Neutrophil adhesion to vessel walls impairs pulmonary circulation in COVID-19 pathology

Neutrophil adhesion to vessel walls impairs pulmonary circulation in COVID-19 pathology | 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 Neutrophil adhesion to vessel walls impairs pulmonary circulation in COVID-19 pathology Yoshihiro Kawaoka, Hiroshi Ueki, I-Hsuan Wang, Maki Kiso, Kenta Horie, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3895679/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Jan, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Microthrombus formation is associated with COVID-19 severity; however, the detailed mechanism remains unclear. In this study, we investigated mouse models with severe pneumonia caused by SARS-CoV-2 infection by using our in vivo two-photon imaging system. In the lungs of SARS-CoV-2-infected mice, increased expression of adhesion molecules in intravascular neutrophils prolonged adhesion time to the vessel wall, resulting in platelet aggregation and impaired lung perfusion. Re-analysis of scRNA-seq data from peripheral blood mononuclear cells from COVID-19 cases revealed increased expression levels of CD44 and SELL in neutrophils in severe COVID-19 cases compared to a healthy group, consistent with our observations in the mouse model. These findings suggest that pulmonary perfusion defects caused by neutrophil adhesion to pulmonary vessels contribute to COVID-19 severity. Health sciences/Diseases/Infectious diseases/Viral infection Biological sciences/Microbiology/Virology/SARS-CoV-2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Main Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative virus of COVID-19 (coronavirus disease 2019) and is transmitted via droplets/aerosols containing the virus. SARS-CoV-2 invasion of the lung is initiated by the binding of the virus to the alveolar epithelium using angiotensin-converting enzyme-2 (ACE-2) as a receptor 1 , 2 . Most cases of COVID-19 are mild with respiratory symptoms such as fever, cough, nasal discharge, and pharyngitis; however, the elderly and those with underlying medical conditions such as obesity, diabetes, and hypertension develop severe viral pneumonia, which often leads to serious complications and even death 3 , 4 . Patients presenting with severe viral pneumonia are treated according to their respiratory and systemic symptoms, but the mechanism of COVID-19 severity is not fully understood and an optimal treatment strategy has not been established. Immunopathology, including neutrophilia and lymphopenia, decreased or delayed type I interferon response, and the cytokine storm caused by dysregulation of monocytes and granulocytes may play role in the pathogenesis of COVID-19 5 . In addition to these immunopathological events, thrombosis has frequently been reported in patients with severe COVID-19, which may also contribute to the severity of the disease 6 – 8 . Platelets bind to neutrophils through neutrophil extracellular traps (NETs) released by the neutrophils to form large thrombus-like aggregates; they also form thrombi independently of neutrophils in response to tissue factors exposed to damaged endothelial cells. Single-cell RNA-seq and proteomic approaches using clinical samples and animal models of SARS-CoV-2 infection have revealed that factors associated with inflammation, coagulation, and NETs release are activated during SARS-CoV-2 infection. The factors involved in thrombus formation are spatiotemporally regulated and the thrombogenic process is thought to be a multistep process; however, these dynamic events are difficult to observe using conventional histological approaches with COVID-19 autopsy samples or SARS-CoV-2-infected animals. Accordingly, it remains unclear how the process of thrombus formation proceeds in patients with severe COVID-19. To establish an effective treatment for patients with severe COVID-19, we must elucidate the mechanisms involved in the severity of the disease. To understand how SARS-CoV-2 affects the responses of platelets and neutrophils in the pulmonary vasculature and the thrombogenic response, we performed two-photon in vivo imaging analysis of cellular responses after SARS-CoV-2 infection by using SARS-CoV-2 strains and lethal mouse models. Our in vivo imaging analysis revealed increased neutrophil numbers in the pulmonary vasculature, prolonged neutrophil adhesion to pulmonary vessels, and enhanced platelet aggregation in SARS-CoV-2-infected mice. The increased expression of adhesion factors on the neutrophils in pulmonary blood vessels promoted thrombus formation and impaired pulmonary blood flow in the lung of the SARS-CoV-2-infected mice. In addition, re-analysis of data from human clinical samples 9 demonstrated the relevance of the findings in mice. Results In vivo imaging of SARS-CoV-2-infected lung. To elucidate the pathological changes of SARS-CoV-2-infected lungs in living animals, we examined K18-human angiotensin converting enzyme 2 transgenic (K18-hACE2 Tg) mice by using an in vivo imaging system we previously established in a biosafety level (BSL) 3 facility 10 , 11 . hACE2 is expressed on the epithelial cells of the lungs, kidneys, liver, intestine, and brain of K18-hACE2 mice, and contributes to the establishment of SARS-CoV-2 infection as a receptor for the S protein 1 , 2 . To visualize SARS-CoV-2-infected cells, K18-hACE2 Tg mice were intranasally inoculated with SARS-CoV-2-Venus, which expresses the Venus fluorescent reporter gene in infected cells. SARS-CoV-2-Venus, generated by reverse genetics in the backbone of pBAC SARS2 WK521 (derived from SARS-CoV-2/JPN/TY/WK-521), exhibited lethal infection with an MLD 50 (mouse lethal dose 50; the dose required to kill 50% of infected mice) value of 10 3.83 plaque-forming units (PFU) in K18-hACE2 Tg mice (Fig. 1 A). To analyze the pathobiological effects of SARS-CoV-2, we infected K18-hACE2 Tg mice with 10 5 PFU of SARS-CoV-2-Venus. Fluorescent dextran as well as fluorochrome-conjugated anti-mouse Ly-6G and CD41 antibodies were also intravenously (i.v.) administered to the mice to visualize lung vascular structures, neutrophils, and platelets, respectively. Virus-infected Venus-positive epithelial cells began to be observed in the lungs of SARS-CoV-2–infected mice on day 4 post infection (p.i.), and their number had visibly increased at day 6 p.i. (Fig. 1 B and Movie 1). The morphology of the Venus-positive infected cells suggested that flat type I alveolar epithelial cells surrounding the alveoli and spherical large type II alveolar epithelial cells were infected with SARS-CoV-2-Venus (Fig. 1 C). Platelets and neutrophils flowed smoothly in the pulmonary capillaries of uninfected mice, whereas in the mice infected with SARS-CoV-2-Venus, their movements were slower in the capillaries (Movie 1). On day 6 p.i., an increase in the number of neutrophils was apparent in the pulmonary capillaries of the infected-K18-hACE2 Tg mice (Fig. 1 D). SARS-CoV-2 alters the motility of intravascular neutrophils and impairs blood flow in infected lung. To analyze the functional changes of neutrophils in SARS-CoV-2-infected lungs, we quantitatively assessed the neutrophil movement. In our previous studies, we reported that pulmonary neutrophils in influenza virus-infected mice show two types of motility 10 : "slow movement", which is considered tethering or rolling, in which neutrophils move along the vessel wall at a speed of 50 µm/s or less; and "rapid motion", which is moving in the blood flow at a speed of more than 50 µm/s. We analyzed the amount of time individual neutrophils engaged in each motion in the lungs of K18-hACE2 Tg mice infected with SARS-CoV-2-Venus. Pulmonary neutrophils observed in SARS-CoV-2-infected lungs were tracked (Fig. 2 A left panels) and ordered according to the amount of time they spent in slow motion, and the total time they spent in slow (Fig. 2 A right panels, blue bars) and rapid (Fig. 2 A right panels, red bars) motion was plotted for each neutrophil (Movie 2). In SARS-CoV-2-Venus-infected lung, the amount of time the neutrophils spent in slow motion was significantly increased compared with uninfected lung (Fig. 2 B). We also found that the percentage of neutrophils that engaged in rapid motion decreased (Fig. 2 B). These results indicate that vascular neutrophils in SARS-CoV-2-infected lungs have increased contact time with the pulmonary vessel wall. Thrombus formation in pulmonary vessels has emerged as a typical clinical feature of COVID-19 pneumonia 12 . To assess whether intravascular neutrophils contribute to thrombus formation during SARS-CoV-2 pathogenesis, we performed an in vivo imaging analysis by co-staining platelets and neutrophils. To quantify the size of the platelet aggregates in the infected lungs, we assessed the fluorescent signals of CD41 as a surface marker of platelets 13 from the original image, measured the area of each platelet, and performed a frequency analysis (Fig. 2 C). CD41 signals occupying a small area (< 50 pixels, which is approximately 8.57 µm 2 in our imaging setting) and flowing in the blood vessels were considered to be single platelets. In SARS-CoV-2-infected lungs, an increased percentage of aggregated platelets with a large area of CD41 signal (≥ 50 pixels and 200 pixels) were also observed to block the vessels as emboli. The percentage of aggregated platelets and thrombi was significantly increased in SARS-CoV-2-infected lungs (Fig. 2 C). Interestingly, aggregated platelets frequently interacted with intravascular neutrophils to form microthrombus-like structures (neutrophil-platelet complexes) in the lungs infected with SARS-CoV-2-Venus (Fig. 2 D and Extended Data Fig. 1 ). These observations suggest that neutrophils adhering to pulmonary vessels in SARS-CoV-2-infected lungs participate in the formation of platelet aggregates. The stagnation of blood neutrophils, aggregation of platelets, and formation of platelet-neutrophil complexes observed in SARS-CoV-2-infected lungs may have a significant effect on pulmonary hemodynamics and oxygen exchange mediated by erythrocytes. To test this concept, we transferred fluorescently labeled erythrocytes into SARS-CoV-2-infected K18-hACE2 Tg mice and observed their trajectories for 10 minutes. The time-lapse images were overlapped every 50 frames to visualize the areas where labeled erythrocytes flowed (Fig. 2 F and Movie 3). In naive mice, the blood vessels visualized by use of fluorescent dextran and the trajectories of the labeled erythrocytes almost overlapped, indicating that most pulmonary vessels were functional for oxygen exchange. In contrast, in SARS-CoV-2-infected lungs, there were areas where the vascular structures and the trajectories of the labeled erythrocytes do not overlap, indicating that the availability of functional capillaries for oxygen exchange was reduced. Blood neutrophil stagnation, platelet aggregation, and platelet-neutrophil complexes may disturb the pulmonary microcirculation in the SARS-CoV-2-infected lungs. Our in vivo imaging data revealed that the functional failure of lung capillaries is likely due to microthrombi formation in mice infected with SARS-CoV-2. Development of a mouse model of severe SARS-CoV-2 pneumonia Although K18-hACE2 Tg mice have been widely used to study SARS-CoV-2 pathogenesis 14 , 15 , intranasal inoculation of these mice with SARS-CoV-2 can cause severe neurological disease, including encephalitis, with features that differ from severe COVID-19 cases 16 , 17 . We previously established a mouse-adapted (MA) SARS-CoV-2 and its derivative expressing the Venus protein (named MA-SARS-CoV-2-Venus), which causes severe lung inflammation in C57BL/6J mice similar to severe COVID-19 cases 18 . To establish an animal model that can mimic the symptoms of severe COVID-19 patients, we used seven mouse strains that are well established disease models [i.e., obesity/diabetes models, C57BL/6 HamSlc- ob/ob (Ob/Ob) mice and KK-A y /Ta Jcl mice; a type 1 diabetes model, Streptozotocin (STZ)-induced diabetic C57BL/6J mice; aged models, SAMP8/TaSlc mice and SAMP10-ΔSglt2 mice; a hyperlipidemia model, B6. B6.KOR/StmSlc- Apoe shl mice; and a hepatitis model, B6-nonalcoholic steato-hepatitis (NASH) mice]. These mice were intranasally infected with 10 3 PFU of MA-SARS-CoV-2 and their survival was assessed. MA-SARS-CoV-2-infected Ob/Ob mice showed significantly reduced survival compared to wild-type mice (Fig. 3 A). MA-SARS-CoV-2 exhibited high pathogenicity with an MLD 50 value of 10 2.5 PFU and caused virus dose-dependent weight loss in Ob/Ob mice (Fig. 3 B). MA-SARS-CoV-2 and MA-SARS-CoV-2-Venus showed comparable virulence in Ob/Ob mice (MLD 50 ; 10 2.5 PFU) (Extended Data Fig. 2 ) Infection of Ob/Ob mice with MA-SARS-CoV-2 resulted in high virus titers in the lungs and nasal turbinates at 2 dpi, and no substantial difference in viral titers in the respiratory organs at this timepoint was observed between Ob/Ob mice and their control groups (Fig. 3 C). Viruses were also recovered from the brain and liver of the infected Ob/Ob mice albeit at low titers (note: the brain samples collected for virus titration included the olfactory bulb). In the lungs of infected Ob/Ob mice at 5 dpi, viral replication was significantly higher than that in the control groups, with a titer of more than 1.0 ×10 7 PFU/g. In contrast, no difference in viral titers in the nasal turbinates was observed between Ob/Ob mice and their control groups, and virus was not detected in any other organs tested at 5 dpi. Micro-CT analysis revealed lung abnormalities in MA-SARS-CoV-2-infected Ob/Ob mice, including patchy and ill-defined regions consistent with COVID-19 pneumonia at 4 dpi (Fig. 3 D). We then examined the histopathologic changes in the organs of Ob/Ob mice after MA-SARS-CoV-2 infection. Histopathological analysis revealed infiltration of inflammatory cells such as neutrophils and mononuclear cells into the alveolar regions in the lungs of both Ob/Ob and their control groups at 2 dpi with MA-SARS-CoV-2 (Fig. 3 E). At the same timepoint, viral antigen was immunohistochemically detected mainly on alveolar and bronchiolar epithelial cells in both groups. On day 5 p.i., the inflammation improved in the control group, but persisted in the Ob/Ob mice. In the control group, there was a clear reduction in the number of virus-positive cells. Conversely, there was no notable reduction in the number of virus-positive cells in the Ob/Ob group. No virus-positive cells or significant morphological changes were detected histopathologically in the brain, heart, liver, spleen, kidney, or intestine (data not shown). Consistent with our histopathologic analysis, flow cytometry showed that the number of neutrophils and monocytes infiltrating the lungs was significantly increased in the MA-SARS-CoV-2 infected lungs compared to the control group (Fig. 3 F; see the Materials and Methods section on the definition of cell types). These results indicate that Ob/Ob mice infected with MA-SARS-CoV-2 or MA-SARS-CoV-2-Venus are a useful model that reflects the pathogenesis of severe COVID-19. Neutrophils cause impaired pulmonary perfusion in SARS-CoV-2-infected lungs. To analyze the pathobiological changes in MA-SARS-CoV-2-infected Ob/Ob mice, the mice were intranasally infected with 10 4 PFU of MA-SARS-CoV-2-Venus. Large numbers of infected cells were observed in the lungs of Ob/Ob mice on day 4 p.i. (Fig. 4 A and Movie 4). Similar to the pathophysiological changes in the infected K-18 hACE2 Tg mice, there was a significant increase in the number of neutrophils, platelet aggregates, and thrombi in the pulmonary vasculature of MA-SARS-CoV-2-Venus-infected Ob/Ob mice (Figs. 4 B and C). To investigate the functional changes in the neutrophils, we quantified the motility of the vascular neutrophils of the infected mice. Ob/Ob mice infected with MA-SARS-CoV-2-Venus showed significantly increased slow neutrophil movement in pulmonary vessels compared to control groups, indicating increased adhesion time of neutrophils to the pulmonary vascular wall (Fig. 4 D). Hemodynamics of the lungs of MA-SARS-CoV-2-Venus-infected Ob/Ob mice showed that pulmonary perfusion was impaired compared with that in infected wild-type mice, with limited capillaries available for oxygen exchange (Fig. 4 F and Movie 5). To clarify whether aggregates of neutrophils and platelets contribute to the impaired pulmonary perfusion observed in SARS-CoV-2-infected lungs, we performed a five-color multiple labeling analysis using our in vivo imaging method 11 . Ob/Ob mice were intranasally infected with MA-SARS-CoV-2-Venus and observed at 4 dpi. At this timepoint, fluorescent dextran, two fluorochrome-conjugated antibodies (Ly-6G and CD41), and fluorescently labeled erythrocytes were i.v. administered and then observed by using our imaging system. In the lungs of Ob/Ob mice infected with MA-SARS-CoV-2-Venus, neutrophil and platelet aggregates were seen obstructing the flow of the transferred erythrocytes, suggesting that these aggregates may be responsible for the impaired pulmonary perfusion (Fig. 4 G and Movie 6). In addition, in SARS-CoV-2-infected lungs, neutrophils adhered to the pulmonary vasculature; this was followed by platelet aggregation and stasis of the transferred erythrocytes, suggesting that neutrophil adhesion to the vessel wall is the point of origin for thrombus formation and subsequent impaired pulmonary perfusion. To assess whether neutrophils are involved in the pathogenesis of MA-SARS-CoV-2, we removed the neutrophils in vivo by using antibodies and analyzed the effect on virus susceptibility. Removal of neutrophils from Ob/Ob mice led to prolonged survival after MA-SARS-CoV-2 infection, indicating that neutrophils contribute to the exacerbation of SARS-CoV-2 pneumonia (Fig. 4 H). These results suggest that thrombi formed upon neutrophil stagnation may exacerbate MA-SARS-CoV-2-Venus pneumonia in Ob/Ob mice. Altered expression of adhesion molecules in pulmonary neutrophils in relation to COVID-19 severity Neutrophils infiltrate the site of infection by interacting with vascular endothelial cells via adhesion molecules 19 . We therefore analyzed the expression levels of molecules (i.e., L-selectin, Cd44, E-selectin, Selplg, and Pecam-1) 20 linked to the interaction with vascular endothelial cells during neutrophil infiltration in SARS-CoV-2-infected mice by flow cytometry. Neutrophils in the pulmonary vasculature of Ob/Ob mice infected with MA-SARS-CoV-2 exhibited upregulated expression of L-selectin, Cd44, and E-selectin (Fig. 5 A). Analysis in this mouse model suggested that SARS-CoV-2 increases neutrophil adhesion to the vessel wall and induces platelet aggregation, which result in impaired pulmonary perfusion in the lungs. Finally, to determine whether the increased expression levels of adhesion molecules on the surface of vascular neutrophils in severe infections observed in SARS-CoV-2-infected mice is relevant to the pathogenesis of COVID-19 pneumonia in humans, we analyzed published data obtained with clinical samples from COVID-19 patients for adhesion-related gene expression. Using two published datasets of scRNA-seq analyses 9 , 21 from peripheral blood mononuclear cells (PBMCs) from healthy volunteers, mild, and severe cases of COVID-19, we analyzed 474 adhesion-related gene 22 expression changes and found that the levels of expression of four genes ( CD44 , SELL , ICAM3 , and CD93) in the two datasets frequently differed between the healthy and severe groups (Figs. 5 B and C). In the subset corresponding to neutrophils, the expression levels of the CD44 and SELL (which encodes L-selectin) genes were significantly increased in COVID-19 severe cases compared to healthy individuals (Fig. 5 D). These findings indicate that the expression of adhesion molecules is also increased in the vascular neutrophils in the lungs of severe COVID-19 cases, supporting the relevance of the data obtained in mice. In contrast, the gene expression of ICAM3 and CD93 was significantly decreased in the severe group compared to that in the healthy group. The expression levels of the SELL gene showed a positive correlation with the severity of COVID-19 in both the Wilk et al. 9 and Xu et al. 21 datasets and the CD44 gene expression level also positively correlated with COVID-19 severity in the Xu et al. 21 dataset; the expression level of the CD44 gene was positively correlated when comparisons were made between healthy and nonventilated patients or ventilated patients in the Wilk et al. 9 dataset. In contrast, the expression levels of the ICAM3 and CD93 genes negatively correlated with severity, suggesting that they could be molecular markers for the severity of COVID-19. Discussion In this study, our in vivo imaging system revealed that the number of neutrophils in pulmonary vessels increases and neutrophil motility decreases in SARS-CoV-2-infected lungs. We also found that platelet aggregation is enhanced in infected lungs, with frequent formation of neutrophil-platelet complexes. The number of blood vessels through which erythrocytes flow for oxygen exchange was reduced in the infected mouse lungs, suggesting that this may contribute to SARS-CoV-2 pathogenesis. Moreover, we found that the expression of adhesion molecules is increased in pulmonary neutrophils in COVID-19 patients and mouse models, suggesting the existence of a common mechanism of SARS-CoV-2 virulence in humans and mice. Our in vivo imaging analysis thus revealed a novel mechanism of SARS-CoV-2 pathogenesis that could not be found using conventional histopathological and histochemical analyses, and flow cytometry. Analysis of mouse models of severe or lethal SARS-CoV-2 infection showed that platelet aggregation is induced by neutrophil adhesion to the pulmonary vascular wall, resulting in impaired pulmonary perfusion. A major mechanism of thrombus formation in COVID-19 patients is thought to be the induction of platelet aggregation and thrombus formation by NETs released from neutrophils 23 . Complexes of neutrophils, platelets, and citrullinated histone H3, a marker of NETs, have been found during lung autopsies of COVID-19 patients 24 . Consistent with these findings, an analysis of human scRNA-seq data revealed a difference in the expression of the PADI4 gene (Extended Data Fig. 3 ), which is responsible for NET release 23 , suggesting that NET release was induced in severe COVID-19 cases. However, under our experimental conditions, in vivo imaging analysis of the lungs of K-18 hACE2 Tg mice on day 6 of infection and obese mice on day 4 of infection did not show NET release from neutrophils after intravenous administration of a NET-labeling reagent (data not shown). In addition, intravascular neutrophils in the infected lungs had a distinct cell membrane shape and slow motility and did not exhibit the apoptotic morphology that is associated with NET release. This discrepancy may reflect the multistep nature of thrombogenesis that has been postulated in vivo 25 ; our findings of thrombus formation induced by neutrophil adhesion to the vessel wall may be an earlier phenomenon than the thrombus formation involving NETs. Since NET release by neutrophils is induced in response to PAMPs and DAMPs 25 , it is likely that NETs are released in response to the DAMPs that are released upon tissue damage and induce thrombus formation in the late stages of infection when the infection is more advanced. Our in vivo imaging analysis likely revealed an early stage of thrombus formation that was not available in the autopsy cases of COVID-19. The expression of the adhesion molecules CD44 and L-selectin was upregulated in the pulmonary intravascular neutrophils of the SARS-CoV-2-infected mice, and the expression levels of the genes encoding these factors were also upregulated in pulmonary neutrophils from severe COVID-19 cases. L-selectin is expressed on leukocytes and is involved in intercellular adhesion; it has been widely implicated in immune cell responses, from cell migration to antigen presentation 26 . In neutrophils, L-selectin is expressed on the membrane surface from the early stage of differentiation from progenitor cells and is involved in adhesion to vascular endothelial cells during migration 19 , 26 . The half-life of mature neutrophils is approximately 6–12 h in the bloodstream, and L-selectin expression decreases with neutrophil aging, which is characterized by plasma membrane instability, activation of apoptotic signaling, and other events 27 . Neutrophil infiltration has been observed in the lungs of patients with COVID-19 and increases in the numbers of both mature and immature neutrophils have been reported 12 , 28 . COVID-19 patients have also been reported to have neutropenia, an increase in the number of neutrophils in the blood 25 , and in our mouse model, SARS-CoV-2 infection increased the number of neutrophils in the pulmonary vessels. Recently, Castanheira et al. 29 reported that SARS-CoV-2 infection of a mouse model ubiquitously expressing hACE2 (CAG-AC-70) increased neutrophil numbers in pulmonary vessels. These findings suggest that SARS-CoV-2 infection promotes neutrophil proliferation in the bone marrow and migration to the infected lungs, and that immature neutrophils expressing high levels of L-selectin are recruited in large numbers to infected pulmonary vessels, leading to susceptibility to thrombus formation and impaired pulmonary perfusion. Consistent with this hypothesis, in the present study, we found that the expression levels of ICAM3, one of the membrane molecules induced by apoptotic signaling 30 , are significantly decreased in neutrophils from patients with severe COVID-19compared to healthy controls. CD44 is also an adhesion factor expressed on leukocytes and is involved in the adhesion and infiltration of neutrophils into the hepatic sinusoids 31 . It has been reported that neutrophil adhesion is suppressed in the hepatic sinusoids of CD44 -deficient mice with LPS-induced liver injury 32 . Although the mechanism of CD44 gene expression in neutrophils remains unclear, we do know that CD44 expression is increased in monocytes in response to inflammatory cytokines 33 . In severe cases of COVID-19, inflammatory mediators induced by SARS-CoV-2 infection may also increase CD44 gene expression in neutrophils, contributing to increased adhesion to pulmonary vessels. Our study has several limitations that require further investigation. Changes in the expression levels of membrane molecules in vascular endothelial cells in SARS-CoV-2-infected lungs were not examined in this study, and it is unclear how vascular endothelial cells are involved in neutrophil adhesion to the pulmonary vessel walls. It has been hypothesized that thrombus formation proceeds because of dysfunctional endothelial responses to SARS-CoV-2 infection 34 , 35 . However, at least in our mouse model, neutrophil adhesion to the vessel wall occurred not only in the proximity of the infected vascular endothelial cells, but also away from SARS-CoV-2-infected cells (Fig. 1 B and 4 A). Another limitation is that it is unclear whether the upregulation of the CD44 and SELL genes in the SARS-CoV-2-infected mouse model and in COVID-19 patients occurs in the entire neutrophil populations or in some subpopulations of neutrophils. There have been many reports of neutrophil heterogeneity 21 , 36 , 37 , suggesting that SARS-CoV-2 infection might occur in a subpopulation of neutrophils that express the CD44 or SELL genes at high levels. Finally, the extent to which impaired pulmonary perfusion due to neutrophil adhesion to the pulmonary vascular wall contributes to the lethal pathogenesis of SARS-COV-2 pneumonia is unknown. Although we observed improved survival after SARS-CoV-2 infection in a neutrophil depletion model using antibodies (Fig. 4 H), a direct contribution of intravascular neutrophils to pathogenesis could not be demonstrated because neutrophils infiltrating lung tissue are removed with intravascular neutrophils in this model. Since adhesion molecules are involved in the migration and infiltration of neutrophils in response to SARS-CoV-2 infection, the dysfunction of adhesion molecules by neutralizing antibodies may also interfere with the protective function of neutrophils against SARS-CoV-2 infection, and this has not been tested. Beyond the acute respiratory infection, symptoms such as shortness of breath, cough, arthralgia, myalgia, fatigue, headache, odor/taste disturbance, and palpitations have been reported as COVID-19 sequelae (also called long COVID) even after the infection has resolved 38 . It has been shown that prolonged post-ischemic symptoms are more likely to occur in groups at high risk for severe COVID-19, particularly the elderly and those with a high BMI (obesity) 39 . It has also been suggested that microembolization-induced tissue damage may contribute to COVID-19 sequelae 40 . In the present study, using in vivo imaging of mouse models, we found that the formation of microthrombi composed of neutrophils and platelets impairs pulmonary blood flow and exacerbates COVID-19 pneumonitis. The pathological mechanisms of COVID-19 revealed by this study will lead to the development of more effective treatments for patients with severe COVID-19 and long COVID. Materials and Methods Viruses. We generated SARS-CoV-2-Venus, in which the fluorescent reporter gene Venus was inserted, by using reverse genetics. For construction of SARS-CoV-2-Venus, we replaced the ORF 8 genes of pBAC SARS2 wk521 with the Venus gene by use of recombination and designated the infectious cDNA clone pBAC SARS-CoV-2-Venus. Mouse-adapted (MA)-SARS-CoV-2 and MA-SARS-CoV-2-Venus were also generated by using reverse genetics are previously described 18 . Virus strains were propagated in VeroE6/TMPRSS2 (JCRB 1819) cells 41 . All experiments with SARS-CoV-2-Venus were performed in enhanced biosafety level 3 (BSL3) containment laboratories at the University of Tokyo, which are approved for such use by the Ministry of Agriculture, Forestry, and Fisheries, Japan. Cells. VeroE6/TMPRSS2 (JCRB 1819) cells 41 were propagated in 1 mg/ml geneticin (G418; Invitrogen) and 5 µg/ml plasmocin prophylactic (Invitrogen) in Dulbecco's modified Eagle's medium (DMEM) containing 10% Fetal Calf Serum (FCS). The cells were regularly tested for mycoplasma contamination by using PCR, and confirmed to be mycoplasma-free. Mice. Eight-week-old hemizygous K18-hACE2 C57BL/6J mice (strain 2B6.Cg-Tg(K18-ACE2)2Prlmn/J), 24-week-old C57BL/6 HamSlc- ob/ob mice, and five-week-old Streptozotocin (STZ)-induced diabetic C57BL/6J mice were purchased from the Jackson Laboratory Japan. In addition, 24-week-old B6-NASH mice, B6.KOR/StmSlc- Apoe shl mice, SAMR1/TaSlc [Senescence-Accelerated Mouse (SAM); senescence-Resistant inbred strains (R)] mice, SAMP8/TaSlc [Senescence-Accelerated Mouse (SAM); senescence-Prone inbred strains (P)] mice, and SAMP10-ΔSglt2 mice were purchased from Japan SLC Inc. KK-A y /Ta Jcl mice (24-week-old) were purchased from CLEA Japan Inc. Age- and sex-matched C57BL/6 mice, which served as controls, were purchased from the same vendor as each disease model mouse strain. Experimental infection of mice. Mice were intranasally inoculated with 10 1 –10 5 PFU of SARS-CoV-2-Venus, MA-SARS-CoV-2, or MA-SARS-CoV-2-Venus. Body weights were measured before infection and then daily. The protocols for the animal studies were approved by the University of Tokyo (approval numbers PA19-72 and PA21-07). Pathological examination. Excised lung tissues were fixed in 4% paraformaldehyde phosphate buffer solution, and processed for paraffin embedding. The paraffin blocks were cut into 3-µm-thick sections and then the sections were stained using a standard hematoxylin and eosin procedure. In addition, tissue sections were stained with a rabbit polyclonal antibody for SARS-CoV nucleocapsid protein (ProSpec; ANT-180, 1:500 dilution, Rehovot) for immunohistochemical analyses. Specific antigen-antibody reactions were visualized by means of 3,3’-diaminobenzidine tetrahydrochloride staining using the Dako Envision system (Dako Cytomation; K4001, 1:1 dilution). Virus titration assay. C57BL/6 mice and HamSlc- ob/ob mice were intranasally inoculated with 10 3 PFU of MA-SARS-CoV-2. Two and five days post-infection (dpi), the animals were euthanized and their organs (lungs, nasal turbinate, brain, heart, liver, spleen, kidneys, and intestine) were collected. Confluent VeroE6/TMPRSS2 cells in 12-well plates were infected with 100 µl of a dilution of the organ homogenate. The virus inoculum was removed after incubation for 1 h at 37°C, and then 1% agarose solution in DMEM was overlaid on the cells. After incubation for 48 h, the agar-covered cells were fixed with 10% neutral buffered formalin. The plaques were counted after removal of the agar. Micro-CT imaging. C57BL/6 mice and HamSlc- ob/ob mice were inoculated intranasally with 10 3 PFU of MA-SARS-CoV-2. Lungs of infected mice were imaged by using an in vivo micro-CT scanner (CosmoScan FX; Rigaku). Under ketamine-xylazine anesthesia, the animals were placed in the image chamber and scanned for 2 min at 90 kV, 88 µA, FOV 45 mm, and pixel size 90.0 µm. After scanning, the lung images were reconstructed by using the CosmoScan Database software of the micro-CT (Rigaku Corporation) and analyzed using the manufacturer-supplied software as described previously 42 . In vivo imaging of mouse lung. The in vivo imaging was performed by using an LSM 980 NLO (Carl Zeiss) equipped with an infrared laser (Chameleon Vision II; Coherent) as described previously 10 , 11 . K18-hACE2 mice, C57BL/6J mice, and C57BL/6 HamSlc- ob/ob mice were infected with 10 5 PFU of SARS-CoV-2-Venus or 10 4 PFU of MA-SARS-CoV-2-Venus. The infected mice were intubated under anesthesia and ventilated at a respiratory rate of 120 breaths per minute. Isoflurane was continuously delivered at 2% to maintain anesthesia. The left lung lobe of the mice was exposed and gently immobilized with a custom-made thoracic suction window. In all experiments, Texas red dextran (70,000 Da; Invitorogen), Phycoerythrin (PE)-conjugated rat anti-mouse CD41 antibody (MWReg30; BD Biosciences), and Alexa Fluor 594-conjugated rat anti-mouse Ly-6G antibody (1A8; Biolegend) were injected i.v. before imaging to visualize the lung vascular structures, platelets, and vascular neutrophils, respectively. For the analyses of pulmonary perfusion, mice infected with SARS-CoV-2-Venus or MA-SARS-CoV-2-Venus were i.v. inoculated with Dio-labeled erythrocytes. A maximal intensity projection of the indicated frames (0–10 min) was generated to show the functional capillary perfused by the erythrocytes, as described previously 43 . To acquire images in spectral imaging mode, lasers at wavelengths of 488 nm, 543 nm, and 910 nm were used for simultaneous excitation of fluorochromes and Venus. All emitted light between 490- and 695-nm wavelengths was detected by using a 20× water-immersion lens (Carl Zeiss). Spectral separation of the acquired lambda stacks was achieved by using the linear unmixing function of the LSM software ZEN blue (Carl Zeiss). Processing, assays, and data visualization were performed using CellProfiler (Broad Institute), Imaris (Carl Zeiss), and in-house MATLAB scripts (MathWorks). Tracking of the neutrophils in the denoised movies was performed by TrackMate (ImageJ; NIH). Flow cytometry. Mouse lungs were dissociated using a Lung Dissociation Kit (Miltenyi) and gentleMACS Dissociator (Miltenyi) according to the manufacturer's instructions for flow cytometry (FCM). Samples were then filtered through a 70-µm filter (Miltenyi) after red blood cell lysis and resuspended for subsequent FCM staining. For experiments staining intravascular neutrophils, mice were injected i.v. with PE–conjugated rat anti-mouse Ly-6G antibody (1A8; Biolegend) 5 min before lung collection. For surface staining, cells were stained for 10 min with antibodies in PBS containing 0.5% BSA and 2 mM EDTA. The following antibody clones were used in this studies: Vio-Green-CD45 (REA737, Miltenyi), PE-NK1.1 (REA1162, Miltenyi), PE-Vio615-CD4 (REA604, Miltenyi), PE-Vio770-B220 (REA755, Miltenyi), APC-CD3 (REA641, Miltenyi), APC-Vio770-CD8a (REA601, Miltenyi), Vio-Blue-MHC class II (REA813, Miltenyi), FITC-Ly6C (REA796, Miltenyi), PE-Vio615-Ly-6G (REA526, Miltenyi), PE-Vio770-CD11c (REA754, Miltenyi), APC-Siglec-F (REA798, Miltenyi), APC-Vio770-CD11b (REA592, Miltenyi), APC-CD44 (IM7 Biolegend), APC- Pecam1 (W18222B, Biolegend), APC-CD62L (MEL-14, Proteintech), APC-CD62E (P2H3, Invitorogen), and APC-CD162 (4RA10, Elabscience). APC conjugation to the anti-CD62E mouse IgG1 antibody was performed using the APC Labeling Kit-NH2 (Wako) according to the manufacturer's protocol. Populations of immune cells were defined as follows: B cells (CD45 + CD3 − B220 + ), NK cells (CD45 + CD3 − B220 − NK1.1 + ), CD4 T cells (CD45 + CD3 + B220 − CD4 + CD8 − ), CD8 T cells (CD45 + CD3 + B220 − CD4 − CD8 + ), alveolar macrophages (CD45 + CD11b dim Siglec-F + CD11c + MHC class II + ), dendritic cells (CD45 + CD11b − Siglec-F − CD11c + ), neutrophils (CD45 + CD11b high Ly-6G + ), eosinophils (CD45 + CD11b high Siglec-F + Ly-6G − CD11c − ), and monocytes (CD45 + CD11b high Siglec-F − Ly-6G − MHC class II − Ly-6C high ). Samples were analyzed on a flow cytometer (MACSQuant Tyto, Miltenyi). Neutrophil motility analysis. To track the movement of neutrophils, Alexa Fluor 594-conjugated rat anti-mouse Ly-6G antibody was injected i.v. into the mice. Neutrophils were imaged at approximately 4 fps for 230 s. All movies were corrected for respiratory motion artifacts and denoised as described previously 10 . Single object tracking was performed by using TrackMate (ImageJ; NIH) to obtain the trajectories of individual neutrophils. For each neutrophil, speeds were measured for individual steps in its trajectory and subsequently defined as slow (≤ 50 µm/s) or rapid (> 50 µm/s) as described previously 10 . We then examined whether a neutrophil performed rapid movement, and calculated the durations it engaged in continuous slow movements without being interrupted by the rapid movement. Quantification of platelet aggregates. CD41 signals were detected in a semi-automated manner by using CellProfiler (Broad Institute), and then divided into three populations according to their sizes: signals covering < 8.57 µm 2 (50 pixels) were defined as a “single platelet”, signals ≥ 8.57 µm 2 and < 34.28 µm 2 (200 pixels) as “aggregated platelets”, and signals ≥ 34.28 µm 2 as “thrombocytes”. The frequency analysis of the CD41 signals was conducted using in-house MATLAB scripts (MathWorks). In vivo depletion of neutrophils. C57BL/6 HamSlc- ob/ob were administered 100 µg of anti-rat Kappa immunoglobulin (clone MAR 18.5, #BE0122) daily for two days prior to infection as described previously 44 . In addition, 50 µg of Anti-Ly6G (clone 1A8, #BP0075-1) and corresponding isotype control (#BP0089) were administered every other day from one day prior to infection. When mice were sequentially injected with two antibodies, an interval of more than 2 hours was set between injections. scRNA-seq data re-analysis. We reanalyzed two published PBMC scRNA-seq datasets for healthy and SARS-CoV-2-infected humans 9 , 21 . For the published data from Wilk et al. 9 , pre-processed scRNA-seq count data with embedding, clustering, and cell type assignment from the previous study 9 were obtained as an RDS file from the COVID-19 Cell Atlas ( https://www.covid19cellatlas.org/#wilk20 ) hosted by the Wellcome Sanger Institute. For the published data from Xu et al. 21 , we downloaded transcript-by-cell matrices output by Cell Ranger from NCBI (GSE216020) and preprocessed using the Seurat 45 package and DoubletFinder 46 , which identifies and removes potential doublets, as described in a previous study 21 . Cells with less than 500 UMI counts, 200 detected genes, and more than 20% mitochondrial gene counts were removed as low-quality cells, as well as potential doublets. Data integration of all samples was performed using the FindIntegrationAnchors and IntegrateData functions in Seurat with the top 3000 most variable genes selected by FindVariableFeatures function. Cell-type annotation was based on marker genes of each cluster defined by FindAllMarkers functions. The subset corresponding to neutrophils in both datasets (Wilk et al. and Xu et al.) was used for differential gene expression analysis related to cell adhesion 22 and neutrophil extracellular trap formation (KEGG: hsa04613), respectively. Statistical analysis. GraphPad Prism was used to analyze all data. Student’s t test, log-rank (Mantel-Cox) tests, and an ANOVA with a multiple corrections post-test were performed, and differences were considered to be statistically significant when the p -value was less than 0.05. For the differential gene expression analysis of the scRNA-seq data, the Wilcoxon signed rank test was used and the p -values were corrected by using the Benjamini-Hochberg Procedure. Declarations Acknowledgements We thank S. Watson for editing the manuscript. We also thank Yuko Sato and Seiya Ozono for their technical assistance. This research was supported by a Research Program on Emerging and Re-emerging Infectious Diseases from the Japan Agency for Medical Research and Development (AMED) (JP19fk0108113, JP20fk0108412, JP21fk0108552, JP233fa627001) by a Japan Program for Infectious Diseases Research and Infrastructure from AMED (JP23wm0125002), the Japan Society for the Promotion of Science (JSPS) (21K14984), the Japan Science and Technology Agency (JST) (Moonshot R&D) (JPMJMS2025), and by the NIAID-funded Center for Research on Influenza Pathogenesis (CRIP; HHSN272201400008C). H.U. was supported by GSK Japan Research Grant 2020, the Astellas Foundation for Research on Metabolic Disorders, the Naito Foundation, the Sumitomo Foundation, the Ichiro Kanehara Foundation, the Uehara Memorial Foundation, the Okinaka Memorial Institute for Medical Research, a Japanese Respiratory Foundation Grant, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, the SENSHIN Medical Research Foundation, and the Takeda Science Foundation. IH.W., HW. H., and CH.W. were supported by the National Science and Technology Council, Taiwan (MOST-110-2320-B-001-005-MY3). Author Contributions H.U., M.K., S.I., S.M., and T.S. performed the mouse infection experiments, titrated virus in organs, and analyzed pathology. M.U. analyzed the micro-CT images. H.U. performed the in vivo imaging analysis and flow cytometry. H.U., IH.W., HW. H., and CH.W. performed image data analyses. W.K. generated the Venus-expressing SARS-CoV-2. 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Cell Systems 8 , 329-337.e324 (2019). https://doi.org:https://doi.org/10.1016/j.cels.2019.03.003 Additional Declarations Yes there is potential Competing Interest. Yoshihiro Kawaoka has ongoing unrelated collaborations and/or sponsored research agreements with Daiichi Sankyo Pharmaceutical, Toyama Chemical, Tauns Laboratories, Inc., Shionogi & Co. Ltd, Otsuka Pharmaceutical, and KM Biologics and has received royalties from MedImmune and Integrated Biotherapeutics. Supplementary Files Movie1.mp4 Movie 1 In vivo imaging of K18-hACE2 C57BL/6J mice infected with SARS-CoV-2-Venus. K18-hACE2 C57BL/6J mice were infected with 10 5 PFU of SARS-CoV-2-Venus and observed by using our in vivo imaging system. Green indicates virus-infected cells. At the indicated timepoints, fluorescent dextran (white), fluorochrome-conjugated anti-mouse Ly-6G antibody (red), and anti-mouse CD41 (Cyan) were i.v. administered to visualize the lung architecture, neutrophils, and platelets, respectively. Movie2.mp4 Movie 2 Trafficking analyses of pulmonary neutrophils in SARS-CoV-2-infected mouse lung. K18-hACE2 C57BL/6J mice were infected with 10 5 PFU of SARS-CoV-2-Venus and observed by using our in vivo imaging system. At the indicated timepoint, fluorescent dextran (blue) and fluorochrome-conjugated anti-mouse Ly-6G antibody (red) were i.v. administered to visualize the lung architecture and neutrophils, respectively. Trajectories of individual neutrophils are displayed in white. Movie3.mp4 Movie 3 In vivo imaging of pulmonary blood perfusion in SARS-CoV-2-Venus-infected mice. K18-hACE2 C57BL/6J mice were infected with 10 5 PFU of SARS-CoV-2-Venus and observed by using our in vivo imaging system. To visualize blood vessels through which erythrocytes passed, SARS-CoV-2-Venus-infected K18-hACE2 C57BL/6J mice were i.v. inoculated with fluorescently labeled erythrocytes (red) at 6 dpi. The mice were also i.v. inoculated with fluorescent dextran (blue) to visualize the lung architecture. A maximal intensity projection of the indicated frames (0–10 min) was generated to show the functional capillaries perfused by the erythrocytes. White circles indicate vessels through which the transferred erythrocytes did not pass during the observation period in the lungs of the SARS-CoV-2-Venus-infected K18-hACE2 C57BL/6J mice. Movie4.mp4 Movie 4 In vivo imaging of Ob/Ob mice infected with MA-SARS-CoV-2-Venus. Ob/Ob mice were infected with 10 4 PFU of MA-SARS-CoV-2-Venus and observed by using our in vivo imaging system. Green indicates virus-infected cells. At the indicated timepoints, fluorescent dextran (white), fluorochrome-conjugated anti-mouse Ly-6G antibody (red), and anti-mouse CD41 (Cyan) were i.v. administered to visualize the lung architecture, neutrophils, and platelets, respectively. Movie5.mp4 Movie 5 In vivo imaging of pulmonary blood perfusion in MA-SARS-CoV-2-Venus-infected mice. Ob/Ob mice were infected with 10 4 PFU of MA-SARS-CoV-2-Venus and observed by using our in vivo imaging system. To visualize blood vessels through which erythrocytes passed, MA-SARS-CoV-2-Venus-infected Ob/Ob mice were i.v. inoculated with fluorescently labeled erythrocytes (red) at 4 dpi. The mice were also i.v. inoculated with fluorescent dextran (blue) to visualize the lung architecture. A maximal intensity projection of the indicated frames (0–10 min) was generated to show the functional capillaries perfused by the erythrocytes. White circles indicate vessels through which the transferred erythrocytes did not pass during the observation period in the lungs of the MA-SARS-CoV-2-Venus-infected Ob/Ob mice. Movie6.mp4 Movie 6 Representative movie of impaired pulmonary microcirculation caused by neutrophil-platelet complexes in MA-SARS-CoV-2-Venus-infected Ob/Ob mice. Ob/Ob mice were intranasally infected with MA-SARS-CoV-2-Venus and observed at 4 dpi. At this timepoint, fluorescent dextran (white), fluorochrome-conjugated anti-mouse Ly-6G antibody (fuschia), and anti-mouse CD41 (Cyan) were i.v. administered to visualize the lung architecture, neutrophils, and platelets, respectively. The mice were also i.v. inoculated with fluorescently labeled erythrocytes (red). White arrows indicate a neutrophil adhering to a pulmonary vessel wall. Yellow arrowheads indicate platelet aggregation, and white arrowheads indicate erythrocytes blocked from passing through the vessels. SARSCoV2ExtendedDataFigure1.tif Extended Data Figure 1 Images of platelet-neutrophil complexes in pulmonary capillaries of SARS-CoV-2-infectedK18-hACE2 C57BL/6J mice. K18-hACE2 C57BL/6J mice were intranasally infected with SARS-CoV-2-Venus and observed at 6 dpi. At this timepoint, fluorescent dextran (white), fluorochrome-conjugated anti-mouse Ly-6G antibody (red), and anti-mouse CD41 (Cyan) were i.v. administered to visualize the lung architecture, neutrophils, and platelets, respectively. Arrows indicate platelet aggregates. Scale bars, 50 µm. SARSCoV2ExtendedDataFigure2.tif Extended Data Figure 2 Survival and body weight changes in MA-SARS-CoV-2-Venus-infected mice. Five mice per group of Ob/Ob mice were infected with 10 2 to 10 5 PFU of MA-SARS-CoV-2-Venus, and survival and body weight were monitored daily for 10 days. The results are expressed as the mean ± SD. SARSCoV2ExtendedDataFigure3.tif Extended Data Figure 3 Violin plots of PADI4 and GSDMD gene expression in PBMC neutrophils. NonVent (non-ventilated) and Vent (ventilated) indicate mild and severe cases of COVID-19, respectively href="#_ENREF_9" title="Wilk, 2020 #1039"> 9 . The asterisks indicate p < 0.05. 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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-3895679","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":270311178,"identity":"7ed670fe-5469-4828-b166-e465af88b9eb","order_by":0,"name":"Yoshihiro Kawaoka","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIie3QsUrDQBjA8U+Ey5Im64WA8REuBKJiHiaHEJeUvkDQg0K6OZvFZ8joeOUgWc49pUtdOjkogrSIYlpTsJCDjg73X/LBlx+5C4BO9z9DQBnePjcRMBjw7cgPJiY/gBwx+ENw3C0UhNST5eL58XxkA6LvZhYFdvHiT1cZnFhN3E+kPCNU4ouCIeGaVRLieUqEWUHgqEiTIkxzTAg3mJsiEcF8WIr2qLRUkuvljow/028RebOncrpmcKsmcdgRVLnDXISkGZR8wCAmCuJI+UucMUouv+6SwJej1/Yu2C/kopdY7R9z1vkNsY08mN1/RP5DXV29rbLIs+r+r5zy3XS8v8C9r2/ymHKl0+l0uq4f22tlbJE0q2MAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-5061-8296","institution":"University of Wisconsin-Madison","correspondingAuthor":true,"prefix":"","firstName":"Yoshihiro","middleName":"","lastName":"Kawaoka","suffix":""},{"id":270311180,"identity":"bedf3cc4-47bd-4adf-a330-25ba1700d2c5","order_by":1,"name":"Hiroshi Ueki","email":"","orcid":"https://orcid.org/0000-0002-6557-0771","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Ueki","suffix":""},{"id":270311181,"identity":"2d482824-db40-42f1-a16c-62194c379a7e","order_by":2,"name":"I-Hsuan Wang","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"I-Hsuan","middleName":"","lastName":"Wang","suffix":""},{"id":270311183,"identity":"7b215f73-a149-4fec-aa81-77d0c5c13823","order_by":3,"name":"Maki Kiso","email":"","orcid":"","institution":"Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Maki","middleName":"","lastName":"Kiso","suffix":""},{"id":270311185,"identity":"b192d994-f206-401c-aca2-f9d9953c9f5d","order_by":4,"name":"Kenta Horie","email":"","orcid":"https://orcid.org/0000-0003-4175-1014","institution":"Chiba University","correspondingAuthor":false,"prefix":"","firstName":"Kenta","middleName":"","lastName":"Horie","suffix":""},{"id":270311186,"identity":"19b174fd-7071-4965-a276-a55a74dad13b","order_by":5,"name":"Shun Iida","email":"","orcid":"https://orcid.org/0000-0003-2258-9031","institution":"National Institute of Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Shun","middleName":"","lastName":"Iida","suffix":""},{"id":270311188,"identity":"145f0888-d39f-430b-a341-7a4f0a482c32","order_by":6,"name":"Sohtaro Mine","email":"","orcid":"","institution":"National Institute of Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Sohtaro","middleName":"","lastName":"Mine","suffix":""},{"id":270311190,"identity":"1fa2f4ad-2b37-4c78-b69c-c930ed5b5c5d","order_by":7,"name":"Michiko Ujie","email":"","orcid":"","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Michiko","middleName":"","lastName":"Ujie","suffix":""},{"id":270311192,"identity":"a4693e76-cc72-4660-ad3d-7619fa3c1a8a","order_by":8,"name":"Hung-Wei Hsu","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Hung-Wei","middleName":"","lastName":"Hsu","suffix":""},{"id":270311194,"identity":"ea2ce275-67c9-4a5a-9055-8d21ea5f7b4c","order_by":9,"name":"Chen-Hui Henry","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Chen-Hui","middleName":"","lastName":"Henry","suffix":""},{"id":270311195,"identity":"2f9df287-2920-4da2-b48e-311cb972ee57","order_by":10,"name":"Masaki Imai","email":"","orcid":"https://orcid.org/0000-0001-6988-1975","institution":"University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Masaki","middleName":"","lastName":"Imai","suffix":""},{"id":270311196,"identity":"4d5fe0bf-4915-41ec-954d-61af72606ccc","order_by":11,"name":"Tadaki Suzuki","email":"","orcid":"https://orcid.org/0000-0002-3820-9542","institution":"National Institute of Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Tadaki","middleName":"","lastName":"Suzuki","suffix":""},{"id":270311197,"identity":"1b243c2c-91e7-4895-bd91-132c4c335549","order_by":12,"name":"Wataru Kamitani","email":"","orcid":"","institution":"Gunma University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wataru","middleName":"","lastName":"Kamitani","suffix":""},{"id":270311198,"identity":"8410dc0e-2ef2-465c-978c-ae1eb5b31408","order_by":13,"name":"Eiryo Kawakami","email":"","orcid":"","institution":"Chiba University","correspondingAuthor":false,"prefix":"","firstName":"Eiryo","middleName":"","lastName":"Kawakami","suffix":""}],"badges":[],"createdAt":"2024-01-25 02:10:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3895679/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3895679/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-55272-0","type":"published","date":"2025-01-13T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50816896,"identity":"0a891e4f-dfb5-418c-b61c-a4da28960051","added_by":"auto","created_at":"2024-02-07 19:50:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1282381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e imaging of SARS-CoV-2-infected K18-hACE2 C57BL/6J mice.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Survival and body weight changes of SARS-CoV-2-Venus-infected mice. Six K18-hACE2 C57BL/6J mice per group were infected with 10\u003csup\u003e1\u003c/sup\u003e to 10\u003csup\u003e5\u003c/sup\u003e PFU of SARS-CoV-2-Venus, and survival and body weight were monitored daily for 10 days. The results are expressed as the mean ± SD. (\u003cstrong\u003eB\u003c/strong\u003e) Pathophysiological changes in SARS-CoV-2-infected lungs. K18-hACE2 C57BL/6J mice were intranasally infected with SARS-CoV-2-Venus and observed at the indicated timepoints. (\u003cstrong\u003eC\u003c/strong\u003e) \u003cem\u003eIn vivo\u003c/em\u003e imaging of SARS-CoV-2-infected cells on 6-day post-infection (dpi). (i) and (ii) are enlarged images of the leftmost image. (\u003cstrong\u003eD\u003c/strong\u003e) The number of intravascular neutrophils in SARS-CoV-2-infected lungs. On 6 dpi, SARS-CoV-2-Venus-infected K18-hACE2 C57BL/6J mice were observed, and neutrophils in the pulmonary capillaries in the microscopic field of view were counted. n = 20. (\u003cstrong\u003eB\u003c/strong\u003e, \u003cstrong\u003eC\u003c/strong\u003e, and \u003cstrong\u003eD\u003c/strong\u003e) Green indicates virus-infected cells. At the indicated timepoints, fluorescent dextran (white), fluorochrome-conjugated anti-mouse Ly-6G antibody (red), and anti-mouse CD41 (Cyan) were i.v. administered to visualize lung architecture, neutrophils, and platelets, respectively. The asterisk indicates \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3895679/v1/649fbc761ec05ebf607b06f3.jpg"},{"id":50817032,"identity":"5d8f41b0-a7a8-41aa-8428-5dc199124683","added_by":"auto","created_at":"2024-02-07 19:58:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1207256,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of pathophysiological changes in SARS-CoV-2-infected lungs.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Motility of pulmonary intravascular neutrophils in response to SARS-CoV-2 infection. To track neutrophil movement, a fluorochrome-conjugated anti-mouse Ly-6G antibody was injected i.v. into SARS-CoV-2-infected K18-hACE2 C57BL/6J mice. Tracking analyses were performed to obtain the trajectories of individual neutrophils (left panel, vascular structures are shown in white, and individual trajectories are coded in different colors). For each neutrophil, speeds were measured for individual steps in its trajectory and subsequently defined as slow (≤50 µm/s) or rapid (\u0026gt;50 µm/s). All neutrophils observed under the same conditions were arranged along the y-axis according to the duration of their slow motion. The amount of time each neutrophil was engaged in slow (blue) or rapid (red) motion was plotted along the x-axis (right panel). n = 6. (\u003cstrong\u003eB\u003c/strong\u003e) Assessment of neutrophil behaviors defined in terms of slow and rapid motion. The average time for which neutrophils engaged in slow movement (left panel) and the average percentage of neutrophils with rapid movement (right panel) in SARS-CoV-2-Venus-infected lung. n = 6. (\u003cstrong\u003eC\u003c/strong\u003e) Quantitative assessment of platelet aggregates in SARS-CoV-2-infected lungs. Histograms were plotted based on the size of the detected platelet (CD41-positive objects) signals at the indicated timepoints (leftmost panel). The population of platelets was divided into three groups according to their sizes: signals covering \u0026lt; 50 pixels (approximately 8.57 μm\u003csup\u003e2\u003c/sup\u003e) were defined as a “single platelet”, signals ≥ 50 pixels \u003csup\u003e\u0026nbsp;\u003c/sup\u003eand\u003csup\u003e \u003c/sup\u003e\u0026lt; 200 pixels (approximately 34.28 μm\u003csup\u003e2\u003c/sup\u003e) as “aggregated platelets”, and signals ≥ 200 pixels as “thrombocytes”. Then, the mean of each population was plotted in a violin plot. n = 4–8. \u0026nbsp;(\u003cstrong\u003eD\u003c/strong\u003e) Representative images of platelet aggregates and platelet-neutrophil complexes in pulmonary capillaries of SARS-CoV-2-infected K18-hACE2 C57BL/6J mice. K18-hACE2 C57BL/6J mice were intranasally infected with SARS-CoV-2-Venus and observed at 6 dpi. At this timepoint, fluorescent dextran (white), fluorochrome-conjugated anti-mouse Ly-6G antibody (red), and anti-mouse CD41 (Cyan) were i.v. administered to visualize the lung architecture, neutrophils, and platelets, respectively. Arrows indicate platelet aggregates. (\u003cstrong\u003eF\u003c/strong\u003e) Pulmonary blood perfusion in SARS-CoV-2-Venus-infected mice. To visualize blood vessels through which erythrocytes passed, SARS-CoV-2-Venus-infected K18-hACE2 C57BL/6J mice were i.v. inoculated with fluorescently labeled erythrocytes (red) at the indicated timepoint. The mice were also i.v. inoculated with fluorescent dextran (blue) to visualize the lung architecture. A maximal intensity projection of the indicated frames (0–10 min) was generated to show the functional capillaries perfused by the erythrocytes\u003ca href=\"#_ENREF_25\" title=\"Gorog, 2022 #1044\"\u003e\u003csup\u003e25\u003c/sup\u003e\u003c/a\u003e. White circles indicate vessels in which the transferred erythrocytes did not pass during the observation period in the lungs of the SARS-CoV-2-Venus-infected K18-hACE2 C57BL/6J mice. The asterisks indicate \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3895679/v1/bd83fa5d8ec963fc7949e6df.jpg"},{"id":50816894,"identity":"1a3927fa-b7db-431a-aef0-b664c74215df","added_by":"auto","created_at":"2024-02-07 19:50:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2241194,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of Ob/Ob mice infected with MA-SARS-CoV-2. (\u003cstrong\u003eA\u003c/strong\u003e) Virulence of MA-SARS-CoV-2 in mouse models of underlying diseases. Five mice per group of Ob/Ob mice, Streptozotocin (STZ)-induced diabetic C57BL/6J mice, KK-A\u003csup\u003ey\u003c/sup\u003e/Ta Jcl mice, SAMP8/TaSlc mice, SAMP10-ΔSglt2 mice, B6.KOR/StmSlc-\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003eshl\u003c/em\u003e\u003c/sup\u003e mice, and B6-NASH mice were infected with 10\u003csup\u003e3\u003c/sup\u003e PFU of MA-SARS-CoV-2, and survival was monitored daily for the indicated timepoints. Age- and sex-matched C57BL/6 mice served as controls. SAMR1/TaSlc mice were used as controls for SAMP8/TaSlc mice and SAMP10-ΔSglt2 mice. (\u003cstrong\u003eB\u003c/strong\u003e) Survival and body weight changes. Five mice per group of Ob/Ob mice were infected with 10\u003csup\u003e1\u003c/sup\u003e to 10\u003csup\u003e4\u003c/sup\u003e PFU of MA-SARS-CoV-2, and survival and body weight were monitored daily for 10 days. The results are expressed as the mean ± SD. (\u003cstrong\u003eC\u003c/strong\u003e) Virus titers in organs of MA-SARS-CoV-2-infected mice. Four mice per group were euthanized and virus titers in the lungs, nasal turbinate, brain, heart, liver, spleen, kidney, and intestine were determined by using plaque assays in VeroE6/TMPRSS2 cells. Dashed lines in the panels indicate the detection limit of the assay for each organ. (\u003cstrong\u003eD\u003c/strong\u003e) Micro-CT imaging of the lungs of infected mice. Axial CT images of the thorax in MA-SARS-CoV-2-infected C57BL/6 mice and Ob/Ob mice. On day 4 post-infection, the MA-SARS-CoV-2-infected Ob/Ob mice had a higher degree of lung abnormalities than the infected C57BL/6 mice, highlighted by the red circle. (\u003cstrong\u003eE\u003c/strong\u003e) Pathological features of MA-SARS-CoV-2-infected Ob/Ob mice. Representative images of lungs are shown. Left and middle columns show hematoxylin and eosin staining. Right column shows immunohistochemistry using a rabbit polyclonal antibody that detects SARS-CoV-2 nucleocapsid protein. Scale bars, 500 µm in the left column; 100 µm in the middle and right columns. (\u003cstrong\u003eF\u003c/strong\u003e) Population of immune cells in the lungs of MA-SARS-CoV-2-infected mice. Lung immune cell numbers were determined in whole lung at 4 dpi in C57BL/6 mice and Ob/Ob mice by flow cytometry (n = 5–6). The asterisks indicate \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3895679/v1/575b44a3c8fa483fdd2c4ac5.jpg"},{"id":50816895,"identity":"8de35950-1774-4569-804c-6e71ed774c48","added_by":"auto","created_at":"2024-02-07 19:50:30","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2185564,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e imaging of SARS-CoV-2-infected Ob/Ob mice. (\u003cstrong\u003eA\u003c/strong\u003e) Pathophysiological changes in SARS-CoV-2-infected Ob/Ob mouse lungs. Ob/Ob mice were intranasally infected with 10\u003csup\u003e4\u003c/sup\u003e PFU of MA-SARS-CoV-2-Venus and observed at 4 days post-infection (dpi). Green indicates virus-infected cells. Fluorescent dextran (white), fluorochrome-conjugated anti-mouse Ly-6G antibody (red), and anti-mouse CD41 (Cyan) were i.v. administered to visualize lung architecture, neutrophils, and platelets, respectively. (\u003cstrong\u003eB\u003c/strong\u003e) Infiltrating and vascular neutrophils in MA-SARS-CoV-2-infected lungs. Ob/Ob mice were infected intranasally with 10\u003csup\u003e3\u003c/sup\u003e PFU of MA-SARS-CoV-2 and infected lungs were analyzed by flow cytometry at 4 dpi (n = 4). (\u003cstrong\u003eC\u003c/strong\u003e) Quantitative assessment of platelet aggregates in MA-SARS-CoV-2-Venus-infected lungs. Histograms were plotted based on the size of the detected platelet (CD41-positive objects) signals at the indicated timepoints (leftmost panel). The population of platelets was divided into three groups according to their sizes: signals covering \u0026lt; 50 pixels (approximately 8.57 μm\u003csup\u003e2\u003c/sup\u003e) were defined as a “single platelet”, signals ≥ 50 pixels and\u003csup\u003e \u003c/sup\u003e\u0026lt; 200 pixels (approximately 34.28 μm\u003csup\u003e2\u003c/sup\u003e) as “aggregated platelets”, and signals ≥ 200 pixels as “thrombocytes”. Then, the mean of each population was plotted in a violin plot. n = 10–22. (\u003cstrong\u003eD\u003c/strong\u003e) Motility of pulmonary intravascular neutrophils in MA-SARS-CoV-2-infected mice. To track neutrophil movement, a fluorochrome-conjugated anti-mouse Ly-6G antibody was injected i.v. into the MA-SARS-CoV-2-Venus-infected Ob/Ob mice. Tracking analyses were performed to obtain the trajectories of individual neutrophils (left panel, vascular structures are shown in white, and individual trajectories are coded in different colors). For each neutrophil, speeds were measured for individual steps in its trajectory and subsequently defined as slow (≤50 µm/s) or rapid (\u0026gt;50 µm/s). All neutrophils observed under the same conditions were arranged along the y-axis according to the duration of their slow motion. The amount of time each neutrophil was engaged in slow (blue) or rapid (red) motion was plotted along the x-axis (right panel). n = 5–6. (\u003cstrong\u003eE\u003c/strong\u003e) Assessment of neutrophil behaviors defined in terms of slow and rapid motion. The average time for which neutrophils engaged in slow movement (left panel) and the average percentage of neutrophils with rapid movement (right panel) in MA-SARS-CoV-2-Venus-infected lung. n = 5–6. (\u003cstrong\u003eF\u003c/strong\u003e) Pulmonary blood perfusion in MA-SARS-CoV-2-Venus-infected mice. To visualize blood vessels through which erythrocytes passed, MA-SARS-CoV-2-Venus-infected Ob/Ob mice were i.v. inoculated with fluorescently labeled erythrocytes (red) at the indicated timepoint. The mice were also i.v. inoculated with fluorescent dextran (blue) to visualize the lung architecture. A maximal intensity projection of the indicated frames (0–10 min) was generated to show the functional capillaries perfused by the erythrocytes. White circles indicate vessels in which the transferred erythrocytes did not pass during the observation period in the lungs of MA-SARS-CoV-2-Venus-infected Ob/Ob mice. (\u003cstrong\u003eG\u003c/strong\u003e) Representative images of impaired pulmonary microcirculation caused by neutrophil-platelet complexes in MA-SARS-CoV-2-Venus-infected Ob/Ob mice. Ob/Ob mice were intranasally infected with MA-SARS-CoV-2-Venus and observed at 4 dpi. At this timepoint, fluorescent dextran (white), fluorochrome-conjugated anti-mouse Ly-6G antibody (red), and anti-mouse CD41 (Cyan) were i.v. administered to visualize the lung architecture, neutrophils, and platelets, respectively. The mice were also i.v. inoculated with fluorescently labeled erythrocytes (red). White arrows indicate a neutrophil (fuschia) adhering to a pulmonary vessel wall. Yellow arrowheads indicate platelet (cyan) aggregation and white arrowheads indicate erythrocytes that have been blocked from passing through the vessels. (\u003cstrong\u003eH\u003c/strong\u003e) Survival and body weight changes of neutrophil-depleted Ob/Ob mice infected with MA-SARS-CoV-2. Seven Ob/Ob mice per group were injected with anti-Kappa immunoglobulin antibody and anti-Ly-6G antibody to deplete neutrophils. The mice were infected with 10\u003csup\u003e3\u003c/sup\u003e PFU of SARS-CoV-2-Venus, and survival and body weight were monitored daily for 14 days. The results are expressed as the mean ± SD. The asterisks indicate \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05. n = 7.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3895679/v1/c61cdda7bb412a8023cf205e.jpg"},{"id":50816887,"identity":"49b96f0b-c5c1-4a2c-a1ad-c344a1e58e58","added_by":"auto","created_at":"2024-02-07 19:50:29","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1307146,"visible":true,"origin":"","legend":"\u003cp\u003eInvolvement of adhesion molecules in pulmonary neutrophils in COVID-19 pneumonia. (\u003cstrong\u003eA\u003c/strong\u003e) Expression of adhesion molecules on vascular neutrophils in the lungs of MA-SARS-CoV-2-infected mice. Positive rates of the indicated adhesion molecules on pulmonary vascular neutrophils were determined in the lungs of C57BL/6 mice and Ob/Ob mice at 4 dpi. For intravascular neutrophil staining, mice were injected i.v. with PE-conjugated anti-Ly-6G antibody 5 min before lung harvesting and then analyzed by flow cytometry. (n = 3–6). Bars in each panel indicate expression levels of adhesion factors determined to be positive. (B, C and D) Analysis of published sc-RNAseq datasets from COVID-19 clinical samples [Wilk et al.\u003ca href=\"#_ENREF_9\" title=\"Wilk, 2020 #1039\"\u003e\u003csup\u003e9\u003c/sup\u003e\u003c/a\u003e and Xu et al.\u003ca href=\"#_ENREF_21\" title=\"Xu, 2022 #1062\"\u003e\u003csup\u003e21\u003c/sup\u003e\u003c/a\u003e]. (\u003cstrong\u003eB\u003c/strong\u003e) Peripheral blood mononuclear cells from COVID-19 patients were clustered based on the gene expression of each cell and plotted as UMAP (upper panel). Comparison of UMAP between healthy individuals and COVID-19 patients (bottom panel). (\u003cstrong\u003eC\u003c/strong\u003e) Volcano plots of adhesion-related genes in PBMC neutrophils from COVID-19 patients. A total of 474 adhesion-related genes were analyzed in clusters corresponding to the neutrophils in (B). Purple plots indicate genes with significantly altered expression between healthy and severe cases and the dashed grey line indicates that the BH-corrected \u003cem\u003ep\u003c/em\u003e-value is 0.05. (\u003cstrong\u003eD\u003c/strong\u003e) Violin plots of gene expression in PBMC neutrophils for four adhesion-related genes whose expression was significantly frequently altered in the two datasets between healthy and severe cases. NonVent (non-ventilated) and Vent (ventilated) indicate mild and severe cases of COVID-19, respectively\u003ca href=\"#_ENREF_9\" title=\"Wilk, 2020 #1039\"\u003e\u003csup\u003e9\u003c/sup\u003e\u003c/a\u003e. The asterisks indicate \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3895679/v1/53cae2eff3b48b2d9e750bfb.jpg"},{"id":73740684,"identity":"cb39810d-4af7-47f0-bc44-849e09608e79","added_by":"auto","created_at":"2025-01-14 08:07:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9264518,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3895679/v1/588434f4-ec32-4eb8-bf24-de631767ee6b.pdf"},{"id":50816888,"identity":"a968f9dd-f0a2-44b7-a283-7fb2275f9e90","added_by":"auto","created_at":"2024-02-07 19:50:29","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5681089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMovie 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e imaging of K18-hACE2 C57BL/6J mice infected with SARS-CoV-2-Venus.\u003c/p\u003e\n\u003cp\u003eK18-hACE2 C57BL/6J mice were infected with 10\u003csup\u003e5\u003c/sup\u003e PFU of SARS-CoV-2-Venus and observed by using our \u003cem\u003ein vivo\u003c/em\u003e imaging system. Green indicates virus-infected cells. At the indicated timepoints, fluorescent dextran (white), fluorochrome-conjugated anti-mouse Ly-6G antibody (red), and anti-mouse CD41 (Cyan) were i.v. administered to visualize the lung architecture, neutrophils, and platelets, respectively.\u003c/p\u003e","description":"","filename":"Movie1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3895679/v1/40230f10df85865959ca1ff5.mp4"},{"id":50816901,"identity":"60eec16c-7c5a-4a33-9e3c-3f20bc0af427","added_by":"auto","created_at":"2024-02-07 19:50:33","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":25354909,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMovie 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTrafficking analyses of pulmonary neutrophils in SARS-CoV-2-infected mouse lung.\u003c/p\u003e\n\u003cp\u003eK18-hACE2 C57BL/6J mice were infected with 10\u003csup\u003e5\u003c/sup\u003e PFU of SARS-CoV-2-Venus and observed by using our \u003cem\u003ein vivo\u003c/em\u003e imaging system. At the indicated timepoint, fluorescent dextran (blue) and fluorochrome-conjugated anti-mouse Ly-6G antibody (red) were i.v. administered to visualize the lung architecture and neutrophils, respectively. Trajectories of individual neutrophils are displayed in white.\u003c/p\u003e","description":"","filename":"Movie2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3895679/v1/076a018c0c227d4248d51a3e.mp4"},{"id":50816891,"identity":"801cf715-20c3-4293-841d-6d3585a3a73e","added_by":"auto","created_at":"2024-02-07 19:50:29","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":29835041,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMovie 3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e imaging of pulmonary blood perfusion in SARS-CoV-2-Venus-infected mice. K18-hACE2 C57BL/6J mice were infected with 10\u003csup\u003e5\u003c/sup\u003e PFU of SARS-CoV-2-Venus and observed by using our \u003cem\u003ein vivo\u003c/em\u003e imaging system. To visualize blood vessels through which erythrocytes passed, SARS-CoV-2-Venus-infected K18-hACE2 C57BL/6J mice were i.v. inoculated with fluorescently labeled erythrocytes (red) at 6 dpi. The mice were also i.v. inoculated with fluorescent dextran (blue) to visualize the lung architecture. A maximal intensity projection of the indicated frames (0–10 min) was generated to show the functional capillaries perfused by the erythrocytes. White circles indicate vessels through which the transferred erythrocytes did not pass during the observation period in the lungs of the SARS-CoV-2-Venus-infected K18-hACE2 C57BL/6J mice.\u003c/p\u003e","description":"","filename":"Movie3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3895679/v1/0ba5928be107c356db705d21.mp4"},{"id":50816893,"identity":"a7118a59-d90e-4f0b-bf80-de8ba8a13727","added_by":"auto","created_at":"2024-02-07 19:50:30","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":9355153,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMovie 4\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e imaging of Ob/Ob mice infected with MA-SARS-CoV-2-Venus. Ob/Ob mice were infected with 10\u003csup\u003e4\u003c/sup\u003e PFU of MA-SARS-CoV-2-Venus and observed by using our \u003cem\u003ein vivo\u003c/em\u003e imaging system. Green indicates virus-infected cells. At the indicated timepoints, fluorescent dextran (white), fluorochrome-conjugated anti-mouse Ly-6G antibody (red), and anti-mouse CD41 (Cyan) were i.v. administered to visualize the lung architecture, neutrophils, and platelets, respectively.\u003c/p\u003e","description":"","filename":"Movie4.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3895679/v1/9d2281e0687634bc66941f1d.mp4"},{"id":50816826,"identity":"938782d2-3b88-4267-832a-51999007f1fa","added_by":"auto","created_at":"2024-02-07 19:50:28","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":17941422,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMovie 5\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e imaging of pulmonary blood perfusion in MA-SARS-CoV-2-Venus-infected mice. Ob/Ob mice were infected with 10\u003csup\u003e4\u003c/sup\u003e PFU of MA-SARS-CoV-2-Venus and observed by using our \u003cem\u003ein vivo\u003c/em\u003e imaging system. To visualize blood vessels through which erythrocytes passed, MA-SARS-CoV-2-Venus-infected Ob/Ob mice were i.v. inoculated with fluorescently labeled erythrocytes (red) at 4 dpi. The mice were also i.v. inoculated with fluorescent dextran (blue) to visualize the lung architecture. A maximal intensity projection of the indicated frames (0–10 min) was generated to show the functional capillaries perfused by the erythrocytes. White circles indicate vessels through which the transferred erythrocytes did not pass during the observation period in the lungs of the MA-SARS-CoV-2-Venus-infected Ob/Ob mice.\u003c/p\u003e","description":"","filename":"Movie5.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3895679/v1/76f5f6b87632e5328a119b74.mp4"},{"id":50817394,"identity":"127fff61-c542-401d-9acd-0e01c94a9eca","added_by":"auto","created_at":"2024-02-07 20:06:29","extension":"mp4","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":4241009,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMovie 6\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative movie of impaired pulmonary microcirculation caused by neutrophil-platelet complexes in MA-SARS-CoV-2-Venus-infected Ob/Ob mice. Ob/Ob mice were intranasally infected with MA-SARS-CoV-2-Venus and observed at 4 dpi. At this timepoint, fluorescent dextran (white), fluorochrome-conjugated anti-mouse Ly-6G antibody (fuschia), and anti-mouse CD41 (Cyan) were i.v. administered to visualize the lung architecture, neutrophils, and platelets, respectively. The mice were also i.v. inoculated with fluorescently labeled erythrocytes (red). White arrows indicate a neutrophil adhering to a pulmonary vessel wall. Yellow arrowheads indicate platelet aggregation, and white arrowheads indicate erythrocytes blocked from passing through the vessels.\u003c/p\u003e","description":"","filename":"Movie6.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3895679/v1/d94d671e3db6c90a2603ed6f.mp4"},{"id":50816885,"identity":"830bf793-913f-42c1-9ac6-aec5a497715f","added_by":"auto","created_at":"2024-02-07 19:50:29","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1750000,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended Data Figure 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImages of platelet-neutrophil complexes in pulmonary capillaries of SARS-CoV-2-infectedK18-hACE2 C57BL/6J mice. K18-hACE2 C57BL/6J mice were intranasally infected with SARS-CoV-2-Venus and observed at 6 dpi. At this timepoint, fluorescent dextran (white), fluorochrome-conjugated anti-mouse Ly-6G antibody (red), and anti-mouse CD41 (Cyan) were i.v. administered to visualize the lung architecture, neutrophils, and platelets, respectively. Arrows indicate platelet aggregates. Scale bars, 50 µm.\u003c/p\u003e","description":"","filename":"SARSCoV2ExtendedDataFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3895679/v1/ef36a502a13a0134217fa865.tif"},{"id":50816799,"identity":"49d1d4c4-451a-4e2e-b883-b95b7d1edfa2","added_by":"auto","created_at":"2024-02-07 19:50:28","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":543370,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended Data Figure 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSurvival and body weight changes in MA-SARS-CoV-2-Venus-infected mice. Five mice per group of Ob/Ob mice were infected with 10\u003csup\u003e2\u003c/sup\u003e to 10\u003csup\u003e5\u003c/sup\u003e PFU of MA-SARS-CoV-2-Venus, and survival and body weight were monitored daily for 10 days. The results are expressed as the mean ± SD.\u003c/p\u003e","description":"","filename":"SARSCoV2ExtendedDataFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3895679/v1/f414c0b4e0c200277accc4cd.tif"},{"id":50816897,"identity":"50f782c6-1411-49cc-beac-34884db5460b","added_by":"auto","created_at":"2024-02-07 19:50:30","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":781448,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended Data Figure 3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViolin plots of \u003cem\u003ePADI4\u003c/em\u003e and \u003cem\u003eGSDMD\u003c/em\u003e gene expression in PBMC neutrophils. NonVent (non-ventilated) and Vent (ventilated) indicate mild and severe cases of COVID-19, respectively\u003ca href=\"#_ENREF_9\" title=\"Wilk, 2020 #1039\"\u003e\u003csup\u003e9\u003c/sup\u003e\u003c/a\u003e. The asterisks indicate \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"SARSCoV2ExtendedDataFigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-3895679/v1/610b1be14b289a268bdf5081.tif"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nYoshihiro Kawaoka has ongoing unrelated collaborations and/or sponsored research agreements with Daiichi Sankyo Pharmaceutical, Toyama Chemical, Tauns Laboratories, Inc., Shionogi \u0026 Co. Ltd, Otsuka Pharmaceutical, and KM Biologics and has received royalties from MedImmune and Integrated Biotherapeutics.","formattedTitle":"Neutrophil adhesion to vessel walls impairs pulmonary circulation in COVID-19 pathology","fulltext":[{"header":"Main","content":"\u003cp\u003eSevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative virus of COVID-19 (coronavirus disease 2019) and is transmitted via droplets/aerosols containing the virus. SARS-CoV-2 invasion of the lung is initiated by the binding of the virus to the alveolar epithelium using angiotensin-converting enzyme-2 (ACE-2) as a receptor\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Most cases of COVID-19 are mild with respiratory symptoms such as fever, cough, nasal discharge, and pharyngitis; however, the elderly and those with underlying medical conditions such as obesity, diabetes, and hypertension develop severe viral pneumonia, which often leads to serious complications and even death\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Patients presenting with severe viral pneumonia are treated according to their respiratory and systemic symptoms, but the mechanism of COVID-19 severity is not fully understood and an optimal treatment strategy has not been established.\u003c/p\u003e\u003cp\u003eImmunopathology, including neutrophilia and lymphopenia, decreased or delayed type I interferon response, and the cytokine storm caused by dysregulation of monocytes and granulocytes may play role in the pathogenesis of COVID-19\u003csup\u003e5\u003c/sup\u003e. In addition to these immunopathological events, thrombosis has frequently been reported in patients with severe COVID-19, which may also contribute to the severity of the disease\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Platelets bind to neutrophils through neutrophil extracellular traps (NETs) released by the neutrophils to form large thrombus-like aggregates; they also form thrombi independently of neutrophils in response to tissue factors exposed to damaged endothelial cells. Single-cell RNA-seq and proteomic approaches using clinical samples and animal models of SARS-CoV-2 infection have revealed that factors associated with inflammation, coagulation, and NETs release are activated during SARS-CoV-2 infection. The factors involved in thrombus formation are spatiotemporally regulated and the thrombogenic process is thought to be a multistep process; however, these dynamic events are difficult to observe using conventional histological approaches with COVID-19 autopsy samples or SARS-CoV-2-infected animals. Accordingly, it remains unclear how the process of thrombus formation proceeds in patients with severe COVID-19.\u003c/p\u003e\u003cp\u003eTo establish an effective treatment for patients with severe COVID-19, we must elucidate the mechanisms involved in the severity of the disease. To understand how SARS-CoV-2 affects the responses of platelets and neutrophils in the pulmonary vasculature and the thrombogenic response, we performed two-photon \u003cem\u003ein vivo\u003c/em\u003e imaging analysis of cellular responses after SARS-CoV-2 infection by using SARS-CoV-2 strains and lethal mouse models. Our \u003cem\u003ein vivo\u003c/em\u003e imaging analysis revealed increased neutrophil numbers in the pulmonary vasculature, prolonged neutrophil adhesion to pulmonary vessels, and enhanced platelet aggregation in SARS-CoV-2-infected mice. The increased expression of adhesion factors on the neutrophils in pulmonary blood vessels promoted thrombus formation and impaired pulmonary blood flow in the lung of the SARS-CoV-2-infected mice. In addition, re-analysis of data from human clinical samples\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e demonstrated the relevance of the findings in mice.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003eimaging of SARS-CoV-2-infected lung.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the pathological changes of SARS-CoV-2-infected lungs in living animals, we examined K18-human angiotensin converting enzyme 2 transgenic (K18-hACE2 Tg) mice by using an \u003cem\u003ein vivo\u003c/em\u003e imaging system we previously established in a biosafety level (BSL) 3 facility\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. hACE2 is expressed on the epithelial cells of the lungs, kidneys, liver, intestine, and brain of K18-hACE2 mice, and contributes to the establishment of SARS-CoV-2 infection as a receptor for the S protein\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. To visualize SARS-CoV-2-infected cells, K18-hACE2 Tg mice were intranasally inoculated with SARS-CoV-2-Venus, which expresses the Venus fluorescent reporter gene in infected cells. SARS-CoV-2-Venus, generated by reverse genetics in the backbone of pBAC SARS2 WK521 (derived from SARS-CoV-2/JPN/TY/WK-521), exhibited lethal infection with an MLD\u003csub\u003e50\u003c/sub\u003e (mouse lethal dose 50; the dose required to kill 50% of infected mice) value of 10\u003csup\u003e3.83\u003c/sup\u003e plaque-forming units (PFU) in K18-hACE2 Tg mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). To analyze the pathobiological effects of SARS-CoV-2, we infected K18-hACE2 Tg mice with 10\u003csup\u003e5\u003c/sup\u003e PFU of SARS-CoV-2-Venus. Fluorescent dextran as well as fluorochrome-conjugated anti-mouse Ly-6G and CD41 antibodies were also intravenously (i.v.) administered to the mice to visualize lung vascular structures, neutrophils, and platelets, respectively. Virus-infected Venus-positive epithelial cells began to be observed in the lungs of SARS-CoV-2\u0026ndash;infected mice on day 4 post infection (p.i.), and their number had visibly increased at day 6 p.i. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB and Movie 1). The morphology of the Venus-positive infected cells suggested that flat type I alveolar epithelial cells surrounding the alveoli and spherical large type II alveolar epithelial cells were infected with SARS-CoV-2-Venus (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Platelets and neutrophils flowed smoothly in the pulmonary capillaries of uninfected mice, whereas in the mice infected with SARS-CoV-2-Venus, their movements were slower in the capillaries (Movie 1). On day 6 p.i., an increase in the number of neutrophils was apparent in the pulmonary capillaries of the infected-K18-hACE2 Tg mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSARS-CoV-2 alters the motility of intravascular neutrophils and impairs blood flow in infected lung.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyze the functional changes of neutrophils in SARS-CoV-2-infected lungs, we quantitatively assessed the neutrophil movement. In our previous studies, we reported that pulmonary neutrophils in influenza virus-infected mice show two types of motility\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e: \"slow movement\", which is considered tethering or rolling, in which neutrophils move along the vessel wall at a speed of 50 \u0026micro;m/s or less; and \"rapid motion\", which is moving in the blood flow at a speed of more than 50 \u0026micro;m/s. We analyzed the amount of time individual neutrophils engaged in each motion in the lungs of K18-hACE2 Tg mice infected with SARS-CoV-2-Venus. Pulmonary neutrophils observed in SARS-CoV-2-infected lungs were tracked (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA left panels) and ordered according to the amount of time they spent in slow motion, and the total time they spent in slow (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA right panels, blue bars) and rapid (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA right panels, red bars) motion was plotted for each neutrophil (Movie 2). In SARS-CoV-2-Venus-infected lung, the amount of time the neutrophils spent in slow motion was significantly increased compared with uninfected lung (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). We also found that the percentage of neutrophils that engaged in rapid motion decreased (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). These results indicate that vascular neutrophils in SARS-CoV-2-infected lungs have increased contact time with the pulmonary vessel wall.\u003c/p\u003e\n\u003cp\u003eThrombus formation in pulmonary vessels has emerged as a typical clinical feature of COVID-19 pneumonia\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. To assess whether intravascular neutrophils contribute to thrombus formation during SARS-CoV-2 pathogenesis, we performed an \u003cem\u003ein vivo\u003c/em\u003e imaging analysis by co-staining platelets and neutrophils. To quantify the size of the platelet aggregates in the infected lungs, we assessed the fluorescent signals of CD41 as a surface marker of platelets\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e from the original image, measured the area of each platelet, and performed a frequency analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). CD41 signals occupying a small area (\u0026lt;\u0026thinsp;50 pixels, which is approximately 8.57 \u0026micro;m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e in our imaging setting) and flowing in the blood vessels were considered to be single platelets. In SARS-CoV-2-infected lungs, an increased percentage of aggregated platelets with a large area of CD41 signal (\u0026ge;\u0026thinsp;50 pixels and \u0026lt;\u0026thinsp;200 pixels, which is approximately 34.28 \u0026micro;m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e in our imaging setting) and thrombi (\u0026gt;\u0026thinsp;200 pixels) were also observed to block the vessels as emboli. The percentage of aggregated platelets and thrombi was significantly increased in SARS-CoV-2-infected lungs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). Interestingly, aggregated platelets frequently interacted with intravascular neutrophils to form microthrombus-like structures (neutrophil-platelet complexes) in the lungs infected with SARS-CoV-2-Venus (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD and Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). These observations suggest that neutrophils adhering to pulmonary vessels in SARS-CoV-2-infected lungs participate in the formation of platelet aggregates.\u003c/p\u003e\n\u003cp\u003eThe stagnation of blood neutrophils, aggregation of platelets, and formation of platelet-neutrophil complexes observed in SARS-CoV-2-infected lungs may have a significant effect on pulmonary hemodynamics and oxygen exchange mediated by erythrocytes. To test this concept, we transferred fluorescently labeled erythrocytes into SARS-CoV-2-infected K18-hACE2 Tg mice and observed their trajectories for 10 minutes. The time-lapse images were overlapped every 50 frames to visualize the areas where labeled erythrocytes flowed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF and Movie 3). In naive mice, the blood vessels visualized by use of fluorescent dextran and the trajectories of the labeled erythrocytes almost overlapped, indicating that most pulmonary vessels were functional for oxygen exchange. In contrast, in SARS-CoV-2-infected lungs, there were areas where the vascular structures and the trajectories of the labeled erythrocytes do not overlap, indicating that the availability of functional capillaries for oxygen exchange was reduced. Blood neutrophil stagnation, platelet aggregation, and platelet-neutrophil complexes may disturb the pulmonary microcirculation in the SARS-CoV-2-infected lungs. Our \u003cem\u003ein vivo\u003c/em\u003e imaging data revealed that the functional failure of lung capillaries is likely due to microthrombi formation in mice infected with SARS-CoV-2.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eDevelopment of a mouse model of severe SARS-CoV-2 pneumonia\u003c/h2\u003e\n\u003cp\u003eAlthough K18-hACE2 Tg mice have been widely used to study SARS-CoV-2 pathogenesis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, intranasal inoculation of these mice with SARS-CoV-2 can cause severe neurological disease, including encephalitis, with features that differ from severe COVID-19 cases\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. We previously established a mouse-adapted (MA) SARS-CoV-2 and its derivative expressing the Venus protein (named MA-SARS-CoV-2-Venus), which causes severe lung inflammation in C57BL/6J mice similar to severe COVID-19 cases\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo establish an animal model that can mimic the symptoms of severe COVID-19 patients, we used seven mouse strains that are well established disease models [i.e., obesity/diabetes models, C57BL/6 HamSlc-\u003cem\u003eob/ob\u003c/em\u003e (Ob/Ob) mice and KK-A\u003csup\u003ey\u003c/sup\u003e/Ta Jcl mice; a type 1 diabetes model, Streptozotocin (STZ)-induced diabetic C57BL/6J mice; aged models, SAMP8/TaSlc mice and SAMP10-\u0026Delta;Sglt2 mice; a hyperlipidemia model, B6. B6.KOR/StmSlc-\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003eshl\u003c/em\u003e\u003c/sup\u003e mice; and a hepatitis model, B6-nonalcoholic steato-hepatitis (NASH) mice]. These mice were intranasally infected with 10\u003csup\u003e3\u003c/sup\u003e PFU of MA-SARS-CoV-2 and their survival was assessed. MA-SARS-CoV-2-infected Ob/Ob mice showed significantly reduced survival compared to wild-type mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). MA-SARS-CoV-2 exhibited high pathogenicity with an MLD\u003csub\u003e50\u003c/sub\u003e value of 10\u003csup\u003e2.5\u003c/sup\u003e PFU and caused virus dose-dependent weight loss in Ob/Ob mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). MA-SARS-CoV-2 and MA-SARS-CoV-2-Venus showed comparable virulence in Ob/Ob mice (MLD\u003csub\u003e50\u003c/sub\u003e; 10\u003csup\u003e2.5\u003c/sup\u003e PFU) (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003eInfection of Ob/Ob mice with MA-SARS-CoV-2 resulted in high virus titers in the lungs and nasal turbinates at 2 dpi, and no substantial difference in viral titers in the respiratory organs at this timepoint was observed between Ob/Ob mice and their control groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). Viruses were also recovered from the brain and liver of the infected Ob/Ob mice albeit at low titers (note: the brain samples collected for virus titration included the olfactory bulb). In the lungs of infected Ob/Ob mice at 5 dpi, viral replication was significantly higher than that in the control groups, with a titer of more than 1.0 \u0026times;10\u003csup\u003e7\u003c/sup\u003e PFU/g. In contrast, no difference in viral titers in the nasal turbinates was observed between Ob/Ob mice and their control groups, and virus was not detected in any other organs tested at 5 dpi. Micro-CT analysis revealed lung abnormalities in MA-SARS-CoV-2-infected Ob/Ob mice, including patchy and ill-defined regions consistent with COVID-19 pneumonia at 4 dpi (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\n\u003cp\u003eWe then examined the histopathologic changes in the organs of Ob/Ob mice after MA-SARS-CoV-2 infection. Histopathological analysis revealed infiltration of inflammatory cells such as neutrophils and mononuclear cells into the alveolar regions in the lungs of both Ob/Ob and their control groups at 2 dpi with MA-SARS-CoV-2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE). At the same timepoint, viral antigen was immunohistochemically detected mainly on alveolar and bronchiolar epithelial cells in both groups. On day 5 p.i., the inflammation improved in the control group, but persisted in the Ob/Ob mice. In the control group, there was a clear reduction in the number of virus-positive cells. Conversely, there was no notable reduction in the number of virus-positive cells in the Ob/Ob group. No virus-positive cells or significant morphological changes were detected histopathologically in the brain, heart, liver, spleen, kidney, or intestine (data not shown). Consistent with our histopathologic analysis, flow cytometry showed that the number of neutrophils and monocytes infiltrating the lungs was significantly increased in the MA-SARS-CoV-2 infected lungs compared to the control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF; see the \u003cspan class=\"InternalRef\"\u003eMaterials and Methods\u003c/span\u003e section on the definition of cell types).\u003c/p\u003e\n\u003cp\u003eThese results indicate that Ob/Ob mice infected with MA-SARS-CoV-2 or MA-SARS-CoV-2-Venus are a useful model that reflects the pathogenesis of severe COVID-19.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeutrophils cause impaired pulmonary perfusion in SARS-CoV-2-infected lungs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyze the pathobiological changes in MA-SARS-CoV-2-infected Ob/Ob mice, the mice were intranasally infected with 10\u003csup\u003e4\u003c/sup\u003e PFU of MA-SARS-CoV-2-Venus. Large numbers of infected cells were observed in the lungs of Ob/Ob mice on day 4 p.i. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA and Movie 4). Similar to the pathophysiological changes in the infected K-18 hACE2 Tg mice, there was a significant increase in the number of neutrophils, platelet aggregates, and thrombi in the pulmonary vasculature of MA-SARS-CoV-2-Venus-infected Ob/Ob mice (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB and C). To investigate the functional changes in the neutrophils, we quantified the motility of the vascular neutrophils of the infected mice. Ob/Ob mice infected with MA-SARS-CoV-2-Venus showed significantly increased slow neutrophil movement in pulmonary vessels compared to control groups, indicating increased adhesion time of neutrophils to the pulmonary vascular wall (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). Hemodynamics of the lungs of MA-SARS-CoV-2-Venus-infected Ob/Ob mice showed that pulmonary perfusion was impaired compared with that in infected wild-type mice, with limited capillaries available for oxygen exchange (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF and Movie 5).\u003c/p\u003e\n\u003cp\u003eTo clarify whether aggregates of neutrophils and platelets contribute to the impaired pulmonary perfusion observed in SARS-CoV-2-infected lungs, we performed a five-color multiple labeling analysis using our \u003cem\u003ein vivo\u003c/em\u003e imaging method\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Ob/Ob mice were intranasally infected with MA-SARS-CoV-2-Venus and observed at 4 dpi. At this timepoint, fluorescent dextran, two fluorochrome-conjugated antibodies (Ly-6G and CD41), and fluorescently labeled erythrocytes were i.v. administered and then observed by using our imaging system. In the lungs of Ob/Ob mice infected with MA-SARS-CoV-2-Venus, neutrophil and platelet aggregates were seen obstructing the flow of the transferred erythrocytes, suggesting that these aggregates may be responsible for the impaired pulmonary perfusion (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG and Movie 6). In addition, in SARS-CoV-2-infected lungs, neutrophils adhered to the pulmonary vasculature; this was followed by platelet aggregation and stasis of the transferred erythrocytes, suggesting that neutrophil adhesion to the vessel wall is the point of origin for thrombus formation and subsequent impaired pulmonary perfusion.\u003c/p\u003e\n\u003cp\u003eTo assess whether neutrophils are involved in the pathogenesis of MA-SARS-CoV-2, we removed the neutrophils \u003cem\u003ein vivo\u003c/em\u003e by using antibodies and analyzed the effect on virus susceptibility. Removal of neutrophils from Ob/Ob mice led to prolonged survival after MA-SARS-CoV-2 infection, indicating that neutrophils contribute to the exacerbation of SARS-CoV-2 pneumonia (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH). These results suggest that thrombi formed upon neutrophil stagnation may exacerbate MA-SARS-CoV-2-Venus pneumonia in Ob/Ob mice.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eAltered expression of adhesion molecules in pulmonary neutrophils in relation to COVID-19 severity\u003c/h2\u003e\n\u003cp\u003eNeutrophils infiltrate the site of infection by interacting with vascular endothelial cells via adhesion molecules\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. We therefore analyzed the expression levels of molecules (i.e., L-selectin, Cd44, E-selectin, Selplg, and Pecam-1)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e linked to the interaction with vascular endothelial cells during neutrophil infiltration in SARS-CoV-2-infected mice by flow cytometry. Neutrophils in the pulmonary vasculature of Ob/Ob mice infected with MA-SARS-CoV-2 exhibited upregulated expression of L-selectin, Cd44, and E-selectin (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Analysis in this mouse model suggested that SARS-CoV-2 increases neutrophil adhesion to the vessel wall and induces platelet aggregation, which result in impaired pulmonary perfusion in the lungs.\u003c/p\u003e\n\u003cp\u003eFinally, to determine whether the increased expression levels of adhesion molecules on the surface of vascular neutrophils in severe infections observed in SARS-CoV-2-infected mice is relevant to the pathogenesis of COVID-19 pneumonia in humans, we analyzed published data obtained with clinical samples from COVID-19 patients for adhesion-related gene expression. Using two published datasets of scRNA-seq analyses\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e from peripheral blood mononuclear cells (PBMCs) from healthy volunteers, mild, and severe cases of COVID-19, we analyzed 474 adhesion-related gene\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e expression changes and found that the levels of expression of four genes (\u003cem\u003eCD44\u003c/em\u003e, \u003cem\u003eSELL\u003c/em\u003e, \u003cem\u003eICAM3\u003c/em\u003e, and \u003cem\u003eCD93)\u003c/em\u003e in the two datasets frequently differed between the healthy and severe groups (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB and C). In the subset corresponding to neutrophils, the expression levels of the \u003cem\u003eCD44\u003c/em\u003e and \u003cem\u003eSELL\u003c/em\u003e (which encodes L-selectin) genes were significantly increased in COVID-19 severe cases compared to healthy individuals (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). These findings indicate that the expression of adhesion molecules is also increased in the vascular neutrophils in the lungs of severe COVID-19 cases, supporting the relevance of the data obtained in mice. In contrast, the gene expression of \u003cem\u003eICAM3\u003c/em\u003e and \u003cem\u003eCD93\u003c/em\u003e was significantly decreased in the severe group compared to that in the healthy group. The expression levels of the \u003cem\u003eSELL\u003c/em\u003e gene showed a positive correlation with the severity of COVID-19 in both the Wilk et al.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and Xu et al.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e datasets and the \u003cem\u003eCD44\u003c/em\u003e gene expression level also positively correlated with COVID-19 severity in the Xu et al.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e dataset; the expression level of the \u003cem\u003eCD44\u003c/em\u003e gene was positively correlated when comparisons were made between healthy and nonventilated patients or ventilated patients in the Wilk et al.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e dataset. In contrast, the expression levels of the \u003cem\u003eICAM3\u003c/em\u003e and \u003cem\u003eCD93\u003c/em\u003e genes negatively correlated with severity, suggesting that they could be molecular markers for the severity of COVID-19.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, our \u003cem\u003ein vivo\u003c/em\u003e imaging system revealed that the number of neutrophils in pulmonary vessels increases and neutrophil motility decreases in SARS-CoV-2-infected lungs. We also found that platelet aggregation is enhanced in infected lungs, with frequent formation of neutrophil-platelet complexes. The number of blood vessels through which erythrocytes flow for oxygen exchange was reduced in the infected mouse lungs, suggesting that this may contribute to SARS-CoV-2 pathogenesis. Moreover, we found that the expression of adhesion molecules is increased in pulmonary neutrophils in COVID-19 patients and mouse models, suggesting the existence of a common mechanism of SARS-CoV-2 virulence in humans and mice. Our \u003cem\u003ein vivo\u003c/em\u003e imaging analysis thus revealed a novel mechanism of SARS-CoV-2 pathogenesis that could not be found using conventional histopathological and histochemical analyses, and flow cytometry.\u003c/p\u003e\u003cp\u003eAnalysis of mouse models of severe or lethal SARS-CoV-2 infection showed that platelet aggregation is induced by neutrophil adhesion to the pulmonary vascular wall, resulting in impaired pulmonary perfusion. A major mechanism of thrombus formation in COVID-19 patients is thought to be the induction of platelet aggregation and thrombus formation by NETs released from neutrophils\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Complexes of neutrophils, platelets, and citrullinated histone H3, a marker of NETs, have been found during lung autopsies of COVID-19 patients\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Consistent with these findings, an analysis of human scRNA-seq data revealed a difference in the expression of the \u003cem\u003ePADI4\u003c/em\u003e gene (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which is responsible for NET release\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, suggesting that NET release was induced in severe COVID-19 cases. However, under our experimental conditions, \u003cem\u003ein vivo\u003c/em\u003e imaging analysis of the lungs of K-18 hACE2 Tg mice on day 6 of infection and obese mice on day 4 of infection did not show NET release from neutrophils after intravenous administration of a NET-labeling reagent (data not shown). In addition, intravascular neutrophils in the infected lungs had a distinct cell membrane shape and slow motility and did not exhibit the apoptotic morphology that is associated with NET release. This discrepancy may reflect the multistep nature of thrombogenesis that has been postulated \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e; our findings of thrombus formation induced by neutrophil adhesion to the vessel wall may be an earlier phenomenon than the thrombus formation involving NETs. Since NET release by neutrophils is induced in response to PAMPs and DAMPs\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, it is likely that NETs are released in response to the DAMPs that are released upon tissue damage and induce thrombus formation in the late stages of infection when the infection is more advanced. Our \u003cem\u003ein vivo\u003c/em\u003e imaging analysis likely revealed an early stage of thrombus formation that was not available in the autopsy cases of COVID-19.\u003c/p\u003e\u003cp\u003eThe expression of the adhesion molecules CD44 and L-selectin was upregulated in the pulmonary intravascular neutrophils of the SARS-CoV-2-infected mice, and the expression levels of the genes encoding these factors were also upregulated in pulmonary neutrophils from severe COVID-19 cases. L-selectin is expressed on leukocytes and is involved in intercellular adhesion; it has been widely implicated in immune cell responses, from cell migration to antigen presentation\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In neutrophils, L-selectin is expressed on the membrane surface from the early stage of differentiation from progenitor cells and is involved in adhesion to vascular endothelial cells during migration\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The half-life of mature neutrophils is approximately 6\u0026ndash;12 h in the bloodstream, and L-selectin expression decreases with neutrophil aging, which is characterized by plasma membrane instability, activation of apoptotic signaling, and other events\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Neutrophil infiltration has been observed in the lungs of patients with COVID-19 and increases in the numbers of both mature and immature neutrophils have been reported\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. COVID-19 patients have also been reported to have neutropenia, an increase in the number of neutrophils in the blood \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, and in our mouse model, SARS-CoV-2 infection increased the number of neutrophils in the pulmonary vessels. Recently, Castanheira et al.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e reported that SARS-CoV-2 infection of a mouse model ubiquitously expressing hACE2 (CAG-AC-70) increased neutrophil numbers in pulmonary vessels. These findings suggest that SARS-CoV-2 infection promotes neutrophil proliferation in the bone marrow and migration to the infected lungs, and that immature neutrophils expressing high levels of L-selectin are recruited in large numbers to infected pulmonary vessels, leading to susceptibility to thrombus formation and impaired pulmonary perfusion. Consistent with this hypothesis, in the present study, we found that the expression levels of ICAM3, one of the membrane molecules induced by apoptotic signaling\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, are significantly decreased in neutrophils from patients with severe COVID-19compared to healthy controls.\u003c/p\u003e\u003cp\u003eCD44 is also an adhesion factor expressed on leukocytes and is involved in the adhesion and infiltration of neutrophils into the hepatic sinusoids\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. It has been reported that neutrophil adhesion is suppressed in the hepatic sinusoids of \u003cem\u003eCD44\u003c/em\u003e-deficient mice with LPS-induced liver injury\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Although the mechanism of \u003cem\u003eCD44\u003c/em\u003e gene expression in neutrophils remains unclear, we do know that \u003cem\u003eCD44\u003c/em\u003e expression is increased in monocytes in response to inflammatory cytokines\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In severe cases of COVID-19, inflammatory mediators induced by SARS-CoV-2 infection may also increase \u003cem\u003eCD44\u003c/em\u003e gene expression in neutrophils, contributing to increased adhesion to pulmonary vessels.\u003c/p\u003e\u003cp\u003eOur study has several limitations that require further investigation. Changes in the expression levels of membrane molecules in vascular endothelial cells in SARS-CoV-2-infected lungs were not examined in this study, and it is unclear how vascular endothelial cells are involved in neutrophil adhesion to the pulmonary vessel walls. It has been hypothesized that thrombus formation proceeds because of dysfunctional endothelial responses to SARS-CoV-2 infection\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. However, at least in our mouse model, neutrophil adhesion to the vessel wall occurred not only in the proximity of the infected vascular endothelial cells, but also away from SARS-CoV-2-infected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Another limitation is that it is unclear whether the upregulation of the \u003cem\u003eCD44\u003c/em\u003e and \u003cem\u003eSELL\u003c/em\u003e genes in the SARS-CoV-2-infected mouse model and in COVID-19 patients occurs in the entire neutrophil populations or in some subpopulations of neutrophils. There have been many reports of neutrophil heterogeneity\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, suggesting that SARS-CoV-2 infection might occur in a subpopulation of neutrophils that express the \u003cem\u003eCD44\u003c/em\u003e or \u003cem\u003eSELL\u003c/em\u003e genes at high levels. Finally, the extent to which impaired pulmonary perfusion due to neutrophil adhesion to the pulmonary vascular wall contributes to the lethal pathogenesis of SARS-COV-2 pneumonia is unknown. Although we observed improved survival after SARS-CoV-2 infection in a neutrophil depletion model using antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), a direct contribution of intravascular neutrophils to pathogenesis could not be demonstrated because neutrophils infiltrating lung tissue are removed with intravascular neutrophils in this model. Since adhesion molecules are involved in the migration and infiltration of neutrophils in response to SARS-CoV-2 infection, the dysfunction of adhesion molecules by neutralizing antibodies may also interfere with the protective function of neutrophils against SARS-CoV-2 infection, and this has not been tested.\u003c/p\u003e\u003cp\u003eBeyond the acute respiratory infection, symptoms such as shortness of breath, cough, arthralgia, myalgia, fatigue, headache, odor/taste disturbance, and palpitations have been reported as COVID-19 sequelae (also called long COVID) even after the infection has resolved\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. It has been shown that prolonged post-ischemic symptoms are more likely to occur in groups at high risk for severe COVID-19, particularly the elderly and those with a high BMI (obesity) \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. It has also been suggested that microembolization-induced tissue damage may contribute to COVID-19 sequelae\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In the present study, using \u003cem\u003ein vivo\u003c/em\u003e imaging of mouse models, we found that the formation of microthrombi composed of neutrophils and platelets impairs pulmonary blood flow and exacerbates COVID-19 pneumonitis. The pathological mechanisms of COVID-19 revealed by this study will lead to the development of more effective treatments for patients with severe COVID-19 and long COVID.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eViruses.\u003c/b\u003e We generated SARS-CoV-2-Venus, in which the fluorescent reporter gene Venus was inserted, by using reverse genetics. For construction of SARS-CoV-2-Venus, we replaced the ORF 8 genes of pBAC SARS2 wk521 with the Venus gene by use of recombination and designated the infectious cDNA clone pBAC SARS-CoV-2-Venus. Mouse-adapted (MA)-SARS-CoV-2 and MA-SARS-CoV-2-Venus were also generated by using reverse genetics are previously described\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Virus strains were propagated in VeroE6/TMPRSS2 (JCRB 1819) cells\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. All experiments with SARS-CoV-2-Venus were performed in enhanced biosafety level 3 (BSL3) containment laboratories at the University of Tokyo, which are approved for such use by the Ministry of Agriculture, Forestry, and Fisheries, Japan.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCells.\u003c/b\u003e VeroE6/TMPRSS2 (JCRB 1819) cells\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e were propagated in 1 mg/ml geneticin (G418; Invitrogen) and 5 \u0026micro;g/ml plasmocin prophylactic (Invitrogen) in Dulbecco's modified Eagle's medium (DMEM) containing 10% Fetal Calf Serum (FCS). The cells were regularly tested for mycoplasma contamination by using PCR, and confirmed to be mycoplasma-free.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMice.\u003c/b\u003e Eight-week-old hemizygous K18-hACE2 C57BL/6J mice (strain 2B6.Cg-Tg(K18-ACE2)2Prlmn/J), 24-week-old C57BL/6 HamSlc-\u003cem\u003eob/ob\u003c/em\u003e mice, and five-week-old Streptozotocin (STZ)-induced diabetic C57BL/6J mice were purchased from the Jackson Laboratory Japan. In addition, 24-week-old B6-NASH mice, B6.KOR/StmSlc-\u003cem\u003eApoe\u003c/em\u003e\u003csup\u003e\u003cem\u003eshl\u003c/em\u003e\u003c/sup\u003e mice, SAMR1/TaSlc [Senescence-Accelerated Mouse (SAM); senescence-Resistant inbred strains (R)] mice, SAMP8/TaSlc [Senescence-Accelerated Mouse (SAM); senescence-Prone inbred strains (P)] mice, and SAMP10-ΔSglt2 mice were purchased from Japan SLC Inc. KK-A\u003csup\u003ey\u003c/sup\u003e/Ta Jcl mice (24-week-old) were purchased from CLEA Japan Inc. Age- and sex-matched C57BL/6 mice, which served as controls, were purchased from the same vendor as each disease model mouse strain.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperimental infection of mice.\u003c/b\u003e Mice were intranasally inoculated with 10\u003csup\u003e1\u003c/sup\u003e\u0026ndash;10\u003csup\u003e5\u003c/sup\u003e PFU of SARS-CoV-2-Venus, MA-SARS-CoV-2, or MA-SARS-CoV-2-Venus. Body weights were measured before infection and then daily. The protocols for the animal studies were approved by the University of Tokyo (approval numbers PA19-72 and PA21-07).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePathological examination.\u003c/b\u003e Excised lung tissues were fixed in 4% paraformaldehyde phosphate buffer solution, and processed for paraffin embedding. The paraffin blocks were cut into 3-\u0026micro;m-thick sections and then the sections were stained using a standard hematoxylin and eosin procedure. In addition, tissue sections were stained with a rabbit polyclonal antibody for SARS-CoV nucleocapsid protein (ProSpec; ANT-180, 1:500 dilution, Rehovot) for immunohistochemical analyses. Specific antigen-antibody reactions were visualized by means of 3,3\u0026rsquo;-diaminobenzidine tetrahydrochloride staining using the Dako Envision system (Dako Cytomation; K4001, 1:1 dilution).\u003c/p\u003e\u003cp\u003e\u003cb\u003eVirus titration assay.\u003c/b\u003e C57BL/6 mice and HamSlc-\u003cem\u003eob/ob\u003c/em\u003e mice were intranasally inoculated with 10\u003csup\u003e3\u003c/sup\u003e PFU of MA-SARS-CoV-2. Two and five days post-infection (dpi), the animals were euthanized and their organs (lungs, nasal turbinate, brain, heart, liver, spleen, kidneys, and intestine) were collected. Confluent VeroE6/TMPRSS2 cells in 12-well plates were infected with 100 \u0026micro;l of a dilution of the organ homogenate. The virus inoculum was removed after incubation for 1 h at 37\u0026deg;C, and then 1% agarose solution in DMEM was overlaid on the cells. After incubation for 48 h, the agar-covered cells were fixed with 10% neutral buffered formalin. The plaques were counted after removal of the agar.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicro-CT imaging.\u003c/b\u003e C57BL/6 mice and HamSlc-\u003cem\u003eob/ob\u003c/em\u003e mice were inoculated intranasally with 10\u003csup\u003e3\u003c/sup\u003e PFU of MA-SARS-CoV-2. Lungs of infected mice were imaged by using an \u003cem\u003ein vivo\u003c/em\u003e micro-CT scanner (CosmoScan FX; Rigaku). Under ketamine-xylazine anesthesia, the animals were placed in the image chamber and scanned for 2 min at 90 kV, 88 \u0026micro;A, FOV 45 mm, and pixel size 90.0 \u0026micro;m. After scanning, the lung images were reconstructed by using the CosmoScan Database software of the micro-CT (Rigaku Corporation) and analyzed using the manufacturer-supplied software as described previously\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eimaging of mouse lung.\u003c/b\u003e The \u003cem\u003ein vivo\u003c/em\u003e imaging was performed by using an LSM 980 NLO (Carl Zeiss) equipped with an infrared laser (Chameleon Vision II; Coherent) as described previously\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. K18-hACE2 mice, C57BL/6J mice, and C57BL/6 HamSlc-\u003cem\u003eob/ob\u003c/em\u003e mice were infected with 10\u003csup\u003e5\u003c/sup\u003e PFU of SARS-CoV-2-Venus or 10\u003csup\u003e4\u003c/sup\u003e PFU of MA-SARS-CoV-2-Venus. The infected mice were intubated under anesthesia and ventilated at a respiratory rate of 120 breaths per minute. Isoflurane was continuously delivered at 2% to maintain anesthesia. The left lung lobe of the mice was exposed and gently immobilized with a custom-made thoracic suction window. In all experiments, Texas red dextran (70,000 Da; Invitorogen), Phycoerythrin (PE)-conjugated rat anti-mouse CD41 antibody (MWReg30; BD Biosciences), and Alexa Fluor 594-conjugated rat anti-mouse Ly-6G antibody (1A8; Biolegend) were injected i.v. before imaging to visualize the lung vascular structures, platelets, and vascular neutrophils, respectively. For the analyses of pulmonary perfusion, mice infected with SARS-CoV-2-Venus or MA-SARS-CoV-2-Venus were i.v. inoculated with Dio-labeled erythrocytes. A maximal intensity projection of the indicated frames (0\u0026ndash;10 min) was generated to show the functional capillary perfused by the erythrocytes, as described previously\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. To acquire images in spectral imaging mode, lasers at wavelengths of 488 nm, 543 nm, and 910 nm were used for simultaneous excitation of fluorochromes and Venus. All emitted light between 490- and 695-nm wavelengths was detected by using a 20\u0026times; water-immersion lens (Carl Zeiss). Spectral separation of the acquired lambda stacks was achieved by using the linear unmixing function of the LSM software ZEN blue (Carl Zeiss). Processing, assays, and data visualization were performed using CellProfiler (Broad Institute), Imaris (Carl Zeiss), and in-house MATLAB scripts (MathWorks). Tracking of the neutrophils in the denoised movies was performed by TrackMate (ImageJ; NIH).\u003c/p\u003e\u003cp\u003e\u003cb\u003eFlow cytometry.\u003c/b\u003e Mouse lungs were dissociated using a Lung Dissociation Kit (Miltenyi) and gentleMACS Dissociator (Miltenyi) according to the manufacturer's instructions for flow cytometry (FCM). Samples were then filtered through a 70-\u0026micro;m filter (Miltenyi) after red blood cell lysis and resuspended for subsequent FCM staining. For experiments staining intravascular neutrophils, mice were injected i.v. with PE\u0026ndash;conjugated rat anti-mouse Ly-6G antibody (1A8; Biolegend) 5 min before lung collection. For surface staining, cells were stained for 10 min with antibodies in PBS containing 0.5% BSA and 2 mM EDTA. The following antibody clones were used in this studies: Vio-Green-CD45 (REA737, Miltenyi), PE-NK1.1 (REA1162, Miltenyi), PE-Vio615-CD4 (REA604, Miltenyi), PE-Vio770-B220 (REA755, Miltenyi), APC-CD3 (REA641, Miltenyi), APC-Vio770-CD8a (REA601, Miltenyi), Vio-Blue-MHC class II (REA813, Miltenyi), FITC-Ly6C (REA796, Miltenyi), PE-Vio615-Ly-6G (REA526, Miltenyi), PE-Vio770-CD11c (REA754, Miltenyi), APC-Siglec-F (REA798, Miltenyi), APC-Vio770-CD11b (REA592, Miltenyi), APC-CD44 (IM7 Biolegend), APC- Pecam1 (W18222B, Biolegend), APC-CD62L (MEL-14, Proteintech), APC-CD62E (P2H3, Invitorogen), and APC-CD162 (4RA10, Elabscience). APC conjugation to the anti-CD62E mouse IgG1 antibody was performed using the APC Labeling Kit-NH2 (Wako) according to the manufacturer's protocol.\u003c/p\u003e\u003cp\u003ePopulations of immune cells were defined as follows: B cells (CD45\u003csup\u003e+\u003c/sup\u003e CD3\u003csup\u003e\u0026minus;\u003c/sup\u003e B220\u003csup\u003e+\u003c/sup\u003e), NK cells (CD45\u003csup\u003e+\u003c/sup\u003e CD3\u003csup\u003e\u0026minus;\u003c/sup\u003e B220\u003csup\u003e\u0026minus;\u003c/sup\u003e NK1.1\u003csup\u003e+\u003c/sup\u003e), CD4 T cells (CD45\u003csup\u003e+\u003c/sup\u003e CD3\u003csup\u003e+\u003c/sup\u003e B220\u003csup\u003e\u0026minus;\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e\u0026minus;\u003c/sup\u003e), CD8 T cells (CD45\u003csup\u003e+\u003c/sup\u003e CD3\u003csup\u003e+\u003c/sup\u003e B220\u003csup\u003e\u0026minus;\u003c/sup\u003e CD4\u003csup\u003e\u0026minus;\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e), alveolar macrophages (CD45\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003edim\u003c/sup\u003e Siglec-F\u003csup\u003e+\u003c/sup\u003e CD11c\u003csup\u003e+\u003c/sup\u003e MHC class II\u003csup\u003e+\u003c/sup\u003e), dendritic cells (CD45\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e\u0026minus;\u003c/sup\u003e Siglec-F\u003csup\u003e\u0026minus;\u003c/sup\u003e CD11c\u003csup\u003e+\u003c/sup\u003e), neutrophils (CD45\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003ehigh\u003c/sup\u003e Ly-6G\u003csup\u003e+\u003c/sup\u003e), eosinophils (CD45\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003ehigh\u003c/sup\u003e Siglec-F\u003csup\u003e+\u003c/sup\u003e Ly-6G\u003csup\u003e\u0026minus;\u003c/sup\u003e CD11c\u003csup\u003e\u0026minus;\u003c/sup\u003e), and monocytes (CD45\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003ehigh\u003c/sup\u003e Siglec-F\u003csup\u003e\u0026minus;\u003c/sup\u003e Ly-6G\u003csup\u003e\u0026minus;\u003c/sup\u003e MHC class II\u003csup\u003e\u0026minus;\u003c/sup\u003e Ly-6C\u003csup\u003ehigh\u003c/sup\u003e). Samples were analyzed on a flow cytometer (MACSQuant Tyto, Miltenyi).\u003c/p\u003e\u003cp\u003e\u003cb\u003eNeutrophil motility analysis.\u003c/b\u003e To track the movement of neutrophils, Alexa Fluor 594-conjugated rat anti-mouse Ly-6G antibody was injected i.v. into the mice. Neutrophils were imaged at approximately 4 fps for 230 s. All movies were corrected for respiratory motion artifacts and denoised as described previously\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Single object tracking was performed by using TrackMate (ImageJ; NIH) to obtain the trajectories of individual neutrophils. For each neutrophil, speeds were measured for individual steps in its trajectory and subsequently defined as slow (\u0026le;\u0026thinsp;50 \u0026micro;m/s) or rapid (\u0026gt;\u0026thinsp;50 \u0026micro;m/s) as described previously\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. We then examined whether a neutrophil performed rapid movement, and calculated the durations it engaged in continuous slow movements without being interrupted by the rapid movement.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantification of platelet aggregates.\u003c/b\u003e CD41 signals were detected in a semi-automated manner by using CellProfiler (Broad Institute), and then divided into three populations according to their sizes: signals covering\u0026thinsp;\u0026lt;\u0026thinsp;8.57 \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e (50 pixels) were defined as a \u0026ldquo;single platelet\u0026rdquo;, signals\u0026thinsp;\u0026ge;\u0026thinsp;8.57 \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and \u0026lt;\u0026thinsp;34.28 \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e (200 pixels) as \u0026ldquo;aggregated platelets\u0026rdquo;, and signals\u0026thinsp;\u0026ge;\u0026thinsp;34.28 \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e as \u0026ldquo;thrombocytes\u0026rdquo;. The frequency analysis of the CD41 signals was conducted using in-house MATLAB scripts (MathWorks).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003edepletion of neutrophils.\u003c/b\u003e C57BL/6 HamSlc-\u003cem\u003eob/ob\u003c/em\u003e were administered 100 \u0026micro;g of anti-rat Kappa immunoglobulin (clone MAR 18.5, #BE0122) daily for two days prior to infection as described previously\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In addition, 50 \u0026micro;g of Anti-Ly6G (clone 1A8, #BP0075-1) and corresponding isotype control (#BP0089) were administered every other day from one day prior to infection. When mice were sequentially injected with two antibodies, an interval of more than 2 hours was set between injections.\u003c/p\u003e\u003cp\u003e\u003cb\u003escRNA-seq data re-analysis.\u003c/b\u003e We reanalyzed two published PBMC scRNA-seq datasets for healthy and SARS-CoV-2-infected humans\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. For the published data from Wilk et al.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, pre-processed scRNA-seq count data with embedding, clustering, and cell type assignment from the previous study\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e were obtained as an RDS file from the COVID-19 Cell Atlas (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.covid19cellatlas.org/#wilk20\u003c/span\u003e\u003cspan address=\"https://www.covid19cellatlas.org/#wilk20\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) hosted by the Wellcome Sanger Institute. For the published data from Xu et al.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, we downloaded transcript-by-cell matrices output by Cell Ranger from NCBI (GSE216020) and preprocessed using the Seurat \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e package and DoubletFinder\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, which identifies and removes potential doublets, as described in a previous study\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Cells with less than 500 UMI counts, 200 detected genes, and more than 20% mitochondrial gene counts were removed as low-quality cells, as well as potential doublets. Data integration of all samples was performed using the FindIntegrationAnchors and IntegrateData functions in Seurat with the top 3000 most variable genes selected by FindVariableFeatures function. Cell-type annotation was based on marker genes of each cluster defined by FindAllMarkers functions. The subset corresponding to neutrophils in both datasets (Wilk et al. and Xu et al.) was used for differential gene expression analysis related to cell adhesion\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and neutrophil extracellular trap formation (KEGG: hsa04613), respectively.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analysis.\u003c/b\u003e GraphPad Prism was used to analyze all data. Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test, log-rank (Mantel-Cox) tests, and an ANOVA with a multiple corrections post-test were performed, and differences were considered to be statistically significant when the \u003cem\u003ep\u003c/em\u003e-value was less than 0.05. For the differential gene expression analysis of the scRNA-seq data, the Wilcoxon signed rank test was used and the \u003cem\u003ep\u003c/em\u003e-values were corrected by using the Benjamini-Hochberg Procedure.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe thank S. Watson for editing the manuscript. We also thank Yuko Sato and Seiya Ozono for their technical assistance. This research was supported by a Research Program on Emerging and Re-emerging Infectious Diseases from the Japan Agency for Medical Research and Development (AMED) (JP19fk0108113, JP20fk0108412, JP21fk0108552, JP233fa627001) by a Japan Program for Infectious Diseases Research and Infrastructure from AMED (JP23wm0125002), the Japan Society for the Promotion of Science (JSPS) (21K14984), the Japan Science and Technology Agency (JST) (Moonshot R\u0026amp;D) (JPMJMS2025), and by the NIAID-funded Center for Research on Influenza Pathogenesis (CRIP; HHSN272201400008C). H.U. was supported by GSK Japan Research Grant 2020, the Astellas Foundation for Research on Metabolic Disorders, the Naito Foundation, the Sumitomo Foundation, the Ichiro Kanehara Foundation, the Uehara Memorial Foundation, the Okinaka Memorial Institute for Medical Research, a Japanese Respiratory Foundation Grant, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, the SENSHIN Medical Research Foundation, and the Takeda Science Foundation. IH.W., HW. H., and CH.W. were supported by the National Science and Technology Council, Taiwan (MOST-110-2320-B-001-005-MY3).\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eH.U., M.K., S.I., S.M., and T.S. performed the mouse infection experiments, titrated virus in organs, and analyzed pathology. M.U. analyzed the micro-CT images. H.U. performed the \u003cem\u003ein vivo\u003c/em\u003e imaging analysis and flow cytometry. H.U., IH.W., HW. H., and CH.W. performed image data analyses. W.K. generated the Venus-expressing SARS-CoV-2. K.H. and E.K. analyzed the single-cell RNA-seq data. H.U., M.I, T.S., and Y.K. obtained funding. H.U., and Y.K. conceived the study and supervised the research. H.U., and Y.K. wrote the initial draft, with all other authors providing editorial comments.\u003c/p\u003e\n\u003ch2\u003eCompeting financial interests\u003c/h2\u003e\n\u003cp\u003eYoshihiro Kawaoka has ongoing unrelated collaborations and/or sponsored research agreements with Daiichi Sankyo Pharmaceutical, Toyama Chemical, Tauns Laboratories, Inc., Shionogi \u0026amp; Co. Ltd, Otsuka Pharmaceutical, and KM Biologics and has received royalties from MedImmune and Integrated Biotherapeutics.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJackson, C. B., Farzan, M., Chen, B. \u0026amp; Choe, H. Mechanisms of SARS-CoV-2 entry into cells. \u003cem\u003eNature reviews. 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S., Murrow, L. M. \u0026amp; Gartner, Z. J. DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors. \u003cem\u003eCell Systems\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 329-337.e324 (2019). https://doi.org:https://doi.org/10.1016/j.cels.2019.03.003\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3895679/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3895679/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicrothrombus formation is associated with COVID-19 severity; however, the detailed mechanism remains unclear. In this study, we investigated mouse models with severe pneumonia caused by SARS-CoV-2 infection by using our \u003cem\u003ein vivo\u003c/em\u003e two-photon imaging system. In the lungs of SARS-CoV-2-infected mice, increased expression of adhesion molecules in intravascular neutrophils prolonged adhesion time to the vessel wall, resulting in platelet aggregation and impaired lung perfusion. Re-analysis of scRNA-seq data from peripheral blood mononuclear cells from COVID-19 cases revealed increased expression levels of \u003cem\u003eCD44\u003c/em\u003e and \u003cem\u003eSELL\u003c/em\u003e in neutrophils in severe COVID-19 cases compared to a healthy group, consistent with our observations in the mouse model. These findings suggest that pulmonary perfusion defects caused by neutrophil adhesion to pulmonary vessels contribute to COVID-19 severity.\u003c/p\u003e","manuscriptTitle":"Neutrophil adhesion to vessel walls impairs pulmonary circulation in COVID-19 pathology","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-07 19:50:18","doi":"10.21203/rs.3.rs-3895679/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e9d353db-900c-4027-a46d-65d613949865","owner":[],"postedDate":"February 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28492796,"name":"Health sciences/Diseases/Infectious diseases/Viral infection"},{"id":28492797,"name":"Biological sciences/Microbiology/Virology/SARS-CoV-2"}],"tags":[],"updatedAt":"2025-01-14T08:07:21+00:00","versionOfRecord":{"articleIdentity":"rs-3895679","link":"https://doi.org/10.1038/s41467-024-55272-0","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-01-13 05:00:00","publishedOnDateReadable":"January 13th, 2025"},"versionCreatedAt":"2024-02-07 19:50:18","video":"","vorDoi":"10.1038/s41467-024-55272-0","vorDoiUrl":"https://doi.org/10.1038/s41467-024-55272-0","workflowStages":[]},"version":"v1","identity":"rs-3895679","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3895679","identity":"rs-3895679","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}