MRC2-mediated collagen internalization is reduced in fibrotic lung fibroblasts and increased upon phenotypic dedifferentiation
Natalie M. Walker, Sean M. Fortier, Jennifer Speth, Steven K. Huang, Sergey Gutor, Timothy S. Blackwell, Marc Peters-Golden
Natalie M. Walker, Sean M. Fortier, Jennifer Speth, Steven K. Huang, Sergey Gutor, Timothy S. Blackwell, Marc Peters-Golden
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Research Article Cell biology Pulmonology

MRC2-mediated collagen internalization is reduced in fibrotic lung fibroblasts and increased upon phenotypic dedifferentiation

Abstract

Idiopathic pulmonary fibrosis (IPF) is characterized by parenchymal scarring reflecting an imbalance between collagen deposition by myofibroblasts (MFs) and its turnover. Although collagen clearance is essential for fibrosis resolution, this process and its potential for therapeutic modulation in IPF are poorly understood. Here we evaluated internalization of degraded collagen and the role of its requisite endocytic receptor mannose receptor C-type 2 (MRC2), in lung tissue and MFs from patients with IPF and bleomycin-injured mice. Fibrotic human and murine lung tissue exhibited an accumulation of degraded collagen, highlighting a failure of its clearance. MFs from fibrotic lung demonstrated a reduced capacity to internalize extracellular degraded collagen, with a concomitant reduction in MRC2 expression and endolysosomal activity. Both diminished collagen uptake and MRC2 expression recovered to baseline levels during spontaneous resolution of bleomycin fibrosis. In vitro treatment of IPF or TGF-β–elicited MFs with a variety of mechanistically distinct agents known to effect phenotypic dedifferentiation restored defective collagen internalization. Although enhanced uptake was MRC2 dependent, it involved increased endolysosomal activity rather than increased MRC2 expression. These results implicate defective MRC2-dependent collagen internalization and endolysosomal function in MFs as important factors contributing to fibrosis that may be therapeutically targeted to promote resolution.

Authors

Natalie M. Walker, Sean M. Fortier, Jennifer Speth, Steven K. Huang, Sergey Gutor, Timothy S. Blackwell, Marc Peters-Golden

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Figure 2

Fibrotic MFs demonstrate reduced gelatin internalization capacity that is enhanced upon treatment with dedifferentiation agents and upon fibrosis resolution.

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Fibrotic MFs demonstrate reduced gelatin internalization capacity that i...
(A) Schematic demonstrating experimental protocol utilized for assessing gelatin internalization. Normal fibroblasts and IPF MFs were treated with Oregon Green 488–conjugated gelatin for 1 hour (5 μg/mL) followed by flow cytometric analysis. (B) Confocal images (×100, oil) demonstrating gelatin internalization in normal fibroblasts and IPF MFs, with gelatin+ puncta quantitated using CellProfiler (C). (D) Schematic demonstrating experimental protocol employed for assessing gelatin internalization after dedifferentiating agent treatment. IPF MFs were treated with dedifferentiating agents for 2 hours followed by addition of conjugated gelatin 1 hour prior to flow analysis for internalization. (E) Confocal imaging (×100) and gelatin puncta quantification along with EEA1+ early endosomal colocalization in IPF MFs after treatment with dedifferentiating agents and conjugated gelatin. Scale bars: 10 μm (B and E). Normal fibroblasts treated with TGF-β for 48 hours to establish MFs followed by flow cytometric analysis of gelatin internalization capacity at baseline (F) and (G) after treatment with dedifferentiating agents. (H) Schemata demonstrating bleomycin injury model timeline and experimental protocol used to assess fibroblasts’ gelatin internalization over time. Mouse fibroblasts isolated and grown in vitro from murine lungs of saline or bleomycin-injured mice at time points indicated were passaged 3 times, then treated with conjugated gelatin (5 μg/mL) for 1 hour and internalization capacity determined by flow cytometry. Each individual data point in A and C–E is derived from independent patient cell lines. Significance for A, C, and F was determined by 2-tailed t test, while a 1-way ANOVA was used in D, E, G, and H followed by Dunnett’s multiple comparisons test (D, E, and G) and Šidák’s multiple comparisons test (H). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.