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Single cell RNA sequencing of mesenteric lymph node dendritic cells in segmented filamentous bacteria(SFB)-colonized C57BL/6 mice

ABSTRACT: Purpose: The goal of this study is to identify gut dendritic cells that induces CD4 Th17 cells that play an critical role in mucosal immunity Methods: C57BL/6 mice were orally gavaged with fecal material derived from SFB-positive donor mice. Five days post oral gavage, mesenteric lymph nodes were harvested and dendritic cells were FACS-sorted. cDNA library from migratory dendritic cells (MHCII-highCD11c+) and resident dendritic cells (MHCII+CD11c-high) were generated using Chromium Single Cell 3' Library v2 Kit (10x Genomics). RNA sequencing was performed using NovaSeq S4 flow cell (Illumina). Data was processed using the Cell Ranger software (10x Genomics) followed by analysis using the R-based package Seurat. Results: Gut colonization with SFB increased the migration of type 2 but not type 1 dendritic cells to the mesenteric lymph nodes. Based on transcriptomic profiles, migratory type 2 dendritic cells consist of 2 major cell clusters (Cd40-Ccl22 and Cd1d1-Tnfrsf4-enriched cells). The Th17 response inducing cytokine IL-6 co-localized with the Cd40-Ccl22-expressing cells. Conclusion: Our study represents the first to carefully characterize the mucosal dendritic cells at steady state and during acute CD4 Th17 response. Transcriptomic profiling enabled the subsetting of dendritic cells based on functional markers (such as cytokines and chemokines) that are otherwise difficult to measure at the protein level. These results provide a focused look at mucosal dendritic cells and their roles in dictating different T cell responses.

ORGANISM(S): Mus musculus

PROVIDER: GSE184423 | GEO | 2022/07/13

REPOSITORIES: GEO

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Project description:Gene expression profile of dendritic cells (DC) of mesenteric lymph node (mLN) and lamina propria of small intestine (SI-LP) of control mice and mice lacking RelB expression in DCs at steady state, four days and 14 days after infection with Heligmosomoides polygyrus bakeri (Hpb) Dendritic cells (DCs) are crucial for initiating protective immune responses and have also been implicated in the generation and regulation of Foxp3+ regulatory T cells (Tregs). Specific DC subsets or DC-intrinsic pathways regulate immunity against pathogens but also tolerance to harmless antigens derived from food or microbiota at barrier sites, but underlying mechanisms in the intestinal tract remain poorly defined. Here, we provide evidence that the alternative NF-B family member RelB controls a defined transcriptional program in migratory DC subsets of mesenteric lymph nodes and the small intestinal lamina propria. Functionally, ablation of RelB in dendritic cells result in increased Foxp3+ Treg cell numbers but decreased RORt peripheral Treg cell numbers maintained even under inflammatory conditions. Single-cell RNA-sequencing revealed a complete RelB dependency for the differentiation of cryptopatches and isolated lymphoid follicles-associated DCs (CIA-DCs) in the lamina propria of the small intestine. In addition, we show a RelB-dependent signature of migratory DCs in mesenteric lymph nodes favoring DC-Treg cell interaction by affecting the expression of chemokines (Ccl22, Ccl17), migration behavior (Cd63), co-stimulatory molecule (Cd80, Cd40, Cd200, Tnfsf4), and tolerance-related integrin (Itgb5, Itgb8) expression. Functionally, increased Treg cell numbers in DC-specific RelB knockout animals did not show any risk of increased reactions in a model of food allergy but instead prevented protective Th2 immune responses in the intestines after infection with Heligmosomoides polygyrus bakeri despite their slight steady-state type 2 immune bias. This protection was dependent on elevated Treg cell frequencies during primary infection as a result of bystander immune tolerance. Thus, RelB expression in conventional DCs acts as a rheostat to establish a tolerogenic set point that is maintained even during infection and strong type 2 immune conditions and thereby is a key regulator of intestinal homeostasis.

Project description:Vertebrates typically harbor a rich gastrointestinal microbiota, which has co-evolved with the host over millennia and is essential for several of its physiological functions, in particular maturation of the immune system. Recent studies have highlighted the importance of a single bacterial species, segmented filamentous bacteria (SFB), in inducing a robust T helper (Th)17 population in the small intestinal lamina propria (SI-LP) of the mouse gut. Consequently, SFB can promote IL-17-dependent immune and autoimmune responses, gut-associated as well as systemic, including inflammatory arthritis and experimental autoimmune encephalomyelitis. Here, we exploit the incomplete penetrance of SFB colonization of NOD mice in our animal facility to explore its impact on the incidence and course of type-1 diabetes in this prototypical, spontaneous model. There was a strong co-segregation of SFB-positivity and diabetes protection in females, but not in males, which remained relatively disease-free regardless of the SFB status. In contrast, insulitis did not depend on SFB colonization. SFB-positive, but not SFB-negative, females had a substantial population of Th17 cells in the SI-LP, which was the only significant, repeatable difference in the examined T cell compartments of the gut, pancreas or systemic lymphoid tissues. Th17 signature transcripts dominated the very limited SFB-induced molecular changes detected in SI-LP CD4+ T cells. Thus, a single bacterium, and the gut immune system alterations associated with it, can either promote or protect from autoimmunity in predisposed mouse models, likely reflecting their variable dependence on different Th subsets. All gene expression profiles were obtained from highly purified T cell populations sorted by flow cytometry. Cells were sorted from individual mice with at least four replicates generated for all groups. RNA from 1-5 x 104 cells was amplified, labeled, and hybridized to Affymetrix Mo GENE 1.0ST microarrays. Raw data were preprocessed with the RMA algorithm in GenePattern, and averaged expression values were used for analysis.

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Single cell RNA sequencing of mesenteric lymph node dendritic cells in segmented filamentous bacteria(SFB)-colonized C57BL/6 mice

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