Serum soluble Fas ligand is a severity and mortality prognostic marker for COVID-19 patients

Abstract

Finding cytokine storm initiator factors associated with uncontrolled inflammatory immune response is necessary in COVID-19 patients. The aim was the identification of Fas/Fas Ligand (FasL) role in lung involvement and mortality of COVID-19 patients. In this case-control study, mild (outpatient), moderate (hospitalized), and severe (ICU) COVID-19 patients and healthy subjects were investigated. RNA isolated from PBMCs for cDNA synthesis and expression of mFas/mFasL mRNA was evaluated by RT-PCR. Serum sFas/sFasL protein by ELISA and severity of lung involvement by CT-scan were evaluated. Also, we docked Fas and FasL via Bioinformatics software (in silico) to predict the best-fit Fas/FasL complex and performed molecular dynamics simulation (MDS) in hyponatremia and fever (COVID-19 patients), and healthy conditions. mFasL expression was increased in moderate and severe COVID-19 patients compared to the control group. Moreover, mFas expression showed an inverse correlation with myalgia symptom in COVID-19 patients. Elevation of sFasL protein in serum was associated with reduced lung injury and mortality. Bioinformatics analysis confirmed that blood profile alterations of COVID-19 patients, such as fever and hyponatremia could affect Fas/FasL complex interactions. Our translational findings showed that decreased sFasL is associated with lung involvement; severity and mortality in COVID-19 patients. We think that sFasL is a mediator of neutrophilia and lymphopenia in COVID-19. However, additional investigation is suggested. This is the first report describing that the serum sFasL protein is a severity and mortality prognostic marker for the clinical management of COVID-19 patients.

Keywords: COVID-19; Fas; hyperinflammation; immunoinformatics; viral Immunology.

Figure 1

Fas and FasL mRNA expression in PBMC samples mRNA expression of Fas and FasL is shown for (A, B) COVID-19 (n = 93) and control group (n = 27). (C, D), mild, moderate, and severe COVID-19 (n = 31 per group), and control group (n = 27). (E, F) Heatmaps graphically demonstrate expression level; *p<0.05, **p< 0.01, ***p<0.001, ****p< 0.0001. Outlier data which were abnormal were excluded for each gene expression analysis. *Outlier values are displayed as blanked in heatmaps. Higher values than graph range exceed the plot and may not be shown.

Figure 2

Correlation matrix for Fas/FasL and clinical disease properties.

Figure 3

Fas and FasL protein level in serum samples. Serum levels of Fas and FasL is shown for (A, B) COVID-19 (n = 66) and control group (n = 22). (C, D), mild, moderate, and severe COVID-19, and control group (n = 22 per group); *p<0.05, **p< 0.01, ***p<0.001, ****p< 0.0001.

Figure 4

Lung CT of COVID-19 patients representing mild, moderate, and severe respiratory injury. (A) Mild, Multiple peripheral and central ground glass opacities (GGOs) in right upper lobe and left lower lobe; Band atelectasis in left lower lobe; (B) Moderate, Subpleural GGOs near costal and mediastinal pleura of both lungs, Consolidation with air-bronchogram in lower lobes of both lungs, multiple observations of band atelectasis; (C) Severe, Peripheral patchy GGOs, Diffuse and mostly peripheral GGOs, Multiple fibrotic bands.

Figure 5

Correlation of lung involvement CT scan and sFas/sFasL in COVID-19 patients. Lung has two right and lobes, that includes upper, middle, and lower lobes. Each lobe is about one-sixth of lung area, and percent of involvement is determined by this method in our department.

Figure 6

Regression analysis and prognostic performance of sFasL for COVID-19 mortality. (A) Regression analysis showed lower sFasL is associated with an increased risk for mortality (n =66). (B) AUC in ROC was 0.70. (C) sFasL levels were lower in dead compared to alive COVID-19 patients. *p<0.05, **p< 0.01, ***p<0.001, ****p< 0.0001.

Figure 7

Protein quality verification for Fas tertiary structure. In this illustration, the proteins (Fas/FasL) are evaluated based on structural Bioinformatics rules. After refinement, Fas structures was favorable. (A, B) In the Ramachandran plot, more residues are located in favorable regions. (C, D) ERRAT score was improved from 36.296% to 85.849%. A score over 80% shows the refined structure has good quality. (E, F) ProSA Z-score was improved from -3.41 to -4.08 which is within the acceptable range for structures with the same aminoacid length.

Figure 8

Protein quality verification for FasL tertiary structure. After refinement, FasL structure was favorable (A, B) In the Ramachandran plot, more residues are located in favorable regions. (C, D) ERRAT score was improved from 70.833% to 88.696%. A score over 80% shows the refined structure has good quality. (E, F) ProSA Z-score was improved from -4.3 to -4.93 which is within the acceptable range for structures with the same aminoacid length.

Figure 9

Refinement of Fas and FasL tertiary structure. Protein structure refinement is the determination and improving of a protein’s structural properties. In this figure (A; Fas and B; FasL), by beige/blue colors, the initial and refined structure have been matched to show how the 3D structures, such as beta-sheets, alpha-helices and coils have been altered to improve the protein quality. Also, a significant portion of the refined structure is made of beta-sheets or alpha-helices indicating good structural stability.

Figure 10

Docked Fas/FasL complex. In this figure, molecular docking has been shown. Docking is the Bioinformatics assessment of how two proteins (Fas and FasL) fit best as a complex. Fas and FasL are shown with distinct colors (purple and beige). Fas/FasL complex contacts are highlighted with yellow lines.

Figure 11

Molecular dynamics simulation (MDS) analyses for Fas/FasL in blood MDS is a simulation approach for analysis of the physical movement of atoms. Here, a biological system of Fas/FasL was virtually set up and equilibrated in blood-like conditions, and was allowed a limited time to evolve. (A) Energy minimization (EM) was set-up for the Fas/FasL system. (B) During the NVT equilibration, the temperature was matched to blood-like conditions. (C) During the NPT equilibration, the pressure reached 0 bar. (D) Density of the Fas/FasL system was also equilibrated in this step. After production MDS of the Fas/FasL system: (E) Root-mean square fluctuation (RMSF) analysis did not show fluctuation in the Fas/FasL system and the proteins did not denature. (F) Root-mean square deviation (RMSD) values shows that the Fas/FasL complex reached stability within the blood-like simulation conditions.