p38α promotes rapid cytokine production through regulating mRNA nuclear export in ILC3s. (A and B) ILC3s sorted from murine small intestine and colon (gated in 7-ADD−CD45lowCD90+) were treated with IL-1β at different time points. (A) Protein levels of total and phosphorylated p38α were shown. Tubulin was detected as a loading control. (B) IL-22 in the culture supernatant was examined by ELISA. The concentration of IL-22 and activation of p38α comparison in time course. (C) LPLs from the small intestine and colon 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 were detected by ELISA after 18 h stimulation. (D) ILC3s were sorted from the small intestine and colon LPLs of Rag1−/−Mapk14fl/fl and Rag1−/−VavicreMapk14fl/fl mice and then were stimulated with IL-1β and IL-23 for 18 h. The protein (left) and mRNA expression levels (right) of IL-22 and GM-CSF were examined by ELISA and qPCR. (E) The diagram of TurboID-based proximity labeling for capturing the p38α-interactome. (F) The GO differential analysis of p38α-interactome between control and IL-1β treatment group. (G) Representative immunofluorescence images of p-p38 in ILC3s. The sorted ILC3s (gated in 7-ADD−CD45lowCD90high) were stimulated with or without IL-1β for 5 min in vitro. Scale bars, 4 μm. (H) ILC3s (gated in 7-ADD−CD45lowCD90high) sorted from the small intestine and colon of Rag1−/−Mapk14fl/fl and Rag1−/−VavicreMapk14fl/fl mice were stimulated with or without IL-1β. The nuclear and cytosol mRNA were fractionated and the distribution of transcripts were analyzed by qPCR. The cytosol fractions in each group were compared for statistical analysis. (I and J) The vectors containing Il22/Csf2 CDS or CDS+3′UTR were delivered into 293T cells. (I) The mRNA and protein expression levels of IL-22 and GM-CSF were examined by qPCR (left) and ELISA (right). (J) The nuclear and cytosol mRNA were fractionated and analyzed by qPCR. Data are representative of one of two independent experiments (A–D and G–J). Each dot (C, D, and I) represents one biological replicate. Data are mean ± SEM and statistical significance was tested by two-tailed Student’s unpaired t test (C, D, and H–J), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, no significant difference. Source data are available for this figure: SourceData F3.
Figure 3.

p38α promotes rapid cytokine production through regulating mRNA nuclear export in ILC3s. (A and B) ILC3s sorted from murine small intestine and colon (gated in 7-ADD−CD45lowCD90+) were treated with IL-1β at different time points. (A) Protein levels of total and phosphorylated p38α were shown. Tubulin was detected as a loading control. (B) IL-22 in the culture supernatant was examined by ELISA. The concentration of IL-22 and activation of p38α comparison in time course. (C) LPLs from the small intestine and colon 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 were detected by ELISA after 18 h stimulation. (D) ILC3s were sorted from the small intestine and colon LPLs of Rag1−/−Mapk14fl/fl and Rag1−/−VavicreMapk14fl/fl mice and then were stimulated with IL-1β and IL-23 for 18 h. The protein (left) and mRNA expression levels (right) of IL-22 and GM-CSF were examined by ELISA and qPCR. (E) The diagram of TurboID-based proximity labeling for capturing the p38α-interactome. (F) The GO differential analysis of p38α-interactome between control and IL-1β treatment group. (G) Representative immunofluorescence images of p-p38 in ILC3s. The sorted ILC3s (gated in 7-ADD−CD45lowCD90high) were stimulated with or without IL-1β for 5 min in vitro. Scale bars, 4 μm. (H) ILC3s (gated in 7-ADD−CD45lowCD90high) sorted from the small intestine and colon of Rag1−/−Mapk14fl/fl and Rag1−/−VavicreMapk14fl/fl mice were stimulated with or without IL-1β. The nuclear and cytosol mRNA were fractionated and the distribution of transcripts were analyzed by qPCR. The cytosol fractions in each group were compared for statistical analysis. (I and J) The vectors containing Il22/Csf2 CDS or CDS+3′UTR were delivered into 293T cells. (I) The mRNA and protein expression levels of IL-22 and GM-CSF were examined by qPCR (left) and ELISA (right). (J) The nuclear and cytosol mRNA were fractionated and analyzed by qPCR. Data are representative of one of two independent experiments (A–D and G–J). Each dot (C, D, and I) represents one biological replicate. Data are mean ± SEM and statistical significance was tested by two-tailed Student’s unpaired t test (C, D, and H–J), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, no significant difference. Source data are available for this figure: SourceData F3.

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