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: 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.

Fig 4: 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.

Fig 5: 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.