Treatment Of Inflammatory Lung Diseases Research Articles

Production of functional recombinant roseltide rT1 antimicrobial peptide in tobacco plants

Plant-derived peptides represent a promising group of natural compounds with broad industrial and pharmaceutical applications. Low-efficiency production level is the major obstacle to the commercial production of such bioactive peptides. Today, recombinant techniques have been developed for fast and cost-effective production of high-quality peptides for various applications in the chemical and food industries. The roseltide rT1 is a plant peptide with different antimicrobial properties and therapeutic applications in the prevention and treatment of inflammatory lung diseases by inhibiting human neutrophil elastases. Here, we report the expression of functional recombinant roseltide rT1 peptide in tobacco plants. Transgenic plants were generated by the Agrobacterium-mediated transformation method followed by molecular analysis of transgenic plants to demonstrate successful integration and expression of recombinant rT1 peptide. Protein extracts of transgenic plants expressing a single-copy rT1 gene showed efficient antimicrobial properties as verified by growth inhibition of different bacterial strains. Our results illustrate that plant-derived recombinant rT1 peptide is a promising alternative for rapid and cost-effective production of this important antimicrobial peptide for application in therapeutic and food industries.

Advances in nano-based drug delivery systems for the management of cytokine influx-mediated inflammation in lung diseases.

Asthma, lung cancer, cystic fibrosis, tuberculosis, acute respiratory distress syndrome, chronic obstructive pulmonary disease, and COVID-19 are few examples of inflammatory lung conditions that cause cytokine release syndrome. It can initiate a widespread inflammatory response and may activate several inflammatory pathways that cause multiple organ failures leading to increased number of deaths and increased prevalence rates around the world. Nanotechnology-based therapeutic modalities such as nanoparticles, liposomes, nanosuspension, monoclonal antibodies, and vaccines can be used in the effective treatment of inflammatory lung diseases at both cellular and molecular levels. This would also help significantly in the reduction of patient mortality. Therefore, nanotechnology could be a potent platform for repurposing current medications in the treatment of inflammatory lung diseases. The aim and approach of this article are to highlight the clinical manifestations of cytokine storm in inflammatory lung diseases along with the advances and potential applications of nanotechnology-based therapeutics in the management of cytokine storm. Further in-depth studies are required to understand the molecular pathophysiology, and how nanotechnology-based therapeutics can help to effectively combat this problem.

Heparin, Low Molecular Weight Heparin, and Non-Anticoagulant Derivatives for the Treatment of Inflammatory Lung Disease.

Unfractionated heparin has multiple pharmacological activities beyond anticoagulation. These anti-inflammatory, anti-microbial, and mucoactive activities are shared in part by low molecular weight and non-anticoagulant heparin derivatives. Anti-inflammatory activities include inhibition of chemokine activity and cytokine synthesis, inhibitory effects on the mechanisms of adhesion and diapedesis involved in neutrophil recruitment, inhibition of heparanase activity, inhibition of the proteases of the coagulation and complement cascades, inhibition of neutrophil elastase activity, neutralisation of toxic basic histones, and inhibition of HMGB1 activity. This review considers the potential for heparin and its derivatives to treat inflammatory lung disease, including COVID-19, ALI, ARDS, cystic fibrosis, asthma, and COPD via the inhaled route.

A new hope? Possibilities of therapeutic IgA antibodies in the treatment of inflammatory lung diseases.

Inflammatory lung diseases represent a persistent burden for patients and the global healthcare system. The combination of high morbidity, (partially) high mortality and limited innovations in the last decades, have resulted in a great demand for new therapeutics. Are therapeutic IgA antibodies possibly a new hope in the treatment of inflammatory lung diseases? Current research increasingly unravels the elementary functions of IgA as protector against infections and as modulator of overwhelming inflammation. With a focus on IgA, this review describes the pathological alterations in mucosal immunity and how they contribute to chronic inflammation in the most common inflammatory lung diseases. The current knowledge of IgA functions in the circulation, and particularly in the respiratory mucosa, are summarized. The interplay between neutrophils and IgA seems to be key in control of inflammation. In addition, the hurdles and benefits of therapeutic IgA antibodies, as well as the currently known clinically used IgA preparations are described. The data highlighted here, together with upcoming research strategies aiming at circumventing the current pitfalls in IgA research may pave the way for this promising antibody class in the application of inflammatory lung diseases.

The NADPH Oxidase Inhibitors Apocynin and Diphenyleneiodonium Protect Rats from LPS-Induced Pulmonary Inflammation

Inflammation is the body's response to insults, for instance, lung inflammation is generally caused by pathogens or by exposure to pollutants, irritants and toxins. This process involves many inflammatory cells such as epithelial cells, monocytes, macrophages and neutrophils. These cells produce and release inflammatory mediators such as pro-inflammatory cytokines, lipids and reactive oxygen species (ROS). Lung epithelial cells and phagocytes (monocytes, macrophages and neutrophils) produce ROS mainly by the NADPH oxidase NOX1 and NOX2, respectively. The aim of this study was to investigate the effects of two NADPH oxidase inhibitors, apocynin and diphenyleneiodonium (DPI), on lipopolysaccharide (LPS)-induced lung inflammation in rats. Our results showed that apocynin and DPI attenuated the LPS-induced morphological and histological alterations of the lung, reduced edema and decreased lung permeability. The evaluation of oxidative stress markers in lung homogenates showed that apocynin and DPI inhibited LPS-induced NADPH oxidase activity, and restored superoxide dismutase (SOD) and catalase activity in the lung resulting in the reduction in LPS-induced protein and lipid oxidation. Additionally, apocynin and DPI decreased LPS-induced MPO activity in bronchoalveolar liquid and lung homogenates, TNF-α and IL-1β in rat plasma. NADPH oxidase inhibition could be a new therapeutic strategy for the treatment of inflammatory lung diseases.

Nanotechnology-based advancements inNF-κB pathway inhibition for the treatment of inflammatory lung diseases.

NanomedicineAhead of Print CommentaryNanotechnology-based advancements in NF-κB pathway inhibition for the treatment of inflammatory lung diseasesAsif Ahmad Bhat, Gaurav Gupta, Santosh Kumar Singh, Hemant KS Yadav, Mahendra Saini, Roshan Salfi, Sachin Kumar Singh & Kamal DuaAsif Ahmad BhatSchool of Pharmacy, Suresh Gyan Vihar University, Jagatpura, Mahal Road, 302017, Jaipur, IndiaSearch for more papers by this author, Gaurav Gupta *Author for correspondence: E-mail Address: gauravpharma25@gmail.comhttps://orcid.org/0000-0001-7941-0229School of Pharmacy, Suresh Gyan Vihar University, Jagatpura, Mahal Road, 302017, Jaipur, IndiaDepartment of Pharmacology, Saveetha Dental College, Saveetha Institute of Medical & Technical Sciences, Saveetha University, Chennai, 602105, IndiaUttaranchal Institute of Pharmaceutical Sciences, Uttaranchal University, Dehradun, 248007, IndiaSearch for more papers by this author, Santosh Kumar SinghSchool of Pharmacy, Suresh Gyan Vihar University, Jagatpura, Mahal Road, 302017, Jaipur, IndiaSearch for more papers by this author, Hemant KS YadavSchool of Pharmacy, Suresh Gyan Vihar University, Jagatpura, Mahal Road, 302017, Jaipur, IndiaSearch for more papers by this author, Mahendra SainiSchool of Pharmacy, Suresh Gyan Vihar University, Jagatpura, Mahal Road, 302017, Jaipur, IndiaSearch for more papers by this author, Roshan SalfiDeccan School of Pharmacy, Darussalam, Aghapura, Nampally, Hyderabad, 500001, IndiaYenepoya Pharmacy College & Research Centre, Yenepoya University, Mangalore, Karnataka, 575023, IndiaSearch for more papers by this author, Sachin Kumar SinghSchool of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab, 144411, IndiaFaculty of Health, Australian Research Center in Complementary & Integrative Medicine, University of Technology Sydney, Ultimo, NSW 2007, AustraliaSearch for more papers by this author & Kamal Dua https://orcid.org/0000-0002-7507-1159Faculty of Health, Australian Research Center in Complementary & Integrative Medicine, University of Technology Sydney, Ultimo, NSW 2007, AustraliaDiscipline of Pharmacy, Graduate School of Health, University of Technology Sydney, NSW 2007, AustraliaSearch for more papers by this authorPublished Online:21 Feb 2023https://doi.org/10.2217/nnm-2022-0220AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleKeywords: asthmaCOPDlungnanotechnologyNF-κB pathway

Leucine improves the aerosol performance of dry powder inhaler formulations of siRNA-loaded nanoparticles

Thermostable dry powder inhaler (DPI) formulations with high aerosol performance are attractive inhalable solid dosage forms for local treatment of inflammatory lung diseases. We recently demonstrated that lipidoid-polymer hybrid nanoparticles (LPNs) loaded with small interfering RNA (siRNA) directed against tumor necrosis factor alpha (TNF-α) mediate efficient intracellular siRNA delivery and reduce inflammation in vivo. Here, we show that mixtures of the stabilizing excipients trehalose (Tre) and dextran (Dex), in combination with the shell-forming dispersion enhancer leucine (Leu), stabilize TNF-α siRNA-loaded LPNs during spray drying into nanocomposite microparticles, and result in DPI formulations with high aerosol performance. At low Leu content (0 to 10%, w/w), the DPI formulations were amorphous, and exhibited poor aerosol performance. When the Leu content was increased from 20 to 60% (w/w), the surface content of Leu increased from 39.2 to 68.1 mol%, and the flowability was significantly improved. Microscopy analysis suggest that the improved powder dispersibility is the result of a wrinkled surface morphology, which reduces the surface area available for interparticle interactions. Increasing the Leu content further (to above 10%, w/w) did not influence the aerosol performance, and the aerosol yield was maximal at 30–40% Leu (w/w). Formulations containing 40% Leu and a Tre:Dex ratio of 10:90 (w/w) displayed a high fine particle fraction and aerosol properties suitable for inhalation. The chemical integrity of TNF-α siRNA was preserved in the solid state, and biodistribution studies in mice showed that pulmonary administration of DPI formulations with high aerosol performance resulted in homogenous deep lung deposition. Our results demonstrate that at optimal ratios, ternary excipient mixtures of Leu, Tre and Dex protect TNF-α siRNA-loaded LPNs during spray drying. Hence, this study shows that microparticles with an amorphous Tre/Dex matrix and a crystalline Leu shell efficiently stabilize the nanocomposite LPNs in the solid state, and ensure aerosol properties suitable for inhalation.

Nanotechnology-based therapeutics for targeting inflammatory lung diseases.

The physiochemical properties of drugs used in treating inflammation-associated lung diseases (i.e., asthma, chronic obstructive pulmonary disease, pulmonary fibrosis) play an important role in determining the effectiveness of formulations. Most commonly used drugs are associated with low solubility, low stability and rapid clearance, thus resulting in low bioavailability and therapeutic index. This review focuses on current trends and development of drugs (i.e., corticosteroids, long-acting β-agonists and biomacromolecules such as DNA, siRNA and mRNA) employed to treat inflammatory lung diseases. In addition, this review includes the current challenges of and future perspective with regard to nanotechnology in the treatment of inflammatory lung diseases.

Effects of 3'-isovaleryl-4'-senecioylkhellactone from Peucedanum japonicum Thunberg on PMA-Stimulated Inflammatory Response in A549 Human Lung Epithelial Cells.

Peucedanum japonicum Thunberg (PJT) has been used in traditional medicine to treat colds, coughs, fevers, and other inflammatory diseases. The goal of this study was to investigate whether 3'-isovaleryl-4'-senecioylkhellactone (IVSK) from PJT has anti-inflammatory effects on lung epithelial cells. The anti-inflammatory effects of IVSK were evaluated using phorbol 12-myristate 13-acetate (PMA)-stimulated A549 cells and regular human lung epithelial cells as a reference. IVSK reduced the secretion of the inflammatory mediators interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1), and the mRNA expression of IL-6, IL-8, MCP-1, and IL-1β. Additionally, it inhibited the phosphorylation of IκB kinase (IKK), p65, Iκ-Bα, and mitogen-activated protein kinases (MAPKs) p38, JNK, and ERK in A549 cells stimulated with PMA. Moreover, the binding affinity of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) was significantly reduced in the luciferase assay, while nuclear translocation was markedly inhibited by IVSK in the immunocytochemistry. These findings indicate that IVSK can protect against inflammation through the AP-1 and NF-κB pathway and could possibly be used as a lead compound for the treatment of inflammatory lung diseases.

Inhaled [D-Ala2]-Dynorphin 1-6 Prevents Hyperacetylation and Release of High Mobility Group Box 1 in a Mouse Model of Acute Lung Injury.

COVID-19 is a respiratory infection caused by the SARS-CoV-2 virus that can rapidly escalate to life-threatening pneumonia and acute respiratory distress syndrome (ARDS). Recently, extracellular high mobility group box 1 (HMGB1) has been identified as an essential component of cytokine storms that occur with COVID-19; HMGB1 levels correlate significantly with disease severity. Thus, the modulation of HMGB1 release may be vital for treating COVID-19. HMGB1 is a ubiquitous nuclear DNA-binding protein whose biological function depends on posttranslational modifications, its redox state, and its cellular localization. The acetylation of HMGB1 is a prerequisite for its translocation from the nucleus to the cytoplasm and then to the extracellular milieu. When released, HMGB1 acts as a proinflammatory cytokine that binds primarily to toll-like receptor 4 (TLR4) and RAGE, thereby stimulating immune cells, endothelial cells, and airway epithelial cells to produce cytokines, chemokines, and other inflammatory mediators. In this study, we demonstrate that inhaled [D-Ala2]-dynorphin 1-6 (leytragin), a peptide agonist of δ-opioid receptors, significantly inhibits HMGB1 secretion in mice with lipopolysaccharide- (LPS-) induced acute lung injury. The mechanism of action involves preventing HMGB1's hyperacetylation at critical lysine residues within nuclear localization sites, as well as promoting the expression of sirtuin 1 (SIRT1), an enzyme known to deacetylate HMGB1. Leytragin's effects are mediated by opioid receptors, since naloxone, an antagonist of opioid receptors, abrogates the leytragin effect on SIRT1 expression. Overall, our results identify leytragin as a promising therapeutic agent for the treatment of pulmonary inflammation associated with HMGB1 release. In a broader context, we demonstrate that the opioidergic system in the lungs may represent a promising target for the treatment of inflammatory lung diseases.

Phosphodiesterase 10A Is a Key Mediator of Lung Inflammation.

Cyclic nucleotides cAMP and cGMP are important regulators of immune cell functions. Phosphodiesterases (PDEs) hydrolyze cAMP and/or cGMP and, thus, play crucial roles in cyclic nucleotide homeostasis. Abnormal alterations of PDE expression have been implicated in several diseases. To understand the function of PDEs in macrophages, we screened for all PDE genes in both peritoneal and alveolar macrophages from C57BL/6J mice and found that PDE4B and PDE10A are highly induced by LPS. A number of PDE4 inhibitors have been used clinically for the treatment of inflammatory lung diseases. However, the role of PDE10A in inflammation is still poorly understood. We therefore investigated the role of PDE10A in macrophage inflammatory response in vitro and acute lung inflammation in vivo. We found that LPS induces a sustained PDE10A expression in macrophages, which is different from a transient induction by PDE4B. PDE10A inhibition blocked LPS-induced MCP-1 expression, but not TNF-α, whereas PDE4B inhibition blocked LPS-induced TNF-α expression, but not MCP-1. In addition, PDE10A inhibition or deficiency decreased LPS-induced HIF-1α protein expression and subsequently suppressed MCP-1 expression. In vivo, PDE10A expression was also elevated in lung tissue after LPS exposure. Global PDE10A knockout or systemic administration of the PDE10A inhibitor TP-10 in mice significantly suppressed inflammatory molecule levels in the lung tissue and bronchoalveolar lavage fluid as well as inflammatory cell infiltration. These findings show that PDE10A plays a critical role in lung inflammation by promoting the activation of resident macrophages and infiltration of neutrophils.

Functional Contribution and Targeted Migration of Group-2 Innate Lymphoid Cells in Inflammatory Lung Diseases: Being at the Right Place at the Right Time.

During the last decade, group-2 innate lymphoid cells (ILC2s) have been discovered and successfully established as crucial mediators of lung allergy, airway inflammation and fibrosis, thus affecting the pathogenesis and clinical course of many respiratory diseases, like for instance asthma, cystic fibrosis and chronic rhinosinusitis. As an important regulatory component in this context, the local pulmonary milieu at inflammatory tissue sites does not only determine the activation status of lung-infiltrating ILC2s, but also influences their motility and migratory behavior. In general, many data collected in recent murine and human studies argued against the former concept of a very strict tissue residency of innate lymphoid cells (ILCs) and instead pointed to a context-dependent homing capacity of peripheral blood ILC precursors and the inflammation-dependent capacity of specific ILC subsets for interorgan trafficking. In this review article, we provide a comprehensive overview of the so far described molecular mechanisms underlying the pulmonary migration of ILC2s and thereby the numeric regulation of local ILC2 pools at inflamed or fibrotic pulmonary tissue sites and discuss their potential to serve as innovative therapeutic targets in the treatment of inflammatory lung diseases.

Oxidative Imbalance as a Crucial Factor in Inflammatory Lung Diseases: Could Antioxidant Treatment Constitute a New Therapeutic Strategy?

Inflammatory lung disease results in a high global burden of death and disability. There are no effective treatments for the most severe forms of many inflammatory lung diseases, such as chronic obstructive pulmonary disease, emphysema, corticosteroid-resistant asthma, and coronavirus disease 2019; hence, new treatment options are required. Here, we review the role of oxidative imbalance in the development of difficult-to-treat inflammatory lung diseases. The inflammation-induced overproduction of reactive oxygen species (ROS) means that endogenous antioxidants may not be sufficient to prevent oxidative damage, resulting in an oxidative imbalance in the lung. In turn, intracellular signaling events trigger the production of proinflammatory mediators that perpetuate and aggravate the inflammatory response and may lead to tissue damage. The production of high levels of ROS in inflammatory lung diseases can induce the phosphorylation of mitogen-activated protein kinases, the inactivation of phosphoinositide 3-kinase (PI3K) signaling and histone deacetylase 2, a decrease in glucocorticoid binding to its receptor, and thus resistance to glucocorticoid treatment. Hence, antioxidant treatment might be a therapeutic option for inflammatory lung diseases. Preclinical studies have shown that antioxidants (alone or combined with anti-inflammatory drugs) are effective in the treatment of inflammatory lung diseases, although the clinical evidence of efficacy is weaker. Despite the high level of evidence for the efficacy of antioxidants in the treatment of inflammatory lung diseases, the discovery and clinical investigation of safer, more efficacious compounds are now a priority.

Nasal Delivery of Hesperidin/Chitosan Nanoparticles Suppresses Cytokine Storm Syndrome in a Mouse Model of Acute Lung Injury.

The cytokine storm or cytokine storm syndrome (CSS) is associated with high mortality in patients with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), for example following sepsis or infectious diseases including COVID-19. However, there are no effective treatments for CSS-associated ALI or ALI/ARDS. Thus, there remains an urgent need to develop effective drugs and therapeutic strategies against CSS and ALI/ARDS. Nasal and inhaled drug delivery methods represent a promising strategy in the treatment of inflammatory lung disease as a result of their ability to improve drug delivery to lungs. Improving the nasal mucosa absorption of poorly water-soluble drugs with poor mucosa bioavailability to a therapeutically effective level is another promising strategy in the fight against ALI/ARDS. Here, chitosan nanoparticles loaded with hesperidin (HPD/NPs) were developed for nasal delivery of the anti-inflammatory HPD compound to inflammatory lungs. In vitro and in vivo, HPD/NPs exhibited enhanced cellular uptake in the inflammatory microenvironment compared with free HPD. In a mouse model of inflammatory lung disease, the HPD/NPs markedly inhibited lung injury as evidenced by reduced inflammatory cytokine levels and suppressed vascular permeability compared with free HPD. Collectively, our study demonstrates that nasal delivery of HPD/NPs suppresses CSS and ALI/ARDS in a murine model of inflammatory lung disease, and that nanoparticle-based treatment strategies with anti-inflammatory effects could be used to reduce CSS and ALI in patients with inflammatory lung injury.

LL-37 restored glucocorticoid sensitivity impaired by virus dsRNA in lung

Glucocorticoids play a key role in treatment of inflammatory lung diseases including both airway and parenchymal lung diseases. RNA viral infections are major causes of chronic lung disease exacerbations and can determine glucocorticoid resistance. The antibacterial peptide LL-37, the only member of human cathelicidin family, also functions as antiviral-activity enhancer. However, whether it can alleviate the glucocorticoid resistance caused by RNA viruses remains unclear. Here, we used type I (BEAS-2B) and type II (A549) lung epithelial cells to assess the effect of LL-37 on dsRNA-induced glucocorticoid resistance. We verified that LL-37 and polyinosinic-polycytidylic acid (poly I:C, a mimic of viral dsRNA) interact and enter both cell lines. Co-treatment with LL-37 and poly I:C increased glucocorticoid-induced expression of promyelocytic leukemia zinc finger (PLZF), an anti-inflammatory protein, compared to poly I:C alone. Pre-treatment with LL-37 also restored transactivation of the glucocorticoid response element (GRE). Moreover, LL-37 rescued poly I:C-induced glucocorticoid resistance by increasing phosphorylation and nuclear translocation of glucocorticoid receptor. Importantly, LL-37 downregulated poly I:C-induced Erk and Akt signaling pathways in lung epithelial cells. Finally, we verified our data in vivo, showing that mCRAMP, the mouse LL-37 ortholog, can alleviate poly I:C-induced glucocorticoid insensitivity in a murine asthma model. In summary, this study showed that LL-37 restored glucocorticoid sensitivity impaired by dsRNA possibly by inhibiting Akt pathway, in addition to Erk1/2 pathway. These findings suggest LL-37 as a therapeutic agent for treatment of viral infections in inflammatory pulmonary diseases.

Clinical Application of Mesenchymal Stem Cell-Derived Extracellular Vesicle-Based Therapeutics for Inflammatory Lung Diseases.

It is currently thought that extracellular vesicles (EVs), such as exosomes and microvesicles, play an important autocrine/paracrine role in intercellular communication. EVs package proteins, mRNA and microRNA (miRNA), which have the ability to transfer biological information to recipient cells in the lungs. Depending on their origin, EVs fulfil different functions. EVs derived from mesenchymal stem cells (MSCs) have been found to promote therapeutic activities that are comparable to MSCs themselves. Recent animal model-based studies suggest that MSC-derived EVs have significant potential as a novel alternative to whole-cell therapies. Compared to their parent cells, EVs may have a superior safety profile and can be stored without losing function. It has been observed that MSC-derived EVs suppress pro-inflammatory processes and reduce oxidative stress, pulmonary fibrosis and remodeling in a variety of in vivo inflammatory lung disease models by transferring their components. However, there remain significant challenges to translate this therapy to the clinic. From this view point, we will summarize recent studies on EVs produced by MSCs in preclinical experimental models of inflammatory lung diseases. We will also discuss the most relevant issues in bringing MSC-derived EV-based therapeutics to the clinic for the treatment of inflammatory lung diseases.

The Lung Microbiota of Healthy Mice Are Highly Variable, Cluster by Environment, and Reflect Variation in Baseline Lung Innate Immunity.

The "gut-lung axis" is commonly invoked to explain the microbiome's influence on lung inflammation. Yet the lungs harbor their own microbiome, which is altered in respiratory disease. The relative influence of gut and lung bacteria on lung inflammation is unknown. To determine whether baseline lung immune tone reflects local (lung-lung) or remote (gut-lung) microbe-host interactions. We compared lung, tongue, and cecal bacteria in 40 healthy, genetically identical, 10-week-old mice, using 16S ribosomal RNA gene quantification and sequencing. We measured inflammatory cytokines, using a multiplex assay of homogenized lung tissue. We compared lung bacteria in healthy mice treated with varied durations of systemic antibiotics. Lung bacterial communities are highly variable among mice, cluster strongly by cage, shipment, and vendor, and are altered by antibiotics in a microbiologically predictable manner. Baseline lung concentrations of two key inflammatory cytokines (IL-1α and IL-4) are correlated with the diversity and community composition of lung bacterial communities. Lung concentrations of these inflammatory cytokines correlate more strongly with variation in lung bacterial communities than with that of the gut or mouth. In the lungs of healthy mice, baseline innate immune tone more strongly reflects local (lung-lung) microbe-host interactions than remote (gut-lung) microbe-host interactions. Our results independently confirm the existence and immunologic significance of the murine lung microbiome, even in health. Variation in lung microbiota is likely an important, underappreciated source of experimental and clinical variability. The lung microbiome is an unexplored therapeutic target for the prevention and treatment of inflammatory lung disease.

Combination of urea-crosslinked hyaluronic acid and sodium ascorbyl phosphate for the treatment of inflammatory lung diseases: An in vitro study

This in vitro study evaluated, for the first time, the safety and the biological activity of a novel urea-crosslinked hyaluronic acid component and sodium ascorbyl phosphate (HA-CL – SAP), singularly and/or in combination, intended for the treatment of inflammatory lung diseases. The aim was to understand if the combination HA-CL – SAP had an enhanced activity with respect to the combination native hyaluronic acid (HA) – SAP and the single SAP, HA and HA-CL components. Sample solutions displayed pH, osmolality and viscosity values suitable for lung delivery and showed to be not toxic on epithelial Calu-3 cells at the concentrations used in this study. The HA-CL – SAP displayed the most significant reduction in interleukin-6 (IL-6) and reactive oxygen species (ROS) levels, due to the combined action of HA-CL and SAP. Moreover, this combination showed improved cellular healing (wound closure) with respect to HA – SAP, SAP and HA, although at a lower rate than HA-CL alone. These preliminary results showed that the combination HA-CL - SAP could be suitable to reduce inflammation and oxidative stress in lung disorders like acute respiratory distress syndrome, asthma, emphysema and chronic obstructive pulmonary disease, where inflammation is prominent.

Fisetin inhibits TNF-α/NF-κB-induced IL-8 expression by targeting PKCδ in human airway epithelial cells

Fisetin (3,7,3′,4′-tetrahydroxyflavone), a natural flavonoid, is a therapeutic agent for respiratory inflammatory diseases such as chronic obstructive pulmonary disease (COPD). However, detailed molecular mechanisms regarding the target protein of fisetin remain unknown.Fisetin significantly reduces tumour necrosis factor alpha (TNF-α)-induced interleukin (IL)-8 levels by inhibiting both nuclear factor kappa B (NF-κB) transcriptional activity and the phosphorylation of its upstream effectors. We show that fisetin prevents interactions between protein kinase C (PKC)δ and TNF receptor-associated factor 2 (TRAF2), thereby inhibiting the inhibitor of kappa B kinase (IKK)/NF-κB downstream signalling cascade. Furthermore, we found that fisetin directly binds to PKCδ in vitro. Our findings provide evidence that fisetin inhibits the TNF-α-activated IKK/NF-κB cascade by targeting PKCδ, thereby mediating inflammatory diseases such as COPD.These data suggest that fisetin is a good therapeutic drug for the treatment of inflammatory lung diseases, such as COPD, by inhibiting the TNF-α/NF-κB signalling pathway.

Abstract B055: New potential therapeutic applications of WX-UK1 as a specific and potent inhibitor of human trypsin-2 and human trypsin-3

Abstract WX-UK1, a multi-serine protease inhibitor, was believed to function primarily as a synthetic small-molecule inhibitor of urokinase (uPA). We now reveal that WX-UK1 is a potent and rather specific inhibitor of human trypsin-2 and human trypsin-3, and propose that these new molecular targets offer potential for new indications in oncology and other diseases. Initially, we performed a bioinformatic analysis of the ~200 human trypsin-like serine proteases, most of which play crucial roles in homeostasis and disease. Among these we selected a subset for biochemical analysis based on an inspection of modeled 3D structures of WX-UK1:protease complexes and sequence alignment of binding site residues. Samples of the selected proteases were prepared and characterized biochemically with respect to inhibitory constant (Ki) of inhibition by WX-UK1-1 and dissociation constant (Kd). The Ki values were determined from enzymatic activity assays (37°C), and Kd values (affinity) by surface plasmon resonance (BIACORE) (25°C). Serine protease(human)Ki (nM)Mean ± SD (n)Kd (nM)Mean ± SD (n)Urokinase (uPA)874 ± 95 (3)720 ± 300 (3)Trypsin(-1)190 ± 10 (6)74 ± 18 (3)Trypsin-275 ± 3 (6)N.D.Trypsin-319 ± 4 (6)6 ± 4 (11) While previously described as an inhibitor of bovine trypsin, we now show WX-UK1 to be a specific competitive inhibitor of both human trypsin-2 and human trypsin-3 with Ki of 75 and 19 nM, respectively. With a Kd of 6 nM, WX-UK-1 binds human trypsin-3 with a >100-fold binding specificity relative to most other trypsin-like serine proteases tested so far, including uPA, and 10-fold over the classical human trypsin. WX-UK1 (and its oral prodrug, upamostat) was originally developed as an inhibitor of uPA and other members of the S1 family of trypsin-like serine proteases, essential to aspects of tissue remodeling associated with tumor invasion and metastasis. However, with the identification of human trypsin-3 and human trypsin-2 as high-affinity molecular targets, new disease indications on top of oncology indications may be feasible. As a low-nanomolar inhibitor of human trypsin-3, WX-UK1 may have potential utility in the treatment of inflammatory digestive diseases, including irritable bowel syndrome, inflammatory bowel disease, and pancreatitis, for the latter of which there are currently no approved therapies. Furthermore, WX-UK1, as a human trypsin-2 inhibitor, could potentially be used in the treatment of inflammatory lung diseases, including acute respiratory distress syndrome, acute lung injury, α-1 antitrypsin deficiency, and chronic obstructive pulmonary disease (COPD)–a major unmet medical need. Citation Format: Emil Oldenberg, Christine R. Schar, Eva L. Lange, Terry F. Plasse, Danielle T. Abramson, Mark L. Levitt, Reza Fathi, Jan K. Jensen. New potential therapeutic applications of WX-UK1 as a specific and potent inhibitor of human trypsin-2 and human trypsin-3 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B055.