(B-43) Investigating the influence of macrophage co-culture on fibroblast mechanotransduction

Introduction:: Pulmonary fibrosis is a pathological wound healing process that disrupts proper lung function and can ultimately lead to organ failure. Characterized by increased tissue stiffness¹, decreased stress relaxation², and an aberrant immune response mediated by macrophages³, pulmonary fibrosis is driven by the chronic activation of fibroblasts that build up scar tissue in the lung interstitial space. While infiltration of ‘classically activated’ M1 and ‘alternatively activated’ M2 macrophages is crucial for normal wound resolution, the prolonged presence of M2 macrophages has been implicated in persistent fibrogenesis⁴. This complex microenvironment makes it challenging to delineate the dynamic between macrophage-fibroblast communication and the changing mechanical environment, motivating the need to create a controlled in vitro model system to investigate the influence of macrophage signaling on fibroblast mechanobiology. Here, we utilized hyaluronic-acid (HA) based hydrogels to recapitulate mechanical properties of healthy and fibrotic lung tissue and investigated the influence of macrophage co-culture on fibroblast activation.


Materials and Methods:: HA was functionalized with norbornenes to allow UV-mediated crosslinking via thiol-ene chemistry. Viscoelasticity was introduced through physical guest-host interactions using β-cyclodextrin-modified HA and a thiolated adamantane peptide. Elastic or viscoelastic hydrogels matching either normal or fibrotic lung tissue mechanics were fabricated in a high-throughput 96-well plate array (Fig. 1A). Hydrogel mechanics were characterized using shear oscillatory rheology. M0, M1, or M2 macrophages derived from human THP-1 monocytes were directly or indirectly cultured with human lung fibroblasts on soft viscoelastic (~ 1 kPa) or stiff elastic (~ 50 kPa) hydrogels. Fibroblast activation was assessed by immunocytochemical staining and quantitative image analysis of F-actin cytoskeletal organization and collagen I expression as well as the colocalization of cadherin-11 and focal adhesions (paxillin).


Results, Conclusions, and Discussions:: We found that fibroblast cell area nearly doubled when cultured on stiff, elastic substrates compared with those cultured on soft, viscoelastic substrates. This behavior was independent of macrophage phenotype or co-culture method (Fig. 1B,C). Additionally, collagen I expression on stiff substrates was an order of magnitude higher than that of fibroblasts cultured on soft, viscoelastic substrates regardless of macrophage phenotype or co-culture method. Notably, fibroblasts directly co-cultured with M2 macrophages exhibited this activated phenotype even on soft viscoelastic hydrogels, with no significant difference observed in cell area, collagen I expression, or cadherin-11 expression between those cultured on soft viscoelastic or stiff elastic hydrogels (Fig. 1D,E). These findings support previous results that implicate these macrophages in fibroblast activation⁵. Additionally, we did not observe this phenotype-dependent activation when fibroblasts were indirectly cultured with macrophage media. These results indicate that direct cell crosstalk could be necessary for fibroblast activation on soft viscoelastic substrates, highlighting the importance of considering both environmental mechanical cues and macrophage-fibroblast signaling in pulmonary fibrosis progression.



Here we fabricated HA-based hydrogels that recapitulate mechanical properties of healthy and fibrotic lung tissue to probe how essential healthy tissue behavior like viscoelasticity influences macrophage-fibroblast crosstalk and mechanotransduction. We show that stiff, elastic substrates activate fibroblasts regardless of macrophage phenotype or co-culture method. Additionally, we found that M2 macrophages stimulate fibroblasts even on soft, viscoelastic substrates when these cells are physically able to interact and have identified a potential mediator in these interactions through cadherin-11. Ongoing work includes exploring the mechanism of fibroblast activation we have described here through pathways involving cadherin-11.




Acknowledgements (Optional): : This work was supported by the NIH MIRA Award (R35 GM138187) and the NSF Graduate Research Fellowship.


References (Optional): : [1] Wells, R. G., Biochim. Biophys. Acta, 2014.; [2] Hilster, R. H. J, Burgess, J. K., Am. J. Physiol. Lung Cell Mol. Physiol., 2020.; [3] Kishore and Petrek, Front. Immunol., 2021; [4] Hashimoto, D., Merad, M., Immunity., 2013. [5] Lodyga, M., et al., Science signaling, 2019.