Search

Asthma is a heterogenous disease strongly associated with inflammation that has many different causes and triggers. Current asthma treatments target symptoms such as bronchoconstriction and airway inflammation. Despite recent advances in biological therapies, there remains a need for new classes of therapeutic agents with novel, upstream targets. The proteinase-activated receptor-2 (PAR2) has long been implicated in allergic airway inflammation and asthma and it remains an intriguing target for novel therapies. Here, we describe the actions of C781, a newly developed low MW PAR2 biased antagonist, in vitro and in vivo in the context of acute allergen exposure.A human bronchial epithelial cell line expressing PAR2 (16HBE14o- cells) was used to evaluate the modulation in vitro, by C781, of physiological responses to PAR2 activation and downstream β-arrestin/MAPK and Gq/CaC781 reduced in vitro physiological signalling in response to ligand and proteinase activation. C781 effectively antagonized β-arrestin/MAPK signalling without significant effect on Gq/CaOur work demonstrates the first biased PAR2 antagonist for β-arrestin/MAPK signalling. C781 is efficacious as a prophylactic treatment for allergen-induced airway hyperresponsiveness and inflammation in mice. It exemplifies a key pharmacophore for PAR2 that can be optimized for clinical development.

Measuring chromatin accessibility is a powerful method to identify cell types and states. Performed at single-cell resolution, this assay has generated catalogs of genome-wide DNA regulatory sites, whole-organism cell atlases, and dynamic chromatin reorganization through development. However, the limited throughput of current single-cell approaches poses a challenge for implementing proper study designs, population-scale profiling, and/or very deep profiling of complex samples. To this end, we developed a 10X-compatible combinatorial indexing ATAC sequencing (“txci-ATAC-seq”), which is a combinatorial indexing framework that initially indexes (“pre-indexes”) chromatin within nuclei with barcoded transposases followed by encapsulation and further barcoding using a commercialized droplet-based microfluidics platform (10X Genomics). Leveraging this molecular hashing strategy, we demonstrate that txci-ATAC-seq enables the indexing of up to 200,000 nuclei across multiple samples in a single emulsion reaction, representing a ∼22-fold increase in throughput compared to the standard workflow at the same collision rate. To improve the efficiency of this new technique, we further developed a faster version of the protocol (“Fast-txci-ATAC-seq”) that separates sample pre-processing from library generation and has the potential to profile up to 96 samples simultaneously. We initially benchmarked our assay by generating chromatin accessibility profiles for 230,018 cells from five native tissues across three experiments, including human cortex (28,513 cells), mouse brain (48,997 cells), human lung (15,799 cells), mouse lung (73,280 cells), and mouse liver (63,429 cells). We also applied our method to a club cell secretory protein knockout (CC16-/-) mouse model to examine the biological and technical limitations of the mouse line. By characterizing DNA regulatory landscapes in 76,498 wild-type and 77,638 CC16-/-murine lung nuclei, our investigations uncovered previously unappreciated residual genetic deviations from the reference strain that resulted from the method of gene targeting, which employed embryonic stem cells from the 129 strain. We found that these genetic remnants from the 129 strain led to profound cell-type-specific changes in chromatin accessibility in regulatory elements near a host of genes. Collectively, we defined single-cell chromatin signatures in 384,154 nuclei from 13 primary samples across different species, organs, biological replicates, and genetic backgrounds, establishing txci-ATAC-seq as a robust, high-quality, and highly multiplexable single-cell assay for large-scale chromatin studies.

RATIONALE: The identification of novel molecules associated with asthma may provide insights into mechanisms of disease and their potential clinical implications.OBJECTIVES: To conduct a screening of circulating proteins in childhood asthma and to study proteins emerged from human studies in a mouse model of asthma.METHODS: We included 2,264 children from eight birth cohorts from the MeDALL project and the Tucson TCRS Study. In cross-sectional analyses, we tested 46 circulating proteins for association with asthma in the selection stage and carried significant signals forward to a validation and replication stage. As creatine kinase (CK) was the only protein consistently associated with asthma, we also compared whole blood CK gene expression between subjects with and without asthma (n=249) and used a house dust mite (HDM) challenged mouse model to gain insights into CK lung expression and its role in resolution of asthma phenotypes.MEASUREMENTS AND MAIN RESULTS: As compared with the lowest CK tertile, children in the highest tertile had significantly lower odds for asthma in selection (adjOR: 0.31, 0.15-0.65; p=0.002), validation (0.63, 0.42-0.95; p=0.03), and replication (0.40, 0.16-0.97; p = 0.04) stages. Both cytosolic CK forms (CKM, CKB) were under-expressed in blood from asthmatics compared to controls (p=0.01 and 0.006, respectively). In the lungs of HDM-challenged mice, Ckb expression was reduced and, following HDM challenge, a CKB inhibitor blocked the resolution of airway hyperresponsiveness (AHR) and reduction of airway mucin.CONCLUSIONS: Circulating levels and gene expression of CK are inversely associated with childhood asthma. Mouse models support a possible direct involvement of CK in asthma protection via inhibition of AHR and reduction of airway mucin.

Rationale: Cigarette smoke (CS) inhalation triggers oxidative stress and inflammation, leading to accelerated lung aging, apoptosis, and emphysema, as well as systemic pathologies. Metformin is beneficial for protecting against aging-related diseases. Objectives: We sought to investigate whether metformin may ameliorate CS-induced pathologies of emphysematous chronic obstructive pulmonary disease (COPD). Methods: Mice were exposed chronically to CS and fed metformin-enriched chow for the second half of exposure. Lung, kidney, and muscle pathologies, lung proteostasis, endoplasmic reticulum (ER) stress, mitochondrial function, and mediators of metformin effects in vivo and/or in vitro were studied. We evaluated the association of metformin use with indices of emphysema progression over 5 years of follow-up among the COPDGene (Genetic Epidemiology of COPD) study participants. The association of metformin use with the percentage of emphysema and adjusted lung density was estimated by using a linear mixed model. Measurements and Main Results: Metformin protected against CS-induced pulmonary inflammation and airspace enlargement; small airway remodeling, glomerular shrinkage, oxidative stress, apoptosis, telomere damage, aging, dysmetabolism in vivo and in vitro; and ER stress. The AMPK (AMP-activated protein kinase) pathway was central to metformin’s protective action. Within COPDGene, participants receiving metformin compared with those not receiving it had a slower progression of emphysema (−0.92%; 95% confidence interval [CI], −1.7% to −0.14%; P = 0.02) and a slower adjusted lung density decrease (2.2 g/L; 95% CI, 0.43 to 4.0 g/L; P = 0.01). Conclusions: Metformin protected against CS-induced lung, renal, and muscle injury; mitochondrial dysfunction; and unfolded protein responses and ER stress in mice. In humans, metformin use was associated with lesser emphysema progression over time. Our results provide a rationale for clinical trials testing the efficacy of metformin in limiting emphysema progression and its systemic consequences.

IntroductionHow cigarette smoke (CS) and chronic obstructive pulmonary disease (COPD) affect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and severity is controversial. We investigated the protein and mRNA expression of SARS-CoV-2 entry receptor ACE2 and proteinase TMPRSS2 in lungs from COPD patients and controls, and lung tissue from mice exposed acutely and chronically to CS. Also, we investigated the effects of CS exposure on SARS-CoV-2 infection in human bronchial epithelial cells.MethodsIn Cohort 1, ACE2-positive cells were quantified by immunostaining in FFPE sections from both central and peripheral airways. In Cohort 2, we quantified pulmonary ACE2 protein levels by immunostaining and ELISA, and both ACE2 and TMPRSS2 mRNA levels by RT-qPCR. In C57BL/6 WT mice exposed to air or CS for up to 6 months, pulmonary ACE2 protein levels were quantified by triple immunofluorescence staining and ELISA. The effects of CS exposure on SARS-CoV-2 infection were evaluated after 72hr in vitro infection of Calu-3 cells. After SARS-CoV-2 infection, the cells were fixed for IF staining with dsRNA-specific J2 monoclonal Ab, and cell lysates were harvested for WB of viral nucleocapsid (N) protein. Supernatants (SN) and cytoplasmic lysates were obtained to measure ACE2 levels by ELISA.ResultsIn both human cohorts, ACE2 protein and mRNA levels were decreased in peripheral airways from COPD patients versus both smoker and NS controls, but similar in central airways. TMPRSS2 levels were similar across groups. Mice exposed to CS had decreased ACE2 protein levels in their bronchial and alveolar epithelia versus air-exposed mice exposed to 3 and 6 months of CS. In Calu3 cells in vitro, CS-treatment abrogated infection to levels below the limit of detection. Similar results were seen with WB for viral N protein, showing peak viral protein synthesis at 72hr.ConclusionsACE2 levels were decreased in both bronchial and alveolar epithelial cells from uninfected COPD patients versus controls, and from CS-exposed versus air-exposed mice. CS-pre-treatment did not affect ACE2 levels but potently inhibited SARS-CoV-2 replication in this in vitro model. These findings urge to further investigate the controversial effects of CS and COPD on SARS-CoV2 infection.

Rationale CC16 (club cell secretory protein) is a pneumoprotein produced predominantly by pulmonary club cells. Circulating CC16 is associated with protection from the inception and progression of ...

Advanced co-spray drying of fluticasone propionate, salmeterol xinafoate, and d-mannitol leads to high-performing inhalable dry powders as molecular mixtures., Rationale: lactose is the only FDA-approved carrier for dry powder inhaler (DPI) formulations in the US. Lactose carrier-based DPI products are contraindicated in patients with a known lactose allergy. Hence, inhaler formulations without lactose will benefit lactose allergic asthmatics. Objectives: to rationally design and develop lactose carrier-free dry powder inhaler formulations of fluticasone propionate and salmeterol xinafoate that will benefit people with known lactose allergy. The study also aims at improving the aerosol deposition of the dry powder formulation through advanced particle engineering design technologies to create inhalable powders consisting of nanoparticles/microparticles. Methods: advanced DPI nanoparticle/microparticle formulations were designed, developed and optimized using organic solution advanced closed-mode spray drying. The co-spray dried (co-SD) powders were comprehensively characterized in solid-state and in vitro comparative analysis of the aerodynamic performance of these molecularly mixed formulations was conducted with the marketed formulation of Advair® Diskus® interactive physical mixture. Measurements and main results: comprehensive solid-state physicochemical characterization of the powders showed that the engineered co-SD particles were small and spherical within the size range of 450 nm to 7.25 μm. Improved fine particle fraction and lower mass median aerodynamic diameter were achieved by these DPI nanoparticles/microparticles. Conclusions: this study has successfully produced a lactose-free dry powder formulation containing fluticasone propionate and salmeterol xinafoate with mannitol as excipient engineered as inhalable DPI nanoparticles/microparticles by advanced spray drying. Further, co-spray drying with mannitol and using Handihaler® device can generate higher fine particle mass of fluticasone/salmeterol. Mannitol, a mucolytic agent and aerosol performance enhancer, is a suitable excipient that can enhance aerosol dispersion of DPIs.

The lung surfactant proteins are recognized as critical not only for their role in lowering lung surface tension but also in innate host defense. Reports have shown that some asthmatic patients have decreased levels of one member of this protein family in particular, surfactant protein-A (SP-A). Our studies set out to determine the contribution of SP-A to the response of a key effector cytokine in asthma, IL-13. Our studies employ both animal models sufficient and deficient in SP-A challenged with IL-13 and primary epithelial cells from participants with asthma that are exogenously treated with SP-A in the context of IL-13 challenge. The inflammatory response and mucin production were assessed in both model systems. As compared with WT mice, we show that the activity of IL-13 is dramatically augmented in SP-A−/− mice, which have significantly increased neutrophil and eosinophil recruitment, mucin production and asthma-associated cytokines in the bronchoalveolar lavage fluid. In parallel, we show asthma-associated factors are attenuated in human cells from asthma subjects when exogenous SP-A is added during IL-13 challenge. Although many of these phenotypes have previously been associated with STAT6 signaling, SP-A inhibited IL-13-induced STAT3 phosphorylation in mice and in human epithelial cells while having little effect on STAT6 phosphorylation. In addition, when either STAT3 or IL-6 were inhibited in mice, the phenotypes observed in SP-A−/− mice were significantly attenuated. These studies suggest a novel mechanism for SP-A in asthma as a modulator of IL-13-induced inflammation via mediating downstream IL-6/STAT3 signaling.

Declines in lung function worsen quality of life and increase the risk of mortality. Obesity and non-alcoholic fatty liver disease are associated with worsened lung function. To investigate this association, we assessed lung function in lean and diet-induced obese conscious mice using our newly developed leak-free head-out plethysmography system. Obesity was associated with increased volume (P

Studies have shown that club cell secretory protein (CC16) plays important protective roles in the lungs, yet its complete biological functions are unclear. We devised a translational mouse model in order to investigate the impact of early life infections, in the context of CC16 deficiency, on lung function in adult mice. CC16 sufficient (WT) and deficient (CC16-/-) mice were infected with Mycoplasma pneumoniae (Mp) as weanlings and assessed as adults ( e arly l ife i nfection m odel; ELIM) and compared to adult mice infected for only three days ( a dult i nfection m odel; AIM). CC16-/- Mp-infected mice had significantly increased airway hyperresponsiveness (AHR) in both models compared to WT mice. However, CC16-/- mice infected in early life (ELIM) displayed significantly increased AHR compared to CC16-/- mice infected in adulthood (AIM). In stark contrast, lung function in ELIM WT mice returned to levels similar to saline-treated controls. While WT mice cleared Mp infection in the ELIM, CC16-/- mice remained colonized with Mp throughout the model, which likely contributed to increased airway remodeling and persistence of Muc5ac expression. When CC16-/- mouse tracheal epithelial cells (MTECs) were infected with Mp, increased Mp colonization and collagen gene expression were also detected compared to WT cells, suggesting that CC16 plays a protective role during Mp infection, in part through epithelial-driven host defense mechanisms.

Studies have shown that club cell secretory protein (CC16) plays important protective roles in the lungs, yet its complete biological functions are unclear. We devised a translational mouse model in order to investigate the impact of early life infections, in the context of CC16 deficiency, on lung function in adult mice. CC16 sufficient (WT) and deficient (CC16(−/−)) mice were infected with Mycoplasma pneumoniae (Mp) as weanlings and assessed as adults (early life infection model; ELIM) and compared to adult mice infected for only 3 days (adult infection model; AIM). CC16(−/−) Mp-infected mice had significantly increased airway hyperresponsiveness (AHR) in both models compared to WT mice. However, CC16(−/−) mice infected in early life (ELIM) displayed significantly increased AHR compared to CC16(−/−) mice infected in adulthood (AIM). In stark contrast, lung function in ELIM WT mice returned to levels similar to saline-treated controls. While WT mice cleared Mp infection in the ELIM, CC16(−/−) mice remained colonized with Mp throughout the model, which likely contributed to increased airway remodeling and persistence of Muc5ac expression. When CC16(−/−) mouse tracheal epithelial cells (MTECs) were infected with Mp, increased Mp colonization and collagen gene expression were also detected compared to WT cells, suggesting that CC16 plays a protective role during Mp infection, in part through epithelial-driven host defense mechanisms.

Background and purpose Despite availability of a variety of treatment options, many asthma patients have poorly controlled disease with frequent exacerbations. Proteinase-activated receptor-2 (PAR2) has been identified in pre-clinical animal models as important to asthma initiation and progression following allergen exposure. Proteinase activation of PAR2 induces intracellular Ca2+ , mitogen activated protein kinase (MAPK) and β-arrestin signaling in the airway, leading to both inflammatory and protective effects. We have developed C391, a potent PAR2 antagonist effective in blocking peptidomimetic- and trypsin-induced PAR2 signaling in vitro as well as reducing inflammatory PAR2-associated pain in vivo. We hypothesized that PAR2 antagonism by C391 would attenuate allergen-induced acutely expressed asthma indicators in murine models. Experimental approach We evaluated the ability for C391 to alter Alternaria alternata-induced PAR2 signaling pathways in vitro using a human airway epithelial cell line that naturally expresses PAR2 (16HBE14o-) and a transfected embryonic cell line (HEK 293). We next evaluated the ability for C391 to reduce A. alternata-induced acutely expressed asthma indicators in vivo in two murine strains. Key results C391 blocked A. alternata-induced, PAR2-dependent Ca2+ and MAPK signaling in 16HBE14o- cells, as well as β-arrestin recruitment in HEK 293 cells. C391 effectively attenuated A. alternata-induced inflammation, mucus production, mucus cell hyperplasia and airway hyperresponsiveness in acute allergen-challenge murine models. Conclusions and implications To our knowledge, this is the first demonstration of pharmacological intervention of PAR2 to reduce allergen-induced asthma indicators in vivo. These data support further development of PAR2 antagonists as potential first-in-class allergic asthma drugs.

The peptide hormone, angiotensin (Ang-(1–7)), produces anti-inflammatory and protective effects by inhibiting production and expression of many cytokines and adhesion molecules that are associated with a cytokine storm. While Ang-(1–7) has been shown to reduce inflammation and airway hyperreactivity in models of asthma, little is known about the effects of Ang-(1–7) during live respiratory infections. Our studies were developed to test if Ang-(1–7) is protective in the lung against overzealous immune responses during an infection with Mycoplasma pneumonia (Mp), a common respiratory pathogen known to provoke exacerbations in asthma and COPD patients. Wild type mice were treated with infectious Mp and a subset of was given either Ang-(1–7) or peptide-free vehicle via oropharyngeal delivery within 2 h of infection. Markers of inflammation in the lung were assessed within 24 h for each set of animals. During Mycoplasma infection, one high dose of Ang-(1–7) delivered to the lungs reduced neutrophilia and Muc5ac, as well as Tnf-α and chemokines (Cxcl1) associated with acute respiratory distress syndrome (ARDS). Despite decreased inflammation, Ang-(1-7)-treated mice also had significantly lower Mp burden in their lung tissue, indicating decreased airway colonization. Ang-(1–7) also had an impact on RAW 264.7 cells, a commonly used macrophage cell line, by dose-dependently inhibiting TNF-α production while promoting Mp killing. These new findings provide additional support to the protective role(s) of Ang1-7 in controlling inflammation, which we found to be highly protective against live Mp-induced lung inflammation.

We identify a homozygous mutation in SFTPA1 in patients with idiopathic pulmonary fibrosis (IPF). The mutation causes increased necroptosis of type II alveolar epithelial cells through the IRE1α–JNK axis, which highlights the necroptosis pathway as a therapeutic target for IPF., Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by scattered fibrotic lesions in the lungs. The pathogenesis and genetic basis of IPF remain poorly understood. Here, we show that a homozygous missense mutation in SFTPA1 caused IPF in a consanguineous Japanese family. The mutation in SFTPA1 disturbed the secretion of SFTPA1 protein. Sftpa1 knock-in (Sftpa1-KI) mice that harbored the same mutation as patients spontaneously developed pulmonary fibrosis that was accelerated by influenza virus infection. Sftpa1-KI mice showed increased necroptosis of alveolar epithelial type II (AEII) cells with phosphorylation of IRE1α leading to JNK-mediated up-regulation of Ripk3. The inhibition of JNK ameliorated pulmonary fibrosis in Sftpa1-KI mice, and overexpression of Ripk3 in Sftpa1-KI mice treated with a JNK inhibitor worsened pulmonary fibrosis. These findings provide new insight into the mechanisms of IPF in which a mutation in SFTPA1 promotes necroptosis of AEII cells through JNK-mediated up-regulation of Ripk3, highlighting the necroptosis pathway as a therapeutic target for IPF.

Surfactant protein-A (SP-A) is an important mediator of pulmonary immunity. A specific genetic variation in SP-A2, corresponding to a glutamine (Q) to lysine (K) amino acid substitution at position 223 of the lectin domain, was shown to alter the ability of SP-A to inhibit eosinophil degranulation. Because a large subgroup of asthmatics have associated eosinophilia, often accompanied by inflammation associated with delayed clearance, our goal was to define how SP-A mediates eosinophil resolution in allergic airways and whether genetic variation affects this activity. Wild-type, SP-A knockout (SP-A KO) and humanized (SP-A2 223Q/Q, SP-A2 223K/K) C57BL/6 mice were challenged in an allergic OVA model, and parameters of inflammation were examined. Peripheral blood eosinophils were isolated to assess the effect of SP-A genetic variation on apoptosis and chemotaxis. Five days postchallenge, SP-A KO and humanized SP-A2 223K/K mice had persistent eosinophilia in bronchoalveolar lavage fluid compared with wild-type and SP-A2 223Q/Q mice, suggesting an impairment in eosinophil resolution. In vitro, human SP-A containing either the 223Q or the 223K allele was chemoattractant for eosinophils whereas only 223Q resulted in decreased eosinophil viability. Our results suggest that SP-A aids in the resolution of allergic airway inflammation by promoting eosinophil clearance from lung tissue through chemotaxis, independent of SP-A2 Q223K, and by inducing apoptosis of eosinophils, which is altered by the polymorphism.

BackgroundIncreased exposure to Ozone (O3) is associated with adverse health effects in individuals afflicted with respiratory diseases. Surfactant protein-A (SP-A), encoded bySP-A1andSP-A2, is the largest protein component in pulmonary surfactant and is functionally impaired by O3-oxidation.ObjectiveWe used humanized SP-A2 transgenic mice with allelic variation corresponding to a glutamine (Q) to lysine (K) amino acid substitution at position 223 in the lectin domain to determine the impact of this genetic variation in regards to O3exposure.MethodsMice were exposed to 2ppm O3or Filtered Air (FA) for 3 hours and 24 hrs post-challenge pulmonary function tests and other parameters associated with inflammation were assessed in the bronchoalveolar lavage (BAL) fluid and lung tissue. Additionally, mouse tracheal epithelial cells were cultured and TEER measurements recorded for each genotype to determine baseline epithelial integrity.ResultsCompared to FA, O3exposure led to significantly increased sensitivity to methacholine challenge in all groups of mice. SP-A2 223Q variant mice were significantly protected from O3-induced AHR compared to SP-A-/-and SP-A2 223K mice. Neutrophilia was observed in all genotypes of mice post O3-exposure, however, SP-A2 223Q mice had a significantly lower percentage of neutrophils compared to SP-A-/-mice. Albumin levels in BAL were unchanged in O3-exposed SP-A2 223Q mice compared to their FA controls, while levels were significantly increased in all other genotypes of O3-exposed mice. SP-A 223Q MTECS has significant higher TEER values than all other genotypes, and WT MTECS has significantly higher TEER than the SP-A KO and SP-A 223K MTECS.SignificanceTaken together, our study suggests that expression of a glutamine (Q) as position 223 in SP-A2, as opposed to expression of lysine (K), is more protective in acute exposures to ozone and results in attenuated O3-induced AHR, neutrophilia, and vascular permeability.

Human surfactant protein-A2 (hSP-A2) is a component of pulmonary surfactant that plays an important role in the lung's immune system by interacting with viruses, bacteria, and fungi to facilitate pathogen clearance and by downregulating inflammatory responses after an allergic challenge. Genetic variation in SP-A2 at position Gln223Lys is present in up to ∼30% of the population and has been associated with several lung diseases, such as asthma, pulmonary fibrosis, and lung cancer (M. M. Pettigrew, J. F. Gent, Y. Zhu, E. W. Triche, et al., BMC Med Genet 8:15, 2007, https://bmcmedgenet.biomedcentral.com/articles/10.1186/1471-2350-8-15; Y. Wang, P. J. Kuan, C. Zing, J. T. Cronkhite, et al., Am J Hum Genet 84:52-59, 2009, https://www.cell.com/ajhg/fulltext/S0002-9297(08)00595-8). Previous work performed by our group showed differences in levels of SP-A binding to non-live mycoplasma membrane fractions that were dependent on the presence of a lysine (K) or a glutamine (Q) at amino acid position 223 in the carbohydrate region of SP-A2. On the basis of these differences, we have derived 20-amino-acid peptides flanking this region of interest in order to test the ability of each to regulate various immune responses to live Mycoplasma pneumoniae in SP-A knockout mice and RAW 264.7 cells. In both models, the 20-mer containing 223Q significantly decreased both tumor necrosis factor alpha (TNF-α) mRNA levels and protein levels in comparison to the 20-mer containing 223K during M. pneumoniae infection. While neither of the 20-mer peptides (223Q and 223K) had an effect on p38 phosphorylation during M. pneumoniae infection, the 223Q-20mer peptide significantly reduced NF-κB p65 phosphorylation in both models. Taken together, our data suggest that small peptides derived from the lectin domain of SP-A2 that contain the major allelic variant (223Q) maintain activity in reducing TNF-α induction during M. pneumoniae infection.

CC16 (club cell secretory protein-16), a member of the secretoglobin family, is one of the most abundant proteins in normal airway secretions and has been described as a serum biomarker for obstructive lung diseases.To determine whether low CC16 is a marker for airway pathology or is implicated in the pathophysiology of progressive airway damage in these conditions.Using human data from the birth cohort of the Tucson Children's Respiratory Study, we examined the relation of circulating CC16 levels with pulmonary function and responses to bronchial methacholine challenge from childhood up to age 32 years. In wild-type and CC16We observed that Tucson Children's Respiratory Study participants in the lowest tertile of serum CC16 had significant deficits in their lung function and enhanced airway hyperresponsiveness to methacholine challenge from 11 years throughout young adult life. Similarly, CC16Our findings support clinical observations by providing evidence that lack of CC16 in the lung results in dramatically altered pulmonary function and structural alterations consistent with enhanced remodeling.

Background Chronic rhinosinusitis (CRS) is a common inflammatory disorder of the upper airway characterized by chronic inflammation and significant sinonasal remodeling. CRS is comprised of 2 major subgroups, based on whether polyps are present or absent. In some cases, it is characterized by colonization with opportunistic pathogens such as Pseudomonas aeruginosa (PA), Staphylococcus aureus, and other bacteria. The innate immune system of the sinonasal epithelium is the first line of defense against inhaled pathogens. Surfactant protein A (SP-A) is a member of the collectin family secreted by the airway epithelia and plays a critical role in airway innate immunity, as it can aggregate bacteria. We hypothesized that SP-A plays a role in bacterial CRS. Methods Air-liquid interface (ALI) cultures of nasal epithelial cells were derived from human ex-vivo healthy and CRS sinus tissues (n = 26) and challenged with PA. SP-A levels were measured with western blot and quantitative reverse transcript-polymerase chain reaction (qRT-PCR) in ALI and sinus tissues. Results We determined that SP-A: (i) mRNA and protein levels are increased significantly in CRS tissues compared with healthy sinuses; (ii) although primarily expressed in the lung, it is also synthesized and expressed in sinonasal epithelia; (ii) is expressed in the sinuses of an SP-A humanized transgenic mouse but not in SP-A knockout mice; (iv) mRNA levels are upregulated significantly during PA challenge, but protein levels are downregulated 4 hours postchallenge and upregulated at 12 hours. Conclusion Our data suggest that SP-A is expressed in the sinuses and that it plays a role in the sinus innate immune responses during bacterial infections.

Chitinase 3–like-1 (Chi3l1) and IL-13 are both ligands of IL-13 receptor α2 (IL-13Rα2). The binding of the former activates mitogen-activated protein kinase, AKT, and Wnt/β-catenin signaling, and plays important roles in innate and adaptive immunity, cellular apoptosis, oxidative injury, allergic inflammation, tumor metastasis and wound healing, fibrosis, and repair in the lung. In contrast, the latter binding is largely a decoy event that diminishes the effects of IL-13. Here, we demonstrate that IL-13Rα2 N-glycosylation is a critical determinant of which ligand binds. Structure–function evaluations demonstrated that Chi3l1–IL-13Rα2 binding was increased when sites of N-glycosylation are mutated, and studies with tunicamycin and Peptide:N-glycosidase F (PNGase F) demonstrated that Chi3l1–IL-13Rα2 binding and signaling were increased when N-glycosylation was diminished. In contrast, structure–function experiments demonstrated that IL-13 binding to IL-13Rα2 was dependent on each of the four sites of N-glycosylation in IL-13Rα2, and experiments with tunicamycin and PNGase F demonstrated that IL-13–IL-13Rα2 binding was decreased when IL-13Rα2 N-glycosylation was diminished. Studies with primary lung epithelial cells also demonstrated that Chi3l1 inhibited, whereas IL-13 stimulated, N-glycosylation as evidenced by the ability of Chi3l1 to inhibit and IL-13 to stimulate the subunits of the oligosaccharide complex A and B (STT3A and STT3B). These studies demonstrate that N-glycosylation is a critical determinant of Chi3l1 and IL-13 binding to IL-13Rα2, and highlight the ability of Chi3l1 and IL-13 to alter key elements of the N-glycosylation apparatus in a manner that would augment their respective binding.

The negative immune regulator Tollip inhibits the proinflammatory response to rhinovirus (RV) infection, a contributor to airway neutrophilic inflammation and asthma exacerbations, but the underlying molecular mechanisms are poorly understood. Tollip may inhibit IRAK1, a signaling molecule downstream of ST2, the receptor of IL-33. This study was carried out to determine whether Tollip downregulates ST2 signaling via inhibition of IRAK1, but promotes soluble ST2 (sST2) production, thereby limiting excessive IL-8 production in human airway epithelial cells during RV infection in a type 2 cytokine milieu (e.g., IL-13 and IL-33 stimulation). Tollip- and IRAK1-deficient primary human tracheobronchial epithelial (HTBE) cells and Tollip knockout (KO) HTBE cells were generated using the shRNA knockdown and CRISPR/Cas9 approaches, respectively. Cells were stimulated with IL-13, IL-33, and/or RV16. sST2, activated IRAK1, and IL-8 were measured. A Tollip KO mouse model was utilized to test if Tollip regulates the airway inflammatory response to RV infection in vivo under IL-13 and IL-33 treatment. Following IL-13, IL-33, and RV treatment, Tollip-deficient (vs. -sufficient) HTBE cells produced excessive IL-8, accompanied by decreased sST2 production but increased IRAK1 activation. IL-8 production following IL-13/IL-33/RV exposure was markedly attenuated in IRAK1-deficient HTBE cells, as well as in Tollip KO HTBE cells treated with an IRAK1 inhibitor or a recombinant sST2 protein. Tollip KO (vs. wild-type) mice developed exaggerated airway neutrophilic responses to RV in the context of IL-13 and IL-33 treatment. Collectively, these data demonstrate that Tollip restricts excessive IL-8 production in type 2 cytokine-exposed human airways during RV infection by promoting sST2 production and inhibiting IRAK1 activation. sST2 and IRAK1 may be therapeutic targets for attenuating excessive neutrophilic airway inflammation in asthma, especially during RV infection. NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [1U19AI125357, R01HL122321, R01AI106287, R01HL125128] This work was supported by the following NIH grants: 1U19AI125357, R01HL122321, R01AI106287, and R01HL125128. These funding sources were not involved in the preparation of data or the manuscript.

Background: Surfactant protein A (SP-A) is well known for its protective role in pulmonary immunity. Studies from our lab show that mice deficient in SP-A have delayed clearance of eosinophils in the bronchoalveolar lavage after experimental ovalbumin challenge as compared to WT mice. Here, we wanted to investigate the mechanism by which SP-A induces eosinophil apoptosis. Methods: Purified eosinophils from asthmatic volunteers were incubated with SP-A for 16 hours. Induction of eosinophil apoptosis and cell death was measured by quantifying caspase-3 cleavage using Western blotting and Annexin V/Propidium Iodide (PI) staining using flow cytometry. To identify potential binding partners for SP-A, eosinophil lysates were subjected to SP-A pull-down and tandem mass spectrometry (MS/MS) analysis. The pro-apoptotic potential of the identified candidate SP-A membrane receptor was then tested by using a specific antibody to block protein binding capacity prior to direct incubation with SP-A, and subsequently analyzed by flow cytometry. Results: Direct incubation of eosinophils with SP-A resulted in increased cleavage of caspase-3 compared to vehicle control. The percentage of apoptotic eosinophils (Annexin V+, PI-) was also significantly increased upon SP-A treatment. Tandem mass spectrometry identified one membrane-bound protein, myeloid-associated differentiation marker (MYADM), as a candidate SP-A binding partner. Pre-treatment with an antibody to MYADM diminished SP-A-induced eosinophil apoptosis compared to those treated with SP-A only. Conclusions: Our results have identified a novel binding partner for SP-A on eosinophils, MYADM, through which we believe SP-A promotes eosinophil apoptosis.

Preformulation studies on tofacitinib citrate, a small molecule JAK3 specific inhibitor, have not been previously reported in literature. We therefore conducted several preformulation studies on tofacitinib citrate, and its free base, to better understand factors that affect its solubility, stability, and solid-state characteristics. Further, the results of the preformulation studies helped facilitate the development of a nebulized formulation of tofacitinib citrate for inhalational delivery to house dust mite allergen-challenged, BALB/c mice as a potential treatment for eosinophilic asthma. The preformulation results indicated tofacitinib having a basic pKa of 5.2, with its stability dependent on pH, ionic strength, and temperature. Degradation of tofacitinib follows apparent first-order kinetics. In order to maximize stability of the drug, ionic strength and temperature should be minimized, with an optimal range pH between 2.0 and 5.0. Additionally, our findings demonstrate that tofacitinib citrate can successfully be nebulized at a suitable droplet size for inhalation (1.2 ± 0.2 μm MMAD) through a nose-only chamber. Animals dosed with tofacitinib citrate demonstrated marked reductions in BAL eosinophils and total protein concentrations following HDM challenge. These data suggest that tofacitinib citrate represents the potential to be an effective therapy for eosinophilic asthma.

SummaryBackground Rhinovirus (RV) infection in asthma induces varying degrees of airway inflammation (e.g. neutrophils), but the underlying mechanisms remain unclear. Objective The major goal was to determine the role of genetic variation [e.g. single nucleotide polymorphisms (SNPs)] of Toll-interacting protein (Tollip) in airway epithelial responses to RV in a type 2 cytokine milieu. Methods DNA from blood of asthmatic and normal subjects was genotyped for Tollip SNP rs5743899 AA, AG and GG genotypes. Human tracheobronchial epithelial (HTBE) cells from donors without lung disease were cultured to determine pro-inflammatory and antiviral responses to IL-13 and RV16. Tollip knockout and wild-type mice were challenged with house dust mite (HDM) and infected with RV1B to determine lung inflammation and antiviral response. Results Asthmatic subjects carrying the AG or GG genotype (AG/GG) compared with the AA genotype demonstrated greater airflow limitation. HTBE cells with AG/GG expressed less Tollip. Upon IL-13 and RV16 treatment, cells with AG/GG (vs. AA) produced more IL-8 and expressed less antiviral genes, which was coupled with increased NF-κB activity and decreased expression of LC3, a hallmark of the autophagic pathway. Tollip co-localized and interacted with LC3. Inhibition of autophagy decreased antiviral genes in IL-13- and RV16-treated cells. Upon HDM and RV1B, Tollip knockout (vs. wild-type) mice demonstrated higher levels of lung neutrophilic inflammation and viral load, but lower levels of antiviral gene expression. Conclusions and Clinical Relevance Our data suggest that Tollip SNP rs5743899 may predict varying airway response to RV infection in asthma.

BackgroundThe Amish and Hutterites are U.S. agricultural populations whose lifestyles are remarkably similar in many respects but whose farming practices, in particular, are distinct; the former follow traditional farming practices whereas the latter use industrialized farming practices. The populations also show striking disparities in the prevalence of asthma, and little is known about the immune responses underlying these disparities. MethodsWe studied environmental exposures, genetic ancestry, and immune profiles among 60 Amish and Hutterite children, measuring levels of allergens and endotoxins and assessing the microbiome composition of indoor dust samples. Whole blood was collected to measure serum IgE levels, cytokine responses, and gene expression, and peripheral-blood leukocytes were phenotyped with flow cytometry. The effects of dust extracts obtained from Amish and Hutterite homes on immune and airway responses were assessed in a murine model of experimental allergic asthma. ResultsDespite the...

Background: The integration of new scientific discoveries into clinical practice costs considerable time and resources. With the increased use of social media for scientific communication, new opportunities arise to ‘bridge the gap’ in translational medicine. The present study aimed to investigate how medical professionals access scientific information and understand their view on the role of social media in translational medicine. Methods: A questionnaire regarding (i) the use of social media for scientific updates, (ii) the opportunities and challenges of social media for translational medicine, (iii) social media function Chatbot, and (iv) participant demographics was developed. The survey link was posted online from February, 2018, until April, 2018. Results: A total of 555 professionals responded to the survey. Respondents identified themselves predominantly as researcher/scientists (27%) or medical/biomedical students (15%). The majority of participants was employed at a university or research institute (59%), and most practiced either in Europe (48%) or in Asia (37%). Seventy-eight percent of respondents reported receiving most of scientific news and updates via non-social media options, such as journal websites and newspapers. Fifty-one percent of respondents believed that social media could contribute to closing the gap between scientific discovery and translation to medical application. The most crucial opportunity created by social media was found to be “connecting the right scientist to the right clinician.” Participants rated “the translation of scientific finding to clinical practice is too fast before the safety is properly demonstrated” as the most crucial challenge. Half of the respondents were aware of their institutions policy on the professional use of social media. Only 2% of respondents had previously used Chatbot. Conclusions: Overall, medical professionals were positive about the idea that social media could contribute to the progress of translational medicine. However, it is clear that they are still being cautious about using social media for professional purposes. To fully harness the potential of social media on translational medicine, the medical community needs to be provided with educational programs, guidelines, and support infrastructure within social media.