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. 2022 Aug 8;13(8):693. doi: 10.1038/s41419-022-05138-4

Fig. 7. In vitro studies revealed that NETs promoted macrophage-to-myofibroblast transition.

Fig. 7

A Representative images of immunofluorescence staining of GSDMD (green). The neutrophils were isolated from the bone marrow of wild-type mice. Neutrophils were primed with very low dose of PMA 20 nM for 3 h and then treated with hydrops extracted from the obstructed kidney (treatment group) or urine of healthy mice (control group) for 8 hours. GSDMD translocalization to the cell membranes was induced in the treatment group. Scale bar: 50 μm. n = 4. B, C Representative images of immunofluorescence staining of Cit-H3 and MPO and quantification of neutrophils undergoing NETs formation characterized by Cit-H3 positive. The neutrophils were isolated from the bone marrow of Gsdmd+/+ mice and Gsdmd−/− mice and treated with as A described. n = 6. Scale bar: 50 μm. D Representative images of immunofluorescence staining of the expression of α-SMA. The macrophages were treated with NETs, NETs plus DNase I(10 U/ml), NETs plus NE inhibitor (Alvelestat, 10 μmol/ml) or NETs plus antiTGF-β1 antibody for 96 h. Scale bar: 50 μm. E Western blot analysis for α-SMA and phosphorylated Smad3 expression of macrophages, which was corresponding to the five condition of D. n = 4. F, G Representative images of immunofluorescence staining of p65. Macrophages were treated with NETs, or NETs plus DNase I. Scale bar: 50μm. H, I Macrophages were treated as described in D. TNF-α and TGF-β1 production in macrophages were detected by ELISA. n = 4. J Macrophages were treated with 1 μg/mL LPS and 500 ng/ml HMGB1 for 18 h. IL-1β production in macrophages were detected by ELISA. The macrophages were isolated from the bone marrow of Gsdmd+/+ mice and Gsdmd−/− mice and treated with HMGB1 or not. n = 4. ** P < 0.01.