Endoplasmic Reticulum Stress Response Pathway Activation Patterns in Lung Fibrosis in End-stage IPF and COVID-ARDS

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Abstract Rationale: Severe COVID frequently leads to the Acute Respiratory Distress Syndrome (ARDS), which can lead to the development of irreversible fibrotic lung disease where the definitive treatment remains lung transplantation. During periods of respiratory distress and lung injury, there may be an accumulation of misfolded proteins leading to endoplasmic reticulum (ER) stress, which in turn has been linked to worse outcomes. The unfolded protein response (UPR) becomes activated to reduce protein synthesis in order to reduce ER stress. In our study, explanted lung specimens from patients with COVID fibrosis, idiopathic pulmonary fibrosis (IPF), and controls were evaluated to analyze the role of UPR/ER stress in the pathogenesis of lung injury. Methods: Patients undergoing lung transplant underwent informed consent, and 1cm cubes of explanted lung tissue (average ischemic time: 8 minutes) were immediately preserved in 20mL of 4% paraformaldehyde. Tissues were paraffin embedded, sectioned and mounted on glass slides. Samples from COVID-ARDS, IPF, and controls (n=3/group) were incubated with 5% goat serum and antibodies against: XBP-1, KRT5, KRT8, KRT17, ATF-4, ATF-6, and p21, followed by incubation with secondary antibodies prior to imaging and quantification of staining by fluorescent microscopy. Results: XBP-1, a marker of ER stress, was present in 66% of IPF (n=3), 100% of COVID-ARDS (n=3), and 100% of normal controls (n=2). KRT5 and KRT17 were present, primarily in airway basal cells, in 100% of IPF and normal control samples, and in 33% of COVID-19 samples. Increased KRT8 was identified in 100% of COVID-ARDS samples, in both airway and alveolar epithelial cells, and in 66% of IPF samples. ATF-4 and ATF-6 were not detected in any samples. There were increased levels of p21 in the distal fibrotic regions of IPF and COVID-ARDS tissues relative to controls. Conclusions: We found increased KRT8 and p21 in fibrotic areas of both IPF and COVID-19 lung explant tissues, indicating a commonality in the end-fibrosis stage of these two different pulmonary diseases. KRT8 identifies transitional cells that have been suggested to be a hallmark of pathologic repair in both COVID-ARDS and IPF. Furthermore, expression of p21 implicates senescence, together suggesting overlapping mechanisms in these conditions. Further studies are needed to further investigate common and disparate molecular pathway activation in IPF versus COVID-ARDS, with the goal of ultimately halting fibrosis and preventing the need for lung transplant.

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