Fig 2: FGA, Hsc70, and TRIM21-mediated PA induced the cytosolic oxidized CD11b and CD18 degradation. (A) Reciprocal co-IP of CD11b and CD18 with FGA in dHL-60 cells. (B) Knockdown of FGA blocked the degradation of CD11b and CD18 in dHL-60 cells (n = 3). Data represent the mean ± s.e.m. (**p < 0.01 versus the control group, ## p < 0.01 and versus the PA-treated group; significance calculated using one-way ANOVA). (C) Reciprocal co-IP of CD11b and CD18 with Hsc70 in dHL-60 cells. (D) Knockdown of Hsc70 blocked the degradation of CD11b and CD18 in dHL-60 cells (n = 3). Data represent the mean ± s.e.m. (**p < 0.01 versus the control group, ## p < 0.01 and versus the PA-treated group; significance calculated using one-way ANOVA). (E) Reciprocal co-IP of CD11b and CD18 with TRIM21 in dHL-60 cells. (F) Knockdown of TRIM21 blocked the degradation of CD11b and CD18 in dHL-60 cells (n = 3). Data represent the mean ± s.e.m. (**p < 0.01 versus the control group, ## p < 0.01 and versus the PA-treated group; significance calculated using one-way ANOVA). (G) Immunoblot for the analysis of CD11b and CD18 oxidation. (H) NAC relieved PA-triggered by the degradation of CD11b and CD18. Data represent the mean ± s.e.m. (**p < 0.01 versus the control group, ## p < 0.01 and versus the PA-treated group; significance calculated using one-way ANOVA).

Fig 3: (A) Cytokine released by human monocyte-derived macrophages (hMDMs) blocked for complement receptor-3 (CR3) and challenged with Sporothrix species. Sporothrix yeasts were made to interact with hMDMs blocked for CR3 (with monoclonal anti-CD11b antibodies) or unblocked (infected control) in a culture medium supplemented with whole human serum (wHS) for 18 h at 37°C and 5% CO2. Non-infected controls were hMDMs blocked with irrelevant antibodies and treated with culture medium supplemented with wHS. The culture supernatants were collected and analyzed for tumor necrosis factor (TNF)-α and interleukin (IL)-1β. Results indicate the mean ± SEM of at least two independent experiments in duplicate or triplicate (n = 4 or 6). ANOVA with Tukey’s multiple comparison posttest was performed for the statistical analysis (***p < 0.001, **p < 0.001). (B) Uptake of Sporothrix yeasts by hMDMs after CR3 blockage. hMDMs were incubated with monoclonal anti-CD11b antibodies for 1 h and challenged with FITC-labeled yeasts of Sporothrix schenckii or Sporothrix brasiliensis. After 4 h of interaction in the presence of medium containing wHS, hMDMs were analyzed by FACS to determine the change in mean binding index (MBI) compared to control (=CD11b unblocked hMDMs interacted with FITC-labeled Sporothrix yeasts). Results are the mean ± SD of an experiment performed in duplicate.

Fig 4: Resident F4/80HighCD206− peritoneal macrophages bind fibrin clots via CD11b.a, b Representative confocal image of the whole-mount (a) and cross-section (b) immunofluorescent staining of the implanted exogenous fibrin clot on day 1 after implantation. n = 3 mice. Scale bars, 100 μm. (c) Representative confocal images of PLA between CD11b of F4/80+ macrophages and fibrin on the ischemic button surface. PLA signals are shown in red. n = 3 independent experiments. Scale bars, 5 μm. d, e Representative images of abdominal adhesions (d) and adhesion scores (e) on day 1 post-ischemic button creation with an intraperitoneal injection of control IgG2b antibody (IgG2b group, n = 6 mice) or anti-CD11b blocking antibody (Anti-CD11b group, n = 7 mice). f, g Representative cross-section immunohistostaining images of the ischemic button (f) and quantification of the coverage of exposed fibrin clots by F4/80+ cells (g) on day 1 after surgery with an intraperitoneal injection of control IgG2b antibody or anti-CD11b blocking antibody. n = 5 mice in each group. Scale bar, 100 μm. Data represent the mean ± SEM. *P < 0.05, two-tailed Student’s t-test.

Fig 5: Autophagy-mediated PA triggered the degradation of ubiquitinated CD11b and CD18 in dairy cow neutrophils. (A) Immunoblot and quantitative analysis for LC3B, p62, CD11b, and CD18 in control and PA-treated bovine neutrophils (treated or not treated with BafA1, CQ or MG132). The protein degradation was blocked by BafA1, CQ, or MG132 in control and PA-treated bovine neutrophils. Quantitative analysis of LC3B, p62, CD11b, and CD18 (n = 3) was performed. Data represent the mean ± s.e.m. (*p < 0.05 and **p < 0.01 versus the control group, ## p < 0.01 versus the PA-treated group; significance calculated using two-way ANOVA). (B) Immunoblot and quantitative analysis for LC3B, p62, CD11b, and CD18 in control and PA-treated dHL-60 cells (infected or not infected with LV-GFP-shATG5). Data represent mean ± s.e.m. Note: (**p < 0.01 versus the control group, ## p < 0.01versus the PA-treated group). Data represent the mean ± s.e.m. (**p < 0.01 versus the control group, ## p < 0.01 versus the PA-treated group; significance calculated using two-way ANOVA). (C) Reciprocal co-IP of CD11b and CD18 with polyubiquitin chains (Lys48). (D) Determination of ubiquitination sites in the CD11b fragment (571-ITGSQISPK (GlyGly)-579) by MS/MS spectra. (E) Determination of ubiquitination sites in the CD18 fragments (311-LAESNIQPIFAVTKK(GlyGly)-325, 333-LTEIIPK (GlyGly)-339) by MS/MS spectra.