Cytokine profiles dynamics in COVID-19 patients: a longitudinal analysis of disease severity and outcomes

Categories: Disease & Virus

April 23, 2025

Characteristics of participants

The demographic information of the study population classified based on disease severity is demonstrated in Table 1. By mixing cross-sectional and cohort studies, 392 patients with acute SARS-CoV-2 infection who were admitted to several hospitals in the different provinces of Iran, 417 outpatients, and 224 healthy individuals were included in this study (Fig. 1A). The hospitalized patients were divided into moderate, severe, and critical categories according to their need for supplementary oxygen and admission to the ICU (Fig. 1B). We conducted 1514 collections and follow-up assessments on the patient population, ranging from one to three longitudinal time points occurring 1–50 days after symptom start (Fig. 1C). As presented in Table 1, the age was higher among patients with moderate, severe, and critical diseases (P < 0.001). There was no significant disparity in gender and body mass index (BMI) between the patients and control groups. The median interval from first symptoms on admission was not significantly different between hospitalized patients. The distribution of coexisting disorders, including diabetes, hypertension, cardiovascular disease, renal disease, and cancer, was higher among patients with moderate, severe, and critical diseases (all P < 0.05). Initial presenting symptoms demonstrated a preponderance of fever (39.33-62.16%) and fatigue (39.09-53.69%), with no significant difference between patients with mild to critical disease. The frequency of dry cough and dyspnea was significantly higher in inpatients with moderate, severe, and critical disease compared with patients with mild disease (all P < 0.001). However, myalgia was observed more in mild (42.21%) and moderate patients (40.15%) than in those who developed deleterious clinical manifestations (32.43-37.78%, P < 0.001). Finally, mortality and length of stay in the hospital were significantly higher in patients who were classified into severe and critical groups than in those who were not (P < 0.001).

Table 1 Characteristics of COVID-19 patients with diverse disease severity and control group.

Cytokine profile in COVID-19 patients

To gain insights into the main differences in the crucial cytokines between COVID-19 patients with diverse disease severity, we analyzed the serum levels of 12 cytokines across all sample collection time points. We observed that the inflammatory cytokines, including IL-1β, IL-1Ra, TNF-α, IL-6, IL-2, IL-8, and IL-18, in all COVID-19 subgroups were significantly elevated compared to the control group. IL-1Ra, IL-6, IL-2, IL-18, and IL-17 displayed more robust associations with illness severity. (Fig. 2). However, the results of other cytokines were different. IFN-γ levels were not significantly different between study groups. IL-12 levels were significantly higher in mild patients than in healthy individuals and hospitalized patients. In patients with moderate and critical disease, we observed an elevation of IFN-β compared to mild patients. In addition, we found that levels of IFN-α were significantly elevated in critical patients as compared to those with mild and moderate conditions (Fig. 2).

Longitudinal analysis of cytokines in COVID-19 patients

We investigate whether temporal pro-inflammatory cytokine patterns are diverse between the various patient groups. The difference between subgroups is more visible in the case of IL-6 than in other cytokines. This finding is either due to the high importance of the changes and function of this cytokine at different time points of COVID-19 or the fact that they are often measured with in vitro diagnostic (IVD) kits, and due to their high sensitivity, the changes are better displayed. IL-6 levels in all subgroups were higher than the control group at all time points except the mild group at day > 14 (Fig. 3). In the mild and moderate groups, IL-6 concentrations were at their highest level in the first week after the symptom onset and then exhibited a decreasing trend. However, elevated concentrations of IL-6 are linked to disease severity. IL-6 was produced higher in moderate, severe, and critical subgroups compared to the mild group and in moderate compared to the critical group at all studied time points (Fig. 3).

It seems that the pattern of changes in IL-1β and IL-1Ra in COVID-19 patients is similar. Remarkably, in the mild group, the amount of these cytokines increased at the day 1–7, reached a peak at the day 8–14, and diminished after > 14 days. While in other groups, from the beginning (day 1–7), they were produced more compared to the mild group, and their levels remained high even after more than 14 days (Fig. 3). However, the variations of TNF-α were the same between subgroups. TNF-α levels elevated at the day 1–7 and 8–14 times intervals, then decreased at the day > 14. We detected that IL-8 was significantly elevated in all COVID-19 subgroups at three studied time intervals compared to the control group. IL-8 was significantly higher in critical patients than moderate subjects (Fig. 3).

Since the amount of IFN-β is undetectable in many samples with COVID-19, no difference between diverse groups has been observed in previous studies^9,23,24. However, since the sample size is remarkable in most groups in this study, IFN-β changes have been determined. We found that although there was no difference in the production of IFN-β in all patients with COVID-19 compared to the control group at the day 1–7, IFN-β levels were higher in moderate, severe, and critical subjects at the day 8–14 or > 14 compared to the healthy control and themselves at the day 1–7 (Fig. 3).

Notably, we detected that while patients with mild and moderate COVID-19 produced IFN-α levels that were significantly lower at all time intervals compared to the healthy group, IFN-α levels did not change in those with severe and critical illnesses compared to the control group (Fig. 3). Furthermore, the patients with critical COVID-19 exhibited significantly elevated levels of IFN-α at the day 1–7 time interval compared to mild and moderate patients and at the day 8–14 and > 14 time intervals compared to moderate patients (Fig. 3).

The patients with severe COVID-19 at second- (day 8–14) and third- (day > 14) times intervals and the subjects with critical COVID-19 at the third-time intervals had significantly elevated levels of IFN-γ compared to the healthy control group (Fig. 3). In intragroup comparison, severe patients had higher levels of IFN-γ at the third-time interval compared to the first- and second-time intervals. Furthermore, at the third-time interval, IFN-γ levels were higher in severe individuals compared to moderate patients (Fig. 3). IL-12 reached its maximum level at the day > 14 in mild patients. It was significantly increased in mild COVID-19 at the day 8–14 and > 14 compared to the control and other COVID-19 subgroups. In contrast, there was no difference in serum concentrations of this cytokine between moderate, severe, and critical patients with the control group. Interestingly, the results of IL-18 were opposite those of IL-12. IL-18 was significantly increased in mild COVID-19 patients only at the day 1–7, and in other COVID-19 patients at all time intervals compared to control group. In addition, IL-18 had a significant association with severity at the second and third weeks after symptom onset (Fig. 3).

All patients with varying degrees of COVID-19 severity showed significantly reduced IL-17 induction at all time intervals examined. However, severe and critical patients exhibited significantly higher concentrations of IL-17 than mild and moderate subjects in the first two weeks after symptom onset.

Distinct Temporal cytokine patterns associated with COVID-19 severity

Given that temporal cytokine patterns were diverse between the COVID-19 patients with varying severity, we employed Spearman’s rho and PCA to investigate further crucial overall cytokine profile differences temporally.

Longitudinal cytokine correlations, measured at diverse time points after symptom onset, indicated that correlations of cytokines IL-1β, IL-1Ra, TNF-α, and IL-8 could be seen in many categories, including at all mild and moderate time intervals, at the first week in severe and the second and third weeks in critical, with the difference that there was a simultaneous increase at the first and second week and a simultaneous decrease at the third week in the mild group, while these inflammatory cytokines were increasing together until the third week in the other groups (Fig. 4). The point to consider in this analysis was the opposite correlation of IFN-α with IL-1β, IL-1Ra, and IL-8 in the mild group rather than other groups. In the mild group, IFN-α had a direct correlation with IL-1β and IL-8 at the first week, while in other groups, these correlations were inverse at different time intervals. In addition, our finding demonstrated that IFN-β was strongly associated with IFN-γ in hospitalized patients at different time intervals.

This scenario is consistent with PCA findings. PCA revealed three components with an eigenvalue > 1.0, explaining 50–57% of the variance in the 12 cytokines of COVID-19 patients (Fig. 5). At the day 1–7, the main contributors to PC1 were IL-1β, TNFα, IFN-α, and IL-12. PC1 was significantly higher in individuals with mild disease compared to those with moderate and critical COVID-19 when evaluated across different disease severity categories (both p < 0.001, Fig. 5A). There was no significant difference in PC2 across the study groups, while PC3, including IFN-β and IFN-γ, was higher in those with moderate vs. mild COVID-19.

At the day 8–14 time interval, PC1 with the greatest contribution of IL-1β, TNFα, IL-1Ra, and IL-8 was significantly higher in mild vs. moderate COVID-19 (Fig. 5B), but PC2 and PC3 did not significantly differ between COVID-19 subgroups. At the day > 14 time interval, IL-2, IL-8, and IL-1Ra contributed in PC1 (Fig. 5C). This component was significantly higher in critical vs. moderate and mild patients (P = 0.003 and P = 0.018, respectively). To better knowledge the results of IFN-α, we divided IFN-α concentrations into three levels and checked the frequency of people based on them (Fig. 6A). Most patients (more than 80%) had a low concentration of IFN-α (0–15 pg/mL). A low percentage of critically ill patients (16%) had moderate levels of IFN-α (15.01-50 pg/mL) at the day time interval > 14, which was significantly higher than other groups (Fig. 6A). According to the obtained results, it seems that delayed increase of type I IFN can impact the disease aggravation.

Based on our previous result that IL-1β and IFN-α were placed in PC1 and had a diverse correlation between mild and critical patients, we assessed the ratio of IL-1β to IFN-α. We observed that this ratio significantly declined in critical patients at the day 1–7 and 8–14 time intervals compared to mild and moderate groups, respectively (Fig. 6B).

To predict illness severity, logistic regression models were employed, adjusting for age and sex. The cytokines that had the highest score on PC1 and were linked to disease severity were input. The logistic regression model reveals that age, Log IL-1β, Log IL-1Ra, Log IL-6, and Log IFNα are significant predictors of hospitalization of COVID-19 patients at the first week after symptom onset. The findings indicate that elevated levels of Log IL-1β were associated with declined odds (OR:0.31 [0.155–0.622]) of hospitalization, whereas higher levels of Log IL-6 (OR: 3.112 [1.749–5.540]), Log IFNα (OR: 2.198 [1.220–3.960]), and Log IL-1Ra (OR: 1.663 [1.160–2.383]) were linked to decreased odds of hospitalization (Table 2). Using the formulation below, the panel of IFN-α2, IL-1β, IL-1Ra, and IL-6 at the first week after symptom onset with the accuracy of 76% and sensitivity of 79% was the suggestive combination to predict hospitalization. However, this combination did not have high sensitivity for invasive mechanical ventilation (33%) and mortality (50%).

Table 2 Multivariate logistic regression analysis: impact of cytokine levels at the first week after symptoms unset on hospitalization.

$$\log {\text{it}}\left( p \right)=\log \left( {\frac{p}{{1 – p}}} \right)$$

= 0.089*sex (male=1)+0.038*age(years: 17-80)-1.171*log (IL-1β(pg/mL:3.9-280)) +0.509*log (IL-1Ra(pg/mL:39-2500))+1.135*log (IL-6(pg/mL:2-1000)) +0.787*log (IFNα2(pg/mL:3.12-200)) – 3.573.

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