Fig 2: 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.

Fig 3: 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.

Fig 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+Ly6Gvariable 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.

Fig 5: 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+Ly6Ghigh), and Gate P2 shows the pathologic aberrant neutrophils (CD45+CD11b+Ly6Gvariable). 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.