Fig. 3

TREM-1-mediated IL-1β induction by M1 macrophages elicits IL-22 production by ILC3. a Contour plot of FACS analysis data showed the purity of AutoMACS-purified ILCs b ELISA determination of IL-22 production by AutoMACS-purified WT Lin⁻CD45⁺ ILCs that were co-cultured with WT GM-BMDMs in the presence or absence of the indicated murine recombinant cytokines. Culture supernatants were harvested after 48 h. Mock, no treatment. ILCs treated with IL-1β + IL-23 in the absence of macrophages served as positive control and IFNγ + LPS or IL-4 + IL-13 in the absence of macrophages served as negative control. Data are the mean ± SEM and representative of four independent experiments performed in triplicate. c ELISA determination of IL-22 production by AutoMACS-purified WT Lin⁻CD45⁺ ILCs that were co-cultured with WT or TREM-1 KO cells (GM-BMDMs or neutrophils, respectively) in the presence of LPS or LPS plus anti-TREM-1 agonistic antibodies (TR1 Ab) or isotype antibodies, respectively. Data are mean ± SEM and representative of 3 independent experiments performed in triplicate. Histogram of FACS analysis data showed TREM-1 levels on M- or GM- BMDMs. d-e Real-time PCR analysis of mRNA level of IL-1β and ELISA determination of IL-1β production by WT- or TREM-1 KO- GM-BMDMs that were cultured in the presence of the polarizing cytokines (IFNγ + LPS for M1 or IL-4 + IL-13 for M2). Culture supernatants were harvested after 24 h. Mock, no treatment. For (b-e), for comparisons between WT and TREM-1 KO, differences were evaluated using an unpaired t test with Welch’s correction: *, p < 0.05; **, p < 0.01; ***, p < 0.001 or ****, p < 0.0001. For comparison to the mock group, differences were evaluated using an unpaired t test with Welch’s correction: #, p < 0.05, ##, p < 0.01 or ###, p < 0.001