Supplementary MaterialsS1 Fig: Single cell RNA sequencing reveals 17 unique cell classes of CD45+Linneg mononuclear cells in the liver and extrahepatic bile duct. ST2+ vs. ST2- CD45+Linneg mononuclear cells isolated from PBS-treated liver (reddish histogram) and IL-33 treated liver (blue histogram) and EHBD (green histogram) is usually shown; histograms are representative of 3 impartial experiments.(TIF) pone.0215481.s002.tif (396K) GUID:?A73402BB-E448-41E4-A660-3AF2B3B29471 S3 Fig: CCR1, a BIM cell class associated protein, is not detected in hepatobiliary Linneg mononuclear cells. Mice were treated with either PBS or IL-33 for 4 days, after which mononuclear cells were isolated from liver as explained in Methods, stained with fluorescent antibodies, and analyzed by circulation cytometry. Cells were gated as shown in Fig 7 to identify CD45+LinnegST2+ vs. ST2- mononuclear cells in liver after APD-356 enzyme inhibitor PBS- or IL-33 treatment. Relative expression of the BIM cell associated marker CCR1 in Tmem2 ST2+ vs. ST2- CD45+Linneg mononuclear cells isolated from PBS-treated liver (reddish histogram), IL-33 treated liver (blue histogram) EHBD (green histogram) is usually shown; histograms are representative of 3 impartial experiments.(TIF) pone.0215481.s003.tif (404K) GUID:?1F62FC56-8970-43F9-A54C-904B3E7C329F S1 Table: Total cell yield of liver and EHBD mononuclear cells for single-cell RNA sequencing. (DOCX) pone.0215481.s004.docx (12K) GUID:?3BDE2086-AEA3-4C60-B51B-006E0BA2FC5D S1 File: PBS and IL33 treated whole Liver and BD gene expression matrix TPM value. (ZIP) pone.0215481.s005.zip (1.1M) GUID:?669CA0D1-CB65-49C7-879C-64B52AB32EE8 S2 File: APD-356 enzyme inhibitor PBS and IL33 treated single cell gene expression matrix TPM value. (ZIP) pone.0215481.s006.zip (5.4M) GUID:?77503C35-CBD9-4CC5-B0EE-0FA2CACA502B Data Availability StatementAll relevant data are within the manuscript and its Supporting Information files. Abstract IL-33 promotes type 2 immunity, epithelial repair, and tissue fibrosis by activating group 2 innate lymphoid cells (ILC2). ILC2 lack all known surface markers of mature T, B, NK, and myeloid cell lineages (Linneg), express the IL-33 receptor ST2, and release type 2 cytokines which contribute to cholangiocyte proliferation and activation of hepatic stellate cells. This pathway results in massive proliferation of the extrahepatic bile duct (EHBD) but also exacerbates liver fibrosis, suggesting that there may be tissue-specific subpopulations of IL-33-induced ILC. To determine the tissue-specific subsets of ILC in the hepatobiliary system, we analyzed CD45+Linneg mononuclear cells from IL-33 treated adult Balb/c mouse liver or EHBD by single cell RNA sequencing. Principal component analysis identified 6 major CD45+Linneg cell classes, two of which were restricted to the EHBD. One of these classes, biliary immature myeloid (BIM) cells, was predicted to interact with ILC2 by a network of shared receptor-ligand pairs. BIM highly expressed Gp49 and ST2 receptors around the cell surface while lacking surface expression of markers for mature myeloid cells. In conclusion, single cell RNA sequencing recognized IL-33 responsive cell groups regionally confined to the liver or extrahepatic bile duct, including a novel population of CD45+Linneg Gp49-expressing mononuclear cells. Introduction Innate lymphoid cells (ILC) are distributed at epithelial sites early in life to uniquely respond to tissue injury and initiate and participate in immune responses. ILC express CD45, IL-7R and other immune activation markers but lack all known lineage markers (Linneg) for T, B, myeloid, and NK cells [1C3]. Among ILCs, the group 2 innate lymphoid cells (ILC2) respond to IL-33, a member of the IL-1 family APD-356 enzyme inhibitor APD-356 enzyme inhibitor of cytokines released upon epithelial damage to promote type 2 immunity to parasites, epithelial repair, and tissue fibrosis in both mice and humans in various tissues including skin, lung, GI tract, liver and bile duct [4,5]. ILC2s release IL-13 and APD-356 enzyme inhibitor other type 2 cytokines, which obvious parasitic infections but play pathogenic functions in exacerbating asthma and allergic immune responses [6]. Within the hepatobiliary system, we as well as others have shown that IL-33 activates hepatic ILC2 to produce IL-13, which induces massive proliferative expansion of the epithelium and peribiliary glands (PBG) of the extrahepatic bile duct (EHBD). This molecular circuit is usually protective in a mouse model of biliary atresia, as evidenced by the fact that 1) a subset of patients with biliary atresia overexpress IL-33, 2) blockade of IL-33 signaling in a mouse model of biliary atresia induced by Rhesus rotavirus (RRV) contamination exacerbates disease, and 3) administration of IL-33 to RRV-infected mice is usually protective against EHBD obstruction [7]. In humans biliary atresia prospects to rapidly progressive biliary cirrhosis, often requiring liver transplantation for long-term survival [8]. Experimentally, IL-33 also promotes the development of cholangiocarcinoma in genetically predisposed mice [7,9]. In this context, previous reports.