Project description:Total RNAof pTh17 cells from wild type (WT) and RASA3 KO (RASA3KO) mouse (C57BL/6) was extracted and RNA-seq libraries were generated. Reads (32-45 Million reads per sample) were analyzed with Salmon software to align and quantify the transcript expression. R packages in Bioconductor, tximport and tximportData were used to aggregate transcript-level quantifications to the gene level, with the R package biomaRt for gene and transcripts mapping. The option "lengthScaledTPM" for countsFromAbundance in tximport was used to obtain the estimated counts at the gene level using abundance estimates scaled based on the average transcript length over samples and the library size. R function voom in limma package was used to transform the estimated count data into log2 scale and estimate the mean-variance relationship so that it can be used to compute appropriate observation level weights, followed by linear modelling. For gene-level differential expression analysis, a linear model was fitted to the log scaled expression data with the genotypes (knockout and wild-type) as one covariate using empirical Bayes moderated t-statistics. The false discovery rate (FDR) was controlled using the Benjamini and Hochberg algorithm. Probes with FDR < 0.05 and fold-change > 2 were judged to be differentially expressed. 317 and 35 genes were identified to be significantly up- and down-regulated respectively. The R function CAMERA was used to determine whether each gene set was differentially expressed in the comparisons as a set. The differential expression patterns and to calculate the enrichment scores of Th1/Th2 (KEGG mmu04658) and Th17 (KEGG mmu04659) related genes in the knockout vs. wild-type comparisons. Our analysis demonstrated that increase of Th2 program and decrease Th17 program in RASA3 KO pTh17 cells, with no change of Th1 related genes. This study indicated that RASA3 is vital for the balance of pTh17-Th2 reciprocal programs.
Project description:Adipose tissue (AT) contains mesenchymal stromal cells (MSC) in stages of commitment to becoming specialized tissue cells, including adipocytes and fibroblasts, and immune cells which support tissue homeostasis. How MSC and immune cells interact during infection is poorly understood. We show that during intestinal helminth infection MSC in mesenteric AT (mAT) become enriched in non-differentiated progenitor cells. This is accompanied by MSC-intrinsic metabolic reprogramming supporting increased secretion of extracellular matrix (ECM), IL-33, and TSLP. In parallel, Th2 resident memory (Th2RM) cells populate the mAT and persist after infection is resolved. These cells express Areg, TGFβ and IL-5, and are necessary to promote infection induced changes within mAT, including MSC reprogramming and tissue eosinophilia. In turn, IL-33 and TSLP from MSC facilitate Th2RM activation and maintenance. Our findings link Th2RM cells to mAT remodeling during intestinal infection, underscoring the reciprocal dependence of stroma and resident immune cells for lasting tissue immunity.
Project description:Deletion of the gene encoding Foxa2, a winged helix transcription factor selectively expressed in respiratory epithelial cells, caused spontaneous pulmonary eosinophilic inflammation and goblet cell metaplasia. Loss of Foxa2 induced the recruitment and activation of myeloid dendritic cells (mDCs) and Th2 cells in the lung, and was associated with the increased production of T helper 2 (Th2) cytokines and chemokines. mRNA microarray analysis demonstrated that deletion of Foxa2 induced the expression of a number of mRNAs regulating pulmonary dendritic cell activation, Th2 mediated inflammation, and goblet cell differentiation. The spontaneous pulmonary inflammation and goblet cell metaplasia caused by loss of Foxa2 was inhibited by treatment of newborn Foxa2â??/â?? mice with monoclonal IL-4Ralpha antibody. Expression of Foxa2 in non-ciliated secretory cells (Clara cells) in vivo inhibited goblet cell differentiation induced by pulmonary allergen exposure. The respiratory epithelium plays a central role in the regulation of Th2-mediated inflammation and innate immunity in the developing lung in a process regulated by Foxa2. To investigate the role of Foxa2 and its downstream targets associated with the Th2 inflammation and goblet cell hyperplasia, RNAs were isolated from the lungs of Foxa2-/- and control littermates at PN15. Lung cRNA was hybridized to the murine genome MOE430 V2 chips.
Project description:Cancer is a heterocellular disease composed of tumor cells and stromal cells. Although stromal cells are known to regulate cancer progression, oncogene-dependent signalling through heterocellular cancer systems remains poorly elucidated. Here, we describe KRASG12D-dependent ‘reciprocal’ signalling across tumor and stromal Pancreatic Ductal Adenocarcinoma (PDA) cells. Heterocellular multivariate phosphoproteomics demonstrates how an oncogenic cue (KRASG12D), a trans-cellular signal (SHH), and stromal cells drive a reciprocal response in tumor cells. KRASG12D-dependent reciprocal signalling regulates the tumor cell phosphoproteome, total proteome, and mitochondria activity via an IGFR1/AXL-AKT axis. The reciprocal KRASG12D signalling state requires a heterocellular context and is unreachable by cell-autonomous oncogenic KRAS alone. These findings provide evidence that oncogenic KRAS regulates tumor cells via heterocellular reciprocation. Comparison between FACS resolved iKRAS cells (previously in co-culture with PSCs) pertubed with a SHH antibody
Project description:Cancer is a heterocellular disease composed of tumor cells and stromal cells. Although stromal cells are known to regulate cancer progression, oncogene-dependent signalling through heterocellular cancer systems remains poorly elucidated. Here, we describe KRASG12D-dependent ‘reciprocal’ signalling across tumor and stromal Pancreatic Ductal Adenocarcinoma (PDA) cells. Heterocellular multivariate phosphoproteomics demonstrates how an oncogenic cue (KRASG12D), a trans-cellular signal (SHH), and stromal cells drive a reciprocal response in tumor cells. KRASG12D-dependent reciprocal signalling regulates the tumor cell phosphoproteome, total proteome, and mitochondria activity via an IGFR1/AXL-AKT axis. The reciprocal KRASG12D signalling state requires a heterocellular context and is unreachable by cell-autonomous oncogenic KRAS alone. These findings provide evidence that oncogenic KRAS regulates tumor cells via heterocellular reciprocation.
Project description:Deletion of the gene encoding Foxa2, a winged helix transcription factor selectively expressed in respiratory epithelial cells, caused spontaneous pulmonary eosinophilic inflammation and goblet cell metaplasia. Loss of Foxa2 induced the recruitment and activation of myeloid dendritic cells (mDCs) and Th2 cells in the lung, and was associated with the increased production of T helper 2 (Th2) cytokines and chemokines. mRNA microarray analysis demonstrated that deletion of Foxa2 induced the expression of a number of mRNAs regulating pulmonary dendritic cell activation, Th2 mediated inflammation, and goblet cell differentiation. The spontaneous pulmonary inflammation and goblet cell metaplasia caused by loss of Foxa2 was inhibited by treatment of newborn Foxa2∆/∆ mice with monoclonal IL-4Ralpha antibody. Expression of Foxa2 in non-ciliated secretory cells (Clara cells) in vivo inhibited goblet cell differentiation induced by pulmonary allergen exposure. The respiratory epithelium plays a central role in the regulation of Th2-mediated inflammation and innate immunity in the developing lung in a process regulated by Foxa2.
Project description:Th2 cell is a subset of CD4+ T helper (Th) cells, which regulates the immunity to extracellular parasites and allergic inflammation. Th2 cells specifically secrete Type 2 cytokines such as IL-4, IL-5, IL-13, and also express many universal cytokines like IL-2 and GM-CSF. While theses cytokines are indispensable for Th2 cell mediated response, there is complex heterogeneity in the pattern of cytokine production among Th2 cell population. As previous studies were mainly conducted at population levelref, here we took advantage of single-cell RNA-seq to explore heterogeneity in in vitro differentiated Th2 cells. Although a few single cell researches about Th2 cells was conducted, there is no comprehensive understanding of the heterogeneity of Th2 lineage yet, inspiring us to make some difference on this issue. Our studies identify that cytokine genes contributed most to the transcriptome heterogeneity of Th2 cells; expression of universal cytokines showed bimodality, but not Th2 specific cytokines.