Investigation of lactotransferrin messenger RNA expression levels as an anti-type 2 asthma biomarker.

BACKGROUND: Lactotransferrin (LTF) has an immunomodulatory function, and its expression levels are associated with asthma susceptibility. OBJECTIVES: We sought to investigate LTF messenger RNA (mRNA) expression levels in human bronchial epithelial cells (BECs) as an anti-type 2 (T2) asthma biomarker. METHODS: Association analyses between LTF mRNA expression levels in BECs and asthma-related phenotypes were performed in the Severe Asthma Research Program (SARP) cross-sectional (n = 155) and longitudinal (n = 156) cohorts using a generalized linear model. Correlation analyses of mRNA expression levels between LTF and all other genes were performed by Spearman correlation. RESULTS: Low LTF mRNA expression levels were associated with asthma susceptibility and severity (P < .025), retrospective and prospective asthma exacerbations, and low lung function (P < 8.3 × 10-3). Low LTF mRNA expression levels were associated with high airway T2 inflammation biomarkers (sputum eosinophils and fractional exhaled nitric oxide; P < 8.3 × 10-3) but were not associated with blood eosinophils or total serum IgE. LTF mRNA expression levels were negatively correlated with expression levels of TH2 or asthma-associated genes (POSTN, NOS2, and MUC5AC) and eosinophil-related genes (IL1RL1, CCL26, and IKZF2) and positively correlated with expression levels of TH1 and inflammation genes (IL12A, MUC5B, and CC16) and TH17-driven cytokines or chemokines for neutrophils (CXCL1, CXCL6, and CSF3) (P < 3.5 × 10-6). CONCLUSIONS: Low LTF mRNA expression levels in BECs are associated with asthma susceptibility, severity, and exacerbations through upregulation of airway T2 inflammation. LTF is a potential anti-T2 biomarker, and its expression levels may help determine the balance of eosinophilic and neutrophilic asthma.

Application of Metabolomics across the Spectrum of Pulmonary and Critical Care Medicine.

In recent years, metabolomics, the systematic study of small-molecule metabolites in biological samples, has yielded fresh insights into the molecular determinants of pulmonary diseases and critical illness. The purpose of this article is to orient the reader to this emerging field by discussing the fundamental tenets underlying metabolomics research, the tools and techniques that serve as foundational methodologies, and the various statistical approaches to analysis of metabolomics datasets. We present several examples of metabolomics applied to pulmonary and critical care medicine to illustrate the potential of this avenue of research to deepen our understanding of pathophysiology. We conclude by reviewing recent advances in the field and future research directions that stand to further the goal of personalizing medicine to improve patient care.

Cooperation of immune regulators Tollip and surfactant protein A inhibits influenza A virus infection in mice.

BACKGROUND: Influenza A virus (IAV) infection is a significant risk factor for respiratory diseases, but the host defense mechanisms against IAV remain to be defined. Immune regulators such as surfactant protein A (SP-A) and Toll-interacting protein (Tollip) have been shown to be involved in IAV infection, but whether SP-A and Tollip cooperate in more effective host defense against IAV infection has not been investigated. METHODS: Wild-type (WT), Tollip knockout (KO), SP-A KO, and Tollip/SP-A double KO (dKO) mice were infected with IAV for four days. Lung macrophages were isolated for bulk RNA sequencing. Precision-cut lung slices (PCLS) from WT and dKO mice were pre-treated with SP-A and then infected with IAV for 48 h. RESULTS: Viral load was significantly increased in bronchoalveolar lavage (BAL) fluid of dKO mice compared to all other strains of mice. dKO mice had significantly less recruitment of neutrophils into the lung compared to Tollip KO mice. SP-A treatment of PCLS enhanced expression of TNF and reduced viral load in dKO mouse lung tissue. Pathway analysis of bulk RNA sequencing data suggests that macrophages from IAV-infected dKO mice reduced expression of genes involved in neutrophil recruitment, IL-17 signaling, and Toll-like receptor signaling. CONCLUSIONS: Our data suggests that both Tollip and SP-A are essential for the lung to exert more effective innate defense against IAV infection.

SURFACTANT PROTEIN A: AN INNATE IMMUNE MODULATOR AND THERAPEUTIC IN ASTHMA.

Surfactant Protein A (SP-A) is an innate immune modulator produced by the lung with known protective effects against bacteria and viruses. Its role in asthma, an inflammatory lung disease that affects 10% of the world's population, is not entirely known. In this review, we demonstrate that SP-A confers protection against exposure to interleukin-13, a type 2 cytokine integral to eosinophilic asthma, in a mouse model of SP-A deficiency, a house dust mite model of asthma, and in human bronchial epithelial cells from participants with asthma. We also show that small peptides derived from SP-A, such as the major allele of single nucleotide polymorphism (SNP) rs1965708, which includes the carbohydrate recognition domain of SP-A2 at position 223, reduce airway hyperresponsiveness, airway eosinophils, and mucus in a mouse model of asthma. These data suggest that SP-A has beneficial effects relevant to asthma and that an SP-A peptide may have a new therapeutic use in asthma.

Circulating CC16 and Asthma: A Population-based, Multicohort Study from Early Childhood through Adult Life.

Rationale: Club cell secretory protein (CC16) is an antiinflammatory protein highly expressed in the airways. CC16 deficiency has been associated with lung function deficits, but its role in asthma has not been established conclusively. Objectives: To determine 1) the longitudinal association of circulating CC16 with the presence of active asthma from early childhood through adult life and 2) whether CC16 in early childhood predicts the clinical course of childhood asthma into adult life. Methods: We assessed the association of circulating CC16 and asthma in three population-based birth cohorts: the Tucson Children's Respiratory Study (years 6-36; total participants, 814; total observations, 3,042), the Swedish Barn/Children, Allergy, Milieu, Stockholm, Epidemiological survey (years 8-24; total participants, 2,547; total observations, 3,438), and the UK Manchester Asthma and Allergy Study (years 5-18; total participants, 745; total observations, 1,626). Among 233 children who had asthma at the first survey in any of the cohorts, baseline CC16 was also tested for association with persistence of symptoms. Measurements and Main Results: After adjusting for covariates, CC16 deficits were associated with increased risk for the presence of asthma in all cohorts (meta-analyzed adjusted odds ratio per 1-SD CC16 decrease, 1.20; 95% confidence interval [CI], 1.12-1.28; P < 0.0001). The association was particularly strong for asthma with frequent symptoms (meta-analyzed adjusted relative risk ratio, 1.40; 95% CI, 1.24-1.57; P < 0.0001), was confirmed for both atopic and nonatopic asthma, and was independent of lung function impairment. After adjustment for known predictors of persistent asthma, children with asthma in the lowest CC16 tertile had a nearly fourfold increased risk for having frequent symptoms persisting into adult life compared with children with asthma in the other two CC16 tertiles (meta-analyzed adjusted odds ratio, 3.72; 95% CI, 1.78-7.76; P < 0.0001). Conclusions: Circulating CC16 deficits are associated with the presence of asthma with frequent symptoms from childhood through midadult life and predict the persistence of asthma symptoms into adulthood. These findings support a possible protective role of CC16 in asthma and its potential use for risk stratification.

Low CC16 mRNA Expression Levels in Bronchial Epithelial Cells Are Associated with Asthma Severity.

Rationale: CC16 is a protein mainly produced by nonciliated bronchial epithelial cells (BECs) that participates in host defense. Reduced CC16 protein concentrations in BAL and serum are associated with asthma susceptibility. Objectives: Few studies have investigated the relationship between CC16 and asthma progression, and none has focused on BECs. In this study, we sought to determine if CC16 mRNA expression levels in BECs are associated with asthma severity. Methods: Association analyses between CC16 mRNA expression levels in BECs (242 asthmatics and 69 control subjects) and asthma-related phenotypes in Severe Asthma Research Program were performed using a generalized linear model. Measurements and Main Results: Low CC16 mRNA expression levels in BECs were significantly associated with asthma susceptibility and asthma severity, high systemic corticosteroids use, high retrospective and prospective asthma exacerbations, and low pulmonary function. Low CC16 mRNA expression levels were significantly associated with high T2 inflammation biomarkers (fractional exhaled nitric oxide and sputum eosinophils). CC16 mRNA expression levels were negatively correlated with expression levels of Th2 genes (IL1RL1, POSTN, SERPINB2, CLCA1, NOS2, and MUC5AC) and positively correlated with expression levels of Th1 and inflammation genes (IL12A and MUC5B). A combination of two nontraditional T2 biomarkers (CC16 and IL-6) revealed four asthma endotypes with different characteristics of T2 inflammation, obesity, and asthma severity. Conclusions: Our findings indicate that low CC16 mRNA expression levels in BECs are associated with asthma susceptibility, severity, and exacerbations, partially through immunomodulation of T2 inflammation. CC16 is a potential nontraditional T2 biomarker for asthma development and progression.

Creatine Kinase Is Decreased in Childhood Asthma.

Rationale: The identification of novel molecules associated with asthma may provide insights into the mechanisms of disease and their potential clinical implications. Objectives: To conduct a screening of circulating proteins in childhood asthma and to study proteins that emerged from human studies in a mouse model of asthma. Methods: We included 2,264 children from eight birth cohorts from the Mechanisms of the Development of ALLergy project and the Tucson Children's Respiratory 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 CK (creatine kinase) 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 the 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 (adjusted odds ratio, 95% confidence interval: 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 and CKB) were underexpressed in blood from asthmatics compared with control subjects (P = 0.01 and 0.006, respectively). In the lungs of HDM-challenged mice, Ckb expression was reduced, and after the HDM challenge, a CKB inhibitor blocked the resolution of airway hyperresponsiveness and reduction of airway mucin. Conclusions: Circulating concentrations 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 airway hyperresponsiveness and reduction of airway mucin.

CC16 Deficiency in the Context of Early-Life Mycoplasma pneumoniae Infection Results in Augmented Airway Responses in Adult Mice.

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.

Surfactant Protein-A Protects against IL-13-Induced Inflammation in Asthma.

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.

CC16 Binding to α4ß1 Integrin Protects against Mycoplasma pneumoniae 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 the two most common obstructive lung diseases (asthma and chronic obstructive pulmonary disease). Objectives Although exact mechanisms remain elusive, studies consistently suggest a causal role of CC16 in mediating antiinflammatory and antioxidant functions in the lung. We sought to determine any novel receptor systems that could participate in CC16's role in obstructive lung diseases. Methods Protein alignment of CC16 across species led to the discovery of a highly conserved sequence of amino acids, leucine-valine-aspartic acid (LVD), a known integrin-binding motif. Recombinant CC16 was generated with and without the putative integrin-binding site. A Mycoplasma pneumoniae mouse model and a fluorescent cellular adhesion assay were used to determine the impact of the LVD site regarding CC16 function during live infection and on cellular adhesion during inflammatory conditions. Measurements and Main Results CC16 bound to integrin α4ß1), also known as the adhesion molecule VLA-4 (very late antigen 4), dependent on the presence of the LVD integrin-binding motif. During infection, recombinant CC16 rescued lung function parameters both when administered to the lung and intravenously but only when the LVD integrin-binding site was intact; likewise, neutrophil recruitment during infection and leukocyte adhesion were both impacted by the loss of the LVD site. Conclusions We discovered a novel receptor for CC16, VLA-4, which has important mechanistic implications for the role of CC16 in circulation as well as in the lung compartment.

Metformin: Experimental and Clinical Evidence for a Potential Role in Emphysema Treatment.

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.

Genetic Variation in Surfactant Protein-A2 Delays Resolution of Eosinophilia in Asthma.

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.

A 20-Mer Peptide Derived from the Lectin Domain of SP-A2 Decreases Tumor Necrosis Factor Alpha Production during Mycoplasma pneumoniae Infection.

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.

Club Cell Secretory Protein Deficiency Leads to Altered Lung Function.

RATIONALE: 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. OBJECTIVES: To determine whether low CC16 is a marker for airway pathology or is implicated in the pathophysiology of progressive airway damage in these conditions. METHODS: 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 CC16-/- mice, we set out to comprehensively examine pulmonary physiology, inflammation, and remodeling in the naive airway. MEASUREMENTS AND MAIN RESULTS: We 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, CC16-/- mice had significant deficits in lung function and enhanced airway hyperresponsiveness to methacholine as compared with wild-type mice, which were independent of inflammation and mucin production. As compared with wild-type mice, CC16-/- mice had significantly elevated gene expression of procollagen type I, procollagen type III, and α-smooth muscle actin, areas of pronounced collagen deposition and significantly enhanced smooth muscle thickness. CONCLUSIONS: Our 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.

Innate Immunity and Asthma Risk in Amish and Hutterite Farm Children.

BACKGROUND: The 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. METHODS: We 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. RESULTS: Despite the similar genetic ancestries and lifestyles of Amish and Hutterite children, the prevalence of asthma and allergic sensitization was 4 and 6 times as low in the Amish, whereas median endotoxin levels in Amish house dust was 6.8 times as high. Differences in microbial composition were also observed in dust samples from Amish and Hutterite homes. Profound differences in the proportions, phenotypes, and functions of innate immune cells were also found between the two groups of children. In a mouse model of experimental allergic asthma, the intranasal instillation of dust extracts from Amish but not Hutterite homes significantly inhibited airway hyperreactivity and eosinophilia. These protective effects were abrogated in mice that were deficient in MyD88 and Trif, molecules that are critical in innate immune signaling. CONCLUSIONS: The results of our studies in humans and mice indicate that the Amish environment provides protection against asthma by engaging and shaping the innate immune response. (Funded by the National Institutes of Health and others.).

Obese asthmatic patients have decreased surfactant protein A levels: Mechanisms and implications.

BACKGROUND: Eosinophils are prominent in some patients with asthma and are increased in the submucosa in a subgroup of obese patients with asthma (OAs). Surfactant protein A (SP-A) modulates host responses to infectious and environmental insults. OBJECTIVE: We sought to determine whether SP-A levels are altered in OAs compared with a control group and to determine the implications of these alterations in SP-A levels in asthmatic patients. METHODS: Bronchoalveolar lavage fluid from 23 lean, 12 overweight, and 20 obese subjects were examined for SP-A. Mouse tracheal epithelial cells grown at an air-liquid interface were used for mechanistic studies. SP-A-/- mice were challenged in allergen models, and exogenous SP-A therapy was given after the last challenge. Eosinophils were visualized and quantitated in lung parenchyma by means of immunostaining. RESULTS: Significantly less SP-A (P = .002) was detected in samples from OAs compared with those from control subjects. A univariable regression model found SP-A levels were significantly negatively correlated with body mass index (r = -0.33, P = .014), whereas multivariable modeling demonstrated that the correlation depended both on asthma status (P = .017) and the interaction of asthma and body mass index (P = .008). Addition of exogenous TNF-α to mouse tracheal epithelial cells was sufficient to attenuate SP-A and eotaxin secretion. Allergen-challenged SP-A-/- mice that received SP-A therapy had significantly less tissue eosinophilia compared with mice receiving vehicle. CONCLUSIONS: SP-A functions as an important mediator in resolving tissue and lavage fluid eosinophilia in allergic mouse models. Decreased levels of SP-A in OAs, which could be due to increased local TNF-α levels, might lead to impaired eosinophil resolution and could contribute to the eosinophilic asthma phenotype.

Preformulation and Evaluation of Tofacitinib as a Therapeutic Treatment for Asthma.

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.

Genetic variation in SP-A2 leads to differential binding to Mycoplasma pneumoniae membranes and regulation of host responses.

Mycoplasma pneumoniae is an extracellular pathogen that colonizes mucosal surfaces of the respiratory tract and is associated with asthma exacerbations. Previous reports demonstrate that surfactant protein-A (SP-A) binds live M. pneumoniae and mycoplasma membrane fractions (MMF) with high affinity. Humans express a repertoire of single-amino acid genetic variants of SP-A that may be associated with lung disease, and our findings demonstrate that allelic differences in SP-A2 (Gln223Lys) affect the binding to MMF. We show that SP-A(-/-) mice are more susceptible to MMF exposure and have significant increases in mucin production and neutrophil recruitment. Novel humanized SP-A2-transgenic mice harboring the hSP-A2 223K allele exhibit reduced neutrophil influx and mucin production in the lungs when challenged with MMF compared with SP-A(-/-) mice. Conversely, mice expressing hSP-A2 223Q have increased neutrophil influx and mucin production that are similar to SP-A(-/-) mice. Using tracheal epithelial cell cultures, we show that enhanced mucin production to MMF occurs in the absence of SP-A and is not dependent upon neutrophil recruitment. Increased phosphorylation of the epidermal growth factor receptor (EGFR) was evident in the lungs of MMF-challenged mice when SP-A was absent. Pharmacologic inhibition of EGFR prior to MMF challenge dramatically reduced mucin production in SP-A(-/-) mice. These findings suggest a protective role for SP-A in limiting MMF-stimulated mucin production that occurs through interference with EGFR-mediated signaling. SP-A interaction with the EGFR signaling pathway appears to occur in an allele-specific manner that may have important implications for SP-A polymorphisms in human diseases.