Intestinal-derived ILCs migrating in lymph increase IFNγ production in response to Salmonella Typhimurium infection

Innate lymphoid cells (ILCs) are enriched in mucosae and have been described as tissue-resident. Interestingly, ILCs are also present within lymph nodes (LNs), in the interfollicular regions, the destination for lymph-migratory cells. We have previously shown that LN ILCs are supplemented by peripheral tissue-derived ILCs. Using thoracic duct cannulations, we here enumerate the intestinal lymph ILCs that traffic from the intestine to the mesenteric LNs (MLNs). We provide, for the first time, a detailed characterisation of these lymph-migratory ILCs. We show that all ILC subsets migrate in lymph, and while global transcriptional analysis reveals a shared signature with tissue-resident ILCs, lymph ILCs express migration-associated genes including S1PRs, SELL (CD62L) and CCR7. Interestingly, we discovered that while Salmonella Typhimurium infections do not increase the numbers of migrating ILCs, infection changes their composition and cytokine profile. Infection increases the proportions of RORyt+ T-bet+ ILCs, levels of IFNγ, and IFNγ/GM-CSF co-expression. Infection-induced changes in migratory ILCs are reflected in colon-draining MLN ILCs, where RORyt+ T-bet+ ILCs accumulate and display corresponding increased cytokine expression. Thus, we reveal that ILCs respond rapidly to intestinal infection and can migrate to the MLN where they produce cytokines

Antibiotics induce sustained dysregulation of intestinal T cell immunity by perturbing macrophage homeostasis

Macrophages in the healthy intestine are highly specialized and usually respond to the gut microbiota without provoking an inflammatory response. A breakdown in this tolerance leads to inflammatory bowel disease (IBD), but the mechanisms by which intestinal macrophages normally become conditioned to promote microbial tolerance are unclear. Strong epidemiological evidence linking disruption of the gut microbiota by antibiotic use early in life to IBD indicates an important role for the gut microbiota in modulating intestinal immunity. Here, we show that antibiotic use causes intestinal macrophages to become hyperresponsive to bacterial stimulation, producing excess inflammatory cytokines. Re-exposure of antibiotic-treated mice to conventional microbiota induced a long-term, macrophage-dependent increase in inflammatory T helper 1 (T 1) responses in the colon and sustained dysbiosis. The consequences of this dysregulated macrophage activity for T cell function were demonstrated by increased susceptibility to infections requiring T 17 and T 2 responses for clearance (bacterial and helminth infections), corresponding with increased inflammation. Short-chain fatty acids (SCFAs) were depleted during antibiotic administration; supplementation of antibiotics with the SCFA butyrate restored the characteristic hyporesponsiveness of intestinal macrophages and prevented T cell dysfunction. Butyrate altered the metabolic behavior of macrophages to increase oxidative phosphorylation and also promoted alternative macrophage activation. In summary, the gut microbiota is essential to maintain macrophage-dependent intestinal immune homeostasis, mediated by SCFA-dependent pathways. Oral antibiotics disrupt this process to promote sustained T cell-mediated dysfunction and increased susceptibility to infections, highlighting important implications of repeated broad-spectrum antibiotic use

Intestinal migratory innate lymphoid cells

Innate lymphoid cells (ILCs) mainly reside at mucosal sites, where they produce effector cytokines upon activation. ILCs are also found, at lower frequencies, in secondary lymphoid tissues. ILCs in the lymph nodes (LN) are located in the interfollicular area, which is rich in migratory dendritic cells (DCs) and recently activated T cells and B cells. We recently discovered that some intestinal ILCs traffic from the intestinal lamina propria to mesenteric lymph nodes (MLN). However, the functions of ILCs in the LNs are hardly understood. Here we provide for the first time a detailed description of the migratory ILCs in intestinal lymph of mice in homeostasis and immunity. To isolate migratory ILCs, we obtained lymph by thoracic duct cannulations of mice with or without a previous mesenteric lymphadenectomy. The ILC populations were then analysed by flow cytometry, and their transcriptomic profile was assessed by performing RNA sequencing. Our results show that a small number of migratory ILCs continuously traffic from the intestine to the MLNs. Whole genome analyses in the steady state revealed a shared global ILC signature between migratory ILCs in lymph and intestinal resident ILCs. We then demonstrated that all subsets of ILCs migrate in intestinal lymph, with ILC1 and ILC2 being most frequent. We compared the transcriptomic profile of migratory DCs, T cells and ILCs in afferent lymph and clearly identified a core migratory signature shared by all cell types. In order to investigate the impact of inflammation on this migratory ILC population, we used several infection and inflammation models, and assessed changes in the migratory ILC populations. We detected phenotypic changes of migratory ILCs following Salmonella infections of mice. Furthermore, although Salmonella infection did not increase total numbers of migratory ILCs in lymph, we observed an increase in T-bet+Roryt+ co-expressing ILCs in lymph of infected mice. After infection, changes in ILCs transcriptomic profile indicate an activated phenotype. Corresponding to the lymph ILC data, we also observed an accumulation of T-bet+Roryt+ co-expressing ILCs in the colonic draining LN (cMLN). Our data clearly demonstrate, for the first time, that inflammation can alter migratory ILC populations. This might be important for the initiation and regulation of adaptive immune responses in the draining LN. This first characterisation of migratory ILCs is important as it helps to understand how they contribute to shaping adaptive immune responses in the interfollicular area of the LNs

Salmonella enterica serovar Typhimurium travels to mesenteric lymph nodes both with host cells, and autonomously

Salmonella infection is a globally important cause of gastroenteritis and systemic disease, and is a useful tool to study immune responses in the intestine. Although mechanisms leading to immune responses against Salmonella have been extensively studied, questions remain about how bacteria travel from the intestinal mucosa to the mesenteric lymph nodes (MLN), a key site for antigen presentation. Here, we used a mouse model of infection with Salmonella enterica serovar Typhimurium (STM) to identify changes in intestinal immune cells induced during early infection. We then used fluorescently-labelled STM to identify interactions with immune cells, from the site of infection, through migration in lymph, to the MLN. We show that viable STM can be carried in the lymph by any subset of migrating dendritic cells, but not by macrophages. Moreover, approximately half of the STM in lymph are not associated with cells at all, and travel autonomously. Within the MLN, STM associates with dendritic cells and B cells, but predominantly with MLN-resident macrophages. In conclusion, we describe the routes used by STM to spread systemically in the period immediately after infection. This deeper understanding of the infection process could open new avenues for controlling it