Multiplex array analysis of circulating cytokines and chemokines in COVID-19 patients during the first wave of the SARS-CoV-2 pandemic in Milan, Italy

Abstract

Background: The systemic inflammatory syndrome called "cytokine storm" has been described in COVID-19 pathogenesis, contributing to disease severity. The analysis of cytokine and chemokine levels in the blood of 21 SARS-CoV-2 positive patients throughout the phases of the pandemic has been studied to understand immune response dysregulation and identify potential disease biomarkers for new treatments. The present work reports the cytokine and chemokine levels in sera from a small cohort of individuals primarily infected with SARS-CoV-2 during the first wave of the COVID-19 pandemic in Milan (Italy).

Results: Among the 27 cytokines and chemokines investigated, a significant higher expression of Interleukin-9 (IL-9), IP-10 (CXCL10), MCP-1 (CCL2) and RANTES (CCL-5) in infected patients compared to uninfected subjects was observed. When the change in cytokine/chemokine levels was monitored over time, from the hospitalization day to discharge, only IL-6 and IP-10 showed a significant decrease. Consistent with these findings, a significant negative correlation was observed between IP-10 and anti-Spike IgG antibodies in infected individuals. In contrast, IL-17 was positively correlated with the production of IgG against SARS-CoV-2.

Conclusions: The cytokine storm and the modulation of cytokine levels by SARS-CoV-2 infection are hallmarks of COVID-19. The current global immunity profile largely stems from widespread vaccination campaigns and previous infection exposures. Consequently, the immunological features and dynamic cytokine profiles of non-vaccinated and primarily-infected subjects reported here provide novel insights into the inflammatory immune landscape in the context of SARS-CoV-2 infection, and offer valuable knowledge for addressing future viral infections and the development of novel treatments.

Keywords: Anti-SARS-CoV-2 IgG; COVID-19; Primarily-infected subjects; SARS-CoV-2; Serum cytokine/chemokines levels.

Fig. 1

Cytokine and chemokine levels of SARS-CoV-2-positive and SARS-CoV-2-negative patients. Cytokines/chemokines (A: IL-9; B: IP-10; C: MCP-1; D: RANTES) were measured in serum samples by multiplex immunoassays and analyzed related to SARS-CoV-2 infection status. Data are presented as log10 (Log) of concentrations (picograms per milliliter, pg/ml) and shown as median with interquartile ranges. Statistical analyses were performed using an unpaired t test. *p < 0.05; **p < 0.01

Fig. 2

Kinetics of IL-6 (A) and IP-10 (B) in SARS-CoV-2 positive patients during hospitalization. The concentrations of cytokines/chemokines are represented as log10 (Log) of concentrations (picograms per milliliter, pg/ml). Statistical analyses were performed using one-way ANOVA and Tukey’s multiple comparison test. *p < 0.05; ****p < 0.001

Fig. 3

Effect of early treatment on IP-10 and IL-6 plasma levels between T0 and T1. A-D) IP-10 levels following treatment with azithromycin (AZ), hydroxychloroquine (HCQ), their combination (AZ + HCQ) and other treatments (Other), respectively. E-H) Variations in IL-6 serum levels post-treatment with AZ, HCQ, their combination and other therapeutic regimens, respectively. Concentrations of IL-6 and IP-10 are provided as log10 (Log) of concentrations (picograms per milliliter, pg/ml). Statistical analyses were performed using paired t test. Statistical significance is indicated above the graphs

Fig. 4

Correlations between serum cytokine/chemokine levels and anti-SARS-CoV-2 IgG antibodies. (A) IgG titers in SARS-CoV-2 infected patients at T0, T1 and T2. One-way ANOVA and Tukey’s multiple comparison test (****p < 0.001; ns, not significant). (B) Heat map representing Spearman’s correlation coefficients for cytokines/chemokines and IgG antibody titers. C-D) Scatter plots showing the correlations between IgG and IP-10 (C), and IL-17 (D). Each plot includes individual data points and a fitted line to indicate the correlation trend. R: correlation coefficient

Fig. 5

Dynamic Th2/Th1 ratios during infection period. Comparative Th2/Th1 ratios based on anti-inflammatory cytokines IL-4 (A, B), IL-6 (C, D) and IL-10 (E, F) vs. pro-inflammatory cytokines IFN-γ (A, C, E) and TNF-α (B, D, F). Box-and-whisker plots display the median and interquartile ranges