Evidence of the pathogenic HERV-W envelope expression in T lymphocytes in association with the respiratory outcome of COVID-19 patients

Abstract

Background: Despite an impressive effort in clinical research, no standard therapeutic approach for coronavirus disease 2019 (COVID-19) patients has been established, highlighting the need to identify early biomarkers for predicting disease progression and new therapeutic interventions for patient management. The present study aimed to evaluate the involvement of the human endogenous retrovirus -W envelope (HERV-W ENV) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection considering recent findings that HERVs are activated in response to infectious agents and lead to various immunopathological effects. We analysed HERV-W ENV expression in blood cells of COVID-19 patients in correlation with clinical characteristics and have discussed its potential role in the outcome of the disease.

Methods: We analysed HERV-W ENV expression in blood samples of COVID-19 patients and healthy donors by flow cytometry and quantitative reverse transcriptase PCR analysis, and evaluated its correlation with clinical signs, inflammatory markers, cytokine expression, and disease progression.

Findings: HERV-W ENV was highly expressed in the leukocytes of COVID-19 patients but not in those of healthy donors. Its expression correlated with the markers of T-cell differentiation and exhaustion and blood cytokine levels. The percentage of HERV-W ENV-positive lymphocytes correlated with inflammatory markers and pneumonia severity in COVID-19 patients. Notably, HERV-W ENV expression reflects the respiratory outcome of patients during hospitalization.

Interpretation: Given the known immuno- and neuro-pathogenicity of HERV-W ENV protein, it could promote certain pathogenic features of COVID-19 and therefore serve as a biomarker to predict clinical progression of disease and open to further studies for therapeutic intervention.

Funding: Information available at the end of the manuscript.

Keywords: COVID-19; Cytokine storm; HERV-W. human endogenous retroviruses; Inflammation; Respiratory outcome; T-cell exhaustion.

Fig. 1

Pathogenic HERV-W ENV mRNA expression and the percentage of HERV-W ENV protein positive cells are significantly expressed in blood cells of COVID-19 patients versus healthy donors. HERV-W expression in blood samples and the percentage of HERV-W ENV-positive cells were analysed in thirty hospitalized COVID-19 patients (COV, gray box plots) and seventeen healthy donors (HD, white box plots). (a) HERV-W ENV expression (mRNA) and HERV-W ENV protein were analysed in leukocytes by qRT-PCR and flow cytometry, respectively. (b) Scatter Plots of the HERV-W ENV mRNA levels (Y-axis) and the percentage HERV-W ENV-positive leukocytes (left) and lymphocytes (right) (X-axis). Pairwise associations between continuous variables were tested through the Spearman correlation coefficient (Rho). Statistically significant values were considered when p <0.050. (c) ROC Curve of the% HERV-W ENV in Lymphocytes (green line), leukocytes (blue line) and HERV-W ENV mRNA levels (orange line). (d) Different subpopulations of leukocytes were analysed according to the morphology, and cell phenotype using specific phenotypic markers (see the gating strategy in supplementary data Fig.S1). The percentage of HERV-W ENV-positive cells has been evaluated in lymphocytes, monocytes, granulocytes, CD3+ (T Lymphocytes), CD4+ (T Helper cells), CD8+ (T Cytotoxic cells), CD19+ (B Lymphocytes), CD56+ (Natural Killer cells) and CD14+ (monocytes) of COVID-19 patients and healthy donors. Non-parametric Mann–Whitney test was used to compare the two groups. Statistically significant values were considered when p <0.050.

Fig. 2

The percentage of HERV-W ENV-positive lymphocytes correlates with markers of T cell differentiation and exhaustion. Scatter plot of the percentage of HERV-W ENV-positive lymphocytes (X axis) and the expression of markers of differentiation and exhaustion in CD3+CD4+ T cells (a) and CD3+CD8+ T cells (b) (Y axis) in COVID-19 patients (n = 30, black dots) and healthy donors (n = 17, white dots). The gating strategy to analyze markers related to differentiation, activation status, senescence, and exhaustion in T cells was provided by Beckman Coulter (Duraclone), specifically, naïve (NAIVE) (CCR7+CD45RA+CD28+CD27+), central memory (CM) (CCR7−CD45RA+CD28+CD27+/−), effector memory (EM) (CCR7−CD45RA−CD28+/−CD27+/−), terminal effector memory (TEM) (CCR7−CD45RA+CD28−CD27+/−), PD1+ exhausted and CD57+ senescent T cells. Pairwise associations between continuous variables were tested through the Spearman correlation coefficient. Statistically significant values were considered when p <0.050.

Fig. 3

HERV-W ENV mRNA expression correlates with the expression of several pro-inflammatory cytokines and innate immunity mediators in blood samples of COVID-19 patients. Scatter plots of the HERV-W ENV mRNA levels (X-axis) and cytokine/cytokine receptor expression (Y-axis: IL-6, IL-10, IL-17, IL-17RA, TNFα, INFγ, MCP1, CXCR1, CXCL6), obtained by qRT-PCR analysis. Pairwise associations between continuous variables were tested through the Spearman correlation coefficient (Rho). Statistically significant values were considered when p <0.050 (a). PBMCs from Healthy donors(n = 4) stimulated with SARS-CoV2 spike protein (1 or 5 nM) and  IL-6 protein (10 ng) for 3,24 h and 5 days(Box plots of HERV-W ENV (b) and IL-6 (c) mRNA levels, obtained by qRT-PCR analysis, d) Box plot of the percentage of HERV-W ENV-positive in CD3+ T lymphocytes after 5 days of stimulation with SARS-CoV-2 spike protein and  IL-6 protein. For comparison the Bonferroni's post-hoc multiple comparison ANOVA test was utilized.

Fig. 4

HERV-W ENV protein expression in the leukocytes of COVID-19 patients was associated with disease severity. The COVID-19 patients have been stratified based on clinical status as asymptomatic and pauci-symptomatic (AS/PS, n = 15), mild, moderate and severe (Mild/Mod/Sev, n = 15).The percentage of HERV-W ENV-positive cells has been analysed in leukocytes, lymphocytes, monocytes, granulocytes and in T cell, B cell and NK lymphocyte subtypes. IL-6 plasma concentration was also evaluated. Data are represented as box plots (white box: healthy donors, HD; gray box: all patients positive for SARS-CoV-2). Non-parametric Kruskall Wallis test and Bonferroni's correction was used to compare groups (Table 4).

Fig. 5

Elevated percentage of HERV-W ENV-positive lymphocytes is associated with pulmonary involvement and correlates with biochemical markers in COVID-19 patients. Patients were stratified into five groups based on pulmonary status: no pneumonia or non-interstitial pneumonia (None+P, n = 7), monolateral or minimal interstitial pneumonia (MiP, n = 6), bilateral or severe pneumonia (BiP, n = 14) and pneumonia with bacterial co-infection (BiP+Bact, n = 3). (a) The percentage of HERV-W ENV-positive lymphocytes was represented as a box plot in all the groups examined and statistical difference was shown. (b) Scatter plot of the percentage of HERV-W ENV-positive CD4+ T cells (X axis) and the biochemical markers (Y axis) in COVID-19 patients. The biochemical markers examined were prothrombin time (PT, sec), prothrombin time internationl normalized ratio (PT-INR, sec), prothrombin activity percentage (PT%), d-DIMERs (ng/ml), fibrinogen (mg/dl) and lactate dehydrogenase (LDH, U/liter). Non-parametric Kruskall Wallis and Bonferroni's correction were used to compare groups; pairwise associations between the continuous variables were tested through the Spearman correlation coefficient. Statistically significant values were considered when p <0.050.

Fig. 6

Levels of HERV-W ENV mRNA expression and the percentage of positive lymphocytes reflect respiratory outcome of COVID-19 patients. The COVID-19 patients were stratified according to respiratory needs during hospitalization: no oxygen support needed (None; n = 16), oxygen support with nasal cannula or ventimask (NC/VMK; n = 8), oxygen support by non-invasive ventilation, continuous positive airway pressure or orotracheal intubation (NIV/C-PAP/OTI; n = 6). (a) The expression of HERV-W ENV mRNA in leukocytes and the percentage of HERV-W ENV-positive cells in leukocytes, lymphocytes, monocytes, granulocytes, CD4+ T cells and IL-6 in plasma were represented as box plots. Non-parametric Kruskall Wallis test and Bonferroni's correction were used to compare groups and statistically significant values were considered when p <0.050. (b) ROC Curve of the% HERV-W ENV Lymphocytes and CD3+ T cells, and IL6 plasma concentration in COV (n = 30) respect to HD (n = 17) left panel; ROC Curve of the% HERV-W ENV in Lymphocytes and in CD4+ T cells and IL-6 plasma concentration respect to the respiratory outcome. (c) Association of high expression of HERV-W ENV protein in the blood cells of COVID-19 patients, and several clinical and biological parameters linked to patient status at hospitalization and disease progression.