Fig. 6: CD62L^dim neutrophils are induced via TLR2 by HMGB1 to form NETs.

a MFI of TLR2 expression on lung infiltrating neutrophils of naive mice and tumor-bearing mice by flow. b MFI of TLR2 expression on lung infiltrating CD62L^dim and CD62L^hi neutrophils of tumor-bearing mice by flow. c Flow analysis (left) and the quantification (right) of CD62L expression on neutrophils from naive mice BM treated with various TLR agonists, which included Pam3CSK4, FSL-1, Poly(I:C), MPLA-SM, FLA-ST, R848, and TLR9 Agonist Kit. d Purified BM neutrophils of naive mice were pre-treated with O-Vanillin (TLR2 inhibitor) and TAK242 (TLR4 inhibitor), TTP488 (RAGE inhibitor) followed by stimulation with rHMGB1. Proportions of CD62L^dim neutrophils were analyzed by flow. e Gel electrophoresis patterns of TLR2^−/− and TLR4^−/− mice. f Quantification of CD62L expression on neutrophils from BM neutrophils of different naive mice treated with rHMGB1 for 4 h in vitro. g Cell immunofluorescent analysis of neutrophils extracellular traps (NETs) of lung infiltrating CD62L^dim neutrophils and CD62L^hi neutrophils of 2-week tumor-bearing mice. NETs staining according to NE (green), Cytox Orange (red) and DAPI (blue). Data are mean ± SEM of one representative experiments. Similar results were seen in three independent experiments. Unpaired Student’s t tests, ns not significant. *p < 0.05, **p < 0.01, ***p < 0.001. See also Fig. S5.