Fig 2: Aging exacerbates A59 (mCoV) infection.(A) Schematic of A59 (mCoV) infection experiment with young (2–6 months) and old mice (20–24 months). (B) Probability of survival of young (n = 6) and old (n = 6) infected mice. Survival of mice was examined after infection with high dose of virus (PFU 7e3) up to 14 days. (C–E) Young (n = 8) and old mice (n = 8) were infected with low dose of virus (PFU 7e2), and weight change (%) (C), O2 saturation (D), and daily food intake (E) were recorded. (F) Plaque assay of lung from uninfected, infected young and old mice. (G–L) Flow cytometry analysis of CD4 T cell (G), CD4/CD8 T cell ratio (H), ?d T cell (I), neutrophil (J), Ly6Chi cell (K), and CD64+ MerTK+ cell (L) on day 8 post (PFU 7e2) infection. Error bars represent the mean ± S.E.M. Two-tailed unpaired t-tests were performed for statistical analysis. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig 3: Muscle leukocyte analysis in spironolactone- and prednisolone-treated mdx quadriceps.(A–F) Representative flow cytometry gating dot plots displaying CD45+CD11b+ myeloid cells (A), CD45+CD11b+LY6G+ neutrophils (B), CD45+CD11b+LY6G–CD64–LY6Chi infiltrating monocytes (C), CD45+CD11b+LY6G–CD64+ macrophages (D), CD45+CD11b+LY6G–CD64+CD206+ macrophages (E), and CD45+CD11b+LY6G–CD64+F4/80hi macrophages (F) from spironolactone-treated (SPR-treated) (n = 13) and prednisolone-treated (PRD-treated) (n = 13) 4.5-week-old mdx quadriceps compared with vehicle (VEH) (n = 12) controls. (G) Quantification of myeloid cells, neutrophils (Nf), infiltrating monocytes (Inf. MO), macrophages (MF), CD206+ macrophages (CD206+), and F4/80hi macrophages represented as dot plots for cells per milligram of muscle (Cells/mg). (H) Bar graphs with individual data points as percentages of total CD45+ leukocytes (%CD45+), CD45+ CD11b+ myeloid cells (%CD45+CD11b+) or macrophages (%CD45+CD11b+LY6G–CD64+) comparing SPR- and PRD-treated mice with VEH controls. Experimental replicates are denoted by black and gray dots within the graphs. Statistics used were ANOVA with the BKY test *P = 0.05.

Fig 4: CLEC5A is critical for P. aeruginosa–induced cell infiltration and inflammation in the lung.WT and Clec5a–/– mice were intratracheally inoculated with median lethal dose (LD50) of P. aeruginosa (Xen41, PAO1 strain); WT mice were injected with DNase I (4 U/mouse) via i.p. route for comparison. (A) Lung tissue was collected at 24 hours after infection for H&E staining. Scale bar: 200 µm. (B) Lung tissue was harvested at 8 hours after infection for measurement of cytokine and chemokine mRNA levels. Data are mean ± SEM (mock: WT, n = 6; WT + DNase 1, n = 5; Clec5a–/–, n = 5; Xen41LD50: WT, n = 4; WT + DNase 1, n = 3; Clec5a–/–, n = 5). (C) BALF was collected at 15 hours after infection, and cytokines and chemokines were quantified by ELISA. Data are mean ± SEM (mock: WT, n = 3; WT + DNase 1, n = 3; Clec5a–/–, n = 3; Xen41LD50: WT, n = 5; WT + DNase 1, n = 5; Clec5a–/–, n = 7). (D) Lung cell suspensions from WT and Clec5a–/– mice were stained with PE/Cy7-conjugated anti-CD45 Ab, PerCP/Cy5.5-conjugated anti-CD64 Ab, APC/Cy7-conjugated anti-IA/IE Ab, BV421-conjugated anti–Siglec-F Ab, BV510-conjugated anti–TNF-a Ab, PE-conjugated anti–pro–IL1ß Ab, APC-conjugated anti–IL-6 Ab, PE-conjugated anti-RANTES Ab to measure intracellular TNF-a, IL-6, pro–IL-1ß, and RANTES in alveolar macrophage (AM?: CD45+CD64+IA/IE+Siglec-F+). Cytokine/chemokine MFIs were normalized to mock for WT and Clec5a–/– mice, and the data (fold change of MFI) are presented as mean ± SEM (mock: WT, n = 3; Clec5a–/–, n = 3; Xen41LD50: WT, n = 4; Clec5a–/–, n = 4). Statistical calculations were performed with 2-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5: CLEC5A is a potential therapeutic target in P. aeruginosa–induced cell infiltration and lethality.WT mice were intratracheally injected with a sublethal dose (LD90) of P. aeruginosa Xen41 (1 × 106 PFU) and injected with isotype control (mIgG1, 100 µg per mouse, i.v.), anti-mCLEC5A mAb (clone 4A12D5, 100 µg per mouse, i.v.), ciprofloxacin (Cip, 4 mg/kg, i.p.), or anti-mCLEC5A mAb (i.v.) combined with ciprofloxacin (i.p.) at 15 hours after infection. Ciprofloxacin was further administered every 12 hours. (A) Survival rate was monitored up to 120 hours after infection. (B) Lung tissues were collected, and formalin-fixed paraffin-embedded sections were prepared. Cellular infiltration was visualized by H&E staining. Scale bar: 200 m. (C) Tissue sections were stained with anti-CD64 Ab (green), anti-CD11b Ab (red), anti–Siglec-F Ab (yellow), and anti-F4/80 Ab (light blue); fluorescence images were captured using a Leica confocal microscope with white light laser system (TCS-SP8-MP-SMD) and analyzed using Leica Application Suite X software. Scale bar: 200 µm. (D and E) The cell population was analyzed with MetaMorph software to determine the percentages of interstitial macrophages (interstitial M?, CD64+CD11b+Siglec-F–F4/80+), alveolar macrophages (AM?, CD64+CD11b–Siglec-F+F4/80+), eosinophils (Eos, CD64–CD11b+Siglec-F+F4/80+), and neutrophils/DCs (PMN/DC, CD11b+Siglec-F–F4/80–CD64–) (ciprofloxacin, n = 11; ciprofloxacin + anti-mCLEC5A mAb, n = 10, from 4 independent experiments) (F) Lung cell suspensions were incubated with BV650-conjugated anti-Ly6G Ab, BUV805-conjugated anti-CD45 Ab, BUV395-conjugated anti-CD11b Ab, BV480-conjugated anti-CD11c Ab, BV605-conjugated anti-CD64 Ab, PE-Cy5-conjugated anti-CD25 Ab, and BUV496-conjugated anti-IA/IE Ab to analyze the percentage of neutrophil (PMN), alveolar macrophage (AM?), interstitial macrophage (IM?), and eosinophils after P. aeruginosa inoculation (ciprofloxacin, n = 4; ciprofloxacin + anti-mCLEC5A mAb, n = 4, from 3 independent experiments). Survival rate was assessed using the log-rank (Mantel-Cox) test with GraphPad Prism software. ***P < 0.005. Statistical analysis for D and E was calculated with unpaired and nonparametric Student’s t test with Mann-Whitney U test. *P < 0.05.