TNF-α exacerbates SARS-CoV-2 infection by stimulating CXCL1 production from macrophages

Abstract

Since most genetically modified mice are C57BL/6 background, a mouse-adapted SARS-CoV-2 that causes lethal infection in young C57BL/6 mice is useful for studying innate immune protection against SARS-CoV-2 infection. Here, we established two mouse-adapted SARS-CoV-2, ancestral and Delta variants, by serial passaging 80 times in C57BL/6 mice. Although young C57BL/6 mice were resistant to infection with the mouse-adapted ancestral SARS-CoV-2, the mouse-adapted SARS-CoV-2 Delta variant caused lethal infection in young C57BL/6 mice. In contrast, MyD88 and IFNAR1 KO mice exhibited resistance to lethal infection with the mouse-adapted SARS-CoV-2 Delta variant. Treatment with recombinant IFN-α/β at the time of infection protected mice from lethal infection with the mouse-adapted SARS-CoV-2 Delta variant, but intranasal administration of recombinant IFN-α/β at 2 days post infection exacerbated the disease severity following the mouse-adapted ancestral SARS-CoV-2 infection. Moreover, we showed that TNF-α amplified by type I IFN signals exacerbated the SARS-CoV-2 infection by stimulating CXCL1 production from macrophages and neutrophil recruitment into the lung tissue. Finally, we showed that intravenous administration to mice or hamsters with TNF protease inhibitor 2 alleviated the severity of SARS-CoV-2 and influenza virus infection. Our results uncover an unexpected mechanism by which type I interferon-mediated TNF-α signaling exacerbates the disease severity and will aid in the development of novel therapeutic strategies to treat respiratory virus infection and associated diseases such as influenza and COVID-19.

Fig 1. Generation of a mouse-adapted ancestral SARS-CoV-2.

(A) Schematic representation of experimental setup. (B) Total RNAs were extracted from lung washes at 3 days p.i. and SARS-CoV-2 N gRNA levels were assessed by quantitative reverse transcription PCR. (C) Schematic diagram of SARS-CoV-2 genome and all the adaptive mutations identified in the ancestral P80 virus. Nonsynonymous mutations were compared to the original ancestral SARS-CoV-2 (WT). Each symbol indicates individual values (B). Data are mean ± s.e.m. (B).

Fig 2. Pathogenesis of a mouse-adapted ancestral SARS-CoV-2 in laboratory mice.

(A-F) BALB/c, C3H, and C57BL/6 mice were infected intranasally with 1×10⁵ pfu of the ancestral P80 virus. Weight loss (A, C and E) and mortality (B, D and F) were monitored for 14 days. (G) The lung washes were collected at 3 days p.i. and viral titers were determined by standard plaque assay. Each symbol indicates individual values (A, C, E and G). Data are mean ± s.e.m. (A, C, E and G). Statistical significance was analyzed by two-way analysis of variance (ANOVA) (A, C, E and G). *P < 0.05, ***P < 0.001, n.s., not significant.

Fig 3. The Delta P80 virus causes lethal infection in young C57BL/6 mice.

(A-G) Six-week-old C57BL/6 mice were infected intranasally with 1×10⁵ pfu of the Delta P18 (A, F and G), P29 (B, F and G), P41 (C, F and G), P61 (D, F and G), or P80 virus (E, F and G). Weight loss (A-E) and mortality (F) were monitored for 14 days. The dashed line indicates the limit of endpoint (D and E). The lung washes were collected at 3 days p.i. and viral titers were determined by standard plaque assay (G). (H) VeroE6/TMPRSS2 cells were infected with the original SARS-CoV-2 Delta variant, mouse-adapted Delta P18, P29, P41, P61, or P80 virus. Cell-free supernatants were collected at 48 h p.i. and analyzed for virus titer by standard plaque assay using VeroE6/TMPRSS2 cells. (I) Schematic diagram of SARS-CoV-2 genome and all the adaptive mutations identified in the Delta P18, P29, P41, P61, and P80 virus. Nonsynonymous mutations were compared to the original SARS-CoV-2 Delta variant (WT). Each symbol indicates individual values (A-E, G and H). Statistical significance was analyzed by two-way analysis of variance (ANOVA) (A-E and G), or two-sided log-rank (Mantel-Cox) test (F). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4. The Delta P80 virus causes severe pneumonia in young C57BL/6 mice.

Six-week-old C57BL/6 mice were infected intranasally with 1×10⁵ pfu of the ancestral or Delta P80 virus. (A and B) Weight loss (A) and mortality (B) were monitored for 14 days. The dashed line indicates the limit of endpoint (A). (C and D) Gross lung pathology (C) and total lung weight (D) of mice infected with the ancestral or Delta P80 viruses at 4 days p.i.. (E) The lung washes were collected at 3 days p.i. and viral titers were determined by standard plaque assay. (F-J) The lung washes were collected at indicated time points and analyzed for IFN-α (F), IFN-β (G), IL-6 (H), TNF-α (I), and CXCL1 (J) by ELISA. (K) The lung was collected from the virus-infected mice at 3 days p.i.. The single-cell-suspensions of lung samples were stained with anti-SARS-CoV-2 nucleoprotein and anti-CD45.2 antibodies. The ratio of the nucleoprotein-positive cells among CD45.2-positive or CD45.2-negative cells are shown. (L and M) Total RNAs were extracted from indicated tissues at 3 (L) and 5 (M) days p.i. The levels of SARS-CoV-2 N gRNA were assessed by quantitative reverse transcription PCR. Each symbol indicates individual values (A, D-M). Statistical significance was analyzed by two-tailed unpaired Student’s t test (A, E-J, L, and M), two-sided log-rank (Mantel-Cox) test (B), or two-way analysis of variance (ANOVA) (D and K). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5. MyD88 and IFNAR1 KO mice are resistant to Delta P80 virus infection.

(A and B) Six-week-old C57BL/6 WT or MyD88 mice were infected intranasally with 1×10⁵ pfu of the ancestral P80 virus. Weight loss (A) and mortality (B) were monitored for 14 days. (C-M) Six-week-old C57BL/6 WT, MyD88, or IFNAR1 KO mice were infected intranasally with 1×10⁵ pfu of the Delta P80 virus. Weight loss (C and E) and mortality (D and F) were monitored for 14 days. The dashed line indicates the limit of endpoint (C and E). The lung washes were collected at 3 days p.i. and viral titers were determined by standard plaque assay (G and H). The lung washes were collected at 2 days p.i. and analyzed for IFN-α (I), IFN-β (J), IFN-λ (K), TNF-α (L), and IL-6 (M) by ELISA. Each symbol indicates individual values (A, C, E and G-M). Statistical significance was analyzed by two-tailed unpaired Student’s t test (A, C, E, G and H), two-sided log-rank (Mantel-Cox) test (B, D and F), or two-way analysis of variance (ANOVA) (I-M). *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.

Fig 6. Protective and detrimental roles of type I IFNs in SARS-CoV-2 infection.

(A-C) Six-week-old C57BL/6 mice were administered intranasally with 1×10⁵ pfu of the ancestral or Delta P80 virus together with PBS or recombinant mouse IFN-α (1,250 unit) and IFN-β (1.25 ng) (arrow). Weight loss (A and B) and mortality (C) were monitored for 14 days. The dashed line indicates the limit of endpoint (B). (D-H) Six-week-old C57BL/6 WT (D-G), IFNAR1 (G), or MyD88 KO (H) mice infected with 1×10⁵ pfu of the ancestral (D-G) or Delta P80 virus (H) were administered intranasally with PBS or recombinant mouse IFN-α (1,250 unit) and IFN-β (1.25 ng) at 2 (D, G and H), 3 (E), or 4 (F) days p.i. (arrow). Mortality was monitored for 14 days. Each symbol indicates individual values (A and B). Statistical significance was analyzed by two-tailed unpaired Student’s t test (A and B), or two-sided log-rank (Mantel-Cox) test (C-H). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 7. TNF-α-CXCL1 axis exacerbates ancestral P80 virus infection.

(A) Bone marrow-derived macrophages were stimulated with indicated recombinant mouse cytokines. Cell-free supernatants were collected at 24 h p.i. and analyzed for CXCL1 by ELISA. (B-D) Six-week-old C57BL/6 mice infected with 1×10⁵ pfu of the ancestral P80 virus were administered intranasally with PBS or recombinant mouse CXCL1 (2.5 μg) at 6 hours (B), 2 (C), or 3 (D) days p.i. (allow). Mortality was monitored for 14 days. (E-G) Six-week-old C57BL/6 WT (E-G), MyD88 (F), or IFNAR1 KO (G) mice were infected intranasally with 1×10⁵ pfu of the Delta P80 virus. The lung washes were collected at 2 (E-G) or 3 (E) days p.i. and analyzed for CXCL1 by ELISA. (H) Six-week-old C57BL/6 WT mice infected with the ancestral P80 virus were administered intranasally with PBS or recombinant mouse TNF-α (2.5 μg) at 2 days p.i. (arrow). Mortality was monitored for 14 days. (I and J) Six-week-old C57BL/6 WT mice were administered intranasally with a recombinant mouse TNF-α (2.5 μg). The lung washes were collected at indicated time points and analyzed for CXCL1by ELISA (I). Three days later, leukocytes were isolated from the lung. The number of Ly6C⁺ Ly6G⁺ neutrophils were analyzed by flow cytometry (J). (K) Six-week-old C57BL/6 WT mice infected with the Delta P80 virus were administered intraperitoneally with a recombinant DNase I at indicated time points (arrow). Mortality was monitored for 14 days. Each symbol indicates individual values (E-G, I, and J). Statistical significance was analyzed by two-sided log-rank (Mantel-Cox) test (B-D, H and K), two-way analysis of variance (ANOVA) (E-G, and I), or two-tailed unpaired Student’s t test (J). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 8. TAPI-2 alleviates SARS-CoV-2-associated mortality.

(A-F) Six-week-old C57BL/6 mice infected with 1×10⁵ pfu of the Delta P80 virus were administered intravenously with saline or TAPI-2 (2.5 μg) at 0, 1, and 2 days p.i. (allow). Mortality was monitored for 14 days (A). The lung washes were collected at indicated time points and analyzed for TNF-α (B) or IL-6 (C) by ELISA. Total RNAs were extracted from lung washes and SARS-CoV-2 N gRNA levels were assessed by quantitative reverse transcription PCR (D). Viral titers were determined by standard plaque assay (E). Five days later, leukocytes were isolated from the lung. The number of Ly6C⁺ Ly6G⁺ neutrophils were analyzed by flow cytometry (F). (G-J) Four-week-old Syrian hamsters infected with 8×10⁶ pfu of wild-type SARS-CoV-2 Delta variant were administered intravenously with saline or TAPI-2 (3 μg) at 0, 1, 2, and 3 days p.i. (allow). Mortality was monitored for 14 days (G). The lung washes were collected at indicated time points and viral titers were determined by standard plaque assay (H). Gross lung pathology (I) and total lung weight (J) of hamsters infected with wild-type SARS-CoV-2 Delta variant at 3 days p.i.. Each symbol indicates individual values (B-F, H, and J). Statistical significance was analyzed by two-tailed unpaired Student’s t test (B, C, D, E, and H), two-sided log-rank (Mantel-Cox) test (A and G), or two-way analysis of variance (ANOVA) (F and J). *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.