Fig 2: The injury-induced inflammatory response at the site of injury triggers HO but not bone loss at 1 dpi. a White blood cells (WBCs), monocytes, basophils, lymphocytes, and neutrophils in the peripheral blood of six- to eight-week-old WT and NSE-BMP4 mice at 1 day after injury. (n = 4-5 per group). Data are presented as the mean ± s.d. of biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed t test). b Schematic outline of the study design depicting the workflow of target tissue collection at different time points for RNA-seq. c GO enrichment analysis of differentially expressed genes (DEGs) in the tibial muscle of the NSE-BMP4 mice (n = 3 per group). Enrichment analysis of immune cell genes in the NSE-BMP4 mice, presented in a KEGG pathway dot plot (d) and heatmap (e) (n = 3 per group). IF staining images (f) and statistical analysis (g) of F4/80+ macrophages in HO lesions after CTX injury. The white dotted line outlines the region of mature HO. Scale bar, 200 μm (n = 3 per group, n = 3 ROI/mouse). Data are presented as the mean ± s.d. of biological replicates. *P < 0.05, ***P < 0.001, ****P < 0.000 1. N.S. indicates no significance (unpaired two-tailed t test). Immunostaining images (h) and statistical analysis (i) of CTSK+ osteoclasts in bone from the NSE-BMP4 mice after CTX injury. The white dotted line outlines the region of CTSK+ cells. Scale bar, 200 μm (n = 3 per group, n = 3 ROI/mouse). Data are presented as the mean ± s.d. of biological replicates. ****P < 0.000 1. N.S. indicates no significance (unpaired two-tailed t test)

Fig 3: Increased Th1 response in PTPN2fl/flxCD11cCre mice in acute colitis. RNA was isolated from whole colon pieces of PTPN2fl/fl (WT) and PTPN2fl/flxCD11cCre (KO) mice: (A,B) mRNA expression of the indicated; (A) Th-cell-associated transcription factors; (B) cytokines and activation markers in whole colon lysates; (C) frequencies of IFNγ+ and IL17+ CD4+ T cells in lamina propria; (D) IHC staining for F4/80+ macrophages in the colon. * p < 0.05, ** p < 0.01; unpaired Mann–Whitney test. Data are representative for one out of two independent experiments with 4–5 mice per experimental group.

Fig 4: Loss of PTPN2 in DCs affects immune cell populations in the lamina propria. Immune cells were analyzed in PTPN2fl/fl (WT) and PTPN2fl/flxCD11cCre (KO) mice: (A,B) t-SNE maps displaying live, CD45+ singlet cells in lamina propria lymphocytes (LPL). Colors correspond to FlowSOM-guided clustering of cell populations. Pie charts represent relative numbers among CD45+ cells; (A) t-SNE maps of CD45+ cells in LPL; (B) total counts of indicated cell populations in LPL; (C) IHC staining for F4/80+ macrophages in colon; (D) relative numbers of Tregs, IFNγ- or TNF-producing CD4+ and CD8+ T cells. * p < 0.05, ** p < 0.01; unpaired Mann–Whitney test, n = 5–7 per group.

Fig 5: BLPs Contain Epithelial Cell Membranes and Are Enriched in Endolysosomal Compartments(A) BLPs (arrow) with thin membranous extensions (arrowheads). Maximum z-projection of a 7 µm (left) and a single slice (right) of distal colonic ??s from CD11c: Cre/R26mTmG mouse; mGFP (green); F-actin (red); DNA (blue); deconvolved. Scale bars: 10 µm, 1 µm on the magnified inset.(B) Inner compartments of BLPs are filled with membranous material, derived from CD11c-negative cells (top panel, distal colon of CD11c: Cre/R26mTmG mouse), in part from the epithelium (bottom panel, distal colon of Villin: Cre-ERT2/R26mTmG mouse; intestinal epithelial cells express membranous GFP [green]), BLPs (MHC class II staining [red]). Maximum z-projections of 1 µm, deconvolved. Scale bar: 1 µm.(C) Correlative electron microscopy (left) of BLP from distal colon of CD11c: Cre/R26mTmG mouse; arrow indicates a BLPs found on the confocal image (mGFP [green], membrane tdTomato, boxed [red]) and overlaid with the low-magnification TEM image; borders or the same BLPs are highlighted with yellow dashed line on the higher-magnification TEM image (right). Scale bar: 1 µm.(D) LAMP2, LAMP1, Rab7A, and Rab11 staining of BLPs, maximum z-projections of 1–2 µm, deconvolved. Scale bars: 2 µm.(E) MHCII, CD74, and F4/80 staining of BLPs. Maximum z-projection of 1–2 µm, deconvolved. Scale bars: 2 µm. See also Video S2.