Ablation of LRP6 in alpha-smooth muscle actin-expressing cells abrogates lung inflammation and fibrosis upon bleomycin-induced lung injury

Abstract

Tissue fibrosis is a progressive pathological process with excessive deposition of extracellular matrix proteins (ECM). Myofibroblasts, identified by alpha-smooth muscle actin (αSMA) expression, play an important role in tissue fibrosis by producing ECM. Here, we found that the Wnt antagonist Dickkopf1 (DKK1) induced gene expressions associated with inflammation and fibrosis in lung fibroblasts. We demonstrated that genetic deletion of LRP6, a receptor for Wnt ligands and DKK1, in αSMA-expressing cells using Acta2-cre Lrp6^fl/fl (Lrp6^AKO) mice abrogated the bleomycin (BLM)-induced lung inflammation and fibrosis phenotype, suggesting an important role for LRP6 in modulating inflammation and fibrotic processes in the lung. Our results highlight the crucial role of LRP6 in fibroblasts in regulating inflammation and fibrosis upon BLM-induced lung injury.

Keywords: LRP6; fibrosis; inflammation.

Fig. 1

DKK1 induces gene expressions for inflammation and tissue repair in lung fibroblasts. (A–C) Lung fibroblasts were treated with DKK1 for 24 h prior to bulk RNA seq. KEGG pathway enrichment analysis (A) and Gene Ontology (GO) enrichment analysis (B) were conducted to identify significantly upregulated pathways in DKK1‐treated group compared to untreated group. DEG analysis between untreated and DKK1‐treated group is visualized in the form of a heatmap (C).

Fig. 2

Lrp6 ^AKO mice showed normal hematological phenotypes. (A) LRP6 expressions in αSMA⁺ or αSMA⁻ populations were quantified by flow cytometry from the lungs of Lrp6 ^AKO and Lrp6 ^fl/fl mice. (B) Inflammatory cells in the peripheral blood from Acta2cre‐Lrp6 ^fl/fl and Lrp6 ^fl/fl mice were analyzed by Hemavet. Shown are white blood cells (WBC), lymphocytes, monocytes, neutrophils, eosinophils, basophils, red blood cells (RBC), platelets, and mean corpuscular volume (MCV) (n = 5). Data are presented as the mean ± SD. Student's t‐test. ns, not significant.

Fig. 3

Lrp6 ^AKO mice showed reduced BLM‐induced lung fibrosis phenotypes. (A–E) Lrp6 ^AKO and their littermate controls Lrp6 ^fl/fl mice were challenged with BLM on day 0. Two weeks later, the lungs were harvested. (A) Representative images of Masson staining are shown. Scale bar = 800 μm (top), 100 μm (bottom). (B) Hydroxyproline concentration was measured (n = 5). (C) αSMA protein levels were analyzed by IHC and imagej (n = 5). (D) Representative images of IHC are shown. Scale bar = 100 μm. (E) The ratio of αSMA, DKK1 and IL‐11 to Gapdh was quantified by western blot and imagej (n = 3). Data are represented as means ± SD. One‐way ANOVA with Tukey's post hoc test. ***P < 0.001, **P < 0.005, *P < 0.05, ns, not significant.

Fig. 4

Lrp6 ^AKO mice showed reduced BLM‐induced lung inflammation. (A, B) Lrp6 ^AKO and their littermate controls Lrp6 ^fl/fl mice were challenged with BLM. Two weeks later, the lungs were harvested. (A) Representative images of H&E staining are shown. Scale bar = 800 μm (top), 100 μm (bottom). (B) CD45⁺ leukocytes, macrophages, neutrophils, CD4⁺ T cells, CD8⁺ T cells and Treg cells were quantitated by flow cytometry. Data are represented as means ± SD (n = 4 or 6). One‐way ANOVA with Tukey's post hoc test. ***P < 0.001, **P < 0.005, *P < 0.05, ns, not significant.