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Platelets inhibit apoptotic lung epithelial cell death and protect mice against infection-induced lung injury

William Bain, Tolani Olonisakin, Minting Yu, Yanyan Qu, Mei Hulver, Zeyu Xiong, Huihua Li, Joseph Pilewski, Rama K. Mallampalli, Mehdi Nouraie, Anuradha Ray, Prabir Ray, Zhenyu Cheng, Robert M. Q. Shanks, Claudette St. Croix, Roy L. Silverstein and Janet S. Lee

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Thrombocytopenic Mpl−/−mice sustain severe lung injury after IT PA exposure. (A) Mpl−/− and Mpl+/+ mice survival following IT inoculation with PA (P < .0001, log-rank [Mantel-Cox] test). In separate experiments, BAL neutrophil counts/mL (B), BAL protein concentrations (mg/mL) (C), and lung bacterial CFU/mL (D) were measured 20 hours post-PA infection (n = 8 Mpl+/+ mice, n = 9 Mpl−/− mice). Whole-lung images (E) and hematoxylin and eosin–stained lung tissue sections (F; scale bars represent 250 μm) from WT and Mpl−/− mice 20 hours following PA infection. (G) Circulating platelet counts obtained from WT and Mpl−/− mice at 0 and 20 hours post-PA infection. Gross appearance of BAL fluid (H), BAL fluid hemoglobin (Hgb) concentration (g/dL) at 0 and 20 hours (I), BAL fluid OD540 measurements at 0 and 20 hours (J) (n = 5/group at 0 hours, n = 8/group at 20 hours, 1 death in the Mpl−/− group prior to 20 hours). (K) Evans blue dye measurements of lung tissue homogenates at OD620 (adjusted for hemoglobin; n = 7 mice/group). (L) BAL OD540 measurements taken at the 20-hour time point from WT or Mpl−/− mice transfused with either vehicle or WT platelets (109) 1 hour post-PA infection (n = 4 mice/group). For all graphs, each tube or point represents an individual mouse, and the group median is displayed. Statistical comparison by Mann-Whitney U test. *P < .05, ***P < .001, and ****P < .0001.

  • Figure 2.

    Pathogenic KP does not induce hemorrhagic lung injury, but the cell-free SN of PA is sufficient to induce neutrophil airspace influx and hemorrhagic injury in thrombocytopenic Mpl−/−mice. (A) Survival of Mpl−/− and Mpl+/+ mice following IT inoculation with pathogenic KP by log-rank (Mantel-Cox) test (n = 9 mice/group). In separate experiments, total BAL leukocytes/mL (B), BAL neutrophil counts/mL (C), BAL protein concentrations (mg/mL) (D), lung CFU/mL (E), and gross appearance of BAL fluid (F) (representative tube displayed; n = 9 mice/group) at the 48-hour time point. (G) Gross appearance of BAL fluid from representative Mpl−/− mice 20 hours after IT administration of either vehicle (LB) or PA cell-free SN with total BAL neutrophils/mL (H) and BAL protein concentrations (mg/mL) (I) (n = 4 mice/group). Each tube or point represents an individual mouse, and the group median is displayed. Statistical comparison by Mann-Whitney U test. *P < .05.

  • Figure 3.

    Reduction of neutrophil airspace influx does not significantly attenuate alveolar barrier disruption in thrombocytopenic Mpl−/−mice following either live PA challenge or PA SN-induced sterile injury. (A) Schematic of experimental design: Mpl−/− mice were administered Ly-6G or isotype control antibody (Ab) at −24 hours and inoculated with PA at 0 hours, and necropsy was performed 20 hours after PA lung infection. (B) Reduction of circulating neutrophil counts (109/L blood) after anti-Ly6G. In separate experiments, mice were given either 106 live bacteria (C-G) or mice were given cell-free PA SN and a second dose of antibody at t = 0 hours (H-K). Following live bacterial challenge, lung CFU/mL (C), BAL neutrophil counts/mL (D), gross appearance of BAL fluid (E), BAL OD540 (F), and BAL IgM concentrations (ng/mL) (G) were obtained at necropsy 20 hours after 106 CFU IT PA. n = 6 mice/group. There was 1 death in the isotype control group prior to the 20-hour time point. Following PA SN-induced sterile injury, BAL neutrophil counts/mL (H), gross appearance of BAL fluid (I), BAL OD540 (J), and BAL IgM concentration (ng/mL) (K) were obtained at necropsy. n = 6 mice/group. There was 1 death in the isotype control group prior to the 20-hour time point. Each tube or point represents an individual mouse, and the group median is displayed. Statistical comparison by Mann-Whitney U test. **P < .01.

  • Figure 4.

    PA SN induces lung injury after antibody-mediated platelet depletion of WT mice, and PA SN-induced injury can be attenuated by partial reconstitution of platelet counts in natively thrombocytopenic Mpl−/−mice. WT mice were administered anti-GP1bα or control antibody 24 hours prior to IT PA SN, and necropsy was performed at 20 hours with peripheral blood platelet counts (109/L) (A). At necropsy 20 hours after IT PA SN inoculation, gross appearance of BAL fluid (B), BAL OD540 measurements (C), BAL protein concentrations (mg/mL) (D), BAL IgM concentrations (ng/mL) (E), and BAL neutrophil counts/mL (F) are shown (n = 6 mice/group; 2 deaths occurred in the anti-GP1bα group prior to the 20-hour time point). In separate experiments, vehicle or 109 platelets from WT mice were transfused into Mpl−/− mice 1 hour following IT PA SN administration. Blood platelet counts (109/L) (G), gross appearance of BAL fluid (H), and BAL OD540 measurements (I) were obtained 20 hours post-PA infection. Each tube or point represents a single mouse (n = 5 for vehicle, n = 7 for the WT platelet group; peripheral blood was not successfully obtained from 1 mouse in the vehicle group), and the group median is displayed. Statistical comparison by Mann-Whitney U test. *P < .05 and **P < .01.

  • Figure 5.

    PA exoproducts are sufficient to induce apoptotic lung epithelial cell death, in part through a secreted T2SS protein. Single-plane confocal images showing Mpl−/− mouse lung tissue at 0 hours (A) and 6 hours (B) after IT PA SN. Tissue was labeled with 4′,6-diamidino-2-phenylindole (nuclei, blue), TUNEL (red), phalloidin (actin, white), and anti-pro-SPC (type 2 cells, green). There was an increase in the number of TUNEL-positive cells at 6 hours, the majority of which were also positive for pro-SPC (indicated by white arrows). Magenta arrows indicate TUNEL-positive cells likely to be endothelial cells based on separate staining for CD31 as well as the shape and size of the nucleus. Scale bars represent 100 μm. (C) Kinetics of cell death as measured by a lactate dehydrogenase assay in MLE cells incubated with vehicle or PA SN. (D) Live-cell imaging showing cellular blebbing and annexin V staining (green) followed by propidium iodide staining (red). Scale bars represent 10 μm. (E) Fold change in MLE cytotoxicity 16 hours following the addition of 10% PA SN, where cells were pretreated with vehicle (dimethyl sulfoxide), pan-caspase inhibitor (Z-VAD-fmk), or RIP1-inhibitor (Nec-1s). Each point represents the median cytotoxicity of 3 to 4 technical replicates from a single trial relative to PA SN and vehicle treatment, which is set at 1.0. (F) Flow cytometry of MLE cells positive for a fluorogenic caspase-3/7 substrate 6 hours after the addition of 10% PA SN. (G) Cleaved caspase-3, total caspase-3, and β-actin protein expression in MLE cells after the addition of 10% PA SN at the indicated time points. Staurosporine served as a positive control for caspase cleavage in panels F and G. (H) Fold change in MLE cytotoxicity after the addition of PA SN or equal parts PA SN denatured for 30 minutes at 100°C. (I) Fold change in MLE cytotoxicity after the addition of 50% PA SN from the PA14 parent strain, PA14ΔxcpQ, or PA14ΔexoTUY. Each point represents median cytotoxicity from a single trial as described above. Comparison by Kruskal-Wallis test with Dunn’s test for multiple comparisons (E; the line represents post hoc analysis) or Mann-Whitney U test (I). *P < .05.

  • Figure 6.

    Genetic deletion of PA T2SS (ΔxcpQ), but not T3SS exotoxins (ΔexoTUY), attenuates alveolar–capillary barrier disruption in thrombocytopenic Mpl−/−mice. Gross appearance of BAL fluid (A), BAL OD540 measurements (B), BAL protein concentrations (mg/mL) (C), BAL IgM (ng/mL) (D), BAL neutrophil counts/mL (E), or lung MPO activity (U/left lung) (F) were obtained in Mpl−/− mice 20 hours following IT administration of vehicle or cell-free SN from PA14 or PA14ΔxcpQ strain (n = 5 mice in vehicle group, n = 8 mice in PA14 and PA14ΔxcpQ groups). In separate experiments, gross appearance of BAL fluid (G), BAL OD540 measurements (H), and BAL protein concentrations (mg/mL) (I) were obtained in Mpl−/− mice 20 hours following IT administration of vehicle or cell-free SN from PA14 or PA14ΔexoTUY (n = 4 mice in vehicle group, n = 8 mice in PA14 and PA14ΔexoTUY groups). Each tube or point represents an individual mouse, and the group median is displayed. Statistical comparison by Kruskal-Wallis test with Dunn’s test for multiple comparisons (lines represent post hoc analysis). *P < .05, **P < .01, and ***P < .001.

  • Figure 7.

    PR attenuate PA SN-triggered lung cell death and limits PA SN-mediated lung injury in thrombocytopenic Mpl−/−mice. (A) WT mice BAL platelet counts (109/L) at baseline (0 hours, n = 5) and 20 hours after IT 106 CFU PA (n = 8). (B) BAL thrombin–antithrombin complexes (nM) in WT and Mpl−/− mice at 0 and 4 hours after IT PA SN (n = 4 each group). In separate experiments using MLE cells, fold change in MLE cytotoxicity 6 hours after PA SN in the presence or absence of pooled thrombin-stimulated PR from WT mice (C) (n = 4 trials, where 3 of the 4 trials included 10 µg group) or denatured PR (D). Cleaved caspase-3, total caspase-3, and β-actin expression in MLE cells treated with PA SN in the presence or absence of increasing PR (100 and 300 µg total protein) at 4 hours (E), fold change in MLE cytotoxicity 6 hours after PA SN in the presence or absence of apyrase (0.2 U) or PR from WT mice (F), or thrombin-stimulated platelets pooled from healthy volunteers (G) (n = 4). For panels C-D and F-G, each point represents the median cytotoxicity of 3 to 4 technical replicates from a single trial, relative to PA SN and vehicle treatment cytotoxicity, which is set at 1.0. In separate experiments with Mpl−/− mice 20 hours after IT PA SN with either vehicle or 100 µg PR, lung tissue expression of cleaved PARP and β-actin is shown, n = 3 in each group (H). Fold change in BAL protein concentration. n = 14 (vehicle) and n = 17 (PR-treated group) from 3 separate experiments (I). Each point represents an individual mouse, and the group median is displayed. Statistical comparison by Mann-Whitney U test (*P < .05 and ***P < .001) (A-B,I) or Kruskal-Wallis test with Dunn’s test for multiple comparisons (lines represent post hoc analysis; *P < .05 (C-D,F).