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

Moe Kobayashi; Nene Kobayashi; Kyoka Deguchi; Seira Omori; Minami Nagai; Ryutaro Fukui; Isaiah Song; Shinji Fukuda; Kensuke Miyake; Takeshi Ichinohe

doi:10.1371/journal.ppat.1012776

. 2024 Dec 9;20(12):e1012776. doi: 10.1371/journal.ppat.1012776

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

Moe Kobayashi ¹, Nene Kobayashi ¹, Kyoka Deguchi ¹, Seira Omori ¹, Minami Nagai ¹, Ryutaro Fukui ², Isaiah Song ³, Shinji Fukuda ^3,^4,^5,⁶, Kensuke Miyake ², Takeshi Ichinohe ^1,^*

Editor: Jie Sun⁷

PMCID: PMC11658697 PMID: 39652608

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.

Author summary

Coronavirus disease 2019 (COVID-19) cause severe morbidity and mortality worldwide. Although mounting evidence indicates that the virus-induced cytokine storm associates with severity of COVID-19, the pathological role of inflammatory cytokines in severe COVID-19 remains unknown. Here, we demonstrated that the virus-induced pulmonary TNF-α amplified by MyD88 and type I interferon receptor signals exacerbated the SARS-CoV-2 infection by stimulating CXCL1 production from macrophages. In addition, we found that TNF-α protease inhibitor 2 alleviated the severity of SARS-CoV-2 and influenza virus infection in mice and hamsters. Our results provide important insights into the role of type I interferon-mediated TNF-α signaling in neutrophil-induced lung pathology and will aid the development of novel therapeutic strategies to treat respiratory viral infections and associated diseases.

Introduction

While human ACE2 (hACE2) transgenic mice are widely used worldwide in SARS-CoV-2 research, studying innate immune signals required for protection against SARS-CoV-2 infection in vivo requires crossing hACE2 transgenic mice with innate immune-related gene-deficient mice [1]. A mouse model of SARS-CoV-2 based on adeno-associated virus (AAV)-mediated expression of hACE2 in respiratory tract is useful model for COVID-19 pathogenesis and protection [2]. Similarly, a mouse-adapted SARS-CoV-2 is convenient for studying innate immune signals required for protection against SARS-CoV-2 infection in vivo. However, previously established mouse-adapted SARS-CoV-2 causes lethal infection only in aged Balb/c or C57BL/6 mice but not 6-week-old young C57BL/6 mice [3–6]. Since most genetically modified mice are C57BL/6 background, a mouse-adapted SARS-CoV-2 that causes lethal infection in young C57BL/6 mice could be more powerful tool for studying innate immune signals required for protection against SARS-CoV-2 infection.

A previous study using a mouse model of SARS-CoV-2 based on AAV-mediated expression of hACE2 demonstrated that type I interferon (IFN) signaling do not control SARS-CoV-2 replication in vivo but are significant drivers of pathological responses [2]. In contrast, Ogger et al. showed that type I IFN signaling is essential for suppressing SARS-CoV-2 replication and inflammatory myeloid cell recruitment to the lung of AAV-hACE2-transduced mice following SARS-CoV-2 infection [7]. In addition, a recent study demonstrated that the STING-deficient K18-hACE2 mice show no difference in weight change and survival following SARS-CoV-2 infection [1]. However, the role of other innate immune signals in severity of SARS-CoV-2 infection in vivo remains unknown.

The objective of this study is to establish a lethal SARS-CoV-2 infection model in 6-week-old mice, which serves as a critical platform for testing novel therapeutics and vaccines, as well as to elucidate the role of innate immune signals in the severity of COVID-19. Unlike models in 10–12-week-old C57BL/6J mice [3,5], which may still be influenced by age-related factors, the use of younger mice minimizes potential confounding effects while providing a standardized and reproducible system. Additionally, this model allows for the investigation of severe disease mechanisms relevant to younger populations, including rare but significant cases of severe COVID-19 in pediatric and adolescent patients. Therefore, we attempted to establish a mouse-adapted SARS-CoV-2 strain that causes lethal infection in young mice. In this study, we establish mouse-adapted ancestral SARS-CoV-2 and the Delta variants by serial passaging 80 times in C57BL/6 mice (named ancestral P80 and Delta P80 viruses, respectively). We demonstrate that MyD88 and type I IFN signaling exacerbates the Delta P80 virus infection. In addition, type I IFN signaling amplifies secretion of pulmonary inflammatory cytokines including tumor necrosis factor alpha (TNF-α), which in turn stimulates CXCL1 production from macrophages and stimulates neutrophil recruitment into the lung tissue. Further, we find that intravenous administration to mice or hamsters with TNF-α protease inhibitor 2 alleviated the severity of SARS-CoV-2 and influenza virus infection.

Results

Generation of a mouse-adapted ancestral SARS-CoV-2

Leist et al. demonstrated that 1-year-old mice were highly susceptible to mouse-adapted SARS-CoV-2 [4]. Thus, we first infected 83-weeks-old aged C57BL/6 mice intranasally with an ancestral SARS-CoV-2 (SARS-CoV-2/UT-NCGM02/human/2020) to generate a mouse-adapted virus [8]. Then, we collected the lung washes at 3 days post infection (p.i.) and inoculated them into VeroE6/TMPRSS2 cells to propagate the SARS-CoV-2 (refer to as P1) (Fig 1A). We confirmed that these viruses were gradually adapted in aged C57BL/6 mice during 5 serial passages by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (Fig 1B). After 5 serial passages in aged mice, we next infected 6-weeks-old young C57BL/6 mice intranasally with the P5 virus (Fig 1A). Then, we collected the lung washes at 3 days p.i. and inoculated them into VeroE6/TMPRSS2 cells to propagate the P6 virus (Fig 1A). Although the levels of SARS-CoV-2 nucleoprotein RNA in the lung washes at 3 days p.i. were gradually reduced during 5 serial passages in young mice, these viruses were gradually adapted in young mice during additional 3 passages (Fig 1B). After 10 serial passages in young mice, we serially passaged the lung washes of the virus-infected young mice every 3 days without virus propagation in VeroE6/TMPRSS2 cells to generate the ancestral P80 virus (Fig 1A). The ancestral P80 viral stocks used in the experiments were propagated in VeroE6/TMPRSS2 cells. Next-generation sequencing analysis revealed that the ancestral P80 virus contained 27 amino acid substitutions that were distributed within the ORF1ab, S, 3a, E, M, 7a, N and 10 genes, respectively (Fig 1C).

Fig 1 — **(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).

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

We next examined pathogenesis of mouse-adapted the ancestral P80 virus in standard laboratory mice. After the ancestral P80 virus infection, young Balb/c and C3H mice succumbed to disease by 5 days p.i. (Fig 2A–2D). In contrast, young C57BL/6 mice exhibited ~25% weight loss and recovered by 10 days p.i. (Fig 2E and 2F). The virus titers were significantly elevated in the lung of young Balb/c mice compared with those of young C57BL/6 mice (Fig 2G).

Fig 2 — **(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.

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

Since the ancestral P80 virus did not cause lethal infection in young C57BL/6 mice, we next tried to generate a mouse-adapted SARS-CoV-2 Delta variant (hCoV-19/Japan/TY11-927-P1/2021) [9]. As described in Fig 1A, we first passed a SARS-CoV-2 Delta variant five times in aged mice and then additional 10 times in young mice. After the Delta P18 or P29 virus infection, young C57BL/6 mice did not reduce their body weight (Fig 3A and 3B). In addition, young C57BL/6 mice infected with the Delta P41 virus exhibited ~10% weight loss and recovered by 6 days p.i. (Fig 3C). In contrast, the Delta P61 and P80 viruses caused lethal infection in young C57BL/6 mice (Fig 3D–3F). The Delta P80 virus was found to exhibit enhanced replication in the lung of young C57BL/6 mice compared with the mouse-adapted Delta P18, P29, P41, or P61 variants without affecting viral replication in VeroE6/TMPRSS2 cells (Fig 3G and 3H). Next-generation sequencing analysis revealed that the Delta P80 virus contained 27 amino acid substitutions that were distributed within the ORF1ab, S, E, M and N genes, respectively (Fig 3I).

Fig 3 — **(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.

So far, we established two mouse-adapted SARS-CoV-2 variants by serial passaging 80 times in C57BL/6 mice. Next, we compared pathogenesis of the ancestral and Delta P80 viruses in young C57BL/6 mice. While young C57BL/6 mice were resistant to the ancestral P80 virus infection, the Delta P80 virus caused lethal infection (Fig 4A and 4B). In addition, the Delta P80 virus caused severe pulmonary edema in young C57BL/6 mice, while the ancestral P80 virus caused partial lung inflammation (Fig 4C and 4D). The virus titers, virus-induced proinflammatory cytokines, and neutrophil recruitment were significantly elevated in the lung of the Delta P80 virus-infected mice compared with those of ancestral P80 virus infected mice (Figs 4E–4J and S1). While the primary target of these two variants was the CD45.2 negative epithelial cells in the lung (Figs 4K and S2), the Delta P80 virus also infected a large number of alveolar macrophages and dendritic cells in the lung tissue (S3–S7 Figs). Next, we examined the possibility that the Delta P80 virus infects tissues other than the lung in mice. However, we were unable to detect viral RNA in the brain, heart, liver or kidney of mice infected with the ancestral or Delta P80 viruses (Fig 4L and 4M). These results suggest that the differences in pathogenicity between these two variants in young C57BL/6 mice are likely due to differences in viral replication or virus-induced inflammatory responses in the lung, and that the Delta variant has not acquired the ability to infect other organs outside the lung.

Fig 4 — 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.

MyD88 and IFNAR1 signals exacerbate SARS-CoV-2 infection

To examine innate immune signals required for protection against SARS-CoV-2 infection, we took advantage of the ancestral and Delta P80 viruses with different pathogenicity in young C57BL/6 mice. We first infected wild-type (WT) and MyD88 KO mice with the ancestral P80 virus. However, both WT and MyD88 KO mice recovered from ancestral P80 virus infection (Fig 5A and 5B). Similarly, no significant differences were observed in body weight change or survival rate between MyD88-deficient and WT mice following a sublethal dose (1×10⁴ pfu) of the Delta P80 virus infection (S8 Fig). Next, we infected WT and MyD88 KO mice with a lethal dose (1×10⁵ pfu) of the Delta P80 virus. Interestingly, MyD88 KO mice exhibited resistance to lethal infection with the Delta P80 virus infection (Fig 5C and 5D). Similarly, IFNAR1 KO mice were highly resistant to the Delta P80 virus infection (Fig 5E and 5F). Although the virus titers were comparable between WT and IFNAR1 or MyD88 KO mice (Fig 5G and 5H), the levels of IFN-α, IFN-β, IFN-λ, and TNF-α, but not IL-6 were significantly reduced in the lungs of MyD88 and IFNAR1 KO mice compared with those of WT mice at 2 days p.i. (Fig 5I–5M). In contrast to these proinflammatory cytokines, the Delta P80 virus did not induce detectable levels of IL-1β in the lungs of WT, MyD88, or IFNAR1 KO mice at 2 days p.i. (S9 Fig). These data suggest that the virus-induced proinflammatory cytokines rather than virus burden in the lung may contribute lethality in the Delta P80 virus-infected mice.

Fig 5 — **(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.

Thus far, our data indicated that the virus-induced proinflammatory cytokines amplified by type I IFN signals exacerbate SARS-CoV-2 infection. Thus, we next examined the role of type I IFNs in disease severity of SARS-CoV-2 infection. Intranasal administration to mice with recombinant IFN-α and IFN-β (IFN-α/β) at the time of infection completely protected mice from the ancestral or Delta P80 virus infection, highlighting the importance of type I IFNs in defense against SARS-CoV-2 infection (Fig 6A–6C). In contrast, intranasal administration to mice with recombinant IFN-α/β at later time points (2 to 4 days p.i.) exacerbated the ancestral P80 virus infection in WT but not IFNAR1 KO mice (Fig 6D–6G). Further, MyD88 KO mice, which were resistant to the Delta P80 virus infection (Fig 5C and 5D), exacerbated the disease after intranasal administration of IFN-α/β at 2 days post infection (Fig 6H).

Fig 6 — **(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.

TNF-α-CXCL1 axis exacerbates SARS-CoV-2 infection

Next, we wished to determine the mechanism by which type I IFN signals following SARS-CoV-2 infection exacerbate severity of the disease. Previous studies have indicated the detrimental role of neutrophils in severe COVID-19 [10–14]. Because the levels of IFN-α, IFN-β, IFN-λ, and TNF-α were significantly elevated in the lungs of the Delta P80 virus-infected WT mice compared with those of MyD88 and IFNAR1 KO mice (Fig 5I–5L), we first examined whether these cytokines are involved in production of CXCL1, which is one of the major chemoattractant of neutrophils, from macrophages. Although IL-1β is an important mediator of CXCL1 production [15,16], it is unknown whether other cytokines stimulate CXCL1 production. Interestingly, treatment of bone marrow-derived macrophages with TNF-α significantly stimulated CXCL1 production in a dose-dependent manner (Fig 7A). We next examined the role of CXCL1 in disease severity of SARS-CoV-2 infection. Indeed, intranasal administration of CXCL1 at later but not early time points exacerbated the ancestral P80 virus infection (Fig 7B–7D). Following the Delta P80 virus infection, the levels of CXCL1 in the lung washes became apparent around day 2 and 3 p.i. (Fig 7E). In contrast, the levels of pulmonary CXCL1 were significantly suppressed in the lung washes of MyD88 or IFNAR1 KO mice compared with WT mice at 2 days p.i. (Fig 7F and 7G). We next examined whether the CXCL1-mediated exacerbation of SARS-CoV-2 infection can be reproduced by intranasal administration of TNF-α. Intranasal administration of TNF-α at 2 days p.i. exacerbated the ancestral P80 virus infection by stimulating CXCL1 production and neutrophil recruitment into the lung tissue (Fig 7H–7J). In addition, MyD88 KO mice, which were resistant to the Delta P80 virus infection, exacerbated the disease after intranasal administration of TNF-α at 2 days p.i. (S10 Fig). Further, neutrophil extracellular traps digestion in vivo resulted in prolonged survival of mice after the Delta P80 virus infection (Fig 7K). Together, these data suggested that TNF-α-stimulated CXCL1 from macrophages may enhance neutrophil recruitment, lung tissue damage and mortality following the Delta P80 virus infection.

Fig 7 — **(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.

Inhibition of TNF-α alleviates the Delta P80 virus-associated mortality

We next examined whether inhibition of TNF-α secretion can alleviate the Delta P80 virus-associated mortality. To this end, we first injected mice intravenously or intranasally with TNF protease inhibitor 2 (TAPI-2), a broad-spectrum inhibitor of matrix metalloprotease and TNF converting enzyme, after the Delta P80 virus infection. Intravenous, but not intranasal (S11 Fig), administration of TAPI-2 to mice resulted in improved survival after Delta P80 infection compared to controls (Fig 8A). The levels of pulmonary TNF-α and neutrophil recruitment into the lung tissue were significantly reduced in mice treated with TAPI-2 intravenously, whereas no significant reduction was observed in pulmonary IL-6 and viral load (Fig 8B–8F). Interestingly, the effect of TAPI-2 appears to be more pronounced in aged mice (S12 Fig), probably due to enhanced inflammatory responses in aged mice compared to younger mice (S13 Fig). Further, TAPI-2-treated hamsters had improved survival and pulmonary edema relative to control group following wild-type SARS-CoV-2 Delta variant infection without affecting pulmonary virus titers (Fig 8G–8J). In addition, we examined the effects of TAPI-2 treatment on influenza virus-induced mortality. Following influenza virus infection, the levels of pulmonary TNF-α but not IL-6 were significantly suppressed in TAPI-2-treated group (S14A and S14B Fig). In addition, the TAPI-2-treated mice had improved survival relative to control group following influenza virus infection without affecting pulmonary virus titers (S14C and S14D Fig). Finally, we investigated whether the administration of anti-TNF-α antibodies improves the survival rate of the Delta P80 virus-infected mice. Intravenous, but not intranasal (S15 Fig), administration of anti-TNF-α antibodies to mice significantly reduced pulmonary TNF-α levels and improved survival after the Delta P80 virus infection compared to control group, without affecting pulmonary viral load (S16 Fig). Taken together, our data show that pulmonary TNF-α amplified by type I IFN signals exacerbated the SARS-CoV-2 infection by stimulating CXCL1 production from macrophages. Under such conditions, protease inhibitors that block TNF-α secretion or anti-TNF-α antibodies might be a possible therapeutic drug to reduce the tissue damage and severity of the disease.

Fig 8 — **(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.

Discussion

The pathogenicity of mouse-adapted SARS-CoV-2 in mice depends on their genetic background, age, and sex [4,17–19]. Thus far, several groups established mouse-adapted SARS-CoV-2 that cause lethal infection in aged Balb/c or C57BL/6 mice but not 6-week-old young C57BL/6 mice [3–6,17–20]. Here we established, for the first time, a mouse-adapted SARS-CoV-2 Delta variant that cause lethal infection in 6-week-old young C57BL/6 mice by serial passaging 80 times in mice. Although the mouse-adapted ancestral SARS-CoV-2 acquired 27 amino acid substitutions throughout the viral genome during 80 serial passages in mice, it did not cause lethal infection in 6-week-old young C57BL/6 mice. Similarly, the mouse-adapted SARS-CoV-2 Delta variants acquired 27 amino acid substitutions throughout the viral genome during 80 serial passages in mice. In contrast to the ancestral P80 virus, the Delta P80 virus caused severe pulmonary edema and lethal infection in 6-week-old young C57BL/6 mice. These data suggest that serial 80 passages do not simply increase the pathogenicity of the mouse adapted SARS-CoV-2 Delta variant, but the Delta P80 virus may maintain the virological characteristics and pathogenicity of the parental SARS-CoV-2 Delta variant during 80 serial passages in mice. It is noteworthy that the nsp9-T35I substitution (Orf1a T4175I), which was identified in the Delta P80 virus, is a characteristic mutation of the currently circulating EG.5 and BA.2.86/JN.1 variants [21,22]. In addition, E-T30I substitution, which was found in the ancestral P80, Delta P18, P29, P41, P61, and P80 viruses, was determined to be the second most frequent recurrently occurring mutation arising in persistent infection [23]. Importantly, a single variant lineage, B.1.616 that is associated with high lethality [24], does contain E-T30I as a lineage-defining mutation [23].

The Delta P80 virus significantly enhanced viral replication in the lung of young C57BL/6 mice compared to the mouse-adapted Delta P18, P29, P41, or P61 variants without affecting viral replication in VeroE6/TMPRSS2 cells. In addition, the Delta P80 virus caused severe pulmonary edema and lethal infection in 6-week-old young C57BL/6 mice. The Delta P80 virus acquired 7 new amino acid substitutions during an additional 19 passages from the P61 virus. These 7 amino acid substitutions include nsp2, nsp3, S, and M genes. Since multiple SARS-CoV-2 proteins including nsp2, nsp3, and M inhibit Ifnb1 transcription [25,26], these amino acid substitutions in nsp2, nsp3, and M proteins of the Delta P80 virus could contribute to inhibit host interferon responses and efficient viral replication in vivo. In this study, we showed that treatment with recombinant IFN-α/β at the time of infection completely protected mice from lethal Delta P80 virus infection. These results suggest that host interferon responses at the time of infection are important for suppressing SARS-CoV-2 replication in vivo. It has been reported that among patients with severe COVID-19, some have deficiencies in toll-like receptor 3 (TLR3), TLR7, or interferon (IFN) signaling, or possess autoantibodies against type I IFNs [27–29]. However, our data also showed that intranasal administration to mice with recombinant IFN-α/β at 2 days p.i. exacerbated the ancestral P80 virus infection. In addition, the pulmonary virus titers were comparable between WT and IFNAR1 KO mice. These results indicate that host type I IFN signals do not control a lethal dose (1×10⁵ pfu) of the Delta P80 virus infection and exacerbate the disease severity. The discrepancies in these results may be explained by the timing of type I IFN responses or the differences in infectious doses across the experimental condition. Indeed, we observed no significant differences in body weight change or survival rate between MyD88-deficient and WT mice following a sublethal dose (1×10⁴ pfu) of the Delta P80 virus infection. In contrast, MyD88 and IFNAR1 KO mice exhibited resistance to a lethal dose (1×10⁵ pfu) of the Delta P80 virus infection. In a lethal dose (3×10⁴ pfu) of SARS-CoV infection, Channappanavar and colleagues have demonstrated that the disease severity is ameliorated in Ifnar^-/- BALB/c mice [30]. These observations suggest that host IFNs can block SARS-CoV-2 infections when viral burdens are low, such as during a physiological dose infection in humans or when mice are infected with the ancestral P80 virus, as demonstrated in this study. However, if innate antiviral immune system deficiencies allow low viral burdens to overcome host antiviral defenses—for example, in individuals with deficiencies in innate immune signals or those with autoantibodies against type I IFNs—or if infections involve high viral loads, as seen in this study where a lethal dose (1×10⁵ pfu) was administered intranasally to mice, the IFN response becomes insufficient to control initial viral replication. This insufficiency may lead to excessive inflammation and lung injury. [31]. Recent studies have demonstrated that SARS-CoV-2 induces delayed type I IFN responses [26,32,33]. In addition, the induction of type I IFN in response to viral infection may be impaired in older hosts [34]. Together, the virological characteristics, host interferon responses, and infectious doses may have important implications for SARS-CoV-2 pathogenesis [31,35,36]. Future studies using physiological doses of mouse-adapted SARS-CoV-2 variants in aged, immunocompromised, or obese mice will facilitate the development of efficacious interventions and treatments for severe COVID-19.

Our data have demonstrated that MyD88 or IFNAR1 KO mice are highly resistant to lethal the Delta P80 virus infection compared with WT mice. Pulmonary virus titers were comparable between WT and MyD88 or IFNAR1 KO mice. Instead, the levels of IFN-α, IFN-β, IFN-λ, and TNF-α were significantly reduced in the lungs of MyD88 and IFNAR1 KO mice compared with those of WT mice. Consistent with a previous report [37], we found that treatment of bone marrow-derived macrophages with TNF-α significantly stimulated CXCL1 production. In addition, we showed that intranasal administration of CXCL1 or TNF-α at 2 days p.i. exacerbated the ancestral P80 virus infection. CXCL1 is a well-known major chemoattractant of neutrophils. It has increasingly become evident that excess neutrophil recruitment into the lung exacerbates SARS-CoV-2 infection [10–14]. In fact, we demonstrated that nasal administration of CXCL1 enhanced neutrophil recruitment into the lung. In addition, previous studies have demonstrated that combination of TNF-α and IFN-γ synergistically induce inflammatory cell death and exacerbates SARS-CoV-2-induced mortality [5,38]. In addition to inflammatory role of TNF-α in severity of SARS-CoV-2 infection, TNF-α is known to cause bronchial hyperreactivity, narrowing of the airways, damage to the respiratory epithelium, stimulation of collagen synthesis and fibrosis in the respiratory system [39]. Recently, it has demonstrated that aged TNF KO mice exhibit resistance to lethal infection with SARS-CoV-2 N501Y P21 virus infection without affecting virus titers in the lung compared with aged WT mice [3]. Similarly, we found that intravenous treatment of the Delta P80 virus-infected mice with an anti-TNF-α antibody significantly improved survival relative to control group without affecting pulmonary virus titers. Together, these data suggest that type I IFNs signals may exacerbate SARS-CoV-2 infection by stimulating TNF-α-induced neutrophils recruitment and/or TNF-α/IFN-γ-induced inflammatory cell death in the lung tissue. Thus, TNF-α could be considered a potential therapeutic target for severe COVID-19 [40].

It is difficult to directly compare the relative virulence of the ancestral and delta variants in humans, given the potential influence of vaccination on the disease severity. A previous study demonstrated that the Delta variant is more virulent than the ancestral strain in dwarf hamsters [41]. Consistent with this observation, we showed that the Delta P80 virus exhibited a higher mortality rate in young C57BL/6 mice compared to the ancestral P80 virus. There are several possible explanations for how specific mutations observed in the Delta P80 virus contribute to MyD88/IFNAR1-mediated severe disease outcomes. First, the Delta P80 virus retained mutations characteristic of the Delta variant spike protein, specifically L452R and P681R, which are important for increasing the fusogenicity of the spike protein and the formation of syncytia in infected cells [42,43]. Syncytial death via apoptosis, pyroptosis, or TNF-mediated necroptosis can release the pathogen-associated molecular patterns (PAMPs) and enhance excessive inflammatory responses at the site of infection [44]. Second, interferon induced transmembrane protein 1 (IFITM1), induced in a MyD88 and IFNAR1 signaling dependent manner [45], is involved in the inhibition of syncytium formation in SARS-CoV-2 infected cells [46]. It is possible that specific mutations observed in the Delta P80 virus may contribute to inhibiting MyD88/IFNAR1-mediated IFITM1 expression [31], which could potentially lead to an increase in syncytium formation in SARS-CoV-2-infected cells and an exacerbation of the syncytial death-mediated inflammatory response. Third, the ancestral and Delta P80 viruses primarily target CD45.2-negative epithelial cells in the lung, but the specific mutations observed in the Delta P80 virus spike protein may have resulted in an increased infection rate of alveolar macrophages and dendritic cells in the lung tissue. This may potentially result in a MyD88/IFNAR1-dependent excessive inflammatory responses and the development of severe pulmonary edema in young C57BL/6 mice. However, treatment of mice with TAPI-2, anti-TNF-α antibody, or DNase I had a limited, but significant, effect on increasing survival after the Delta P80 virus infection. Therefore, the identification of more efficacious methods for the suppression of TNF-α production or neutrophil recruitment may facilitate the development of superior therapeutic interventions for the treatment of severe cases of COVID-19.

In summary, our findings substantially expand our understanding of how innate antiviral immune signals exacerbate SARS-CoV-2 infection. In addition, we established, for the first time, a mouse-adapted SARS-CoV-2 Delta variant that cause lethal infection in 6-week-old young C57BL/6 mice by serial passaging 80 times in mice. The Delta P80 virus infection enhanced pulmonary proinflammatory cytokines production including TNF-α in a MyD88- and IFNAR1-dependent manner. The TNF-α stimulated CXCL1 production from bone marrow-derived macrophages, which may enhance lung tissue damage and the disease severity following the Delta P80 virus infection (S17 Fig). Because decreased IFN and elevated proinflammatory cytokines are a common characteristic of the innate immune system in older humans [34], our results imply a possible effect of SARS-CoV-2-induced proinflammatory cytokines in severity of the diseases in elderly people. In addition, it will be important to determine whether type I IFNs therapy can be used for severe COVID-19.

Materials and methods

Ethics statement

All experiments with SARS-CoV-2 were performed in enhanced biosafety level 3 (BSL3) containment laboratories at the University of Tokyo, in accordance with the institutional biosafety operating procedures. All animal experiments including generation of mouse-adapted SARS-CoV-2 variants were performed in accordance with University of Tokyo’s Regulations for Animal Care and Use, which were approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo (PA22-33).

Animals

Six-week-old female C57BL/6JJmsSlc, BALB/cCrSIc, C3H/HeYokSlc mice, and 4-week-old female Syrian hamsters obtained from Japan SLC, Inc. were used as WT controls. For some experiments we used aged (64- to 83-week-old) female C57BL/6JJcl mice obtained from CLEA Japan, Inc. MyD88-deficient C57BL/6 mice were purchased from Oriental Bioservice (Kyoto, Japan). IFNAR1-deficient C57BL/6 mice were described previously [47].

Cell culture

VeroE6 cells stably expressing transmembrane protease serine 2 (VeroE6/TMPRSS2; JCRB Cell Bank 1819) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (low-glucose) (Nacalai Tesque, 08456–65) supplemented with 10% v/v fetal bovine serum (FBS), 1% v/v penicillin (100 units/ml)/streptomycin (100 μg/ml), and G418 (1 mg ml^-1; Nacalai Tesque, 16512–94). L929 cells were maintained in DMEM (high-glucose) (Nacalai Tesque, 08458–45) supplemented with 10% v/v FBS and 1% v/v penicillin/streptomycin (P/S). To prepare bone marrow-derived macrophages, bone marrows from the tibia and femur were obtained by flushing with DMEM. Bone marrow cells were cultured with DMEM supplemented with 10% FBS, L-glutamine, 1% P/S, and 30% L929 supernatant containing the macrophage colony-stimulating factor at 37°C for 5 days [48].

Viruses

An ancestral SARS-CoV-2 strain bearing aspartic acid at position 614 of spike (S) protein (S-614D) [8] and the Delta variant hCoV-19/Japan/TY11-927-P1/2021 (lineage B.1.617.2, GISAID ID: EPI_ISL_2158617) [9] variant were grown in VeroE6/TMPRSS2 cells for 2 days at 37°C. Viral titers were quantified by a standard plaque assay using VeroE6/TMPRSS2 cells and viral stock was stored at -80°C.

For generation of mouse-adapted SARS-CoV-2 variants, 64- to 83-week-old

C57BL/6 mice were intranasally infected with an ancestral SARS-CoV-2 (SARS-CoV-2/UT-NCGM02/human/2020) [8] or the Delta variant hCoV-19/Japan/TY11-927-P1/2021 (lineage B.1.617.2, GISAID ID: EPI_ISL_2158617) [9]. The lung washes were collected at 3 days p.i.. Then VeroE6/TMPRSS2 cells were inoculated with the lung washes to propagate the mouse-adapted SARS-CoV-2 (refer to as P1) (Fig 1A). After 5 serial passages in aged mice, 6-weeks-old young C57BL/6 mice were intranasally infected with the P5 virus (Fig 1A). The lung washes were collected at 3 days p.i. and VeroE6/TMPRSS2 cells were inoculated with the lung washes to propagate the P6 virus (Fig 1A). After 10 serial passages in young mice, we serially passaged the lung washes of the virus-infected young mice every 3 days without virus propagation in VeroE6/TMPRSS2 cells (Fig 1A). The ancestral and the Delta P80 viral stocks used in the experiments were grown in VeroE6/TMPRSS2 cells for 2 days at 37°C. Viral titers were quantified by a standard plaque assay using VeroE6/TMPRSS2 cells and viral stock was stored at -80°C.

A mouse-adapted influenza A virus strain A/Puerto Rico/8/1934 (PR8) was grown in allantoic cavities of 10-d-old fertile chicken egg for 2 days at 35°C. Viral titers were quantified by a standard plaque assay using MDCK cells and viral stock was stored at –80°C.

For intranasal infection, mice were infected by intranasal application of 50 μL of virus suspension (1×10⁵ pfu of mouse-adapted SARS-CoV-2 variants or 1×10³ pfu of PR8 in PBS) under isoflurane anaesthesia. Syrian hamsters were infected by intranasal application of 400 μL of virus suspension (8×10⁶ pfu of wild-type SARS-CoV-2 Delta variant in PBS).

Reagents

A recombinant mouse IFN-α (Cat#HC1040-10) was purchased from Hycult Biotech. A recombinant mouse IFN-β (Cat#8234-MB-010) was obtained from R&D Systems. Recombinant mouse CXCL1 (Cat#250–11) and TNF-α (Cat#315-01A) were from PeproTech. TAPI-2 (Cat#INH-3852-PI) was purchased from Biosynth. Monoclonal antibody against mouse TNF-α (XT3.11, Cat#BE0058) and rat IgG1 isotype control (HRPN, Cat#BE0088) were purchased from Bio X Cell.

Quantitative PCR

Total RNA was extracted from lung washes using TRIzol reagent (Invitrogen, 15596018) and reverse transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen, 18080085) with a SARS-CoV-2 N reverse primer (5’- tctggttactgccagttgaatctg-3’). TB Green Premix Ex Taq II (TaKaRa, RR820A) and a LightCycler 1.5 instrument (Roche Diagnostics) were used for quantitative PCR with the following primers: SARS-CoV-2 N forward, 5’-gaccccaaaatcagcgaaat-3’, and reverse, 5’-tctggttactgccagttgaatctg-3’; influenza virus NP forward, 5’-agaacatctgacatgaggac-3’, and reverse, 5’-gtcaaaggaaggcacgatc-3’ [16,49].

ELISA

Cell-free supernatants or lung washes were analyzed for the presence of IFN-α (Hycult Biotech, HM1001; PBL Assay Science, 32100–1), IL-1β (eBioscience, 14-7012-85 and 13-7112-85), IL-6 (eBioscience, 14-7061-85 and 13-7062-85), and TNF-α (eBioscience, 14-7423-85 and 13-7341-85) using an enzyme-linked immunosorbent assay (ELISA) utilizing paired antibodies [50]. IFN-β (PBL Assay Science, 42400–1), IFN-λ (PBL Assay Science, 62830–1), or CXCL1 (Proteintech, KE10019) ELISA was performed according to the manufacturer’s instructions. Absorbance at 450 nm was measured by using Microplate Manager version 6 (Bio-Rad).

Flow cytometry

The single-cell suspensions of lung samples were prepared as previously described [51]. Briefly, lungs were perfused with 10 ml PBS through the right ventricle, minced using razor blades, and incubated in HBSS containing 2.5 mM Hepes and 1.3 mM EDTA at 37°C for 30 min. The cells were resuspended in RPMI containing 5% FBS, 1 mM CaCl₂, 1 mM MgCl₂, 2.5 mM Hepes, and 0.5 mg/ml collagenase D (Roche) and incubated at 37°C for 60 min. A single-cell suspension was prepared after red blood cell lysis. The resulting cells were filtered through a 70-μm cell strainer (BD). For neutrophil staining, cells were incubated with APC-labeled anti-Ly6G (Invitrogen, 17-9668-82; 1:200) and eFluor 450-labeled anti-Ly6C (Invitrogen, 48-5932-82; 1:200) (S18 Fig). For the detection of SARS-CoV-2-infected cells, cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences, 554714), and intracellulary stained with PE-labeled rabbit anti-SARS-CoV-2 nucleocapsid (abcam, ab283244; 1:200) antibody. Flow cytometric analysis was performed with a FACSVerse flow cytometer (BD Biosciences). The final analysis and graphical output were performed using FlowJo software (Tree Star, Inc.).

Statistical analysis

Statistical significance was tested using nonparametric one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test, non-parametric Mann-Whitney t test, or Student’s two-tailed, unpaired t test where indicated in the figure legend, using PRISM software (version 5; GraphPad software). P < 0.05 was considered statistically significant.

Supporting information

S1 Fig. Neutrophil recruitment into the lung after the ancestral or Delta P80 virus infection.

Six-week-old C57BL/6 mice were infected intranasally with 1×10⁵ pfu of the ancestral or Delta P80 virus. Five days later, leukocytes were isolated from the lung. The number of Ly6C⁺ Ly6G⁺ neutrophils were analyzed by flow cytometry. Each symbol indicates individual values. Statistical significance was analyzed by two-way analysis of variance (ANOVA). **P < 0.01.

(TIF)

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S2 Fig. Gating strategy to assess the frequency of the SARS-CoV-2-infected CD45.2⁺ or CD45.2^– cells.

Six-week-old C57BL/6 mice were infected intranasally with 1×10⁵ pfu of the ancestral or Delta P80 virus. Leukocytes were isolated from the lung at 3 days post infection, and intracellularly stained with the nucleoprotein-specific antibody. The frequency of the SARS-CoV-2-infected CD45.2⁺ or CD45.2^– cells were analyzed by flow cytometry.

(TIF)

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S3 Fig. Gating strategy to assess the frequency of the SARS-CoV-2-infected alveolar macrophages.

(A and B) Six-week-old C57BL/6 mice were infected intranasally with 1×10⁵ pfu of the ancestral or Delta P80 virus. Leukocytes were isolated from the lung washes at 3 days post infection, and intracellularly stained with the nucleoprotein-specific antibody. To identify alveolar macrophage population, leucocytes were gated by forward and side scatter. Doublet signals are excluded by plotting forward scatter area versus forward scatter height. CD11b and CD11c double-positive cells were identified as alveolar macrophages (A). Frequency of nucleoprotein⁺ cells in alveolar macrophages are shown (B). (C) The population was confirmed to be macrophages by depletion with clodronate liposomes. Each symbol indicates individual values. Statistical significance was analyzed by two-way analysis of variance (ANOVA) (B). ***P < 0.001.

(TIF)

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S4 Fig. Gating strategy to assess the frequency of the SARS-CoV-2-infected dendritic cells in the lung.

(A and B) Six-week-old C57BL/6 mice were infected intranasally with 1×10⁵ pfu of the ancestral or Delta P80 virus. Leukocytes were isolated from the lung at 3 days post infection, and intracellularly stained with the nucleoprotein-specific antibody. To identify dendritic cell population, leucocytes were gated by forward and side scatter. Doublet signals are excluded by plotting forward scatter area versus forward scatter height. CD11c-positive cells were identified as dendritic cells (A). Frequency of nucleoprotein⁺ cells in CD11c⁺ dendritic cells are shown (B). Each symbol indicates individual values. Statistical significance was analyzed by two-way analysis of variance (ANOVA) (B). ***P < 0.001, n.s., not significant.

(TIF)

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S5 Fig. Gating strategy to assess the frequency of the SARS-CoV-2-infected B cells in the lung.

(A and B) Six-week-old C57BL/6 mice were infected intranasally with 1×10⁵ pfu of the ancestral or Delta P80 virus. Leukocytes were isolated from the lung at 3 days post infection, and intracellularly stained with the nucleoprotein-specific antibody. To identify B cell population, leucocytes were gated by forward and side scatter. Doublet signals are excluded by plotting forward scatter area versus forward scatter height. CD19 and B220 double-positive cells were identified as B cells (A). Frequency of nucleoprotein⁺ cells in B cells are shown (B). Each symbol indicates individual values. Statistical significance was analyzed by two-way analysis of variance (ANOVA) (B). ***P < 0.001, n.s., not significant.

(TIF)

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S6 Fig. Gating strategy to assess the frequency of the SARS-CoV-2-infected CD4⁺ or CD8⁺ T cells in the lung.

(A-C) Six-week-old C57BL/6 mice were infected intranasally with 1×10⁵ pfu of the ancestral or Delta P80 virus. Leukocytes were isolated from the lung at 3 days post infection, and intracellularly stained with the nucleoprotein-specific antibody. To identify CD4⁺ and CD8⁺ T cell population, leucocytes were gated by forward and side scatter. Doublet signals are excluded by plotting forward scatter area versus forward scatter height. CD3 and CD4 or CD3 and CD8 double-positive cells were identified as CD4⁺ and CD8⁺ T cells, respectively (A). Frequency of nucleoprotein⁺ cells in CD4⁺ (B) and CD8⁺ T cells (C) are shown. Each symbol indicates individual values. Statistical significance was analyzed by two-way analysis of variance (ANOVA) (B and C). **P < 0.01, ***P < 0.001, n.s., not significant.

(TIF)

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S7 Fig. Gating strategy to assess the frequency of the SARS-CoV-2-infected neutrophils in the lung.

(A and B) Six-week-old C57BL/6 mice were infected intranasally with 1×10⁵ pfu of the ancestral or Delta P80 virus. Leukocytes were isolated from the lung at 3 days post infection, and intracellularly stained with the nucleoprotein-specific antibody. To identify neutrophil population, leucocytes were gated by forward and side scatter. Doublet signals are excluded by plotting forward scatter area versus forward scatter height. Then, B cells and T cells were excluded based on B220 and CD3 expression, respectively. Ly6C and Ly6G double-positive cells were identified as neutrophils (A). Frequency of nucleoprotein⁺ cells in neutrophils are shown (B). Each symbol indicates individual values. Statistical significance was analyzed by two-way analysis of variance (ANOVA) (B). *P < 0.05, ***P < 0.001.

(TIF)

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S8 Fig. Effects of MyD88 deficiency on body weight changes and survival after sublethal dose of Delta P80 virus infection.

(A and B) Six-week-old C57BL/6 WT or MyD88 mice were infected intranasally with 1×10⁴ pfu of the Delta P80 virus. Weight loss (A) and mortality (B) were monitored for 14 days. Statistical significance was analyzed by two-tailed unpaired Student’s t test (A) or two-sided log-rank (Mantel-Cox) test (B).

(TIF)

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S9 Fig. Infection of mice with Delta P80 virus does not stimulate detectable levels of IL-1β in BALF.

C57BL/6 WT, MyD88, or IFNAR1 KO mice were infected intranasally with 1×10⁵ pfu of the Delta P80 virus. The lung washes were collected at 2 days p.i. and analyzed for IL-1β by ELISA. Each symbol indicates individual values.

(TIF)

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S10 Fig. TNF-α exacerbates Delta P80 virus infection in MyD88 mice.

Six-week-old MyD88 KO mice infected with the Delta 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. Statistical significance was analyzed by two-sided log-rank (Mantel-Cox) test.

(TIF)

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S11 Fig. Intranasal administration of TAPI-2 has no effect on the survival rate of the Delta P80 virus-infected mice.

Six-week-old C57BL/6 mice infected with 1×10⁵ pfu of the Delta P80 virus were administered intranasally with saline or TAPI-2 (0.5 μg) at 1 and 2 days p.i. (allow). Mortality was monitored for 14 days. Statistical significance was analyzed by two-sided log-rank (Mantel-Cox) test.

(TIF)

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S12 Fig. TNF protease inhibitor 2 alleviates SARS-CoV-2-accosiated mortality in aged mice.

(A and B) Aged (21-week-old) C57BL/6 mice were infected intranasally with 100 pfu of the ancestral P80 virus. Then, infected mice were administered intravenously with saline or TAPI-2 (2.5 μg) at indicated time points (arrows). Mortality was monitored for 14 days (A). The lung washes were collected at 3 days p.i. and viral titers were determined by standard plaque assay (B). Each symbol indicates individual values (B). Statistical significance was analyzed by two-sided log-rank (Mantel-Cox) test (A), or two-tailed unpaired Student’s t test (B). n.s., not significant.

(TIF)

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S13 Fig. TNF protease inhibitor 2 suppresses SARS-CoV-2-induced inflammatory responses in aged mice.

(A-C) Aged (55-week-old) C57BL/6 mice were infected intranasally with 1×10⁵ pfu of the Delta P80 virus. Then, infected mice were administered intravenously with saline or TAPI-2 (2.5 μg) at 0, 1, and 2 days p.i.. The lung washes were collected at 2 days p.i. and analyzed for TNF-α (A), IL-6 (B), or CXCL1 (C) by ELISA. Each symbol indicates individual values. Statistical significance was analyzed by two-way analysis of variance (ANOVA). ***P < 0.001, n.s., not significant.

(TIF)

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S14 Fig. TAPI-2 alleviates influenza virus-associated mortality in mice.

Six-week-old C57BL/6 mice infected with 1×10³ pfu of the PR8 virus were administered intravenously with saline or TAPI-2 (2.5 μg) at 0, 1, and 2 days p.i. (allow). (A-C) The lung washes were collected at 2 days p.i. and analyzed for TNF-α (A) or IL-6 (B) by ELISA. Total RNAs were extracted from lung washes and influenza virus NP RNA levels were assessed by quantitative reverse transcription PCR (C). (D) Mortality was monitored for 14 days. Each symbol indicates individual values (A-C). Statistical significance was analyzed by two-tailed unpaired Student’s t test (A-C), or two-sided log-rank (Mantel-Cox) test (D). ***P < 0.001, n.s., not significant.

(TIF)

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S15 Fig. Intranasal administration of anti-TNF-α antibodies has no effect on the survival rate of the Delta P80 virus-infected mice.

Six-week-old C57BL/6 mice infected with 1×10⁵ pfu of the Delta P80 virus were administered intranasally with isotype rat IgG (2.5 μg) or anti-TNF-α antibodies (2.5 μg) at 1 day p.i. (allow). Mortality was monitored for 14 days. Statistical significance was analyzed by two-sided log-rank (Mantel-Cox) test.

(TIF)

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S16 Fig. Intravenous administration of anti-TNF-α antibodies alleviates Delta P80 virus-associated mortality.

(A-C) Six-week-old C57BL/6 mice infected with 1×10⁵ pfu of the Delta P80 virus were administered intravenously with isotype rat IgG (10 μg) or anti-TNF-α antibodies (10 μg) at 1-day p.i. (allow). Mortality was monitored for 14 days (A). The lung washes were collected at 2 days p.i. and analyzed for TNF-α by ELISA (B). Total RNAs were extracted from lung washes and SARS-CoV-2 N gRNA levels were assessed by quantitative reverse transcription PCR (C). Each symbol indicates individual values (B and C). Statistical significance was analyzed by two-sided log-rank (Mantel-Cox) test (A), two-way analysis of variance (ANOVA) (B), or two-tailed unpaired Student’s t test (C). ***P < 0.001, n.s., not significant.

(TIF)

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S17 Fig. Proposed mechanism by which type I IFN signals exacerbate SARS-CoV-2 infection.

Infection with a lethal dose (1×10⁵ pfu) of the Delta P80 virus enhances type I IFNs and proinflammatory cytokines production in a MyD88- and IFNAR1-dependent manner. TNF-α stimulates CXCL1 production from macrophages, which may enhance lung tissue damage by neutrophils and the disease severity following the Delta P80 virus infection.

(TIF)

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S18 Fig. Gating strategy for identifying neutrophils in the lung.

To identify neutrophil population, leucocytes were gated by forward and side scatter. Doublet signals are excluded by plotting forward scatter area versus forward scatter height. Then, B cells and T cells were excluded based on B220 and CD3 expression, respectively. Ly6C and Ly6G double-positive cells were identified as neutrophils.

(TIF)

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S1 Data. Raw data of main figures.

(XLSX)

ppat.1012776.s019.xlsx^{(49.7KB, xlsx)}

S2 Data. Raw data of supporting information figures.

(XLSX)

ppat.1012776.s020.xlsx^{(27.8KB, xlsx)}

Acknowledgments

We thank Yoshihiro Kawaoka (University of Wisconsin and University of Tokyo) for providing SARS-CoV-2/UT-NCGM02/Human/2020/Tokyo, Ken Maeda (National Institute of Infectious Diseases) for providing SARS-CoV-2 Delta variant, Takara Bio Inc. and Rhelixa Inc. for next-generation sequencing analysis. Flow cytometric analysis was performed in the IMSUT FACS Core laboratory.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported in part by research grants from the Japan Agency for Medical Research and Development (AMED) (JP233fa627001 to T.I. and K.M, JP22gm1010009 to S.F.), JSPS KAKENHI (22H03541 to S.F.), JST ERATO (JPMJER1902 to S.F.), the Food Science Institute Foundation (to S.F.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Pathog. doi: 10.1371/journal.ppat.1012776.r001

Decision Letter 0

Jie Sun, Sonja M Best

26 Jul 2024

Dear Dr. Ichinohe,

Thank you very much for submitting your manuscript "TNF-α exacerbates SARS-CoV-2-infection by stimulating CXCL-1 production from macrophages" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Jie Sun, Ph.D.

Academic Editor

PLOS Pathogens

Sonja Best

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Deguchi et al's manuscript "TNF-α exacerbates SARS-CoV-2-infection by stimulating CXCL-1 production from macrophages" describes 2 mouse adapted SARS-CoV-2 infection models and investigates the pathogenesis of SARS-CoV-2 infection based on these models. Their work provides potential tools for further identify the pathology of COVID-19. However, some substantial concerns need to be addressed.

Reviewer #2: This manuscript titled “TNF exacerbates SARS-CoV-2 infection by stimulating CXCL-1 production from macrophages” by Deguchi K et al. has two primary objectives: a) to develop a reliable mouse-adapted (MA)-virus that causes severe disease in commonly used young C57BL/6 mice, and b) to investigate the basis for SARS-CoV-2-induced severe disease using the novel MA virus. Here, the authors examine underlying basis for SARS-CoV-2-induced cytokine storm and severe disease using MA SARS-CoV-2 viruses derived from different human variants. The authors show that high-passaged (p80) MA SARS-CoV-2 derived from delta variant, but not the ancestral variant, caused lethal disease in young B6 mice, while P80 virus from both backgrounds caused severe disease in BALB/c and C3H mice. In young B6 mice, the P80 MA-delta virus replicated to high titers, caused lung pathology, and triggered a robust cytokine response. The authors also showed that Myd88 and IFN-I signaling were pathogenic, early IFN-β treatment protected, and the delayed IFN-β administration caused pathology in P80 MA-delta virus-infected B6 mice. Mechanistically, the IFN-I-induced TNF-mediated CXCL-1 response was associated with severe disease in P80 MA-delta virus-infected young B6 mice.

Strengths:

This is an interesting study wherein the authors have developed a novel MA virus on the Delta variant background for studying SARS-CoV-2 pathogenesis in B6 mice. The study also highlights the SARS-CoV-2 variant-specific role of IFN-I and Myd88 in cytokine storm and severe disease. Additionally, the work identifies the role of the TNF-CXCL-1 axis in causing severe lung inflammation and lethal pneumonia. However, there are several major concerns that diminish the enthusiasm for this work in its current form. Comments are listed below.

Weaknesses:

1. Study implications: It is now well-established that the lack of IFN-I and TLR3/7 signaling is associated with severe COVID19. As a result, significance and implications of the differential role of IFN-I and Myd88 in MA-ancestral and MA-delta virus-infected mice, and how these results correlate with COVID-19 outcomes in humans, are not clear, nor are well articulated. Specifically, it is unclear whether the loss of IFN and Myd88 signaling is associated with less severe disease in humans infected with the Delta variant.

2. MA viruses developed by different laboratories (including MA-10 by Baric lab and MA-30 by Perlman lab) are extensively used to study SARS-CoV-2 pathogenesis. Although these viruses cause mild disease in young 6-week B6 mice, these viruses (specifically MA30) cause lethal disease in adult (16-20 week or older) B6 mice. Therefore, the premise of having to develop a MA virus specifically to induce severe disease in 6-week mice is not justified. Additionally, 6-week mice, although extensively used, are too young for SARS-CoV-2 studies, a virus that causes mild disease in young individuals.

3. The differential disease outcomes following MA-ancestral and MA-delta virus infections are novel. However, it would be interesting to know if specific mutations observed in MA-delta contribute to IFN-I/Myd88 mediated severe disease outcomes.

4. The protective and detrimental roles of early and delayed IFN-β treatment, respectively, are well described for SARS-CoV, MERS-CoV, and SARS-CoV-2 infection by several investigators, making these observations less novel. However, the role of IFN-I mediated TNF signaling in CXCL-1-induced lung pathology is novel and significant.

5. Figure 7: CXCL-1 and TNF treatment enhanced disease severity in ancestral and delta P80 MA virus-infected mice. However, TAPI treatment marginally, albeit significantly, enhanced survival. These results show that perhaps using knockout mice or blocking TNF and CXCL-1 using specific monoclonal antibodies is a better approach compared to using an inhibitor.

6. The authors postulate that TNF-mediated CXCL-1-induced neutrophils cause inflammation and pathology in delta-MA infected mice. However, no neutrophil data (FACS or histology) is available to support these conclusions.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: Major concerns

1 In this work, SARS-CoV-2 viruses were adapted in murine system by serial passages, which is expected to lead to new mutations as shown in Fig. 1 and 3. Authors need to provide further interpretations on these mutations, especially their clinical relevance. If these mutations are identified in SARS-CoV-2 variants circulating in human population as well, the value of these models will be significantly improved.

2 In the pathogenesis investigation, ancestral P80 and delta P80 were used as moderate and lethal models, respectively, which need to be clarified to avoid confusions. Based on the severity caused by different viruses, the role of MyD88 and IFNAR1, as well as different treatment of type I IFN were used correspondingly. Again, these designs are understandable, but the generated data need to be interpreted separately because the pathology of COVID-19 caused by different variants, especially those carrying multiple mutations, could be distinct. In addition, the authors are encouraged to establish different severity model using the same virus by optimizing the infectious doses.

3 The author claim the critical role of CXCL-1 in TNF-induced inflammation but failed to provide direct evidence. In vivo TNF-dependent CXCL-1 expression by macrophage is warranted. Meanwhile, the underlying mechanisms contributing to CXCL-1-mediated inflammation (by recruiting neutrophil as shown in Fig. s4? If so, the neutrophil accumulation in affected lung need to be determined) need to be further identified.

Reviewer #2: 1. The differential disease outcomes following MA-ancestral and MA-delta virus infections are novel. However, it would be interesting to know if specific mutations observed in MA-delta contribute to IFN-I/Myd88 mediated severe disease outcomes.

2. The protective and detrimental roles of early and delayed IFN-β treatment, respectively, are well described for SARS-CoV, MERS-CoV, and SARS-CoV-2 infection by several investigators, making these observations less novel. However, the role of IFN-I mediated TNF signaling in CXCL-1-induced lung pathology is novel and significant.

3. Figure 7: CXCL-1 and TNF treatment enhanced disease severity in ancestral and delta P80 MA virus-infected mice. However, TAPI treatment marginally, albeit significantly, enhanced survival. These results show that perhaps using knockout mice or blocking TNF and CXCL-1 using specific monoclonal antibodies is a better approach compared to using an inhibitor.

4. The authors postulate that TNF-mediated CXCL-1-induced neutrophils cause inflammation and pathology in delta-MA infected mice. However, no neutrophil data (FACS or histology) is available to support these conclusions.

5. The authors show increased virus titers in MA-delta (p80) infected mice compared to MA-ancestral delta (p80). As shown, it is not obvious whether severe disease upon P80-MA-delta virus-infected mice is due to high virus titers or virus-induced inflammation or both. A side-by-side comparison of lung inflammation in MA-delta and ancestral-MA virus-infected mice/lungs is required to support these conclusions.

6. It would be interesting to know whether MA-delta and ancestral-MA viruses have differential cell tropism within the lungs and in extrapulmonary tissues. Perhaps this would also explain the basis for differential outcomes.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Minor concerns

The introduction need to be polished. Some information, such as "experimental techniques for tracheal surgery are required for intratracheal inoculation of the AAV vector" is not accurate because AAV transfection can be accomplished well by intranasal inoculation.

Reviewer #2: 1. Introduction lacks a clear rationale. It appears that the goal of the study is to develop an MA virus that causes severe disease in 6-week-old B6 mice.

2. The rationale for using aged mice to initially adapt the viruses is not clear. While most of the advanced variants do bind to murine ACE2, the ACE2 binding ability of the ancestral variant (spike) used in this study is not provided.

3. Figure 2 and elsewhere: the authors estimate titers in BALF. Why not estimate titers in lungs?

4. Figure 4: The authors rely on lung edema and lung weight for inflammation studies. A thorough histopathological and flow cytometry evaluation of lungs/lung cells from MA-delta and ancestral-MA virus-infected mice is critical to establish lung inflammation.

5. Lines 304-317: The discussion is not overtly relevant and needs to be more aligned with the study objectives to support the results.

6. Discuss the relevance of these findings with human COVID-19 following ancestral and delta variant infections.

**********

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Reviewer #1: Yes: Jian Zheng

Reviewer #2: No

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PLoS Pathog. 2024 Dec 9;20(12):e1012776. doi: 10.1371/journal.ppat.1012776.r002

Author response to Decision Letter 0

26 Sep 2024

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ppat.1012776.s021.docx^{(33.9KB, docx)}

PLoS Pathog. doi: 10.1371/journal.ppat.1012776.r003

Decision Letter 1

Jie Sun, Sonja M Best

26 Oct 2024

PPATHOGENS-D-24-01041R1TNF-α exacerbates SARS-CoV-2-infection by stimulating CXCL1 production from macrophagesPLOS Pathogens Dear Dr. Ichinohe, Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript within 30 days Dec 25 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript:* A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. We look forward to receiving your revised manuscript. Kind regards, Jie Sun, Ph.D.Academic EditorPLOS Pathogens Sonja BestSection EditorPLOS Pathogens Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064 Journal Requirements: Additional Editor Comments (if provided): Please add additional discussion or "limitations of the study" section to address remaining concerns of the reviewer 2. [Note: HTML markup is below. Please do not edit.] Reviewers' Comments: Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The current revision is satisfactory and I would like to endorse its publication.

Reviewer #2: TNF exacerbates SARS-CoV-2 infection by stimulating CXCL-1 production from macrophages” by Deguchi K et al. has two primary objectives: a) to develop a reliable mouse-adapted (MA)-virus that causes severe disease in commonly used young C57BL/6 mice, and b) to investigate the basis for SARS-CoV-2-induced severe disease using the novel MA virus. Here, the authors examine underlying basis for SARS-CoV-2-induced cytokine storm and severe disease using MA SARS-CoV-2 viruses derived from different human variants. The authors show that high-passaged (p80) MA SARS-CoV-2 derived from delta variant, but not the ancestral variant, caused lethal disease in young B6 mice, while P80 virus from both backgrounds caused severe disease in BALB/c and C3H mice. In young B6 mice, the P80 MA-delta virus replicated to high titers, caused lung pathology, and triggered a robust cytokine response. The authors also showed that Myd88 and IFN-I signaling were pathogenic, early IFN-β treatment protected, and the delayed IFN-β administration caused pathology in P80 MA-delta virus-infected B6 mice. Mechanistically, the IFN-I-induced TNF-mediated CXCL-1 response was associated with severe disease in P80 MA-delta virus-infected young B6 mice

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Reviewer #1: No.

Reviewer #2: The authors have provided explanation for the comments raised during previous iteration. However, the authors have failed to address majority of the concerns expressed by this reviewer.

Original Comment 1: Study implications- The authors were suggested to explain the implications of the current results to human WA and Delta SARS-CoV-2 infection. Although the authors mention that they included an explanation, those modifications do not discuss implications of the current results with the outcomes in humans.

This is a missed opportunity, since highlighting the differential role of IFN-I and Myd88 in Delta MA vs WA-MA virus infected mice and providing including implications to human infections would have informed clinicians and scientist alike about differential role of these signaling pathways in different SARS-CoV-2 variant infections. The authors could have also re-written the study to highlight that severe disease upon delta variant infection could be due to differential and pathogenic role of the above mentioned pathway.

Original Comment 2: The authors were asked to provide rationale for developing a MA virus that causes severe disease in 6-week old mice as opposed to MA-virus by other labs that cause severe disease 12-20 week or older old mice. The justification of eliminating age as a factor and cellular senescence are not satisfactory, as 12-20 week mice are not old mice and they likely do not have senescent cells, unlike 20month old mice.

Original Comment 3: No explanation provided to explain why IFN-I and Myd88 may cause severe disease upon delta MA infection compared to WA-MA infection.

Original Comment 5: The authors were suggested to use TNF-/- mice or anti-TNF and anti-CXCL-1 mAb to show direct and endogenous role of these mediators in disease pathogenesis, as exogenous administration TNF, CXCL-1, and other inflammatory mediators will likely have adverse outcomes. Therefore, it is critical to block/neutralize endogenous levels of these mediators to show their clinical relevance. However, the authors could not do these studies and cite expense associated with the neutralizing antibodies as key reason to not perform the studies. These antibodies (anti-TNA) are available through BioXcell and Leinco technologies at affordable rate.

The authors instead use TAPI-2, a inhibitor of matrix matalloproteases (targets several MMPs) and TACE (targets TNF). The results obtained using TAPI-2 are not specific to TNF, and therefore the conclusions are not well justified. Moreover, TAPI-2 is given via IP route instead of IN route. Additionally, DNAse could have several off target effects.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: No.

Reviewer #2: (No Response)

**********

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Reviewer #1: Yes: Jian Zheng

Reviewer #2: No

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PLoS Pathog. 2024 Dec 9;20(12):e1012776. doi: 10.1371/journal.ppat.1012776.r004

Author response to Decision Letter 1

20 Nov 2024

Attachment

Submitted filename: 241119 Response to Reviewers.pdf

ppat.1012776.s022.pdf^{(262.1KB, pdf)}

PLoS Pathog. doi: 10.1371/journal.ppat.1012776.r005

Decision Letter 2

Jie Sun, Sonja M Best

25 Nov 2024

Dear Dr. Ichinohe,

We are pleased to inform you that your manuscript 'TNF-α exacerbates SARS-CoV-2 infection by stimulating CXCL1 production from macrophages' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Jie Sun, Ph.D.

Academic Editor

PLOS Pathogens

Sonja Best

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #2: (No Response)

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Reviewer #2: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #2: (No Response)

**********

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Reviewer #2: No

PLoS Pathog. doi: 10.1371/journal.ppat.1012776.r006

Acceptance letter

Jie Sun, Sonja M Best

3 Dec 2024

Dear Dr. Ichinohe,

We are delighted to inform you that your manuscript, "TNF-α exacerbates SARS-CoV-2 infection by stimulating CXCL1 production from macrophages," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Neutrophil recruitment into the lung after the ancestral or Delta P80 virus infection.

(TIF)

ppat.1012776.s001.tif^{(2.9MB, tif)}

S2 Fig. Gating strategy to assess the frequency of the SARS-CoV-2-infected CD45.2⁺ or CD45.2^– cells.

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S3 Fig. Gating strategy to assess the frequency of the SARS-CoV-2-infected alveolar macrophages.

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S4 Fig. Gating strategy to assess the frequency of the SARS-CoV-2-infected dendritic cells in the lung.

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S5 Fig. Gating strategy to assess the frequency of the SARS-CoV-2-infected B cells in the lung.

(A and B) Six-week-old C57BL/6 mice were infected intranasally with 1×10⁵ pfu of the ancestral or Delta P80 virus. Leukocytes were isolated from the lung at 3 days post infection, and intracellularly stained with the nucleoprotein-specific antibody. To identify B cell population, leucocytes were gated by forward and side scatter. Doublet signals are excluded by plotting forward scatter area versus forward scatter height. CD19 and B220 double-positive cells were identified as B cells (A). Frequency of nucleoprotein⁺ cells in B cells are shown (B). Each symbol indicates individual values. Statistical significance was analyzed by two-way analysis of variance (ANOVA) (B). ***P < 0.001, n.s., not significant.

(TIF)

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S6 Fig. Gating strategy to assess the frequency of the SARS-CoV-2-infected CD4⁺ or CD8⁺ T cells in the lung.

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S7 Fig. Gating strategy to assess the frequency of the SARS-CoV-2-infected neutrophils in the lung.

(A and B) Six-week-old C57BL/6 mice were infected intranasally with 1×10⁵ pfu of the ancestral or Delta P80 virus. Leukocytes were isolated from the lung at 3 days post infection, and intracellularly stained with the nucleoprotein-specific antibody. To identify neutrophil population, leucocytes were gated by forward and side scatter. Doublet signals are excluded by plotting forward scatter area versus forward scatter height. Then, B cells and T cells were excluded based on B220 and CD3 expression, respectively. Ly6C and Ly6G double-positive cells were identified as neutrophils (A). Frequency of nucleoprotein⁺ cells in neutrophils are shown (B). Each symbol indicates individual values. Statistical significance was analyzed by two-way analysis of variance (ANOVA) (B). *P < 0.05, ***P < 0.001.

(TIF)

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S8 Fig. Effects of MyD88 deficiency on body weight changes and survival after sublethal dose of Delta P80 virus infection.

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S9 Fig. Infection of mice with Delta P80 virus does not stimulate detectable levels of IL-1β in BALF.

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S10 Fig. TNF-α exacerbates Delta P80 virus infection in MyD88 mice.

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S11 Fig. Intranasal administration of TAPI-2 has no effect on the survival rate of the Delta P80 virus-infected mice.

(TIF)

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S12 Fig. TNF protease inhibitor 2 alleviates SARS-CoV-2-accosiated mortality in aged mice.

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S13 Fig. TNF protease inhibitor 2 suppresses SARS-CoV-2-induced inflammatory responses in aged mice.

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S14 Fig. TAPI-2 alleviates influenza virus-associated mortality in mice.

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S15 Fig. Intranasal administration of anti-TNF-α antibodies has no effect on the survival rate of the Delta P80 virus-infected mice.

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S16 Fig. Intravenous administration of anti-TNF-α antibodies alleviates Delta P80 virus-associated mortality.

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S17 Fig. Proposed mechanism by which type I IFN signals exacerbate SARS-CoV-2 infection.

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S18 Fig. Gating strategy for identifying neutrophils in the lung.

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S1 Data. Raw data of main figures.

(XLSX)

ppat.1012776.s019.xlsx^{(49.7KB, xlsx)}

S2 Data. Raw data of supporting information figures.

(XLSX)

ppat.1012776.s020.xlsx^{(27.8KB, xlsx)}

Attachment

Submitted filename: 240925 Response to Reviewers.docx

ppat.1012776.s021.docx^{(33.9KB, docx)}

Attachment

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ppat.1012776.s022.pdf^{(262.1KB, pdf)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

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

Moe Kobayashi

Nene Kobayashi

Kyoka Deguchi

Seira Omori

Minami Nagai

Ryutaro Fukui

Isaiah Song

Shinji Fukuda

Kensuke Miyake

Takeshi Ichinohe

Roles

Abstract

Author summary

Introduction

Results

Generation of a mouse-adapted ancestral SARS-CoV-2

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

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

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

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

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

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

MyD88 and IFNAR1 signals exacerbate SARS-CoV-2 infection

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

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

TNF-α-CXCL1 axis exacerbates SARS-CoV-2 infection

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

Inhibition of TNF-α alleviates the Delta P80 virus-associated mortality

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

Discussion

Materials and methods

Ethics statement

Animals

Cell culture

Viruses

Reagents

Quantitative PCR

ELISA

Flow cytometry

Statistical analysis

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Jie Sun

Sonja M Best

Roles

Author response to Decision Letter 0

Decision Letter 1

Jie Sun

Sonja M Best

Roles

Author response to Decision Letter 1

Decision Letter 2

Jie Sun

Sonja M Best

Roles

Acceptance letter

Jie Sun

Sonja M Best

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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