Figure 4.

HSC myeloid bias induced by B. abortus Omp25/CD150 interaction is hematopoietic cell autonomous. (a) Experimental scheme: BM cells from CD150^−/− CD45.1 mice and wt CD45.2 mice were isolated from mouse tibiae and femurs and transplanted in a 1:1 ratio into lethally irradiated recipient CD45.2 mice. 12 wk after transplantation, wt CD45.2:CD150^−/− CD45.1 chimeric mice were intraperitoneally injected with PBS (Mock, unfilled circle) or inoculated with 1 × 10⁶ CFU of Ba WT, B. abortus ∆omp25 (Ba ∆omp25). Blood and BM were analyzed 8 d later. (b) Overall blood chimerism was obtained 8 and 12 wk after reconstitution and 1 wk after infection with Ba WT (black square) and Ba Δomp25 (gray square). (c–f) Myeloid (CD45⁺, CD11b⁺ cells) to lymphoid (CD45⁺, CD3e⁺; and CD45⁺, CD19⁺) ratio in the blood (from left to right: n = 8, 8, 8, 8, 7, 7); frequency of (d) GMP (from left to right: n = 6, 6, 7, 7, 9, 9), (e) MPP2-3 (from left to right: n = 6, 6, 6, 6, 9, 9), and (f) MPP4 (from left to right: n = 6, 6, 8, 8, 9, 9) in BM lin⁻ cells of wt CD45.2 (circle) and CD150^−/− CD45.1 (square) compartment is shown for chimeric mice intraperitoneal injected with PBS (Mock, unfilled symbols) or inoculated with 1 × 10⁶ CFU of Ba WT (symbol filled in black) or Ba Δomp25 (symbol filled in gray). (g and h) CFU count per gram of organ at day 30 after infection of wt CD45.1 and CD150^−/− CD45.1 mice for (g) BM (from left to right: n = 18, 9, 8, 12, 6, 6) and (h) spleen (from left to right: n = 14, 18, 7, 7, 8, 6) infected with Ba WT (black square), Ba Δomp25 (gray square), or B. abortus Δomp25 complemented with p:Omp25 (Ba Δomp25c; unfilled square). (i) Fold change of HSC absolute number in mice at day 30 after infection (D30 p.i.) with indicated bacteria (from left to right: n = 9, 14, 11, 8, 11, 11). Data were obtained from distinct samples from three independent experiments. (j) Experimental scheme: wt CD45.1 mice were intraperitoneally inoculated with 1 × 10⁶ CFU of Ba WT. BM cells were isolated from femurs and tibiae of the infected mice at day 30 after infection, and lin⁻ cells were sorted and analyzed by FACS and transplanted in a 1:1 ratio with lin⁻ cells from wt CD45.2 non-infected mice into previously lethally irradiated wt CD45.2-recipient mice. FACS analyses on blood were performed at 4, 6, and 8 wk after transplantation. (k) Contribution of CD45.1 cells from infected mice to blood chimerism in CD45.2 recipients at 4, 6, and 8 wk after transplantation (mock, n = 5; Ba WT, n = 7; Ba Δomp25, n = 7). Data were obtained from two independent experiments. Mean ± SEM is represented by a horizontal bar. Significant differences from mock are shown. ***, P < 0.001; **, P < 0.01; *, P < 0.05. Absence of P value or ns, non-significant. Since data did not follow a normal distribution, P values were generated using Kruskal–Wallis followed by Dunn’s test.