In a recent study published in the journal Science Translational Medicine, researchers characterized the long-term pulmonary consequences of mouse-adapted strain (MA10) infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in BALB laboratory mice / c.
Study: SARS-CoV-2 infection produces chronic dysfunction of lung epithelial and immune cells with fibrosis in mice. Image credit: eamesBot / Shutterstock
Fund
Due to the lack of longitudinal tissue samples, the mechanistic basis for post-acute sequelae of SARS-CoV-2-related lung abnormalities (PASC) is barely understood. In addition, little is known about the underlying mechanisms governing chronic non-viral active pneumonia (CAP) or pulmonary fibrosis (PF) in humans, providing incomplete roadmaps for SARS-CoV-2 lung pathogenesis studies. Here it should also be noted that human autopsy samples are heterogeneous and describe the disease only at a specific time. Therefore, they do not elucidate the pathogenesis of post-SARS-CoV-2 lung disease.
Mice infected with MA10 suffer from an acute respiratory distress syndrome (ARDS) similar to that of humans. Therefore, BALB / c mice present an opportunity to investigate the pathogenesis of PASC from the acute recovery phases to the clinic. In addition, this murine model facilitates countermeasures testing to improve PASC. Previous studies have also not described PASC-like disease phenotypes in the lung after virus clearance.
About the study
In the present study, researchers inoculated 103 plaque-forming units (PFUs) of SARS-CoV-2 MA10 into one-year-old female BALB / c mice to induce severe acute disease. Similarly, they inoculated 10-week-old mice with a higher inoculum of MA10 (104 PFU) to induce a similar severity of the disease.
According to the recommendations to diagnose different phases of COVID-19 in humans, the team performed an autopsy of these mice at two, seven, 15, 30, 60, and 120 days after infection (dpi). They used the recovered samples for the analysis of the study.
The team used complementary virological and histological methods to assess lung damage in surviving mice. In addition, they used the digital spatial profile (DSP) to identify transcriptional profiles during the acute and chronic disease phases to characterize tissue damage and repair in mice and humans. The team complemented these techniques with immunohistochemistry (IHC) exploration and computed tomography (CT). They also used in situ ribonucleic acid hybridization (RNA-ISH) to validate data obtained from DSP analyzes. Finally, they investigated measures to identify early biomarkers to identify PASC and evaluated countermeasures to prevent lung disease during PASC.
Study results
Larger mice that survived MA10 infection eliminated infection at 15 dpi. Like humans, they had damaged lung epithelia that turned into persistent lung lesions, and micro-CT also revealed subpleural opacity and fibrosis.
The lesions were heterogeneous and ranged in severity from 30 to 120 dpi. In addition, these mice had type II alveolar epithelial cells (AT2) and interstitial macrophages that repaired abnormally along with persistent lung lesions. In the subpleural regions, they had myofibroblast proliferation, accumulated lymphoid cells, and deposited interstitial collagen.
SARS-CoV-2 MA10 infection causes lung damage in aged surviving mice. One-year-old BALB / c mouse females were infected with 103 PFU SARS-CoV-2 MA10 (n = 74) or PBS (n = 24) and controlled for (A) percent initial weight and (B) ) survival. (C) Lung titles of registry-transformed infectious viruses were analyzed at the indicated time points. The dotted line represents the LOD. Undetected samples are represented at half the LOD. (D to F) Pulmonary function was assessed by full-body plethysmography for (D) PenH, (E) Rpef, and (F) EF50. (G) Histopathological analysis of the lungs is shown at the indicated time points. H&E indicates hematoxylin and eosin staining. SMA indicates DAB-labeled immunohistochemistry (brown) for α-smooth muscle actin. Picrosirius red stain (bright pink-red) highlights collagen fibers. Image scale bars represent 1000 μm for low magnification and 100 μm for 400X images. (H) The disease incidence score is shown for the indicated time points: 0 = 0% of the total area of the examined section, 1 = less than 5%; 2 = 6 to 10%; 3 = 11 to 50%; 4 = 51 to 95%; 5 = more than 95%. The graphs represent necropsied individuals at each time point (C and H), with the mean value of each treatment and the error bars represent the standard error of the mean. Simulated infected animals represented by open black circles and animals infected with SARS-CoV-2 MA10 are represented by closed red circles.
These mice also had elevated levels of various proinflammatory and profibrotic cytokines. These include interleukin-1Beta (IL-1β), IL-33, IL-17A, tumor necrosis factor-alpha (TNF-α), granulocyte-macrophage colony stimulating factor (GM-CSF), and tumor growth factor- beta. TGF-β).
Although most cytokines returned to their normal levels at 30 dpi, subpleural fibrotic regions showed prolonged regulation of TGF-β signaling, as observed during DSP and RNA-ISH. Previous studies have observed heterogeneous cellular and fibrotic characteristics similar to the subpleural regions of patients with late-stage COVID-19.
Bronchial infection, especially in the subpleural regions, provided clues about the etiology of the late-stage CAP / PF alveolar response. Despite similar infections, the bronchioles were repaired without any evidence of fibrotic sequelae. In all likelihood, tissue-specific ISG responses protected the bronchioles from this adverse fate.
In addition, CD4 + and CD8 + T cell populations increased in SARS-CoV-2 diseased areas of mouse lungs and peripheral lymphoid aggregations characterized chronic disease. According to human studies, DSP and flow cytometry data confirmed the expansion of the interstitial macrophage population. Most importantly, the study data confirmed that replication cells are defective for replication and proinflammatory, including the alveolar differentiation intermediate (ADI) / the transient parent associated with damage (DATP) / pre-AT1 (PATS) transition cell status emerges soon after SARS-CoV. -2 infection and persists with continued inflammation and repair failure.
The authors first observed these two-dpi cells in the test animals and persisted up to 30 dpi in diseased but not morphologically intact alveolar regions. Histological studies showed the activation of extracellular matrix pathways related to ADI / DATP / PATS cells in the subpleural areas.
Early treatment with molnupiravir weakened chronic PASC in the SARS-CoV-2 MA10 mouse model. Similarly, early administration of direct-acting antivirals, Nintedanib also reduced maximal fibrotic responses to SARS-CoV-2 by between seven and 15 dpi. However, additional studies could confirm these findings and evaluate other antifibrotic drugs for PASC treatments.
Conclusions
Overall, the current study modeled chronic SARS-CoV-2 to help longitudinally study the molecular pathways that mediate long-term COVID-19 lung sequelae to evaluate treatments for human PASC. The study’s findings also provided clues about the role of host genetics in defining PASC outcomes. In terms of countermeasures, the SARS-CoV-2 MA10 model could help to rapidly test agents that may counteract the CAP / PF lung effects of COVID-19 during longer clinical trials.
Magazine reference:
- Kenneth H. Dinnon Iii, Sarah R. Leist, Kenichi Okuda, Hong Dang, Ethan J. Fritch, Kendra L. Gully, Gabriela De La Cruz, Mia D. Evangelista, Takanori Asakura, Rodney C. Gilmore, Padraig Hawkins, Satoko Nakano , Ande West, Alexandra Schäfer, Lisa E. Gralinski, Jamie L. Everman, Satria P. Sajuthi, Mark R. Zweigart, Stephanie Dong, Jennifer Mcbride, Michelle R. Cooley, Jesse B. Hines, Miriya K. Lovesteve D. Groshong , Alison Vanschoiack, Stefan J. Phelan, Tyler Hether, Michael Leon, Ross E. Zumwalt, Lisa M. Barton, Eric J. Duval, Sanjay Mukhopadhyay, Edana Stroberg, Alain Borczuk, Leigh B. Thorne, Muthu K. Sakthivel, Yueh Z. Lee, James S. Hagood, Jason R. Mock, Max A. Seibold, Wanda K. O’neal, Stephanie A. Montgomery, Richard C. Boucher, Ralph S. Baric, SARS-CoV-2 infection produces a chronic lung disease dysfunction of epithelial and immune cells with fibrosis in mice, Science Translational Medicine 2022, DOI: 10.1126 / scitranslmed.abo5070,