Murine Airway Research Articles

Contribution of Postjunctional M2 Muscarinic Receptors to Cholinergic Nerve-Mediated Contractions of Murine Airway Smooth Muscle.

Postjunctional M2Rs on airway smooth muscle (ASM) outnumber M3Rs by a ratio of 4:1 in most species, however, it is the M3Rs that are thought to mediate the bronchoconstrictor effects of acetylcholine. In this study, we describe a novel and profound M2R-mediated hypersensitization of M3R-dependent contractions of ASM at low stimulus frequencies.. Contractions induced by 2Hz EFS were augmented by > 2.5-fold when the stimulus interval was reduced from 100 to 10 s. This effect was reversed by the M2R antagonists, methoctramine, and AFDX116, and was absent in M2R null mice. The M3R antagonist 4-DAMP abolished the entire response in both WT and M2R KO mice. The M2R-mediated potentiation of EFS-induced contractions was not observed when the stimulus frequency was increased to 20 Hz. A subthreshold concentration of carbachol enhanced the amplitude of EFS-evoked contractions in WT, but not M2R null mice. These data highlight a significant M2R-mediated potentiation of M3R-dependent contractions of ASM at low frequency stimulation that could be relevant in diseases such as asthma and COPD.

Ginger metabolites and metabolite-inspired synthetic products modulate intracellular calcium and relax airway smooth muscle.

Asthma affects millions of people worldwide and its prevalence is increasing. It is characterized by chronic airway inflammation, airway remodeling, and pathologic bronchoconstriction, and it poses a continuous treatment challenge with very few new therapeutics available. Thus, many asthmatics turn to plant-based complementary products, including ginger, for better symptom control, indicating an unmet need for novel therapies. Previously, we demonstrated that 6-shogaol (6S), the primary bioactive component of ginger, relaxes human airway smooth muscle (hASM) likely by inhibition of phosphodiesterases (PDEs) in the β-adrenergic (cyclic nucleotide PDEs), and muscarinic (phospholipase C, PLC) receptor pathways. However, oral 6S is extensively metabolized and it is unknown if the resulting metabolites remain bioactive. Here, we screened all the known human metabolites of 6S and several metabolite-based synthetic derivatives to better understand their mechanism of action and structure-function relationships. We demonstrate that several metabolites and metabolite-based synthetic derivatives are able to prevent Gq-coupled stimulation of intracellular calcium [Ca2+]i and inositol trisphosphate (IP3) synthesis by inhibiting PLC, similar to the parent compound 6S. We also show that these compounds prevent recontraction of ASM after β-agonist relaxation likely by inhibiting PDEs. Furthermore, they potentiate isoproterenol-induced relaxation. Importantly, moving beyond cell-based assays, metabolites also retain the functional ability to relax Gq-coupled-contractions in upper (human) and lower (murine) airways. The current study indicates that, although oral ginger may be metabolized rapidly, it retains physiological activity through its metabolites. Moreover, we are able to use naturally occurring metabolites as inspiration to develop novel therapeutics for brochoconstrictive diseases.

Hemagglutinins of Avian Influenza Viruses Are Proteolytically Activated by TMPRSS2 in Human and Murine Airway Cells.

Cleavage of the influenza A virus (IAV) hemagglutinin (HA) by host proteases is indispensable for virus replication. Most IAVs possess a monobasic HA cleavage site cleaved by trypsin-like proteases. Previously, the transmembrane protease TMPRSS2 was shown to be essential for proteolytic activation of IAV HA subtypes H1, H2, H7, and H10 in mice. In contrast, additional proteases are involved in activation of certain H3 IAVs, indicating that HAs with monobasic cleavage sites can differ in their sensitivity to host proteases. Here, we investigated the role of TMPRSS2 in proteolytic activation of avian HA subtypes H1 to H11 and H14 to H16 in human and mouse airway cell cultures. Using reassortant viruses carrying representative HAs, we analyzed HA cleavage and multicycle replication in (i) lung cells of TMPRSS2-deficient mice and (ii) Calu-3 cells and primary human bronchial cells subjected to morpholino oligomer-mediated knockdown of TMPRSS2 activity. TMPRSS2 was found to be crucial for activation of H1 to H11, H14, and H15 in airway cells of human and mouse. Only H9 with an R-S-S-R cleavage site and H16 were proteolytically activated in the absence of TMPRSS2 activity, albeit with reduced efficiency. Moreover, a TMPRSS2-orthologous protease from duck supported activation of H1 to H11, H15, and H16 in MDCK cells. Together, our data demonstrate that in human and murine respiratory cells, TMPRSS2 is the major activating protease of almost all IAV HA subtypes with monobasic cleavage sites. Furthermore, our results suggest that TMPRSS2 supports activation of IAV with a monobasic cleavage site in ducks. IMPORTANCE Human infections with avian influenza A viruses upon exposure to infected birds are frequently reported and have received attention as a potential pandemic threat. Cleavage of the envelope glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. In this study, we identify the transmembrane protease TMPRSS2 as the major activating protease of avian influenza virus HAs of subtypes H1 to H11, H14 and H15 in human and murine airway cells. Our data demonstrate that inhibition of TMPRSS2 activity may provide a useful approach for the treatment of human infections with avian influenza viruses that should be considered for pandemic preparedness as well. Additionally, we show that a TMPRSS2-orthologous protease from duck can activate avian influenza virus HAs with a monobasic cleavage site and, thus, represents a potential virus-activating protease in waterfowl, the primary reservoir for influenza A viruses.

Alkaloids from Lime Flower (Tiliae flos) Exert Spasmodic Activity on Murine Airway Smooth Muscle Involving Acetylcholinesterase.

Lime flower (Tiliae flos) is traditionally used either for treatment of the common cold or to relieve symptoms of mental stress. Recently, the presence of a new class of piperidine and dihydro-pyrrole alkaloids from lime flower has been described. The present study aimed to investigate the pharmacological activity of hydroacetonic lime flower extracts, alkaloid-enriched lime flower fractions, and isolated alkaloids on the murine airway smooth muscle and the cholinergic system. While a hydroacetonic lime flower extract did not show any pharmacological activity, enriched Tilia alkaloid fractions potentiated acetylcholine-induced contractions of the trachea by ~ 30%, showing characteristics comparable to galanthamine. Effects were abrogated by atropine, indicating an involvement of muscarinic receptors. The dihydro-pyrrole alkaloid tiliine A, the piperidine alkaloid tiliamine B, and the acetylated piperidine alkaloid tilacetine A were characterized as acetylcholinesterase inhibitors. The positive control galanthamine (IC50 = 2.0 µM, 95% CI 1.7 to 2.2 µM) was approximately 100 times more potent compared to tiliine A (IC50 = 237 µM, 95% CI 207 to 258 µM) and tiliamine B (IC50 = 172 µM, 95% CI 158 to 187 µM). Neither DNA synthesis of HepG2 liver cells, HaCaT keratinocytes, and Caco-2 intestinal epithelial cells nor cell viability of primary human fibroblasts was reduced by the alkaloids. The indirect cholinergic activity of the alkaloids might explain some aspects of the traditional use of lime flowers and may extend the portfolio of compounds with regard to diseases involving parasympathetic malfunction or central cholinergic imbalance.

LasR-regulated proteases in acute vs. chronic lung infection: a double-edged sword.

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen capable of causing both acute and chronic infections, particularly in individuals with compromised host defenses. The quorum sensing transcriptional activator LasR is widely recognized for its role in regulating the expression of acute virulence factors, notably several secreted proteases which cause direct host damage and subvert host immunity in acute infections. Paradoxically, lung infections caused by LasR-deficient variants, which are found in at least a third of cystic fibrosis (CF) patients with chronic P. aeruginosa infections, are associated with accelerated lung disease and increased markers of inflammation compared to infections caused by strains with a functional LasR system. While the loss of LasR function often (although not always) results in impaired production of LasR-controlled acute virulence factors, the implication of this pathoadaptation on host-pathogen interactions and chronic disease pathology is less well recognized. We recently observed that loss of LasR function in lasR variants, which results in impaired secreted protease production, led to increased expression of the membrane-bound surface adhesion molecule mICAM-1 in the airway epithelium, and increased neutrophilic inflammation. Specifically, human airway epithelial cells stimulated with lasR variants had higher mICAM-1 expression and greater neutrophil binding in vitro compared to stimulation with wild-type P. aeruginosa. In a subacute non-lethal P. aeruginosa lung infection model, lasR variant infection also induced higher mICAM-1 expression in the murine airway epithelium and was associated with increased neutrophilic pulmonary inflammation in vivo. Here, we discuss how (loss of) LasR function and LasR-regulated proteases affect host immunity, inflammation and tissue pathology in acute vs. chronic P. aeruginosa lung infection.

Airway basal stem cells reutilize the embryonic proliferation regulator, Tgfβ-Id2 axis, for tissue regeneration.

During development, quiescent airway basal stem cells are derived from proliferative primordial progenitors through the cell-cycle slowdown. In contrast, basal cells contribute to adult tissue regeneration by shifting from slow cycling to proliferating and subsequently back to slow cycling. Although sustained proliferation results in tumorigenesis, the molecular mechanisms regulating these transitions remain unknown. Using temporal single-cell transcriptomics of developing murine airway progenitors and genetic validation experiments, we found that TGF-β signaling decelerated cell cycle by inhibiting Id2 and contributed to slow-cycling basal cell specification during development. In adult tissue regeneration, reduced TGF-β signaling restored Id2 expression and initiated regeneration. Id2 overexpression and Tgfbr2 knockout enhanced epithelial proliferation; however, persistent Id2 expression drove basal cell hyperplasia that resembled a precancerous state. Together, the TGF-β-Id2 axis commonly regulates the proliferation transitions in basal cells during development and regeneration, and its fine-tuning is critical for normal regeneration while avoiding basal cell hyperplasia.

Transcriptome profiling and protease inhibition experiments identify proteases that activate H3N2 influenza A and influenza B viruses in murine airways

Cleavage of influenza virus hemagglutinin (HA) by host proteases is essential for virus infectivity. HA of most influenza A and B (IAV/IBV) viruses is cleaved at a monobasic motif by trypsin-like proteases. Previous studies have reported that transmembrane serine protease 2 (TMPRSS2) is essential for activation of H7N9 and H1N1pdm IAV in mice but that H3N2 IAV and IBV activation is independent of TMPRSS2 and carried out by as-yet-undetermined protease(s). Here, to identify additional H3 IAV- and IBV-activating proteases, we used RNA-Seq to investigate the protease repertoire of murine lower airway tissues, primary type II alveolar epithelial cells (AECIIs), and the mouse lung cell line MLE-15. Among 13 candidates identified, TMPRSS4, TMPRSS13, hepsin, and prostasin activated H3 and IBV HA in vitro. IBV activation and replication was reduced in AECIIs from Tmprss2/Tmprss4-deficient mice compared with WT or Tmprss2-deficient mice, indicating that murine TMPRSS4 is involved in IBV activation. Multicycle replication of H3N2 IAV and IBV in AECIIs of Tmprss2/Tmprss4-deficient mice varied in sensitivity to protease inhibitors, indicating that different, but overlapping, sets of murine proteases facilitate H3 and IBV HA cleavages. Interestingly, human hepsin and prostasin orthologs did not activate H3, but they did activate IBV HA in vitro. Our results indicate that TMPRSS4 is an IBV-activating protease in murine AECIIs and suggest that TMPRSS13, hepsin, and prostasin cleave H3 and IBV HA in mice. They further show that hepsin and prostasin orthologs might contribute to the differences observed in TMPRSS2-independent activation of H3 in murine and human airways.

A search for influenza A and B virus hemagglutinin‐activating host cell proteases with trypsin‐like activity in the murine airways

A search for influenza A and B virus hemagglutinin‐activating host cell proteases with trypsin‐like activity in the murine airways

Contribution of opioid and neurotensin receptors in the anti-inflammatory activity of PK20 hybrid compound in murine airways.

PK20 is an anti-inflammatory hybrid compound, composed of an endomorphin-2-like and neurotensin-like fragments. The aim of the present study is to assess the contribution of particular pharmacophores to the activity of the hybrid tested. For this purpose, airway hyperresponsiveness, accumulation of inflammatory cells in bronchoalveolar lavage fluid (BALF), concentration of mouse mast cell protease, malondialdehyde and secretory phospholipase 2 activity in lung tissue, as well as production of pro-inflammatory cytokines in BALF and lung were determined by using murine model of non-atopic asthma. Blocking either neurotensin receptors or mu opioid receptors did not alter the potential of PK20 in reducing airway hyperresponsiveness. In studies of inflammatory cells, the beneficial effect of the entire peptide occurs to be mediated by the stimulation of neurotensin receptors. However, regarding cytokine and biochemical assays, pretreatment with both receptor antagonists resulted in a different effect on its activity depending on the parameter studied. To conclude, the activation of both the opioid and neurotensin receptors seems to be necessary to induce the full anti-inflammatory activity of the hybrid compound.

Tectochrysin ameliorates murine allergic airway inflammation by suppressing Th2 response and oxidative stress

Tectochrysin, a flavonoid compound, can be isolated from propolis, Alpinia oxyphylla Miq, and Lychnophora markgravii. This study evaluated the efficacy of tectochrysin in the treatment of shrimp tropomyosin (ST)-induced mouse asthma. Mice were sensitized with intraperitoneal (i.p.) injection of ST together with aluminum hydroxide as an adjuvant to establish a mouse model of asthma. Mice were i.p.-treated daily with tectochrysin. IgE levels in plasma, Th2 cytokines from both bronchoalveolar lavage (BAL) fluid and splenocytes, and CD200R on basophils in peripheral blood were measured. Histological analyses of lung tissues and accumulation of leukocytes in BAL fluid were performed. Lung eosinophil peroxidase, catalase and glutathione peroxidase activities were examined. ST was found to markedly increase eosinophilic inflammation and Th2 response in mice. Tectochrysin treatment reduced the level of IgE in plasma, the percentage of eosinophils in total white blood cells in peripheral blood, the total number of cells in BAL fluid, and eosinophil peroxidase activity in lung tissues. Tectochrysin attenuated ST-induced infiltration of eosinophils and epithelial mucus secretion in lung tissues and suppressed the overproduction of Th2 cytokines (IL-4 and IL-5) in BAL fluid. Tectochrysin also attenuated Th2 cytokine (IL-4 and IL-5) production from antigen-stimulated murine splenocytes in vitro, decreased the expression of CD200R on basophils in peripheral blood of asthmatic mice and inhibited IL-4 secretion from IgE-sensitized RBL-2H3 cells. In addition, tectochrysin enhanced catalase and glutathione peroxidase activities in lung tissues. Our findings demonstrate that TEC ameliorates allergic airway inflammation by suppressing Th2 response and oxidative stress.

Comparative analysis of the role of mast cells in murine asthma models using Kit-sufficient mast cell-deficient animals.

Asthma is a frequent chronic disease that can potentially severely affect the respiratory capacity and well-being of patients. Mast cells (MCs) are regarded as major players in human asthma due to their capacity to release crucial inflammatory mediators following allergen exposure. However, unambiguous characterization of their role in animal models has long been hindered by the unavailability of specific MC-deficient models lacking confounding MC-unrelated effects. This study aims to examine the role of MCs in Kit-sufficient MC-deficient Cpa3Cre/+ mice. We used a variety of models of acute and chronic asthma employing distinct routes and regimes of sensitization. These sensitizations were done via the peritoneal cavity, the skin, or the lung. Additionally, different allergens, i.e. ovalbumin and house dust mite extract, were used. Our results show that the absence of MCs had no impact on the severity of allergic airway inflammation in any of the tested mouse models, as measured by leukocyte infiltration in the airways, cytokine expression, antibody production, airway hyper-responsiveness and mucus production. This indicates that MCs do not play a major role in murine allergic airway inflammation.

An Improved Murine Premalignant Squamous Cell Model: Tobacco Smoke Exposure Augments NTCU-Induced Murine Airway Dysplasia.

Tobacco smoke-induced squamous cell lung cancer (SCC) develops from endobronchial dysplastic lesions that progress to invasive disease. A reproducible murine model recapitulating histologic progression observed in current and former smokers will advance testing of new preventive and therapeutic strategies. Previous studies show that prolonged topical application of N-nitroso-tris-chloroethylurea (NTCU) generates a range of airway lesions in sensitive mice similar to those induced by chronic tobacco smoke exposure in humans. To improve the current NTCU model and better align it with human disease, NTCU was applied to mice twice weekly for 4-5 weeks followed by a recovery period before cigarette smoke (CS) or ambient air (control) exposure for an additional 3-6 weeks. Despite the short time course, the addition of CS led to significantly more premalignant lesions (PML; 2.6 vs. 0.5; P < 0.02) and resulted in fewer alveolar macrophages (52,000 macrophages/mL BALF vs. 68,000; P < 0.05) compared with control mice. This improved NTCU + CS model is the first murine SCC model to incorporate tobacco smoke and is more amenable to preclinical studies because of the increased number of PML, decreased number of mice required, and reduced time needed for PML development.

Abscisic acid suppresses the activation of NLRP3 inflammasome and oxidative stress in murine allergicairwayinflammation.

Abscisic acid (ABA), a well-known natural phytohormone reportedly exerts anti-inflammatory and anti-oxidative properties in diabetes and colitis. However, the efficacy of ABA against allergic airway inflammation and the underlying mechanism remain unknown. Herein, an OVA-induced murine allergic airway inflammation model was established and treated with ABA in the presence or absence of PPAR-γ antagonist GW9662. The results showed that ABA effectively stunted the development of airway inflammation, and concordantly downregulated OVA-induced activation of NLRP3 inflammasome, suppressed oxidative stress and decreased the expression of mitochondrial fusion/fission markers including Optic Atrophy 1 (OPA1), Mitofusion 2 (Mfn2), dynamin-related protein 1 (DRP1) and Fission 1 (Fis1). Moreover, ABA treatment further increased OVA-induced expression of PPAR-γ, while GW9662 abrogated the inhibitory effect of ABA on allergic airway inflammation as well as on the activation of NLRP3 inflammasome and oxidative stress. Consistently, ABA inhibited the activation of NLRP3 inflammasome, suppressed oxidative stress and mitochondrial fusion/fission in LPS-stimulated Raw264.7 cells via PPAR-γ. Collectively, ABA ameliorates OVA-induced allergic airway inflammation in a PPAR-γ dependent manner, and such effect of ABA may be associated with its inhibitory effect on NLRP3 inflammasome and oxidative stress. Our results suggest the potential of ABA or ABA-rich food in protecting against asthma.

Root extract of Angelica reflexa B.Y.Lee reduces allergic lung inflammation by regulating Th2 cell activation

Ethnopharmacological relevanceTraditionally, the roots of Angelica reflexa B.Y.Lee (AR) have been used to treat cough, phlegm, neuralgia, and arthralgia in Northeast Asia. Aim of the studyThe anti-asthmatic effect of AR root extract (ARE) was determined using a murine airway allergic inflammation model and the primary T cell polarization assay. Materials and methodsTo evaluate the anti-asthmatic effect of ARE, inflammatory cell infiltration was determined histologically and inflammatory mediators were measured in bronchoalveolar lavage fluid (BALF). Furthermore, the effects of AREs on Th2 cell differentiation and activation were determined by western blotting and flow cytometry. ResultsAsthmatic phenotypes were alleviated by ARE treatment, which reduced mucus production, inflammatory cell infiltration (especially eosinophilia), and type 2 cytokine levels in BALF. ARE administration to mice reduced the number of activated Th2 (CD4+CD25+) cells and level of GATA3 in the lungs. Furthermore, ARE treatment inhibited the differentiation of Th2 cells in primary cell culture systems via interferon regulatory factor 4 (IRF4) signaling. ConclusionsOur findings indicate that the anti-asthmatic effect of AREs is mediated by the reduction in Th2 cell activation by regulating IRF4.

Antigen- and Epitope-Delivering Nanoparticles Targeting Liver Induce Comparable Immunotolerance in Allergic Airway Disease and Anaphylaxis as Nanoparticle-Delivering Pharmaceuticals

The targeting of natural tolerogenic liver sinusoidal endothelial cells (LSEC) by nanoparticles (NPs), decorated with a stabilin receptor ligand, is capable of generating regulatory T-cells (Tregs), which can suppress antigen-specific immune responses, including to ovalbumin (OVA), a possible food allergen. In this regard, we have previously demonstrated that OVA-encapsulating poly(lactic-co-glycolic acid) (PLGA) nanoparticles eliminate allergic airway inflammation in OVA-sensitized mice, prophylactically and therapeutically. A competing approach is a nanocarrier platform that incorporates pharmaceutical agents interfering in mTOR (rapamycin) or NF-κB (curcumin) pathways, with the ability to induce a tolerogenic state in nontargeted antigen-presenting cells system-wide. First, we compared OVA-encapsulating, LSEC-targeting tolerogenic nanoparticles (TNPs) with nontargeted NPs incorporating curcumin and rapamycin (Rapa) in a murine eosinophilic airway inflammation model, which is Treg-sensitive. This demonstrated roughly similar tolerogenic effects on allergic airway inflammation by stabilin-targeting NPOVA versus nontargeted NPs delivering OVA plus Rapa. Reduction in eosinophilic inflammation and TH2-mediated immune responses in the lung was accompanied by increased Foxp3+ Treg recruitment and TGF-β production in both platforms. As OVA incorporates IgE-binding as well as non-IgE-binding epitopes, the next experiment explored the possibility of obtaining immune tolerance by non-anaphylactic T-cell epitopes. This was accomplished by incorporating OVA323-339 and OVA257-264 epitopes in liver-targeting NPs to assess the prophylactic and therapeutic impact on allergic inflammation in transgenic OT-II mice. Importantly, we demonstrated that the major histocompatibility complex (MHC)-II binding (former) but not the MHC-I binding (latter) epitope interfered in allergic airway inflammation, improving TNPOVA efficacy. The epitope-specific effect was transduced by TGF-β-producing Tregs. In the final phase of experimentation, we used an OVA-induced anaphylaxis model to demonstrate that targeted delivery of OVA and its MHC-II epitope could significantly suppress the anaphylaxis symptom score, mast cell release, and the late-phase inflammatory response. In summary, these results demonstrate comparable efficacy of LSEC-targeting versus pharmaceutical PLGA nanoparticles, as well as the ability of T-cell epitopes to achieve response outcomes similar to those of the intact allergens.

Airway Tissue Stem Cells Reutilize the Embryonic Proliferation Regulator, Tgfβ-Id2 Axis, for Tissue Regeneration

During development, quiescent basal stem cells are derived from proliferative primordial progenitors through the cell cycle slowdown. In contrast, quiescent basal cells contribute to tissue repair during adult tissue regeneration by shifting from slow-cycling to proliferating and subsequently back to slow-cycling. Although sustained basal cell proliferation results in tumorigenesis, the molecular mechanisms regulating these transitions remain unknown. Using temporal single-cell transcriptomics of developing murine airway progenitors and in vivo genetic validation experiments, we found that Tgfβ signaling slowed down cell cycle by inhibiting Id2 expression in airway progenitors and contributed to the specification of slow-cycling basal cell population during development. In adult tissue regeneration, reduced Tgfβ signaling restored Id2 expression and initiated epithelial regeneration. Id2 overexpression and Tgfbr2 knockout enhanced epithelial proliferation; however, persistent Id2 expression in basal cells drove hyperplasia at a rate that resembled a precancerous state. Together, the Tgfβ-Id2 axis commonly regulates the proliferation transitions in airway basal cells during development and regeneration, and its fine-tuning is critical for normal regeneration while avoiding basal cell hyperplasia.

SCRINSHOT enables spatial mapping of cell states in tissue sections with single-cell resolution.

Changes in cell identities and positions underlie tissue development and disease progression. Although single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single-Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRINSHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity, and quantitative qualities of SCRINSHOT facilitate single-cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions.

Lung-targeting lentiviral vector for passive immunisation against influenza

When recombinant simian immunodeficiency virus (SIV) is pseudotyped with the F and HN glycoproteins from murine respiratory Sendai virus (rSIV.F/HN), it provides efficient lung cell targeting and lifelong transgene expression...

Sojadodamgangki-tang attenuates allergic lung inflammation by inhibiting T helper 2 cells and Augmenting alveolar macrophages

Ethnopharmacological relevanceSojadodamgangki-tang (SDG) is a traditional East-Asian herbal medicine mainly composed of Pinellia ternate (Thunb.) Makino, Perilla frutescens (L.) Britt and 10 kinds of medicinal herbs. It has been used to treat asthma and mucus secretion including lung and bronchi. Aim of the studyThe aim of this study was to investigate the anti-inflammatory effects of Sojadodamgangki-tang (SDG) on allergic lung inflammation in vitro and in vivo as well as the underlying mechanisms. Materials and methodsWe used an ovalbumin (OVA)-induced murine allergic airway inflammation model. Five groups of 8-week-old female BALB/C mice were divided into the following groups: saline control group, the vehicle (allergic) group that received OVA only, groups that received OVA and SDG (200 mg/kg or 400 mg/kg), and a positive control group that received OVA and Dexamethasone (5 mg/kg). In vitro experiments include T helper 2 (TH2) polarization system, murine macrophage cell culture, and human bronchial epithelial cell line (BEAS-2B) culture. ResultsSDG administration reduced allergic airway inflammatory cell infiltration, especially of eosinophils, mucus production, Th2 cell activation, OVA-specific immunoglobulin E (IgE), and total IgE production. Moreover, the activation of alveolar macrophages, which leads to immune tolerance in the steady state, was promoted by SDG treatment. Interestingly, SDG treatment also reduced the production of alarmin cytokines by the human bronchial epithelial cell line BEAS-2B stimulated with urban particulate matter. ConclusionOur findings indicate that SDG has potential as a therapeutic drug to inhibit Th2 cell activation and promote alveolar macrophage activation.

Transcriptome profiling and protease inhibition experiments identify proteases that activate H3N2 influenza A and influenza B viruses in murine airways

Cleavage of influenza virus hemagglutinin (HA) by host proteases is essential for virus infectivity. HA of most influenza A and B (IAV/IBV) viruses is cleaved at a monobasic motif by trypsin-like proteases. Previous studies have reported that transmembrane serine protease 2 (TMPRSS2) is essential for activation of H7N9 and H1N1pdm IAV in mice but that H3N2 IAV and IBV activation is independent of TMPRSS2 and carried out by as-yet-undetermined protease(s). Here, to identify additional H3 IAV- and IBV-activating proteases, we used RNA-Seq to investigate the protease repertoire of murine lower airway tissues, primary type II alveolar epithelial cells (AECIIs), and the mouse lung cell line MLE-15. Among 13 candidates identified, TMPRSS4, TMPRSS13, hepsin, and prostasin activated H3 and IBV HA in vitro IBV activation and replication was reduced in AECIIs from Tmprss2/Tmprss4-deficient mice compared with WT or Tmprss2-deficient mice, indicating that murine TMPRSS4 is involved in IBV activation. Multicycle replication of H3N2 IAV and IBV in AECIIs of Tmprss2/Tmprss4-deficient mice varied in sensitivity to protease inhibitors, indicating that different, but overlapping, sets of murine proteases facilitate H3 and IBV HA cleavages. Interestingly, human hepsin and prostasin orthologs did not activate H3, but they did activate IBV HA in vitro Our results indicate that TMPRSS4 is an IBV-activating protease in murine AECIIs and suggest that TMPRSS13, hepsin, and prostasin cleave H3 and IBV HA in mice. They further show that hepsin and prostasin orthologs might contribute to the differences observed in TMPRSS2-independent activation of H3 in murine and human airways.