COVID-19 Patients Upregulate Toll-like Receptor 4-mediated Inflammatory Signaling That Mimics Bacterial Sepsis

Abstract

Background: Observational studies of the ongoing coronavirus disease 2019 (COVID-19) outbreak suggest that a 'cytokine storm' is involved in the pathogenesis of severe illness. However, the molecular mechanisms underlying the altered pathological inflammation in COVID-19 are largely unknown. We report here that toll-like receptor (TLR) 4-mediated inflammatory signaling molecules are upregulated in peripheral blood mononuclear cells (PBMCs) from COVID-19 patients, compared with healthy controls (HC).

Methods: A total of 48 subjects including 28 COVID-19 patients (8 severe/critical vs. 20 mild/moderate cases) admitted to Chungnam National University Hospital, and age/sex-matched 20 HC were enrolled in this study. PBMCs from the subjects were processed for nCounter Human Immunology gene expression assay to analyze the immune related transcriptome profiles. Recombinant proteins of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) were used to stimulate the PBMCs and monocyte-derived macrophages, and real-time polymerase chain reaction was performed to quantify the mRNA expressions of the pro-inflammatory cytokines/chemokines.

Results: Among the most highly increased inflammatory mediators in severe/critically ill patients, S100A9, an alarmin and TLR4 ligand, was found as a noteworthy biomarker, because it inversely correlated with the serum albumin levels. We also observed that recombinant S2 and nucleocapsid proteins of SARS-CoV-2 significantly increased pro-inflammatory cytokines/chemokines and S100A9 in human primary PBMCs.

Conclusion: These data support a link between TLR4 signaling and pathological inflammation during COVID-19 and contribute to develop therapeutic approaches through targeting TLR4-mediated inflammation.

Keywords: Cytokines; Inflammation; S100A9; SARS-CoV-2.

Fig. 1. Transcriptome analysis reveals that immune gene expression profiles of COVID-19 patients are distinct to HC. (A) Schematic diagram of the immune transcriptome analysis in this study. (B) A result of principal component analysis of log2-transformed 579 immune gene expression levels. (C) The scatter plots representing 579 immune genes with the log2-transformed FPKM for COVID-19 patients compared to HC. (D) The ten most significantly enriched KEGG pathways of the 298 DEiGs from COVID-19 patients compared to HC. (E) Log2-transformed fold changes of chemokine and chemokine receptor genes from MILD (x-axis) and SEVERE (y-axis) vs. HC. (F) Expression levels (FPKM) of marked chemokines in (E). Error bar indicates standard error of mean. P values were calculated using Mann-Whitney U test and adjusted P values (FDR) were shown.

COVID-19 = coronavirus disease 2019, HC = healthy controls, FPKM = fragments per kilobase exon-model per million reads mapped, KEGG = Kyoto Encyclopedia of Genes and Genomes, DEiG = differentially expressed immune gene, MILD = mild/moderate, SEVERE = severe/critical, FDR = false discovery rate, IBD = inflammatory bowel disease, TNF = tumor necrosis factor, CCL = C-C motif chemokine ligand, CXCL = C-X-C motif chemokine ligand. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

Fig. 2. COVID-19 infection boosts NF-κB signaling pathway. (A) The left (MILD) and right (SEVERE) sides of box represent the mean fold change in mRNA levels, compared with HC. The NF-κB signaling pathway was adopted from KEGG database (accession number: hsa04064). (B) The expression levels of IRF3, TLR3, TLR7, TLR8 and TLR9 were represented by FPKM. Error bar indicates standard error of mean. P values were calculated using Mann-Whitney U test and adjusted P values (FDR) were shown.

COVID-19 = coronavirus disease 2019, NF = nuclear factor, MILD = mild/moderate, SEVERE = severe/critical, HC = healthy controls, KEGG = Kyoto Encyclopedia of Genes and Genomes, FPKM = fragments per kilobase exon-model per million reads mapped, FDR = false discovery rate, TLR = toll-like receptor, IRF = interferon regulatory factor. ^*P < 0.05.

Fig. 3. Top 10 most significantly up- and down-regulated DEiGs in SEVERE patients. (A) Log2-transformed fold changes of 579 immune-genes from MILD vs. HC (x-axis) and SEVERE vs. HC (y-axis). The genes for red and blue colors indicate up- and down-regulated DEiGs in SEVERE patients, respectively. (B, C) Comparisons of expression levels of top 10 up- (B) and down- (C) regulated DEiGs. The expression level was represented by FPKM. (D) Correlation analysis between S100A9 expression level and serum albumin in MILD and SEVERE patients. Error bar indicates standard error of mean. P values were calculated using Mann-Whitney U test and adjusted P values (FDR) were shown (B, C) and Spearman's correlation is shown (D).

DEiG = differentially expressed immune gene, MILD = mild/moderate, SEVERE = severe/critical, HC = healthy controls, FPKM = fragments per kilobase exon-model per million reads mapped, FDR = false discovery rate, CCL = C-C motif chemokine ligand, CXCL = C-X-C motif chemokine ligand. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

Fig. 4. Recombinant NC and S2 ECD proteins of SARS-CoV-2 robustly induce the expression of pro-inflammatory cytokines/chemokines in human primary PBMCs. (A) Schematic diagram of interaction between host cell and SARS-CoV-2 antigen. (B, C) RT-qPCR analysis of indicated genes in human primary PBMCs treated with recombinant NC, S2 ECD, S1 subunit, or RBD antigen (B) and NC or S2 ECD (C) (2 μg/mL each; for 6 hours). (D) Level of IL-6 in cell supernatant from (B) measured by ELISA. (E) Immunoblot analysis of phospho-p65 (NF-κB) in human primary PBMCs treated with S2 ECD (2 μg/mL) for indicated time. (F) RT-qPCR analysis of IL1B in human primary PBMCs treated with recombinant S2 ECD in presence or absence of indicated doses of S100A9 for 6 hours. Welch's t-test (B-D) and One-way analysis of variance (F) were used to measure the significance. Values are mean ± standard deviation. from a representative of two independent experiments performed in triplicate (B, C, F) or mean ± standard error of mean. of pooled data from two independent experiments (D).

NC = nucleocapsid, S = spike, ECD = extracellular domain, SARS-CoV-2 = severe acute respiratory syndrome coronavirus-2, PBMC = peripheral blood mononuclear cell, RT-qPCR = real-time quantitative polymerase chain reaction, RBD = receptor binding domain, IL = interleukin, ELISA = enzyme-linked immunosorbent assay, NF = nuclear factor, IFN = interferon, CCL = C-C motif chemokine ligand, CXCL = C-X-C motif chemokine ligand, RBD = receptor binding domain, UN = untreated. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.