PMC

Lung leukocytes were isolated from infected MARCO^+/+ and MARCO^-/- mice. (A) Frequency of moDC (CD11c⁺MHCII⁺CD64⁺) was significantly lower in MARCO^-/- mice than MARCO^+/+ mice. (B) MARCO has no effect on the frequencies of CD11b⁺ cDC (CD11c⁺MHCII⁺CD64^-SIRPα⁺) and CD103⁺ cDC (CD11c⁺MHCII⁺CD64^-XCR1⁺). (C) MARCO^-/- mice had significantly lower total number of moDC but not CD11b⁺cDC and CD103⁺ cDC in the infected lungs relative to WT control. Results represent mean ± SEM (n = 5 mice for each time point). * p<0.05 in comparison between MARCO^+/+ and MARCO^-/- mice.

Lung leukocytes isolated from infected MARCO^+/+ and MARCO^-/- mice were analyzed using flow cytometry as per Material and Methods. Frequency and total number of moDC that express CD80 (A and B) and CD206 (C and D) at 7 dpi were assessed. Note the total number of moDC expressing CD80 and the frequency of moDC expressing CD206 were affected by MARCO expression. Gene expression of iNOS (E) and Arg1 (F) by pulmonary leukocytes at 4 and 7 dpi was evaluated by RT-qPCR. Note that the expression of iNOS was affected by MARCO deletion, but the expression of Arg1 were similar between MARCO^+/+ and MARCO^-/- groups. Results represent mean ± SEM (n = 5 mice for each time point). * p < 0.05 in comparison between MARCO^+/+ and MARCO^-/- mice.

Expression of VSIG4, MARCO and CD49a on freshly isolated PBMCs and IHLs (A) (left) Representative histogram of VSIG4 expression on gated CD49a⁻ PBMCs (red), CD49a⁻ ihMφ (light blue), and CD49a⁺ ihMφ (dark blue). (right) Scatter plot summarizing VSIG4 MdFI on CD49a⁻ PBMCs (red), CD49a⁻ ihMφ (light blue) and CD49a⁺ ihMφ (dark blue) (n = 5). (B) (left) Representative histogram of MARCO expression on gated CD49a⁻ PBMCs (red), CD49a⁻ ihMφ (light blue) and CD49a⁺ ihMφ (dark blue). (right) Scatter plot summarizing MARCO MdFI on CD49a⁻ PBMCs (red), CD49a⁻ ihMφ (light blue) and CD49a⁺ ihMφ (dark blue) (n = 6). (C) (left) Representative histogram of CD49a expression on gated VSIG4⁻ PBMCs (red), VSIG4⁻ ihMφ (light blue) and VSIG4⁺ ihMφ (dark blue). (right) Scatter plot summarizing CD49a MdFI on VSIG4⁻ PBMCs (red), VSIG4⁻ ihMφ (light blue) and VSIG4⁺ ihMφ (dark blue) (n = 6). (D) (left) Representative histogram of MARCO expression on gated VSIG4⁻ PBMCs (red), VSIG4⁻ ihMφ (light blue) and VSIG4⁺ ihMφ (dark blue). (right) Scatter plot summarizing MARCO MdFI on VSIG4⁻ PBMCs (red), VSIG4⁻ ihMφ (light blue) and VSIG4⁺ ihMφ (dark blue) (n = 6). (E) (left) Representative histogram of CD49a expression on gated MARCO⁻ PBMCs (red), MARCO⁻ ihMφ (light blue) and MARCO⁺ ihMφ (dark blue). (right) Scatter plot summarizing CD49a MdFI on MARCO⁻ PBMCs (red), MARCO⁻ ihMφ (light blue) and MARCO⁺ ihMφ (dark blue) (n = 6 for PBMCs and MARCO⁻ IHLs, n = 5 for MARCO⁺ IHLs). (F) (left) Representative histogram of VSIG4 expression on gated MARCO⁻ PBMCs (red), MARCO⁻ ihMφ (light blue) and MARCO⁺ ihMφ (dark blue). (right) Scatter plot summarizing VSIG4 MdFI on MARCO⁻ PBMCs (red), MARCO⁻ ihMφ (light blue) and MARCO⁺ ihMφ (dark blue) (n = 5 for PBMCs and MARCO⁻ IHLs, n = 4 for MARCO⁺ IHLs). One sample was excluded due to the low proportion of MARCO⁺ cells. Median is depicted and error bars indicate the min-max range, p-value determined by Mann-Whitney test (PBMCs vs. IHLs) and Wilcoxon matched-pairs signed rank test when comparing IHL populations.

Actin binding mediated in part by MARCO scavenger receptor. Ex vivo wild-type (WT) and MARCO^−/− BALF AMs were collected from lung lavage and preincubated (37°C, 40 min) with or without DS (100 μg/ml). AMs were washed and incubated in 20 μg/ml A647-actin, 20 μg/ml A647-BSA, or vehicle alone (4°C, 30 min) and washed in preparation for flow cytometry. A: mean fluorescence (FL) index (%parent × A647-fluorescence) observed via flow cytometry for WT, MARCO^−/− (± DS) with A647-actin. B: mean FL index for WT, MARCO^−/− (± DS) with A647-BSA. C: histograms for graph in A: WT, MARCO^−/− (± DS) with A647-actin. Dark gray, WT + GAB; light gray, MARCO^−/− + GAB; magenta, WT + actin, purple, WT + DS + actin; green, MARCO^−/− + actin; red, MARCO^−/− + DS + actin. D: histograms for graph in B: WT, MARCO^−/− (± DS) with A647-BSA. Light gray, WT + BSA; green, WT + DS + BSA; purple, MARCO^−/− + BSA; red, MARCO^−/− + DS + BSA; magenta, WT + actin. All histograms represent A647-fluorescence intensity counts of 10,000 events/sample (n = 3); *P < 0.0001. In A and B, ○, ●, ■, ∇, ▼, and ◆ correspond to samples collected from groups defined in the x-axes.