Fig. 3: Piezo1^−/− neutrophil NET DNA inhibits M1 macrophage differentiation through TLR9 and cGAS signalling.

A–C Wild-type (WT) and Piezo1^−/− mice were infected with the PR8 virus for 48 h. A TLR9 and cGAS expression in CD11b⁺F4/80⁺ macrophages in the BALF was measured via flow cytometry. Dot plots present representative data from flow cytometry analysis (left), and the statistical results are shown (right). B mRNA expression of the indicated genes in CD11b⁺F4/80⁺ macrophages sorted from BALF by flow cytometry. C Western blot analysis of STING and IRF3 in sorted CD11b⁺F4/80⁺ macrophages in the BALF via flow cytometry. D, E Neutrophils were isolated from mouse spleens and stimulated with virus in vitro for 6 h, and NET DNA was purified and collected for subsequent experiments. Bone marrow-derived macrophages were transfected with shRNA control (ctrl), shRNA-mediated cGAS (shcGAS) or shRNA-mediated STING (shSTING) and treated with NET DNA (1 ng/µl) from neutrophils stimulated with virus for 6 h. The levels of TNFα, IL-10 (D), NOS2 and CD206 (E) in macrophages were determined via flow cytometry. Fluorescence Minus One control, FMO. F–I Neutrophils of the same number were isolated from WT and Piezo1^−/− mouse spleens and stimulated with virus in vitro for 6 h, after which NET DNA was purified and collected for subsequent experiments. BMDMs were transfected with shRNA control (ctrl), shRNA cGAS (shcGAS) or shRNA STING (shSTING) and treated with NET DNA from WT and Piezo1^−/− neutrophils in the presence of virus for 6 h (F). Intracellular staining of TNFα (G), NOS2 (H) and CD206 (I) in macrophages was determined by flow cytometry. The graph shows data from three independent experiments with three to four mice per group. ***P < 0.001, compared with the indicated groups.