Study subjects
A total of 442 subjects with COVID-19, from three reference hospitals in Karachi from November 2020 to July 2021, were recruited for the study (supplementary material) . Inclusion criteria was Covid 19 infection and one or more symptoms of COVID-19 including fever, headache, cough, sore throat, sputum production, fatigue, shortness of breath (SOB), nausea, vomiting, diarrhea, nasal congestion, conjunctival congestion, myalgia, and arthralgia. A set of 50 healthy volunteers were enrolled in the study. This group is referred as “control”. The patients were divided into two groups “Covid 19 positive patients with comorbidities” including diabetes and hypertension and “Covid 19 positive patients without comorbidities”. Each group was further sub-divided into two sub-groups, Without comorbidities as “Mild” and “Severe” and with comorbidities as “C-Mild” and “C-Severe”. The disease severity criteria found in supplementary material. Ethical approvals were taken from Ethical Review Board of Sohail University. The study was conducted in accordance with the Declaration of Helsinki. Written consent was taken from the patients.
COVID-19 PCR Testing: The patients were tested for COVID-19 in their nasopharyngeal sample, by a quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) by using a COVID-19 Nucleic Acid Detection Kit according to the manufacturer’s protocol (Genesig, Primerdesign Ltd, Chandler’s Ford, UK) Patients with a confirmed diagnosis of SARS-CoV-2 infection were included in the study.
Sample collection: A total of 10 mL peripheral blood was collected for laboratory experiments. Nasopharyngeal swab was taken for SARS-CoV-2 testing. Sera and plasma were isolated from the blood accordingly and stored at −80 °C until further assay.
Cell isolation:
Peripheral blood was drawn in sodium heparinized tubes and allowed to ‘rest’ at room temp for at least 1 hour. Peripheral Blood Mononuclear Cells (PBMCs) were prepared from Buffy- Coats. PBMCs were isolated by gradient density centrifugation using histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA). The cell pellets were used for RNA extraction and protein extraction. Following density gradient centrifugation, PBMCs were also harvested for culturing.
Cell stimulation
PBMCs were cultured at 20 × 106/mL in Iscove Modified Dulbecco's Medium (IMDM) (Sigma-Aldrich, Schnelldorf, Germany). PBMCs were stimulated in the presence or absence of TNF-alpha or IL-10 for various time points. PBMCs were incubated in a humidified atmosphere at 37°C with 5% CO2.
Nuclear Extraction: After incubation, PBMCs were washed with PBS containing 10% endotoxin-free FBS (Gibco, New York, USA). Washed cells were then treated with a nuclear extract kit (Abcam, Shanghai, China) according to the manufacturer's instructions. A cytoplasmic extract and a nuclear extract for each culture condition was obtained and conserved at -80°C until further assays.
Immunological Assays
Serum HMGB1, S-100B and CML levels were measured by the quantitative sandwich enzyme immunoassay technique. using an enzyme-linked immunosorbent assay kit according to the manufacturer’s instructions (BioVision Inc., CA, USA). Following kits were used to analyze the inflammatory cytokines: Human IL-10 Quantikine ELISA kit Immunoassay, Human IL-6 Quantikine ELISA kit Immunoassay, Human TNF-alpha Quantikine ELISA kit Immunoassay (R&D System, Minneapolis, MN, USA),
Gene expression by real time PCR
The mRNA expression levels of RAGE, NF-kB and STAT-3 were quantified, using quantitative PCR (Startagene MX 3000P, Agilent technologies, Germany) as described, previously [8]. Using Trizol reagent (Invitrogen, CA, USA), RNA was extracted from the peripheral blood. cDNA synthesis was carried out using 1 microgram RNA by SuperScript III First-Strand Synthesis System (Thermoscientific Karlsruhe, Germany). Quantitative PCR was performed by SYBR Green Realtime PCR Master Mix (Thermoscientific Mannheim, Germany) in accordance with manufacturer’s instruction. Each measurement was performed in triplicate. The mRNA expression levels were normalized to beta-actin. Primer sequences are available in supplementary material.
Western Blot
PBMCs were isolated and homogenized at 4 °C (Stuart Homogeniser, UK) in lysis buffer. The cells were then processed as described previously [9]. Briefly, homogenates were solubilized on ice for 30 min followed by centrifugation at 12,000 × g for 15 min. The supernatant was separated and protein content was determined by the Bradford method. 400 µg of protein was used for immunoprecipitation. Immunoprecipitated proteins were separated by SDS-PAGE, and western blot analysis was performed as described [10] using anti RAGE antibody (Abcam, Cambridge, UK).
Measurement of Activation of NF-kB and STAT-3
Phosphorylated STAT3 and phosphorylated NF-kB levels were measured, in nuclear extracts of PBMCs after stimulation, by transcription factor ELISA kit, Phospho-Stat3 (pTyr705 ) and pan-Stat3 ELISA Kit (Sigma-Aldrich, St. Louis, MO, USA), total and phosphorylated NFkB p65 by human InstantOne ELISA Kit (ThermoFisher, MA USA).
Measurement of oxidative stress parameters
For measurement of AOPP, plasma samples were diluted in PBS and levels of AOPP were measured by colorimetry using OxiSelect™ AOPP Assay Kit, (Cell Biolabs Inc San Diego, USA) as per manufacturer instructions by comparison with Chloramine standard curve. The optical density was read at 340 nm using a Multimode Microplate Reader (Varioskan™ LUX multimode microplate reader, USA). The levels of Thiobarbituric Acid Reactive Substances (TBARS) were measured by fluorometry using TBARS assay kit (Cayman Chemical MI, USA).
Statistical Analysis
Statistical analysis was performed using SPSS version 26 software (SPSS Inc., Chicago, IL). The results were adjusted for age through propensity score matching. Groups of data were compared, after calculating mean +/- SEMs, either using two way ANOVA, followed by post hoc analysis (using Bonferroni test) or one-way ANOVA, followed by post hoc analysis (using Dunnett’s multiple comparison tests). The Student's t tests and chi-squared test was used to compare the distribution of a categorical variables, when appropriate. A p-value ≤ 0.05 was taken to indicate statistical significance.