Figure 5. IL-33 expands liver resident type-2 related ILC that produce IL-13.

(A) Flow cytometric analysis of isolated hepatic immune cell populations from naïve IL-4/eGFP reporter mice or 5d after treatment with mcIL-33. (N=3-4/group; **p<0.01) (B) Hepatic immune cell populations of mock or mcIL-33 injected Rag1^−/− mice were isolated and depleted from lin⁺ (CD5⁺, B220⁺, CD11b⁺, Gr1⁺, 7/4⁺, Ter119⁺) cells. The expression of IL-7Rα and ST2 was analyzed by flow cytometry. Gated IL-7Rα⁺/ST2⁺ cells were further analyzed for expression of CD45, Thy1.2, CD44, ICOS and Sca-1. Blue histograms represent isotype controls. Numbers of lin⁻ IL-7Rα⁺/ST2⁺ cells in livers of controls or mcIL-33 injected Rag1^−/− mice. (N=4-8/group; ***p<0.001) (C) Hepatic immune cells from 4get/Rag1^−/− mice were stimulated for 4h with PMA and Ionomycin. Surface expression of Sca-1 and ICOS and intracellular IL-13 expression were analyzed by flow cytometry (D) Total liver RNA from 5d control or mcIL-33 treated mice was isolated and used for RORα specific qPCR analysis. (E) Total RNA from sort purified liver ILC2 isolated from Rag1^−/− mice treated for 5d with mcIL-33 was used for RORα and RORγ specific qPCR (*p<0.05). (F) Flow cytometry -based quantification of lin⁻Sca-1⁺ICOS⁺ cells in irradiated mice reconstituted with BM from WT or sg/sg mice. (N=2-4/group) (G) Flow cytometry analysis of lin⁻, eGFP⁺, ICOS⁺ cells in livers of 4get mice treated for 4 weeks with CCL₄. (H) Il1rl1^+/+ WT or Il1rl1^−/− mice were treated for 4 weeks with CCL₄ before hepatic immune cells were isolated and analyzed by flow cytometry for the presence of lin⁻Sca-1⁺ICOS⁺ cells. Data are representative of at least two different experiments with 5 mice/group with similar results. (I) CD1 mice were infected with ~ 200 S. mansoni cercaria or left uninfected. 3 weeks later mice liver ILC2 numbers were determined by flow cytometry. (J) CD1 mice were infected with ~ 30 S. mansoni cercaria. 4 weeks later they were HD injected with a expression construct for sST2. 4 weeks later mice were sacrificed and liver ILC2 numbers were determined. Hepatic Rora mRNA was quantified by qPCR. n=5/group; *p<0.05).