In this study, nasopharyngeal samples from mild and severe patients were used and associated changes in viral loads and the expression of various cytokines in nasopharyngeal samples with COVID-19 disease severity were explored . It is important to mention that this study is part of the larger study assessing immunological response against SARS-CoV-2 in HIV-positive and HIV-negative individuals. We found a significant difference in cytokine expression in nasopharyngeal samples from (HIV-negative) mild and severe groups.
According to clinical observations, SARS-CoV-2 infection can vary from asymptomatic infection to a mild respiratory disease with rising fever and dry cough to a severe form of disease accompanied by the development of acute respiratory distress syndrome or/and unusual upper respiratory tract pneumonia . In severely sick patients with pneumonia, aberrant and uncontrolled cytokine production has been seen . On January 30, 2020, the first guidelines for the diagnosis and treatment of SARS-CoV-2-infected pneumonia were released, which for the first time recommended monitoring of cytokine expression, to increase the curative rate and minimize mortality . Following the evidence of the "cytokine storm's" pathogenic significance, numerous investigations were performed to see if different chemokines may contribute to promoting COVID-19 development. The results of these investigations suggest the role of increasing or decreasing levels of one or more specific chemokines in COVID-19 patients . Such studies have also highlighted the need for biomarkers that can be used to predict the likelihood of developing a mild or severe form of the disease. However, most current approaches rely on invasive blood sampling, which may overlook early viral-host processes in infection that aid in viral illness progression. Estimation in cytokines in the nasopharyngeal region can be particularly beneficial, as nasopharyngeal swabs/samples provide a non-invasive way of studying early changes in viral titers and immunological biomarkers that can be linked to illness severity.
In this study, we analyzed the differential viral loads and expression of eight cytokines (IL-1, IL-2, IL-4, IL-6, IL-10 IFN-γ, TNF-α, and TGF-β-) in a total of 118 laboratory-confirmed cases, both mild and severe, of COVID-19. We found no significant difference in viral loads of the mild and severe groups, suggesting a lack of association between viral titers and disease severity. A similar result was reported by Julia R. et al, who found the total quantity of respiratory viral titer is indifferent to the severity of the COVID-19 disease .
We estimated the differential expression of cytokines in mild and severe groups. We found the expression of IL-2, TNF-α, IFN-gamma to be significantly higher in severe cases as compared to mild cases. Similar studies have been done which reported that IL-2 and TNF-alpha levels are higher in serum of patients with severe COVID-19 disease as compared to the mild patients and healthy group [13, 18–21]. Another study by Bastard et al, identified high titers of neutralizing autoantibody against IFN-γ in roughly 10% of patients with severe COVID-19 pneumonia. These antibodies were not present in infected patients who were asymptomatic or had a milder disease or healthy people.. Similarly, another study also reported high expression of pro-inflammatory serum cytokines (IFN-alpha and IFN-γ) and interferon-stimulated genes CXCL10 and CCL-2 in patients with severe COVID-19 disease as compared to healthy people or those with mild-moderate sickness [23, 24]. Boan Li et al, investigating the differential serum cytokine expression in individuals with severe and mild COVID-19 disease, reported IL-6 and IL-10 to be significantly increased in COVID-19 of the severe group as compared to the mild group . This observation is in contrast to our results where we found the expression of IL-6 and IL-10 to be significantly decreased in both groups. This difference may be due to the type of samples used in the study (serum vs nasal swabs) and may suggest that downregulation of IL-6 and IL-10 in the nasopharyngeal milieu and not serum may be linked to disease severity. Our results are, however, supported by the study by Suxin Wan et al, who reported dysregulation of IL-6 levels in more than 50% of their mild patients . Our results also showed decreased levels of IL-10 in both groups. One of the previous studies reported that patients with the severe illness show extremely low levels of anti-inflammatory cytokines IL-10. .
In the correlation analysis, we found a positive correlation between IL-1 and TNF-α, as well as a positive correlation between-6 and TNF-α. A previous study on acute-phase cytokines in the nasopharynx also reported cytokines IL-1, IL-6, and TNF-α to be positively correlated with each other . A study on COVID-19 infected pregnant women indicated that IFN-Ɣ and IL-6 have statistically significant positive relationships with disease severity . Another study was conducted by Meira et al., showed the positive correlation between IL-2 and IFN-Ɣ which is the Th1 subset's definer. In contrast to the study by Meira et al., in COVID-19 nasopharyngeal swab samples, we observed a strong positive correlation of IL-2 with IFN-γ, IL-4, IL-10, TNF-α, and TGF-β1 . Another study indicated a positive correlation between IL-4 and TNF-α, but a negative correlation between IL-4 and IL-6 after ERCP (Endoscopic Retrograde Cholangio Pancreatography) . We also observed a positive correlation between IL-4 and TNF-α but no correlation was observed between IL-4 and IL-6.
In conclusion, we found the nasopharyngeal expression of certain pro-inflammatory cytokines to be significantly higher in the severe group as compared to the mild group. These findings were demonstrated that high expression of pro-inflammatory cytokines IL-2, IFN-γ, and TNF-α in the nasopharyngeal milieu may initiate the development of cytokine storm, which may later be observed in serum and may correlate with the severe form of the disease. Early detection of high expression of these cytokines may, therefore, be useful in predicting the development of the severe form of the disease in patients with COVID-19, and such patients can be provided with appropriate therapy (for example, cytokine inhibitors or anti-inflammatory agents), which may stop the disease from taking a severe form and can provide patients with a chance of early recovery.