Fig. 9: Effects of TGF-β mRNA-IAJD34 bleomycin-induced injury.

BALB/c mice were exposed to PBS or bleomycin (ITB) and subsequently received either an empty IAJD34 or 10 µg TGF-β mRNA-IAJD34. Cell-free BAL fluid, BAL cells, and large aggregate surfactant fractions were collected 3 days post-exposure and injection. A Cell-free BAL fluid was evaluated for total protein content using a BCA assay (n/group = 7/PBS + Empty-IAJD34, 6/PBS + TGF-β mRNA+IAJD34, 10/ITB+Empty-IAJD34, 8/ITB + TGF-β mRNA+IAJD34). Data were evaluated for normality using a Shaprio-Wilks normality test. PBS and ITB were compared using a 2-way ANOVA. B Total phospholipids were determined from the large aggregate surfactant fraction (n/group = 4/PBS + Empty-IAJD34, 3/PBS + TGF-β mRNA + IAJD34, 5/ITB + Empty-IAJD34, 5/ITB + TGF-β mRNA + IAJD34). Data were compared using a 2-way ANOVA. C Total BAL cells were counted using a colter counter (n/group = 8/PBS + Empty-IAJD34, 6/PBS + TGF-β mRNA + IAJD34, 10/ITB + Empty-IAJD34, 10/ITB + TGF-β mRNA+IAJD34). Data were compared using a 2-way ANOVA with Šídák’s multiple comparisons test. D, E BAL cells were immunostained for flow cytometric analysis. Cells that were positively stained for both Siglec F and F4/80 were determined to be alveolar macrophages (AMs). Resident macrophages (CD11c⁺ /CD11b⁻), can be differentiated from recruited (CD11c⁻/CD11b⁺) or migratory macrophages (CD11c⁺/CD11b⁺) (n/group = 7/PBS + Empty-IAJD34, 6/PBS + TGF-β mRNA + IAJD34, 10/ITB+Empty-IAJD34, 8/ITB + TGF-β mRNA+IAJD34). Resident AMs exposed to PBS and ITB were compared using a 2-way ANOVA. Recruited AMs were compared using a two-tailed Wilcoxon Signed Rank test. F Cells from digested lung tissue were immunomagnetically separated based upon CD45 expression. CD45⁺ cells were isolated, immunostained, and analyzed. Cells that expressed F4/80 and CD11b in the absence of Siglec F were categorized as interstitial macrophages (IMs) and were analyzed for Ly6c expression (n/group = 7/PBS + Empty-IAJD34, 6/PBS + TGF-β mRNA + IAJD34, 10/ITB + Empty-IAJD34, 8/ITB + TGF-β mRNA + IAJD34). Ly6c⁺ IMs exposed to PBS and ITB were compared using a 2-way ANOVA. All data were evaluated for normality using a Shaprio-Wilks normality test and are presented as Mean ± SE, n = 3–10/group. * indicates a significant difference from the control, p < 0.05. ((A): PBS vs ITB p = 0.0486; (C): PBS/Empty-IAJD34 vs ITB/Empty-IAJD34 p = 0.0087; (D): PBS vs ITB p = 0.0360; (E): PBS/Empty-IAJD34 vs ITB/Empty-IAJD34 p = 0.0156; (F): PBS vs ITB p = 0.0089). Source data are provided as a Source Data file.