Fig 2: MHC-II−S100A9+ monocytes exert immunosuppressive function on T cell immunity. Monocytes from spleen of WT or S100a9−/− mice undergoing CLP surgery for 72 h were isolated, followed by co-culture with CD3/CD28 activated naïve CD4+ T cells isolated from unmanipulated murine spleens. Supernatants and CD4+ T cells were assayed after 3 d of coculture. a Histogram with quantitative bar plot exhibited and compared the proliferative activity of naïve CD4+ T cells cocultured with monocytes in each group based on CFSE assay. b Contour plots with quantitative bar chart revealed the proportion of CD4+CD25+Foxp3+ Tregs. c Quantitative bar charts showed levels of IL-2, IL-4, IL-10, and IFN-γ in the cocultured supernatants, with ratio of IFN-γ to IL-4. Two-way ANOVA with Tukey HSD test was used to determine the statistical significance between groups. Data are shown as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. MHC major histocompatibility complex, WT wild-type, CLP cecal ligation and puncture, CFSE carboxyl fluorescein succinyl ester staining, Tregs regulatory T cells, IL interleukins, IFN-γ interferon-γ, ANOVA analysis of variance, SD standard deviation

Fig 3: MHC-II−S100A9+ monocytes are associated with immunocompromised state in late sepsis. a–c At different intervals after CLP surgery (0, 24, and 72 h), circulating monocytes were isolated and subjected to the subsequent experiments. Counter plots with quantitative bar charts showed the proportion of MHC-II−S100A9+ monocytes at different time points after CLP operation (a). Representative confocal immunofluorescence images of S100A9+ monocytes in each group (Scale bar = 50 μm, 20 μm) (b). Western blotting analysis indicated the protein expression of S100A9 at various time points (c). d–g PBMCs and plasma were collected from WT mice subjected to CLP surgery across different sampling time points (0, 24, and 72 h). Histogram and quantitative bar charts indicated the percentage of CD3+ cells at different time points after CLP surgery (d). Counter plots with quantitative bar charts were compared the proportion of CD3+CD4+ cells between groups (e). Representative counter plots with quantitative bar plots showed CD3+CD4+Foxp3+ Tregs proportion at different time points (f). Quantitative bar plots showed circulating levels of IL-2, IL-4, IL-10, IFN-γ, TGF-β and the ratio of IFN-γ to IL-4 in various groups (g). Statistics were analyzed by One-way ANOVA with Tukey HSD test for comparison of two groups. Data are shown as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. CLP cecal ligation and puncture, PBMCs peripheral blood mononuclear cells, WT wild-type, IL interleukins, IFN-γ interferon-γ, TGF-β transforming growth factor-β, DAPI 4′, 6′-diamidino-2-phenylindole, Treg regulatory T cells, MHC major histocompatibility complex, ANOVA analysis of variance, SD standard deviation

Fig 4: S100a8/a9 induces overwhelming inflammatory responses and acute lung injury, both of which could be alleviated by S100a8/a9 inhibitor.A, B The changes of S100A9+ neutrophil proportion were analyzed by flow cytometry (n = 3 in each group); C–F ELISA results showed the variations of S100a8/a9 and inflammatory cytokines concentrations in serum (n = 6 in each group). G The expression of S100a8 + S100a9 protein in lung tissues measured by Western blot (n = 6 in each group. The antibody we used is Rabbit recombinant multiclonal [RM1038] to S100A8 + S100A9 (Abcam; ab288715). The observed band size of S100A8 + S100A9 is 11, 14 kDa. But in some cell types, only one band can be observed, and the band size of S100A8 + S100A9 is 13 kDa approximately.); H–J The mRNA expression levels of inflammatory mediators detected by RT-qPCR (n = 3 in each group); K Representative immunofluorescence images showed S100a8/a9 expression in lung tissues; Scale bar: 40 µm; L Photos of dissected lung from mice; M Images of H&E and Masson staining of lung tissues to assess the degree of lung injury and fibrosis; Scale bar: 100 µm; N Wet/dry ratio was used to evaluate the extent of lung edema (n = 6 in each group); O The degree of lung injury was measured by lung injury score (n = 3 in each group); P Images of TUNEL staining of lung tissues were used to assess cell apoptosis; Scale bar: 40 µm (n = 3 in each group). Each bar showed means ± SEM. Unpaired t-test was used for the comparison between two groups. Comparison among three or more groups was analyzed by one-way ANOVA. *p < 0.05, **p < 0.01 versus sham group. #p < 0.05, ##p < 0.01 versus sepsis-24h group.

Fig 5: Increased numbers of S100A8/A9hi neutrophils exist in lung tissues from CLP mice, and showed enhanced interactions with endothelial cells.A The main workflow of the scRNA-seq; B The UMAP plot was based on scRNA-seq data, and it showed five identified immune cell types; C The sankey diagram showed the percentages of five immune cells in sham and CLP groups; D Five clusters of neutrophils were identified in the UMAP plot; E The changes of five neutrophil subclusters percentage were shown on the sankey diagram; F Volcano map showed upregulated genes in S100A8/A9hi neutrophils compared with other subpopulations; G The expression levels of S100A8 and S100A9 in five neutrophil subclusters on dot plot; H The prediction of neutrophil differentiation trajectories using pseudotime analysis; I The expression levels of S100A8 and S100A9 of neutrophils in sham and CLP groups; J, K The number and strength of interactions among neutrophils, endothelial cells and epithelial cells analyzed by CellChat; L The expression levels of ligand-receptor pairs analyzed by CellChat (Since there is no ligand-receptor pair with high expression among epithelial cells in sham group, the dot plot only shows the expressions of ligand-receptor pairs among epithelial cells in CLP group.); M, N The distribution of S100A8/A9hi neutrophils and NET-related gene+ neutrophils on the UMAP plot. Wilcoxon rank sum test was used for the comparison between two groups. *p < 0.05, **p < 0.01 versus sham group.