Journal: Research

Article Title: Splenic PD-L1 + Myeloid-Derived Suppressor Cells Induce Contact-Dependent Alveolar Macrophage Dysfunction in Early Sepsis

doi: 10.34133/research.1407

Figure Lengend Snippet: LPS-stimulated AMs cocultured with PD-L1 + PMN-MDSCs exhibit immunosuppressive and exhausted profiles. (A) Principal component analysis ( n = 3). (B) GO enrichment analysis. (C) Volcano plot of differentially expressed genes. (D) Flow cytometric sorting strategy for murine alveolar macrophages. (E) Flow cytometric sorting strategy for alveolar macrophages from bronchoalveolar lavage fluid (BALF) of clinical patients. (F) Protein levels of C1q, LAG3, CD200, S100A8, S100A9, Wnt5a, and LEF1 in LPS-stimulated AMs cocultured or not with PD-L1 + PMN-MDSCs. (G) Protein levels of KLF9, PD-1, C1q, LAG3, and CD200 in flow-sorted AMs from BALF of septic and nonseptic patients. (H) Correlation network of PD-1, Wnt, and classical complement pathways. (I to K) GSEA of complement regulation (I), chemokine receptor binding (J), and PD-1 signaling (K). (L) Flow cytometric gating strategy for identifying PMN-MDSCs (defined as CD45 + CD11b + CD15 + CD14 − CD33 + HLA-DR l ° w ) and the CXCR2 + PD-L1 + subpopulation in the peripheral blood of clinical patients. (M and N) Absolute number of total PMN-MDSCs (M) and CXCR2 + PD-L1 + PMN-MDSCs (N) in peripheral blood from septic and nonseptic patients ( n = 6). Values are mean ± SD. Panels (M) and (N) were analyzed by Student t test. * P < 0.05, ** P < 0.01.

Article Snippet: Membranes were incubated overnight at 4 °C with antibodies against C1qa (1:1,000, Proteintech, #11602-1-AP), LAG3 (1:1,000, Abclonal, #A2996), CD200 (1:1,000, Abclonal, #A23050), S100A8 (1:1,000, Abclonal, #A15315), S100A9 (1:1,000, Abclonal, #A9842), Wnt5A (1:1,000, Zen Bio, #619919), LEF1 (1:500, Proteintech, #14972-1-AP), KLF9 (1:1,000, Abclonal, #A7196), PD-1 (1:1,000, Abclonal, #A11973), PD-L1 (1:1,000, Abclonal, #A21443), IL-6 (1:1,000, Abcam, #ab229381), TFDP1 (1:1,000, Abclonal, #A5422), IL-10 (1:1,000, Abclonal, #a12255), TGF-β (1:1,000, Abcam, #ab64715), TNF-α (1:1,000, Abclonal, #A11534), PCNA (1:500, Proteintech, #10205-2-AP), or β-actin (1:4,000, Proteintech, #66009-1-Ig).

Techniques: Binding Assay

Journal: Research

Article Title: Splenic PD-L1 + Myeloid-Derived Suppressor Cells Induce Contact-Dependent Alveolar Macrophage Dysfunction in Early Sepsis

doi: 10.34133/research.1407

Figure Lengend Snippet: Myeloid PD-1 deficiency improves outcomes after polymicrobial sepsis and Pseudomonas aeruginosa infection. (A) Protein levels of PD-1, C1qa, CD200, and LAG3 in flow-sorted AMs from Pdcd1 flox/flox and Lyz2 Cre Pdcd1 flox/flox mice post-CLP ( n = 6). (B) Kaplan–Meier survival analysis following P. aeruginosa infection ( n = 21). (C to F) Core body temperature ( n = 16 to 18) (C), bacterial lung burden ( n = 7) (D), and BALF levels of TNF-α ( n = 7) (E) and IL-6 ( n = 7) (F), in septic mice 1 d after P. aeruginosa infection. (G) Lung H&E images and histological injury scoring ( n = 7). (H) Immunofluorescence images and semiquantitative analysis of NET formation using Cit-H3 and MPO staining ( n = 6). Scale bar, 50 μm. (I) Parabiosis and CLP workflow. (J and K) Example FCM plots (J) and proportions of partner-derived neutrophils, IMs, and AMs in lung ( n = 5) (K). (L) Schematics of parabiosis of Pdcd1 flox/flox and Lyz2 Cre Pdcd1 flox/flox mice. (M) BALF TNF-α and IL-6 in septic parabionts after P. aeruginosa infection ( n = 7). Values are mean ± SD. Panel (A) was analyzed by one-way ANOVA with Tukey’s post hoc test; panel (B) was analyzed by log-rank (Mantel–Cox) test; panels (C) to (H) and (M) were analyzed by Student t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns, not significant.

Article Snippet: Membranes were incubated overnight at 4 °C with antibodies against C1qa (1:1,000, Proteintech, #11602-1-AP), LAG3 (1:1,000, Abclonal, #A2996), CD200 (1:1,000, Abclonal, #A23050), S100A8 (1:1,000, Abclonal, #A15315), S100A9 (1:1,000, Abclonal, #A9842), Wnt5A (1:1,000, Zen Bio, #619919), LEF1 (1:500, Proteintech, #14972-1-AP), KLF9 (1:1,000, Abclonal, #A7196), PD-1 (1:1,000, Abclonal, #A11973), PD-L1 (1:1,000, Abclonal, #A21443), IL-6 (1:1,000, Abcam, #ab229381), TFDP1 (1:1,000, Abclonal, #A5422), IL-10 (1:1,000, Abclonal, #a12255), TGF-β (1:1,000, Abcam, #ab64715), TNF-α (1:1,000, Abclonal, #A11534), PCNA (1:500, Proteintech, #10205-2-AP), or β-actin (1:4,000, Proteintech, #66009-1-Ig).

Techniques: Infection, Immunofluorescence, Staining, Derivative Assay

Journal: Research

Article Title: Splenic PD-L1 + Myeloid-Derived Suppressor Cells Induce Contact-Dependent Alveolar Macrophage Dysfunction in Early Sepsis

doi: 10.34133/research.1407

Figure Lengend Snippet: TFDP1 and S100A9 promote expansion and immunosuppression of splenic PD-L1 + PMN-MDSCs in early sepsis. (A and B) Western blot and quantification of TFDP1 (A) ( n = 6) and S100A9 (B) ( n = 6) in spleens. (C) Schematic of CLP modeling, Adv-shRNA (Tfdp1) treatment, and tissue collection in WT mice. (D) Example FCM plots and percentage of Ki67 in splenic PD-L1 + PMN-MDSCs ( n = 8). (E) Example FCM plots and percentage of PD-L1 in pulmonary PMN-MDSCs ( n = 6). (F) Schematic of CLP modeling, Adv-shRNA ( S100a9 ) treatment, and tissue collection in WT mice. (G) Example FCM plots and percentage of PD-L1 in splenic PMN-MDSCs ( n = 9). (H) Schematic of CLP modeling, Adv-shRNA ( S100a9 ) treatment, and tissue collection in Kaede-tg mice. (I) Example FCM plots and percentage of Kaede red + in pulmonary PD-L1 + PMN-MDSCs ( n = 7 to 8). (J) FCM analysis using t-SNE algorithm showing expressions of PD-1, Lag3, and CD200 in pulmonary AMs ( n = 9 to 10). Values are shown as mean ± SD; statistical testing was determined using Student t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Membranes were incubated overnight at 4 °C with antibodies against C1qa (1:1,000, Proteintech, #11602-1-AP), LAG3 (1:1,000, Abclonal, #A2996), CD200 (1:1,000, Abclonal, #A23050), S100A8 (1:1,000, Abclonal, #A15315), S100A9 (1:1,000, Abclonal, #A9842), Wnt5A (1:1,000, Zen Bio, #619919), LEF1 (1:500, Proteintech, #14972-1-AP), KLF9 (1:1,000, Abclonal, #A7196), PD-1 (1:1,000, Abclonal, #A11973), PD-L1 (1:1,000, Abclonal, #A21443), IL-6 (1:1,000, Abcam, #ab229381), TFDP1 (1:1,000, Abclonal, #A5422), IL-10 (1:1,000, Abclonal, #a12255), TGF-β (1:1,000, Abcam, #ab64715), TNF-α (1:1,000, Abclonal, #A11534), PCNA (1:500, Proteintech, #10205-2-AP), or β-actin (1:4,000, Proteintech, #66009-1-Ig).

Techniques: Western Blot, shRNA