Transcriptional profilling of In vitro-primed C.rodentium-specific Th17 cells, cytokine-differentiated Th17 cells and ex vivo Th17 cells following C.rodentium infection
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PROVIDER: PRJNA591908 | ENA |
REPOSITORIES: ENA
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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
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.
2020-01-10 | GSE141062 | GEO
Project description:T helper 17 (Th17) cells are a distinct subset of CD4+ T cells necessary for maintaining gut homeostasis and have prominent roles in autoimmunity and inflammation. Th17 cells have unique metabolic features, including a stem cell-like signature and reliance on mitochondrial respiratory chain function and tricarboxylic acid (TCA) cycle to coordinate metabolic and epigenetic remodeling. Dynamic changes in mitochondrial membrane morphology are key to sustain organelle function. However, it remains unclear whether mitochondrial membrane remodeling orchestrates metabolic and differentiation events in Th17 cells. Here we demonstrate that mitochondrial membrane fusion and tight cristae organization are required for Th17 cell function (i.e. cytokine expression). As a genetic model system we employ Th17 specific deletion of optic atrophy 1 (OPA1), a gene that encodes a protein involved in mitochondrial inner membrane fusion and cristae organization. As a result, we find that Th17 cells rely on mitochondrial fusion (due to their low metabolic activity). Here, we carry out DIA-based differential quantitative proteomic analysis of murine wild-type and OPA1 knock-out Th17 cells. Through Ingenuity pathway analysis and together with transcriptional and metabolomic profiling we identify the serine/threonine kinase liver associated kinase B1 (LKB1/STK11) as an essential node coupling mitochondrial function to IL-17A cytokine expression in T cells.
2022-10-13 | PXD036162 | Pride
Project description:T helper cells integrate signals from their microenvironment to acquire distinct specialization programs for efficient clearance of diverse pathogens or for immunotolerance. Ionic signals have recently been demonstrated to affect T cell polarization and function. Sodium chloride (NaCl) was proposed to accumulate in peripheral tissues upon dietary intake and to promote autoimmunity via the Th17 cell axis. Here we demonstrate that high NaCl conditions induced a stable, pathogen-specific, anti-inflammatory Th17 cell fate in human T cells in vitro. The p38/MAPK pathway, involving NFAT5 and SGK1, regulated FoxP3 and interleukin (IL)-17-expression in high-NaCl conditions. The NaCl-induced acquisition of an anti-inflammatory Th17 cell fate was confirmed in vivo in an experimental autoimmune encephalomyelitis (EAE) mouse model, which demonstrated strongly reduced disease symptoms upon transfer of T cells polarized in high NaCl conditions. However, NaCl was coopted to promote murine and human Th17 cell pathogenicity, if T cell stimulation occurred in a pro-inflammatory and TGF--low cytokine microenvironment. Taken together, our findings reveal a context-dependent, dichotomous role for NaCl in shaping Th17 cell pathogenicity. NaCl might therefore prove beneficial for the treatment of chronic inflammatory diseases in combination with cytokine-blocking drugs.
2020-04-15 | GSE148669 | GEO
Project description:Transcriptional profilling of In vitro-primed C.rodentium-specific WT or Caspase-1 deficient Th cells
| PRJNA591907 | ENA
Project description:Dysregulated Th17 cell responses underlie multiple inflammatory and autoimmune diseases, including autoimmune uveitis and its animal model, EAU. However, clinical trials targeting IL-17A in uveitis were not successful. Here, we found that Th17 cells were regulated by their own signature cytokine, IL-17A. Loss of IL-17A in autopathogenic Th17 cells did not reduce their pathogenicity and instead elevated their expression of the Th17 cell cytokines GM-CSF and IL-17F. Mechanistic in vitro studies revealed a Th17 cell-intrinsic autocrine loop triggered by binding of IL-17A to its receptor, leading to activation of transcription factor NFκB and induction of IL 24, which repressed the Th17 cytokine program. In vivo, IL-24 treatment ameliorated Th17-induced EAU, whereas silencing of IL-24 in Th17 cells enhanced disease. This regulatory pathway also operated in human Th17 cells. Thus, IL-17A limits pathogenicity of Th17 cells by inducing IL-24. These findings may explain the disappointing therapeutic effect in targeting IL-17A in uveitis.
2020-07-22 | GSE152534 | GEO
Project description:Non-canonical STAT3 activity sustains pathogenic Th17 proliferation and cytokine response to antigen
2020-07-01 | GSE151447 | GEO
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.
2020-03-25 | PXD016633 | Pride
Project description:Tumor- and cytokine-primed human natural killer cells exhibit distinct phenotypic and transcriptional signatures
| PRJNA534523 | ENA
Project description:Th17 cells are known to exert pathogenic and non-pathogenic functions. Although the cytokine transforming growth factor β1 (TGF-β1) is instrumental for Th17 cell differentiation, it is dispensable for generation of pathogenic Th17 cells. Here, we examined the T cell-intrinsic role of Activin-A, a TGF-β superfamily member closely related to TGF-β1, in pathogenic Th17 cell differentiation. Activin-A expression was increased in individuals with relapsing-remitting multiple sclerosis and in mice with experimental autoimmune encephalomyelitis. Stimulation with interleukin-6 and Activin-A induced a molecular program that mirrored that of pathogenic Th17 cells and was inhibited by blocking Activin-A signaling. Genetic disruption of Activin-A and its receptor ALK4 in T cells impaired pathogenic Th17 cell differentiation in vitro and in vivo. Mechanistically, extracellular-signal-regulated kinase (ERK) phosphorylation, which was essential for pathogenic Th17 cell differentiation, was suppressed by TGF-β1-ALK5 but not Activin-A-ALK4 signaling. Thus, Activin-A drives pathogenic Th17 cell differentiation, implicating the Activin-A-ALK4-ERK axis as a therapeutic target for Th17 cell-related diseases.
2021-01-11 | GSE151533 | GEO