Fig 1: Macrophages control Mtb survival and promote osteogenic differentiation of MSCs via BMP/SMAD/RUNX2 pathway.a Lung CT scan from patients TB-7 and TB-3. Arrows indicate calcific densities in pathologic lesions. b, c Representative images of histologic analysis and Von Kossa staining (b), and p-SMAD1/5/8 and RUNX2 immunohistochemical analysis (c) of NC and TB lung sections from TB patients as in a. Single arrowheads, the osteoid with mass deposits of calcium (black); double arrowheads, dispersed deposits of calcium (brown); asterisks, the necrotizing foci. OC osteocyte; OB osteoblast; MGC multinucleated giant cell. Scale bars, 200 µm. d, e Quantitative analysis of p-SMAD1/5/8 (d) and RUNX2 (e) immunostaining as in c. f Immunoblot analysis of p-SMAD1/5/9, SMAD1/5/9, RUNX2, and ß-Actin in six independent NC or calcified TB lung samples. g Immunoblot analysis of BMP2/4, p-SMAD1/5/9, SMAD1/5/9, RUNX2, and GAPDH in U937 cells. Cells were treated with 500 nM LDN-193189, 1 µM K02288, 10 µM Galunisertib or control DMSO, and infected with Mtb for 0–48 h. h Survival of Mtb in U937 cells treated as in g. i, j ELISA of TNF (i) and iNOS (j) from U937 cells treated with LDN-193189 or control DMSO and infected with Mtb as in g. k Representative images of CD29 and CD68 immunohistochemical analysis of NC and TB lung sections. Arrowheads indicate the gathering of CD29+ cells (indicating MSCs) and CD68+ cells (indicating macrophages). Scale bars, 200 µm. l Representative images of Alizarin Red S staining of BMSCs. BMSCs were cultured with conditional media derived from BMDMs (CM), Mtb-infected BMDMs (Mtb-CM), LDN-193189-pretreated and Mtb-infected BMDMs (LDN-Mtb-CM) or control media for 0–14 d. Arrowheads, the mineralized nodules. Scale bars, 200 µm. m– o Quantitative PCR analysis of Alp (m), Bglap (n), and Runx2 (o) mRNAs in BMSCs treated as in l. P > 0.05, not significant (ns); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (mean ± s.e.m. of n = 4 in d, e, h, and m–o, and n = 3 in i, j, two-way ANOVA). Results are representatives from at least three independent experiments.
Supplier Page from RayBiotech for Human TNF-alpha ELISA