Impact of endoplasmic reticulum stress on lung tissue predisposition to pulmonary fibrosis and its development


Idiopathic pulmonary fibrosis (IPF) is a rare lung disease with no cure. Despite much research, the precise pathomechanism of this disease remains uncovered. Nevertheless, according to the central paradigm, apoptosis of alveolar epithelial cells type II (AECIIs) plays a crucial role in triggering the event cascade that leads to fibrosis. Several publications confirmed this hypothesis, showing that dramatic loss of AECIIs in mice leads to lung fibrosis. However, the reason for the high level of apoptosis and AECII loss in IPF lungs remains unclear. One of the mechanisms involved in fibrosis pathology that can cause cell death is the unfolded protein response (UPR) triggered by endoplasmic reticulum (ER) stress. Therefore, this thesis aimed to decipher the role of the activation of different UPR branches in AECII in the development of pulmonary fibrosis. To that end, three UPR mediators, XBP1s, ATF6p50, and CHOP, were conditionally overexpressed in the AECIIs of mouse lungs. Western blotting, immunofluorescence (IF) staining, and flow cytometry analysis showed that overexpression of XBP1s and ATF6p50 did not induce AECII apoptosis and did not change the ECM protein level (smooth muscle actin, collagen I, fibronectin) even after 12 months of the transgene overexpression. At the same time, the overexpression of CHOP induced cell death and led to reduced AECIIs number up to 40 % on the 28th day of transgene expression. Thus, CHOP may be a possible link between maladaptive ER stress and apoptosis, leading to the loss of AECIIs. We also analyzed the influence of overexpression of UPR mediators on mouse lung morphology and mechanics in the bleomycin-induced fibrosis model. According to IF staining, Masson-Goldner staining, and western blot analysis, neither XBP1s, ATF6p50, nor CHOP overexpression induced significant changes in the level of injury (CK5pos patches, apoptosis level) and ECM proteins (smooth muscle actin, fibronectin, and collagen I) after bleomycin treatment. Interestingly, tissue morphometric analysis showed a significant decrease in septal thickness and alveolar mean linear intercept in ATF6p50 overexpressing mice after bleomycin application. At the same time, no significant change in these parameters was observed in the lungs of XBP1s and CHOP overexpressing mice. Nevertheless, despite the absence of substantial morphological changes, XBP1s and CHOP overexpressing mice showed a clear trend toward worsening lung mechanics. Tissue elastance was increased, and inspiratory capacity and static compliance were decreased. Such changes can possibly indicate early pro-fibrotic alterations in the lungs. In addition, since viral infections are considered a potential trigger for acute exacerbations in IPF, and viruses can also activate UPR to their advantage, it is essential to know how the presence of the activated UPR may influence the level of infection and alveolar epithelial cell (AEC) death during this infection. To this end, we infected precision-cut lung slices (PCLS) from ATF6p50, XBP1s, and CHOP overexpressing mice with Influenza virus and analyzed the infection level and epithelial apoptosis by flow cytometry. Interestingly, ATF6p50 and XBP1 overexpression had no significant effect on influenza infection or AEC death. At the same time, CHOP overexpression positively affected the number of infected and apoptotic AEC. This fact may also explain the acute exacerbations in patients with IPF after infection. Altogether, our findings demonstrate that 1. Overexpression of ATF6p50 and XBP1s in the AECII of mouse lungs does not affect the number of AECII, does not induce their apoptosis, and does not upregulate pro-fibrotic markers. At the same time, overexpression of CHOP leads to a 40% reduction of AECII. 2. When using a low dose of bleomycin and short-term activation of the transgene, overexpression of UPR mediators does not change lung morphology but tends to worsen respiratory mechanics. 3. CHOP overexpression can increase the level of influenza virus infection and the number of apoptotic AEC during the infection.




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