Alteration of the gut microbiota has been associated with different gastrointestinal disorders. Normobiosis restoration by faecal microbiota transplantation (FMT) is considered a promising therapeutic approach, even if the mechanisms underlying its efficacy are at present largely unknown. Here we sought to elucidate the functional effects of therapeutic FMT administration during experimental colitis on innate and adaptive immune responses in the intestinal mucosa. We show that therapeutic FMT reduces colonic inflammation and initiates the restoration of intestinal homeostasis through the simultaneous activation of different immune-mediated pathways, ultimately leading to IL-10 production by innate and adaptive immune cells, including CD4+ T cells, iNKT cells and Antigen Presenting Cells (APC), and reduces the ability of dendritic cells, monocytes and macrophages to present MHCII-dependent bacterial antigens to colonic T cells. These results demonstrate the capability of FMT to therapeutically control intestinal experimental colitis and poses FMT as a valuable therapeutic option in immune-related pathologies.

The gut mucosa is continuously exposed to a vast community of microorganisms, collectively defined as microbiota, establishing a mutualistic relationship with the host and contributing to shape the immune system. Gut microbiota is acquired at birth, and its composition is relatively stable during the entire adult life. Intestinal dysbiosis, defined as a microbial imbalance of gut bacterial communities, can be caused by several factors, including bacterial infections and antibiotic use, and has been associated with an increased risk to develop or exacerbate immune-mediated pathologies, such as allergic reactions, asthma, and inflammatory bowel diseases. Still, the mechanisms by which antibiotic-induced gut dysbiosis may lead to development of mucosal inflammation are still matter of debate. To this end, we aimed to evaluate the impact of antibiotic treatment on phenotype and functions of intestinal immune cell populations, including invariant natural killer T (iNKT) cells, a subset of lipid-specific T cells profoundly influenced by alterations on the commensal microbiota. To this aim, a cocktail of broad-spectrum antibiotics was administered for 2 weeks to otherwise healthy mice before re-colonization of the intestinal microbial community with oral gavage of eubiotic or dysbiotic mucosa-associated bacteria and luminal colonic content, followed or not by intestinal inflammation induction. Here. we showed that short-term antibiotic treatment alters frequency and functions of intestinal iNKT cells, even in the absence of intestinal inflammation. The presence of a dysbiotic microbiota after antibiotic treatment imprints colonic iNKT and CD4+ T cells toward a pro-inflammatory phenotype that collectively contributes to aggravate intestinal inflammation. Nonetheless, the inflammatory potential of the dysbiotic microbiota decreases over time opening the possibility to temporally intervene on the microbial composition to re-equilibrate dysbiosis, thus controlling concomitantly mucosal immune T cell activations.

Animal models are powerful tools to expand our understanding of human diseases. Histopathological evaluation of murine experimental models is often required to support further research; thus, a more rigorous evaluation of murine histological samples is strongly advocated. Indeed, the overall quality of tissue sections is critical to draw reliable and accurate conclusions. As several methodological variables may reduce the reliability of the pathological analysis, a standardization of the procedural steps required for the processing of histological murine tissues is advisable. Here, we describe a method to standardize the technical procedure from the initial preparation to the paraffin embedding of murine samples. Specifically, we have implemented an automated inclusion system, that is, the SAKURA Tissue-Tek inclusion instrument, which is routinely used for paraffin inclusion of human samples, to process murine specimens of intestinal inflammation. Colitis severity was assessed in chronically Dextran Sodium Sulphate (DSS)-treated mice by cytofluorimetric analysis of colonic cellular infiltrates, expression of inflammatory genes and histopathological analysis of tissue samples, comparing manual and automated tissue preparation systems. We here conclude that implementation of this technique can significantly increase the quality and the reliability of histopathological examination of murine tissues.