Increased sHLA-G Is Associated with Improved COVID-19 Outcome and Reduced Neutrophil Adhesion

Abstract

Human leukocyte antigen (HLA) is a group of molecules involved in inflammatory and infective responses. We evaluated blood sHLA-E and sHLA-G levels in hospitalized COVID-19 patients with respiratory failure and their relationship with clinical evolution, changes in endothelial activation biomarker profile, and neutrophil adhesion. sHLA-E, sHLA-G, and endothelial activation biomarkers were quantified by ELISA assay in plasma samples. Neutrophil adhesion to endothelium was assessed in the presence/absence of patients' plasma samples. At admission, plasma levels of sHLA-G and sHLA-E were significantly higher in COVID-19 patients with respiratory failure compared to controls. COVID-19 clinical improvement was associated with increased sHLA-G plasma levels. In COVID-19, but not in control patients, an inverse correlation was found between serum sICAM-1 and E-selectin levels and plasma sHLA-G values. The in vitro analysis of activated endothelial cells confirmed the ability of HLA-G molecules to control sICAM-1 and sE-selectin expression via CD160 interaction and FGF2 induction and consequently neutrophil adhesion. We suggest a potential role for sHLA-G in improving COVID-19 patients' clinical condition related to the control of neutrophil adhesion to activated endothelium.

Keywords: CD160; COVID-19; E-selectin; HLA-G; ICAM-1; neutrophil.

Figure 1

sHLA-G and sHLA-E concentration in blood of COVID-19 patients and controls. Blood sHLA-G and sHLA-E levels in COVID-19 patients and relationships with disease progression and clinical outcome: (A,C) Plasma sHLA-G and sHLA-E levels at baseline (T1) and at follow-up (T2 and T3; 7 ± 2 day interval between assessments) in non-survivor patients (red histograms) or survivor patients (blue histograms) during the study period. (B,D) Plasma sHLA-G and sHLA-E levels at baseline (T1) and after 7 ± 2 days (T2 and T3) on the basis of worsening, stability, or improving of the clinical manifestation of the disease (* p < 0.05, Student’s t-test). (E,F) Plasma sHLA-G and sHLA-E levels at baseline (T1) and at follow-up (T2 and T3; 7 ± 2 day interval between assessments) in non-survivor (red dots) or survivor (blue dots) control patients during the study period.

Figure 2

Correlations between sHLA-G concentration and sICAM and E-Selectin levels. Correlations between blood sHLA-G and (A) sICAM and (B) sE-Selectin levels in COVID-19 patients. Circles: single samples’ values; Line: fitted linear regression line; Dots: data distribution.

Figure 3

Endothelial cell in vitro assays. HUVEC endothelial cells were treated with TNF-alpha (0.625, 1.25, 2.5, 5.0 ng/mL) for 4 h and cultured overnight. (A) The levels of sICAM and sE-Selectin were assessed in culture supernatants in the absence or presence of FGF2 inhibitor. HUVEC endothelial cells were treated with TNF-alpha (0.625, 1.25, 2.5, 5.0 ng/mL) for 4 h and treated overnight with HLA-G molecules (20, 40, 80 ng/mL). (B–D) The levels of sICAM and sE-Selectin were assessed in culture supernatants in the absence or presence of anti-HLA-G (10 ng/mL) or anti-CD160 (20 ng/mL) antibodies. (E) Fold increase expression of FGF2 in TNF-alpha (2.5 ng/mL) activated HUVEC in the presence of HLA-G or anti-HLA-G, anti-CD160 antibodies in comparison with untreated HUVEC. (F) HUVEC endothelial cells were treated with TNF-alpha (2.5 ng/mL) for 4 h and cultured overnight in the presence of anti-E-Selectin (20 ng/mL), anti-ICAM1 (10 ng/mL), HLA-G molecule (40 ng/mL), and plasma samples (100 μL) from survivor and non-survivor patients in the absence (untreated) or presence of TNF-alpha treatment (2.5 ng/mL). The cells were then co-cultured with peripheral blood neutrophils from non-COVID-19 healthy controls and analyzed for neutrophil cells adhesion. anti-CD160 (10 ng/mL) was used to confirm the effect of interaction between plasmatic sHLA-G and CD160 in controlling neutrophil cells adhesion. A control IgG was used. * p < 0.05; Student’s t-test. (G) Images of BioTracker 488 Green Nuclear Dye-labeled neutrophils bound to untreated and TNFα-treated HUVEC monolayers incubated with plasma sample (100 μL) from survivor (left panel) and non-survivor (right panel) patients.

Figure 4

Representation of the molecular interaction on the basis of neutrophil cell adhesion to endothelial cells. (A) Neutrophils interact with E-selectin, enhancing ICAM-1 recognition and adhesion to endothelial cells. Both these molecules are induced by FGF2 (fibroblast growth factor 2). (B) In the presence of COVID-19 there is an increase in sHLA-G molecules interacting with CD160, which inhibits FGF2-dependent induction of E-selectin and ICAM-1. The reduction of E-selectin and ICAM-1 expression reduces neutrophil adhesion to endothelial cells. This condition might improve clinical conditions, reducing neutrophils activation.