Fig 2: A, Absolute number of AMs in bronchoalveolar lavage fluid (BALF). B, Representative flow cytometry plots of the gating strategy, (upper panel), live CD45+ and Gr1- were then analyzed for CD11b and F4/80 expression (lower panel). Gates were set based on background staining in fluorescence minus one controls. C, Data from three independent experiments are summarized as mean ± SEM and plotted in the bar graph (control, n = 5; vehicle, n = 8; clodronate IV n = 8; clodronate IT, n = 7). *P < .05, **P < .01, ****P < .001 (one-way ANOVA, Tukey's multiple comparisons). D, Representative histological sections of the lungs showing Ki67 positive cellular staining. Scale bar = 100 µm, insert 20 µm. E, Percentage of Ki67+ cells. Ten regions of interest of 1 mm2 per mouse were analyzed with a minimum of 100.000 cells counted per condition. Values represent mean ± SEM; n = 4 mice per group. AM, alveolar macrophage; ANOVA, analysis of variance; IT, intratracheal; IV, intravenously

Fig 3: Representative flow cytometry analysis of HoxB8 MPP differentiation into CDP and DC subsets.(A) HoxB8 MPP are cultured in HoxB8 growth medium without E2 for 3 days and analyzed by flow cytometry (section 1.1.2.3.3, step 5). MPP: Gr1- CD117+ CD135-; CDP: Gr1- CD117int/low CD135+ CD115+. (B) HoxB8 MPP are cultured in HoxB8 growth medium without E2 for 8 days and analyzed by flow cytometry (see below section 1.1.2.3.5.2, step 7). cDC1: Gr1- CD11c+ CD11blow/- XCR1+; cDC2: Gr1- CD11c+ CD11b+ XCR1-; pDC: Gr1- CD11c+ CD11b- B220+.

Fig 4: IR-61 preferentially accumulates in macrophages at infection sites and promotes bacterial clearance. (a)E. coli or PBS was intratracheally administered into the lungs of mice, and then major organs were imaged using a NIR imaging system after i.v. injection of IR-61 at 12, 24, and 120 h (n = 3 mice per group). (b) Staining indicating the IR-61 levels in macrophages (CD11b+F4/80+), neutrophils (CD11b+Gr1+), monocytes (CD11b+Gr1low), dendritic cells (DCs) (CD11c+Gr1low), B cells (CD19+), and T cells (CD3+) isolated from normal lungs and infected lungs (unstained: n = 3 mice; non-infection: n = 5 mice; infection: n = 5 mice). (c) The amount of internalized IR-61 in various immune cells was measured by MFI (infection group: Macrophages vs monocytes p < 0.0001, Macrophages vs DCs p < 0.0001, Macrophages vs neutrophils p < 0.0001, Macrophages vs T cells p < 0.0001, Macrophages vs B cells p < 0.0001; unstained: n = 3 mice; non-infection: n = 5 mice; infection: n = 5 mice). (d) The levels of Tnf, IL-6, IL-1ß, and IL-10 in BALF of mice infected with E. coli at 24 h after the treatment with vehicle or IR-61 (Vehicle vs IR-61 p = 0.0017 (Tnf), p = 0.0049 (IL-6), p < 0.0001 (IL-1ß), p = 0.0053 (IL-10); n = 5 mice per group). (e) Representative images of H&E-stained lungs of E. coli-infected mice 24 h after the treatment with vehicle or IR-61 (bars represent 250 µm; n = 5 mice per group). (f) CFUs of bacteria in BALF of mice infected with E. coli at 24 h after the treatment with vehicle or IR-61 (Vehicle vs IR-61 p = 0.0009; n = 5 mice per group). Results are presented as the mean ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001; one-way ANOVA with Bonferroni post-hoc test (graphs in panel c); two-sided Student's t-test (graphs in panels d and f)).

Fig 5: Representative flow cytometry analysis of HoxB8 MPP. HoxB8 MPP were cultured in basic HoxB8 culture medium and subjected to flow cytometry analysis with specific antibodies. MPP: Gr1- CD117+ CD135-; CDP: Gr1- CD117int/low CD135+ CD115+.