Background: Systemic rheumatic diseases frequently involve the lungs in the form of interstitial lung disease (ILD), a debilitating manifestation leading to increased mortality across all connective tissue diseases (CTDs). Mortality further increases in progressive ILD, stressing the urgency of understanding and targeting progressive ILD. Identifying patients at risk of progression early in the disease course is critical for improving clinical outcomes and is a cornerstone of the 2025 ERS/EULAR clinical practice guideline for CTD-ILD. Current methods of diagnosing progressive ILD (e.g. forced vital capacity decline, high-resolution CT (HRCT), etc.) cannot detect if fibrosis is actively occurring at the time of assessment and is a poor predictor of future progression. In contrast, novel molecular diagnostic methods could identify and predict active progressive fibrosis earlier, before loss of lung function. Development of such methods will require an improved molecular understanding of the mechanisms underlying progressive fibrosis in ILD. However, identification of molecular markers and drivers of early stage CTD-ILD progression have been hindered by research emphasis on late-stage disease with explanted lung tissue. Therefore, we aim to identify key mechanisms driving progression early in the disease course using upper and lower airway tissues from early CTD-ILD patients.

Objectives: To identify functional molecular drivers of early CTD-ILD, and to assess nasal mucosa for display of these signatures.

Methods: Lung tissue samples collected and stored in FFPE from early CTD-ILD patients and healthy controls managed at the University Hospital Zurich, with associated clinical and HRCT data, were identified. Global spatial transcriptomics data (Visium HD) have been generated on two samples (n=1 CTD-ILD, n=1 healthy), with additional data from 13 CTD-ILD and 5 healthy controls actively being generated. Data have been processed with the R package Seurat, and analysis includes cell type composition, gene set enrichment, differential expression, and spatial niche analysis. To assess these findings in the upper airway, we used nasal mucosa samples collected via the Copan Flocked Swab from patients with early CTD-ILD (n = 26) (Oslo University Hospital) and from healthy controls (n = 10) (UCLA and Oslo). We assessed the transcriptome of nasal mucosa samples with the NanoString Autoimmune Profiling panel to identify unique molecular profiles of CTD-ILD compared to healthy controls.

Results: We observed a higher abundance of myofibroblasts in CTD-ILD tissue with the spatial transcriptomics datasets. Gene Ontology (GO) enrichment analysis of distinct spatial clusters revealed biologically relevant processes, including immune signatures localized to spatial domains, which likely represent the inflammatory niches characteristic of ILD. Notably, alveolar regions showed enrichment for interferon (IFN) response pathways. The nasal mucosa of CTD-ILD patients was also enriched for IFN type 1 and 2 response pathways as well as key components of the MAPK signaling pathway, including transcription factors JUN and FOS, and crucial signaling molecules such as STAT1, TGFB1, and TNF. These MAPK signatures may be indicative of IFN crosstalk, which has been previously reported in the lung. This finding is interesting as it provides a direct link to the prominent inflammatory signatures we independently identified in the distal lung of CTD-ILD patients and supports the use of nasal mucosa samples to study and manage CTD-ILD.

Conclusions: The convergence between distal lung tissue and the nasal mucosa suggests that the pro-fibrotic MAPK activity previously observed in the lung is reflected in the upper airways, validating our nasal swab findings and supporting the united airway hypothesis in CTD-ILD. Complementing this, our spatial transcriptomic dataset (Visium HD) independently captured interferon pathway signatures within alveolar regions of early CTD-ILD tissue, providing the precise inflammatory context in which MAPK activation occurs. Thus, our findings coherently point to a model where MAPK signaling is a foundational mechanism driving the progression of early CTD-ILD. This possibility will be further explored with our expanded Visium HD dataset described above.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: Philip Stauffer: None declared, Vyacheslav Palchevskiy: None declared, Laura Much: None declared, Phuong Phuong Diep: None declared, Magdalena Maria Aabel: None declared, Natasha Moe Boehringer Ingelheim, Thomas Gaisl: None declared, Carolin Steinack: None declared, Sharzad Lari: None declared, Elena Pachera: None declared, Øyvind Molberg: None declared, Oliver Distler Actelion, Boehringer-Ingelheim, Iten-Kohaut, Walter und Gertrud Siegenthaler Fellowship, the LOOP Zurich, Pfizer, Actelion, Amgen, Otsuka, Samuel S Weigt: None declared, John Belperio: None declared, Anna-Maria Hoffmann-Vold Boehringer Ingelheim, Janssen, Medscape, Merck Sharp & Dohme, Novartis, Roche, AbbVie, Avalyn, Astra Zeneca, Boehringer Ingelheim, Bristol Myers Squibb, Calluna Pharma, Genentech, Janssen, Medscape, Merck Sharp & Dohme, Pliant, Roche, Werfen, Astra Zeneca, Boehringer Ingelheim, Janssen.