Given the significance of inflammatory cytokines in predicting disease severity and outcome in SARS-CoV-2 patients, the present study undertook a task to evaluate a battery of inflammatory cytokines for determining the signature inflammatory cytokine pattern of prognostic and/predictive relevance in SARS-CoV-2 patients from Kashmir. Here we established the enzyme linked immunosorbent assay (ELISA) for cytokine profiling of IL-10, IL-6, IL8, IL-1-a, TNF-a, VEGF cytokines, known markers of inflammation and organ damage. Correlation analysis followed by ROC analysis, univariant and multivariant, after adjusting for age, gender and other inflammatory markers, revealed raised IL-8, IL-10, IL-6 and decreased VEGF as particularly significant signature inflammatory cytokine panel as prognosticators of disease severity and / predictors of poor disease outcome in SARS-CoV-2 patients.
The cytokine storm is an interesting feature of SARS-CoV-2 infection. Upon the binding of SARS-CoV-2 spike(S) to angiotensin converting enzyme (ACE) on type II pulmonary alveolar epithelial cells, it triggers the release of inflammatory cytokines such as IL-6. These cytokines then activate the innate and adaptive immune response, involving macrophages, monocytes, T cells, and B cells.The inflammatory immune cells upon invasion of the alveoli cause lung inflammation and lung damage [15–18]. The signature inflammatory cytokine panel found here included IL-6, IL10, VEGF and IL-8. of various inflammatory cytokines of the signature panel, IL6 is the key mediator of the inflammatory immunological responses in covid-19. In the present cohort, multivariant ROC analysis revealed 3-fold raised IL-6 level ( = > 80pg/ml) as an independent predictors of disease severity and poor disease outcome (Table 7,8). This finding is inconsistent to the meta-analysis (9 studies) reporting > 3fold increase (≥ 80pg/ml) in IL-6 level as a mortality risk factor in SARS-CoV-2 infection [19] (Fig. 2b;Table 5). Eighty two percent (82%)of the patient in this study received Dexamethasone. The efficacy of the dexamethasone treatment could be explained by underlying mechanism of reduction of IL-6 levels in moderate and severe SARS patients as proved by Ledford [20]. Patients with very high IL-6 levels anti IL-6 monoclonal antibody therapy is, therefore, an optimal treating option to prevent poor outcome in these patients. Similarly IL-10 proved to be equally significant independent predictor of disease severity and poor disease outcome after nullifying the risk contribution of variables like age, gender and other inflammatory markers for Covid-19 (Table 7,8). The finding is inconsistent to various other studies including that of Lu et al and Aber et al [21, 22]. IL-10 is a pleiotropic cytokine produced by various immune cells with strong anti-inflammatory and immunosuppressive effects. It typically suppresses pro-inflammatory signals by activating the JAK1-TYK2-STAT3 pathway, leading to STAT3-mediated transcription of genes that limit the inflammatory response.[23, 24]. During the acute phase of viral infection, IL-10 suppresses the activity of T cells, macrophages, and natural killer cells, preventing successful viral elimination and minimizing collateral tissue damage. However, in rare cases, such as in SARS-COV-2 infection, particularly in patients with comorbidities (e.g., diabetes, cancer) or those on immunosuppressants, or elderly patients, resistance to IL-10 has been observed, impairing its anti-inflammatory function and instead potentiates its pro-inflammatory action. This "IL-10 resistance" can be described as the potential evasion of effector immune cells from IL-10's inhibitory anti-inflammatory action, leading to an overwhelming counterstrike of inflammatory cytokines [25]. High levels of IL-10 is also known to augment the proinflammatory reactions to bacterial lipopolysaccharides (LPS) in human endotoxemia [26]. This points to the possibility that the combination of elevated IL-10 and bacterial products (which are abundant in severe Covid-19 cases) could empower the inflammatory machinery in Covid-19 [25]. In support of this hypothesis, we have reported here resistance to IL-10’s anti-inflammatory action under hyperglycemic conditions i.e in individuals with diabetes [27]. Significantly, diabetes has been shown to be a contributing factor to the increased severity and mortality of Covid-19. The resistance of IL-10 may provide a potential link between diabetes and negative outcomes in Covid-19. IL-10 may have dual roles in the progression of Covid-19. Initially, the rise in IL-10 during early infection may indicate a protective anti-inflammatory response to control the action of proinflammatory cytokines. However, as the disease advances, the anti-inflammatory function of IL-10 is inhibited, and it may instead act as a proinflammatory mediator, contributing to the hyper-inflammation associated with viral sepsis and exacerbating the cytokine storm in severe cases of Covid-19 [28].
IL-8 is normally secreted by multiple cells including neutrophils exposed to stimuli and is considered the primary molecule of acute inflammation. Several studies have shown Neutrophilia and increased NLR(neutrophil lymphocyte ratio) as indicators of covid-19 severity (29), so there is every possibility of increase in IL-8, an inflammatory cytokine secreted by neutrophils. In consistent to this, in the present study increased IL-8 level has been associated with covid-19 severity (p = 0.01) and poor outcome (Fig. 2d; Table 5) (AUC = 0.69). The possible mechanism is that Neutrophils elicits immune response to danger signals in the form of Neutrophil Extracellular Traps (NETs), a networks of extracellular fibers primarily composed of DNA from neutrophils, that slow down and binds to pathogens. In a process known as "NETosis," NETs enable neutrophils to eliminate external pathogens while minimizing damage to the host cells. However, excessive activation can lead to damage to the host cells, either through an excessive of NET formation or the impaired removal of NETs, both of which can result in toxic effects for the host, as seen in patients with SARS-CoV-2. (30). NET degradation products have been detected in patient plasma and correlate with lung distress in Covid-19 (31). Since IL-8 is expressed by neutrophils, therefore, in consistent to present study, IL-8 overexpression, by potentiating neutrophil chemotaxis and promoting angiogenesis, proves to be a poor prognostic biomarker for the SARS-CoV-2 patients (Table 5).
VEGF is a vascular factor with various biological functions, including angiogenesis and vascular permeability, which are the most well-known. Apart from promoting angiogenesis, VEGF also effectively increases vascular permeability through the VEGFR2 receptor (vascular endothelial growth factor receptor) receptor-mediated alterations of vascular fenestration and inter- endothelial junction [32] During a Covid-19 infection, patients frequently experience difficulty breathing, known as dyspnea, due to lung inflammation, plasma leakage, collapsed air sacs, and lung collapse. Dyspnea leads to reduced oxygen levels in the lung tissue and throughout the body, and induces a series of pathological responses through the activation of hypoxia-inducible factor-1alpha (HIF-1α) [33] among which VEGF is one of the key HIF-targeted genes [34]. In consistent with previous studies an increasing trend in VEGF levels similar to other inflammatory markers (IL-6, IL-10, IL8) was found here in SARS-CoV-2 patients on their arrival at hospital i.e at earlier stage, possibility to compensate for hypoxia by increasing the angiogenesis and vascular permeability (Fig. 2f). However decreasing trend in VEGF was found in patients of stage 3 severity (as later stage) (Fig. 2f ) as compensatory mechanism of hypoxia induced VEGF had either failed or could be due to anti VEGF medications given in patients at risk of pulmonary edema. Therefore, decreasing VEGF level proved to be an independent predictor of poor disease outcome (ROC = 0.69) after adjusting for variables like age, gender and other inflammatory markers imposing risk for Covid-19. Ultimately, in theses patients lung atelectasis aggravates dyspnea, hypoxia resulting in acute respiratory distress syndrome. Thus hypoxia-induced VEGF mediated (increase followed by decrease) vascular leakage plays a central role in causing a myriad of pathological changes that occur in severe SARS-CoV-2 patients.
Regarding ferritin, while as high levels of ferritin [≥ 1500 ng/ml ie > 3fold] was found in patients with pneumonia, cough was more common in patients with mild and moderate levels of ferritin. A relationship between disease severity and ferritin levels was found here as percent increase in number of SARS-CoV-2 patients of stage 3 disease severity was found as ferritin levels increased from mild, moderate to high level. Since ferritin was found significantly higher in diabetic patients which explains risk of Covid-19 severity and poor outcome in these patients. Further ROC analysis revealed ferritin as predictors of negative disease outcome (AUC = 0.66). This is in consistent to world wide report that suggest ferritin as prognosticator of disease severity and very high ferritin (> 1500ng/ml) level as indicator of hyperinflammatory phenotype [36]. The possible mechanism is that hyperferritinemia causes inflammatory states in SARS-CoV-2 infection particularly in the lungs, as demonstrated by the presence of a high number of macrophages in the lung parenchyma of SARS-CoV-2 patients and high ferritin also potentiates the production of IL6 [37]. Also a feed back mechanism between ferritin and cytokines is known to control pro-inflammatory and anti-inflammatory responses with cytokines triggering ferritin expression and ferritin promoting both pro-inflammatory and anti-inflammatory cytokine expression[38] .
The present study was limited by the number of samples. The results of the study, therefore, should be substantiated by similar future studies conducted on a large sample size. Also the reference range of some of the cytokines was not known so we took the samples from healthy controls in order to determine reference range.