We examined expression levels of nine cytokine-coding genes among ICU-admitted COVID-19 patients, non-ICU-admitted ones and healthy subjects. We detected over-expression of IFN-G, IL-2, IL-4, IL-6, IL-17, TGF-B, IL-8 and IL-1B in COVID-19 patients compared with healthy subjects and in both female and male patients compared with sex-matched controls. However, expression of none of these cytokines was different between ICU-admitted patients and other patients except for IL-6 whose expression was lower in the former group compared with the latter. Expression of TNF-A was not different between COVID-19 patients and healthy subjects.
Levels of several cytokines have been assessed in different subgroups of COVID-19 patients. For instance, Chen et al. have reported remarkable over-expression of IL-2R and IL-6 in the critically ill COVID-19 patients compared with severe and mild groups (11). However, we detected a trend toward under-expression of IL-6 in the ICU-admitted patients compared with the other group of patients. This finding is not reliable as sex-based comparisons did not verify the difference in the expression of this cytokine between ICU-admitted and non-ICY admitted subjects. Moreover, this finding is in contrast with our previous study demonstrating higher median levels of IL-6 protein in the serum of ICU-admitted patients (12). However, it is worth mentioning that the current study varies with our previous study in the terms of applied technique and source of expression assessment. Anyway, we recommend assessment of expression of IL-6 in larger cohorts of Iranian patients to unravel the possible difference in its expression between Iranian patients and patients from other populations.
Chen et al. did not detect difference in the serum levels of TNF-α, IL-1 and IL-8 among the critical, severe and moderate groups COVID-19 patients (11). This finding is in accordance with our finding regarding similar levels of these cytokine between ICU-admitted and non-ICU-admitted COVID-19 patients.
The observed over-expression of IFN-G in COVID-19 patients is in line with the recently reported augmented nucleoprotein-induced IFN-γ release in these patients (13). Hu et al. have demonstrated lower probability of lung fibrosis at discharge in patients who had higher baseline levels of IFN-γ (14). Although we did not assess the presence of lung fibrosis in the admitted patients, we demonstrated similar levels of IFN-G between ICU-admitted and non-ICU-admitted patients. Over-expression of IL-17 in COVID-19 patients has also been reported in other populations (15). In addition, MERS-CoV has been shown to stimulate expression of this cytokine in humans (16). Consistently, Th17 cells have been reported to participate in the cytokine storm stimulated by SARS-CoV-2 (17). Up-regulation of IL-17, IL-2 and IL-4 levels have also been reported in COVID-19 patients with lung lesions (15).
Similar levels of TNF-A between three study subgroups in the current investigation raises the possibility of ethnic-based differences in the immunological responses in the context of COVID-19 infection, since this cytokine has been repeatedly reported to be increased in these patients and has been suggested as a target of immune-modulatory options (18). SARS-CoV-2 has also been suggested to activate of IL-1β, which consecutively induces other pro-inflammatory cytokines, such as IL-6 and TNF-α (18). However, while we detected over-expression of IL-1B and IL-6 in COVID-19 patients, we could not demonstrate any significant difference in the expression of TNF-A.
Then, we measured diagnostic power of cytokine transcripts in diagnosing COVID-19 patients from healthy controls. The AUC value was highest for IL-2 and IL-1B. After combining the transcript levels of all cytokines, AUC, sensitivity and specificity values reached 1.0, 1.0 and 0.99, respectively. Therefore, cytokine levels can be used for distinguishing disease status. For differentiation between ICU-admitted patients and other patients, IL-4 with AUC value of 0.68, had the best diagnostic power among cytokine coding genes. Therefore, these molecules cannot differentiate subgroups of COVID-19 patients. Besides, expression of none of cytokine coding genes was correlated with the assessed clinical/demographic data including age, gender, ICU admission, or CRP/ESR levels.
Lastly, we evaluated the correlation between the transcript levels of cytokine coding genes among three study subgroups. Patterns of correlation between expression levels of genes were more similar between non-ICU admitted patients and healthy controls, implying the altered regulatory mechanisms of cytokines expression in severely affected patients.
In brief, we demonstrated altered levels of several cytokine coding genes in Iranian patients with COVID-19 infection. Our study provides further evidence for contribution of “cytokine storm” in the pathogenesis of moderate/severe forms of COVID-19.