Extracellular Vesicles In COVID-19 Pneumonia and Progression to Acute Respiratory Failure

Extracellular Vesicles In COVID-19 Pneumonia and Progression to Acute Respiratory Failure

Abstract
Extracellular vesicles (EVs) and their miRNA load are major players in intercellular communication1 and regulate the immune response and and disease progression in COVID-192. For our studies, EVs were isolated from serum of patients with variable degrees of disease severity (n=40) and from healthy controls (n=20) and total RNA isolated from EVs. Small RNA was profiled by RNA sequencing and evaluated by differential gene expression analysis (DGE). Differentially regulated miRNAs between groups fulfilling stringent selection criteria (BaseMean ≥ 50 reads, |log2FC| ≥ 1.8, padj ≤ 0.05) were then in-silico analyzed (Ingenuity Pathway Analysis, IPA, Qiagen Digital Insights, Redwood, USA), used for identifying target mRNA transcripts of miRNAs and applied for constructing signaling networks.
When patients with COVID-19 pneumonia (SARS-CoV-2 alpha variant) were compared to non-vaccinated healthy controls, DGE analysis revealed 43 significantly and differentially expressed miRNAs (25 downregulated). Network analysis for this comparison showed upregulated miR-3168, among others, targeting IL-6 and OR52N2, an olfactory smell receptor in the nasal epithelium. The comparison of patients with COVID-19 pneumonia to those who progressed to severe pulmonary failure (COVID-19 acute respiratory distress syndrome, ARDS) requiring ICU treatment and mechanical ventilation revealed twenty significantly regulated miRNAs (15 downregulated) in COVID-19 ARDS patients in comparison to those with COVID-19 pneumonia. Here, miR-3168 was significantly downregulated in COVID-19 ARDS and targeted IL-8 (CXCL8) in a network which was completely activated. Toll-like receptor 4 (TLR4) was inhibited in COVID-19 pneumonia by miR-146a-5p and upregulated in ARDS by let-7e-5p. CXCL8 regulates neutrophil recruitment into the lung causing epithelial damage whereas activated TLR4, to which SARS-CoV-2 spike protein binds strongly, increases cell surface ACE2 expression and destroys type II alveolar cells that secrete pulmonary surfactants; both resulting in pulmonary-capillary leakage and ARDS. In contrast to patients with active disease, the comparison between vaccinated (ChAdOx1 nCoV-19 vector vaccine or BNT162b2 mRNA vaccine) and healthy unvaccinated individuals without exposure to the virus did not show any significant differences in expression values of EV derived miRNAs (n=6 in each group).
Our studies reveal that EV-associated miRNAs and their targeted cellular transcripts have an important regulatory function in the immune response to SARS-CoV-2 and the progression to severe disease. They may serve as biomarker candidates or targets for pharmacologic interventions. EVs may also be useful as biocompatible carriers for future generations of COVID-19 vaccine molecules with a theoretical possibility for oral application.
1Kalluri R et al, Science 2020; 367; 2Meidert AS et al. Frontiers in Immunology 2021; 12