Figure 4. Increased circulating monocytes in severe IAV infection is matched by high levels of monocyte-derived macrophages in lungs but reduced resident alveolar macrophages.

(A) Appearance of lungs from day 4 of severe IAV infection (PR8) showing hemorrhagic areas. (B–G) Gating of monocytes and neutrophils in blood of mild and severe murine models: classical Ly6G⁺ neutrophils (i) and CCR2⁺Ly6C^hiLy6G^– monocytes (ii) (equivalent to human CD14^hi monocytes) on FACS plot of red cell–lysed blood on day 3 of mild (X-179A) and severe (PR8) IAV infection. Alveolar resident macrophages are CD11c⁺SiglecF⁺ and monocytes or MDMs in BAL are CCR2⁺. Populations of cells in BAL on day 3 after infection: Ly6C^midF4/80^– neutrophils (i), Ly6C^hiF4/80^mid differentiating monocytes/monocyte-derived macrophages (MDM) (ii), and Ly6C^– F4/80^hi resident alveolar macrophages (iii). Positive expression was defined against FMO samples to accommodate autofluorescence. (H and I). Alveolar macrophages (iii) express M2 markers, while monocytes/MDMs (ii) express M1 markers and are low in M2 expression. (J–L) Absolute numbers of monocytes and neutrophils in blood and monocytes/MDMs and neutrophils in BAL and lung digests on day 3 (D3) after infection with X-179A (mild) or PR8 (severe) and uninfected mice. Findings are from 2 experiments; n = 6 mice in total. Statistical comparison between monocytes and neutrophils in PR8 infection in J–L was performed separately and showed higher levels of monocytes/MDMs in BAL and lung digests compared with neutrophils (Mann-Whitney). (M and N) Number of Siglec F⁺ alveolar resident macrophages on day 3 in BAL and lung digests of mice. Statistical significance measured using 1-way ANOVA with Tukey test. Horizontal lines and error bars represent mean ± SEM for normally distributed sets and median ± interquartile range for nonnormal distribution. *P < 0.05; **P < 0.01; ****P < 0.0001. BAL, bronchoalveolar lavage; FMO, fluorescence minus one controls; AM, alveolar macrophages.