Neutrophils play key protective roles in influenza infections, yet excessive neutrophilic inflammation is a hallmark of acute lung injury during severe infections. Phenotypic heterogeneity is increasingly recognized in neutrophil populations; however, how functional variation in neutrophils between individuals determine the diverse outcomes of influenza remains unclear. To examine immunologic responses that may drive varying outcomes in influenza, we infected C57BL/6 (B6) and A/J mice with mouse-adapted influenza A virus A/PR/8/34 H1N1. A self-resolving dose in B6 mice was lethal in A/J mice, which had increased viral load throughout infection accompanied by prominent bronchoalveolar neutrophilia and pulmonary vascular leakage preceding mortality. Notably, the B6 mice heavily recruited neutrophils to lungs early in infection while A/J mice failed to do so. Neutrophils from A/J mice additionally displayed reduced neutrophil extracellular trap (NET) release and reactive oxygen species (ROS) generation compared to B6 mice early in infection, suggesting the failure to control virus in A/J mice was a product of deficient neutrophil response. To determine if variation in neutrophils between strains governed viral control and inflammation, we adoptively transferred bone marrow neutrophils from B6 or A/J donors to A/J recipients early in infection and found that the transfer of B6 neutrophils enhanced viral clearance and abrogated the dissemination of CXCL1 and IL-6. The transfer of A/J neutrophils, however, failed to achieve either. Furthermore, B6 neutrophils were capable of greater levels of viral killing in vitro than their A/J counterparts. These results suggest that a key moderator of inflammation in influenza infection is the control of virus by neutrophils early in infection. Thus, host-specific differences in both the recruitment of these cells as well as interindividual variation in neutrophil ability to support viral clearance may in part dictate differing susceptibility to respiratory viral infections.

Abstract Introduction: Type I IFNs play a critical role in the immune response to viral infections. Suppressed type I IFN levels are predictive of severe SARS-CoV-2 infections. Given the critical role of neutrophils in viral-mediated lung injury, we investigated how type I IFNs regulated neutrophil responses during pulmonary coronaviral infections. Methods: IFNAR-/- and C57BL/6 (WT) mice were infected with 2,500PFU of murine beta-coronavirus (MHV-1) and survival determined. At day 3 post infection, cellular recruitment, cytokine profiles and markers of lung injury were evaluated. Neutrophils were evaluated for activation markers, ROS generation and NET formation. Results: All IFNAR-/- mice succumbed within 5 days versus 100% survival for WT mice. IFNAR-/- mice had higher pulmonary levels of GM-CSF (p=0.0079) and 3-fold more recruited neutrophils (p=0.0159) and monocytes (p=0.016). IFNAR-/- lung neutrophils had significantly lower TLR3, TLR4, TLR7, TLR9, CD88, CXCR2, CD69 (p<0.05 for all) but increased levels of PDL-1. IFNAR-/- neutrophils isolated during infection showed significantly decreased ROS and NETs compared to WT. Conclusion:IFNAR-/- mice are highly susceptible to MHV-1, displaying elevated neutrophil recruitment with lower frequency of activation markers, reduced ROS and NETs, and increased expression of PDL-1. Our findings demonstrate mechanisms by which inadequate type I IFN levels subvert critical aspects of neutrophil responses during severe viral respiratory infections.

Abstract Introduction: IL-27 is elevated during COPD, ARDS and high levels are predictive of severe SARS-CoV-2 infection. Despite the association with severe pulmonary outcomes, little is understood on how it affects lung injury or cellular recruitment early in viral pulmonary infection. Methods: C57BL/6 (WT) and IL-27ra-/- mice were infected with 500,000PFU of murine beta-coronavirus (MHV-1) or 500PFU of Influenza (H1N1) and evaluated for survival. At days 3 and 5 post infection kinetics of cellular recruitment, viral load and markers of lung injury were evaluated. Results: IL-27ra-/- mice have increased survival compared to the WT mice when infected with MHV-1 (p= 0.0184) and H1N1 (p=0.0305). Despite no differences in viral load, the IL-27ra-/- had decreased lung injury at days 3 and 5 post infection (p<0.05) for both MHV-1 and H1N1. IL-27ra-/- mice had significant neutrophilia during infection at day 3 and 5 (p<0.05) but with altered neutrophil phenotypes (adhesion, activation and maturation markers) including decreased expression of TLR3, TLR4 and CD49d (p<0.05 for all). Conclusion: The IL-27ra-/- mice are resistant to pulmonary infection with MHV-1 and H1N1 with increased survival but no differences in viral load. Despite high pulmonary neutrophilia in both infections, the IL-27ra-/- mice paradoxically have decreased lung injury. This study highlights IL-27 as a potential early target for altering neutrophil phenotypes to limit lung injury during respiratory viral infections.

Neutrophils are an essential cellular component of innate immunity and control bacterial infections through a combination of intracellular and extracellular killing methods. Although the importance of neutrophils has been established, the exact methods used to handle particular bacterial challenges and the efficiency of bacterial killing remain not well understood. In this study, we addressed how neutrophils eliminate Streptococcus pneumoniae (Spn), a leading cause of community acquired and post-influenza bacterial pneumonia. We analyzed killing methods with variable bacterial:neutrophil concentrations and following priming with PAM3CSK4 (P3CSK), an agonist for Toll-like-receptor 2 (TLR2). Our results show that murine neutrophils display surprisingly weak bactericidal activity against Spn, employing a predominantly extracellular mode of killing at lower concentrations of bacteria, whereas challenges with higher bacterial numbers induce both extracellular and intracellular elimination modes but require TLR2 activation. TLR2 activation increased reactive oxygen species (ROS) and neutrophil extracellular trap (NET) formation in response to Spn. Despite this, supernatants from P3CSK-stimulated neutrophils failed to independently alter bacterial replication. Our study reveals that unstimulated neutrophils are capable of eliminating bacteria only at lower concentrations via extracellular killing methods, whereas TLR2 activation primes neutrophil-mediated killing using both intracellular and extracellular methods under higher bacterial burdens.

BackgroundLung 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.MethodsIn 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.ResultsA/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.ConclusionsThese 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.

Severe respiratory viral infections, including SARS-CoV-2, have resulted in high mortality rates despite corticosteroids and other immunomodulatory therapies. Despite recognition of the pathogenic role of neutrophils, in-depth analyses of this cell population have been limited, due to technical challenges of working with neutrophils. We undertook an unbiased, detailed analysis of neutrophil responses in adult patients with COVID-19 and healthy controls, to determine whether distinct neutrophil phenotypes could be identified during infections compared to the healthy state. Single-cell RNA sequencing analysis of peripheral blood neutrophils from hospitalized patients with mild or severe COVID-19 disease and healthy controls revealed distinct mature neutrophil subpopulations, with relative proportions linked to disease severity. Disruption of predicted cell-cell interactions, activated oxidative phosphorylation genes, and downregulated antiviral and host defense pathway genes were observed in neutrophils obtained during severe compared to mild infections. Our findings suggest that during severe infections, there is a loss of normal regulatory neutrophil phenotypes seen in healthy subjects, coupled with the dropout of appropriate cellular interactions. Given that neutrophils are the most abundant circulating leukocytes with highly pathogenic potential, current immunotherapies for severe infections may be optimized by determining whether they aid in restoring an appropriate balance of neutrophil subpopulations.

Background: SARS-CoV-2, the virus causing COVID-19 infection, can significantly contaminate environmental surfaces and can remain viable on surfaces for up to 9 days. Although respiratory route remains the most significant mode of transmission, fomites and environmental sources of infection remain a concern for healthcare personnel who are working in dedicated COVID-19 units. We investigated the extent of detectable SARS-CoV-2 contamination in the environment of COVID-19 patients at a single VA hospital, with the intent of identifying potential high-touch surfaces at risk for viral contamination, which could be used to inform the development of simple COVID-19 prevention strategies. Methods: We conducted a cohort study at 1 VA hospital in a unit housing adult veterans admitted with COVID-19 between October and December 2020. In total, 11 swab specimens were collected for PCR analysis (SARS-CoV-2 env gene) from environmental surfaces inside and just outside the rooms of COVID-19 patients one time. Retrospective chart reviews were conducted to provide the SARS-CoV-2 epidemiologic context for environmental detection. Results: In total, 297 swabs were collected from the unit and environmental areas surrounding 27 hospitalized patients: average age, 72.5 years (range, 34–94); 100% male; 92% non-Hispanic white; average comorbidities, 1.8 (SD, 1.1). Of 297 swabs, 80 (27%) were positive for SARS-CoV-2 and 19 (70%) of 27 patients had at least 1 positive site. The most contaminated site was the floor just outside the patient room (78% positive samples), followed by the patient’s bedrail (37%) and chair handle (37%) (Fig. 1). Traditionally high-touch surfaces, such as the door handle (outside patient room) and the light switch, did not have high positivity rates (<15%). Interestingly, both the personal protective equipment (PPE) cart outside patient’s room (33%) and the double doors leading out of the unit (19%) were positive, which are surfaces often touched with bare hands after handwashing. Analyses of clinical data are underway to examine whether specific care needs, based on activities of daily living disability, comorbidities, and clinical presentation of COVID-19, predict SARS-CoV-2 environmental contamination. Conclusions: The presence of environmental contamination by SARS-CoV-2 highlights the importance of transmission via direct or indirect contact. Studies targeting high-risk populations are needed to better understand the transmission of SARS-CoV-2 between infected patients and their environment. Our findings also suggest that handwashing and attention to using disinfecting wipes may mitigate the risk of transmission of virus from surfaces that one might consider safe to touch.Funding: NoneDisclosures: None

Severe cases of COVID-19 are characterized by dysregulated immune responses, but specific mechanisms contributing to the most severe outcomes remain unclear. Neutrophils are the most abundant leukocyte population in human hosts and reach markedly high numbers during severe COVID-19. However, a detailed examination of their responses has been largely overlooked in the COVID-19 literature to date. Here, we report for the first time a dedicated study of neutrophil responses using single-cell RNA sequencing (scRNA-Seq) of fresh leukocytes from 11 hospitalized adult patients with mild and severe COVID-19 disease and 5 healthy controls. We observed that neutrophils display a pronounced inflammatory profile, with dramatic disruption of predicted cell-cell interactions as the severity of the disease increases. We also identified unique mature and immature neutrophil subpopulations based on transcriptomic profiling, including an antiviral phenotype, and changes in the proportion of each population linked to the severity of the disease. Finally, pathway analysis revealed increased markers of oxidative phosphorylation and ribosomal genes, along with downregulation of many antiviral and host defense pathway genes during severe disease compared to mild infections. Collectively, our findings indicate that neutrophils are capable of mounting effective antiviral defenses but adopt a form of immune dysregulation characterized by excess cellular stress, thereby contributing to the pathogenesis of severe COVID-19.

Influenza and other respiratory viral infections are the most common type of acute respiratory infection. Viral infections predispose patients to secondary bacterial infections, which often have a more severe clinical course. The mechanisms underlying post-viral bacterial infections are complex, and include multifactorial processes mediated by interactions between viruses, bacteria, and the host immune system. Studies over the past 15 years have demonstrated that unique microbial communities reside on the mucosal surfaces of the gastrointestinal tract and the respiratory tract, which have both direct and indirect effects on host defense against viral infections. In addition, antiviral immune responses induced by acute respiratory infections such as influenza are associated with changes in microbial composition and function (“dysbiosis”) in the respiratory and gastrointestinal tract, which in turn may alter subsequent immune function against secondary bacterial infection or alter the dynamics of inter-microbial interactions, thereby enhancing the proliferation of potentially pathogenic bacterial species. In this review, we summarize the literature on the interactions between host microbial communities and host defense, and how influenza, and other acute respiratory viral infections disrupt these interactions, thereby contributing to the pathogenesis of secondary bacterial infections.