In the present study, we investigated the phenotype and plasticity of hepatic ILC2 in response to liver inflammation-induced cytokines and effector CD4 + T cells in order to identify mechanisms of ILC2 regulation in liver disease. Material and Methods Mice. C57BL/6 mice and OT-II mice were bred in the University Medical Center Hamburg-Eppendorf animal facility (Hamburg, Germany). Mouse experiments were conducted according to the German animal protection law and approved by the institutional review board (Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany; G44/15). Mice received humane care according to the national guidelines of the National Institutes of Health in Germany. Animal treatment. C57BL/6 mice received intraperitoneally recombinant murine (rm)IL-33 (0.3 μg; BioLegend, San Diego, CA) daily on four consecutive days. ConA (7 mg/kg; Sigma-Aldrich, St. Louis, MO) was injected intravenously into C57BL/6 mice that were analyzed 8 and 24 hours later. Heart blood was drawn from individual mice and liver injury was quantified by automated measurement of plasma activities of alanine transaminase (ALT) using a COBAS Mira System (Roche Diagnostic, Mannheim, Germany). Immunohistochemistry. Liver samples were fixed with 4% formalin and embedded in paraffin. 3 μm liver sections were cut, stained with H&E following standard protocols, and analyzed by light microscopy. Isolation of hepatic ILC2 and CD4 + T cells. Hepatic non-parenchymal cells were isolated from livers of naive and IL-33-treated mice by Percoll density gradient centrifugation. Single cell suspensions were stained with the Lineage Antibody Cocktail (APC; BD Pharmingen, Heidelberg, Germany) as well as anti-Sca-1 (Pacific Blue; D7; BioLegend) and anti-ST2 (PerCP-eFluor 710; RMST2-2; ThermoFisher Scientific, Waltham, MA) antibodies. Lineage-negative (lin −) cells were enriched by magnetic cell sorting and lin − Sca-1 + ST2 + ILC2 were purely isolated by FACS (BD FACSAriaTM III sorter, BD Biosciences). Ovalbumin (OVA)-specific CD4 + T cells were isolated from spleen and lymph nodes of OT-II mice. Therefore, CD4 + T cells were enriched from single cell suspensions using the CD4 + T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Subsequently, cells were stained with anti-TCRβ (PE-Cy7; H57-597) and anti-CD4 (BV421; GK1.5; both BioLegend) antibodies and TCRβ + CD4 + T cells were purified by FACS.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.