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Influenza infections result in a significant number of severe illnesses annually, many of which are complicated by secondary bacterial super-infection. Primary influenza infection has been shown to increase susceptibility to secondary methicillin-resistant Staphylococcus aureus (MRSA) infection by altering the host immune response, leading to significant immunopathology. Type III interferons (IFNs), or IFNλs, have gained traction as potential antiviral therapeutics due to their restriction of viral replication without damaging inflammation. The role of IFNλ in regulating epithelial biology in super-infection has recently been established; however, the impact of IFNλ on immune cells is less defined. In this study, we infected wild-type and IFNLR1-/- mice with influenza A/PR/8/34 followed by S. aureus USA300. We demonstrated that global IFNLR1-/- mice have enhanced bacterial clearance through increased uptake by phagocytes, which was shown to be cell-intrinsic specifically in myeloid cells in mixed bone marrow chimeras. We also showed that depletion of IFNLR1 on CX3CR1 expressing myeloid immune cells, but not neutrophils, was sufficient to significantly reduce bacterial burden compared to mice with intact IFNLR1. These findings provide insight into how IFNλ in an influenza-infected lung impedes bacterial clearance during super-infection and show a direct cell intrinsic role for IFNλ signaling on myeloid cells.

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Asthma is characterized by airway remodeling and hyperreactivity. Our earlier studies determined that the nitric oxide (NO)-soluble guanylyl cyclase (sGC)-cGMP pathway plays a significant role in human lung bronchodilation. However, this bronchodilation is dysfunctional in asthma due to high NO levels, which cause sGC to become heme-free and desensitized to its natural activator, NO. In order to determine how asthma impacts the various lung segments/lobes, we mapped the inflammatory regions of lungs to determine whether such regions coincided with molecular signatures of sGC dysfunction. We demonstrate using murine models of asthma (OVA and CFA/HDM) that the inflamed segments of these murine lungs can be tracked by upregulated expression of HO1 and these regions in turn overlap with regions of heme-free sGC as evidenced by a decreased sGC-α1ß1 heterodimer and an increased response to heme-independent sGC activator, BAY 60-2770, relative to naïve uninflamed regions. We also find that NO generated from iNOS upregulation in the inflamed segments has a higher impact on developing heme-free sGC as increasing iNOS activity correlates linearly with elevated heme-independent sGC activation. This excess NO works by affecting the epithelial lung hemoglobin (Hb) to become heme-free in asthma, thereby causing the Hb to lose its NO scavenging function and exposing the underlying smooth muscle sGC to excess NO, which in turn becomes heme-free. Recognition of these specific lung segments enhances our understanding of the inflamed lungs in asthma with the ultimate aim to evaluate potential therapies and suggest that regional and not global inflammation impacts lung function in asthma.

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Respiratory tract virus infections cause millions of hospitalizations worldwide each year. Severe infections lead to lung damage that coincides with persistent inflammation and a lengthy repair period. Vaccination and antiviral therapy help to mitigate severe infections before or during the acute stage of disease, but there are currently limited specific treatment options available to individuals experiencing the long-term sequelae of respiratory viral infection. Herein, C57BL/6 mice were infected with influenza A/PR/8/34 as a model for severe viral lung infection and allowed to recover for 21 days. Mice were treated with rapamycin, a well-characterized mammalian target of rapamycin complex 1 (mTORC1) inhibitor, on days 12 to 20 after infection, a time period after viral clearance. Persistent inflammation following severe influenza infection in mice was primarily driven by macrophages and T cells. Uniform manifold approximation and projection analysis of flow cytometry data revealed that lung macrophages had high activation of mTORC1, an energy-sensing kinase involved in inflammatory immune cell effector functions. Rapamycin treatment reduced lung inflammation and the frequency of exudate macrophages, T cells, and B cells in the lung, while not impacting epithelial progenitor cells or adaptive immune memory. These data highlight mTORC1's role in sustaining persistent inflammation following clearance of a viral respiratory pathogen and suggest a possible intervention for post-viral chronic lung inflammation.

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RESUMEN

Asthma is characterized by airway remodeling and hyperreactivity. Our earlier studies determined that the Nitric Oxide (NO)-soluble Guanylyl Cyclase (sGC)-cGMP pathway plays a significant role in human lung bronchodilation. However this bronchodilation is dysfunctional in asthma due to high NO levels which cause sGC to become heme-free and desensitized to its natural activator, NO. In order to determine how asthma impacts the various lung segments/lobes we mapped the inflammatory regions of lungs to determine whether such regions coincided with molecular signatures of sGC dysfunction. We demonstrate using models of mouse asthma (OVA, CFA/HDM) that the inflammed segments of the mouse asthma lungs can be tracked by upregulated expression of HO1 and these regions in-turn overlap with regions of heme-free sGC as evidenced by a decreased sGC-α1ß1 heterodimer and an increased response to heme-independent sGC activator, BAY 60-2770 relative to naïve uninflamed regions. We also find that NO generated from iNOS upregulation in the inflamed segments has a higher impact in developing heme-free sGC as increasing iNOS activity correlates linearly with elevated heme-independent sGC activation. This excess NO works by affecting the epithelial lung hemoglobin (Hb) to become heme-free in asthma thereby causing the Hb to lose its NO scavenging function and exposing the underlying smooth muscle sGC to excess NO, which in-turn becomes heme-free. Recognition of these specific lung segments enhance our understanding of the inflammed lungs in asthma with the ultimate aim to evaluate potential therapies and suggests that regional and not global inflammation impacts lung function in asthma.

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Polytherapy with antiseizure medications (ASMs) is often used to control seizures in patients suffering from epilepsy, where about 30% of patients are pharmacoresistant. While drug combinations are intended to be beneficial, the consequence of CYP-dependent drug interactions on apoptotic protein levels and mitochondrial function in the epileptic brain remains unclear. We examined the interactions of ASMs given prior to surgery in surgically resected brain tissues and of three ASMs (lacosamide, LCM; oxcarbazepine, OXC; levetiracetam LEV) in isolated brain cells from patients with drug-resistant epilepsy (n = 23). We divided the patients into groups-those who took combinations of NON-CYP + CYP substrate ASMs, NON-CYP + CYP inducer ASMs, CYP substrate + CYP substrate or CYP substrate + CYP inducer ASMs-to study the 1) pro- and anti-apoptotic protein levels and other apoptotic signaling proteins and levels of reactive oxygen species (reduced glutathione and lipid peroxidation) in brain tissues; 2) cytotoxicity at blood-brain barrier epileptic endothelial cells (EPI-ECs) and subsequent changes in mitochondrial membrane potential in normal neuronal cells, following treatment with LCM + OXC (CYP substrate + CYP inducer) or LCM + LEV (CYP substrate + NON-CYP-substrate) after blood-brain barrier penetration, and 3) apoptotic and mitochondrial protein targets in the cells, pre-and post-CYP3A4 inhibition by ketoconazole and drug treatments. We found an increased BAX (pro-apoptotic)/Bcl-XL (anti-apoptotic) protein ratio in epileptic brain tissue after treatment with CYP substrate + CYP substrate or inducer compared to NON-CYP + CYP substrate or inducer, and subsequently decreased glutathione and elevated lipid peroxidation levels. Further, increased cytotoxicity and Mito-ID levels, indicative of compromised mitochondrial membrane potential, were observed after treatment of LCM + OXC in combination compared to LCM + LEV or these ASMs alone in EPI-ECs, which was attenuated by pre-treatment of CYP inhibitor, ketoconazole. A combination of two CYP-mediated ASMs on EPI-ECs resulted in elevated caspase-3 and cytochrome c with decreased SIRT3 levels and activity, which was rescued by CYP inhibition. Together, the study highlights for the first time that pro- and anti-apoptotic proteins levels are dependent on ASM combinations in epilepsy, modulated via a CYP-mediated mechanism that controls free radicals, cytotoxicity and mitochondrial activity. These findings lead to a better understanding of future drug selection choices offsetting pharmacodynamic CYP-mediated interactions.

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The glucocorticoid receptor (GR) at the blood−brain barrier (BBB) is involved in the pathogenesis of drug-resistant epilepsy with focal cortical dysplasia (FCD); however, the roles of GR isoforms GRα and GRß in the dysplastic brain have not been revealed. We utilized dysplastic/epileptic and non-dysplastic brain tissue from patients who underwent resective epilepsy surgery to identify the GRα and GRß levels, subcellular localization, and cellular specificity. BBB endothelial cells isolated from the dysplastic brain tissue (EPI-ECs) were used to decipher the key BBB proteins related to drug regulation and BBB integrity compared to control and transfected GRß-overexpressed BBB endothelial cells. GRß was upregulated in dysplastic compared to non-dysplastic tissues, and an imbalance of the GRα/GRß ratio was significant in females vs. males and in patients > 45 years old. In EPI-ECs, the subcellular localization and expression patterns of GRß, Hsp90, CYP3A4, and CYP2C9 were consistent with GRß+ brain endothelial cells. Active matrix metalloproteinase levels and activity increased, whereas claudin-5 levels decreased in both EPI-ECs and GRß+ endothelial cells. In conclusion, the GRß has a major effect on dysplastic BBB functional proteins and is age and gender-dependent, suggesting a critical role of brain GRß in dysplasia as a potential biomarker and therapeutic target in epilepsy.

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