on both the scientific community and the society. Produced results have contributed towards science, innovation and generation of knowledge in the Immunology field. Our data identify a novel pathway for immune regulation of NKT cells mediated by CD1d expression on ILC3s. Notably, this pathway could have implications for a variety of immune responses where CD1d-dependent immunity plays a central role, including intestinal immunity. It is particularly relevant in the context of several immune disorders, such as IBD. Thus, generated knowledge offers to scientific community new possibilities to explore novel targets for immune intervention in intestinal disorders. Therefore, these findings certainly can contribute to public health and wellbeing. Engagement of CD1d on ILC3s induces IL-22 production. ILC3s internalize and present lipid antigens to iNKT cells. CD1d expression on ILCs.