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. 2018 Apr 4;9:677. doi: 10.3389/fimmu.2018.00677

Figure 3.

Figure 3

Recombinant Rv0888NS/MS sphingomyelinase induces neutrophil extracellular traps (NETs) formation in lung. (A) Immunofluorescence staining of lung sections from mice 48 h after intraperitoneal injection of recombinant Rv0888NS/MS, D438A/MS, and H481N/MS (n = 3 mice per group) administration was performed and compared with the control section PBS and recombinant pMV262/MS (n = 3 mice per group) for DNA (blue), CitH3 (green), and myeloperoxidase (MPO) (red). Images are representative of one of three independent experiments with three mice per group. The recombinant Rv0888NS/MS and D438A/MS groups’ merge showed co-localization of MPO with CitH3, indicating NETs’ formation. Scale bars = 50 µm. The higher magnification views of the insets (1 and 2), which were randomly chosen, show co-localization of DNA (blue), CitH3 (green), and MPO (red) in NETs structures. Scale bars = 10 µm. (B) Western blotting analysis of MPO, histone H4, and CitH3 protein levels in the bronchoalveolar lavage fluid. Images are representative of one of three independent experiments with three mice per group. (C) Reactive oxygen species (ROS) were measured in the serum and lung homogenates of infected mice using a Mouse ROS enzyme-linked immunosorbent assay (ELISA) Kit. (D) Ceramide in the serum and lung homogenates of infected mice was determined by the Mouse Ceramide ELISA Kit. The error bars show the SEM of three independent experiments with three mice per group. (C,D) One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001, ns, non-significant.