Fig 1: mregDCs are enriched upon sepsis induction with dual immunoregulatory and immunogenic functions on CD4+ T cell responses. A. Representative contour plots elucidating flow cytometry-based gating strategy for spleen-derived cDCs in wild-type mice. cDCs were defined as CD45+ Lin (CD3e, CD19, CD49b, Ly6C)- CD11c+ MHC-II+ cells, in which cDC1 and cDC2 were defined CD45+ Lin- CD11c+ MHC-II+ CD8a+ CD11b- and CD45+ Lin- CD11c+ MHC-II+ CD8a- CD11b+ cells, respectively. B, C. Frequency of splenic mregDCs as a proportion of total cDCs at disparate time points after CLP operation. mregDCs were defined as CD45+ Lin- CD11c+ MHC-II+ CD274hi CD80hi cells. Contour plots B. and quantitative bar charts C. showing alterations in mregDCs proportion upon sepsis induction measured by flow cytometry analysis. D. Scatter plot showing mregDCs proportion in cDC1 and cDC2 subsets independently between sham and sepsis groups. E. Quantitative bar plots displaying and comparing mregDCs proportion between wild-type, Batf3-/- and Irf4-/- mice. F. Histograms with quantitative bar charts showing proportion of CCR7, CD80, and CD274 positive cells measured by flow cytometry, across distinct time points after onset of sepsis, as a percentage of total cDCs. G. Multiplex immunofluorescence images demonstrating the in situ existence of mregDCs in spleen after septic challenge, using antibodies, including CD11c, MHC-II, CD80, and CD274. Scale bar, 100 µm. H. Quantitative bar charts showing the level of multiple cytokines in supernatants between mregDCs and non-mregDCs groups, including interleukin (IL)-2, IL-4, IL-10, IL-12, and IFN-?. I. Quantitative bar chart displaying the results from cell counting kit-8 (CCK-8) assay. J. Histogram with quantitative bar plot illustrating and comparing the proliferative activity of naïve CD4+ T cells co-cultured with either mregDCs or non-mregDCs based on carboxyl fluorescein succinyl ester staining (CFSE) assay. K. Contour plots with quantitative bar chart showing the proportion of CD4+CD25+Foxp3+ Tregs between mregDCs and non-mregDCs groups. One-way ANOVA with Tukey HSD test C, F, J, K; Unpaired two-sided Student's t test D, H, I. Data were expressed as means ± SEM. *P<0.05; **P <0.01; ***P<0.001; ****P<0.0001.
Fig 2: Sepsis-associated mregDC program is initiated in a TNFRSF-NF-κB and IFNGR2-STAT3 dependent manner. A. Histogram indicating the relative expression level of selected genes related to JAK-STAT and NF-κB pathways from bulk RNA-seq analyses (blue, low expression level; red, high expression level). B. Histogram showing the cell-cell communication between mregDCs and other immune cell types, based on selected ligand-receptor pairs in association with TNFRSF-TNFSF and IFN-γ- IFNGR2. Statistical significance (P<0.05) was determined by permutation test from CellPhoneDB, with color of grey indicating no statistical significance. The color gradient indicated the level of interaction (blue, low level of interaction; red, high level of interaction). C. Representative Western blotting images of splenic CD11c+ DCs isolated from wild-type mice undergone sham or CLP operation at distinct time points. D. Quantitative bar charts displaying the results of Western blotting analyses. The values represent protein levels relative to the unphosphorylated form or β-actin level. The data shown are representative of 3 independent experiments. E. Inhibitory experiments were performed to validate the effect of TNFRSF-NF-κB and IFNGR2-STAT3 pathways on mregDC program using STAT3 and NK-κB inhibitors, JSH-23 and SH-4-54, respectively. Contour plots (left panel) with quantitative bar chart (upper right panel) showing the proportion of mregDCs upon inhibition of STAT3 and NK-κB. Western blot analysis of PD-L1 expression in splenic CD11c+ DCs after treatment with inhibitors (lower right panel). F. Proposed model of mregDC program upon sepsis induction. Graphs were created with BioRender.com. Statistical significance was calculated using one-way ANOVA with Tukey HSD test D, E. Data were expressed as means ± SEM. *P<0.05; **P<0.01; ***P<0.001.
Fig 3: Ogr1 deletion enhances the infiltration of CD8+ T cells and contributes to tumor regression.A Representative IHC staining was applied to assess the expression of NK cells (CD49b), macrophages (F4/80), B cells (B220) as well as dendritic cells (CD11c) in xenograft melanomas. B The representative immunohistochemistry and immunofluorescence staining were applied to assess the number of CD4+ T cells and CD8+ T cells infiltration in xenograft melanomas. C Staining intensities for CD4+ T cells and CD8+ T cells in melanomas were determined by the assignment of semiquantitative scores. D Schematic of mouse models of conditional knockout of Ogr1 in CD4+ and CD8+ T cells. When Ogr1 flox mice mated with CRE mice, the specific exon of Ogr1 was deleted, thus realizing the conditional knockout of Ogr1 gene. E Ogr1 expression in T cells was detected by RT-PCR. Tumor growth (F) and mean tumor volume (Day 20) (G) of subcutaneous B16-F10 (5 × 105 in 100 µl PBS) in WT mice versus Ogr1flox/floxCD4Cre+/- or Ogr1flox/floxCD8Cre+/- mice. In A and B, original magnification ×20; scale bar = 100 µm. F, G experiments were repeated three times. The results are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, as obtained by unpaired test.
Fig 4: STING deficiency moderated mtDNA‐mediated immunoparalysis of spleen DCs in vivo. (A) Representative image of spleens and proportion of CD11b+CD11c+ cells of mice treated with PBS (ip, 200 μl), mtDNA (iv, 100 μg/mice), LPS (ip, 10 mg/kg) + DNase I (iv, 5 mg/kg) or LPS (ip) + mtDNA (iv, 100 μg/mice) (n = 4). (B) Proportion of CD11c+ CD40+ cells in spleen of mice treated as (A) (n = 6). (C) Proportion of CD11c+ CD80+ and CD11c+ CD86+ cells in spleen of mice treated as (A) (n = 6). (D) IL‐10 and IL‐12p70 in plasma of mice treated as (A) (n = 6). (E) Immunofluorescence staining of CD80 in spleen of mice treated as (A). Scale bar: 100 μm. (F) Plasma mtDNA levels of STING−/− mice treated with PBS (ip, 200 μl) or LPS (ip, 10 mg/kg) (n = 6). (G) Plasma IL‐10 levels of WT and STING−/− mice treated as (F) (n = 6). (H) Proportion of CD11c+ CD40+, CD11c+ CD80+ and CD11c+ CD86+ cells in spleen of WT and STING−/− mice treated with LPS (ip, 10 mg/kg) + mtDNA (iv, 100 μg/mice) (n = 4). (I) Plasma IL‐10 levels of WT and STING−/− mice treated as (H) (n = 4). (J) Survival rate of WT and STING−/− mice treated with LPS (ip, 10 mg/kg) or LPS (ip, 10 mg/kg) + DNase I (5 mg/kg) or LPS (ip, 10 mg/kg) + mtDNA (iv, 100 μg/mice) (n = 10–12). Data are shown as the mean ± SEM or SE. *p < 0.05; **p < 0.01; ***p < 0.001. DC, dendritic cell; LPS, lipopolysaccharide; mtDNA, mitochondrial DNA; WT, wild type.
Fig 5: Sepsis associated elevated peripheral mtDNA correlates with disease severity of sepsis and contributes to immunoparalysis of spleen DCs. Intraperitoneal injection of LPS (10 mg/kg) and CLP were applied to established endotoxin mice model. (A) Clinical scores of mice treated with LPS (ip, n = 6–12). (B) Peripheral mtDNA levels of mice treated with LPS (ip, n = 6) or PBS (ip, n = 6). (C) Clinical scores of mice after CLP (n = 5). (D) Peripheral mtDNA levels of mice after CLP (n = 5). (E) Correlation analysis between peripheral mtDNA and clinical scores. (F) Representative image of spleen and CD11b+ CD11c+ cells in spleen (n = 6). (G) CD11c+, CD80+ and CD11c+ CD86+ cells in spleen (n = 6). (H) Proliferation of CD4+ T cells co-cultured with spleen DCs (n = 3). Data were shown as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. CFSE, 5,6-carboxyfluorescein diacetate succinimidyl ester; CLP, cecal ligation and puncture; DC, dendritic cell; LPS, lipopolysaccharide; mtDNA, mitochondrial DNA.
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