Lower peripheral blood Toll-like receptor 3 expression is associated with an unfavorable outcome in severe COVID-19 patients

Abstract

The role of innate immunity in COVID-19 is not completely understood. Therefore, this study explored the impact of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection on the expression of Pattern Recognition Receptors (PRRs) in peripheral blood cells and their correlated cytokines. Seventy-nine patients with severe COVID-19 on admission, according to World Health Organization (WHO) classification, were divided into two groups: patients who needed mechanical ventilation and/or deceased (SEVERE, n = 50) and patients who used supplementary oxygen but not mechanical ventilation and survived (MILD, n = 29); a control group (CONTROL, n = 17) was also enrolled. In the peripheral blood, gene expression (mRNA) of Toll-like receptors (TLRs) 3, 4, 7, 8, and 9, retinoic-acid inducible gene I (RIGI), NOD-like receptor family pyrin domain containing 3 (NLRP3), interferon alpha (IFN-α), interferon beta (IFN-β), interferon gamma (IFN-γ), interferon lambda (IFN-λ), pro-interleukin(IL)-1β (pro-IL-1β), and IL-18 was determined on admission, between 5-9 days, and between 10-15 days. Circulating cytokines in plasma were also measured. When compared to the COVID-19 MILD group, the COVID-19 SEVERE group had lower expression of TLR3 and overexpression of TLR4.

Figure 1

Quantitative mRNA of Pattern Recognition Receptors (PRRs) in circulating leukocytes of patients with COVID-19 on admission day. Expression of PRRs (mRNA) was measured in circulating leukocytes. Compared to the CONTROL group (N = 17 patients), increased mRNA transcription was observed for TLRs 3, 7, 9, RIGI, and NLRP3 in COVID-19 patients, while TLR8 was decreased. No significant difference in mRNA transcription for TLRs 7, 8 and 9, RIG, or NLRP3 was observed between COVID-19 MILD (N = 29 patients) and COVID-19 SEVERE (N = 50 patients) groups. On the other hand, TLR3 mRNA was decreased, while TLR4 mRNA was increased in COVID-19 SEVERE group when compared to COVID-19 MILD group. P values were calculated using the nonparametric Kruskal–Wallis test in R software (version 4.0.3 for macOS).

Figure 2

Quantitative mRNA of cytokines in circulating leukocytes of patients with COVID-19 on admission day. Expression of mRNAs of cytokines was measured in circulating leukocytes. IFN-α, IFN-β, IFN-λ, and IL-18 were increased in COVID-19 patients (N = 79 patients; 29 MILD and 50 SEVERE) when compared to CONTROL subjects (N = 17 patients). Pro-IL-1β mRNA was decreased in COVID-19 patients when compared to CONTROL subjects. No difference in IFN-γ expression was observed between CONTROL and COVID-19 patients. P values were calculated using the nonparametric Kruskal–Wallis test in R software (version 4.0.3 for macOS).

Figure 3

Association between Toll-like Receptors (TLRs) 3 and 4 and cytokine expression. The expression of IFN-γ and IFN-β was positively associated with TLR3 expression (Panels A and B) with R² = 0.263 and 0.282, respectively. Expression of pro-IL-1β and IL-18 was positively associated with the expression of TLR4 (Panels C and D), with R² = 0.239 and 0.713, respectively. Pearson correlation coefficient was used in R software (version 4.0.3 for macOS).

Figure 4

Quantitative mRNA of Pattern Recognition Receptors (PRRs) in circulating leukocytes of patients with COVID-19 during hospital stay. Expression of PRRs (mRNA) was measured in circulating leukocytes. Blood samples were collected every five days as feasible during hospitalization. The difference between COVID-19 MILD (N = 17 patients) and COVID-19 SEVERE (N = 24 patients) TLR3 and TLR4 mRNA expression at admission widened during follow up. Controls are also shown (17 patients). P values were calculated using the nonparametric Kruskal–Wallis test in R software (version 4.0.3 for macOS).

Figure 5

Quantitative mRNA of cytokines in circulating leukocytes of patients with COVID-19 during hospital stay. Expression of cytokines (mRNA) was measured in circulating leukocytes. Blood samples were collected every five days as feasible during hospitalization. IFN-γ mRNA expression was lower in COVID-19 SEVERE (N = 24 patients) in comparison to COVID-19 MILD (N = 17 patients), and widened during the hospital internment. Controls are also shown (17 patients). P values were calculated using the nonparametric Kruskal–Wallis test in R software (version 4.0.3 for macOS).

Figure 6

Serum cytokine levels during hospitalization. Circulating cytokines in plasma from the COVID-19 patients was also measured, and the profile was similar to that observed for mRNA. IFN-γ was lower in the SEVERE COVID-19 group (N = 50 patients) when compared to COVID-19 MILD (N = 29 patients), while pro-inflammatory cytokine levels (IL-6, TNF-α, and MCP-1) were higher. P values were calculated using the nonparametric Kruskal–Wallis test in R software (version 4.0.3 for macOS).