RESUMO
Humans homozygous for inactivating LRBA (lipopolysaccharide (LPS)-responsive beige-like anchor) mutations or with compound heterozygous mutations exhibit a spectrum of immune-related pathologies including inflammatory bowel disease (IBD). The cause of this pathology remains undefined. Here we show that disruption of the colon epithelial barrier in LRBA-deficient mice by dextran sulfate sodium (DSS) consumption leads to severe and uniformly lethal colitis. Analysis of bone marrow (BM) chimeras showed that susceptibility to lethal colitis is primarily due to LRBA deficiency in the immune compartment and not the gut epithelium. Further dissection of the immune defect in LRBA-deficient hosts showed that LRBA is essential for the expression of CTLA4 by Treg cells and IL22 and IL17 expression by ILC3 cells in the large intestine when the gut epithelium is compromised by DSS. We further show that SHIP1 agonism partially abrogates the severity and lethality of DSS-mediated colitis. Our findings indicate that enteropathy induced by LRBA deficiency has multiple causes and that SHIP1 agonism can partially abrogate the inflammatory milieu in the gut of LRBA-deficient hosts.
Assuntos
Colite , Imunodeficiência de Variável Comum , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Colite/induzido quimicamente , Colite/genética , Camundongos , Mutação , Linfócitos T ReguladoresRESUMO
Since their discovery, innate lymphoid cells (ILCs) have gradually been gaining greater relevance in the field of immunology due to their multiple functions in the innate immune response. They can mainly be found in mucosal and barrier organs like skin, gut, and lungs, and have been classified into five main types (NKs, ILC1s, ILC2s, ILC3s, and Lti cells) according to their function and development. They all play major roles in functions such as tissue homeostasis, early pathogen defense, regulation of inflammation, or tissue remodeling. ILCs are mostly tissue-resident cells tightly bound to the tissue structure, a fact that requires long and complex protocols that do not always provide sufficient yield for analysis. This suggests the need for optimized approaches aimed at ensuring that enriched and viable ILC samples are obtained, in order to furnish quality results. Herein a detailed protocol is established for obtaining a single-cell suspension highly enriched in lymphoid cells from mouse gut in order to identify the different subsets of ILCs by means of flow cytometry. The cell marker panel and flow cytometry gating strategies for identification and quantification of all the different ILC populations are provided for simultaneous analysis. Moreover, the protocol described includes a procedure for studying the different cytokines produced by ILC3s involved in maintaining the integrity of the gut barrier and defending against extracellular pathogens. As a result, herein an efficient method is presented for studying mouse ILCs within the lamina propria of the small intestine and colon; this can constitute a useful tool for future investigations in the field.