Induction of alarmin S100A8/A9 mediates activation of aberrant neutrophils in the pathogenesis of COVID-19

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic poses an unprecedented public health crisis. Evidence suggests that SARS-CoV-2 infection causes dysregulation of the immune system. However, the unique signature of early immune responses remains elusive. We characterized the transcriptome of rhesus macaques and mice infected with SARS-CoV-2. Alarmin S100A8 was robustly induced in SARS-CoV-2-infected animal models as well as in COVID-19 patients. Paquinimod, a specific inhibitor of S100A8/A9, could rescue the pneumonia with substantial reduction of viral loads in SARS-CoV-2-infected mice. Remarkably, Paquinimod treatment resulted in almost 100% survival in a lethal model of mouse coronavirus infection using the mouse hepatitis virus (MHV). A group of neutrophils that contributes to the uncontrolled pathological damage and onset of COVID-19 was dramatically induced by coronavirus infection. Paquinimod treatment could reduce these neutrophils and regain anti-viral responses, unveiling key roles of S100A8/A9 and aberrant neutrophils in the pathogenesis of COVID-19, highlighting new opportunities for therapeutic intervention.

Keywords: Paquinimod; S100A8/A9; SARS-CoV-2; aberrant neutrophils.

Graphical abstract

Figure 1

S100A8 and neutrophil-related gene expressions were significantly induced in the early stage of SARS-CoV-2 infection (A) A flow chart depicting the process of animal experiments with rhesus macaques. Rhesus macaques (3–4 years old) were challenged with 10⁶ TCID50 SARS-CoV-2 virus by intratracheal routes. (B) Go analysis of the differences in rhesus macaques infected with SARS-CoV-2 in comparison with mock (fold change [FC] > 4 or < 0.25; p value < 0.05). (C) Analysis of neutrophil marker genes expression. n = 4. (D) Volcano plots show differentially expressed genes in rhesus macaques infected with SARS-CoV-2 at 3 and 5 dpi. S100A8 expression is significantly increased after SARS-CoV-2 infection. (E) Analysis of all known alarmins showing that S100A8 is the most significantly induced one. (F) qRT-PCR analysis for viral loads and the expression of S100A8 and S100A9 in the lungs of SARS-CoV-2-infected rhesus macaques at 3 and 5 dpi. n = 3. (G) Heatmap shows alarmins in the blood from rhesus macaques infected with SARS-CoV-2 at 5 dpi (left). The expression of neutrophil marker genes was analyzed (FC to mock, right). n = 4. (H) Heatmap depicting the expression of alarmins of the lung samples from healthy control and post-mortem lung samples from COVID-19 patients (left). The expression of neutrophil marker genes analyzed (FC to healthy control, right). Data from the lungs of COVID-19 patients and healthy control correspond to GEO: GSE147507. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. Error bars, SD.

Figure 2

Anti-viral innate immune disorder and aberrant S100a8 expression induced by coronavirus but no other viruses (A) A flow chart depicting the process of animal experiments with mice. All the mice were inoculated intranasally with viruses. Virus doses: SARS-CoV-2 (10⁵ TCID50), IAV (10⁵ TCID50), EMCV (10⁷ plaque-forming units [PFU]), HSV-1 (10⁶ PFU), and MHV-A59 (10⁵ PFU). (B) RNA-seq analysis of lungs from mice infected with SARS-CoV-2 or IAV. Go analysis was performed with the differentially expressed genes and compared with mock (FC > 4 or < 0.25; p value < 0.05). n = 3. (C) qRT-PCR analysis for the expression of S100a8, Ly6g, Ifnb, and Isg15 in the lungs of mice at different time points after IAV or SARS-CoV-2 infection. n = 3. (D) Heatmap depicting the expression differences of neutrophil marker genes in the lungs of mice infected with IAV or SARS-CoV-2. (E) qRT-PCR analysis for the expression of S100a8 and Ly6g in the blood and lungs of mice infected with different viruses. n = 3. (F) Post-infection survival curves of wild-type mice and Ifnar^−/− mice infected with MHV. n = 10. (G) RNA-seq analysis of the lungs of Ifnar^−/− mice infected with MHV at 5 dpi. Go and KEGG analysis were performed with the differentially expressed genes and compared with mock (FC > 4 or < 0.25; p value < 0.05). (H) qRT-PCR analysis for the expression of Ifnb, Isg15, S100a8, and Ly6g in the lungs of Ifnar^−/− mice infected with IAV or MHV at 5 dpi. n = 3. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. Error bars, SD.

Figure 3

A group of immature aberrant neutrophils emerged in coronavirus-infected mice (A) Immunohistochemical analysis of the location and expression of S100A8 in the lung tissue of mice infected with SARS-CoV-2 or MHV at 5 dpi. The S100A8⁺ cells in the lungs of mice infected with coronavirus were increased significantly. The red arrows indicate the S100A8⁺ cells. n = 5. Scale bars, 50 μm. (B) Flow cytometry analysis of neutrophils in lungs from mice infected with SARS-CoV-2 and MHV at 5 dpi. Control group means mice treated with vehicle. Gate P1 shows the conventional neutrophils (CD45⁺CD11b⁺Ly6G^high), and Gate P2 shows the pathologic aberrant neutrophils (CD45⁺CD11b⁺Ly6G^variable). Aberrant neutrophils (P2) in the lungs of mice infected with coronavirus were significantly increased. n = 3. (C) Flow cytometry analysis of neutrophils in lungs of mice challenged with IAV, EMCV, HSV-1, and LPS at 5 dpi. The results showed that these treatments did not induce an increase in aberrant neutrophils. n = 3. (D) Flow cytometry analysis of neutrophils in bone marrow from mice infected with SARS-CoV-2 and MHV at 5 dpi. n = 3. (E) qRT-PCR-analyzed related gene expression of aberrant neutrophils in bone marrow of mice infected with MHV at 5 dpi and identified the differentiated types of aberrant neutrophils. n = 3. ^∗∗∗p < 0.001. Error bars, SD.

Figure 4

Paquinimod rescues the mice infected by SARS-CoV-2 and MHV (A) A flow chart depicting the process of a drug rescue experiment. (B) Analysis of weight and survival rate of mice infected with SARS-CoV-2 and MHV after Paquinimod treatment. n = 6. (C) Analysis of the rescue effect of Paquinimod by H&E staining and pathology score of lung tissue in mice infected with SARS-CoV-2 or MHV at 5 dpi. Paquinimod treatment significantly prevented the bleeding and fibrosis in lung tissue. A number of pulmonary H&E staining images were randomly selected for pathological scoring. n = 10. Scale bars, 100 μm. (D) Immunohistochemical analysis of the S100A8⁺ cells in the lung tissue of mice infected with SARS-CoV-2 or MHV at 5 dpi after Paquinimod treatment. The S100A8⁺ cells in the lungs of mice infected with coronavirus were decreased significantly after Paquinimod treatment. The red arrows indicate the S100A8⁺ cells. n = 5. Scale bars, 50 μm. (E) qRT-PCR analysis for the expression of S100a8 and Ly6g in the lung of mice infected with SARS-CoV-2 at 5 dpi after Paquinimod treatment. n = 3. (F) qRT-PCR analysis of viral loads in the lungs of mice infected with coronavirus SARS-CoV-2 and MHV at 5 dpi after Paquinimod treatment. n = 3. (G) Flow cytometry analysis of neutrophils in lungs, blood, and bone marrow from mice infected with SARS-CoV-2 at 5 dpi after Paquinimod treatment. CD45⁺CD11b⁺Ly6G^variable aberrant neutrophils (P2) in the mice infected with SARS-CoV-2 were significantly decreased by Paquinimod treatment. n = 3. (H) Heatmap depicting a decrease in B cell-related gene expression in the lungs of mice infected with SARS-CoV-2. (I) qRT-PCR analysis for the expression of B cell marker gene Cd19 in the peripheral blood of mice infected with SARS-CoV-2 after Paquinimod treatment. n ≥ 5. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. Error bars, SD.

Figure 5

Blocking TLR4 signal can alleviate coronavirus fatal infection (A and B) Flow cytometry analysis of neutrophils in lungs, blood, and bone marrow from mice infected with SARS-CoV-2 (A) and MHV (B) at 5 dpi after Resatorvid treatment. Aberrant neutrophils (P2) in the mice infected with coronavirus were significantly decreased by Resatorvid treatment. n = 3. (C and D) qRT-PCR analysis for the expression of S100a8, Ly6g, and viral loads in the lungs of mice infected with SARS-CoV-2 (C) and MHV (D) at 5 dpi after Resatorvid treatment. n = 3. (E) qRT-PCR analysis for the effect of recombinant S100A8/A9 on S100a8 and Cxcl2 expression through TLR4 pathway. n = 3. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. Error bars, SD.