Figure 6.

TLR4 deletion protects from experimental BM fibrosis in mice. (A) The TPO^high model was established in wt/wt and Tlr4^−/− mice. (B and C) Total numbers of LSK, LT-HSC, ST-HSC, MMP, CMP, GMP, and MEP cells in the BM (B) and spleens (C) of wt/wt and Tlr4^−/− mice after treatment with TPO. n = 3. (D) Immunofluorescence of BM CD41⁺ Mk’s in wt/wt and Tlr4^−/− mice after treatment with TPO. Nuclei were stained with Hoechst 33258. Bar, 50 µm. Objective, 20×. (E) Flow cytometry quantification of CD45/41⁺ Mk’s in BM cells of wt/wt and Tlr4^−/− mice after treatment with TPO. n = 4. (F and G) Flow cytometry analysis of CD45/41⁺ Mk’s (F) and relative quantification (G) in spleen cells of wt/wt and Tlr4^−/− mice after treatment with TPO. n = 3. (H and I) Quantification of IL-6 (n = 7; H) and TNF-α (n = 6; I) in BM cell-free fluids of wt/wt and Tlr4^−/− mice treated with TPO. (J–L) Flow cytometry histograms (J) and relative mean fluorescence intensity (MFI) quantification of intracellular IL-6 (K) and TNF-α (L) of CD41⁺ Mk’s in saline and TPO treated wt/wt and Tlr4^−/− mice. n = 3. (M and N) Representative Gomori stain (M) and quantitative assessment of reticulin deposition (N) in BM sections of wt/wt and Tlr4^−/− mice after experimental fibrosis. Bar, 50 µm. Objective, 20×. n = 3. (O and P) Immunofluorescence staining (O) and quantitative assessment (P) of FN and type III collagen in BM sections of wt/wt and Tlr4^−/− mice after experimental fibrosis. Bar, 50 µm. Objective, 60×. (Q) Representative spleens of saline and TPO treated wt/wt and Tlr4^−/− mice. (R) Spleen/body weight ratios in wt/wt and Tlr4^−/− mice treated with saline or TPO. n = 4. (S) H&E stain of spleen sections of wt/wt and Tlr4^−/− mice after experimental fibrosis. Bar, 50 µm. (T–V) Peripheral platelet (PLT; T), white (WBC; U), and red blood (RBC; V) cell counts of wt/wt and Tlr4^−/− mice after TPO treatment. n = 5. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are shown as mean ± SD.