Single-cell RNA sequencing (scRNA-seq) of all IL-23R+ cell types in the small intestine of healthy mice
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE229976 | GEO | 2024/04/23
REPOSITORIES: GEO
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2020-03-17 | GSE114749 | GEO
Project description:Single-cell RNA sequencing (scRNA-seq) of all IL-23R+ cell types in the small intestine of healthy mice
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Project description:Interleukin (IL)-23 mediated signal transduction represents a major molecular mechanism underlying the pathology of inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC). In addition, emerging evidence supports the role of IL-23-driven Th17 cells in inflammation. Components of the IL-23 signalling pathway, such as IL23R, JAK2 and STAT3, have been characterised, but elements unique to this network as compared to other interleukins have not been readily addressed. In this study, we have undertaken a multi-stage phosphoproteomics approach to better characterise downstream signalling events. To this end, we performed and compared phosphopeptide and phosphoprotein enrichment methodologies after activation of T lymphocytes by IL-23. We demonstrate the complementary nature of the two phosphor-enrichment approaches by maximising the capture of phosphorylation events. A total of 8069 unique phosphopeptides (containing 8344 unique sites), and 4317 unique phosphorylated proteins were identified, amongst which STAT3, PKM2, CDK6 and LASP-1 clearly showed induction of specific phosphorylation sites that were not readily observed after IL-2 stimulation. Interestingly, we observed subsequent translocation of STAT3 and PKM2 to the nucleus as well as increased phosphorylation especially of the nuclear portion at ~30 min after IL-23 stimulation, suggesting a wide range of cellular responses including proliferation and metabolic adaptation.
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Project description:Purpose: The goals of this study are to compare RNAs bound by translating ribosomes in the presence or absence of Segmented Filamentous Bacteria (SFB) colonization of the small intestine Methods: EDTA fractions containing small intestine epithelial cells from SFB negative and SFB positive mice were harvested. TRAP assay enriching for ribosome bound fraction were enriched using anti-GFP antibodies against GFP-ribosomeL10 fusion protein. mRNA profiles were generated by deep sequencing, using Illumina HighSeq. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) and TopHat followed by CuffDiff. qRT–PCR validation was performed using SYBR Green assays. Results: SFB colonization upregulate a subset of mRNAs being translated by ribosomes. Conclusions: SFB changes small intestine epithelial cell translation profile.
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Project description:Microarray was used to determine transcriptional differences between Nr1d1+/+ CCR6+ ILC3s and Nr1d1-/- CCR6+ ILC3s isolated from small intestine lamina propria
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Project description:Group 3 innate lymphoid cells (ILC3s) are key players in intestinal homeostasis. Endoplasmic reticulum (ER) stress is linked to inflammatory bowel disease (IBD). Herein, we used cell culture, novel mouse models, and human specimens to examine if ER stress in ILC3s impacts IBD pathophysiology. We show that mouse intestinal ILC3s exhibited a 24h-rhythmic expression pattern of the master ER stress response regulator, IRE1α-XBP1. Proinflammatory cytokine IL-23 selectively stimulated IRE1α-XBP1 in mouse ILC3s through mitochondrial reactive oxygen species (mtROS). IRE1α-XBP1 was activated in ILC3s of mice exposed to experimental colitis and in inflamed human IBD specimens. Mice with Ire1α deletion in ILC3s (Ire1αΔRorc) showed reduced expression of ER stress response and cytokine genes including Il22 in ILC3s and were highly vulnerable to infections and colitis. Administration of IL-22 counteracted their colitis susceptibility. In human ILC3s, IRE1 inhibitors suppressed cytokine production, which was upregulated by an IRE1 activator. Moreover, the frequencies of intestinal XBP1s+ ILC3s in Crohn’s disease patients before administration of ustekinumab, an anti-IL-12/IL-23 antibody, positively correlated with response to treatment. We demonstrate that a non-canonical mtROS-IRE1α-XBP1 pathway augments cytokine production by ILC3s and identify XBP1+ ILC3s as a potential biomarker for predicting response to anti-IL-23 therapies in IBD. Group 3 innate lymphoid cells (ILC3s) have recently emerged as important regulators and potential drug targets for IBD. However, the response of ILC3s to environmental stimuli during intestinal inflammation remains elusive. IRE1a-XBP1 serves as the regulatory hub of the unfolded protein response (UPR) that plays a vital role in intestinal inflammation.
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Project description:The IL-23 receptor (IL-23R) has been found to be associated with small bowel Crohn's disease (CD) in a whole genome association study. Specifically, the rare allele of the R381Q single nucleotide polymorphism (SNP) conferred protection against CD. It is unknown whether IL-23R is associated with IBD in children. The aim was to examine the association of IL-23R with susceptibility to IBD in pediatric patients.DNA was collected from 609 subjects (151 CD and 52 ulcerative colitis [UC] trios). Trios were genotyped for the R381Q SNP of the IL-23R gene and SNP8, SNP12, SNP13, of the CARD15 gene using Taqman. The transmission disequilibrium test (TDT) was used for association to disease using GENEHUNTER 2.0.The rare allele of R381Q SNP was present in 2.7% of CD and 2.9% UC probands. The CARD15 frequency was 31.5% (CD) and 18% (UC). The IL-23R allele was negatively associated with inflammatory bowel disease (IBD): the R381Q SNP was undertransmitted in children with IBD (8 transmitted [T] versus 27 untransmitted [UT]; P = 0.001). This association was significant for all CD patients (6 T versus 19 UT; P = 0.009), especially for non-Jewish CD patients (2 T versus 17 UT; P = 0.0006). TDT showed a borderline association for UC (2 T versus 8 UT; P = 0.06). As expected, CARD15 was associated with CD in children by the TDT (58 T versus 22 UT P = 0.00006), but not with UC.The protective IL-23R R381Q variant was particularly associated with CD in non-Jewish children. Thus, the initial whole genome association study based on ileal CD in adults has been extended to the pediatric population and beyond small bowel CD.
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Project description:Group 3 innate lymphoid cells (ILC3s) are crucial for the maintenance of host-microbiota homeostasis in gastrointestinal mucosal tissues. The mechanisms that maintain lineage identity of intestinal ILC3s, and ILC3s-mediated orchestration of microbiota and mucosal T cell immunity are elusive. Here, we identified BATF as a gatekeeper of ILC3s homeostasis in the gut. Depletion of BATF in ILC3s resulted in excessive interferon-γ production, dysbiosis, aberrant T cell immune responses and spontaneous inflammatory bowel disease (IBD), which was considerably ameliorated by removal of adaptive immunity or antibiotic treatment. Mechanistically, BATF directly regulates ILC3s identity by globally shapes chromatin landscape of ILC3s. BATF directly binds to the cis-regulatory elements of type 1 effector genes, restrains their chromatin accessibility and inhibits their expression. Conversely, BATF promotes chromatin accessibility of genes involved in MHCII antigen processing and presentation pathways. Collectively, our findings reveal BATF is a promising candidate to maintain ILC3s stability and coordinate ILC3s–mediated control of intestinal homeostasis.
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Project description:Group 3 innate lymphoid cells (ILC3s) are crucial for the maintenance of host-microbiota homeostasis in gastrointestinal mucosal tissues. The mechanisms that maintain lineage identity of intestinal ILC3s, and ILC3s-mediated orchestration of microbiota and mucosal T cell immunity are elusive. Here, we identified BATF as a gatekeeper of ILC3s homeostasis in the gut. Depletion of BATF in ILC3s resulted in excessive interferon-γ production, dysbiosis, aberrant T cell immune responses and spontaneous inflammatory bowel disease (IBD), which was considerably ameliorated by removal of adaptive immunity or antibiotic treatment. Mechanistically, BATF directly regulates ILC3s identity by globally shapes chromatin landscape of ILC3s. BATF directly binds to the cis-regulatory elements of type 1 effector genes, restrains their chromatin accessibility and inhibits their expression. Conversely, BATF promotes chromatin accessibility of genes involved in MHCII antigen processing and presentation pathways. Collectively, our findings reveal BATF is a promising candidate to maintain ILC3s stability and coordinate ILC3s–mediated control of intestinal homeostasis.
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