Experimental model reveals possible causes and treatments for long COVID

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In a recent study published in the journal Science Translational Medicineresearchers characterized the long-term pulmonary consequences of infection with the mouse-adapted strain (MA10) of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) in BALB/c laboratory mice.

Study: SARS-CoV-2 infection produces chronic dysfunction of lung epithelium and immune cells with fibrosis in mice. Image Credit: eamesBot / Shutterstock

Background

Due to a lack of longitudinal tissue samples, the mechanistic basis of the post-acute sequelae of SARS-CoV-2-related pulmonary abnormalities (PASC) is barely understood. Moreover, little is known about the underlying mechanisms governing nonviral chronic active pneumonia (CAP) or pulmonary fibrosis (PF) in humans, providing incomplete roadmaps for studies of the pulmonary pathogenesis of the disease. SARS-CoV-2. Here it should also be noted that human autopsy samples are heterogeneous and only describe the disease at a specific time. Therefore, they do not elucidate the pathogenesis of post-SARS-CoV-2 lung disease.

Mice infected with MA10 suffer from acute respiratory distress syndrome (ARDS) similar to humans. Therefore, BALB/c mice present an opportunity to study PASC pathogenesis from acute to clinical recovery phases. Additionally, this mouse model facilitates testing of countermeasures to improve PASC. Previous studies have also not described PASC-like disease phenotypes in the lungs after virus clearance.

About the study

In the present study, the researchers inoculated 103 plaque-forming units (PFU) of SARS-CoV-2 MA10 in 1-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 similar disease severity.

Following recommendations for diagnosing different phases of COVID-19 in humans, the team autopsied these mice at two, seven, 15, 30, 60 and 120 days post infection (dpi). They used the recovered samples for the study analyses.

The team used complementary virological and histological methods to assess lung damage in surviving mice. Additionally, they used digital spatial profiling (DSP) to identify transcript profiles during acute and chronic phases of disease to characterize tissue damage and repair in mice and humans. The team supplemented these techniques with immunohistochemistry (IHC) and computed tomography (CT). They also used ribonucleic acid in situ hybridization (RNA-ISH) to validate the data obtained from the DSP analyses. Finally, they investigated measures to identify early biomarkers to identify PASC and assessed countermeasures to prevent lung disease during PASC.

Study results

Older mice that survived MA10 infection cleared the infection by 15 dpi. Like humans, they had damaged lung epithelia that developed into persistent lung lesions, and micro-CT also revealed subpleural opacities and fibrosis.

The lesions were heterogeneous and varied in severity between 30 and 120 dpi. Additionally, these mice had abnormally reparative type II alveolar epithelial cells (AT2) and interstitial macrophages, as well as persistent lung damage. In the subpleural regions, they showed myofibroblast proliferation, accumulated lymphoid cells, and deposited interstitial collagen.

SARS-CoV-2 MA10 infection causes lung damage in surviving aged mice.  One-year-old female BALB/c mice were infected with 103 PFU SARS-CoV-2 MA10 (n=74) or PBS (n=24) and monitored for (A) percentage of baseline weight and (B ) the survival.  (C) Lung titers of log-transformed infectious virus were assayed at the indicated time points.  The dotted line represents LOD.  Undetected samples are plotted at half LOD.  (D to F) Lung function was assessed by whole body plethysmography for (D) PenH, (E) Rpef and (F) EF50.  (G) Histopathological analysis of the lungs at the indicated time points is shown.  H&E indicates hematoxylin and eosin staining.  SMA indicates DAB labeling immunohistochemistry (brown) for α smooth muscle actin.  The Picrosirius Red stain (bright pink-red) highlights the 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 section examined, 1 = less than 5%;  2 = 6 to 10%;  3 = 11 to 50%;  4 = 51-95%;  5 = greater than 95%.  Graphs represent autopsied individuals at each time point (C and H), with the mean value for each treatment and error bars representing the standard error of the mean.  Simulated infected animals are represented by open black circles and animals infected with SARS-CoV-2 MA10 are represented by closed red circles.SARS-CoV-2 MA10 infection causes lung damage in surviving aged mice. One-year-old female BALB/c mice were infected with 103 PFU SARS-CoV-2 MA10 (n=74) or PBS (n=24) and monitored for (A) percentage of baseline weight and (B ) the survival. (C) Lung titers of log-transformed infectious virus were assayed at the indicated time points. The dotted line represents LOD. Undetected samples are plotted at half LOD. (D to F) Lung function was assessed by whole body plethysmography for (D) PenH, (E) Rpef and (F) EF50. (G) Histopathological analysis of the lungs at the indicated time points is shown. H&E indicates hematoxylin and eosin staining. SMA indicates DAB labeling immunohistochemistry (brown) for α smooth muscle actin. The Picrosirius Red stain (bright pink-red) highlights the 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 section examined, 1 = less than 5%; 2 = 6 to 10%; 3 = 11 to 50%; 4 = 51-95%; 5 = greater than 95%. Graphs represent autopsied individuals at each time point (C and H), with the mean value for each treatment and error bars representing the standard error of the mean. Simulated infected animals are 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 several pro-inflammatory and pro-fibrotic cytokines. These included 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 exhibited prolonged upregulation of TGF-β signaling, as observed during DSP and RNA-ISH. Previous studies have observed similar heterogeneous cellular and fibrotic features in the subpleural regions of advanced COVID-19 patients.

Infection in the bronchioles, particularly in the subpleural regions, provided clues to the etiology of the advanced alveolar CAP/PF response. Despite similar infections, the bronchioles were repaired without any signs of fibrotic sequelae. In all likelihood, tissue-specific ISG responses protected the bronchioles from this undesirable fate.

Additionally, CD4+ and CD8+ T-cell populations increased in areas of the lungs of mice with SARS-CoV-2, and peripheral lymphoid aggregations characterized chronic disease. Consistent with human studies, DSP and flow cytometry data confirmed expansion of the interstitial macrophage population. More importantly, the study data confirmed that replication-defective and pro-inflammatory transitional cells, including alveolar differentiation intermediate (ADI)/damage-associated transient progenitors (DATP)/state pre-AT1 transitional cell (PATS) emerge early after SARS-CoV-2 infection and persist with ongoing inflammation and repair failure.

The authors first observed these cells at two dpi in test animals, and they persisted up to 30 dpi in diseased but not morphologically intact alveolar regions. Histological studies have shown activation of extracellular matrix pathways related to ADI/DATP/PATS cells in subpleural areas.

Early treatment with molnupiravir weakened chronic PASC in the SARS-CoV-2 MA10 mouse model. Similarly, early administration of a direct-acting antiviral, nintedanib, also blunted peak fibrotic responses to SARS-CoV-2 between seven and 15 dpi. However, additional studies could confirm these results and evaluate other anti-fibrotic drugs for PASC treatments.

conclusion

Overall, the current study modeled chronic SARS-CoV-2 to help longitudinally investigate the molecular pathways mediating the long-term pulmonary sequelae of COVID-19 to evaluate treatments for human PASC. The study results also provided clues to the role of host genetics in defining PASC outcomes. As for countermeasures, the SARS-CoV-2 MA10 model could help rapidly test agents that may counter the pulmonary CAP/PF effects of COVID-19 in longer clinical trials.

Journal 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- infection 2 produced chronic lung infection epithelial and immune cell dysfunction with fibrosis in mice, Science Translational Medicine 2022, DOI: 10.1126/scitranslmed.abo5070, https://www.science.org/doi/10.1126/scitranslmed.abo5070
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