Hyperactivated ZBP1-Mediated Necroptosis Exacerbates Pneumonia in Mice Co-Infected with Low-Lethality H1N1 and HCoV-229E

Hyperactivated ZBP1-Mediated Necroptosis Exacerbates Pneumonia in Mice Co-Infected with Low-Lethality H1N1 and HCoV-229E | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Hyperactivated ZBP1-Mediated Necroptosis Exacerbates Pneumonia in Mice Co-Infected with Low-Lethality H1N1 and HCoV-229E Xunlong Shi, Suya Lao, Xin Ai, Yijun Niu, Zichen Tian, Zhixuan Cai, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7426580/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Influenza virus causes millions of infections annually, with severe viral pneumonia accounting for a substantial proportion of associated mortality. Co-infections with other respiratory pathogens further worsen clinical outcomes, yet the underlying mechanisms remain poorly defined. Clinical observations have documented influenza–coronavirus co-infections, but their pathological interplay has not been elucidated. To address this gap, we established a murine model of sequential H1N1 (FM1 or PR8 strains) and human coronavirus 229E (HCoV-229E) infection, which recapitulated the fatal pneumonia observed in patients. Strikingly, co-infected mice exhibited accelerated mortality and exacerbated lung pathology despite reduced viral loads, revealing a paradoxical “low-virus, high-inflammation” state. Targeted transcriptional and functional analyses identified ZBP1-dependent necroptosis as the central driver of pathology. Lung tissues showed robust activation of the ZBP1–RIPK3–MLKL axis, which correlated with cytokine overproduction and histopathological damage. Notably, inhibition of RIPK3—rather than direct antiviral treatment—restored lung function and improved survival, highlighting the causal role of necroptosis independent of viral replication. Furthermore, this ZBP1-driven mechanism was conserved across diverse influenza strains, emphasizing its broad biological relevance. Collectively, our findings reveal a previously unrecognized immunopathological axis in which influenza-primed lungs undergo coronavirus-triggered necroptotic storm, directly linking viral co-infection to fatal pneumonia. These results identify ZBP1 and RIPK3 as promising therapeutic targets for decoupling inflammation from viral clearance in severe respiratory co-infections. Biological sciences/Immunology/Cell death and immune response Biological sciences/Immunology/Infectious diseases Biological sciences/Immunology/Inflammation/Acute inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Influenza A virus (IAV) is a human-adapted respiratory pathogen that causes significant morbidity and mortality worldwide(1). Due to its rapid transmission and broad host range, IAV is the only influenza virus known to have caused pandemics to date(2). Even in non-pandemic years, IAV infects 5–15% of the global population, leading to approximately 500 000 deaths and 3 million severe cases annually(3, 4). Pathogen screening of influenza patients has frequently identified co-existing pathogens, including Streptococcus pneumoniae, Haemophilus influenzae, and respiratory syncytial virus (RSV)(5). Secondary infections with RSV or coronaviruses have been shown to exacerbate influenza severity(6, 7), suggesting that pathogen co-infection is a major contributor to influenza-associated morbidity and mortality. Human coronaviruses (HCoVs) are positive-sense single-stranded RNA viruses with high zoonotic potential and rapid evolutionary dynamics. To date, seven HCoV strains have been identified and classified into two genera: Alphacoronavirus and Betacoronavirus(8). Human coronavirus 229E (HCoV-229E), a member of the Alphacoronavirus genus, is an important cause of community-acquired pneumonia and contributes significantly to respiratory disease burden(9). However, co-infection involving HCoV-229E and IAV has not been reported, and the immunopathological mechanisms underlying such dual infections remain undefined. Viral co-infections often result in atypical clinical manifestations, including increased mortality, neurological complications, and immunosuppression(10), which cannot be explained by single-pathogen infection. They frequently share transmission routes (respiratory or hematogenous) and host tropism(11), facilitating their high transmissibility and cross-infection potential in endemic regions. Current diagnostic approaches—primarily serological testing and virus isolation—are limited(12–14). Serological assays suffer from low sensitivity and cross-reactivity(15), while virus isolation requires permissive cell lines or animal models and may be confounded by viral interference(16, 17). Therapeutically, most available antivirals target single viruses, rendering them ineffective in co-infections. Combination regimens carry risks of drug antagonism, toxicity, and accelerated resistance, ultimately compromising efficacy(18). These limitations underscore the urgent need to elucidate the pathological mechanisms of viral co-infections and to develop integrated therapeutic strategies. Recent studies have implicated necroptosis as a key driver of influenza-associated pathology. Viral infection generates Z-form RNA, which is sensed by the host protein Z-DNA-binding protein 1 (ZBP1). Upon recognition, ZBP1 engages receptor-interacting protein kinase 3 (RIPK3) to form a necroptosis-inducing complex, which phosphorylates mixed lineage kinase domain-like protein (MLKL), thereby triggering necroptosis(19). ZBP1-mediated necroptosis is characterized by inflammatory cell death, tissue damage, and immune dysregulation, and has been implicated in multiple diseases, including viral pneumonia(20). In our experimental model, coronavirus infection secondary to IAV significantly worsened pneumonia severity and increased lethality in mice, though the underlying mechanism was unclear. To address this, we established a physiologically relevant low-dose sequential co-infection model with varied orders of IAV and HCoV-229E exposure. This model recapitulated diverse clinical infection scenarios and revealed that even low-dose HCoV-229E following IAV infection aggravated pneumonia. Further investigation linked this pathological progression to ZBP1-dependent necroptosis. We demonstrated that HCoV-229E potentiates ZBP1–RIPK3–MLKL-mediated necroptosis in influenza-infected lungs. These findings provide a potential explanation for the diagnostic and therapeutic challenges of influenza–coronavirus co-infections and highlight the ZBP1/RIPK3/MLKL signaling axis as a promising therapeutic target for severe viral pneumonia. Results Secondary HCoV-229E Infection Following Primary H1N1 Exacerbates Pulmonary Lesions To investigate the effects of co-infection with H1N1 and HCoV-229E on pulmonary disease, we established co-infected mouse models (Fig. 1 A). Disease pathology was assessed in mice infected with H1N1 FM1 or HCoV-229E individually, simultaneously, or sequentially with a 24-hour interval to determine the impact of infection order. Notably, secondary infection with HCoV-229E following H1N1 (Co-infection-1) resulted in significant weight loss (Fig. 1 B), pulmonary edema, and pronounced lung injury (Fig. 1 C, D), whereas other co-infection modalities produced minimal effects. These observations prompted a detailed investigation of this specific co-infection scenario. After 2 days of sequential infection, co-infected mice exhibited dramatic weight loss compared with mice infected with H1N1 alone, whereas HCoV-229E mono-infected mice maintained body weight similar to the control group (Fig. 1 B). On day 4 post-infection, mice were anesthetized and euthanized to collect lung tissues for lung index measurement and histopathological analysis. The lung index, defined as the ratio of lung weight to body weight, was markedly elevated in co-infected mice (14.5), approximately double that of the control group (6.6), and significantly higher than in H1N1- (9.7) or HCoV-229E-infected mice (6.2) (Fig. 1 C). The elevated lung index indicates severe inflammatory lesions in lung tissues. Gross examination revealed extensive congestion and pronounced tissue edema in co-infected lungs (Fig. 1 D). Histological analysis by H&E staining further demonstrated widespread alveolar atrophy, structural destruction of alveolar lumens, and extensive infiltration of inflammatory cells in co-infected mice (Fig. 1 F). In contrast, lung lesions in mono-infected mice were notably milder, consistent with the observed lung index changes. In a 14-day survival assay, co-infected mice exhibited earlier mortality and a higher overall death rate compared with H1N1 mono-infected mice. The final mortality rate reached 83% in co-infected mice, significantly higher than that of H1N1- (60%) or HCoV-229E-infected mice (0%) (Fig. 1 E). These results indicate that HCoV-229E co-infection markedly exacerbates lung injury in H1N1-infected mice and substantially increases mortality. Severe Pneumonia Results from Hyperactivated Inflammation Rather than Uncontrolled Viral Replication We analyzed two major factors—pathogen and host immune response—that contribute to severe pneumonia during co-infection(21). First, we examined the correlation between severe lung injury and immune responses in the lungs of mice. RT-qPCR analysis of multiple cytokine transcripts in lung tissues revealed that H1N1/HCoV-229E co-infection led to a significant upregulation of inflammatory factors, including IL-1β, TNF-α, and IL-6 (Fig. 2 A), accompanied by increased transcription of IFN-α and IFN-β, compared with H1N1 or HCoV-229E mono-infection (Fig. 2 B). Histopathological analysis of lung tissues showed extensive immune cell infiltration, predominantly innate immune cells such as monocytes and neutrophils (Fig. 1 F). Immunofluorescence staining further demonstrated that co-infection markedly enhanced the recruitment of neutrophils (CD11b + /Ly6G + double-positive) (Fig. 2 D), with a concomitant elevation of neutrophil activation marker MPO, indicating an inflammatory activation state (Fig. 2 C). Flow cytometry revealed a decrease in macrophage numbers in co-infected lungs compared with mono-infected lungs (Fig. 2 E), which was confirmed by immunofluorescence staining of F4/80 + cells (Fig. 2 F). Collectively, these results indicate that H1N1/HCoV-229E co-infection induces a complex immune response characterized by massive neutrophil recruitment and activation, a reduction in macrophages, elevated pro-inflammatory cytokine production, and increased interferon transcription. We next examined viral load as a second factor contributing to severe lung injury. Influenza virus replication in lung tissues was assessed by quantitative RT-qPCR and immunofluorescence. Co-infected mice exhibited significantly lower transcription of the influenza M gene and reduced expression of NA glycoprotein compared with H1N1 mono-infected mice (Figs. 2 G–I), indicating that co-infection did not enhance viral replication. On the contrary, H1N1 replication was markedly suppressed, suggesting that severe lung injury in H1N1/HCoV-229E co-infected mice is not driven by uncontrolled viral proliferation. These findings suggest that the severe pneumonia and high mortality observed in co-infected mice are associated with dysregulated immune responses rather than elevated viral load, highlighting the need for further investigation of key immune signaling pathways. ZBP1-Mediated Necroptosis Promotes Pneumonia Severity in Co-Infection Analysis of cellular morphology in lung tissues by H&E staining revealed pronounced cellular necrosis in co-infected mice compared with those infected with a single virus (Fig. 3 A). Previous studies have reported that Z-DNA/RNA generated by various viruses, including influenza viruses, can bind to the host sensor ZBP1, forming a ZBP1-RIPK3 complex with RIPK3 kinase, which subsequently activates MLKL-mediated necroptosis and triggers inflammatory responses(22–24). To investigate this pathway in our model, we examined key necroptosis signaling targets—ZBP1, RIPK3, and MLKL—at both the transcript and protein levels in mouse lung tissues. Co-infection significantly increased mRNA expression of ZBP1, RIPK3, and MLKL compared with single-virus infection (Figs. 3 B–D), accompanied by elevated protein levels (Figs. 3 E–G). Immunohistochemical analysis further confirmed high expression of necroptosis signaling proteins in the lungs of co-infected mice (Figs. 3 H, 3 I). Collectively, these findings indicate that H1N1/HCoV-229E co-infection activates the ZBP1-RIPK3-MLKL necroptotic pathway in lung tissues, contributing to exacerbated lung injury and inflammation. Necroptosis Is Consistently Induced by Diverse H1N1 Strains in Co-Infection with HCoV-229E To determine whether ZBP1-RIPK3-MLKL-mediated necroptosis is induced by co-infection with specific influenza virus strains and HCoV-229E, we established co-infected mouse models using the influenza A virus H1N1 PR8 strain in combination with HCoV-229E. The results showed that H1N1 PR8/HCoV-229E co-infection caused marked weight loss (Fig. 4 A), more severe lung injury (Figs. 4 B–D), and a significant increase in necroptosis-associated proteins ZBP1, RIPK3, and phosphorylated MLKL in lung tissues (Figs. 4 E–I), consistent with observations using the H1N1 FM1 strain. These experiments confirm that co-infection of HCoV-229E with different influenza A virus strains induces more severe pneumonia, and that disease severity is positively correlated with the level of ZBP1-RIPK3-MLKL-mediated necroptosis. Blockade of Necroptosis Ameliorates Pneumonia in Co-Infected Mice To investigate the role of ZBP1-RIPK3-MLKL-mediated necroptosis in H1N1 /HCoV-229E co-infection-induced pneumonia, we treated co-infected mice with GSK872, a RIPK3 inhibitor that blocks necroptosis initiation(25, 26). The results showed that GSK872 did not affect the transcription of necroptosis-related genes ZBP1, RIPK3, and MLKL (Fig. 5 A), but significantly reduced the protein levels of these key necroptotic factors (Fig. 5 B). Immunohistochemical analysis further confirmed that GSK872 effectively inhibited necroptosis in lung tissues (Fig. 5 C). Treatment with GSK872 significantly reduced the lung index in H1N1/HCoV-229E co-infected mice (Fig. 5 D), alleviated hemorrhagic and edematous changes in the lungs (Fig. 5 E), and suppressed inflammatory cell recruitment, particularly neutrophil recruitment and activation (Figs. 5 F–I). Collectively, these results demonstrate that severe pneumonia induced by H1N1/HCoV-229E co-infection is closely associated with activation of the ZBP1-RIPK3-MLKL necroptosis pathway, and that pharmacological inhibition of necroptosis can mitigate co-infection-associated lung injury. Necroptosis in Alveolar Macrophages and Epithelial Cells Correlates with Interferon Upregulation during Co-Infection We previously demonstrated that H1N1/HCoV-229E co-infection exacerbates pulmonary damage in mice through ZBP1-RIPK3-MLKL-mediated necroptosis. To further determine the specific cell types undergoing necroptosis in lung tissues, we performed immunofluorescence staining to assess co-localization of key necroptotic proteins (ZBP1 and pMLKL) with cell-specific markers. Notably, strong co-localization of necroptotic proteins was observed in macrophages (F4/80⁺/ZBP1⁺/pMLKL⁺) and type II alveolar epithelial cells (Pro-SPC⁺/ZBP1⁺/pMLKL⁺) in co-infected mouse lungs (Fig. 6 A). Subsequent in vitro validation using RAW 264.7 cells further confirmed that co-infection significantly reduced cell viability compared with single-virus infection, indicating enhanced necroptosis in macrophages (Fig. 6 B). Together, these findings establish that ZBP1-RIPK3-MLKL-driven necroptosis predominantly occurs in pulmonary macrophages and alveolar epithelial cells during co-infection. Previously, we observed that interferon expression was markedly elevated in the lungs of co-infected mice (Fig. 2 B). Since viral infection typically induces interferon production, which in turn affects multiple cell types, we hypothesized that interferons play a critical role in the pathogenesis of co-infection. ZBP1, an interferon-inducible protein, has been reported to mediate interferon-driven necroptosis(27). To test this mechanism, we used IFN-α treatment to mimic the effect of HCoV-229E co-infection. Under low-dose HCoV-229E exposure (Fig. 5 F), IFN-α stimulation following H1N1 infection significantly enhanced inflammatory responses (Figs. 5 C, D), suppressed H1N1 replication (Fig. 5 E), and upregulated key necroptotic effector proteins (Figs. 5 G, H). Collectively, these results indicate that in H1N1/HCoV-229E co-infection, necroptosis is regulated by multiple factors, with interferons serving as universal and critical modulators across different cell types. Discussion Influenza A viruses are a major cause of respiratory infections in humans, ranging from mild to severe, and impose a substantial public health burden(28, 29). According to the World Health Organization, seasonal influenza—including influenza A (H1N1, H3N2) and influenza B strains—accounts for 3–5 million cases of severe respiratory illness and 290 000–650 000 deaths globally each year(30). Pathogen screening in patients with severe influenza often identifies multiple pathogens, including influenza viruses, suggesting that secondary viral infections can frequently exacerbate disease severity(31). In the present study, we provide experimental evidence that secondary infection with HCoV-229E aggravates H1N1-induced pneumonia, a process closely linked to activation of the ZBP1-RIPK3-MLKL necroptosis pathway rather than to increased viral replication. These findings offer a mechanistic explanation for the clinical challenges associated with diagnosing influenza-coronavirus co-infections and highlight ZBP1/RIPK3/MLKL-mediated necroptosis as a potential therapeutic target for severe viral pneumonia. In nature, simultaneous or sequential co-infection of a single host by multiple pathogens is common(32, 33). Viral co-infections can complicate disease diagnosis and treatment by producing overlapping clinical symptoms, altering viral pathogenicity and transmissibility, and facilitating viral mutation(34–36). Some studies suggest that viral interference may occur through mechanisms such as interferon-mediated enhancement of innate immunity(37), disruption of viral replication cycles(38, 39), or competition for metabolites and host factors essential for viral replication(40, 41). In contrast, other evidence indicates that viral synergism can arise through processes such as promotion of infected cell membrane fusion(42) or induction of host immunodeficiency(43). Collectively, these findings demonstrate that viral interactions and the immunological impact of co-infection on hosts involve highly complex mechanisms. By establishing a series of influenza A virus (IAV) and coronavirus (HCoV-229E) co-infection models, we observed that secondary HCoV-229E infection exacerbated pneumonia in H1N1-infected mice. Investigation into the underlying cause revealed that this exacerbation was not associated with uncontrolled viral replication, as co-infected mice exhibited a significant reduction in H1N1 M gene transcription and H1N1 NA protein expression compared to mice infected with H1N1 alone. We then examined immune alterations in the host lungs and found that H1N1/HCoV-229E co-infection induced a complex immune response, characterized by massive neutrophil recruitment, inflammatory activation, and a reduction in macrophages, accompanied by elevated transcript levels of multiple pro-inflammatory mediators. Nonetheless, the specific immune signaling pathways driving these changes warrant further investigation. Pathological analysis of lungs from H1N1/HCoV-229E co-infected mice revealed extensive nuclear fragmentation, suggesting that severe pneumonia under co-infection conditions may result from cellular necrosis. Necroptosis has been implicated in multiple viral infections, including herpes simplex virus (HSV)(44), cytomegalovirus (CMV)(45), and influenza A viruses such as H1N1 and H5N1(46). Consistently, our study showed marked upregulation of both transcript and protein levels of necroptosis mediators, including ZBP1, RIPK3, and phosphorylated MLKL (pMLKL). Z-DNA-binding protein 1 (ZBP1) serves as a critical inducer of necroptosis during viral infection by recognizing nuclear Z-RNA and activating the ZBP1-RIPK3-MLKL signaling axis(47), ultimately leading to nuclear membrane rupture and release of nuclear-derived damage-associated molecular patterns (DAMPs)(48). Importantly, pharmacological inhibition of RIPK3 with GSK872 significantly alleviated pneumonia severity in co-infected mice, indicating that disease progression is tightly linked to necroptosis activation. Moreover, co-infection experiments using distinct H1N1 strains (FM1 and PR8) in combination with HCoV-229E consistently demonstrated a correlation between pneumonia severity and activation of the ZBP1-RIPK3-MLKL pathway, underscoring necroptosis as a prevalent and central mechanism in co-infection–induced severe pneumonia. Alveolar macrophages are among the earliest immune cells to participate in host defense, capable of rapidly recognizing respiratory pathogens and regulating local inflammatory responses and immune reactions, thereby playing a critical role in controlling disease progression and severity(49). Pulmonary epithelial cells constitute another essential barrier; damage to this barrier allows bacteria or viruses to disseminate systemically within the host, leading to severe or even fatal outcomes(50). Previous studies have demonstrated that necroptosis plays an important role in acute lung injury (ALI), being activated in both macrophages and alveolar epithelial cells(51–53). Consistent with this, our study revealed marked colocalization of ZBP1/pMLKL in lung macrophages and type II alveolar epithelial cells, indicating widespread necroptosis in these cell populations. Further analyses showed that H1N1/HCoV-229E co-infection significantly reduced the number of lung macrophages, while in vitro experiments confirmed a marked decline in macrophage activity under co-infection conditions. Taken together, these findings indicate that necroptosis is prevalent in both alveolar macrophages and epithelial cells during co-infection and may play a critical role in driving lung injury. Compared with H1N1 infection alone, lungs from H1N1/HCoV-229E co-infected mice exhibited elevated transcription of type I interferons (IFN-α/β) and reduced H1N1 replication, suggesting the involvement of IFNs in this process. Interferons play critical roles in immune regulation and antiviral defense, and previous studies have reported that IFNs participate in necroptosis, with ZBP1 serving as a key mediator of IFN-induced necroptosis(54–56). In our study, co-stimulation with H1N1 and IFN-α further increased the transcription of IFNs and necroptosis-related proteins in mouse lung tissues, while concomitantly reducing H1N1 replication. Based on these findings, we propose that IFNs induced during viral co-infection exert dual effects: high levels of IFNs facilitate viral clearance, but sustained upregulation of ZBP1 drives necroptosis in macrophages and alveolar epithelial cells, thereby exacerbating lung injury. Materials and Methods Mice Female C57BL/6J mice were used in this study. All animal experiments and histological analyses were conducted under blinded conditions. Animals were housed in the specific-pathogen-free (SPF) facility of the special infection animal room, Antiviral Drug Research Laboratory (BSL-2), School of Pharmacy, Fudan University. Mice were maintained under controlled environmental conditions (temperature 20–25°C, relative humidity 40–70%) in individually ventilated cages with a 12-h light/dark cycle. Sterilized chow and water were provided ad libitum. For infection models, mice were anesthetized with isoflurane and intranasally inoculated with 30 µL of viral suspension. Single-virus infection models were established using either H1N1 or HCoV-229E, while co-infection models were generated using both viruses. The median lethal dose (LD 50 ) of H1N1 was determined as 1 × 10⁻⁵, and infection was performed at 4 × LD 50 . HCoV-229E infection was performed using undiluted viral stock solution (dose = 1). Cells and Viruses The RAW 264.7 cell line was obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂. Influenza A virus strains H1N1 A/FM/1/47 and H1N1 A/PR/8/34 were obtained from the Shanghai Centre for Disease Control and Prevention (SCDC) and ATCC, respectively. Human Coronavirus-229E (HCoV-229E) was kindly provided by Prof. Meiqing Feng. All viral stocks were stored at − 80°C in the Antiviral Drug Research Laboratory, School of Pharmacy, Fudan University. Virus Infection and Cell Viability Assay RAW 264.7 cells were seeded in 96-well plates at a density of 4 × 10⁴ cells per well 24 h prior to infection. Cells were washed with phosphate-buffered saline (PBS) and then incubated with H1N1 PR8 virus (1:1000 dilution, TCID₅₀ = 10⁻⁵.⁵/mL) and HCoV-229E virus (1:500 dilution, TCID₅₀ = 10⁻³.⁵/mL) in infection medium consisting of DMEM supplemented with 1% penicillin/streptomycin, at 37°C in a humidified atmosphere containing 5% CO₂. Cell viability was assessed 24 h post-infection using the CellTiter-Lumi™ Plus Luminescent Cell Viability Assay Kit (Beyotime, China; C0068M) according to the manufacturer’s instructions. RT-qPCR The mRNA levels of the indicated genes were quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Total RNA was extracted using TRIzol reagent (Invitrogen™, 15596018) and reverse-transcribed into cDNA using a reverse transcription kit (Vazyme, Nanjing, China; R433-01). Quantitative PCR was performed with SYBR Green Master Mix (Abclonal, Wuhan, China) using gene-specific primers. Relative mRNA expression levels were calculated using the 2⁻ ΔΔ Ct method. The sequences of all indicated primers are listed in the Supplementary Table 1. Hematoxylin and Eosin (H&E) Staining Lung samples were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned at 5 µm thickness. Paraffin sections were deparaffinized in xylene and rehydrated through a graded ethanol series (100%, 95%, 70%) to water. Sections were stained with hematoxylin for 2 min, differentiated briefly in 1% acid alcohol, and rinsed in running water. Sections were then counterstained with eosin for 1 min, dehydrated through graded ethanol solutions, cleared in xylene, and mounted. Images were acquired using an Olympus scanning system. Immunofluorescence Staining Paraffin-embedded sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed by placing the sections in a repair cassette containing EDTA antigen retrieval buffer (pH 8.0; Beijing Solarbio Science & Technology, C1034) and heating in a microwave oven. Sections were blocked with 5% bovine serum albumin (BSA; Beijing Solarbio Science & Technology, A8010) for 30 min, followed by incubation with primary antibodies in a humidified chamber at 4°C overnight. After washing, sections were incubated with species-appropriate secondary antibodies, and nuclei were counterstained with DAPI (Beijing Solarbio Science & Technology, C0065). Slides were mounted and examined under a fluorescence microscope. The antibodies used in this study were as follows: Influenza A H1N1 NA (Invitrogen, PA5-32238), F4/80 (CST, 70076S), Ly6G (Abcam, AB238132), CD11b (Abcam, AB133357), ZBP1 (Proteintech, 13285-1-AP), pMLKL (Affinity, AF7420), and Pro-SPC (Abcam, AB211326). Immunohistochemical Analysis Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed in citrate-based antigen repair buffer (pH 6.0; Wuhan BaiQianDu Biotechnology, B2010). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide (EZ2921B398, B12555), followed by blocking with 3% bovine serum albumin (BSA; DAKO, BioFroxx). Sections were then incubated with primary antibodies in a humidified chamber at 4°C overnight. After washing, the corresponding secondary antibody for DAB (CITOTEST, 2005289) was applied to visualize the target proteins, followed by nuclear counterstaining. Finally, the sections were dehydrated, mounted, and examined under a light microscope. The following primary antibodies were used: ZBP1 (Proteintech, 13285-1-AP), RIPK3 (Abclonal, A5431), MLKL (Abclonal, A21894), and phosphorylated MLKL (pMLKL; Affinity, AF7420). Flow Cytometry Whole lung tissues were collected and mechanically dissociated through a 40 µm cell strainer to generate a single-cell suspension. Red blood cells were lysed using erythrocyte lysis buffer, followed by washing with PBS. Cells were then fixed in 4% formaldehyde. For intracellular staining, cells were incubated with permeabilization solution at room temperature for 5–10 min. After centrifugation, cells were incubated with primary antibodies (100 µL per sample) at pre-determined dilutions for 1 h. Following incubation, unbound antibodies were removed by centrifugation, and cells were washed with PBS and resuspended in PBS for flow cytometric analysis. The following antibodies were used: CD11b (Cell Signaling Technology, 7008S) and F4/80 (Cell Signaling Technology, 52267S). Enzyme-Linked Immunosorbent Assay (ELISA) Mouse lung tissues were homogenized in ice-cold PBS at a ratio of 1 mL per 100 mg tissue. The homogenates were centrifuged at 12,000 × g for 15 min at 4°C, and the supernatants were collected as total protein lysates. Lysates were aliquoted and stored at − 80°C until analysis. Protein concentrations were determined using a BCA assay (Beyotime, P0010). Standards and working solutions were prepared according to the manufacturer’s instructions. Lung tissue samples were appropriately diluted based on pre-experiment optimization. Absorbance at 450 nm was measured for each well, and protein concentrations were calculated using the generated standard curve. Statistical Analysis All statistical analyses were performed using GraphPad Prism software (version 8.0). Data are presented as mean ± SD. Differences between groups were assessed using two-tailed Student’s t-test or one- or two-way analysis of variance (ANOVA) followed by appropriate multiple comparison post-hoc tests. Detailed statistical methods are provided in the figure legends. All experiments were independently performed in triplicate. A p-value < 0.05 was considered statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant). Declarations Data Availability Data will be made available on request. Acknowledgements This work was supported by a grant from the National Key Research and Development Program of China (2023YFC3503400), the National Nature Science Foundation of China [No. 82074097]. Funding Open access funding provided by Fudan University. Author information These authors contributed equally: Suya Lao, Xin Ai. Authors and Affiliations School of Pharmaceutical Sciences, Shanghai Engineering Research Center of Immunotherapeutics, Fudan University, Shanghai 201203, China. Suya Lao, Xin Ai, Zhixuan Cai1, Yijun Niu, Zichen Tian, Weiming Xu, Xiaotong Lin, Chengjie Xia, Haiyan Zhu & Xunlong Shi Contributions X．S． conceived the study and participated in the overall design, supervision, and coordination of the study. S.L. and X.A. performed most experiments and wrote the manuscript. Y.N. and Z.T. participated in animal studies. Z.C., X.L., W.X., C.X. and H.Z. provided the technical supports and revised the manuscript. All authors read and approved the final manuscript. Corresponding author Correspondence to Xunlong Shi (E-mail address: [email protected] ) References Wang J, Sun Y, Liu S. Emerging antiviral therapies and drugs for the treatment of influenza. Expert Opinion on Emerging Drugs. 2022;27(4):389–403. Li R, Qu S, Qin M, Huang L, Huang Y, Du Y, et al. Immunomodulatory and antiviral effects of Lycium barbarum glycopeptide on influenza a virus infection. Microbial Pathogenesis. 2023;176:106030. Bao Y, Gao Y, Shi Y, Cui X. Dynamic gene expression analysis in a H1N1 influenza virus mouse pneumonia model. Virus Genes. 2017;53(3):357 − 66. Ampomah PB, Lim LHK. Influenza A virus-induced apoptosis and virus propagation. Apoptosis. 2020;25(1):1–11. 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JOURNAL OF EXPERIMENTAL MEDICINE. 2017;214(8):2217-29. Thapa RJ, Ingram JP, Ragan KB, Nogusa S, Boyd DF, Benitez AA, et al. DAI Senses Influenza A Virus Genomic RNA and Activates RIPK3-Dependent Cell Death. CELL HOST & MICROBE. 2016;20(5):674 − 81. Gautam A, Boyd DF, Nikhar S, Zhang T, Siokas I, Van de Velde LA, et al. Necroptosis blockade prevents lung injury in severe influenza. NATURE. 2024;628(8009). Ruscitti C, Radermecker C, Marichal T. Journey of monocytes and macrophages upon influenza A virus infection. CURRENT OPINION IN VIROLOGY. 2024;66. Onufer AP, Mell JC, Cort L, Rao A, Mdluli N, Carey AJ. Influenza virus-induced type I interferons disrupt alveolar epithelial repair and tight junction integrity in the developing lung. MUCOSAL IMMUNOLOGY. 2025;18(3):607 − 19. Dong JY, Liu WH, Liu WL, Wen YQ, Liu QK, Wang HT, et al. Acute lung injury: a view from the perspective of necroptosis. INFLAMMATION RESEARCH. 2024;73(6):997–1018. Sha HX, Liu YB, Qiu YL, Zhong WJ, Yang NSY, Zhang CY, et al. Neutrophil extracellular traps trigger alveolar epithelial cell necroptosis through the cGAS-STING pathway during acute lung injury in mice. INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES. 2024;20(12):4713-30. Zhong WJ, Zhang J, Duan JX, Zhang CY, Ma SC, Li YS, et al. TREM-1 triggers necroptosis of macrophages through mTOR-dependent mitochondrial fission during acute lung injury. JOURNAL OF TRANSLATIONAL MEDICINE. 2023;21(1). Takaoka A, Wang Z, Choi MK, Yanai H, Negishi H, Ban T, et al. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. NATURE. 2007;448(7152):501-U14. Li Y, Guo XM, Hu CM, Du Y, Guo CS, Wang D, et al. Type I IFN operates pyroptosis and necroptosis during multidrug-resistant A. baumannii infection. CELL DEATH AND DIFFERENTIATION. 2018;25(7):1304-18. Ingram JP, Thapa RJ, Fisher A, Tummers B, Zhang T, Yin CR, et al. ZBP1/DAI Drives RIPK3-Mediated Cell Death Induced by IFNs in the Absence of RIPK1. JOURNAL OF IMMUNOLOGY. 2019;203(5):1348-55. Additional Declarations There is no duality of interest Supplementary Files SupplementaryTable1.xlsx Supplementary Table 1 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7426580","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":507295842,"identity":"db9564de-d3f6-4310-b9c8-fe1e9d061fd6","order_by":0,"name":"Xunlong Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYHACxgMg0oCZ+cAHBjawiAFBPVAtbIkzSNTCwGNInBaDG7kHDnzcUWtvzs7zsZmnzC6Pgb15mwRDzR2cWiRn5CUcnHnmeOLOZt6NzTznkosZeI6VSTAce4ZTC79EjsFh3rZjCUBy+2PeNubEBokcMwnGhsM4tbBBtdgbHOZ52MzbVp/YIP8GvxaoLTWMGw7zMAK1HAbawoNfi2TPG4ODM9sOJG44zGbYOOfc8cQ2nrRii4RjuLUYHM8xfPCxrc7e4Pzhhw1vyqoT+9kPb7zxoQa3FihAUgCOmgRCGhgY6ggrGQWjYBSMgpELACDSV70axfRhAAAAAElFTkSuQmCC","orcid":"","institution":"Fudan University","correspondingAuthor":true,"prefix":"","firstName":"Xunlong","middleName":"","lastName":"Shi","suffix":""},{"id":507295843,"identity":"e4df927e-c01d-42da-99e6-d42988155eb6","order_by":1,"name":"Suya Lao","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Suya","middleName":"","lastName":"Lao","suffix":""},{"id":507295844,"identity":"385d2a73-8a62-45a5-aa90-bfe879f605c9","order_by":2,"name":"Xin Ai","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Ai","suffix":""},{"id":507295845,"identity":"ca7a8aca-ebd2-4acd-8b39-b0e892931cbc","order_by":3,"name":"Yijun Niu","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yijun","middleName":"","lastName":"Niu","suffix":""},{"id":507295846,"identity":"5fd6fe7d-fd36-4d35-bd99-b460ce64edd3","order_by":4,"name":"Zichen Tian","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Zichen","middleName":"","lastName":"Tian","suffix":""},{"id":507295847,"identity":"7f5a68d9-f8e1-418a-90ac-b32c8c027f7d","order_by":5,"name":"Zhixuan Cai","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Zhixuan","middleName":"","lastName":"Cai","suffix":""},{"id":507295848,"identity":"91aa9378-9ade-4d1d-84f7-e0c0a8218bbf","order_by":6,"name":"Xiaotong Lin","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Xiaotong","middleName":"","lastName":"Lin","suffix":""},{"id":507295849,"identity":"78632f16-988b-4f0e-84af-65d926750665","order_by":7,"name":"Weiming Xu","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Weiming","middleName":"","lastName":"Xu","suffix":""},{"id":507295850,"identity":"6a7ab11d-cb1c-44fc-b4f0-be2a1d7d076a","order_by":8,"name":"Chengjie Xia","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Chengjie","middleName":"","lastName":"Xia","suffix":""},{"id":507295851,"identity":"94d9e8d8-d0c3-4e31-a8cb-b32fb48988c1","order_by":9,"name":"Haiyan Zhu","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Haiyan","middleName":"","lastName":"Zhu","suffix":""}],"badges":[],"createdAt":"2025-08-21 13:11:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7426580/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7426580/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90317677,"identity":"36f638de-5dff-4bb0-9e4a-a2f4748ac983","added_by":"auto","created_at":"2025-09-01 10:28:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4707549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHCoV-229E aggravates lung injury and mortality in H1N1-infected mice. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) C57BL/6J mice were infected with H1N1 (FM1 strain), HCoV-229E, or sequentially with both viruses. Body weight changes (\u003cstrong\u003eB\u003c/strong\u003e) and survival probability (\u003cstrong\u003eE\u003c/strong\u003e) were monitored in real time (n=12). Mice were sacrificed at 4 days post-infection (dpi) for lung tissue collection (n=8). (\u003cstrong\u003eC\u003c/strong\u003e) Lung index was calculated as lung weight (mg) / body weight (g). (\u003cstrong\u003eD\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eGross pathological changes of lung tissues were evaluated.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eF\u003c/strong\u003e) Histopathological changes were assessed by H\u0026amp;E staining. Alveolar epithelial cell hyperplasia (blue), inflammatory cell infiltration (red). Statistical significance was determined by one-way ANOVA. *p \u0026lt; 0.05; **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7426580/v1/015d3abfaaae5789d48c332b.png"},{"id":90317685,"identity":"ea52a513-57f2-458a-ae4d-cd47ef65dc79","added_by":"auto","created_at":"2025-09-01 10:28:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5471675,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eH1N1/HCoV-229E co-infection induces excessive inflammatory responses and suppresses viral replication in mouse lungs.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e,\u003cstrong\u003e B\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eRelative mRNA levels of inflammatory factors and cytokines were measured by RT-qPCR, normalized to β-actin, and expressed as fold change relative to normal controls. (\u003cstrong\u003eC\u003c/strong\u003e) The neutrophil marker MPO in peripheral blood was quantified by ELISA. Neutrophils (\u003cstrong\u003eD\u003c/strong\u003e) and macrophages (\u003cstrong\u003eF\u003c/strong\u003e) in lung tissues were detected by immunofluorescence. (\u003cstrong\u003eE\u003c/strong\u003e) Macrophages in lung tissue homogenates were quantified by flow cytometry. (\u003cstrong\u003eG\u003c/strong\u003e) Relative expression of the H1N1 M gene was measured by RT-qPCR, normalized to GAPDH, and expressed as fold change relative to normal controls. (\u003cstrong\u003eH\u003c/strong\u003e,\u003cstrong\u003e I\u003c/strong\u003e) Expression of the NA glycoprotein in lung tissues was analyzed by immunofluorescence. Statistical significance was determined by one-way ANOVA. *p \u0026lt; 0.05; **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7426580/v1/9893327c8df1a5cc5da14e6a.png"},{"id":90318701,"identity":"bf0e9b03-0cc7-40ce-a43c-11a5561fdc73","added_by":"auto","created_at":"2025-09-01 10:36:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7429104,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eH1N1/HCoV-229E co-infection activates the ZBP1–RIPK3–MLKL signaling pathway to promote necroptosis.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eCell fragmentation in lung tissues was observed by H\u0026amp;E staining.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eB\u003c/strong\u003e-\u003cstrong\u003eD\u003c/strong\u003e) Relative mRNA levels of ZBP1, RIPK3, and MLKL in lung tissues were measured by RT-qPCR, normalized to β-actin, and expressed as fold change relative to normal controls. (\u003cstrong\u003eE\u003c/strong\u003e-\u003cstrong\u003eG\u003c/strong\u003e) Protein levels of ZBP1, RIPK3, and pMLKL in lung tissues were quantified by ELISA. (\u003cstrong\u003eH\u003c/strong\u003e,\u003cstrong\u003e I\u003c/strong\u003e) Expression of pMLKL in lung tissues was detected by immunohistochemistry. Statistical significance was determined by one-way ANOVA. *p \u0026lt; 0.05; **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7426580/v1/e94f863b1ad5bdc4c252c819.png"},{"id":90318699,"identity":"0656f2ed-ac23-411c-9e49-9d1805294a3c","added_by":"auto","created_at":"2025-09-01 10:36:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6888953,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eH1N1 PR8/HCoV-229E co-infection activates the ZBP1–RIPK3–MLKL pathway to induce severe pneumonia. \u003c/strong\u003eC57BL/6J mice were infected with H1N1 (PR8 strain), HCoV-229E, or co-infected with both viruses (n = 6). Mice were sacrificed at 4 days post-infection for lung tissue collection. (\u003cstrong\u003eA\u003c/strong\u003e) Body weight changes were monitored in real time. (\u003cstrong\u003eB\u003c/strong\u003e) Lung index was calculated as lung weight (mg) / body weight (g). (\u003cstrong\u003eC\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eGross pathological changes of lung tissues were evaluated. (\u003cstrong\u003eD\u003c/strong\u003e) Histopathological changes were assessed by H\u0026amp;E staining. Alveolar proteinaceous exudate (yellow), inflammatory cell infiltration (red). (\u003cstrong\u003eE-G\u003c/strong\u003e) Protein levels of ZBP1, RIPK3, and pMLKL in lung tissues were quantified by ELISA.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eH\u003c/strong\u003e, \u003cstrong\u003eI\u003c/strong\u003e) Expression of pMLKL in lung tissues was detected by immunohistochemistry. Statistical significance was determined by one-way ANOVA. *p \u0026lt; 0.05; **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7426580/v1/4aeff537de942dbd434775e7.png"},{"id":90317688,"identity":"6a3fee0a-e830-4743-b5cc-1907c928daca","added_by":"auto","created_at":"2025-09-01 10:28:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":9205500,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlocking necroptosis alleviates lung injury during H1N1/HCoV-229E co-infection by reducing immune overactivation. \u003c/strong\u003eC57BL/6J mice (n = 6) received daily intraperitoneal injections of the RIPK3 inhibitor GSK-872 (10 mg/kg, 0.2 mL per mouse) from day 2 to day 4 post-infection with H1N1 (FM1 strain) or HCoV-229E. (\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eRelative mRNA levels of ZBP1, RIPK3, and MLKL in lung tissues were measured by RT-qPCR, normalized to β-actin, and expressed as fold change relative to normal controls. (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eProtein levels of ZBP1, RIPK3, and pMLKL in lung tissues were quantified by ELISA. (\u003cstrong\u003eC\u003c/strong\u003e) Expression of pMLKL in lung tissues was detected by immunohistochemistry. (\u003cstrong\u003eD\u003c/strong\u003e) Lung index was calculated as lung weight (mg) / body weight (g). (\u003cstrong\u003eE\u003c/strong\u003e) Gross pathological changes of lung tissues were evaluated. (\u003cstrong\u003eF\u003c/strong\u003e) Histopathological changes were assessed by H\u0026amp;E staining Alveolar epithelial cell hyperplasia (blue), inflammatory cell infiltration (red), nuclear fragmentation (black). (\u003cstrong\u003eG\u003c/strong\u003e) The neutrophil marker MPO in peripheral blood was quantified by ELISA. (\u003cstrong\u003eH\u003c/strong\u003e,\u003cstrong\u003e I\u003c/strong\u003e) Macrophages in lung tissues were detected by immunofluorescence. Statistical significance was determined by one-way ANOVA. *p \u0026lt; 0.05; **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7426580/v1/8b58bd4b1b779e10abba18de.png"},{"id":90317690,"identity":"28b4ffa7-4584-4216-92a7-7ff5bd6c8035","added_by":"auto","created_at":"2025-09-01 10:28:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2667921,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZBP1–RIPK3–MLKL-mediated necroptosis occurs in both lung macrophages and alveolar epithelial cells. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) I Immunofluorescence co-localization of macrophages (F4/80\u003csup\u003e+\u003c/sup\u003e) and type II alveolar epithelial cells (Pro-SPC\u003csup\u003e+\u003c/sup\u003e) with necroptotic proteins ZBP1 and pMLKL. F4/80 (green), Pro-SPC (green), ZBP1 (red), and pMLKL (pink). (\u003cstrong\u003eB\u003c/strong\u003e) Raw 264.7 cells were infected with H1N1 (PR8 strain) or HCoV-229E, and cell viability was assessed by an ATP assay. (\u003cstrong\u003eC\u003c/strong\u003e-\u003cstrong\u003eH\u003c/strong\u003e) C57BL/6J mice (n = 6) were intranasally administered a single dose of IFN-α (1 mg/mL, 20 μL per mouse) at 1 day post-infection with H1N1 (FM1 strain). Relative mRNA expression of type I interferons (\u003cstrong\u003eC\u003c/strong\u003e,\u003cstrong\u003e D\u003c/strong\u003e), viral M gene (\u003cstrong\u003eE\u003c/strong\u003e,\u003cstrong\u003e F\u003c/strong\u003e) and necrosis signaling proteins (\u003cstrong\u003eG\u003c/strong\u003e,\u003cstrong\u003e H\u003c/strong\u003e) in lung tissues were measured by RT-qPCR, normalized to β-actin, and expressed as fold change relative to normal controls. Statistical significance was determined by one-way ANOVA. *p \u0026lt; 0.05; **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7426580/v1/84bfb89e52fe025ae3e0d2e2.png"},{"id":91450087,"identity":"e6605025-7ef1-4db1-9630-84aff4da909b","added_by":"auto","created_at":"2025-09-16 15:20:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":32402921,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7426580/v1/d6624aac-35ed-4658-947c-67a5e3ab6394.pdf"},{"id":90318697,"identity":"0f18ca42-3301-40b7-9244-eb3cd087b971","added_by":"auto","created_at":"2025-09-01 10:36:11","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10502,"visible":true,"origin":"","legend":"Supplementary Table 1","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7426580/v1/f22f091679b0ffa845e16359.xlsx"}],"financialInterests":"There is no duality of interest","formattedTitle":"Hyperactivated ZBP1-Mediated Necroptosis Exacerbates Pneumonia in Mice Co-Infected with Low-Lethality H1N1 and HCoV-229E","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInfluenza A virus (IAV) is a human-adapted respiratory pathogen that causes significant morbidity and mortality worldwide(1). Due to its rapid transmission and broad host range, IAV is the only influenza virus known to have caused pandemics to date(2). Even in non-pandemic years, IAV infects 5\u0026ndash;15% of the global population, leading to approximately 500 000 deaths and 3\u0026nbsp;million severe cases annually(3, 4). Pathogen screening of influenza patients has frequently identified co-existing pathogens, including Streptococcus pneumoniae, Haemophilus influenzae, and respiratory syncytial virus (RSV)(5). Secondary infections with RSV or coronaviruses have been shown to exacerbate influenza severity(6, 7), suggesting that pathogen co-infection is a major contributor to influenza-associated morbidity and mortality.\u003c/p\u003e\u003cp\u003eHuman coronaviruses (HCoVs) are positive-sense single-stranded RNA viruses with high zoonotic potential and rapid evolutionary dynamics. To date, seven HCoV strains have been identified and classified into two genera: Alphacoronavirus and Betacoronavirus(8). Human coronavirus 229E (HCoV-229E), a member of the Alphacoronavirus genus, is an important cause of community-acquired pneumonia and contributes significantly to respiratory disease burden(9). However, co-infection involving HCoV-229E and IAV has not been reported, and the immunopathological mechanisms underlying such dual infections remain undefined.\u003c/p\u003e\u003cp\u003eViral co-infections often result in atypical clinical manifestations, including increased mortality, neurological complications, and immunosuppression(10), which cannot be explained by single-pathogen infection. They frequently share transmission routes (respiratory or hematogenous) and host tropism(11), facilitating their high transmissibility and cross-infection potential in endemic regions. Current diagnostic approaches\u0026mdash;primarily serological testing and virus isolation\u0026mdash;are limited(12\u0026ndash;14). Serological assays suffer from low sensitivity and cross-reactivity(15), while virus isolation requires permissive cell lines or animal models and may be confounded by viral interference(16, 17). Therapeutically, most available antivirals target single viruses, rendering them ineffective in co-infections. Combination regimens carry risks of drug antagonism, toxicity, and accelerated resistance, ultimately compromising efficacy(18). These limitations underscore the urgent need to elucidate the pathological mechanisms of viral co-infections and to develop integrated therapeutic strategies.\u003c/p\u003e\u003cp\u003eRecent studies have implicated necroptosis as a key driver of influenza-associated pathology. Viral infection generates Z-form RNA, which is sensed by the host protein Z-DNA-binding protein 1 (ZBP1). Upon recognition, ZBP1 engages receptor-interacting protein kinase 3 (RIPK3) to form a necroptosis-inducing complex, which phosphorylates mixed lineage kinase domain-like protein (MLKL), thereby triggering necroptosis(19). ZBP1-mediated necroptosis is characterized by inflammatory cell death, tissue damage, and immune dysregulation, and has been implicated in multiple diseases, including viral pneumonia(20).\u003c/p\u003e\u003cp\u003eIn our experimental model, coronavirus infection secondary to IAV significantly worsened pneumonia severity and increased lethality in mice, though the underlying mechanism was unclear. To address this, we established a physiologically relevant low-dose sequential co-infection model with varied orders of IAV and HCoV-229E exposure. This model recapitulated diverse clinical infection scenarios and revealed that even low-dose HCoV-229E following IAV infection aggravated pneumonia. Further investigation linked this pathological progression to ZBP1-dependent necroptosis. We demonstrated that HCoV-229E potentiates ZBP1\u0026ndash;RIPK3\u0026ndash;MLKL-mediated necroptosis in influenza-infected lungs. These findings provide a potential explanation for the diagnostic and therapeutic challenges of influenza\u0026ndash;coronavirus co-infections and highlight the ZBP1/RIPK3/MLKL signaling axis as a promising therapeutic target for severe viral pneumonia.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSecondary HCoV-229E Infection Following Primary H1N1 Exacerbates Pulmonary Lesions\u003c/h2\u003e\u003cp\u003eTo investigate the effects of co-infection with H1N1 and HCoV-229E on pulmonary disease, we established co-infected mouse models (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Disease pathology was assessed in mice infected with H1N1 FM1 or HCoV-229E individually, simultaneously, or sequentially with a 24-hour interval to determine the impact of infection order. Notably, secondary infection with HCoV-229E following H1N1 (Co-infection-1) resulted in significant weight loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), pulmonary edema, and pronounced lung injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D), whereas other co-infection modalities produced minimal effects. These observations prompted a detailed investigation of this specific co-infection scenario.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter 2 days of sequential infection, co-infected mice exhibited dramatic weight loss compared with mice infected with H1N1 alone, whereas HCoV-229E mono-infected mice maintained body weight similar to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). On day 4 post-infection, mice were anesthetized and euthanized to collect lung tissues for lung index measurement and histopathological analysis. The lung index, defined as the ratio of lung weight to body weight, was markedly elevated in co-infected mice (14.5), approximately double that of the control group (6.6), and significantly higher than in H1N1- (9.7) or HCoV-229E-infected mice (6.2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The elevated lung index indicates severe inflammatory lesions in lung tissues. Gross examination revealed extensive congestion and pronounced tissue edema in co-infected lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Histological analysis by H\u0026amp;E staining further demonstrated widespread alveolar atrophy, structural destruction of alveolar lumens, and extensive infiltration of inflammatory cells in co-infected mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). In contrast, lung lesions in mono-infected mice were notably milder, consistent with the observed lung index changes.\u003c/p\u003e\u003cp\u003eIn a 14-day survival assay, co-infected mice exhibited earlier mortality and a higher overall death rate compared with H1N1 mono-infected mice. The final mortality rate reached 83% in co-infected mice, significantly higher than that of H1N1- (60%) or HCoV-229E-infected mice (0%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). These results indicate that HCoV-229E co-infection markedly exacerbates lung injury in H1N1-infected mice and substantially increases mortality.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSevere Pneumonia Results from Hyperactivated Inflammation Rather than Uncontrolled Viral Replication\u003c/h3\u003e\n\u003cp\u003eWe analyzed two major factors\u0026mdash;pathogen and host immune response\u0026mdash;that contribute to severe pneumonia during co-infection(21). First, we examined the correlation between severe lung injury and immune responses in the lungs of mice. RT-qPCR analysis of multiple cytokine transcripts in lung tissues revealed that H1N1/HCoV-229E co-infection led to a significant upregulation of inflammatory factors, including IL-1β, TNF-α, and IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), accompanied by increased transcription of IFN-α and IFN-β, compared with H1N1 or HCoV-229E mono-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Histopathological analysis of lung tissues showed extensive immune cell infiltration, predominantly innate immune cells such as monocytes and neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Immunofluorescence staining further demonstrated that co-infection markedly enhanced the recruitment of neutrophils (CD11b\u003csup\u003e+\u003c/sup\u003e/Ly6G\u003csup\u003e+\u003c/sup\u003e double-positive) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), with a concomitant elevation of neutrophil activation marker MPO, indicating an inflammatory activation state (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Flow cytometry revealed a decrease in macrophage numbers in co-infected lungs compared with mono-infected lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), which was confirmed by immunofluorescence staining of F4/80\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Collectively, these results indicate that H1N1/HCoV-229E co-infection induces a complex immune response characterized by massive neutrophil recruitment and activation, a reduction in macrophages, elevated pro-inflammatory cytokine production, and increased interferon transcription.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next examined viral load as a second factor contributing to severe lung injury. Influenza virus replication in lung tissues was assessed by quantitative RT-qPCR and immunofluorescence. Co-infected mice exhibited significantly lower transcription of the influenza M gene and reduced expression of NA glycoprotein compared with H1N1 mono-infected mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG\u0026ndash;I), indicating that co-infection did not enhance viral replication. On the contrary, H1N1 replication was markedly suppressed, suggesting that severe lung injury in H1N1/HCoV-229E co-infected mice is not driven by uncontrolled viral proliferation.\u003c/p\u003e\u003cp\u003eThese findings suggest that the severe pneumonia and high mortality observed in co-infected mice are associated with dysregulated immune responses rather than elevated viral load, highlighting the need for further investigation of key immune signaling pathways.\u003c/p\u003e\n\u003ch3\u003eZBP1-Mediated Necroptosis Promotes Pneumonia Severity in Co-Infection\u003c/h3\u003e\n\u003cp\u003eAnalysis of cellular morphology in lung tissues by H\u0026amp;E staining revealed pronounced cellular necrosis in co-infected mice compared with those infected with a single virus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Previous studies have reported that Z-DNA/RNA generated by various viruses, including influenza viruses, can bind to the host sensor ZBP1, forming a ZBP1-RIPK3 complex with RIPK3 kinase, which subsequently activates MLKL-mediated necroptosis and triggers inflammatory responses(22\u0026ndash;24). To investigate this pathway in our model, we examined key necroptosis signaling targets\u0026mdash;ZBP1, RIPK3, and MLKL\u0026mdash;at both the transcript and protein levels in mouse lung tissues. Co-infection significantly increased mRNA expression of ZBP1, RIPK3, and MLKL compared with single-virus infection (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026ndash;D), accompanied by elevated protein levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u0026ndash;G). Immunohistochemical analysis further confirmed high expression of necroptosis signaling proteins in the lungs of co-infected mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Collectively, these findings indicate that H1N1/HCoV-229E co-infection activates the ZBP1-RIPK3-MLKL necroptotic pathway in lung tissues, contributing to exacerbated lung injury and inflammation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eNecroptosis Is Consistently Induced by Diverse H1N1 Strains in Co-Infection with HCoV-229E\u003c/h3\u003e\n\u003cp\u003eTo determine whether ZBP1-RIPK3-MLKL-mediated necroptosis is induced by co-infection with specific influenza virus strains and HCoV-229E, we established co-infected mouse models using the influenza A virus H1N1 PR8 strain in combination with HCoV-229E. The results showed that H1N1 PR8/HCoV-229E co-infection caused marked weight loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), more severe lung injury (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u0026ndash;D), and a significant increase in necroptosis-associated proteins ZBP1, RIPK3, and phosphorylated MLKL in lung tissues (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u0026ndash;I), consistent with observations using the H1N1 FM1 strain.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese experiments confirm that co-infection of HCoV-229E with different influenza A virus strains induces more severe pneumonia, and that disease severity is positively correlated with the level of ZBP1-RIPK3-MLKL-mediated necroptosis.\u003c/p\u003e\n\u003ch3\u003eBlockade of Necroptosis Ameliorates Pneumonia in Co-Infected Mice\u003c/h3\u003e\n\u003cp\u003eTo investigate the role of ZBP1-RIPK3-MLKL-mediated necroptosis in H1N1 /HCoV-229E co-infection-induced pneumonia, we treated co-infected mice with GSK872, a RIPK3 inhibitor that blocks necroptosis initiation(25, 26). The results showed that GSK872 did not affect the transcription of necroptosis-related genes ZBP1, RIPK3, and MLKL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), but significantly reduced the protein levels of these key necroptotic factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Immunohistochemical analysis further confirmed that GSK872 effectively inhibited necroptosis in lung tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTreatment with GSK872 significantly reduced the lung index in H1N1/HCoV-229E co-infected mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), alleviated hemorrhagic and edematous changes in the lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), and suppressed inflammatory cell recruitment, particularly neutrophil recruitment and activation (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u0026ndash;I).\u003c/p\u003e\u003cp\u003eCollectively, these results demonstrate that severe pneumonia induced by H1N1/HCoV-229E co-infection is closely associated with activation of the ZBP1-RIPK3-MLKL necroptosis pathway, and that pharmacological inhibition of necroptosis can mitigate co-infection-associated lung injury.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eNecroptosis in Alveolar Macrophages and Epithelial Cells Correlates with Interferon Upregulation during Co-Infection\u003c/h2\u003e\u003cp\u003eWe previously demonstrated that H1N1/HCoV-229E co-infection exacerbates pulmonary damage in mice through ZBP1-RIPK3-MLKL-mediated necroptosis. To further determine the specific cell types undergoing necroptosis in lung tissues, we performed immunofluorescence staining to assess co-localization of key necroptotic proteins (ZBP1 and pMLKL) with cell-specific markers. Notably, strong co-localization of necroptotic proteins was observed in macrophages (F4/80⁺/ZBP1⁺/pMLKL⁺) and type II alveolar epithelial cells (Pro-SPC⁺/ZBP1⁺/pMLKL⁺) in co-infected mouse lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Subsequent in vitro validation using RAW 264.7 cells further confirmed that co-infection significantly reduced cell viability compared with single-virus infection, indicating enhanced necroptosis in macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Together, these findings establish that ZBP1-RIPK3-MLKL-driven necroptosis predominantly occurs in pulmonary macrophages and alveolar epithelial cells during co-infection.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePreviously, we observed that interferon expression was markedly elevated in the lungs of co-infected mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Since viral infection typically induces interferon production, which in turn affects multiple cell types, we hypothesized that interferons play a critical role in the pathogenesis of co-infection. ZBP1, an interferon-inducible protein, has been reported to mediate interferon-driven necroptosis(27). To test this mechanism, we used IFN-α treatment to mimic the effect of HCoV-229E co-infection. Under low-dose HCoV-229E exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), IFN-α stimulation following H1N1 infection significantly enhanced inflammatory responses (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D), suppressed H1N1 replication (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), and upregulated key necroptotic effector proteins (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H). Collectively, these results indicate that in H1N1/HCoV-229E co-infection, necroptosis is regulated by multiple factors, with interferons serving as universal and critical modulators across different cell types.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eInfluenza A viruses are a major cause of respiratory infections in humans, ranging from mild to severe, and impose a substantial public health burden(28, 29). According to the World Health Organization, seasonal influenza\u0026mdash;including influenza A (H1N1, H3N2) and influenza B strains\u0026mdash;accounts for 3\u0026ndash;5\u0026nbsp;million cases of severe respiratory illness and 290 000\u0026ndash;650 000 deaths globally each year(30). Pathogen screening in patients with severe influenza often identifies multiple pathogens, including influenza viruses, suggesting that secondary viral infections can frequently exacerbate disease severity(31). In the present study, we provide experimental evidence that secondary infection with HCoV-229E aggravates H1N1-induced pneumonia, a process closely linked to activation of the ZBP1-RIPK3-MLKL necroptosis pathway rather than to increased viral replication. These findings offer a mechanistic explanation for the clinical challenges associated with diagnosing influenza-coronavirus co-infections and highlight ZBP1/RIPK3/MLKL-mediated necroptosis as a potential therapeutic target for severe viral pneumonia.\u003c/p\u003e\u003cp\u003eIn nature, simultaneous or sequential co-infection of a single host by multiple pathogens is common(32, 33). Viral co-infections can complicate disease diagnosis and treatment by producing overlapping clinical symptoms, altering viral pathogenicity and transmissibility, and facilitating viral mutation(34\u0026ndash;36). Some studies suggest that viral interference may occur through mechanisms such as interferon-mediated enhancement of innate immunity(37), disruption of viral replication cycles(38, 39), or competition for metabolites and host factors essential for viral replication(40, 41). In contrast, other evidence indicates that viral synergism can arise through processes such as promotion of infected cell membrane fusion(42) or induction of host immunodeficiency(43). Collectively, these findings demonstrate that viral interactions and the immunological impact of co-infection on hosts involve highly complex mechanisms.\u003c/p\u003e\u003cp\u003eBy establishing a series of influenza A virus (IAV) and coronavirus (HCoV-229E) co-infection models, we observed that secondary HCoV-229E infection exacerbated pneumonia in H1N1-infected mice. Investigation into the underlying cause revealed that this exacerbation was not associated with uncontrolled viral replication, as co-infected mice exhibited a significant reduction in H1N1 M gene transcription and H1N1 NA protein expression compared to mice infected with H1N1 alone. We then examined immune alterations in the host lungs and found that H1N1/HCoV-229E co-infection induced a complex immune response, characterized by massive neutrophil recruitment, inflammatory activation, and a reduction in macrophages, accompanied by elevated transcript levels of multiple pro-inflammatory mediators. Nonetheless, the specific immune signaling pathways driving these changes warrant further investigation.\u003c/p\u003e\u003cp\u003ePathological analysis of lungs from H1N1/HCoV-229E co-infected mice revealed extensive nuclear fragmentation, suggesting that severe pneumonia under co-infection conditions may result from cellular necrosis. Necroptosis has been implicated in multiple viral infections, including herpes simplex virus (HSV)(44), cytomegalovirus (CMV)(45), and influenza A viruses such as H1N1 and H5N1(46). Consistently, our study showed marked upregulation of both transcript and protein levels of necroptosis mediators, including ZBP1, RIPK3, and phosphorylated MLKL (pMLKL). Z-DNA-binding protein 1 (ZBP1) serves as a critical inducer of necroptosis during viral infection by recognizing nuclear Z-RNA and activating the ZBP1-RIPK3-MLKL signaling axis(47), ultimately leading to nuclear membrane rupture and release of nuclear-derived damage-associated molecular patterns (DAMPs)(48). Importantly, pharmacological inhibition of RIPK3 with GSK872 significantly alleviated pneumonia severity in co-infected mice, indicating that disease progression is tightly linked to necroptosis activation. Moreover, co-infection experiments using distinct H1N1 strains (FM1 and PR8) in combination with HCoV-229E consistently demonstrated a correlation between pneumonia severity and activation of the ZBP1-RIPK3-MLKL pathway, underscoring necroptosis as a prevalent and central mechanism in co-infection\u0026ndash;induced severe pneumonia.\u003c/p\u003e\u003cp\u003e Alveolar macrophages are among the earliest immune cells to participate in host defense, capable of rapidly recognizing respiratory pathogens and regulating local inflammatory responses and immune reactions, thereby playing a critical role in controlling disease progression and severity(49). Pulmonary epithelial cells constitute another essential barrier; damage to this barrier allows bacteria or viruses to disseminate systemically within the host, leading to severe or even fatal outcomes(50). Previous studies have demonstrated that necroptosis plays an important role in acute lung injury (ALI), being activated in both macrophages and alveolar epithelial cells(51\u0026ndash;53). Consistent with this, our study revealed marked colocalization of ZBP1/pMLKL in lung macrophages and type II alveolar epithelial cells, indicating widespread necroptosis in these cell populations. Further analyses showed that H1N1/HCoV-229E co-infection significantly reduced the number of lung macrophages, while in vitro experiments confirmed a marked decline in macrophage activity under co-infection conditions. Taken together, these findings indicate that necroptosis is prevalent in both alveolar macrophages and epithelial cells during co-infection and may play a critical role in driving lung injury.\u003c/p\u003e\u003cp\u003eCompared with H1N1 infection alone, lungs from H1N1/HCoV-229E co-infected mice exhibited elevated transcription of type I interferons (IFN-α/β) and reduced H1N1 replication, suggesting the involvement of IFNs in this process. Interferons play critical roles in immune regulation and antiviral defense, and previous studies have reported that IFNs participate in necroptosis, with ZBP1 serving as a key mediator of IFN-induced necroptosis(54\u0026ndash;56). In our study, co-stimulation with H1N1 and IFN-α further increased the transcription of IFNs and necroptosis-related proteins in mouse lung tissues, while concomitantly reducing H1N1 replication. Based on these findings, we propose that IFNs induced during viral co-infection exert dual effects: high levels of IFNs facilitate viral clearance, but sustained upregulation of ZBP1 drives necroptosis in macrophages and alveolar epithelial cells, thereby exacerbating lung injury.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMice\u003c/h2\u003e\u003cp\u003eFemale C57BL/6J mice were used in this study. All animal experiments and histological analyses were conducted under blinded conditions. Animals were housed in the specific-pathogen-free (SPF) facility of the special infection animal room, Antiviral Drug Research Laboratory (BSL-2), School of Pharmacy, Fudan University. Mice were maintained under controlled environmental conditions (temperature 20\u0026ndash;25\u0026deg;C, relative humidity 40\u0026ndash;70%) in individually ventilated cages with a 12-h light/dark cycle. Sterilized chow and water were provided ad libitum.\u003c/p\u003e\u003cp\u003eFor infection models, mice were anesthetized with isoflurane and intranasally inoculated with 30 \u0026micro;L of viral suspension. Single-virus infection models were established using either H1N1 or HCoV-229E, while co-infection models were generated using both viruses. The median lethal dose (LD\u003csub\u003e50\u003c/sub\u003e) of H1N1 was determined as 1 \u0026times; 10⁻⁵, and infection was performed at 4 \u0026times; LD\u003csub\u003e50\u003c/sub\u003e. HCoV-229E infection was performed using undiluted viral stock solution (dose\u0026thinsp;=\u0026thinsp;1).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCells and Viruses\u003c/h2\u003e\u003cp\u003eThe RAW 264.7 cell line was obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS). Cells were cultured at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂.\u003c/p\u003e\u003cp\u003eInfluenza A virus strains H1N1 A/FM/1/47 and H1N1 A/PR/8/34 were obtained from the Shanghai Centre for Disease Control and Prevention (SCDC) and ATCC, respectively. Human Coronavirus-229E (HCoV-229E) was kindly provided by Prof. Meiqing Feng. All viral stocks were stored at \u0026minus;\u0026thinsp;80\u0026deg;C in the Antiviral Drug Research Laboratory, School of Pharmacy, Fudan University.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eVirus Infection and Cell Viability Assay\u003c/h2\u003e\u003cp\u003eRAW 264.7 cells were seeded in 96-well plates at a density of 4 \u0026times; 10⁴ cells per well 24 h prior to infection. Cells were washed with phosphate-buffered saline (PBS) and then incubated with H1N1 PR8 virus (1:1000 dilution, TCID₅₀ = 10⁻⁵.⁵/mL) and HCoV-229E virus (1:500 dilution, TCID₅₀ = 10⁻\u0026sup3;.⁵/mL) in infection medium consisting of DMEM supplemented with 1% penicillin/streptomycin, at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂.\u003c/p\u003e\u003cp\u003eCell viability was assessed 24 h post-infection using the CellTiter-Lumi\u0026trade; Plus Luminescent Cell Viability Assay Kit (Beyotime, China; C0068M) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eRT-qPCR\u003c/h2\u003e\u003cp\u003eThe mRNA levels of the indicated genes were quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Total RNA was extracted using TRIzol reagent (Invitrogen\u0026trade;, 15596018) and reverse-transcribed into cDNA using a reverse transcription kit (Vazyme, Nanjing, China; R433-01). Quantitative PCR was performed with SYBR Green Master Mix (Abclonal, Wuhan, China) using gene-specific primers. Relative mRNA expression levels were calculated using the 2⁻\u003csup\u003eΔΔ\u003c/sup\u003eCt method. The sequences of all indicated primers are listed in the Supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eHematoxylin and Eosin (H\u0026amp;E) Staining\u003c/h2\u003e\u003cp\u003eLung samples were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned at 5 \u0026micro;m thickness. Paraffin sections were deparaffinized in xylene and rehydrated through a graded ethanol series (100%, 95%, 70%) to water. Sections were stained with hematoxylin for 2 min, differentiated briefly in 1% acid alcohol, and rinsed in running water. Sections were then counterstained with eosin for 1 min, dehydrated through graded ethanol solutions, cleared in xylene, and mounted. Images were acquired using an Olympus scanning system.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence Staining\u003c/h2\u003e\u003cp\u003eParaffin-embedded sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed by placing the sections in a repair cassette containing EDTA antigen retrieval buffer (pH 8.0; Beijing Solarbio Science \u0026amp; Technology, C1034) and heating in a microwave oven. Sections were blocked with 5% bovine serum albumin (BSA; Beijing Solarbio Science \u0026amp; Technology, A8010) for 30 min, followed by incubation with primary antibodies in a humidified chamber at 4\u0026deg;C overnight. After washing, sections were incubated with species-appropriate secondary antibodies, and nuclei were counterstained with DAPI (Beijing Solarbio Science \u0026amp; Technology, C0065). Slides were mounted and examined under a fluorescence microscope.\u003c/p\u003e\u003cp\u003eThe antibodies used in this study were as follows: Influenza A H1N1 NA (Invitrogen, PA5-32238), F4/80 (CST, 70076S), Ly6G (Abcam, AB238132), CD11b (Abcam, AB133357), ZBP1 (Proteintech, 13285-1-AP), pMLKL (Affinity, AF7420), and Pro-SPC (Abcam, AB211326).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemical Analysis\u003c/h2\u003e\u003cp\u003eParaffin-embedded tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed in citrate-based antigen repair buffer (pH 6.0; Wuhan BaiQianDu Biotechnology, B2010). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide (EZ2921B398, B12555), followed by blocking with 3% bovine serum albumin (BSA; DAKO, BioFroxx). Sections were then incubated with primary antibodies in a humidified chamber at 4\u0026deg;C overnight. After washing, the corresponding secondary antibody for DAB (CITOTEST, 2005289) was applied to visualize the target proteins, followed by nuclear counterstaining. Finally, the sections were dehydrated, mounted, and examined under a light microscope.\u003c/p\u003e\u003cp\u003eThe following primary antibodies were used: ZBP1 (Proteintech, 13285-1-AP), RIPK3 (Abclonal, A5431), MLKL (Abclonal, A21894), and phosphorylated MLKL (pMLKL; Affinity, AF7420).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eFlow Cytometry\u003c/h2\u003e\u003cp\u003eWhole lung tissues were collected and mechanically dissociated through a 40 \u0026micro;m cell strainer to generate a single-cell suspension. Red blood cells were lysed using erythrocyte lysis buffer, followed by washing with PBS. Cells were then fixed in 4% formaldehyde. For intracellular staining, cells were incubated with permeabilization solution at room temperature for 5\u0026ndash;10 min. After centrifugation, cells were incubated with primary antibodies (100 \u0026micro;L per sample) at pre-determined dilutions for 1 h. Following incubation, unbound antibodies were removed by centrifugation, and cells were washed with PBS and resuspended in PBS for flow cytometric analysis.\u003c/p\u003e\u003cp\u003eThe following antibodies were used: CD11b (Cell Signaling Technology, 7008S) and F4/80 (Cell Signaling Technology, 52267S).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/h2\u003e\u003cp\u003eMouse lung tissues were homogenized in ice-cold PBS at a ratio of 1 mL per 100 mg tissue. The homogenates were centrifuged at 12,000 \u0026times; g for 15 min at 4\u0026deg;C, and the supernatants were collected as total protein lysates. Lysates were aliquoted and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis. Protein concentrations were determined using a BCA assay (Beyotime, P0010). Standards and working solutions were prepared according to the manufacturer\u0026rsquo;s instructions. Lung tissue samples were appropriately diluted based on pre-experiment optimization. Absorbance at 450 nm was measured for each well, and protein concentrations were calculated using the generated standard curve.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using GraphPad Prism software (version 8.0). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Differences between groups were assessed using two-tailed Student\u0026rsquo;s t-test or one- or two-way analysis of variance (ANOVA) followed by appropriate multiple comparison post-hoc tests. Detailed statistical methods are provided in the figure legends. All experiments were independently performed in triplicate. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ns, not significant).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003eData Availability\u003c/h2\u003e\n \u003cp\u003eData will be made available on request.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThis work was supported by a grant from the National Key Research and Development Program of China (2023YFC3503400), the National Nature Science Foundation of China [No. 82074097].\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eOpen access funding provided by Fudan University.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThese authors contributed equally: Suya Lao, Xin Ai.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eSchool of Pharmaceutical Sciences, Shanghai Engineering Research Center of Immunotherapeutics, Fudan University, Shanghai 201203, China.\u003c/p\u003e\n \u003cp\u003eSuya Lao, Xin Ai, Zhixuan Cai1, Yijun Niu, Zichen Tian, Weiming Xu, Xiaotong Lin, Chengjie Xia, Haiyan Zhu \u0026amp; Xunlong Shi\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eX．S．\u0026nbsp;conceived the study and participated in the overall design, supervision, and coordination of the study. S.L. and X.A. performed most experiments and wrote the manuscript. Y.N. and Z.T. participated in animal studies. Z.C., X.L., W.X., C.X. and H.Z. provided the technical supports and revised the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eCorrespondence to Xunlong Shi (E-mail address: [email protected])\u003c/p\u003e\n\u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Wang J, Sun Y, Liu S. Emerging antiviral therapies and drugs for the treatment of influenza. Expert Opinion on Emerging Drugs. 2022;27(4):389\u0026ndash;403.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Li R, Qu S, Qin M, Huang L, Huang Y, Du Y, et al. Immunomodulatory and antiviral effects of Lycium barbarum glycopeptide on influenza a virus infection. 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INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. 2022;23(12).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Guo HY, Gilley RP, Fisher A, Lane R, Landsteiner VJ, Ragan KB, et al. Species-independent contribution of ZBP1/DAI/DLM-1-triggered necroptosis in host defense against HSV1. CELL DEATH \u0026amp; DISEASE. 2018;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Upton JW, Kaiser WJ, Mocarski ES. DAI/ZBP1/DLM-1 Complexes with RIP3 to Mediate Virus-Induced Programmed Necrosis that Is Targeted by Murine Cytomegalovirus vIRA. CELL HOST \u0026amp; MICROBE. 2012;11(3):290-7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Kesavardhana S, Kuriakose T, Guy CS, Samir P, Malireddi RKS, Mishra A, et al. ZBP1/DAI ubiquitination and sensing of influenza vRNPs activate programmed cell death. 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TREM-1 triggers necroptosis of macrophages through mTOR-dependent mitochondrial fission during acute lung injury. JOURNAL OF TRANSLATIONAL MEDICINE. 2023;21(1).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Takaoka A, Wang Z, Choi MK, Yanai H, Negishi H, Ban T, et al. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. NATURE. 2007;448(7152):501-U14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Li Y, Guo XM, Hu CM, Du Y, Guo CS, Wang D, et al. Type I IFN operates pyroptosis and necroptosis during multidrug-resistant A. baumannii infection. CELL DEATH AND DIFFERENTIATION. 2018;25(7):1304-18.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Ingram JP, Thapa RJ, Fisher A, Tummers B, Zhang T, Yin CR, et al. ZBP1/DAI Drives RIPK3-Mediated Cell Death Induced by IFNs in the Absence of RIPK1. JOURNAL OF IMMUNOLOGY. 2019;203(5):1348-55.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7426580/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7426580/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInfluenza virus causes millions of infections annually, with severe viral pneumonia accounting for a substantial proportion of associated mortality. Co-infections with other respiratory pathogens further worsen clinical outcomes, yet the underlying mechanisms remain poorly defined. Clinical observations have documented influenza\u0026ndash;coronavirus co-infections, but their pathological interplay has not been elucidated.\u003c/p\u003e\u003cp\u003eTo address this gap, we established a murine model of sequential H1N1 (FM1 or PR8 strains) and human coronavirus 229E (HCoV-229E) infection, which recapitulated the fatal pneumonia observed in patients. Strikingly, co-infected mice exhibited accelerated mortality and exacerbated lung pathology despite reduced viral loads, revealing a paradoxical \u0026ldquo;low-virus, high-inflammation\u0026rdquo; state.\u003c/p\u003e\u003cp\u003eTargeted transcriptional and functional analyses identified ZBP1-dependent necroptosis as the central driver of pathology. Lung tissues showed robust activation of the ZBP1\u0026ndash;RIPK3\u0026ndash;MLKL axis, which correlated with cytokine overproduction and histopathological damage. Notably, inhibition of RIPK3\u0026mdash;rather than direct antiviral treatment\u0026mdash;restored lung function and improved survival, highlighting the causal role of necroptosis independent of viral replication. Furthermore, this ZBP1-driven mechanism was conserved across diverse influenza strains, emphasizing its broad biological relevance.\u003c/p\u003e\u003cp\u003eCollectively, our findings reveal a previously unrecognized immunopathological axis in which influenza-primed lungs undergo coronavirus-triggered necroptotic storm, directly linking viral co-infection to fatal pneumonia. These results identify ZBP1 and RIPK3 as promising therapeutic targets for decoupling inflammation from viral clearance in severe respiratory co-infections.\u003c/p\u003e","manuscriptTitle":"Hyperactivated ZBP1-Mediated Necroptosis Exacerbates Pneumonia in Mice Co-Infected with Low-Lethality H1N1 and HCoV-229E","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-01 10:28:06","doi":"10.21203/rs.3.rs-7426580/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"199646ce-1abd-4876-95a0-0c13e2dd1b62","owner":[],"postedDate":"September 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53883315,"name":"Biological sciences/Immunology/Cell death and immune response"},{"id":53883316,"name":"Biological sciences/Immunology/Infectious diseases"},{"id":53883317,"name":"Biological sciences/Immunology/Inflammation/Acute inflammation"}],"tags":[],"updatedAt":"2025-09-16T15:12:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-01 10:28:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7426580","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7426580","identity":"rs-7426580","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}