Salivary miRNA Profiles in COVID-19 Patients with Different Disease Severities

Abstract

The oral mucosa is the first site of SARS-CoV-2 entry and replication, and it plays a central role in the early defense against infection. Thus, the SARS-CoV-2 viral load, miRNAs, cytokines, and neutralizing activity (NA) were assessed in saliva and plasma from mild (MD) and severe (SD) COVID-19 patients. Here we showed that of the 84 miRNAs analyzed, 8 were differently expressed in the plasma and saliva of SD patients. In particular: (1) miRNAs let-7a-5p, let-7b-5p, and let-7c-5p were significantly downregulated; and (2) miR-23a and b and miR-29c, as well as three immunomodulatory miRNAs (miR-34a-5p, miR-181d-5p, and miR-146) were significantly upregulated. The production of pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IL-8, IL-9, and TNFα) and chemokines (CCL2 and RANTES) increased in both the saliva and plasma of SD and MD patients. Notably, disease severity correlated with NA and immune activation. Monitoring these parameters could help predict disease outcomes and identify new markers of disease progression.

Keywords: COVID-19; SARS-CoV-2; epigenetic profile; miRNA; non-coding RNA.

Figure 1

SARS-CoV-2 viral replication and neutralizing activity in saliva and plasma samples of MD and SD patients. Viral replications N1 and N2 of SARS-CoV-2 are reported in panel (A). Neutralizing activity (NA) from saliva and plasma of 10 MD patients (light blue bars) and 10 SD patients (blue bars), measured by virus neutralization assay (vNTA), are reported in panels (B) and (C), respectively. The Spearman correlation between the SARS-CoV-2 viral copy and NA level (D). Significance was indicated as follows: * = p < 0.05.

Figure 2

MiRNA expression analysis in saliva samples by PCR array. miRNA profile of SD patients, compared to MD patients (A). Analyses of miRNA with antiviral (B) and immunological functions (C) were performed in saliva samples of 10 HC (white bar), 10 MD patients (light blue bars), and 10 SD patients (blue bars). Results were calculated relative to the arithmetical mean of the references available in the arrays RNU6-2. Values were mean ± SEM. Significance was indicated as follows: * = p < 0.05, and ** = p < 0.01.

Figure 3

miRNA expression analysis in plasma samples by PCR array. miRNA profile of SD patients, compared to MD patients (A). Analyses of miRNA with antiviral (B) and immunological functions (C) were performed on saliva samples of 10 HC (white bar), 10 MD patients (light blue bars), and 10 SD patients (blue bars). Results were calculated relative to the arithmetical mean of the references available in the arrays RNU6-2. Values were mean ± SEM. Significance was indicated as follows: * = p < 0.05, and ** = p < 0.01.

Figure 4

mRNA expression of genes involved in the antiviral/immune response. Quantigene Plex Gene expression technology was applied to quantify gene expression on RNA extracted from PBMCs isolated from 10 MD patients (light blue bars) and 10 SD patients (blue bars). Results were calculated relative to the arithmetical mean of the references available in the panel: GAPDH, b-Actin, and PPI. Only statistically significant p-values from the t-test comparison between SD and MD patients are shown * = p < 0.05, and ** = p < 0.01.

Figure 5

Plasma secretion of cytokines/chemokines that were part of the inflammatory response. The production of 27 cytokines/chemokines regulating immune response was assessed by Luminex assay in saliva (A) and plasma (B) specimens of 10 HC, 10 MD patients, and 10 SD patients. Cytokine/chemokine productions (mean values) are shown as a color scale from white to blue (heatmap). Only statistically significant p-values between at least 2 groups were reported, along with the percentage of difference in lower panels of (A, saliva) and (B, plasma).