Fig 2: IR injury induced progressive changes in innate immune cell populations within the retina. A Representative scatter-graphs showing the flow-cytometric analysis used to quantify immune cell populations in the retina. After gating for single cells, events were gated into CD11b+/CD45low (principally microglia), CD11b+/CD45hi myeloid leukocytes, and CD11bneg/CD45hi lymphocytes. Myeloid leukocytes were further gated into CD11b+/CD45hi/Ly6Chi/Ly6Gneg classical monocytes, CD11b+/CD45hi/Ly6Cneg/Ly6Gneg non-classical monocytes or MDM, and CD11b+/CD45hi/Ly6C+/Ly6G+ granulocytes. Microglia-like cells were further gated to quantify CD11b+/CD45low/Ly6Cneg/Ly6Gneg microglia. B–E At the indicated times following IR injury, flow-cytometric analysis was used to quantify microglia and leukocyte populations in Sham and IR-injured retinas, including: granulocytes (B), classical monocytes (C), non-classical monocytes/MDM (D), and microglia (E). For each analysis, 4 retinas were pooled and analyzed with n = 4 pools of retinas for each group at 1 day, 4 days, 1 week, and 4 weeks following IR injury. *p = 0.05, **p = 0.01, and ***p = 0.001 by one-way ANOVA with Bonferroni and Sidak multiple comparison test. F Representative images obtained by confocal microscopy of the superficial retinal vasculature of at the optic nerve head and periphery of Sham and IR retinas at 24 h after injury. Images show IB4 (green, note: binds to endothelium, with arterial > venus, and to some leukocytes), ColIV (red), and CD45 (magenta). Note that CD45+ leukocytes appear both within vessel lumen and within the retina. G Confocal microscopy images showing IB4 (green), ZO-1 (red), and CD45 (magenta) of a superficial vascular region of an IR-injured retina at 24 h after injury (Sham not shown). Arrows indicate regions where the vessel is exhibiting disorganization of endothelial TJ complexes coinciding with apparent extravasation of CD45+ leukocytes

Fig 3: Virulent Yersinia becomes partially embedded within the macrophage plasma membrane.(A) RAW macrophages were infected with YPIII/pYV for 100 minutes and analyzed by scanning electron microscopy (SEM). Shown is a representative image depicting a single macrophage infected with several YPIII/pYV bacteria. Shown in the enlargements are bacteria partially enveloped within host membrane. The bacterium in panel b is indicated by an arrow. Shown below is a false coloring rendering highlighting the demarcation of the host cell (blue) and YPIII/pYV bacteria (red). (B) Macrophages were infected with YPIII/pYV for 100 minutes and then stained for the plasma membrane marker (CD11b), the early endosomal marker (EEA1), and a lysosomal marker (LAMP1) and analyzed by fluorescent microscopy. The arrow in the enlarged image of the CD11b-stained cells indicates a bacterium closely associated with host cell membrane; 62% of macrophage-associated bacteria (N = 56) were similarly observed. Shown in the right panels of the EEA1- and LAMP1-stained cells is the percentage of bacteria-containing endosomes that stained for either EEA1 or LAMP1 (N = 111 and 52, respectively). The arrow in the enlarged image indicates a bacterium distinct from an EEA1-staining compartment. The data shown is representative of 3 independent experiments with similar results using both RAW macrophages and murine peritoneal macrophages.

Fig 4: Conventional anti-Mac-1 therapy, but not a ligand-specific inhibition by anti-M7, is a potent inducer of integrin outside-in signaling and aberrant cytokine secretion. a, b Mouse macrophages were isolated from the peritoneal cavity of C57Bl/6 mice 72 h after injection of 4% thioglycollate. Peritoneal cells were collected by lavage and the purity was confirmed by flow cytometry (>90% F4/80+ macrophages). Cells were cultured in 5%FCS/RPMI overnight and co-incubated with 10 µg/ml of mouse IgG, anti-human Mac-1 (clone 2LPM19c), anti-mouse Mac-1 (clone M1/70), or anti-M7 for 30 min in the presence of an anti-mouse CD16/CD32 Fc-block. Cells were lysed and total and phosphorylated ERK1/2 and p38 were visualized by western blot and the ratio of phosphorylated fractions was calculated. Values were calculated as relative arbitrary units (AU) normalized to signal of cells stimulated with saline alone. c Saline, or IgG, anti-M7, anti-Mac-1 (clone M1/70) antibodies were injected i.p. in mice and plasma concentrations of IL-6, TNFa, and MCP-1 were measured by cytometric bead array 4 h after injection. d Human macrophages were differentiated from peripheral monocytes and co-incubated with the indicated antibody clones including the anti-human Mac-1 clone 2LPM19c in the presence of an anti-human CD16/CD32 Fc-block for 5 h. Cytokines were quantified by intracellular flow cytometry in CD68+ macrophages. e Human neutrophils were enriched by density centrifugation of peripheral blood. f–i The anti-ß2 antibodies Mab24 (detecting the ß2 high-affinity conformation) and KIM127 (for ß2-extension) and anti-M7 or IgG (10 µg/ml) were added during acquisition in flow cytometry. The mean fluorescence intensity (MFI) for Mab24 (f, g) and KIM127 (h, i) was continuously recorded over 10 min within the neutrophil gate (e) and quantified as % of the starting MFI (g, i). Error bars indicate mean ± SEM. Statistical significance was assessed by a two-sided, unpaired Student’s T-test between the indicated groups. Data are the result of N = 3 independent experiments (a, b). N = 9 mice per group (c) N = 6 human donors (d). N = 3 human donors (f–i). A representative FACS plot is shown in e

Fig 5: Osteosarcoma cell-derived extracellular-vesicles (EVs) promote accumulation of bone marrow-derived cells in the lungs. (A) Schematic illustration of the experimental setup. Mice were treated with either PBS, 143-B EVs or SAOS-2 EVs (10 µg) 2 times a week for 3 weeks after which mice were euthanized. (B) Relative mRNA expression levels of integrin alpha M gene encoding CD11b (Itgam) in bulk lung RNA in mice of indicated treatment groups. Transcript levels are normalized to Gapdh and expressed as fold change relative to control mice. n = 6, mice were pooled from two independent experiments indicated in red and black (C) Representative flow cytometric profile (left panel) and quantification (right panel) of CD11b+ cells isolated from lungs of mice from indicated treatment groups; n = 3 mice per group. (D) Representative immunofluorescence images (left panel) and quantification (right panel) of CD11b+ cells (red) and nuclei (blue) in lungs of mice from indicated treatment groups. Scale bar, 500 µm. (*, p < 0.05, **, p < 0.01, ***, p = 0.0001, ****, p < 0.0001, Bonferroni’s one-way ANOVA test).