Modeling mechanical activation of macrophages during pulmonary fibrogenesis for targeted anti-fibrosis therapy

Abstract

Pulmonary fibrosis is an often fatal lung disease. Immune cells such as macrophages were shown to accumulate in the fibrotic lung, but their contribution to the fibrosis development is unclear. To recapitulate the involvement of macrophages in the development of pulmonary fibrosis, we developed a fibrotic microtissue model with cocultured human macrophages and fibroblasts. We show that profibrotic macrophages seeded on topographically controlled stromal tissues became mechanically activated. The resulting co-alignment of macrophages, collagen fibers, and fibroblasts promoted widespread fibrogenesis in micro-engineered lung tissues. Anti-fibrosis treatment using pirfenidone disrupts the polarization and mechanical activation of profibrotic macrophages, leading to fibrosis inhibition. Pirfenidone inhibits the mechanical activation of macrophages by suppressing integrin αMβ2 and Rho-associated kinase 2. These results demonstrate a potential pulmonary fibrogenesis mechanism at the tissue level contributed by macrophages. The cocultured microtissue model is a powerful tool to study the immune-stromal cell interactions and the anti-fibrosis drug mechanism.

Fig. 1.. Mechanical activation of lung macrophages in a highly remodeled fibrotic tissue microenvironment.

(A) Hematoxylin and eosin (H&E) staining of a highly remodeled region in a fibrotic lung tissue. (B) Confocal fluorescence images of α-SMA and CD206 of a matching region in an adjacent tissue section. Both α-SMA⁺ myofibroblasts and CD206⁺ macrophages adopted elongated morphology and aligned with each other. Scale bars, 100 μm. (C) t-Distributed stochastic neighbor embedding (t-SNE) plot showing the distinct populations of alveolar macrophages in patients with IPF and healthy donors. (D) t-SNE plot showing the preferential expression of ITGAM gene in alveolar macrophages in the patients with IPF illustrated in (C). Data extracted from healthy subjects (n = 8) and patients with IPF (n = 4) based on NCBI GEO dataset GSE122960. (E) Expression of genes linked to the activation of mechanobiological pathways in alveolar macrophages of healthy donors (blue) and patients with IPF (orange). Bubble size corresponds to the average expression level of the gene within each group. The data were extracted from the NCBI GEO datasets GSE122960 and GSE228232. (F) Fold enrichment of Gene Ontology (GO) biological process in genes that are up-regulated in fibrotic lungs compared to healthy donor lungs. The GO enrichment data were collected from dataset GSE122960.

Fig. 2.. Macrophage mechanical activation in response to topographically controlled tissue remodeling.

Phase contrast images of fibroblast and macrophage cocultured lung microtissues formed on a group of flexible micropillars arranged in a spiral pattern (A), a diamond pattern (B), and a square pattern (C). Confocal reflectance images of collagen fibers (cyan) and fluorescence images of CD206 (red) of spiral patterned (D), diamond patterned (E), and square patterned (F) microtissues. (G to I) Enlarged views of boxed regions in (D) to (F) showing the co-alignment between the CD206-positive macrophages and the collagen fibers. (J) Scanning electron microscopy (SEM) images of a fibroblast only microtissue showing the fibroblast alignment. (K) SEM images of a cocultured microtissue showing the co-alignment between fibroblasts and macrophages.

Fig. 3.. Extensive co-alignment between macrophages and fibroblasts promoted a widespread fibrosis response in the microtissue.

(A) Schematic showing the fibrosis induction in NHLF-populated microtissues by pro-fibrotic macrophages. (B) Representative fluorescence images of NHLF only (untreated), TGF-β1–treated NHLF only, and M2 macrophage and NHLF cocultured microtissues (left to right). Microtissues were stained for nuclei (blue), α-SMA (green) and CD206 (red). Scale bar, 200 μm. (C) Fluorescence intensity measurement of α-SMA for different microtissue culture conditions. a.u., arbitrary units; ns, not significant. (D) Contractile force measurement for different microtissue culture conditions. (E) Supernatant active TGF-β level for different microtissue culture conditions. (F) Enlarged view of the boxed region in (B) showing the co-alignment between single myofibroblasts and M2 macrophages. (G) Enlarged view shows positive TGF-β signal co-localizing with CD206 but not with α-SMA. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 when compared to untreated group by one-way ANOVA with Tukey test; 'ns' indicates no significance between two treatment groups using a two-tailed paired Student's t test.

Fig. 4.. The effect of preventative anti-fibrosis treatment on cocultured microtissue.

(A) Schematic showing the preventative anti-fibrosis treatment and results evaluation. PFD was administrated at the beginning of the coculture. (B) Representative fluorescence images of cocultured microtissues treated with PFD (0, 10, or 1000 μg/ml) (left to right). Scale bar, 200 μm. (C) The number of macrophages adhered on the microtissue under different PFD treatments. (D) Fluorescence intensity measurement of α-SMA under different PFD treatment conditions. (E) Contractile force measurement under different PFD treatment conditions. (F) Supernatant active TGF-β level under different PFD treatment conditions. *P ≤ 0.05 and ***P ≤ 0.001 when compared with untreated group by one-way ANOVA with Tukey test; #P ≤ 0.05, ##P ≤ 0.01, and ###P ≤ 0.001 when comparison were performed between treatment groups using a two-tailed paired Student's t test.

Fig. 5.. The effect of therapeutic anti-fibrosis treatment on cocultured microtissue.

(A) Schematic showing the therapeutic anti-fibrosis treatment and results evaluation. PFD was administrated after 3 days of coculture. (B) Representative fluorescence images of cocultured microtissues treated with PFD (1000 μg/ml). Scale bar, 200 μm. (C) The number of macrophages adhered on the microtissue under different PFD conditions. (D) Fluorescence intensity measurement of α-SMA under different PFD treatment conditions. (E) Contractile force measurement under different PFD treatment conditions. (F) Supernatant active TGF-β level under different PFD treatment conditions. ***P ≤ 0.001 when compared with untreated group by one-way ANOVA with Tukey test; 'ns' indicates no significance between two treatment groups using a two-tailed paired Student's t test.

Fig. 6.. The effect of anti-fibrosis pretreatment on cocultured microtissue.

(A) Schematic showing the anti-fibrosis pretreatment and results evaluation. Macrophages were pretreated with PFD for 24 hours and then added to NHLF microtissues to form the coculture. (B) Representative fluorescence images of cocultured microtissues treated with PFD (0, 10 or 1000 μg/ml) (left to right). Scale bar, 200 μm. (C) The number of macrophages adhered on the microtissue under different PFD conditions. (D) Fluorescence intensity measurement of α-SMA under different PFD treatment conditions. (E) Contractile force measurement under different PFD treatment conditions. (F) Supernatant active TGF-β level under different PFD treatment conditions. **P ≤ 0.01, and ***P ≤ 0.001 when compared with untreated group by one-way ANOVA with Tukey test; #P ≤ 0.05 and ###P ≤ 0.001 when comparison were performed between treatment groups using a two-tailed paired Student's t test.

Fig. 7.. PFD suppresses macrophage mechanical activation through inhibition of ROCK2 and integrin αMβ2.

(A) Representative immunofluorescence images of macrophage and fibroblast cocultured microtissues under PFD or ROCK2 inhibitor KD025 treatments. Microtissues were stained for CD206 (red), ROCK2 (yellow), and nuclei (blue) from top to bottom. Fluorescence intensity measurement of ROCK2 in cocultured microtissues under preventative treatment (B) and therapeutic treatment (C). Number of adherent macrophages on cocultured microtissues under preventative treatment (D) and therapeutic treatment (E). Active TGF-β in cell culture supernatant under preventative treatment (F) and therapeutic treatment (G). (H) Representative immunofluorescence images of macrophage and fibroblast cocultured microtissues under different PFD treatment conditions. Microtissues were stained for CD11b (green), CD18 (yellow), and CD206 (red) from top to bottom. Fluorescence intensity measurement of CD11b (I) and CD18 (J) under treatments with or without PFD. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 when compared with untreated group by one-way ANOVA with Tukey test; 'ns' indicates no significance between two treatment groups using a two-tailed paired Student's t-test; #P ≤ 0.05 and ##P ≤ 0.01 between two treatment groups using a two-tailed paired Student's t test.