(396) Lung Extracellular Matrix Remodeling in Human Cystic Fibrosis and Implications for Innate Immunity

Background: Elevated levels of proteolytic enzymes found in sputum of cystic fibrosis (CF) patients often correlate with worsening lung function in CF. However, downstream effects on distal lung extracellular matrix (ECM) and resident immune cell function remain unknown. To address this gap, we: (i) performed a comprehensive characterization of the distal lung ECM in human CF, (ii) utilized extracted lung ECM to prepare human- and patient-specific biomaterials, and (iii) co-cultured CF ECM biomaterials and iPSC-derived healthy and CF macrophages to model CF in vitro.

Methods: Human lung tissue was obtained from patients with end-stage CF (n=8) and from control lungs (n=3). Quantitative characterization of lung ECM was performed with liquid chromatography mass spectrometry (LC-MS/MS) and targets were validated using gene ontology (GO) analysis. iPSCs were obtained from healthy (WTC11) and CF (∆F508/∆F508) donors and differentiated into CD14⁺ monocytes/macrophages (M0). Healthy and CF iPSC-derived M0 were cultured alone or with lung ECM (CF, control). On day 3, M0 activation was assessed using multiplex cytokine arrays and bulk RNA sequencing.

Results: Through quantitative analysis of the distal lung ECM in human CF tissue, we determined that the majority of significantly altered ECM proteins were downregulated in CF (log₂FC < -1, p < 0.05), including key basement membrane constituents: collagens (collagen 4, 5, 6, 11, 21, 12, 26, 18), laminins (laminin 2, 4, 5), and nidogen 1. Elastin was similarly decreased in CF compared to control lung (log₂FC = -1.6, p = 0.026). When filtering for downregulated peptides (log₂FC < -1), GO pathways identified included ECM structural constituent (p = 6.9E-38) and ECM organization (p = 1.0E-18). When filtering for upregulated peptides (log₂FC > 1), identified GO terms included: unfolded protein binding (p = 3.6E-9), response to topologically incorrect protein (p = 2.9E-6), and negative regulation of wound healing (p = 8.2E-3), indicating that pathological alteration to the ECM has downstream effects on cell populations in CF. To further investigate how ECM breakdown impacts immune cell activation in CF, we exposed iPSC-derived M0 from CF and healthy donors (Fig 1A) to CF and control lung ECM (Fig 1B). We determined that loss of CFTR leads to intrinsic abnormalities in M0 activation based on significantly higher baseline expression of IL-8, IL-18, and MCP-1 (p < 0.05) even in the absence of ECM exposure. Furthermore, we determined that CF M0 express lower levels of inflammatory markers in response to CF matrix compared to healthy M0 exposed to the same ECM, suggesting that the ability to respond to the inflammatory CF environment may be compromised in CF M0 (Fig 1C).

Conclusions: We quantified pathological remodeling of the lung ECM in CF and determined downstream effects on M0 activation using an in vitro human tissue model of the CF lung microenvironment. We determined that key structural and basement membrane proteins are downregulated in end-stage CF and that these ECM alterations lead to dysregulation of cell-matrix pathways implicated in lung repair. Finally, we found that CF iPSC-derived M0 exhibit intrinsic inflammatory abnormalities which are exacerbated by exposure to isolated human ECM from CF and control patients. Pathways identified in this study could serve as biomarkers of disease state or therapeutic targets in the future.

Acknowledgements: The authors gratefully acknowledge the CF Foundation for supporting this work (VUNJAK20G0).

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