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. 2023 Apr 17;220(7):e20210567. doi: 10.1084/jem.20210567

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© 2023 Hysenaj et al.

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Figure 2. — B. abortus induces PU.1 upregulation in an Omp25/CD150-dependent manner. (a) Experimental scheme: Pu.1^+/GFP and CD150^−/− Pu.1^+/GFP mice were intraperitoneally injected with PBS (Mock, unfilled circle) or inoculated with 1 × 10⁶ CFU of Ba WT (black square), Ba ∆omp25 (gray square). 2 d later, the percentage of GFP expression in HSC (lin⁻, Sca⁺, cKit⁺, CD48⁻, CD135⁻ CD34⁻) was assessed by flow cytometry. (b) GFP expression in BM HSC (lin⁻, Sca⁺, cKit⁺, CD48⁻, CD135⁻, CD34⁻) assessed by flow cytometry at day 2 after infection. Data (b) were obtained from distinct samples (from left to right, n = 5, 10, 5, 5, 6) from three independent experiments. Mean ± SD is represented by a horizontal bar. Significant differences from mock are shown. ***, P < 0.001; * 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. (c) Experimental scheme: HSC (lin⁻, Sca⁺, cKit⁺, CD48⁻, CD135⁻, CD34⁻) from Pu.1^+/GFP or CD150^−/− Pu.1^+/GFP mice were sorted, then stimulated ex vivo with PBS or B. abortus WT OMVs, B. abortus ∆omp25 OMVs. After 16 h, the level of GFP in cells was assessed by flow cytometry. (d) Percentage of GFP + HSC 16 h after ex vivo stimulation with B. abortus WT OMV (black square), B. abortus ∆omp25 OMV (gray square) compared to mock (Mock, unfilled circle) assessed by flow cytometry (from left to right: n = 6, 6, 6, 5, 5). (e) Percentage of GFP⁺ HSC assessed by flow cytometry 0 h (Mock, unfilled circle, n = 4) or 16 h after ex vivo stimulation with B. abortus WT (black square, n = 4), B. abortus WT OMV and the control peptide (100 µg/ml; black square, n = 2), and blocking peptide of CD150 (100 µg/ml; gray square, n = 2). Data (d and e) were obtained from HSC of a pool of three to four mice, and the pool of cells has been divided by the number of tested conditions. Each dot is a replicative experiment. Mean ± SD is represented by a horizontal bar. Significant differences from mock are shown. **, P < 0.01; *, P < 0.05. P values were generated using Mann–Whitney test.