MSC-extracellular vesicle microRNAs target host cell-entry receptors in COVID-19: in silico modeling for in vivo validation

Abstract

Background: Coronavirus disease 2019 (COVID-19) has created a global pandemic with significant morbidity and mortality. SARS-CoV-2 primarily infects the lungs and is associated with various organ complications. Therapeutic approaches to combat COVID-19, including convalescent plasma and vaccination, have been developed. However, the high mutation rate of SARS-CoV-2 and its ability to inhibit host T-cell activity pose challenges for effective treatment. Mesenchymal stem cells (MSCs) and their extracellular vesicles (MSCs-EVs) have shown promise in COVID-19 therapy because of their immunomodulatory and regenerative properties. MicroRNAs (miRNAs) play crucial regulatory roles in various biological processes and can be manipulated for therapeutic purposes.

Objective: We aimed to investigate the role of lyophilized MSC-EVs and their microRNAs in targeting the receptors involved in SARS-CoV-2 entry into host cells as a strategy to limit infection. In silico microRNA prediction, structural predictions of the microRNA-mRNA duplex, and molecular docking with the Argonaut protein were performed.

Methods: Male Syrian hamsters infected with SARS-CoV-2 were treated with human Wharton's jelly-derived Mesenchymal Stem cell-derived lyophilized exosomes (Bioluga Company)via intraperitoneal injection, and viral shedding was assessed. The potential therapeutic effects of MSCs-EVs were measured via histopathology of lung tissues and PCR for microRNAs.

Results: The results revealed strong binding potential between miRNA‒mRNA duplexes and the AGO protein via molecular docking. MSCs-EVs reduced inflammation markers and normalized blood indices via the suppression of viral entry by regulating ACE2 and TMPRSS2 expression. MSCs-EVs alleviated histopathological aberrations. They improved lung histology and reduced collagen fiber deposition in infected lungs.

Conclusion: We demonstrated that MSCs-EVs are a potential therapeutic option for treating COVID-19 by preventing viral entry into host cells.

Keywords: COVID-19; MSC–EVs; MicroRNAs; Molecular docking; Receptors for viral entry.

Fig. 1

Timeline of the experimental design

Fig. 2

2D structural models of miRNAs) miR-200c-3p, miR-26b-5p and miR-125b-5p (; 2D and 3D structural models of microRNAs duplexes with mRNA of ACE2 gene; Binding sites interactions between amino acid residues of Argonaut protein and microRNAs- ACE2 duplexes and surface shape of argonaut silencing complexes

Fig. 3

2D structural models of miR-17-5p, miR-27b-3p and miR-143-3p; 2D and 3D structural models of different microRNAs duplexes with mRNA of ACE2 gene; Binding sites interactions between amino acid residues of Argonaut protein and microRNAs- ACE2 duplexes and surface shape of argonaut silencing complexes

Fig. 4

2D structural models of miR-98-5p, miR-214-3p and miR-32-3p; 2D and 3D structural models of different microRNAs (miR-98-5p, miR-214-3p and miR-32-3p) duplexes with mRNA of TMPRESS2 gene; Binding sites interactions between amino acid residues of Argonaut protein and microRNAs- TMPRESS2 duplexes and surface shape of argonaut silencing complexes

Fig. 5

2D structural models of miR-98-3p, miR-92a-3p and miR-31-5p; 2D and 3D structural models of miR-98-3p with mRNA of ITGA3 gene and miR-92a-3p, miR-31-5p with mRNA of ITGA5 gene; Binding sites interactions between amino acid residues of Argonaut protein and microRNAs- ITGA duplexes and surface shape of argonaut silencing complexes

Fig. 6

2D structural models of miR-32-5p, miR-30a-5p and let-7f-5p; 2D and 3D structural models of miR-32-5p with mRNA of ITGAV, miR-30a-5p with mRNA of CTSL1, let-7f-5p with mRNA of TMPRESS2 gene; Binding sites interactions between amino acid residues of Argonaut protein and microRNAs- ITGAV, CTSL1 and TMPRESS2 duplexes and surface shape of argonaut silencing complexes

Fig. 7

Viral shedding is unaltered following exosome treatment. Viral shedding from infected and infected + MSCs–EVs was determined at days 0, 2, 4, 6, and 8. The x-axis indicates time (days), and y-axis indicates Viral Shedding (log ct)

Fig. 8

Immunohistochemical reaction for MSCs–EVs localization in lung tissues. a control group: negative immune reaction for CD105, b covid group: negative immune reaction for CD105, c MSCs–EVs group: Strong immune reaction for CD105 (arrow). d Allred score for CD105 expressed as mean ± SEM.***p < 0.001 versus the control group, ###p < 0.001 versus the covid group. e control group: negative immune reaction for CD73, f covid group: negative immune reaction for CD73, g MSCs–EVs group: Strong immune reaction for CD73 (arrow). h) Allred score for CD73 expressed as mean ± SEM.***p < 0.001 versus the control group, ###p < 0.001 versus the covid group

Fig. 9

MSC–EVs administration reduces covid induced inflammatory responses. a Serum CRP; b serum WBCs; c Serum neutrophil /lymphocyte. Data are expressed as mean ± SEM. *p < 0.05 versus the control group, ****p < 0.0001 versus the control group, ##p < 0.01 versus the covid group, ####p < 0.0001 versus the covid group

Fig. 10

MSC–EVs and their role in miRNA expression regulation. a Fold change of ACE2 receptor interacting microRNAs miR-17-5p, miR-26b-5p, miR-27b-3p, miR-125b-5p, miR-143-3p and miR-200c-3p in control, covid and MSC–EVs groups.; b Fold change of TMPRESS2 receptor interacting microRNAs miR-98-5p, miR-214-3p, miR-32-3p and let-7f-5p in control, covid and MSC–EVs groups. c Fold change of ITGA receptors interacting microRNAs miR-31-5p, miR-98-5p, miR-32-5p and miR-92a-3p in control, covid-infected and MSC–EVs groups.; d Fold change of CTSL1 receptor interacting microRNA miR-30a-5p in control, covid and MSC–EVs groups. Data are expressed as mean ± SEM. *p < 0.05 versus the control group, **p < 0.01 versus the control group, ***p < 0.001 versus the control group, ****p < 0.0001 versus the control group, #p < 0.05 versus the covid group, ##p < 0.01 versus the covid group, ###p < 0.001 versus the covid group, ####p < 0.0001 versus the covid group

Fig. 11

Representative photomicrographs of lung immune stained sections for a–c TMPRSS2 expression a Control group: moderate immune expression (bold arrow) b Covid group: intense brown granular cytoplasmic positivity (arrow) in the alveolar cells c) MSCs–EVs group: mild to moderate brown granular cytoplasmic positivity (bold arrow) in the alveolar cells. e–f ACE2 expression e Control group: mild immunoreactivity in some alveolar cells. f Covid group: intense immunoreactivity in a large number of cells. g MSCs–EVs group: moderate reaction in the alveolar cells. d and h Allred score for TMPRSS2 and ACE2 expression. Data are expressed as mean ± SEM. *p < 0.05 versus the control group, **p < 0.01 versus the control group, ***p < 0.001 versus the control group, ###p < 0.001 versus covid group. Allred index (0–1 = negative, 2–3 = mild, 4–6 = moderate, and 7–8 = strongly positive)

Fig. 12

Representative photomicrographs from SARS-CoV-2 immune stained lung sections showing a Control group: negative immune reactivity b Covid group: intense brown granular cytoplasmic immune reactivity (arrow) in the alveolar epithelium c MSCs–EVs group: mild to moderate brown granular cytoplasmic positivity (arrowhead) in the alveolar epithelium. d Allred score for SARS-CoV-2 immune expression. Data are expressed as mean ± SEM. **p < 0.01 versus the control group, ***p < 0.001 versus the control group, ###p < 0.001 versus the covid group,####p < 0.0001 versus the covid group. Allred index (0–1 = negative, 2–3 = mild, 4–6 = moderate, and 7–8 = strongly positive)

Fig. 13

Representative photomicrographs from (a–c) hematoxylin and eosin and (e–g) Masson trichrome stained lung sections. H&E: a Control group: patent alveolar lumens and thin interalveolar septa (double arrow) lined by squamous type I pneumocytes (arrowhead) and large cuboidal type II pneumocytes (short arrow). An alveolar capillary (c) is also seen. b Covid group: Lung consolidation with diffuse thickening of the interalveolar septa, abundant inflammatory cells (if), collapsed lumens (arrow), intra alveolar hemorrhage (asterisk) and congested blood vessels (bv). c MSCs–EVs group: with predominantly patent alveolar lumens and thin interalveolar septa (arrow) and residual thickening of the interalveolar septa with inflammatory cell infiltration (if) and congested blood vessels (bv). d Assessment of lung with the lung injury scoring system. Data are expressed as mean ± SEM.***p < 0.001 versus the control group, ###p < 0.001 versus the covid group. Masson trichrome: e Control group: fine collagen fibers in the interalveolar septa and surrounding the bronchioles and blood vessels. f Covid group: abundant, intensely staining collagen fibers in the thickened interalveolar septa (arrow) and surrounding the bronchioles (double arrow). g MSCs–EVs group: fine, intensely stained collagen fibers in the interalveolar septa (arrow) and around the blood vessels. h Mean area percentage of collagen fibers in the different experimental groups. Data are expressed as mean ± SEM. ***p < 0.001 versus the control group, ###p < 0.001 versus the covid group