Sprouty2 positively regulates T cell function and airway inflammation through regulation of CSK and LCK kinases.

The function of Sprouty2 (Spry2) in T cells is unknown. Using 2 different (inducible and T cell-targeted) knockout mouse strains, we found that Spry2 positively regulated extracellular signal-regulated kinase 1/2 (ERK1/2) signaling by modulating the activity of LCK. Spry2-/- CD4+ T cells were unable to activate LCK, proliferate, differentiate into T helper cells, or produce cytokines. Spry2 deficiency abrogated type 2 inflammation and airway hyperreactivity in a murine model of asthma. Spry2 expression was higher in blood and airway CD4+ T cells from patients with asthma, and Spry2 knockdown impaired human T cell proliferation and cytokine production. Spry2 deficiency up-regulated the lipid raft protein caveolin-1, enhanced its interaction with CSK, and increased CSK interaction with LCK, culminating in augmented inhibitory phosphorylation of LCK. Knockdown of CSK or dislodgment of caveolin-1-bound CSK restored ERK1/2 activation in Spry2-/- T cells, suggesting an essential role for Spry2 in LCK activation and T cell function.

Optimal identification of human conventional and nonconventional (CRTH2-IL7Rα-) ILC2s using additional surface markers.

BACKGROUND: Human type 2 innate lymphoid cells (ILC2s) are identified by coupled detection of CRTH2 and IL7Rα on lineage negative (Lin-) cells. Type 2 cytokine production by CRTH2-IL7Rα- innate lymphoid cells (ILCs) is unknown. OBJECTIVE: We sought to identify CRTH2-IL7Rα- type 2 cytokine-producing ILCs and their disease relevance. METHODS: We studied human blood and lung ILCs from asthmatic and control subjects by flow cytometry, ELISA, RNA sequencing, quantitative PCR, adoptive transfer to mice, and measurement of airway hyperreactivity by Flexivent. RESULTS: We found that IL-5 and IL-13 were expressed not only by CRTH2+ but also by CRTH2-IL7Rα+ and CRTH2-IL7Rα- (double-negative [DN]) human blood and lung cells. All 3 ILC populations expressed type 2 genes and induced airway hyperreactivity when adoptively transferred to mice. The frequency of type 2 cytokine-positive IL7Rα and DN ILCs were similar to that of CRTH2 ILCs in the blood and lung. Their frequency was higher in asthmatic patients than in disease controls. Transcriptomic analysis of CRTH2, IL7Rα, and DN ILCs confirmed the expression of mRNA for type 2 transcription factors in all 3 populations. Unexpectedly, the mRNA for GATA3 and IL-5 correlated better with mRNA for CD30, TNFR2, ICOS, CCR4, and CD200R1 than for CRTH2. By using a combination of these surface markers, especially CD30/TNFR2, we identified a previously unrecognized ILC2 population. CONCLUSIONS: The commonly used surface markers for human ILC2s leave a majority of type 2 cytokine-producing ILC2s unaccounted for. We identified top GATA3-correlated cell surface-expressed genes in human ILCs by RNA sequencing. These new surface markers, such as CD30 and TNFR2, identified a previously unrecognized human ILC2 population. This ILC2 population is likely to contribute to asthma.

New insights in the diagnosis of chronic refractory cough.

BACKGROUND: Chronic Refractory Cough (CRC) is a common condition that significantly impairs patients' quality of life. Unfortunately, in many situations patients continue to experience CRC in spite of following published guidelines for diagnosis and treatment. METHODS: 99 patients were referred to National Jewish Health (NJH), a specialty respiratory center for evaluation of CRC (cough ≥ 8 weeks duration). Study duration occurred over 18 months. Intake evaluation for all patients included history, physical examination, spirometry and fiberoptic laryngoscopy. Testing to confirm causes of CRC were performed. Specific therapy for each potential cause was provided. A visual analog cough scale measured cough response. RESULTS: Ten final diagnostic categories were found in the cohort of 99 patients with CRC: Obstructive sleep apnea (apnea/hypoxia index ≥ 5), rhinosinusitis, Tracheobronchomalacia (≥65% collapse of airway with dynamic expiratory imaging), esophageal dysmotility, gastroesophageal reflux, abnormal swallowing with laryngeal penetration, asthma, COPD, bronchiectasis and paradoxical vocal cord movement. In these patients there were 42 incorrect intake diagnoses and 101 new diagnoses established. Patients with CRC have had multiple diagnoses (3.8 ±â€¯1.6) associated with chronic cough. With directed therapy 71/76 (93%) patients had resolution or improvement in cough symptoms. CONCLUSIONS: Among patients referred to a specialty respiratory center with CRC multiple concomitant diagnoses for cough were common. Certain diagnoses such as OSA and TBM have not been reported in cough guidelines but in this study are commonly associated diagnoses. Targeted therapy for each recognized diagnosis improves patient response.

Steroid resistance of airway type 2 innate lymphoid cells from patients with severe asthma: The role of thymic stromal lymphopoietin.

BACKGROUND: Type 2 innate lymphoid cells (ILC2s) represent an important type 2 immune cell. Glucocorticoid regulation of human ILC2s is largely unknown. OBJECTIVE: We sought to assess steroid resistance of human blood and airway ILC2s from asthmatic patients and to examine its mechanism of induction. METHODS: We studied human blood and lung ILC2s from asthmatic patients and control subjects using flow cytometry and ELISA. RESULTS: Dexamethasone inhibited (P = .04) chemoattractant receptor-homologous molecule expressed on TH2 lymphocytes and type 2 cytokine expression by blood ILC2s stimulated with IL-25 and IL-33. However, it did not do so when ILC2s were stimulated with IL-7 and thymic stromal lymphopoietin (TSLP), 2 ligands of IL-7 receptor α. Unlike blood ILC2s, bronchoalveolar lavage (BAL) fluid ILC2s from asthmatic patients were resistant to dexamethasone. BAL fluid from asthmatic patients had increased TSLP but not IL-7 levels. BAL fluid TSLP levels correlated (r = 0.74) with steroid resistance of ILC2s. TSLP was synergistically induced in epithelial cells by IL-13 and human rhinovirus. Mechanistically, dexamethasone upregulated ILC2 expression of IL-7 receptor α, which augmented and sustained signal transducer and activator of transcription (STAT) 5 signaling by TSLP. TSLP induced mitogen-activated protein kinase kinase (MEK), c-Fos, inhibitor of DNA binding 3, phosphorylated signal transducer and activator of transcription (pSTAT) 3, and pSTAT5, molecules linked to steroid resistance. Dexamethasone inhibited c-Fos, inhibitor of DNA binding 3, and pSTAT3 but not pSTAT5 and MEK. The MEK inhibitor trametinib, the Janus kinase-STAT inhibitor tofacitinib, and the STAT5 inhibitor pimozide reversed steroid resistance of BAL ILC2s. CONCLUSIONS: Dexamethasone inhibited type 2 cytokine production by blood ILC2s. IL-7 and TSLP abrogated this inhibition and induced steroid resistance of ILC2s in a MEK- and STAT5-dependent manner. BAL fluid ILC2s from asthmatic patients with increased TSLP levels were steroid resistant, which was reversed by clinically available inhibitors of MEK and STAT5.

Airway and serum biochemical correlates of refractory neutrophilic asthma.

BACKGROUND: Despite progress in the diagnosis and management of asthma, many patients have poorly controlled or refractory asthma (RA). The mechanism of this RA is not well understood. OBJECTIVE: We sought to explore the relationship between neutrophils and other biomarkers of RA. METHOD: Sixty patients with RA, 30 patients with nonrefractory asthma (NRA), and 20 healthy subjects were enrolled. We performed a comprehensive characterization of these study subjects, which included laboratory and pulmonary function studies, chest computed tomography, and bronchoscopy with bronchoalveolar lavage (BAL). We analyzed BAL fluid and serum for a total of 244 biomolecules using a multiplex assay and correlated them with clinical and other laboratory parameters. RESULTS: RA was significantly different from NRA with regard to pulmonary function indices, bronchial basement membrane thickness, and BAL fluid neutrophil and lymphocyte counts but not eosinophil counts. BAL fluid neutrophil counts negatively and positively correlated with forced vital capacity and age, respectively. Of the 244 biomolecules studied, 52 and 14 biomolecules from BAL fluid and serum, respectively, were significantly different among the study groups. Thirteen of these 52 molecules correlated with BAL fluid neutrophil counts. BAL fluid from 40% of patients with RA was positive for a pathogenic microbe. Infection-negative neutrophilic RA was associated with an increase in levels of select biomarkers of inflammation in the serum, suggesting the presence of systemic inflammation. CONCLUSIONS: RA was associated with increased numbers of neutrophils and proneutrophilic biomolecules in the airways. Subclinical infection was present in 40% of patients with RA, which likely contributed to neutrophilic inflammation. A subgroup of patients with noninfected neutrophilic RA was associated with systemic inflammation.

Mechanism of TH2/TH17-predominant and neutrophilic TH2/TH17-low subtypes of asthma.

BACKGROUND: The mechanism of TH2/TH17-predominant and TH2/TH17-low asthma is unknown. OBJECTIVE: We sought to study the immune mechanism of TH2/TH17-predominant and TH2/TH17-low asthma. METHODS: In a previously reported cohort of 60 asthmatic patients, 16 patients were immunophenotyped with TH2/TH17-predominant asthma and 22 patients with TH2/TH17-low asthma. We examined bronchoalveolar lavage (BAL) fluid leukocytes, cytokines, mediators, and epithelial cell function for these asthma subgroups. RESULTS: Patients with TH2/TH17-predominant asthma had increased IL-1ß, IL-6, IL-23, C3a, and serum amyloid A levels in BAL fluid, and these correlated with IL-1ß and C3a levels. TH2/TH17 cells expressed higher levels of the IL-1 receptor and phospho-p38 mitogen-activated protein kinase. Anakinra, an IL-1 receptor antagonist protein, inhibited BAL TH2/TH17 cell counts. TH2/TH17-low asthma had 2 distinct subgroups: neutrophilic asthma (45%) and pauci-inflammatory asthma (55%). This contrasted with patients with TH2/TH17-predominant and TH2-predominant asthma, which included neutrophilic asthma in 6% and 0% of patients, respectively. BAL fluid neutrophils strongly correlated with BAL fluid myeloperoxidase, IL-8, IL-1α, IL-6, granulocyte colony-stimulating factor, and GM-CSF levels. Sixty percent of the patients with neutrophilic asthma had a pathogenic microorganism in BAL culture, which suggested a subclinical infection. CONCLUSION: We uncovered a critical role for the IL-1ß pathway in patients with TH2/TH17-predminant asthma. A subgroup of patients with TH2/TH17-low asthma had neutrophilic asthma and increased BAL fluid IL-1α, IL-6, IL-8, granulocyte colony-stimulating factor, and GM-CSF levels. IL-1α was directly involved in IL-8 production and likely contributed to neutrophilic asthma. Sixty percent of neutrophilic patients had a subclinical infection.

Persistence of asthma requires multiple feedback circuits involving type 2 innate lymphoid cells and IL-33.

BACKGROUND: Asthma in a mouse model spontaneously resolves after cessation of allergen exposure. We developed a mouse model in which asthma features persisted for 6 months after cessation of allergen exposure. OBJECTIVE: We sought to elucidate factors contributing to the persistence of asthma. METHODS: We used a combination of immunologic, genetic, microarray, and pharmacologic approaches to dissect the mechanism of asthma persistence. RESULTS: Elimination of T cells though antibody-mediated depletion or lethal irradiation and transplantation of recombination-activating gene (Rag1)(-/-) bone marrow in mice with chronic asthma resulted in resolution of airway inflammation but not airway hyperreactivity or remodeling. Elimination of T cells and type 2 innate lymphoid cells (ILC2s) through lethal irradiation and transplantation of Rag2(-/-)γc(-/-) bone marrow or blockade of IL-33 resulted in resolution of airway inflammation and hyperreactivity. Persistence of asthma required multiple interconnected feedback and feed-forward circuits between ILC2s and epithelial cells. Epithelial IL-33 induced ILC2s, a rich source of IL-13. The latter directly induced epithelial IL-33, establishing a positive feedback circuit. IL-33 autoinduced, generating another feedback circuit. IL-13 upregulated IL-33 receptors and facilitated IL-33 autoinduction, thus establishing a feed-forward circuit. Elimination of any component of these circuits resulted in resolution of chronic asthma. In agreement with the foregoing, IL-33 and ILC2 levels were increased in the airways of asthmatic patients. IL-33 levels correlated with disease severity. CONCLUSIONS: We present a critical network of feedback and feed-forward interactions between epithelial cells and ILC2s involved in maintaining chronic asthma. Although T cells contributed to the severity of chronic asthma, they were redundant in maintaining airway hyperreactivity and remodeling.

The role of atypical infections and macrolide therapy in patients with asthma.

For many years, the clinical benefit of macrolide use has been recognized in specific groups of patients with pulmonary disease. Dramatic improvement in survival of patients with diffuse panbronchiolitis is the most striking example of successful macrolide use as well as treatment of community acquired pneumonia caused by the atypical bacteria Mycoplasma, Chlamydophila, and Legionella. There also has been documentation of reduction in the exacerbation rate and of improvement in quality of life in patients with cystic fibrosis, bronchiectasis, chronic obstructive pulmonary disease, and reduction in post-lung transplantation bronchiolitis frequency. There has long been an interest in treating patients with severe asthma by using macrolides, but research results have not shown consistent clinical benefit in their use in the "general" population of patients with severe asthma. Rather, the successful use of macrolides seems to be in those patients with either documented Mycoplasma or Chlamydophila infection, or noneosinophilic asthma. Patients with neutrophil predominant phenotype severe asthma tend to show a decline in exacerbation rate, improved peak expiratory flows, and improved quality of life when treated with macrolides. This article will review the use of macrolides in the treatment of asthma.

Increased frequency of dual-positive TH2/TH17 cells in bronchoalveolar lavage fluid characterizes a population of patients with severe asthma.

BACKGROUND: TH2 cells can further differentiate into dual-positive TH2/TH17 cells. The presence of dual-positive TH2/TH17 cells in the airways and their effect on asthma severity are unknown. OBJECTIVE: We sought to study dual-positive TH2/TH17 cells in bronchoalveolar lavage (BAL) fluid from asthmatic patients, examine their response to glucocorticoids, and define their relevance for disease severity. METHODS: Bronchoscopy and lavage were performed in 52 asthmatic patients and 25 disease control subjects. TH2 and TH2/TH17 cells were analyzed by using multicolor flow cytometry and confocal immunofluorescence microscopy. Cytokines were assayed by means of ELISA. RESULTS: Dual-positive TH2/TH17 cells were present at a higher frequency in BAL fluid from asthmatic patients compared with numbers seen in disease control subjects. High-level IL-4 production was typically accompanied by high-level IL-17 production and coexpression of GATA3 and retinoic acid receptor-related orphan receptor γt. Increased presence of TH2/TH17 cells was associated with increased IL-17 production in lavage fluid. TH2/TH17 cell counts and IL-17 production correlated with PC20 for methacholine, eosinophil counts, and FEV1. TH2/TH17 cells, unlike TH2 cells, were resistant to dexamethasone-induced cell death. They expressed higher levels of mitogen-activated protein-extracellular signal-regulated kinase kinase 1, a molecule that induces glucocorticoid resistance. On the basis of the dominance of BAL fluid TH2 or TH2/TH17 cells, we identified 3 subgroups of asthma: TH2(predominant), TH2/TH17(predominant), and TH2/TH17(low). The TH2/TH17(predominant) subgroup manifested the most severe form of asthma, whereas the TH2/TH17(low) subgroup had the mildest asthma. CONCLUSION: Asthma is associated with a higher frequency of dual-positive TH2/TH17 cells in BAL fluid. The TH2/TH17(predominant) subgroup of asthmatic patients manifested glucocorticoid resistance in vitro. They also had the greatest airway obstruction and hyperreactivity compared with the TH2(predominant) and TH2/TH17(low) subgroups.

An index to objectively score supraglottic abnormalities in refractory asthma: learning, validation, and significance.

BACKGROUND: Patients with refractory asthma frequently have elements of laryngopharyngeal reflux (LPR) with potential aspiration contributing to their poor control. We previously reported on a supraglottic index (SGI) scoring system that helps in the evaluation of LPR with potential aspiration. However, to further the usefulness of this SGI scoring system for bronchoscopists, a teaching system was developed that included both interobserver and intraobserver reproducibility. METHODS: Five pulmonologists with expertise in fiber-optic bronchoscopy but novice to the SGI participated. A training system was developed that could be used via Internet interaction to make this learning technique widely available. RESULTS: By the final testing, there was excellent interreader agreement (κ of at least 0.81), thus documenting reproducibility in scoring the SGI. For the measure of intrareader consistency, one reader was arbitrarily selected to rescore the final test 4 weeks later and had a κ value of 0.93, with a 95% CI of 0.79 to 1.00. CONCLUSIONS: In this study, we demonstrate that with an organized educational approach, bronchoscopists can develop skills to have highly reproducible assessment and scoring of supraglottic abnormalities. The SGI can be used to determine which patients need additional intervention to determine causes of LPR and gastroesophageal reflux. Identification of this problem in patients with refractory asthma allows for personal, individual directed therapy to improve asthma control.

An Index to Objectively Score Supraglottic Abnormalities in Refractory Asthma.

BACKGROUND: Patients with refractory asthma frequently have elements of laryngopharyngeal reflux (LPR) with potential aspiration contributing to their poor control. We previously reported on a supraglottic index (SGI) scoring system that helps in the evaluation of LPR with potential aspiration. However, to further the usefulness of this SGI scoring system for bronchoscopists, a teaching system was developed that included both interobserver and intraobserver reproducibility. METHODS: Five pulmonologists with expertise in fiber-optic bronchoscopy but novice to the SGI participated. A training system was developed that could be used via Internet interaction to make this learning technique widely available. RESULTS: By the final testing, there was excellent interreader agreement (κ of at least 0.81), thus documenting reproducibility in scoring the SGI. For the measure of intrareader consistency, one reader was arbitrarily selected to rescore the final test 4 weeks later and had a κ value of 0.93, with a 95% CI of 0.79 to 1.00. CONCLUSIONS: In this study, we demonstrate that with an organized educational approach, bronchoscopists can develop skills to have highly reproducible assessment and scoring of supraglottic abnormalities. The SGI can be used to determine which patients need additional intervention to determine causes of LPR and gastroesophageal reflux. Identification of this problem in patients with refractory asthma allows for personal, individual directed therapy to improve asthma control.

Macrolides in the treatment of asthma.

PURPOSE OF REVIEW: This review summarizes the importance of macrolide therapy in the treatment of asthma, discusses macrolide mechanisms of action, and outlines new clinical data supporting their use. The effects of macrolides on both the innate and adaptive immune responses are discussed. RECENT FINDINGS: Subacute bacterial infection with both typical and atypical organisms contributes to poor asthma control. Identification of pathogens using polymerase chain reaction (PCR) and cultures from bronchoscopic samples directs antibiotic therapy and improves asthma control. PCR identification of Mycoplasma pneumoniae and Chlamydophila pneumoniae in asthmatics best identifies the macrolide responsive phenotype. SUMMARY: Because of their effect on protein synthesis, macrolides have both antimicrobial and anti-inflammatory properties. Both mechanisms appear to be important in their clinical efficacy in treating a wide variety of pulmonary disorders, including asthma.

Update on infection and antibiotics in asthma.

Asthma pathogenesis seems to be a result of a complex mixture of genetic and environmental influences. There is evidence that Mycoplasma pneumoniae and Chlamydophila pneumoniae (formerly known as Chlamydia pneumoniae) play a role in promoting airway inflammation that could contribute to the onset and clinical course of asthma. Evidence also indicates that when antimicrobial therapy can eradicate or suppress these organisms, it may be possible to alter the course of the disease. Certain macrolide antibiotics have been shown to improve control of asthma symptoms and lung function in patients diagnosed with acute C. pneumoniae or M. pneumoniae infection. Positive polymerase chain reaction studies for C. pneumoniae or M. pneumoniae are needed to select asthma patients for chronic treatment. Macrolide antibiotics may also have independent anti-inflammatory activity that may be useful in the management of asthma and other inflammatory diseases.

Performance of a new screening spirometer at a community health fair.

OBJECTIVE: Compare the results from a new screening spirometer (EasyOne) with the results from a standard laboratory spirometer (Vmax) approved by the American Thoracic Society. SETTING: A health fair at a community hospital. METHODS: We measured forced expiratory volume in the first second (FEV(1)) and forced expiratory volume in the first 6 seconds (FEV(6)). With the screening spirometer, good quality testing was achieved in 359 of 394 subjects (91%), and 115 subjects were also tested with the standard laboratory spirometer. The best test values for FEV(1) and FEV(6) were taken for 3 tests that agreed within 3%. FEV(6) was extrapolated from forced vital capacity on the printouts from the standard laboratory spirometer. RESULTS: Correlations between the screening spirometer results and the standard laboratory spirometer were excellent for FEV(1) (r = 0.93), FEV(6) (r = 0.96), and FEV(1)/FEV(6) (r = 0.72) (p = 0.001 for all comparisons). The 95% limits of agreement (mean difference between the 2 spirometers +/- 1.96 standard deviations) were: -0.18 and 0.69 for FEV(1); -0.24 and 0.81 for FEV(6); and -0.12 and 0.13 for FEV(1)/FEV(6). CONCLUSION: The new screening spirometer is suitable for clinical use.