Background: SSc-associated interstitial lung disease (SSc-ILD) is a key driver of morbidity and the leading cause of death in SSc [1,2]. SSc-ILD is increasingly understood as a maladaptive epithelial-mesenchymal repair response in the peri-alveolar regions. In this model, injury, senescence, and reduced regenerative capacity of type II alveolar epithelial cells (AEC2) drive myofibroblast differentiation and excessive extracellular matrix deposition [3,4]. Current human models of SSc-ILD do not fully recapitulate the distal lung compartments with presence of alveolar cells, distal bronchial epithelial cells and lung fibroblasts.

Objectives: To establish and validate SSc-iPSCs-derived human lung organoids (HLOs) containing the relevant epithelial-mesenchymal compartments as a preclinical model of SSc-ILD.

Methods: HLOs were generated from iPSCs derived from SSc patients using a previously described protocol [5]. The presence of AEC1 and 2, distal bronchial epithelial cells, and fibroblasts was evaluated by immunofluorescence. SSc-HLOs were then exposed to pro-fibrotic stimuli (TGFβ or serum from SSc patients) and to the antifibrotic agent nintedanib. Fibrotic transformation in response to TGFβ or SSc serum, as well as the antifibrotic effects of nintedanib, were quantified using qPCR, immunofluorescence, and bulk RNA sequencing. Single-cell RNA sequencing (scRNA-seq) was performed to identify fibroblast subpopulations and AEC2s in SSc-HLOs exposed to SSc serum, and to assess shifts in cell frequencies relative to healthy HLOs treated with healthy serum.

Results: HLOs derived from SSc-iPSCs demonstrated formation of cell types relevant for SSc-ILD: HOPX+ AEC1, SFTPC+ AEC2, SOX9+ precursors of AEC1/2, E-cadherin+ bronchial epithelial cells (including P63+ basal cells and FOXJ1+ ciliated cells), as well as PDGFRα+ fibroblasts. Exposure of SSc-HLOs to TGFβ or SSc patient serum promoted fibroblast-to-myofibroblast differentiation, evidenced by increased ACTA2 mRNA expression and a rise in αSMA+ myofibroblasts. This was accompanied by excessive extracellular matrix (ECM) deposition, with upregulated COL1A1 and FN1 transcripts and increased collagen type I protein levels. Furthermore, exposure of SSc-HLOs to TGFβ or SSc serum induced transcriptomic changes with upregulation of pathways related to ECM deposition, epithelial cell damage and proinflammatory signaling. Nintedanib attenuated the profibrotic effects of TGFβ or SSc serum, with reduced ACTA2 mRNA expression, αSMA+ myofibroblasts, COL1A1 and FN1 transcripts and collagen type I protein levels, and partially normalized the aberrant transcriptomic profiles induced by these profibrotic stimuli. SSc-HLOs exposed to SSc serum recapitulated fibroblast subpopulations previously described in SSc-ILD [6], including a proliferative myofibroblast subset that was more abundant in SSc-HLOs than in healthy HLOs exposed to healthy serum. In parallel, AEC2 precursors showed increased expression of injury-associated signals and were reduced in frequency in SSc-HLOs.

Conclusions: This study demonstrates that SSc-HLOs recapitulate several key features of SSc-associated interstitial lung disease (SSc-ILD): (1) co-existence of distal lung epithelial and mesenchymal compartments relevant to SSc-ILD; (2) profibrotic responses to TGFβ or SSc patient serum, accompanied by transcriptional programs consistent with epithelial injury and proinflammatory signaling; (3) attenuation of these profibrotic responses by nintedanib treatment; and (4) the presence of fibroblast subpopulations and shifts in cellular composition resembling those observed in SSc-ILD. Together, these findings support SSc-HLOs as a promising preclinical model for mechanistic studies and drug testing in SSc-ILD.

REFERENCES: [1] Volkmann, E.R. & Fischer, A. Update on Morbidity and Mortality in Systemic Sclerosis-Related Interstitial Lung Disease. J Scleroderma Relat Disord 6, 11-20 (2021).

[2] Tyndall, A.J., et al. Causes and risk factors for death in systemic sclerosis: a study from the EULAR Scleroderma Trials and Research (EUSTAR) database. Ann Rheum Dis 69, 1809-1815 (2010).

[3] Goldman, N., Ong, V.H. & Denton, C.P. Pathogenesis of interstitial lung disease in systemic sclerosis. Rheumatol Immunol Res 5, 141-151 (2024).

[4] Nihtyanova, S.I. & Denton, C.P. Pathogenesis of systemic sclerosis associated interstitial lung disease. J Scleroderma Relat Disord 5, 6-16 (2020).

[5] Miller, A.J., et al. Generation of lung organoids from human pluripotent stem cells in vitro. Nature Protocols 14, 518-540 (2019).

[6] Valenzi, E., et al. Single-cell analysis reveals fibroblast heterogeneity and myofibroblasts in systemic sclerosis-associated interstitial lung disease. Annals of the Rheumatic Diseases 78, 1379-1387 (2019).

Acknowledgments: NIL.

Disclosure of Interests: Ranjana Neelagar: None declared, Xuezhi Hong: None declared, Haodong Qi: None declared, Yanhua Xiao: None declared, Yi-Nan Li: None declared, Martin Regensburger: None declared, Franz Marxreiter: None declared, Tim Filla: None declared, Andrea-Hermina Györfi: None declared, James Adjaye: None declared, Juergen Winkler: None declared, Jörg Distler Stock owner of 4D Science and Scientific head of FibroCure, Actelion, Active Biotech, Anamar, ARXX, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB, Anamar, Active Biotech, Array Biopharma, aTyr, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, Novartis, Sanofi-Aventis, RedX, UCB, Alexandru-Emil Matei: None declared.