p38α regulates ILC3 cytokine production. (A–C) The colonic LPLs were isolated from naive RorccreMapk14fl/fl (n = 4) or Mapk14fl/fl (n = 4) littermate mice. Flow cytometry analysis of different subsets (A), cell proliferation by Ki67 (B), and cell viability by fixable cell viability dye (C) in ILC3s (gated in CD45+CD90+TCRβ−RORγt+) were shown. (D) 8-wk-old RorccreMapk14fl/fl and their littermates Mapk14fl/fl mice were given 3% DSS in drinking water for 6 days. The colon tissue was collected for RNA isolation and Il1b, Il6, Ccl2, Il23a, Csf2, and Il22 expression in DSS-treated RorccreMapk14fl/fl (n = 12) and Mapk14fl/fl (n = 7) were measured by qPCR. Each symbol represents an individual mouse from two pooled experiments. (E and F) ILC3s were sorted from the small intestinal and colonic LPLs of Rag1−/−Mapk14fl/fl and Rag1−/−VavicreMapk14fl/fl mice. (E) The mRNA expression of Il1r1, Il1r2, Il1rap, Dusp1, Dusp4, and Dusp8 was measured by qPCR. (F) The protein expression of DUSP1 was examined by western blot. HSP90 was detected as a loading control. (G) LPLs from the intestinal lamina propria were pre-treated with or without α-amanitin and then stimulated with IL-1β and IL-23 in vitro. The production of IL-22 and GM-CSF by ILC3s (gated in eflour780−CD45lowCD90high) were detected by flow cytometry. (H and I) LPLs from RorccreMapk14fl/fl and Mapk14fl/fl mice were stimulated with IL-1β or IL-23 for 18 h. The protein (H) and mRNA expression levels (I) of IL-22 and GM-CSF were examined by ELISA and qPCR. (J and K) LPLs from the small intestinal and colonic lamina propria of RorccreMapk14fl/fl or Mapk14fl/fl littermate mice were stimulated with IL-23 or PMA/Ionomycin in vitro. The production of IL-22 and GM-CSF (J) or IL-17A and IFNγ (K) by ILC3s (gating in eflour780−CD45lowCD90+) was analyzed by flow cytometry. (L) ILC3s were sorted from the small intestinal and colonic LPLs of Rag1−/−Mapk14fl/fl and Rag1−/−VavicreMapk14fl/fl mice and then were stimulated with IL-23 at different time points. The protein (left) and mRNA expression levels (right) of IL-22 were examined by ELISA and qPCR. (M) ILC2s (7-ADD−CD45+CD90+CD3−NK1.1−KLRG1+) and ILC1s (7-ADD−CD45+CD90+ CD3−NK1.1+KLRG1−) were sorted from small intestinal and colonic LPLs, and the expression of IL-5 in ILC2 (left) and IFNγ in ILC1 (right) at both protein (top) and mRNA (bottom) levels were evaluated by ELISA and qPCR. Data are representative of one of two independent experiments (A–C and E–M). Two experiments were pooled together (D). Each dot (A–D) represents one individual mouse. Each dot (G–I) represents one biological replicate. Data are mean ± SEM and statistical significance was tested by two-tailed Student’s unpaired t test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, no significant difference. Source data are available for this figure: SourceData FS2.
Figure S2.

p38α regulates ILC3 cytokine production. (A–C) The colonic LPLs were isolated from naive RorccreMapk14fl/fl (n = 4) or Mapk14fl/fl (n = 4) littermate mice. Flow cytometry analysis of different subsets (A), cell proliferation by Ki67 (B), and cell viability by fixable cell viability dye (C) in ILC3s (gated in CD45+CD90+TCRβ−RORγt+) were shown. (D) 8-wk-old RorccreMapk14fl/fl and their littermates Mapk14fl/fl mice were given 3% DSS in drinking water for 6 days. The colon tissue was collected for RNA isolation and Il1b, Il6, Ccl2, Il23a, Csf2, and Il22 expression in DSS-treated RorccreMapk14fl/fl (n = 12) and Mapk14fl/fl (n = 7) were measured by qPCR. Each symbol represents an individual mouse from two pooled experiments. (E and F) ILC3s were sorted from the small intestinal and colonic LPLs of Rag1−/−Mapk14fl/fl and Rag1−/−VavicreMapk14fl/fl mice. (E) The mRNA expression of Il1r1, Il1r2, Il1rap, Dusp1, Dusp4, and Dusp8 was measured by qPCR. (F) The protein expression of DUSP1 was examined by western blot. HSP90 was detected as a loading control. (G) LPLs from the intestinal lamina propria were pre-treated with or without α-amanitin and then stimulated with IL-1β and IL-23 in vitro. The production of IL-22 and GM-CSF by ILC3s (gated in eflour780−CD45lowCD90high) were detected by flow cytometry. (H and I) LPLs from RorccreMapk14fl/fl and Mapk14fl/fl mice were stimulated with IL-1β or IL-23 for 18 h. The protein (H) and mRNA expression levels (I) of IL-22 and GM-CSF were examined by ELISA and qPCR. (J and K) LPLs from the small intestinal and colonic lamina propria of RorccreMapk14fl/fl or Mapk14fl/fl littermate mice were stimulated with IL-23 or PMA/Ionomycin in vitro. The production of IL-22 and GM-CSF (J) or IL-17A and IFNγ (K) by ILC3s (gating in eflour780−CD45lowCD90+) was analyzed by flow cytometry. (L) ILC3s were sorted from the small intestinal and colonic LPLs of Rag1−/−Mapk14fl/fl and Rag1−/−VavicreMapk14fl/fl mice and then were stimulated with IL-23 at different time points. The protein (left) and mRNA expression levels (right) of IL-22 were examined by ELISA and qPCR. (M) ILC2s (7-ADD−CD45+CD90+CD3−NK1.1−KLRG1+) and ILC1s (7-ADD−CD45+CD90+ CD3−NK1.1+KLRG1−) were sorted from small intestinal and colonic LPLs, and the expression of IL-5 in ILC2 (left) and IFNγ in ILC1 (right) at both protein (top) and mRNA (bottom) levels were evaluated by ELISA and qPCR. Data are representative of one of two independent experiments (A–C and E–M). Two experiments were pooled together (D). Each dot (A–D) represents one individual mouse. Each dot (G–I) represents one biological replicate. Data are mean ± SEM and statistical significance was tested by two-tailed Student’s unpaired t test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, no significant difference. Source data are available for this figure: SourceData FS2.

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