A Differential Signature of Circulating miRNAs and Cytokines Between COVID-19 and Community-Acquired Pneumonia Uncovers Novel Physiopathological Mechanisms of COVID-19

Abstract

Coronavirus Disease 2019 (COVID-19) pneumonia is a life-threatening infectious disease, especially for elderly patients with multiple comorbidities. Despite enormous efforts to understand its underlying etiopathogenic mechanisms, most of them remain elusive. In this study, we compared differential plasma miRNAs and cytokines profiles between COVID-19 and other community-acquired pneumonias (CAP). A first screening and subsequent validation assays in an independent cohort of patients revealed a signature of 15 dysregulated miRNAs between COVID-19 and CAP patients. Additionally, multivariate analysis displayed a combination of 4 miRNAs (miR-106b-5p, miR-221-3p, miR-25-3p and miR-30a-5p) that significantly discriminated between both pathologies. Search for targets of these miRNAs, combined with plasma protein measurements, identified a differential cytokine signature between COVID-19 and CAP that included EGFR, CXCL12 and IL-10. Significant differences were also detected in plasma levels of CXCL12, IL-17, TIMP-2 and IL-21R between mild and severe COVID-19 patients. These findings provide new insights into the etiopathological mechanisms underlying COVID-19.

Keywords: COVID-19; community-acquired pneumonia; microRNAs; plasma; soluble proteins.

Figure 1

miRNA signature in COVID-19 patients. (A) Volcano plot showing differential expression of 179 abundant miRNAs in human plasma between CAP and COVID-19 patients. Log₂ of fold change of normalised relative quantities (NRQ) and statistical significance (–log₁₀ of the p-value) from Mann-Whitney tests for each miRNA were assessed. In red, miRNAs with a corrected p value<0.05 assessed by multivariate statistical analysis. Names of the 15 miRNAs that were further validated are shown. (B) Hierarchical clustering heatmap of 35 differentially expressed miRNAs in plasma of 9 CAP and 38 COVID-19 individuals. Red and blue colour indicate upregulated and downregulated expression of COVID-19, respectively, as compared to CAP. (C) RT-qPCR of 15 validated miRNAs performed in the validation cohort. Box and whisker plots of NRQ for each miRNA are shown. A group of 4 highly stable miRNAs were used as normalisers. Statistical significance was assessed by means of multivariate statistical tests. *p<0.05, **p<0.01, ***p<0.001.

Figure 2

Multivariate regression model for classification of COVID-19 pneumonia and CAP and differences in circulating proteins. (A) Left: final logistic regression model for patient classification based on miRNAs. Odds ratio (OR), confidence interval 95% (95%CI) and p-value for each variable were calculated. Stepwise procedure, with both backward and forward search based on Akaike information criteria, to select the critical variables were employed. Right: ROC curve showing the power of discrimination between CAP and COVID-19 of the variables combination shown in the table. Inside the graph, the AUC and its 95%CI. (B) Left: relative percentage of each biological process associated with the functional pathways with 10 fold enrichment or higher are shown. Pathway enrichment analysis was assessed using Panther Classification System. Right: enrichment of each individual functional pathway within the two most relevant biological processes. (C) Measurement of soluble cytokines in plasma of 24 CAP and 85 COVID-19 individuals. ELISA assays with plasma diluted 1/2 were carried out. Box and whisker plots are shown, statistical significance was assessed by Mann-Whitney tests. *p<0.05, **p<0.01. (D) miRNAs regulating EGFR and CXCL12 according to miRTarBase 8.0. Circles show miRNAs with lower expression in COVID-19 as compared to CAP. Rectangles show miRNAs with higher expression in COVID-19 as compared to CAP. (E) Box and whisker plots of soluble cytokines in plasma of 43 mild and 42 severe COVID-19 patients. ELISA assays with plasma diluted 1/2 were carried out. Statistical significance was assessed by Mann-Whitney tests. **p<0.01, ***p<0.001, ****p<0.0001.