Fig 2: Extravascular choroidal mononuclear phagocytes are increased in Cfh+/−∼HFC mice and are ameliorated by anti-C5a therapy. (A) RPE/choroid leukocytes were gated for an intravascularly injected anti-CD45 antibody to distinguish the intra- and extravascular space. Gating for polymorphonuclear leukocytes (PMNs) and three previously established populations of mononuclear phagocyte (MNP) (Ly6Chi [Class Mo], Ly6Clo, and CD64+) was performed.64 (B) Within the extravascular space significant increases were seen in total leukocytes, PMNs, and all the MNP subpopulations (Class Mo, CD64+ MNP, and Ly6Clo MNP). Interestingly, anti-C5a therapy significantly blocked the extravasation of total leukocytes and PMNs, as well as Class Mo and Ly6Clo MNP subpopulations. *χ2 test for P < 0.05. Data presented represent a single experiment using a pooled sample from three mice per group. A separate independent experiment was performed to confirm trends shown above. Class Mo, classical monocyte.

Fig 3: 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.

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: Gating strategy and absolute numbers from FACS.(A) Gating strategy and representative contour plots used for the identification of immune cells using flow cytometry in half brain of 9.5-mo-old PS19 and WT littermate mice. PBMs were sorted as TCRb− CD11b+ Ly6g− CD45high F4/80+ CD64+ CD206+. PBM phenotypes were identified by the expression of MHCII and/or CD38. (B) Absolute numbers of CD206+ cells, and subtypes of CD38+MHCII+CD206+, CD38-MHCII+CD206+, and CD38+MHCII-CD206+ cells. n = 9–10 mice/group. Data are presented as mean ± SEM. Significance was determined using two-tailed Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Males and females are represented with blue dots and orange dots, respectively.