Fig. 7: CD5L promotes neutrophil phagocytosis.

a, b In vivo analysis of phagocytosis of pHrodo^TM Red E. coli BioParticles^TM. WT mice were injected IP with 15 μl BioParticles and 50 μg of rCD5L, or PBS (0 μg). Peritoneal cells were recovered 3 h later and analyzed by flow cytometry. a Percentage of large peritoneal macrophages (LPMs, CD45⁺CD11b^+/highF4/80^high) or neutrophils (CD45⁺CD11b⁺Ly6G⁺) that internalized BioParticles. Mice per group: 6. b MFI (geometric mean) of pHrodo channel within the pHrodo-positive LPM or neutrophil populations. Statistical comparisons between groups determined by two-tailed unpaired t-tests with Welch’s correction. Mice per group: 6. c, d Thioglycolate-elicited macrophages collected from mouse peritonea 72 h post-broth injection were infected with cecal bacteria pre-coated with 0, 2 or 10 μg of rCD5L. c After coating, a sample of cecal bacteria was boiled and subjected to SDS-PAGE. Detection of rCD5L bound to bacteria was performed via western blotting using primary anti-His tag mAb. d Gentamycin protection assay for phagocytosis analysis after 1-h incubation with pre-coated cecal bacteria at an MOI of 5 and enumeration of CFUs determined by serial dilution plating. Mice per group: 6. a–d Statistical comparisons between groups established by unpaired t-tests with Welch’s correction. Pooled data from two independent experiments. Floating bars show minimum, average, and maximum values within each group. e, f Resident LPMs and thioglycolate-elicited neutrophils were collected from WT mouse peritonea 6 h post-broth injection and incubated with BioParticles pre-coated with 0 or 10 μg rCD5L for the indicated times before phagocytosis analysis by flow cytometry. e Percentage of LPMs (upper left panel) and neutrophils (lower left panel) that internalized BioParticles, and MFI values of pHrodo channel within the pHrodo-positive LPM (upper right panel) and neutrophil (lower right panel) populations. Statistical differences between groups analyzed by two-way ANOVA with Šídák’s multiple comparisons test. Data shown are mean with SEM of n = 3 samples/group from one representative of two independent experiments. f Histogram overlays of representative examples of pHrodo channel fluorescence of LPMs and neutrophils incubated for the indicated times with uncoated (0) or rCD5L-coated (10) BioParticles.