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Transcriptional profilling of In vitro-primed C.rodentium-specific Th17 cells, cytokine-differentiated Th17 cells and ex vivo Th17 cells following C.rodentium infection

ABSTRACT: Transcriptional profilling of In vitro-primed C.rodentium-specific Th17 cells, cytokine-differentiated Th17 cells and ex vivo Th17 cells following C.rodentium infection

PROVIDER: PRJNA591908 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10534666.fastq.gz	Fastqsanger.gz
	SRR10534667.fastq.gz	Fastqsanger.gz
	SRR10534668.fastq.gz	Fastqsanger.gz
	SRR10534669.fastq.gz	Fastqsanger.gz
	SRR10534670.fastq.gz	Fastqsanger.gz

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Transcriptional profilling of In vitro-primed C.rodentium-specific Th17 cells, cytokine-differentiated Th17 cells and ex vivo Th17 cells following C.rodentium infection

Project description:Introduction: We reported a in vitro pathogen-specific priming system to using pathogen lysate stimulated splenic CD11c+ DCs to differentiate pathogen-specific murine T helper cells. This method allowed us to compare side-by-side transcriptional profiles of bulk T helper cells that are specific to distinct pathogens and to study their composition and functionality. Moreover, we can utilize cytokine reporter mice to isolate specific Th subtypes from these in vitro-primed T cells and study their transcriptional regulation. Method: We isolate commited Th17 cells by utilizing IL-17A fate-mapping mice (Il17aCRE; R26tdT). We generated Th17 cells in 3 conditions: 1. We used Cr-stimulated DCs to prime Th17 cells and sorted CD90+CFSE-tdT+ population, dubbed 'ppTh17' cells. CFSE+CD90+ naive T cells from the same culture as controls. 2. We polarized naive Il17aCRE; R26tdT CD4 T cells to Th17 lineage using previously defined cytokine and antibody cocktail (IL-6+TGFb1+IL-1b+IL-23; plate bound anti-CD3+anti-CD28). We sorted CD90+tdT+ population, dubbed 'cdTh17' cells. 3. We intragastrically infected Il17aCRE; R26tdT mice with 5x10^8 C.rodentium and harvested mesenteric lymph nodes at 10 days post infection (dpi). We sorted tdT+ CD4 T cells from mesenteric lymph nodes, dubbed "ex vivo Th17' cells. The four sorted populations were subjected to mRNA-sequencing analysis. Conclusion: We found that compared to cdTh17 cells, ppTh17 cells and ex vivo Th17 cells resemble each other. These two physiologically relevant Th17 populations maintain high levels of heterogeneity and plasticity, as well as posess ability to upregulate memory T cell related genes. cdTh17 cells highly express glycolysis and one-carbon metabolism genes, correlate with their expression of cell cycle genes, suggesting a highly activated state.

2020-01-10 | GSE141061 | GEO

Transcriptional profilling of In vitro-primed C.rodentium-specific WT or Caspase-1 deficient Th cells

Project description:Introduction: We reported that caspase-1 is highly expressed in in vitro-primed C.rodentium-specific Th cells. Caspase-1 deficient (Casp1d10) naïve T cells differentiate into pathogen-specific Th17 cells at an suboptimal level and fail to protect against C.rodentium infection. To gain insight into the regulatory pathways exerted by caspase-1 in Th cells, we performed RNA-seq on in vitro-primed WT and Casp1d10 Th cells. Method: We stimulated WT C57/BL6J splenic CD11c+ DCs with 10ug/ml C.rodentium lysate for 5 hours. Then DCs are washed and co-cultured in 1:5 ratio with naïve WT C57/BL6J or Casp1d10 CD4 T cells for 10 days. CFSE-CD90+ (pathogen-specific Th cells) were FACS-sorted and subjected to mRNA-seq analysis. Conclusion: We found that WT Cr-specific Th cells have higher expression of Th17 genes, including Il17a,f,Il22 and Rorc. Casp1Δ10 Cr-specific Th cells exhibited higher expression of iNOS genes and exogenous lipid metabolism genes such as Cd36, suggesting a distinct cellular metabolism potentially regulated by Caspase-1.

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Transcriptional signature of human pro-inflammatory TH17 cells [ex vivo TH17 cells]

Project description:Transcriptional signature of human pro-inflammatory TH17 cells [ex vivo TH17 cells]

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Transcriptional signature of human pro-inflammatory TH17 cells [ex vivo TH17 cells]

Project description:Human peripheral blood IFN-γ+IL-17+ (TH1/17) and IFN-γ–IL-17+ (TH17) CD4+ T cells display distinct transcriptional profiles in high-throughput transcription analyses. Compared to TH17 cells, TH1/17 cells have similar gene signatures to mouse pathogenic TH17 cells.

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Transcriptional profilling of In vitro-primed C.rodentium-specific WT or Caspase-1 deficient Th cells

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Project description:Interleukin 23 (IL-23) triggers pathogenic features in pro-inflammatory, IL-17-secreting T cells (Th17 and Tγδ17) that play a key role in the development of inflammatory diseases. However, the IL-23 signaling cascade remains largely undefined. Here we used quantitative phosphoproteomics to characterize IL-23 signaling in primary murine Th17 cells. We quantified 6,888 phosphorylation sites in Th17 cells, and found 168 phosphorylations regulated upom IL-23 stimulation. IL-23 increased the phosphorylation of the myosin regulatory light chain (RLC), an actomyosin contractibility marker, in Th17 and Tγδ cells. IL-23-induced RLC phosphorylation required JAK2 and ROCK catalytic activity, and the study of the IL-23/ROCK axis revealed an unexpected role of IL-23 in the migration of Tγδ17 and Th17 cells. Moreover, pharmacological inhibition of ROCK reduced Tγδ17 recruitment to inflamed skin upon challenge with inflammatory agent Imiquimod. This work: i) provides new insights into phosphorylation networks that control Th17 cells, ii) widely expands the current knowledge on IL-23 signaling, and iii) contributes to the increasing list of immune cells subsets characterized by global phosphoproteomic approaches.

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