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. 2023 Sep 27;120(40):e2215421120. doi: 10.1073/pnas.2215421120

Fig. 5.

Fig. 5.

TGFβ receptor type I/II signaling mediates suppression of IL-27p28 in macrophages. (A) Mononuclear phagocytic cells were incubated with LPS (100 ng/mL) ± TGFβ1 (10 ng/mL) followed by detection of IL-27p28 in supernatants; C57BL/6 J-derived AM and PEM, mouse AM cell line (MH-S), C57BL/6 J BMDM and RAW264.7 macrophage cell line, 24 h. LPS was set as 100% for each cell type. (B) TLR4/LPS-activated BMDM were incubated with different concentrations of TGFβ1 before quantification of IL-27p28, 24 h. (C) Time course of IL-27p28 release by LPS-activated BMDM ± TGFβ1. (D) RT-PCR for ebi3 mRNA after LPS ± TGFβ1 in BMDM. (E) RT-PCR for il-27p28 mRNA after LPS ± TGFβ1 in BMDM. (F) Flow cytometry for intracellular IL-27p28 in BMDM, 12 h, pregated on CD11b+ cells. (G) Inhibition of IL-27p28 release by TGFβ1 in BMDM from C57BL/6 J (WT), MyD88−/− or TRIF−/− mice, 24 h. (H) Suppression of IL-27p28 release by TGFβ1 in BMDM activated with several agonists; Zymosan (TLR2), LTA (TLR2), Poly(I:C) (TLR3), LPS (TLR4), 5′ppp-dsRNA (RIG-I) and IFNγ, 24 h. (I) Reversal of TGFβ1-mediated IL-27p28 suppression by increasing concentrations of the TGFβ receptor I signaling inhibitor, SB431542, in TLR4/LPS-activated BMDM, 24 h. LPS alone was used for normalization (=100% value). (J) IL-27p28 from BMDM of TGFβ receptor II floxed (TbRIIfl/fl) mice and ΔMyeTbRII mice after LPS ± TGFβ1, 24 h. (K) Flow cytometry of intracellular IL-27p28 in BMDM from TbRIIfl/fl or ΔMyeTbRII mice, 12 h. (L) Lung histology of bleomycin-induced fibrosis in TbRIIfl/fl or ΔMyeTbRII mice, 4 wk, Masson–Goldner’s trichrome staining (Scale bar, 50 µm). (M) Quantification of lung collagen deposition from scanned whole slides from mice in frame L. (N) Collagen I–V in lungs of TbRIIfl/fl mice and ΔMyeTbRII mice after bleomycin i.t, 4 wk, SIRCOL assay. (O) IL-27p28 in BALF of bleomycin-treated TbRIIfl/fl mice and ΔMyeTbRII mice, 4 wk. Frames A-CG-JO: ELISA. Representatives of three independent experiments (frames A–K) or n ≥ 6 mice/group (frames L–O); Student’s t test or one-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.