Lung microvascular occlusion by platelet-rich neutrophil-platelet aggregates promotes cigarette smoke-induced severe flu.

Cigarette smoking is associated with a higher risk of ICU admissions among patients with flu. However, the etiological mechanism by which cigarette smoke (CS) exacerbates flu remains poorly understood. Here, we show that a mild dose of influenza A virus promotes a severe lung injury in mice preexposed to CS but not room air for 4 weeks. Real-time intravital (in vivo) lung imaging revealed that the development of acute severe respiratory dysfunction in CS- and flu-exposed mice was associated with the accumulation of platelet-rich neutrophil-platelet aggregates (NPAs) in the lung microcirculation within 2 days following flu infection. These platelet-rich NPAs formed in situ and grew larger over time to occlude the lung microvasculature, leading to the development of pulmonary ischemia followed by the infiltration of NPAs and vascular leakage into the alveolar air space. These findings suggest, for the first time to our knowledge, that an acute onset of platelet-driven thrombo-inflammatory response in the lung contributes to the development of CS-induced severe flu.

Influenza Suppresses Neutrophil Recruitment to the Lung and Exacerbates Secondary Invasive Pulmonary Aspergillosis.

Aspergillus fumigatus is an environmental fungus that can cause invasive pulmonary aspergillosis when spores are inhaled into the respiratory tract and invade airway or lung tissue. Influenza is a common respiratory virus that can cause severe respiratory disease, and postinfluenza invasive pulmonary aspergillosis, which is becoming a well-recognized clinical problem, typically occurs in critically ill patients. Mice challenged with influenza A PR/8/34 H1N1 and subsequently challenged with A. fumigatus had increased fungal burden, viral burden, inflammation, and mortality compared with single infected mice. Neutrophil recruitment in the lung of superinfected mice was decreased; however, mice were not neutropenic, and there was no difference in absolute blood neutrophils between groups. Additionally, CXCL1 and CXCL2 were decreased in lungs of superinfected mice compared with controls. IFN levels were increased in mice that received influenza, and deletion of STAT1 resulted in decreased fungal burden, increased airway and lung neutrophils, and increased CXCL1 compared with wild-type mice, whereas deletion of STAT2 did not change fungal burden or airway neutrophilia compared with wild-type mice. These data demonstrate a mechanism by which influenza A-induced STAT1 signaling inhibits neutrophil recruitment and increases susceptibility to postinfluenza invasive pulmonary aspergillosis.

COVID-19 and the Early-Career Physician-Scientist. Fostering Resilience beyond the Pandemic.

The coronavirus disease (COVID-19) pandemic has created significant stressors for the academic and scientific community, with unique challenges for early-career physician-scientists. The pandemic-related disruptions have significantly affected research productivity, access to mentoring, professional development and networking opportunities, funding, and personal wellness. This is especially true for pulmonary and critical care medicine faculty because of the burden of specialized clinical care responsibilities that the COVID-19 pandemic has demanded. Departmental, institutional, and national leadership should foster open dialogue to identify and mitigate these challenges to promote ongoing career development of early-career physician-scientists. Implementation of thoughtful interventions to address these challenges will provide essential support for junior faculty and help retain a generation of physician-scientists.

Survival during influenza-associated bacterial superinfection improves following viral- and bacterial-specific monoclonal antibody treatment.

Postinfluenza bacterial superinfections cause increased morbidity and mortality compared with singular infection with influenza during both pandemics and seasonal epidemics. Vaccines and current treatments provide limited benefit, a rationale to conduct studies utilizing alternative therapies. FY1 and an optimized version, MEDI8852, anti-influenza HA mAbs, have been shown to neutralize influenza virus during singular influenza infection. MEDI4893*, an anti-Staphylococcus aureus α-toxin mAb, has been shown to improve survival when administered prophylactically prior to S. aureus pneumonia. Our objective was to determine if mAbs can improve survival during postinfluenza bacterial pneumonia. We administered FY1 in a murine model of postinfluenza methicillin-resistant S. aureus (MRSA) pneumonia and observed improved survival rates when given early during the course of influenza infection. Our findings indicate decreased lung injury and increased uptake and binding of bacteria by macrophages in the mice that received FY1 earlier in the course of influenza infection, corresponding to decreased bacterial burden. We also observed improved survival when mice were treated with a combination of FY1 and MEDI4893* late during the course of postinfluenza MRSA pneumonia. In conclusion, both FY1 and MEDI4893* prolong survival when used in a murine model of postinfluenza MRSA pneumonia, suggesting pathogen-specific mAbs as a possible therapeutic in the context of bacterial superinfection.

IL-22-binding protein exacerbates influenza, bacterial super-infection.

Secondary bacterial pneumonia is a significant complication of severe influenza infection and Staphylococcus aureus and Streptococcus pneumoniae are the primary pathogens of interest. IL-22 promotes S. aureus and S. pneumoniae host defense in the lung through epithelial integrity and induction of antimicrobial peptides and is inhibited by the soluble decoy receptor IL-22-binding protein (IL-22BP). Little is known about the effect of the IL-22/IL-22BP regulatory pathway on lung infection, and it has not been studied in the setting of super-infection. We exposed wild-type and IL-22BP-/- mice to influenza A/PR/8/34 for 6 days prior to infection with S. aureus (USA300) S. pneumoniae. Super-infected IL-22BP-/- mice had decreased bacterial burden and improved survival compared to controls. IL-22BP-/- mice exhibited decreased inflammation, increased lipocalin 2 expression, and deletion of IL-22BP was associated with preserved epithelial barrier function with evidence of improved tight junction stability. Human bronchial epithelial cells treated with IL-22Fc showed evidence of improved tight junctions compared to untreated cells. This study revealed that IL-22BP-/- mice are protected during influenza, bacterial super-infection, suggesting that IL-22BP has a pro-inflammatory role and impairs epithelial barrier function likely through interaction with IL-22.

IL-33-mediated IL-13 secretion by ST2+ Tregs controls inflammation after lung injury.

Acute respiratory distress syndrome is an often fatal disease that develops after acute lung injury and trauma. How released tissue damage signals, or alarmins, orchestrate early inflammatory events is poorly understood. Herein we reveal that IL-33, an alarmin sequestered in the lung epithelium, is required to limit inflammation after injury due to an unappreciated capacity to mediate Foxp3+ Treg control of local cytokines and myeloid populations. Specifically, Il33-/- mice are more susceptible to lung damage-associated morbidity and mortality that is typified by augmented levels of the proinflammatory cytokines and Ly6Chi monocytes in the bronchoalveolar lavage fluid. Local delivery of IL-33 at the time of injury is protective but requires the presence of Treg cells. IL-33 stimulates both mouse and human Tregs to secrete IL-13. Using Foxp3Cre × Il4/Il13fl/fl mice, we show that Treg expression of IL-13 is required to prevent mortality after acute lung injury by controlling local levels of G-CSF, IL-6, and MCP-1 and inhibiting accumulation of Ly6Chi monocytes. Our study identifies a regulatory mechanism involving IL-33 and Treg secretion of IL-13 in response to tissue damage that is instrumental in limiting local inflammatory responses and may shape the myeloid compartment after lung injury.

Interferon Lambda Inhibits Bacterial Uptake during Influenza Superinfection.

Influenza kills 30,000 to 40,000 people each year in the United States and causes 10 times as many hospitalizations. A common complication of influenza is bacterial superinfection, which exacerbates morbidity and mortality from the viral illness. Recently, methicillin-resistant Staphylococcus aureus (MRSA) has emerged as the dominant pathogen found in bacterial superinfection, with Streptococcus pneumoniae a close second. However, clinicians have few tools to treat bacterial superinfection. Current therapy for influenza/bacterial superinfection consists of treating the underlying influenza infection and adding various antibiotics, which are increasingly rendered ineffective by rising bacterial multidrug resistance. Several groups have recently proposed the use of the antiviral cytokine interferon lambda (IFN-λ) as a therapeutic for influenza, as administration of pegylated IFN-λ improves lung function and survival during influenza by reducing the overabundance of neutrophils in the lung. However, our data suggest that therapeutic IFN-λ impairs bacterial clearance during influenza superinfection. Specifically, mice treated with an adenoviral vector to overexpress IFN-λ during influenza infection exhibited increased bacterial burdens upon superinfection with either MRSA or S. pneumoniae Surprisingly, adhesion molecule expression, antimicrobial peptide production, and reactive oxygen species activity were not altered by IFN-λ treatment. However, neutrophil uptake of MRSA and S. pneumoniae was significantly reduced upon IFN-λ treatment during influenza superinfection in vivo Together, these data support the theory that IFN-λ decreases neutrophil motility and function in the influenza-infected lung, which increases the bacterial burden during superinfection. Thus, we believe that caution should be exercised in the possible future use of IFN-λ as therapy for influenza.

STAT2 Signaling Regulates Macrophage Phenotype During Influenza and Bacterial Super-Infection.

Influenza is a common respiratory virus that infects between 5 and 20% of the US population and results in 30,000 deaths annually. A primary cause of influenza-associated death is secondary bacterial pneumonia. We have previously shown that influenza induces type I interferon (IFN)-mediated inhibition of Type 17 immune responses, resulting in exacerbation of bacterial burden during influenza and Staphylococcus aureus super-infection. In this study, we investigated the role of STAT2 signaling during influenza and influenza-bacterial super-infection in mice. Influenza-infected STAT2-/- mice had increased morbidity, viral burden, and inflammation when compared to wild-type mice. Despite an exaggerated inflammatory response to influenza infection, we found increased bacterial control and survival in STAT2 deficient mice during influenza-MRSA super-infection compared to controls. Further, we found that increased bacterial clearance during influenza-MRSA super-infection is not due to rescue of Type 17 immunity. Absence of STAT2 was associated with increased accumulation of M1, M2 and M1/M2 co-expressing macrophages during influenza-bacterial super-infection. Neutralization of IFNγ (M1) and/or Arginase 1 (M2) impaired bacterial clearance in Stat2-/- mice during super-infection, demonstrating that pulmonary macrophages expressing a mixed M1/M2 phenotype promote bacterial control during influenza-bacterial super-infection. Together, these results suggest that the STAT2 signaling is involved in suppressing macrophage activation and bacterial control during influenza-bacterial super-infection. Further, these studies reveal novel mechanistic insight into the roles of macrophage subpopulations in pulmonary host defense.

The inflammasome potentiates influenza/Staphylococcus aureus superinfection in mice.

Secondary bacterial respiratory infections are commonly associated with both acute and chronic lung injury. Influenza complicated by bacterial pneumonia is an effective model to study host defense during pulmonary superinfection due to its clinical relevance. Multiprotein inflammasomes are responsible for IL-1ß production in response to infection and drive tissue inflammation. In this study, we examined the role of the inflammasome during viral/bacterial superinfection. We demonstrate that ASC-/- mice are protected from bacterial superinfection and produce sufficient quantities of IL-1ß through an apoptosis-associated speck-like protein containing CARD (ASC) inflammasome-independent mechanism. Despite the production of IL-1ß by ASC-/- mice in response to bacterial superinfection, these mice display decreased lung inflammation. A neutrophil elastase inhibitor blocked ASC inflammasome-independent production of IL-1ß and the IL-1 receptor antagonist, anakinra, confirmed that IL-1 remains crucial to the clearance of bacteria during superinfection. Delayed inhibition of NLRP3 during influenza infection by MCC950 decreases bacterial burden during superinfection and leads to decreased inflammatory cytokine production. Collectively, our results demonstrate that ASC augments the clearance of bacteria, but can also contribute to inflammation and mortality. ASC should be considered as a therapeutic target to decrease morbidity and mortality during bacterial superinfection.

Effect of aerosolized antipseudomonals on Pseudomonas positivity and bronchiolitis obliterans syndrome after lung transplantation.

PURPOSE: To describe the effects of aerosolized antipseudomonals (AAPs) on Pseudomonas (PS) culture positivity, bronchiolitis obliterans syndrome (BOS), and acute cellular rejection (ACR) in lung transplant recipients (LTRs). METHODS: Single-center, retrospective cohort study was performed of adult LTRs treated with either AAP for ≥28 days vs no AAP therapy or AAP therapy <28 days, indexed to a matched median date post lung transplantation (LT). Primary outcome was freedom from PS positivity by positive bronchoalveolar lavage or bronchial wash at 1 year. Secondary outcomes were freedom from BOS or BOS progression and ACR burden (defined by the novel composite rejection standardized score. Normality was assessed, and univariate and multivariate parametric and non-parametric statistical tests were used to assess baseline characteristics and outcomes, where appropriate. Freedom from events was compared using the Kaplan-Meier method with log-rank conversion and risk was assigned using multivariable Cox proportional hazards (PH) modeling. RESULTS: In total, 293 LTRs (105 with AAP, 188 with no AAP) were included. Median ages in AAP and control cohorts were 51 (30-63) and 62 (54-67) years (P<.01). Median AAP duration was 198 (interquartile range 94-395) days. Time to median positive PS culture was similar between AAP (median 1.02 [95% confidence interval {CI} 0.74-1.22] years) and control (median 0.96 [95% CI 0.72-1.21] years). Log-rank test for time-to-PS positivity was similar for both groups (log-rank P=.26). Incidence of PS culture positivity at 1 year was similar in APP vs controls (59.0% vs 54.8%, P=.48). In the non-cystic fibrosis (CF) subgroup, AAP use was protective against PS recurrence on univariate Cox PH model (hazard ratio [HR] 0.55, 95% CI 0.38-0.83) and on multivariate Cox PH adjusting for age and induction (HR 0.56, 95% CI 0.38-0.83). Incidence of new-onset BOS or BOS progression in APP vs control at 1 (17.1% vs 14.9%, P=.61) and 3 (38.1% vs 37.8%, P=.96) years was similar. CRSS was similar in APP vs control group at 1 year (0.42 vs 0.33, P=.41). CONCLUSION: AAP use was not associated with less PS positivity, BOS, or ACR in all LTRs. In the non-CF subgroup analysis, treatment with AAPS was associated with protection against recurrent PS. Limitations include retrospective design, heterogeneous AAP therapy among LTRs, and potential convenience sampling of LTRs receiving AAPs for >28 days at our center. Larger assessments and better controlled analyses are required to further define efficacy of AAPs after LT.

Epigenetic and Transcriptomic Regulation of Lung Repair during Recovery from Influenza Infection.

Seasonal and pandemic influenza is a cause of morbidity and mortality worldwide. Most people infected with influenza virus display mild-to-moderate disease phenotypes and recover within a few weeks. Influenza is known to cause persistent alveolitis in animal models; however, little is known about the molecular pathways involved in this phenotype. We challenged C57BL/6 mice with influenza A/PR/8/34 and examined lung pathologic processes and inflammation, as well as transcriptomic and epigenetic changes at 21 to 60 days after infection. Influenza induced persistent parenchymal lung inflammation, alveolar epithelial metaplasia, and epithelial endoplasmic reticulum stress that were evident after the clearance of virus and resolution of morbidity. Influenza infection induced robust changes in the lung transcriptome, including a significant impact on inflammatory and extracellular matrix protein expression. Despite the robust changes in lung gene expression, preceding influenza (21 days) did not exacerbate secondary Staphylococcus aureus infection. Finally, we examined the impact of influenza on miRNA expression in the lung and found an increase in miR-155. miR-155 knockout mice recovered from influenza infection faster than controls and had decreased lung inflammation and endoplasmic reticulum stress. These data illuminate the dynamic molecular changes in the lung in the weeks after influenza infection and characterize the repair process, identifying a novel role for miR-155.

STAT1 Is Required for Suppression of Type 17 Immunity during Influenza and Bacterial Superinfection.

Influenza is an annual, global health care concern. Secondary bacterial pneumonia is a severe complication associated with primary influenza virus infection, often resulting in critical morbidity and mortality. Our laboratory has identified influenza-induced suppression of anti-bacterial Type 17 immunity as a mechanism for enhanced susceptibility to bacterial super-infection. We have shown that influenza-induced type I interferon impairs Type 17 activation. STAT1 is a transcription factor involved in interferon signaling, shared by type I, II, and III interferon. In this work, we investigated the role of STAT1 signaling during influenza, methicillin-resistant Staphylococcus aureus (MRSA) super-infection. STAT1-/- mice had increased morbidity and airway inflammation compared to control mice during influenza mono-infection. Despite this worsened anti-viral response, STAT1-/- mice were protected from super-infection bacterial burden and mortality compared to controls. Type 17 immune activation was increased in lymphocytes in STAT1-/- mice during super-infection. The elevation in Type 17 immunity was not related to increased IL-23 production, as type I interferon could inhibit IL-23 expression in a STAT1 independent manner. STAT1-/- antigen presenting cells were inherently biased towards Type 17 polarization compared to control cells. Further, STAT1-/- dendritic cells produced attenuated IL-6 and TNFα upon heat-killed S. aureus stimulation compared to control. Overall, these data indicate that STAT1 signaling plays a detrimental role in influenza, MRSA super-infection by controlling the magnitude of Type 17 immune activation.

Influenza and Bacterial Superinfection: Illuminating the Immunologic Mechanisms of Disease.

Seasonal influenza virus infection presents a major strain on the health care system. Influenza virus infection has pandemic potential, which was repeatedly observed during the last century. Severe disease may occur in the young, in the elderly, in those with preexisting lung disease, and in previously healthy individuals. A common cause of severe influenza pathogenesis is superinfection with bacterial pathogens, namely, Staphylococcus aureus and Streptococcus pneumoniae. A great deal of recent research has focused on the immune pathways involved in influenza-induced susceptibility to secondary bacterial pneumonia. Both innate and adaptive antibacterial host defenses are impaired in the context of preceding influenza virus infection. The goal of this minireview is to highlight these findings and synthesize these data into a shared central theme of pathogenesis.

Influenza-induced type I interferon enhances susceptibility to gram-negative and gram-positive bacterial pneumonia in mice.

Suppression of type 17 immunity by type I interferon (IFN) during influenza A infection has been shown to enhance susceptibility to secondary bacterial pneumonia. Although this mechanism has been described in coinfection with gram-positive bacteria, it is unclear whether similar mechanisms may impair lung defense against gram-negative infections. Furthermore, precise delineation of the duration of type I IFN-associated susceptibility to bacterial infection remains underexplored. Therefore, we investigated the effects of preceding influenza A virus infection on subsequent challenge with the gram-negative bacteria Escherichia coli or Pseudomonas aeruginosa and the temporal association between IFN expression with susceptibility to Staphylococcus aureus challenge in a mouse model of influenza and bacterial coinfection. Here we demonstrate that preceding influenza A virus led to increased lung E. coli and P. aeruginosa bacterial burden, which was associated with suppression of type 17 immunity and attenuation of antimicrobial peptide expression. Enhanced susceptibility to S. aureus coinfection ceased at day 14 of influenza infection, when influenza-associated type I IFN levels had returned to baseline levels, further suggesting a key role for type I IFN in coinfection pathogenesis. These findings further implicate type I IFN-associated suppression of type 17 immunity and antimicrobial peptide production as a conserved mechanism for enhanced susceptibility to both gram-positive and gram-negative bacterial coinfection during influenza infection.

The immunology of influenza virus-associated bacterial pneumonia.

Infection with influenza virus has been a significant cause of morbidity and mortality for more than a hundred years. Severe disease and increased mortality often results from bacterial super-infection of patients with influenza virus infection. Preceding influenza infection alters the host's innate and adaptive immune responses, allowing increased susceptibility to secondary bacterial pneumonia. Recent advances in the field have helped to define how influenza alters the immune response to bacteria through the dysregulation of phagocytes, antimicrobial peptides, and lymphocytes. Viral-induced interferons play a key role in altering the phenotype of the immune response. Potential genetic modifiers of disease will help to define additional immunologic mechanisms that predispose to viral, bacterial super-infection with the overarching goal of developing effective therapeutic strategies to prevent and treat disease.

The role of IL-27 in susceptibility to post-influenza Staphylococcus aureus pneumonia.

BACKGROUND: Influenza is a common respiratory virus and Staphylococcus aureus frequently causes secondary pneumonia during influenza infection, leading to increased morbidity and mortality. Influenza has been found to attenuate subsequent Type 17 immunity, enhancing susceptibility to secondary bacterial infections. IL-27 is known to inhibit Type 17 immunity, suggesting a potential critical role for IL-27 in viral and bacterial co-infection. METHODS: A murine model of influenza and Staphylococcus aureus infection was used to mimic human viral, bacterial co-infection. C57BL/6 wild-type, IL-27 receptor α knock-out, and IL-10 knock-out mice were infected with Influenza H1N1 (A/PR/8/34) or vehicle for 6 days followed by challenge with Staphylococcus aureus or vehicle for 24 hours. Lung inflammation, bacterial burden, gene expression, and cytokine production were determined. RESULTS: IL-27 receptor α knock-out mice challenged with influenza A had increased morbidity compared to controls, but no change in viral burden. IL-27 receptor α knock-out mice infected with influenza displayed significantly decreased IL-10 production compared to wild-type. IL-27 receptor α knock-out mice co-infected with influenza and S. aureus had improved bacterial clearance compared to wild-type controls. Importantly, there were significantly increased Type 17 responses and decreased IL-10 production in IL-27 receptor α knock-out mice. Dual infected IL-10-/- mice had significantly less bacterial burden compared to dual infected WT mice. CONCLUSIONS: These data reveal that IL-27 regulates enhanced susceptibility to S. aureus pneumonia following influenza infection, potentially through the induction of IL-10 and suppression of IL-17.

A tale of two cytokines: IL-17 and IL-22 in asthma and infection.

The Th17 pathway has recently been shown to play a critical role in host defense, allergic responses and autoimmune inflammation. Th17 cells predominantly produce IL-17 and IL-22, which are two cytokines with broad effects in the lung and other tissues. This review summarizes not only what is currently known about the molecular regulation of this pathway and Th17-related cytokine signaling, but also the roles of these cytokines in pathogen immunity and asthma. In the last 5 years, the Th17 field has rapidly grown and research has revealed that the Th17 pathway is essential in lung pathogenesis in response to exogenous stimuli. As work in the field continues, it is expected that many exciting therapeutic advances will be made for a broad range of diseases.

Influenza A virus exacerbates Staphylococcus aureus pneumonia in mice by attenuating antimicrobial peptide production.

Influenza A represents a significant cause of morbidity and mortality worldwide. Bacterial complications of influenza A confer the greatest risk to patients. TH17 pathway inhibition has been implicated as a mechanism by which influenza A alters bacterial host defense. Here we show that preceding influenza causes persistent Staphylococcus aureus infection and suppression of TH17 pathway activation in mice. Influenza does not inhibit S. aureus binding and uptake by phagocytic cells but instead attenuates S. aureus induced TH17 related antimicrobial peptides necessary for bacterial clearance in the lung. Importantly, exogenous lipocalin 2 rescued viral exacerbation of S. aureus infection and decreased free iron levels in the bronchoalveolar lavage from mice coinfected with S. aureus and influenza. These findings indicate a novel mechanism by which influenza A inhibits TH17 immunity and increases susceptibility to secondary bacterial pneumonia. Identification of new mechanisms in the pathogenesis of bacterial pneumonia could lead to future therapeutic targets.

Influenza A exacerbates Staphylococcus aureus pneumonia by attenuating IL-1ß production in mice.

Pneumonia is a leading cause of death worldwide. Staphylococcal aureus can be a cause of severe pneumonia alone or a common pathogen in secondary pneumonia following influenza. Recently, we reported that preceding influenza attenuated the Type 17 pathway, increasing the lung's susceptibility to secondary infection. IL-1ß is known to regulate host defense, including playing a role in Th17 polarization. We examined whether IL-1ß signaling is required for S. aureus host defense and whether influenza infection impacted S. aureus-induced IL-1ß production and subsequent Type 17 pathway activation. Mice were challenged with S. aureus (USA 300), with or without preceding Influenza A/PR/8/34 H1N1 infection. IL-1R1(-/-) mice had significantly higher S. aureus burden, increased mortality, and decreased Type 17 pathway activation following S. aureus challenge. Coinfected mice had significantly decreased IL-1ß production versus S. aureus infection alone at early time points following bacterial challenge. Preceding influenza did not attenuate S. aureus-induced inflammasome activation, but there was early suppression of NF-κB activation, suggesting an inhibition of NF-κB-dependent transcription of pro-IL-1ß. Furthermore, overexpression of IL-1ß in influenza and S. aureus-coinfected mice rescued the induction of IL-17 and IL-22 by S. aureus and improved bacterial clearance. Finally, exogenous IL-1ß did not significantly rescue S. aureus host defense during coinfection in IL-17RA(-/-) mice or in mice in which IL-17 and IL-22 activity were blocked. These data reveal a novel mechanism by which Influenza A inhibits S. aureus-induced IL-1ß production, resulting in attenuation of Type 17 immunity and increased susceptibility to bacterial infection.