Fig 2: Identification and quantitative analysis of neutrophils in the airways and the lung vessels. (A) The representative three-dimensional image of the lung of mouse that received i.p. RBD injections and the o.ph. application of 100 nm particles in the RBD solution. The large vessels (CD106, magenta), the airways (Streptavidin, grayscale), and 100 nm particles agglomerates (FluoSpheres, red) are represented via volume rendering. Scale bar 1000 µm. (B) The higher magnification of the region boxed in (A) demonstrating the vessel (magenta) and CD11b+ cells (light blue) as the frontal (left image) and the lateral (right image) projections. Scale bar 100 µm. (C) The image of the region boxed in (B) showing the vessel and CD11b+ cells via surface rendering. Scale bar 40 µm. (D,E) Representative three-dimensional images of the regions of conducting airway mucosa of the mouse with induced RBD-specific antibodies (D) and of the PBS-injected mouse (E) 24 h after the application of 100 nm particles in the RBD solution. 100 nm particles (FluoSpheres, red), epithelial and smooth muscle cells (phalloidin, grayscale), neutrophils (Ly6G, light blue) and CD11c+ cells (green) are represented as the frontal (upper images) and the lateral (lower images) projections. Scale bar 30 µm. (F,G) The quantitative analysis of neutrophils in the airway mucosa (F) or BAL (G) of mice that were injected with RBD (gray bars, black circles) or with PBS (open bars, black circles) 24 h after the application of 100 nm particles in the RBD solution, and in intact mice (NM, open bars, open circles). (H,I) The levels of IgA (H) and IgG (I) in the BAL fluids of mice that were injected with RBD (gray bars, black circles) or with PBS (open bars, black circles) 24 h after the application of 100 nm particles in RBD solution, and in the intact mice (open bars, open circles). The data are shown as medians and i.q.r. for n = 4 mice per group. A significant difference between the indicated group and the intact mice was detected using the Mann–Whitney test *: p = 0.05.

Fig 3: RXRa deficiency impacts embryonic development of RTMs.(A) RNA-seq dataset from Mass et al. (3) were analyzed for the expression of Rxra and Vav1. Heatmaps show normalized gene expression (log2) of transcripts for Rxra (average of ENSMUST00000077257, ENSMUST00000100251, ENSMUST00000166775) and Vav1 (average of ENSMUST00000169220, ENSMUST00000005889, ENSMUST00000112870). (B and C) Flow cytometry analyses of E9.5 yolk sac (YS) from Rxrafl/fl (wild-type [WT]) and VavCre+Rxrafl/fl (knockout [KO]) embryos. (B) Representative dot plots and graphs show frequency among CD45+ cells of EMPs (CD45+c-Kit+) and macrophages (CD45+F4/80+). Phenotypes of c-Kit+ and F4/80+ cells are detailed further in Supplemental Fig. 3. (C) Representative dot plots and graphs show frequency among CD45+ cells of precursor macrophages (pMacs) (CD45+CD11blo/+F4/80-) and macrophages (Macs) (CD45+CD11b+F4/80+). (D) Flow cytometry analyses of E14.5 fetal liver (FL) from Rxrafl/fl (WT) and VavCre+Rxrafl/fl (KO) fetuses. Representative flow cytometry dot plots and graphs show frequency among CD45+ cells of E14.5 FL monocytes (CD45+CD11b+F4/80lo Mos) and macrophages (CD45+CD11bloF4/80hi Macs). Data in (B)–(D) are from two independent experiments. (E) Frequency and cell numbers among CD45+ cells of postnatal day 1 (D1) and 8 (D8) spleen (F4/80+VCAM1+), liver (CD11bloF4/80hi), lung (CD11c+Siglec-F+) and skin (CD11b+F4/80+) RTM subsets. Data in (E) are representative of three independent experiments. Error bars are SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: HSCs and LSECs Induce Monocyte Recruitment and Engraftment in the Liver after Kupffer Depletion(A) Principal component analysis of HSCs, LSECs, and hepatocytes at steady state, 12 h or 36 h after DT injection.(B) Heatmap of genes involved in granulocyte and agranulocyte adhesion and diapedesis upregulated by HSCs and/or LSECs after KC depletion according to ingenuity pathway analysis.(C) Analysis by ELISA of CCL2 concentration in the serum of mice after DT injection. Control mice consist of a mix of C57BL/6 DT-injected and Clec4fDTR/+ PBS-injected mice (no differences observed). Pooled data are from 3 experiments. Control mice n = 5 (4h), 6 (10h, 12h, 16h), 9 (8h), and 10 mice (0h). KC-depleted mice n = 5 (0h); 11 (4h, 16h); 12 (12h); 15 (10h) and 16 mice (8h). Two-way ANOVA with Tukey post-test. *p < 0.05; ****p < 0.0001.(D) On the left is the MIP of Clec4fDTR/+-Ccr2GFP/+ mouse liver sections stained for CCL2 (white), Desmin (green), GFP (orange), F4/80 (red), and CD31 (blue) 8 h after DT injection. CCL2 was mainly co-localized with HSCs (arrows), although a small amount could be found in monocytes (arrowheads) or in LSECs (asterisks). Data are representative of 2 experiments. Scale bar, 20 µm.(E) Quantification of CCL2+ cells at the indicated time points and normalized per mm2 of tissue. Dots represent individual pictures. Pooled data are from 2 experiments; n = 4. Two-way ANOVA with Tukey post-test. ****p < 0.0001.(F) Quantification of VCAM-1 and selectin E expression by HSCs, LSECs, and hepatocytes after DT injection. Given that VCAM-1 was already expressed on HSCs at steady state, data are represented as median fluorescence intensity (MFI) fold increase as compared with PBS control. PBS controls were included in each individual experiment. Selectin E was represented as percentage of positive cells in each cell population. Pooled data are from 2–4 experiments; n = 7 (4h), 8 (10h, 16h, 24h, 48h), 9 (12h), 15 (PBS) and 17 mice (8h). One-way ANOVA with Bonferroni post-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Related to Figure S3 and S4.

Fig 5: The CD106+ population is uniquely localized in pancreatic injury and precancerous lesions Representative IHC staining of tumor core and adjacent region in PDAC patient (stage II) for CD106+. The corresponding magnification insets of the tumor core (red dotted line) or adjacent region (green dotted line) are displayed on the right. Scale bar, 500 µm in gross, 20 µm in magnification.Immunofluorescence staining of the pancreas obtained from a 2.5-month-old KC mouse for CD106 (red), a-SMA (green) or CK19 (green), and DAPI (blue). Scale bar, 50 µm, 10 µm.Gating strategy for the identification of the CD106+CD24+CD44+ population in KC and WT mice. FACS analysis of the percentage of CD106+ among CD45-CD31- cells; each dot represents one mouse. The bars are representing the mean. The P-values were calculated using Student's t-test.Immunofluorescence staining of pancreases obtained from a KC mouse (left) and a 2.5-month-old KPC mouse (right) for CD44 (green), CD24 (red), and DAPI (blue). Scale bar, 50, 10 µm. Source data are available online for this figure.