Evaluation of Cellular Senescence in Lung Tissue of Patients with Chronic Obstructive Pulmonary Disease (COPD) and Idiopathic Pulmonary Fibrosis (IPF)

Introduction: Chronic Obstructive Pulmonary Disease (COPD) and Idiopathic Pulmonary Fibrosis (IPF) are respiratory disorders strongly associated with advanced age and premature cellular senescence. Senescent cells are commonly identified by senescence-associated β-galactosidase or by cell cycle inhibitors such as p16 and p21. However, they can also be evaluated by measuring senescence-associated products, such as lipofuscin. Sudan Black B (SBB) has been recently recognized as a specific lipofuscin staining applicable to both cryo‐preserved and archival tissues. Aim of the study: To investigate cellular senescence in COPD and IPF using lipofuscin as the main marker. Specifically, we evaluated whether lung parenchyma from COPD and IPF patients shows a higher density of lipofuscin-positive cells than controls and explored correlations with key clinical characteristics. Methods: This is an observational retrospective single-center study. We recruited patients with COPD and IPF, along with control subjects, at the San Luigi Gonzaga University Hospital (Orbassano, Italy). All subjects had undergone lung resection to eradicate lung cancer or surgical lung biopsy for a diagnostic purpose between April 2006 and July 2024. In COPD patients and control subjects, cellular senescence was assessed using lipofuscin. In IPF patients, it was assessed using lipofuscin together with p16 and p21. Standard SBB was used to stain lipofuscin, while immunohistochemistry was performed to evaluate p16 and p21. Results: In the first part of the study, 20 patients with COPD were compared with 35 control subjects divided into smokers (CTRLs, N=24) and non-smokers (CTRLns, N=11). The density of lipofuscin-positive senescent cells was higher in COPD than in control subjects (median 4.5% in COPD vs 2.5% in CTRLs, with p = 0.0313, and vs 2.8% in CTRLns, with p = 0.0159). In COPD patients, a correlation between the density of senescent cells and a wide spectrum of respiratory function parameters emerged. In the second part of this study, 21 patients with IPF were compared to control subjects. A single control group was created to match age and smoking history found in the IPF group. The density of lipofuscin-positive cells resulted higher in IPF than in control subjects (mean 4.5% vs 3% respectively, p = 0.0042). All IPF patients tested negative for p21 and almost half showed a p16-positivity. Except for higher prevalence of arterial hypertension and longer interval between tissue sampling and testing in the negative group, no significant difference emerged based on p16 positivity. In patients with IPF, we did not find a correlation between the density of lipofuscin-positive cells and a series of clinical variables, but a correlation emerged with the change in respiratory function in the years following biopsy, more robust with the change in forced vital capacity (FVC) in the first year after biopsy (regression coefficient -0.77, 95%CI -1.25 – -0.29, R-squared 0.51, p = 0.0043). No difference in overall survival emerged when comparing patients based on p16 or lipofuscin positivity. Conclusions: This study confirmed an increase in lipofuscin-positive senescent cells in lung parenchyma of patients with COPD and showed it for the first time in IPF. This supports the central role of cellular senescence in both diseases. In COPD, the density of lipofuscin-positive cells correlated with a wide spectrum of respiratory function parameters, while in IPF correlated with the short-term change in FVC, demonstrating to be a possible indicator of a more aggressive disease. The assessment of p16 and p21 appeared less reliable, possibly due to the long time elapsed between tissue sampling and testing. Prospective studies are needed to better investigate the correlation between lipofuscin positivity and functional trends in IPF and COPD.