Antimicrobial mitochondrial reactive oxygen species induction by lung epithelial immunometabolic modulation.

Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infection an urgent need. Manipulating the lungs' intrinsic host defenses by therapeutic delivery of certain pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODN) with mitochondrial voltage-dependent anion channel 1 (VDAC1). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), increases mitochondrial membrane potential (ΔΨm), differentially modulates ETC complex activities and consequently results in leak of electrons from ETC complex III and superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy to broadly protect against pneumonia without reliance on antibiotics.

Development of a Corticosteroid-Immunosuppressed Mouse Model to Study the Pathogenesis and Therapy of Influenza-Associated Pulmonary Aspergillosis.

Influenza-associated pulmonary aspergillosis (IAPA) is a feared complication in patients with influenza tracheobronchitis, especially those receiving corticosteroids. Herein, we established a novel IAPA mouse model with low-inoculum Aspergillus infection and compared outcomes in mice with and without cortisone acetate (CA) immunosuppression. CA was an independent predictor of increased morbidity/mortality in mice with IAPA. Early antifungal treatment with liposomal amphotericin B was pivotal to improve IAPA outcomes in CA-immunosuppressed mice, even after prior antiviral therapy with oseltamivir. In summary, our model recapitulates key clinical features of IAPA and provides a robust preclinical platform to study the pathogenesis and treatment of IAPA.

Redox-Dependent Activation of Lung Epithelial STAT3 Is Required for Inducible Protection against Bacterial Pneumonia.

The lung epithelium is dynamic, capable of considerable structural and functional plasticity in response to pathogen challenges. Our laboratory has demonstrated that an inhaled combination of a Toll-like receptor (TLR) 2/6 agonist and a TLR9 agonist (Pam2ODN) results in robust protection against otherwise lethal pneumonias. We have previously shown that intact epithelial TLR signaling and generation of multisource epithelial reactive oxygen species (ROS) are required for inducible protection. Further investigating the mechanisms underlying this phenomenon of inducible resistance, reverse-phase protein array analysis demonstrated robust STAT3 (signal transducer and activator of transcription 3) phosphorylation following treatment of lung epithelial cells. We show here that Pam2ODN-induced STAT3 phosphorylation is IL-6-independent. We further found that therapeutic epithelial STAT3 activation is required for inducible protection against Pseudomonas aeruginosa pneumonia. Additional studies showed that inhibiting epithelial dual oxidases or scavenging ROS significantly reduced the Pam2ODN induction of STAT3 phosphorylation, suggesting a proximal role for ROS in inducible STAT3 activation. Dissecting these mechanisms, we analyzed the contributions of redox-sensitive kinases and found that Pam2ODN activated epithelial growth factor receptor in an ROS-dependent manner that is required for therapeutically inducible STAT3 activation. Taken together, we demonstrate that epithelial STAT3 is imperative for Pam2ODN's function and describe a novel redox-based mechanism for its activation. These key mechanistic insights may facilitate strategies to leverage inducible epithelial resistance to protect susceptible patients during periods of peak vulnerability.

A molecularly engineered antiviral banana lectin inhibits fusion and is efficacious against influenza virus infection in vivo.

There is a strong need for a new broad-spectrum antiinfluenza therapeutic, as vaccination and existing treatments are only moderately effective. We previously engineered a lectin, H84T banana lectin (H84T), to retain broad-spectrum activity against multiple influenza strains, including pandemic and avian, while largely eliminating the potentially harmful mitogenicity of the parent compound. The amino acid mutation at position 84 from histidine to threonine minimizes the mitogenicity of the wild-type lectin while maintaining antiinfluenza activity in vitro. We now report that in a lethal mouse model H84T is indeed nonmitogenic, and both early and delayed therapeutic administration of H84T intraperitoneally are highly protective, as is H84T administered subcutaneously. Mechanistically, attachment, which we anticipated to be inhibited by H84T, was only somewhat decreased by the lectin. Instead, H84T is internalized into the late endosomal/lysosomal compartment and inhibits virus-endosome fusion. These studies reveal that H84T is efficacious against influenza virus in vivo, and that the loss of mitogenicity seen previously in tissue culture is also seen in vivo, underscoring the potential utility of H84T as a broad-spectrum antiinfluenza agent.

Immune Modulation to Improve Survival of Viral Pneumonia in Mice.

Viral pneumonias remain global health threats, as exemplified in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, requiring novel treatment strategies both early and late in the disease process. We have reported that mice treated before or soon after infection with a combination of inhaled Toll-like receptor (TLR) 2/6 and 9 agonists (Pam2-ODN) are broadly protected against microbial pathogens including respiratory viruses, but the mechanisms remain incompletely understood. The objective of this study was to validate strategies for immune modulation in a preclinical model of viral pneumonia and determine their mechanisms. Mice were challenged with the Sendai paramyxovirus in the presence or absence of Pam2-ODN treatment. Virus burden and host immune responses were assessed to elucidate Pam2-ODN mechanisms of action and to identify additional opportunities for therapeutic intervention. Enhanced survival of Sendai virus pneumonia with Pam2-ODN treatment was associated with reductions in lung virus burden and with virus inactivation before internalization. We noted that mortality in sham-treated mice corresponded with CD8+ T-cell lung inflammation on days 11-12 after virus challenge, after the viral burden had declined. Pam2-ODN blocked this injurious inflammation by minimizing virus burden. As an alternative intervention, depleting CD8+ T cells 8 days after viral challenge also decreased mortality. Stimulation of local innate immunity within the lungs by TLR agonists early in disease or suppression of adaptive immunity by systemic CD8+ T-cell depletion late in disease improves outcomes of viral pneumonia in mice. These data reveal opportunities for targeted immunomodulation to protect susceptible human subjects.

Twelve Tips to Succeed as Health Profession Clinical Educator in Resource-Limited Settings.

Health professions education is one of the pillars of academic medicine; however, clinical educators often lack the appropriate resources to succeed in this field. Examples of these challenges include: lack of support for faculty development, mentorship, and high cost of resources, when available. In addition, challenges such as the Coronavirus disease (COVID-19) pandemic can affect healthcare personnel who are already struggling to provide adequate patient care while attempting to succeed in the role of educator and supervisor of trainees. Clinical educators face more challenges particularly in low-middle income countries as the limitations are more prominent and become key barriers to success. Similarly, due to COVID-19, these challenges can be far more evident in disadvantaged geographical, economic, and academic environments even in the United States. Herein, in this perspective paper, we define resource-limited settings in medical education, provide an overview of the most common barriers to career development as a clinical educator, and offer practical strategies to overcome some of these shortcomings.

The RNA receptor RIG-I binding synthetic oligodeoxynucleotide promotes pneumonia survival.

Pneumonia is a worldwide threat to public health, demanding novel preventative and therapeutic strategies. The lung epithelium is a critical environmental interface that functions as a physical barrier to pathogen invasion while also actively sensing and responding to pathogens. We have reported that stimulating lung epithelial cells with a combination therapeutic consisting of a diacylated lipopeptide and a synthetic CpG oligodeoxynucleotide (ODN) induces synergistic pneumonia protection against a wide range of pathogens. We report here that mice deficient in Toll-like receptor 9 (TLR9), the previously described receptor for ODN, still displayed partial ODN-induced protection. This prompted us to seek an alternate ODN receptor, and we discovered by mass spectroscopy that the RNA sensor RIG-I could also bind DNA-like ODN. ODN binding by RIG-I resulted in MAVS-dependent pneumonia-protective signaling events. While RIG-I is essential to native defenses against viral infections, we report that therapeutic RIG-I activation with ODN promoted pathogen killing and host survival following both viral and bacterial challenges. These data indicate that maximal ODN-induced pneumonia protection requires activation of both TLR9/MyD88 and RIG-I/MAVS signaling pathways. These findings not only identify what we believe to be a novel pattern recognition receptor for DNA-like molecules, but reveal a potential therapeutic strategy to protect susceptible individuals against lethal pneumonias during periods of peak vulnerability.

Correlation and Level of Agreement between the Ocular Surface Disease Index and the Symptom Assessment in Dry Eye Questionnaires: A Survey-Based Study.

PURPOSE: The Ocular Surface Disease Index (OSDI) is the most frequently used dry eye disease (DED) questionnaire, and the Symptom Assessment iN Dry Eye (SANDE) is the simplest and quickest to apply. We analyze the correlation and level of agreement between these two questionnaires in a large DED heterogeneous population to evaluate their performance and potential interchangeability. METHODS: Prospective, multicenter longitudinal survey-based study performed on patients diagnosed with DED by 99 ophthalmologists from 20/32 Mexican states. Questionnaires were applied in two consecutive visits to analyze the correlation between OSDI and SANDE to evaluate patients with DED clinically. Level of agreement was evaluated with Bland-Altman analysis, and internal consistency of instruments was evaluated individually and combined with Cronbach's alpha index. RESULTS: 3421 patients studied: 1996 (58.3%) women and 1425 (41.7%) men, aged 49.5 ± 15.4 years; 995 (29.1%) patients had aqueous-deficient, 1086 (31.7%) evaporative, and 1340 (39.2%) mixed DED subtypes. Normalized baseline scores were 53.7 (OSDI) and 54.1 (SANDE). After 36.3 ± 24.4 days between visits, scores were reduced to 25.2 (OSDI) and 21.8 (SANDE) points (p < .001). A positive correlation between questionnaires was found at baseline (R = 0.592; p < .001), follow-up (R = 0.543; p < .001) and change between visits (R = 0.630; p < .001). Using both questionnaires together improved the overall reliability of symptom evaluation at baseline (α = 0.7), follow-up (α = 0.7), and both (α = 0.7), compared to individual application (OSDI α = 0.5, SANDE α = 0.6)-the same improvements applied to all DED subtypes. Bland-Altman analysis revealed a differential bias of -0.41% at baseline and +3.6% at follow-up visits between OSDI and SANDE. CONCLUSIONS: We validated the correlation (high precision) between questionnaires in a large-scale population, demonstrating improved reliability (high accuracy) in evaluating DED when used together, challenging their interchangeable use. These results open a venue to improve recommendations toward a more precise and accurate diagnostic and therapeutic evaluation of DED by using OSDI and SANDE concurrently.

Antimicrobial mitochondrial reactive oxygen species induction by lung epithelial metabolic reprogramming.

Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infections an urgent need. We have previously shown that manipulating the lungs' intrinsic host defenses by therapeutic delivery of a unique dyad of pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODNs) with mitochondrial voltage-dependent anion channel 1 (VDAC1) without dependence on Toll-like receptor 9 (TLR9). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), enhances mitochondrial membrane potential (Δ Ψm ), and differentially modulates ETC complex activities. These combined effects promote leak of electrons from ETC complex III, resulting in superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy that has the potential to broadly protect patients against pneumonia during periods of peak vulnerability without reliance on currently available antibiotics. Author Summary: Pneumonia is a major cause of death worldwide. Increasing antibiotic resistance and expanding immunocompromised populations continue to enhance the clinical urgency to find new strategies to prevent and treat pneumonia. We have identified a novel inhaled therapeutic that stimulates lung epithelial defenses to protect mice against pneumonia in a manner that depends on production of reactive oxygen species (ROS). Here, we report that the induction of protective ROS from lung epithelial mitochondria occurs following the interaction of one component of the treatment, an oligodeoxynucleotide, with the mitochondrial voltage-dependent anion channel 1. This interaction alters energy transfer between the mitochondria and the cytosol, resulting in metabolic reprogramming that drives more electrons into the electron transport chain, then causes electrons to leak from the electron transport chain to form protective ROS. While antioxidant therapies are endorsed in many other disease states, we present here an example of therapeutic induction of ROS that is associated with broad protection against pneumonia without reliance on administration of antibiotics.

OBIF: an omics-based interaction framework to reveal molecular drivers of synergy.

Bioactive molecule library screening may empirically identify effective combination therapies, but molecular mechanisms underlying favorable drug-drug interactions often remain unclear, precluding further rational design. In the absence of an accepted systems theory to interrogate synergistic responses, we introduce Omics-Based Interaction Framework (OBIF) to reveal molecular drivers of synergy through integration of statistical and biological interactions in synergistic biological responses. OBIF performs full factorial analysis of feature expression data from single versus dual exposures to identify molecular clusters that reveal synergy-mediating pathways, functions and regulators. As a practical demonstration, OBIF analyzed transcriptomic and proteomic data of a dyad of immunostimulatory molecules that induces synergistic protection against influenza A and revealed unanticipated NF-κB/AP-1 cooperation that is required for antiviral protection. To demonstrate generalizability, OBIF analyzed data from a diverse array of Omics platforms and experimental conditions, successfully identifying the molecular clusters driving their synergistic responses. Hence, unlike existing synergy quantification and prediction methods, OBIF is a phenotype-driven systems model that supports multiplatform interrogation of synergy mechanisms.

COVID-19 patients share common, corticosteroid-independent features of impaired host immunity to pathogenic molds.

Patients suffering from coronavirus disease-2019 (COVID-19) are susceptible to deadly secondary fungal infections such as COVID-19-associated pulmonary aspergillosis and COVID-19-associated mucormycosis. Despite this clinical observation, direct experimental evidence for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)-driven alterations of antifungal immunity is scarce. Using an ex-vivo whole blood stimulation assay, we challenged blood from twelve COVID-19 patients with Aspergillus fumigatus and Rhizopus arrhizus antigens and studied the expression of activation, maturation, and exhaustion markers, as well as cytokine secretion. Compared to healthy controls, T-helper cells from COVID-19 patients displayed increased expression levels of the exhaustion marker PD-1 and weakened A. fumigatus- and R. arrhizus-induced activation. While baseline secretion of proinflammatory cytokines was massively elevated, whole blood from COVID-19 patients elicited diminished release of T-cellular (e.g., IFN-γ, IL-2) and innate immune cell-derived (e.g., CXCL9, CXCL10) cytokines in response to A. fumigatus and R. arrhizus antigens. Additionally, samples from COVID-19 patients showed deficient granulocyte activation by mold antigens and reduced fungal killing capacity of neutrophils. These features of weakened anti-mold immune responses were largely decoupled from COVID-19 severity, the time elapsed since diagnosis of COVID-19, and recent corticosteroid uptake, suggesting that impaired anti-mold defense is a common denominator of the underlying SARS-CoV-2 infection. Taken together, these results expand our understanding of the immune predisposition to post-viral mold infections and could inform future studies of immunotherapeutic strategies to prevent and treat fungal superinfections in COVID-19 patients.

Inducible lung epithelial resistance requires multisource reactive oxygen species generation to protect against bacterial infections.

Pneumonia remains a global health threat, in part due to expanding categories of susceptible individuals and increasing prevalence of antibiotic resistant pathogens. However, therapeutic stimulation of the lungs' mucosal defenses by inhaled exposure to a synergistic combination of Toll-like receptor (TLR) agonists known as Pam2-ODN promotes mouse survival of pneumonia caused by a wide array of pathogens. This inducible resistance to pneumonia relies on intact lung epithelial TLR signaling, and inducible protection against viral pathogens has recently been shown to require increased production of epithelial reactive oxygen species (ROS) from multiple epithelial ROS generators. To determine whether similar mechanisms contribute to inducible antibacterial responses, the current work investigates the role of ROS in therapeutically-stimulated protection against Pseudomonas aerugnosa challenges. Inhaled Pam2-ODN treatment one day before infection prevented hemorrhagic lung cytotoxicity and mouse death in a manner that correlated with reduction in bacterial burden. The bacterial killing effect of Pam2-ODN was recapitulated in isolated mouse and human lung epithelial cells, and the protection correlated with inducible epithelial generation of ROS. Scavenging or targeted blockade of ROS production from either dual oxidase or mitochondrial sources resulted in near complete loss of Pam2-ODN-induced bacterial killing, whereas deficiency of induced antimicrobial peptides had little effect. These findings support a central role for multisource epithelial ROS in inducible resistance against a bacterial pathogen and provide mechanistic insights into means to protect vulnerable patients against lethal infections.

Inducible Lung Epithelial Resistance Requires Multisource Reactive Oxygen Species Generation To Protect against Viral Infections.

Viral pneumonias cause profound worldwide morbidity, necessitating novel strategies to prevent and treat these potentially lethal infections. Stimulation of intrinsic lung defenses via inhalation of synergistically acting Toll-like receptor (TLR) agonists protects mice broadly against pneumonia, including otherwise-lethal viral infections, providing a potential opportunity to mitigate infectious threats. As intact lung epithelial TLR signaling is required for the inducible resistance and as these cells are the principal targets of many respiratory viruses, the capacity of lung epithelial cells to be therapeutically manipulated to function as autonomous antiviral effectors was investigated. Our work revealed that mouse and human lung epithelial cells could be stimulated to generate robust antiviral responses that both reduce viral burden and enhance survival of isolated cells and intact animals. The antiviral protection required concurrent induction of epithelial reactive oxygen species (ROS) from both mitochondrial and dual oxidase sources, although neither type I interferon enrichment nor type I interferon signaling was required for the inducible protection. Taken together, these findings establish the sufficiency of lung epithelial cells to generate therapeutically inducible antiviral responses, reveal novel antiviral roles for ROS, provide mechanistic insights into inducible resistance, and may provide an opportunity to protect patients from viral pneumonia during periods of peak vulnerability.IMPORTANCE Viruses are the most commonly identified causes of pneumonia and inflict unacceptable morbidity, despite currently available therapies. While lung epithelial cells are principal targets of respiratory viruses, they have also been recently shown to contribute importantly to therapeutically inducible antimicrobial responses. This work finds that lung cells can be stimulated to protect themselves against viral challenges, even in the absence of leukocytes, both reducing viral burden and improving survival. Further, it was found that the protection occurs via unexpected induction of reactive oxygen species (ROS) from spatially segregated sources without reliance on type I interferon signaling. Coordinated multisource ROS generation has not previously been described against viruses, nor has ROS generation been reported for epithelial cells against any pathogen. Thus, these findings extend the potential clinical applications for the strategy of inducible resistance to protect vulnerable people against viral infections and also provide new insights into the capacity of lung cells to protect against infections via novel ROS-dependent mechanisms.