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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.

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 (ΔΨm), 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 SummaryPneumonia 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.

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.

Significance There is a pressing need for new antiinfluenza therapeutic agents. We show that a molecularly engineered banana lectin (carbohydrate-binding protein) has broad-spectrum activity against all influenza strains tested, including drug-resistant and currently circulating strains; is safe upon repeated administration in mice; and, moreover, is efficacious at treating lethal influenza infection via clinically pertinent routes of administration. We demonstrate that the lectin binds to the viral hemagglutinin glycoprotein and exerts its primary antiviral effect via inhibition of an early stage of the viral life cycle, viral membrane fusion to the host endosomal membrane. Our findings indicate that this engineered lectin, which has a mechanism of action quite distinct from the presently available agents, has potential as an antiinfluenza agent., 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.

Allergic asthma is a chronic inflammatory respiratory disease associated with eosinophilic infiltration, increased mucus production, airway hyperresponsiveness, and airway remodeling. Epidemiologic data reveal that the prevalence of allergic sensitization and associated diseases has increased in the twentieth century. This has been hypothesized to be partly due to reduced contact with microbial organisms (the hygiene hypothesis) in industrialized society. Airway epithelial cells, once considered a static physical barrier between the body and the external world, are now widely recognized as immunologically active cells that can initiate, maintain, and restrain inflammatory responses, such as those that mediate allergic disease. Airway epithelial cells can sense allergens via expression of myriad Toll-like receptors (TLRs) and other pattern-recognition receptors. We sought to determine whether the innate immune response stimulated by a combination of Pam2CSK4 (“Pam2”, TLR2/6 ligand) and a class C oligodeoxynucleotide ODN362 (“ODN”, TLR9 ligand), when delivered together by aerosol (“Pam2ODN”), can modulate the allergic immune response to allergens. Treatment with Pam2ODN 7 days before sensitization to House Dust Mite (HDM) extract resulted in a strong reduction in eosinophilic and lymphocytic inflammation. This Pam2ODN immunomodulatory effect was also seen using Ovalbumin (OVA) and A. oryzae (Ao) mouse models. The immunomodulatory effect was observed as much as 30 days before sensitization to HDM, but ineffective just 2 days after sensitization, suggesting that Pam2ODN immunomodulation lowers the allergic responsiveness of the lung, and reduces the likelihood of inappropriate sensitization to aeroallergens. Furthermore, Pam2 and ODN cooperated synergistically suggesting that this treatment is superior to any single agonist in the setting of allergen immunotherapy.

Allergic asthma is a chronic inflammatory respiratory disease associated with eosinophilic infiltration, increased mucus production, airway hyperresponsiveness (AHR), and airway remodeling. Epidemiologic data has revealed that the prevalence of allergic sensitization and associated diseases has increased in the twentieth century. This has been hypothesized to be partly due to reduced contact with microbial organisms (the hygiene hypothesis) in industrialized society. Airway epithelial cells, once considered a static physical barrier between the body and the external world, are now widely recognized as immunologically active cells that can initiate, maintain, and restrain inflammatory responses, such as those that mediate allergic disease. Airway epithelial cells can sense allergens via myriad expression of Toll-like receptors (TLRs) and other pattern-recognition receptors (PRRs). We sought to determine whether the innate immune response stimulated by a combination of Pam2CSK4 (“Pam2”, TLR2/6 ligand) and a class C oligodeoxynucleotide ODN362 (“ODN”, TLR9 ligand) when delivered together by aerosol (“Pam2ODN”), can modulate the allergic immune response to allergens. Treatment with Pam2ODN 7 days before sensitization to House Dust Mite (HDM) extract resulted in a strong reduction in eosinophilic and lymphocytic inflammation. This Pam2ODN immunomodulatory effect was also seen using Ovalbumin (OVA) and A. oryzae (Ao) mouse models. The immunomodulatory effect was observed as much as 30 days before sensitization to HDM, but ineffective just 2 days after sensitization, suggesting that Pam2ODN immunomodulation lowers the allergic responsiveness of airway epithelial cells. Furthermore, Pam2 and ODN cooperated synergistically suggesting that this treatment is superior to any single agonist in the setting of allergen immunotherapy.One Sentence SummaryA synergistic combination of Toll-like Receptor agonists, delivered directly into the lung mucosa, can attenuate allergic responsiveness of airway epithelial cells and prevent host sensitization to aeroallergens.What is already knownAllergic sensitization has increased in the 20th century due to reduced contact with microbial organisms in industrialized society (ie. hygiene hypothesis)We have previously identified a pharmacological means to stimulate innate immunity of lung epithelial cells.What this study addsActivation of innate immunity in lung epithelial cells attenuates the allergic responsiveness of mice.Synergistic cooperation of pattern recognition receptors induces stronger immunomodulatory responsesWhat is the clinical significanceAerosolized Toll-like Receptor agonists have been demonstrated as safe in human clinical trialsThis study provides proof-of-principle that aerosolized toll-like receptor agonists could have clinical efficacy in the setting of the allergen immunotherapy

Background: Pneumonias exert tremendous worldwide morbidity, especially among immunocompromised populations. Our laboratory has identified an inhaled therapeutic comprised of Toll-like receptor (TLR) 2/6 and TLR9 agonists that protects mice against a broad array of respiratory pathogens. This protection is associated with the generation of an antimicrobial environment in the lungs and requires induction of epithelial reactive oxygen species of both dual oxidase and mitochondrial sources. Objective: To discern how the treatment induces generation of mitochondrial reactive oxygen species (mtROS). Results: Inducible mtROS generation was driven by epithelial exposure to a class C CpG oligodeoxynucleotide (ODN M362), a reported TLR9 ligand. However, ODN M362-induced mtROS generation was not dependent on TLR9 signaling, but required that the oligodeoxynucleotide include phosphorothiorate linkages, rather than the phosphodiester linkages common in the DNA of most species. Phosphorothiorate ODN directly interacted with mitochondrial permeability transition pores, altering the ATP:ADP ratio in both cytosol and mitochondria, activating AMPK and ACC pathways and increasing beta-oxidation. This led to enhancement of electron transport chain complex II activity and reduction of complex III activity, causing increased electron leakage that resulted in superoxide (O2- ·) production. The resultant mtROS both exerted directly microbicidal effects and stabilized Hif1α to support antimicrobial peptide production. Conclusions: These data identify novel means to manipulate mtROS and may provide an opportunity to protect vulnerable patients against pneumonia.

Bioactive molecule library screening strategies may empirically identify effective combination therapies. However, without a systems theory to interrogate synergistic responses, the molecular mechanisms underlying favorable drug-drug interactions remain unclear, precluding rational design of combination therapies. Here, we introduce Omics-Based Interaction Framework (OBIF) to reveal molecular drivers of synergy through integration of statistical and biological interactions in supra-additive biological responses. OBIF performs full factorial analysis of feature expression data from single vs. dual factor exposures to identify molecular clusters that reveal synergy-mediating pathways, functions and regulators. As a practical demonstration, OBIF analyzed a therapeutic dyad of immunostimulatory small molecules that induces synergistic protection against influenza A pneumonia. OBIF analysis of transcriptomic and proteomic data identified biologically relevant, unanticipated cooperation between RelA and cJun that we subsequently confirmed to be required for the synergistic antiviral protection. To demonstrate generalizability, OBIF was applied to data from a diverse array of Omics platforms and experimental conditions, successfully identifying the molecular clusters driving their synergistic responses. Hence, OBIF is a phenotype-driven systems model that supports multiplatform exploration of synergy mechanisms.

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.

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.

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 bacterial pathogens and provide mechanistic insights into means to protect vulnerable patients against lethal 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.

Despite widespread infection prevention efforts, pneumonia remains the leading cause of death among patients with acute leukemia, due to complex disease- and treatment-dependent immune defects. We have reported that a single inhaled treatment with a synergistic combination of Toll-like receptor 2/6 (TLR 2/6) and TLR9 agonists (Pam2-ODN) induces protective mucosal defenses in mice against a broad range of pathogens. As Pam2-ODN-induced protection persists despite depletion of several leukocyte populations, we tested whether it could prevent pneumonia in a mouse model of acute myeloid leukemia (AML) remission induction therapy. Pam2-ODN prevented death due to pneumonia caused by Pseudomonas aeruginosa, Streptococcus pneumoniae, and Aspergillus fumigatus when mice were heavily engrafted with leukemia cells, had severe chemotherapy-induced neutropenia or both. Pam2-ODN also extended survival of pneumonia in NSG mice engrafted with primary human AML cells. Protection was associated with rapid pathogen killing in the lungs at the time of infection and with reduced pathogen burdens at distant sites at the end of observation. Pathogen killing was inducible directly from isolated lung epithelial cells and was not abrogated by the presence of leukemia cells or cytotoxic agents. Pam2-ODN had no discernible effect on replication rate, total tumor population, or killing by chemotherapy of mouse or human leukemia cells, either in vitro or in vivo. Taken together, we report that therapeutic stimulation of lung epithelial defenses robustly protects against otherwise lethal pneumonias despite the profound immune dysfunction associated with acute leukemia and its treatment. These findings may suggest an opportunity to protect this population during periods of peak vulnerability.