Fig 2: Inhibitory effect of Abs against scavenger receptors for AGE uptake RAW264.7 cells seeded at 2 × 105 per well were pre-incubated with or without neutralizing Abs against scavenger receptors at 20 or 10 µg/ml for 1 h followed by culturing with BSA, AGE-2, or AGE-3 at 200 µg/ml for 1 h. (a) Neutralizing effect of anti-CD204 Ab on the uptake of fluorescent AGE-2, AGE-3, and BSA was determined by flow cytometry (n = 3). Goat IgG was used as an isotype-matched control of anti-CD204 Ab. (b) Neutralizing effect of each Ab against RAGE, LOX-1, CD36, CD163, or CD206 on the uptake of fluorescent BSA, AGE-2, or AGE-3 was determined by flow cytometry (n = 3–10). (c) Effect of anti-CD204 Ab on the expression of RAGE, LOX-1, CD36, CD163, and CD206 was determined by flow cytometry (n = 4–5). Each column represents the MFIs of the respective surface membrane proteins relative to the medium only group (Unt, Untreated) after 1 h incubation, which was arbitrarily defined as 100%. Data are presented as the means ± SEM and were analysed using one-way ANOVA followed by Tukey’s test. ***p < 0.001, **p < 0.01 compared with the value for Unt. ###p < 0.001, ##p < 0.01 compared with the value for AGE-2 alone. †††p < 0.001 compared with the value for AGE-3 alone.

Fig 3: Alveolar macrophages exhibit a dysregulated phenotype with LOX-1 inhibition.(A) Age-matched C57BL/6 mice were intratracheally treated with 10 µg anti–LOX-1 IgG or control IgG and E. coli for 6–36 hours (n = 6–8 per group). FACS-sorted alveolar macrophages were subsequently analyzed by flow cytometry for (B) levels of reactive oxygen species (ROS) at 6 hours, (C) IL-6 and CXCL2 expression at 24 hours, or (D) CD206 expression at 18 hours after anti–LOX-1 or IgG treatment and infection. Intracellular flow cytometry was performed on fixed cells at 18–36 hours after anti–LOX-1 or control IgG treatment and E. coli infection to determine (E) Arginase 1, iNOS, MHCII, and CD11b expression (MFI) and (F) total alveolar macrophage numbers. Data are represented as mean ± SEM with individual data points representative of mice from 2–3 independent experiments. Statistical analysis was performed on normalized (log-transformed) IL-6 and CXCL2 gene expression (fold change). ***P < 0.001, *P < 0.05 for 1-way ANOVA with Holm-Šídák post hoc test (B); 2-tailed Welch’s t test (C); 2-tailed, unpaired t test (D); or 2-way ANOVA with Holm-Šídák post hoc test (D and E).

Fig 4: Airspace neutrophils are a significant source of lung LOX-1 during pneumonia.(A and B) Age-matched male and female C57BL/6 mice (n = 10–11) were intraperitoneally treated twice with 500 µg anti-Ly6G to deplete neutrophils and intratracheally infected with E. coli for 24 hours. (A) BALF neutrophil numbers and (B) lung homogenate LOX-1 concentrations were subsequently measured. (C) Gene expression of Olr1 and (D) surface protein expression of LOX-1 was measured in sorted blood and airspace neutrophils (CD45+CD11b+Ly6G+) at 24 hours after intratracheal instillation with E. coli. Data are represented as mean ± SEM with individual data points representative of mice from 1–2 independent experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001 for 2-tailed, unpaired t test (A and B) or ratio paired t test (C and D).

Fig 5: Hematopoietic cells are required for lung LOX-1 accumulation.(A) Bone marrow chimeras were generated from age-matched C57BL/6 (CD45.1) and LOX-1–/– (CD45.2) mice and infected with E. coli for 24 hours at 2 weeks or 10 weeks after bone marrow engraftment (n = 5–17). Engraftment efficiency was measured in LOX-1–/– mice at (A) 2 weeks and (D) 10 weeks posttransplant by measuring CD45.1 expression by flow cytometry on neutrophils and alveolar macrophages in BALF. (B and E) Soluble LOX-1 and (C and F) total protein (as a measure of injury) were determined in BALF following bone marrow transplant and intratracheal infection with E. coli. Data are represented as mean ± SEM with individual data points representative of mice from 2 independent experiments. ****P < 0.0001, *P < 0.05 for 2-tailed, unpaired t test (B and C) or 2-tailed Welch’s t test (D).