Fig 2: scRNAseq analysis of WT BM neutrophils.A The UMAP diagram of three subsets of neutrophils (exhausted NCD177; resolving NCD200R; and intermediate Nint). B Relative percentages of three neutrophil subsets collected from naive WT mice bone marrow. C The violin plot of Cd177 expression in three neutrophil subsets. D The bubble plot analysis of Cd200r and Cd86 expression in three neutrophil subsets. E The UMAP diagram of neutrophils subjected to PBS or 4-PBA treatment. F The bubble plot analysis of Cd200r and Cd86 levels in PBS or 4-PBA-treated neutrophils.

Fig 3: Genetic deletion of TRAM renders constitutive generation of resolving neutrophils.A Flow analyses of CD86 and CD200R expression levels on WT or TRAM KO neutrophils (n = 3). B The levels of Resolvin D1 and SerpinB1 from the supernatant of overnight-cultured WT or TRAM KO neutrophils (n = 3). C Flow analyses of neutrophil phagocytosis of GFP-labeled E. coli. Purified WT or TRAM KO neutrophils were co-incubated with GFP-labeled E. coli for 20 min. Following washing, neutrophils were subjected to flow analyses, and the percentages of GFP-E. coli containing neutrophils were counted and plotted (n = 3). D Analyses of bacterial killing through plating of viable E. coli harvested from lysed neutrophils. WT or TRAM KO neutrophils were co-incubated with GFP-labeled E. coli for 30 min. Following washing, neutrophils were lysed and plated on bacterial culture plates. The numbers of viable E. coli were counted and the CFU were plotted (n = 3). E Analyses of bacterial killing through plating of viable E. coli collected from culture supernatants. WT or TRAM KO neutrophils were co-incubated with GFP-labeled E. coli for 30 minutes. Culture supernatants containing extracellular viable bacteria were plated and counted with the CFU plotted (n = 3). F–H The levels of PPAR?, LMO4, ß-actin, p-STAT3, total STAT3 of overnight-cultured WT or TRAM KO neutrophils were determined by Western blot. Data are representative of at least three independent experiments. Data plotted as mean ± SD. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 using two-sided Student’s t test.

Fig 4: Resolving neutrophils trained by 4-PBA express elevated levels of cell surface CD86/CD200R, increased RvD1/SerpinB1, and reduced TNF-a.A Flow analyses of mature Ly6GhiCD11b+ neutrophils. WT neutrophils treated with PBS or 4-PBA (1 mM) for 24 hours were subjected to flow analyses and the percentages of Ly6GhiCD11b+ neutrophils were plotted. (n = 3). B The mean fluorescent intensity (MFI) levels of Ly6G on WT neutrophils treated with PBS or 4-PBA (1 mM) for 24 hours (n = 3). C The levels of CD86 and CD200R on WT neutrophils treated with PBS or 4-PBA (1 mM) for 24 hours (n = 3). D The expression of RvD1 and SerpinB1 from WT neutrophils treated with PBS or 4-PBA (1 mM) for 24 hours (n = 3). E The secretion of TNF-a from WT neutrophils treated with PBS or 4-PBA (1 mM) for 24 hours followed by PMA priming (100 ng/ml) for 30 min (n = 3). Data plotted as mean ± SD. ****P < 0.0001, ***P < 0.001, **P < 0.01 using two-sided Student’s t test.

Fig 5: CD200R-/- neutrophils show enhanced expression of proteins associated with reactive oxygen species (ROS) clearance and reduced ROS output. Neutrophils were isolated by negative selection from WT and CD200R-/- C57BL/6 mouse bone marrow. a Heatmap of selected genes identified in RNA-seq analysis of primary neutrophils isolated from 3 WT and 3 CD200R-/- mice. Signal intensity represents gene expression change following mean centring scaling. b, c WT and CD200R-/- neutrophils were incubated for 15 min with Dihydrorhodamine 123 (DHR123) (25 ng/ml), followed by 1 h stimulation with Phorbol 12-myristate 13-acetate (PMA) (100 ng/ml). b Representative histograms of FITC DHR123 expression in unstimulated or PMA-treated WT and CD200R-/- neutrophils. c Oxidative index was quantified using the following equation: mean fluorescence intensity (MFI) Stimulated/MFI Unstimulated. Alternatively, neutrophils were infected with F. tularensis MOI 100 and incubated with DHR123 for 24 h. d Representative histograms of FITC DHR123 expression in unstimulated and F. tularensis-infected neutrophils. e Oxidative index was quantified using the following equation: MFI F. tularensis-infected/MFI unstimulated. Data represents b, c four or d, e two independent experiments and is shown as mean ± SD (n = 12 and 6, respectively). Statistical analysis was performed using unpaired t-test (*p < 0.01)