Fig 2: CBM mutation in Sh3bp2 enhances neutrophil-mediated events that contribute to PAMPs-induced jawbone expansion. (A) Increased SH3BP2 expression in sorted Ly6G-positive cells from Sh3bp2 KI/KI bone marrow shown by immunoblotting. (B) Increased percentages of blood neutrophils (Ly6G+CD115-) and monocytes (Ly6G-CD115+) in Sh3bp2 KI/KI mice shown by flow cytometry (n = 5 per group). (C) Detection of MPO (green) and citrullinated H3 (pink) in Sh3bp2 +/+ and Sh3bp2 KI/KI right mandibles 14 days after pulp exposure. Scale bar = 200 µm. Histograms show quantification of MPO, citH3, and colocalization of MPO and citH3 signals normalized to DAPI (blue) from nine periapical and furcation areas in three Sh3bp2 +/+ and Sh3bp2 KI/KI mice. (D) Coronal and axial planes of µCT images of Sh3bp2 KI/KI mice treated with Ly6G antibody or isotype control prior to and after pulp exposure. (E) Quantification of mandible expansion shows significant amelioration after Ly6G antibody treatment in Sh3bp2 KI/KI mice compared to no antibody or isotype control (arrows) antibody. Scale bar = 1 mm. Each dot in graphs represents a single biological sample (n = 3–7 per group). Data are presented as mean ± SD. *p < 0.05, **p < 0.01 indicate significant difference by Student's t test (B), (C) and one-way ANOVA (E).

Fig 3: Migration efficiency of IV transferred monocytes and cMDM to acutely inflamed brain tissue is linked to monocyte-to-macrophage differentiation.(A) MDM cultured from day 0 to day 28 were introduced IV (5×106 cells) into recipient mice bearing LPS-induced acute neuroinflammaiton, and cMDM in vivo brain homing efficiency deceased as the in vitro cultivation time went up. Female gDNA = female mouse brain tissue genomic DNA (negative control). No cell IV = LPS injected control animal that received no cell IV transplant. Final data presented here represents mean values ± SD. Data was analyzed by one-way ANOVA, with resulting p-value <0.05. (B–D) Flow cytometry analysis on cMDM cell populations at different time points post in vitro cultivation. (B) Example of phenotype identification of cMDM using D5 cMDM. The green cluster reflects macrophage phenotype (CD11b+ F4/80hi Ly6Clow to neg), blue cluster reflects Ly6clow monocytes (CD11b+ F4/80low to med Ly6Clow to neg), and yellow cluster reflects Ly6Chi monocytes (CD11b+ F4/80low to med Ly6Chigh). (C) The ratio of macrophages (CD11b+ F4/80high Ly6Cneg) and total monocytes (CD11b+ F4/80low to med Ly6Clow to high), and (C) the ratio of Ly6Chi monocytes (CD11b+ F4/80med Ly6Chigh CCR2+) and Ly6Clo monocytes (CD11b+ F4/80low Ly6Clow CCR2low to neg) in culture were evaluated. (E) Phenotyping of freshly isolated EnMO by flow cytometry analysis. Expression level of CD115, CD11b, F4/80, and Ly6c were measured to determine the purity of the enriched cells. (F) Freshly isolated EnMO showed superior brain homing efficiency over cMDM. 5x106 of EnMO (D0 EnMO), or MDM cultured for 2-, 5-, or 12- days (D2 cMDM, D5 cMDM, D12 cMDM, respectively) were infused IV to animals with acute neuroinlfammation, and the number of donor cells present in the LPS-injected brain hemisphere was quantified at 48 hour following cell IVI. Final data presented here represents mean values ± SD. Data was analyzed by one-way ANOVA, with resulting p-value all < 0.05.

Fig 4: Opposing effects of EPS on murine macrophage cytokine responses to B. breve strains. (A,B) Gating strategy to identify and sort BMDMs. The bone marrow cells were analyzed and sorted by multi-color flow cytometry. Cells were stained for MHC-II, CD11b, CD11c, and CD115. (A) For the adherent cells, MHC-II vs. CD11b contour plots were obtained by gating on MHC-II+CD11c+ cells. Forward size scattered (FSC) vs. CD115 contour plots were obtained by gating on MHC-IIlowCD11bhigh cells. CD115+ cells were sorted and considered as pure macrophages population. (B) For the suspension cells MHC-II vs. CD11b contour plots were obtained by gating on MHC-II+CD11c+ cells. FSC vs. CD115 contour plots were obtained by gating on MHC-IIhighCD11blow cells. CD115– cells were sorted and considered as pure dendritic cells population. (C,D) TNF-a and IL10 secretion from BMDMs co-cultured with B. breve UCC2003, JCM7017 and their isogenic EPS– mutants. (E,F) TNF-a and IL10 secretion from BMDCs co-cultured with B. breve UCC2003, JCM7017 and their isogenic EPS– mutants. Cytokines were analyzed by ELISA. Data is the average of three independent experiments performed in triplicates. Statistical analysis was assessed using the student t-test in GraphPad Prism where *p-value < 0.05 and **p-value < 0.005.

Fig 5: ABCA1 Invalidation Propagates Myelopoiesis and Accelerates Extramedullary Hematopoiesis on a Tet2-Deficient Background(A) Experimental overview. BM from Mx1-Cre+, Mx1-Cre+Abca1fl/fl, Mx1-Cre+Tet2fl/fl, and Mx1-Cre+Tet2,l/flAbca1fl/,fl mice were transplanted into lethally irradiated WT mice, and after a 5-week recovery period, the mice were injected with poly(l:C) and analyzed over a 20-week period.(B) Modulation of Abca1 and Tet2 mRNA expression levels in the BM of the aforementioned mouse models.(C) Quantification of the percentage of peripheral blood myeloid cells determined by hematology cell counter over the course of 20 weeks after poly (I:C) injection in recipient mice transplanted with the BM from Mx1-Cre+, Mx1-Cre+Abca1fl/fl, Mx-Cre+Tet2fl/fl, and Mx1-Cre+Tetfl/fl Abca1fl/fl mice.(D) Peripheral blood myeloid subsets (CD115+Ly6Chi and CD115+Ly6Clo monocytes and CD115-Ly6Chi neutrophils) were also quantified in these mice at the indicated time point(E) Representative H&E staining of paraffin-embedded spleen sections from these mice. Original magnification × 200. Arrows indicate extensive cellular infiltrate.(F and G) Quantification of spleen weight (F) and myeloid subsets (eosinophils, neutrophils, monocytes, and red pulp macrophages [RPMs] in the spleens (G) of recipient mice transplanted with the BM from Mx1-Cre+Abca1fl/fl, Mx1-Cre+Tet2, and Mx1-Cre+Tet2fl/fl Abca1fl/fl mice.(H and I) Quantification of hematopoietic stem (H) and progenitor (MEPs); Lin-Sca1-c-kit+CD34hiFc?Rhi are GMP; and Lin-Sca1-c-kit+CD34hi Fc?Rlow are CMPs.The results are the mean ± SEMs of 5–7 animals per groups ND, not detectable. #p < 0.05 and ##p < 0.001 versus Mx1-Cre+ controls.