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Alterations in mitochondrial function and associated quality control programs, including mitochondrial-specific autophagy, termed mitophagy, are gaining increasing recognition in the context of disease. However, the role of mitophagy in organ transplant rejection remains poorly understood. Using mice deficient in Parkin, a ubiquitin-ligase which tags damaged or dysfunctional mitochondria for autophagic clearance, we assessed the impact of Parkin-dependent mitophagy on skin-graft rejection. We observed accelerated graft loss in Parkin-deficient mice across multiple-skin graft models. Immune cell distributions post-transplantation were largely unperturbed compared to wild-type; however, the CD8+ T cells of Parkin-deficient mice expressed more T-bet, IFNγ and Ki67, indicating greater priming towards effector function. This was accompanied by increased circulating levels of IL-12p70 in Parkin-deficient mice. Using a mixed leukocyte reaction, we demonstrated that naïve Parkin-deficient CD4+ and CD8+ T cells exhibit enhanced activation marker expression and proliferative responses to alloantigen, which were attenuated with administration of a pharmacological mitophagy inducer (p62-mediated mitophagy inducer), known to increase mitophagy in the absence of a functional PINK1-Parkin pathway. These findings indicate a role for Parkin-dependent mitophagy in curtailing skin-graft rejection.
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Obesity is a major risk factor for atrial fibrillation (AF) the most common serious cardiac arrhythmia, but the molecular mechanisms underlying diet-induced AF remain unclear. In this study, we subjected mice to a chronic high-fat diet and acute sympathetic activation ('two-hit' model) to study the mechanisms by which diet-induced obesity promotes AF. Surface electrocardiography revealed that diet-induced obesity and sympathetic activation synergize during intracardiac tachypacing to induce AF. At the cellular level, diet-induced obesity and acute adrenergic stimulation facilitate the formation of delayed afterdepolarizations in atrial myocytes, implicating altered Ca2+ dynamics as the underlying cause of AF. We found that diet-induced obesity does not alter the expression of major Ca2+-handling proteins in atria, including the sarcoplasmic reticulum Ca2+-ATPase (SERCA), a major component of beat-to-beat Ca2+ cycling in the heart. Paradoxically, obesity reduces phospholamban phosphorylation, suggesting decreased SERCA activity, yet atrial myocytes from obese mice showed a significantly increased Ca2+ transient amplitude and SERCA-mediated Ca2+ uptake. Adrenergic stimulation further increases the Ca2+ transient amplitude but does not affect Ca2+ reuptake in atrial myocytes from obese mice. Transcriptomics analysis showed that a high-fat diet prompts upregulation of neuronatin, a protein that has been implicated in obesity and is known to stimulate SERCA activity. We propose a mechanism in which obesity primes SERCA for paradoxical activation, and adrenergic stimulation facilitates AF conversion through a Ca2+-induced Ca2+ release gain in atrial myocytes. Overall, this study links obesity, altered Ca2+ signaling, and AF, and targeting this mechanism may prove effective for treating obesity-induced AF.
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Influenza poses a persistent health burden worldwide. To design equitable vaccines effective across all demographics, it is essential to better understand how host factors such as genetic background and aging affect the single-cell immune landscape of influenza infection. Cytometry by time-of-flight (CyTOF) represents a promising technique in this pursuit, but interpreting its large, high-dimensional data remains difficult. We have developed a new analytical approach, in silico gating annotating training elucidating (iGATE), based on probabilistic support vector machine classification. By rapidly and accurately "gating" tens of millions of cells in silico into user-defined types, iGATE enabled us to track 25 canonical immune cell types in mouse lung over the course of influenza infection. Applying iGATE to study effects of host genetic background, we show that the lower survival of C57BL/6 mice compared with BALB/c was associated with a more rapid accumulation of inflammatory cell types and decreased IL-10 expression. Furthermore, we demonstrate that the most prominent effect of aging is a defective T cell response, reducing survival of aged mice. Finally, iGATE reveals that the 25 canonical immune cell types exhibited differential influenza infection susceptibility and replication permissiveness in vivo, but neither property varied with host genotype or aging. The software is available at https://github.com/UmichWenLab/iGATE.
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Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae , Análise de Célula Única , Animais , Camundongos , Infecções por Orthomyxoviridae/imunologia , Análise de Célula Única/métodos , Pulmão/imunologia , Pulmão/virologia , Pulmão/patologia , Influenza Humana/imunologia , Humanos , Modelos Animais de Doenças , Envelhecimento/imunologia , Envelhecimento/genética , Citometria de Fluxo/métodos , Linfócitos T/imunologia , Simulação por ComputadorRESUMO
Invasive graft biopsies assess the efficacy of immunosuppression through lagging indicators of transplant rejection. We report on a microporous scaffold implant as a minimally invasive immunological niche to assay rejection before graft injury. Adoptive transfer of T cells into Rag2-/- mice with mismatched allografts induced acute cellular allograft rejection (ACAR), with subsequent validation in wild-type animals. Following murine heart or skin transplantation, scaffold implants accumulate predominantly innate immune cells. The scaffold enables frequent biopsy, and gene expression analyses identified biomarkers of ACAR before clinical signs of graft injury. This gene signature distinguishes ACAR and immunodeficient respiratory infection before injury onset, indicating the specificity of the biomarkers to differentiate ACAR from other inflammatory insult. Overall, this implantable scaffold enables remote evaluation of the early risk of rejection, which could potentially be used to reduce the frequency of routine graft biopsy, reduce toxicities by personalizing immunosuppression, and prolong transplant life.
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Aloenxertos , Biomarcadores , Rejeição de Enxerto , Animais , Rejeição de Enxerto/imunologia , Camundongos , Transplante de Pele/efeitos adversos , Transplante de Coração/efeitos adversos , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Tela Subcutânea/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Itaconate has emerged as a critical immunoregulatory metabolite. Here, we examined the therapeutic potential of itaconate in atherosclerosis. We found that both itaconate and the enzyme that synthesizes it, aconitate decarboxylase 1 (Acod1, also known as immune-responsive gene 1 [IRG1]), are upregulated during atherogenesis in mice. Deletion of Acod1 in myeloid cells exacerbated inflammation and atherosclerosis in vivo and resulted in an elevated frequency of a specific subset of M1-polarized proinflammatory macrophages in the atherosclerotic aorta. Importantly, Acod1 levels were inversely correlated with clinical occlusion in atherosclerotic human aorta specimens. Treating mice with the itaconate derivative 4-octyl itaconate attenuated inflammation and atherosclerosis induced by high cholesterol. Mechanistically, we found that the antioxidant transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), was required for itaconate to suppress macrophage activation induced by oxidized lipids in vitro and to decrease atherosclerotic lesion areas in vivo. Overall, our work shows that itaconate suppresses atherogenesis by inducing Nrf2-dependent inhibition of proinflammatory responses in macrophages. Activation of the itaconate pathway may represent an important approach to treat atherosclerosis.
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Doenças da Aorta , Aterosclerose , Succinatos , Camundongos , Humanos , Animais , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Macrófagos/metabolismo , Aterosclerose/tratamento farmacológico , Aterosclerose/genética , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Doenças da Aorta/metabolismoRESUMO
Aging is a strong risk factor for atherosclerosis and induces accumulation of memory CD8+ T cells in mice and humans. Biological changes that occur with aging lead to enhanced atherosclerosis, yet the role of aging on CD8+ T cells during atherogenesis is unclear. In this study, using femle mice, we found that depletion of CD8+ T cells attenuated atherogenesis in aged, but not young, animals. Furthermore, adoptive transfer of splenic CD8+ T cells from aged wild-type, but not young wild-type, donor mice significantly enhanced atherosclerosis in recipient mice lacking CD8+ T cells. We also characterized T cells in healthy and atherosclerotic young and aged mice by single-cell RNA sequencing. We found specific subsets of age-associated CD8+ T cells, including a Granzyme K+ effector memory subset, that accumulated and was clonally expanded within atherosclerotic plaques. These had transcriptomic signatures of T cell activation, migration, cytotoxicity and exhaustion. Overall, our study identified memory CD8+ T cells as therapeutic targets for atherosclerosis in aging.
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Aterosclerose , Placa Aterosclerótica , Humanos , Animais , Camundongos , Idoso , Linfócitos T CD8-Positivos , Células T de Memória , Camundongos Endogâmicos C57BLRESUMO
Advances in healthcare and improvements in living conditions have led to rising life expectancy worldwide. Aging is associated with excessive oxidative stress, a chronic inflammatory state, and limited tissue healing, all of which result in an increased risk of heart failure. In fact, the prevalence of heart failure approaches 40% in the ninth decade of life, with the majority of these cases suffering from heart failure with preserved ejection fraction (HFpEF). In cardiomyocytes (CMs), age-related mitochondrial dysfunction results in disrupted calcium signaling and covalent protein-linked aggregates, which cause cardiomyocyte functional disturbances, resulting in increased stiffness and diastolic dysfunction. Importantly, aging is also associated with chronic low-grade, sterile inflammation, which alters the function of interstitial cardiac cells and leads to cardiac fibrosis. Taken together, cardiac aging is associated with cellular, structural, and functional changes in the heart that contribute to the rising prevalence of heart failure in older people.
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Although aging enhances atherosclerosis, we do not know if this occurs via alterations in circulating immune cells, lipid metabolism, vasculature, or adipose tissue. Here, we examined whether aging exerts a direct pro-atherogenic effect on adipose tissue in mice. After demonstrating that aging augmented the inflammatory profile of visceral but not subcutaneous adipose tissue, we transplanted visceral fat from young or aged mice onto the right carotid artery of Ldlr-/- recipients. Aged fat transplants not only increased atherosclerotic plaque size with increased macrophage numbers in the adjacent carotid artery, but also in distal vascular territories, indicating that aging of the adipose tissue enhances atherosclerosis via secreted factors. By depleting macrophages from the visceral fat, we identified that adipose tissue macrophages are major contributors of the secreted factors. To identify these inflammatory factors, we found that aged fat transplants secreted increased levels of the inflammatory mediators TNFα, CXCL2, and CCL2, which synergized to promote monocyte chemotaxis. Importantly, the combined blockade of these inflammatory mediators impeded the ability of aged fat transplants to enhance atherosclerosis. In conclusion, our study reveals that aging enhances atherosclerosis via increased inflammation of visceral fat. Our study suggests that future therapies targeting the visceral fat may reduce atherosclerosis disease burden in the expanding older population.
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Aterosclerose , Monócitos , Animais , Camundongos , Monócitos/metabolismo , Quimiotaxia , Aterosclerose/metabolismo , Inflamação/metabolismo , Tecido Adiposo/metabolismo , Mediadores da Inflamação/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Aging impairs the immune responses to influenza A virus (IAV), resulting in increased mortality to IAV infections in older adults. However, the factors within the aged lung that compromise host defense to IAV remain unknown. Using a murine model and human samples, we identified prostaglandin E2 (PGE2), as such a factor. Senescent type II alveolar epithelial cells (AECs) are overproducers of PGE2 within the aged lung. PGE2 impairs the proliferation of alveolar macrophages (AMs), critical cells for defense against respiratory pathogens, via reduction of oxidative phosphorylation and mitophagy. Importantly, blockade of the PGE2 receptor EP2 in aged mice improves AM mitochondrial function, increases AM numbers and enhances survival to IAV infection. In conclusion, our study reveals a key mechanism that compromises host defense to IAV, and possibly other respiratory infections, with aging and suggests potential new therapeutic or preventative avenues to protect against viral respiratory disease in older adults.
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Vírus da Influenza A , Influenza Humana , Infecções por Orthomyxoviridae , Camundongos , Humanos , Animais , Idoso , Macrófagos Alveolares/metabolismo , Dinoprostona/metabolismo , MitocôndriasRESUMO
T cell proliferation and cytokine production are bioenergetically and biosynthetically costly. The inability to meet these metabolic demands results in altered differentiation, accompanied by impaired effector function, and attrition of the immune response. Interleukin-17-producing CD4 T cells (TH17s) are mediators of host defense, autoimmunity, and antitumor immunity in the setting of adoptive T cell therapy. TH17s are long-lived cells that require mitochondrial oxidative phosphorylation (OXPHOS) for effector function in vivo. Considering that TH17s polarized under standardized culture conditions are predominately glycolytic, little is known about how OXPHOS regulates TH17 processes, such as their ability to persist and thus contribute to protracted immune responses. Here, we modified standardized culture medium and identified a culture system that reliably induces OXPHOS dependence in TH17s. We found that TH17s cultured under OXPHOS conditions metabolically resembled their in vivo counterparts, whereas glycolytic cultures were dissimilar. OXPHOS TH17s exhibited increased mitochondrial fitness, glutamine anaplerosis, and an antiapoptotic phenotype marked by high BCL-XL and low BIM. Limited mitophagy, mediated by mitochondrial fusion regulator OPA-1, was critical to apoptotic resistance in OXPHOS TH17s. By contrast, glycolytic TH17s exhibited more mitophagy and an imbalance in BCL-XL to BIM, thereby priming them for apoptosis. In addition, through adoptive transfer experiments, we demonstrated that OXPHOS protected TH17s from apoptosis while enhancing their persistence in the periphery and tumor microenvironment in a murine model of melanoma. Together, our work demonstrates how metabolism regulates TH17 cell fate and highlights the potential for therapies that target OXPHOS in TH17-driven diseases.
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Fosforilação Oxidativa , Microambiente Tumoral , Camundongos , Animais , Mitocôndrias/metabolismo , Glicólise/genética , Diferenciação CelularRESUMO
Aging leads to a gradual dysregulation of immune functions, one consequence of which is reduced vaccine efficacy. In this review, we discuss several key contributing factors to the age-related decline in vaccine efficacy, such as alterations within the lymph nodes where germinal center (GC) reactions take place, alterations in the B cell compartment, alterations in the T cell compartment, and dysregulation of innate immune pathways. Additionally, we discuss several methods currently used in vaccine development to bolster vaccine efficacy in older adults. This review highlights the multifactorial defects that impair vaccine responses with aging.
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Envelhecimento , Eficácia de Vacinas , Vacinas , Idoso , Humanos , Linfócitos B , Centro Germinativo , Linfócitos T Auxiliares-Indutores , Linfócitos T , Imunidade InataRESUMO
People with kidney disease are disproportionately affected by atherosclerosis for unclear reasons. Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived mediator of kidney disease, levels of which are strongly associated with cardiovascular outcomes. We assessed suPAR's pathogenic involvement in atherosclerosis using epidemiologic, genetic, and experimental approaches. We found serum suPAR levels to be predictive of coronary artery calcification and cardiovascular events in 5,406 participants without known coronary disease. In a genome-wide association meta-analysis including over 25,000 individuals, we identified a missense variant in the plasminogen activator, urokinase receptor (PLAUR) gene (rs4760), confirmed experimentally to lead to higher suPAR levels. Mendelian randomization analysis in the UK Biobank using rs4760 indicated a causal association between genetically predicted suPAR levels and atherosclerotic phenotypes. In an experimental model of atherosclerosis, proprotein convertase subtilisin/kexin-9 (Pcsk9) transfection in mice overexpressing suPAR (suPARTg) led to substantially increased atherosclerotic plaques with necrotic cores and macrophage infiltration compared with those in WT mice, despite similar cholesterol levels. Prior to induction of atherosclerosis, aortas of suPARTg mice excreted higher levels of CCL2 and had higher monocyte counts compared with WT aortas. Aortic and circulating suPARTg monocytes exhibited a proinflammatory profile and enhanced chemotaxis. These findings characterize suPAR as a pathogenic factor for atherosclerosis acting at least partially through modulation of monocyte function.
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Aterosclerose , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Animais , Camundongos , Aterosclerose/genética , Biomarcadores , Estudo de Associação Genômica Ampla , Monócitos , Pró-Proteína Convertase 9 , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Fatores de Risco , Ativador de Plasminogênio Tipo Uroquinase , HumanosRESUMO
OBJECTIVE: The intersection between immunology and metabolism contributes to the pathogenesis of obesity-associated metabolic diseases as well as molecular control of inflammatory responses. The metabolite itaconate and the cell-permeable derivatives have robust anti-inflammatory effects; therefore, it is hypothesized that cis-aconitate decarboxylase (Acod1)-produced itaconate has a protective, anti-inflammatory effect during diet-induced obesity and metabolic disease. METHODS: Wild-type and Acod1-/- mice were subjected to diet-induced obesity. Glucose metabolism was analyzed by glucose tolerance tests, insulin tolerance tests, and indirect calorimetry. Gene expression and transcriptome analysis was performed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and RNA sequencing. RESULTS: Wild-type and Acod1-/- mice on high-fat diet had equivalent weight gain, but Acod1-/- mice had impaired glucose metabolism. Insulin tolerance tests and glucose tolerance tests after 12 weeks on high-fat diet revealed significantly higher blood glucose levels in Acod1-/- mice. This was associated with significant enrichment of inflammatory gene sets and a reduction in genes related to adipogenesis and fatty acid metabolism. Analysis of naive Acod1-/- mice showed a significant increase in fat deposition at 3 and 6 months of age and obesity and insulin resistance by 12 months. CONCLUSIONS: The data show that Acod1 has an important role in the regulation of glucose homeostasis and obesity under normal and high-fat diet conditions.
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Resistência à Insulina , Insulinas , Animais , Anti-Inflamatórios/uso terapêutico , Carboxiliases , Dieta Hiperlipídica , Glucose/metabolismo , Homeostase , Insulina , Resistência à Insulina/genética , Insulinas/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/complicaçõesRESUMO
Older people exhibit dysregulated innate immunity to respiratory viral infections, including influenza and SARS-CoV-2, and show an increase in morbidity and mortality. Nanoparticles are a potential practical therapeutic that could reduce exaggerated innate immune responses within the lungs during viral infection. However, such therapeutics have not been examined for effectiveness during respiratory viral infection, particular in aged hosts. Here, we employed a lethal model of influenza viral infection in vulnerable aged mice to examine the ability of biodegradable, cargo-free nanoparticles, designated ONP-302, to resolve innate immune dysfunction and improve outcomes during infection. We administered ONP-302 via i.v. injection to aged mice at day 3 after infection, when the hyperinflammatory innate immune response was already established. During infection, we found that ONP-302 treatment reduced the numbers of inflammatory monocytes within the lungs and increased their number in both the liver and spleen, without impacting viral clearance. Importantly, cargo-free nanoparticles reduced lung damage, reduced histological lung inflammation, and improved gas exchange and, ultimately, the clinical outcomes in influenza-infected aged mice. In conclusion, ONP-302 improves outcomes in influenza-infected aged mice. Thus, our study provides information concerning a practical therapeutic, which, if translated clinically, could improve disease outcomes for vulnerable older patients suffering from respiratory viral infections.
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COVID-19 , Doenças Transmissíveis , Influenza Humana , Nanopartículas , Infecções por Orthomyxoviridae , Animais , Humanos , Pulmão/patologia , Camundongos , Monócitos , SARS-CoV-2RESUMO
BACKGROUND: Aging enhances most chronic diseases but its impact on human aortic tissue in health and in thoracic aortic aneurysms (TAA) remains unclear. METHODS: We employed a human aortic biorepository of healthy specimens (n=17) and those that underwent surgical repair for TAA (n=20). First, we performed proteomics comparing aortas of healthy donors to aneurysmal specimens, in young (ie, <60 years of age) and old (ie, ≥60 years of age) subjects. Second, we measured proteins, via immunoblotting, involved in mitophagy (ie, Parkin) and also mitochondrial-induced inflammatory pathways, specifically TLR (toll-like receptor) 9, STING (stimulator of interferon genes), and IFN (interferon)-ß. RESULTS: Proteomics revealed that aging transformed the aorta both quantitatively and qualitatively from health to TAA. Whereas young aortas exhibited an enrichment of immunologic processes, older aortas exhibited an enrichment of metabolic processes. Immunoblotting revealed that the expression of Parkin directly correlated to subject age in health but inversely to subject age in TAA. In TAA, but not in health, phosphorylation of STING and the expression of IFN-ß was impacted by aging regardless of whether subjects had bicuspid or tricuspid valves. In subjects with bicuspid valves and TAAs, TLR9 expression positively correlated with subject age. Interestingly, whereas phosphorylation of STING was inversely correlated with subject age, IFN-ß positively correlated with subject age. CONCLUSIONS: Aging transforms the human aortic proteome from health to TAA, leading to a differential regulation of biological processes. Our results suggest that the development of therapies to mitigate vascular diseases including TAA may need to be modified depending on subject age.
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Aneurisma da Aorta Torácica , Envelhecimento , Aorta/metabolismo , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/metabolismo , Humanos , Interferons , Proteoma , Ubiquitina-Proteína LigasesRESUMO
Phospholamban (PLN) and Sarcolipin (SLN) are homologous membrane proteins that belong to the family of proteins that regulate the activity of the cardiac calcium pump (sarcoplasmic reticulum Ca2+-ATPase, SERCA). PLN and SLN share highly conserved leucine zipper motifs that control self-association; consequently, it has been proposed that both PLN and SLN assemble into stable pentamers in the membrane. In this study, we used molecular dynamics (MD) simulations and Western blot analysis to investigate the precise molecular architecture of the PLN and SLN oligomers. Analysis showed that the PLN pentamer is the predominant oligomer present in mouse ventricles and ventricle-like human iPSC-derived cardiomyocytes, in agreement with the MD simulations showing stable leucine zipper interactions across all protomer-protomer interfaces and MD replicates. Interestingly, we found that the PLN pentamer populates an asymmetric structure of the transmembrane region, which is likely an intrinsic feature of the oligomer in a lipid bilayer. The SLN pentamer is not favorably formed across MD replicates and species of origin; instead, SLN from human and mouse atria primarily populate coexisting dimeric and trimeric states. In contrast to previous studies, our findings indicate that the SLN pentamer is not the predominant oligomeric state populated in the membrane. We conclude that despite their structural homology, PLN and SLN adopt distinct oligomeric states in the membrane. We propose that the distinct oligomeric states populated by PLN and SLN may contribute to tissue-specific SERCA regulation via differences in protomer-oligomer exchange, oligomer-SERCA dynamics, and noise filtering during ß-adrenergic stimulation in the heart.
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Background: Lung inflammation, neutrophil infiltration, and pulmonary vascular leakage are pathological hallmarks of acute respiratory distress syndrome (ARDS) which can lethally complicate respiratory viral infections. Despite similar comorbidities, however, infections in some patients may be asymptomatic while others develop ARDS as seen with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections for example. Methods: In this study, we infected resistant C57BL/6 and susceptible A/J strains of mice with pulmonary administration of murine hepatitis virus strain 1 (MHV-1) to determine mechanisms underlying susceptibility to pulmonary vascular leakage in a respiratory coronavirus infection model. Results: A/J animals displayed increased lung injury parameters, pulmonary neutrophil influx, and deficient recruitment of other leukocytes early in the infection. Moreover, under basal conditions, A/J neutrophils overexpressed primary granule protein genes for myeloperoxidase and multiple serine proteases. During infection, myeloperoxidase and elastase protein were released in the bronchoalveolar spaces at higher concentrations compared to C57BL/6 mice. In contrast, genes from other granule types were not differentially expressed between these 2 strains. We found that depletion of neutrophils led to mitigation of lung injury in infected A/J mice while having no effect in the C57BL/6 mice, demonstrating that an altered neutrophil phenotype and recruitment profile is a major driver of lung immunopathology in susceptible mice. Conclusions: These results suggest that host susceptibility to pulmonary coronaviral infections may be governed in part by underlying differences in neutrophil phenotypes, which can vary between mice strains, through mechanisms involving primary granule proteins as mediators of neutrophil-driven lung injury.