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Pulmonary emphysema is a destructive inflammatory disease primarily caused by cigarette smoking (CS). Recovery from CS-induced injury requires proper stem cell (SC) activities with a tightly controlled balance of proliferation and differentiation. Here we show that acute alveolar injury induced by two representative tobacco carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene (N/B), increased IGF2 expression in alveolar type 2 (AT2) cells to promote their SC function and facilitate alveolar regeneration. Autocrine IGF2 signaling upregulated Wnt genes, particularly Wnt3, to stimulate AT2 proliferation and alveolar barrier regeneration after N/B-induced acute injury. In contrast, repetitive N/B exposure provoked sustained IGF2-Wnt signaling through DNMT3A-mediated epigenetic control of IGF2 expression, causing a proliferation/differentiation imbalance in AT2s and development of emphysema and cancer. Hypermethylation of the IGF2 promoter and overexpression of DNMT3A, IGF2, and the Wnt target gene AXIN2 were seen in the lungs of patients with CS-associated emphysema and cancer. Pharmacologic or genetic approaches targeting IGF2-Wnt signaling or DNMT prevented the development of N/B-induced pulmonary diseases. These findings support dual roles of AT2 cells, which can either stimulate alveolar repair or promote emphysema and cancer depending on IGF2 expression levels. Significance: IGF2-Wnt signaling plays a key role in AT2-mediated alveolar repair after cigarette smoking–induced injury but also drives pathogenesis of pulmonary emphysema and cancer when hyperactivated.

Background/Aims: Neutrophilia is frequently observed in bronchoalveolar lavage fluid (BALF) of idiopathic pulmonary fibrosis (IPF) patients. Granulocyte colony-stimulating factor (G-CSF) is a potent neutrophil-activating glycoprotein. However, the clinical implications of G-CSF remain poorly understood.in patients with IPF. Therefore, we evaluated the relationship between the G-CSF concentration in BALF and the progression of fibrosis, including in terms of the decline in lung function and long-term survival rate.Methods: G-CSF concentrations were measured in BALF using enzyme-linked immunosorbent assay (ELISA). The survival rate was estimated using Kaplan-Meier survival analyses.Results: G-CSF protein levels were significantly higher in IPF (n = 87; 1.88 [0 to 5.68 pg/mL]), nonspecific interstitial pneumonia (n = 22; 0.58 [0 to 11.64 pg/mL]), and hypersensitivity pneumonitis (n = 19; 2.48 [0.46 to 5.71 pg/mL]) patients than in normal controls (n = 33; 0 [0 to 0.68 pg/mL]) (all p < 0.01). A receiver operating characteristic curve showed a difference in G-CSF levels between IPF and NC (area under the curve, 0.769): The G-CSF cut-off of 0.96 pg/mL indicated 84.9% specificity and 63.2% sensitivity for IPF. The survival rate was significantly lower in the group with G-CSF > 2.872 pg/mL than in the group with ≤ 2.872 pg/mL (hazard ratio, 2.69; p = 0.041). The annual decline in diffusing capacity of the lung for carbon monoxide was positively correlated with the G-CSF level (p = 0.018).Conclusions: G-CSF may participate in the development of IPF and be useful for predicting the prognosis of IPF. Therefore, G-CSF should be analyzed in BALF, in addition to differential cell counts.

Novel genetic and epigenetic factors involved in the development and prognosis of idiopathic pulmonary fibrosis (IPF) have been identified. We previously observed that erythrocyte membrane protein band 4.1-like 3 (EPB41L3) increased in the lung fibroblasts of IPF patients. Thus, we investigated the role of EPB41L3 in IPF by comparing the EPB41L3 mRNA and protein expression of lung fibroblast between patients with IPF and controls. We also investigated the regulation of epithelial–mesenchymal transition (EMT) in an epithelial cell line (A549) and fibroblast-to-myofibroblast transition (FMT) in a fibroblast cell line (MRC5) by overexpressing and silencing EPB41L3. EPB41L3 mRNA and protein levels, as measured using RT-PCR, real-time PCR, and Western blot, were significantly higher in fibroblasts derived from 14 IPF patients than in those from 10 controls. The mRNA and protein expression of EPB41L3 was upregulated during transforming growth factor-β-induced EMT and FMT. Overexpression of EPB41L3 in A549 cells using lenti-EPB41L3 transfection suppressed the mRNA and protein expression of N-cadherin and COL1A1. Treatment with EPB41L3 siRNA upregulated the mRNA and protein expression of N-cadherin. Overexpression of EPB41L3 in MRC5 cells using lenti-EPB41L3 transfection suppressed the mRNA and protein expression of fibronectin and α-SMA. Finally, treatment with EPB41L3 siRNA upregulated the mRNA and protein expression of FN1, COL1A1, and VIM. In conclusion, these data strongly support an inhibitory effect of EPB41L3 on the process of fibrosis and suggest the therapeutic potential of EPB41L3 as an anti-fibrotic mediator.

Supplementary Methods, Tables, and Figures

Pulmonary emphysema is a destructive inflammatory disease primarily caused by cigarette smoking (CS). Recovery from CS-induced injury requires proper stem cell (SC) activities with a tightly controlled balance of proliferation and differentiation. Here we show that acute alveolar injury induced by two representative tobacco carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene (N/B), increased IGF2 expression in alveolar type 2 (AT2) cells to promote their SC function and facilitate alveolar regeneration. Autocrine IGF2 signaling upregulated Wnt genes, particularly Wnt3, to stimulate AT2 proliferation and alveolar barrier regeneration after N/B-induced acute injury. In contrast, repetitive N/B exposure provoked sustained IGF2-Wnt signaling through DNMT3A-mediated epigenetic control of IGF2 expression, causing a proliferation/differentiation imbalance in AT2s and development of emphysema and cancer. Hypermethylation of the IGF2 promoter and overexpression of DNMT3A, IGF2, and the Wnt target gene AXIN2 were seen in the lungs of patients with CS-associated emphysema and cancer. Pharmacologic or genetic approaches targeting IGF2-Wnt signaling or DNMT prevented the development of N/B-induced pulmonary diseases. These findings support dual roles of AT2 cells, which can either stimulate alveolar repair or promote emphysema and cancer depending on IGF2 expression levels.Significance:IGF2-Wnt signaling plays a key role in AT2-mediated alveolar repair after cigarette smoking–induced injury but also drives pathogenesis of pulmonary emphysema and cancer when hyperactivated.

Novel genetic and epigenetic factors involved in the development and prognosis of idiopathic pulmonary fibrosis (IPF) have been identified. We previously observed that EPB41L3 increased in the lung fibroblasts of IPF patients. Thus, we investigated the role of EPB41L3 in IPF by comparing EPB41L3 mRNA and protein expression of lung fibroblast between patients with IPF and controls. We also investigated the regulation of the epithelial-mesenchymal transition (EMT) in an epithelial cell line (A549), and the fibroblast-to-myofibroblast transition (FMT) in a fibroblast cell line (MRC5), by overexpressing and silencing EPB41L3. EPB41L3 mRNA and protein levels, as measured by RT-PCR, real-time PCR, and western blot, were significantly higher in fibroblasts derived from 14 IPF patients than in those from 10 controls. The mRNA and protein expression of EPB41L3 was upregulated during transforming growth factor-β-induced EMT and FMT. Overexpression of EPB41L3 in A549 cells using lenti-EPB41L3 transfection suppressed the mRNA and protein expression of N-cadherin and COL1A1. Treatment with EPB41L3 siRNA upregulated mRNA and protein expression of N-cadherin. Overexpression of EPB41L3 in MRC5 cells using lenti-EPB41L3 transfection suppressed mRNA and protein expression of fibronectin and α-SMA. Finally, treatment with EPB41L3 siRNA upregulated mRNA and protein expression of FN1, COL1A1, and VIM. Overall, these data strongly support an inhibitory effect of EPB41L3 on the process of fibrosis and suggest therapeutic potential of EPB41L3 as an anti-fibrotic mediator.

Supplemental figure legends.

Supplemental Figure S1. The effect of oncogenes on DX2 expression. Supplemental Figure S2. Oncogenic property of DX2. Supplemental Figure S3. Small cell lung cancer in DX2 and DK transgenic model. Supplemental Figure S4. DX2 suppresses p14/ARF. Supplemental Figure S5. Screening of specific inhibitor of p14/ARF and DX2. Supplemental Figure S6. SLCB050 as specific binding inhibitor of p14/ARF and DX2. Supplemental Figure S7. The effect of SLCB050 on DX2. Supplemental Figure S8. Anti-tumoral effects of SLCB050 in DK mice. Supplemental Figure S9. Diagram for summary.

Table S1. Clino-pathological information of human samples.

Background. Toluene diisocyanate (TDI) causes occupational asthma by generating oxidative stress, leading to tissue injury and inflammation. Glutathione transferases (GSTs) are detoxifying enzymes that eliminate oxidative stress. We examined whether the genotypes of the GSTM1 and GSTT1 genes are associated with TDI-induced occupational asthma (TDI-OA). Methods. The study population consisted of 26 asthmatics with a positive response to the TDI challenge (TDI-PA) and 27 asthmatics with negative responses (TDI-NA). GSTM1 and GSTT1 null and wild-type genotypes were determined using multiplex PCR. The plasma GSTM1 and GSTT1 protein concentrations were determined using ELISA. Results. The GSTM1 null genotype was more frequent in the TDI-PA than in the TDI-NA (77.8 vs. 50.0%, OR = 3.5, p = 0.03 ), while the frequency of the GSTT1 null genotype tended to be higher in the TDI-PA than in the TDI-NA (59.3 vs. 42.3%, OR = 1.98, p = 0.21 ). When analyzed together, the GSTM1/GSTT1 null genotype was more frequent in the TDI-PA than in the TDI-NA (48.2 vs. 15.3%, OR = 6.5, p = 0.04 ). The decline in the FEV in 1 s after TDI challenge was higher with the GSTM1/GSTT1 null than the GSTM1 wild-type/GSTT1 null genotypes (24.29% vs. 7.47%, p = 0.02 ). The plasma GSTM1 level was lower with the GSTM1 null than with the GSTM1 wild-type genotypes both before (13.7 vs. 16.6 ng/mg, p = 0.04 ) and after (12.9 vs. 17.1 ng/mg, p = 0.007 ) the TDI challenge, while the GSTT1 level was not changed with either the GSTT1 null or wild-type genotype. Conclusions. The GSTM1 null genotype, but not GSTT1 alone, may confer susceptibility to TDI-OA. However, the genetic effect of the GSTM1 null genotype may be enhanced synergistically by the GSTT1 null genotype. The genetic effect of GSTM1 was validated in the plasma as the GSTM1 protein level. Therefore, the GSTM1 and GSTT1 genotypes may be useful diagnostic markers for TDI-OA.

A control group is defined as a group of people used for comparison. Depending on the type of study, it can be a group of healthy people or a group not exposed to risk factors. It is important to allow researchers to select the appropriate control participants. The Korea Biobank Project-sponsored biobanks are affiliated with the Korea Biobank Network (KBN), for which the National Biobank of Korea plays a central coordinating role among KBN biobanks. KBN organized several working groups to address new challenges and needs in biobanking. The “Normal Healthy Control Working Group” developed standardized criteria for three defined control groups, namely, normal, normal-plus, and disease-specific controls. Based on the consensus on the definition of a normal control, we applied the criteria for normal control participants to retrospective data. The main reason for exclusion from the “Normal-plus” group was blood test results beyond 5% of the reference range, including hypercholesterolemia. Subclassification of samples of normal controls by detailed criteria will help researchers select optimal normal controls for their studies.

Background: E-cigarettes are steadily gaining popularity in Korea. However, the characteristics of e-cigarette smokers, especially nicotine dependence and stress susceptibility, have not been evaluated in comparison to those of nonsmokers or combustible cigarette smokers in Korea. Methods: In this study, 28,059 participants from the Korea National Health and Nutrition Examination Survey (2013-2017) were classified into the following three groups: non-smokers, smokers (current smokers and ex-smokers of combustible cigarettes only), and e-smokers (current smokers and ex-smokers of e-cigarettes regardless of combustible cigarette use). Results: Among the participants, 16,980 (60.5%), 9,247 (33.0%), and 1,832 (6.4%) subjects were non-smokers, smokers, and e-smokers, respectively. E-smokers were younger, more educated, and had a higher household income than nonsmokers or smokers. The number of e-smokers who smoked within 5 minutes of waking up (31.5% vs. 19.8%, p

Background/Aims Receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a major regulator of Wnt signaling, which is involved in fibroblast dysfunction. Because its role has not been evaluated in idiopathic pulmonary fibrosis (IPF), we examined the clinical implications of ROR2 expression. Methods ROR2 mRNA expression was measured using reverse transcription polymerase chain reaction in lung tissue-derived fibroblasts from IPF patients (n = 14) and from controls (n = 10). ROR2 protein was measured using enzyme-linked immunosorbent assay in primary fibroblasts from IPF patients (n = 14) and controls (n = 10), and in bronchoalveolar lavage (BAL) fluids obtained from normal controls (NC; n = 30). IPF patients (n = 84), and other patients with interstitial lung diseases, including nonspecific interstitial pneumonia (NSIP; n = 10), hypersensitivity pneumonitis (HP; n = 10), and sarcoidosis (n = 10). Results ROR2 mRNA and protein levels were significantly higher in IPF fibroblasts than in controls (p = 0.003, p = 0.0017, respectively). ROR2 protein levels in BAL fluids from patients with IPF were significantly higher than in those from NC (p < 0.001), and from patients with NSIP (p = 0.006), HP (p = 0.004), or sarcoidosis (p = 0.004). Receiver operating characteristic curves showed a clear difference between IPF and NC in ROR2 protein level (area under the curve, 0.890; confidence interval, 0.829 to 0.950; p < 0.001). ROR2 protein levels were significantly higher in GAP stage III than in GAP stages I and II (p = 0.016). Conclusions ROR2 may be related to the development of IPF, and its protein level may be a useful and severity-dependent candidate marker for IPF.

For the past three decades, more than a thousand of genetic studies have been performed to find out the genetic variants responsible for the risk of asthma. Until now, all of the discovered single nucleotide polymorphisms have explained genetic effects less than initially expected. Thus, clarification of environmental factors has been brought up to overcome the 'missing' heritability. The most exciting solution is epigenesis because it intervenes at the junction between the genome and the environment. Epigenesis is an alteration of genetic expression without changes of DNA sequence caused by environmental factors such as nutrients, allergens, cigarette smoke, air pollutants, use of drugs and infectious agents during pre- and post-natal periods and even in adulthood. Three major forms of epigenesis are composed of DNA methylation, histone modifications, and specific microRNA. Recently, several studies have been published on epigenesis in asthma and allergy as a powerful tool for research of genetic heritability in asthma albeit epigenetic changes are at the starting point to obtain the data on specific phenotypes of asthma. In this presentation, we mainly review the potential role of DNA CpG methylation in the risk of asthma and its sub-phenotypes including nonsteroidal anti-inflammatory exacerbated respiratory diseases.

Background Asthma exacerbation threatens patient's life. Several genetic studies have been conducted to determine the risk factors for asthma exacerbation, but this information is still lacking. We aimed to determine whether genetic variants of Oxidative Stress Responsive Kinase 1 (OXSR1), a gene with functions of salt transport, immune response, and oxidative stress, are associated with exacerbation of asthma. Methods Clinical data were obtained from 1454 asthmatics and single nucleotide polymorphisms (SNPs) of OXSR1 were genotyped. Genetic associations with annual exacerbation rate were analyzed depending on smoking status. Results Eleven SNPs were selected using Asian data in the International HapMap database. The common allele of rs1384006 C > T of OXSR1 showed a significantly higher annual exacerbation rate than the rare allele in non-smoking asthmatics (CC vs. CT vs. TT: 0.43 ± 0.04 vs. 0.28 ± 0.03 vs. 0.31 ± 0.09, P = 0.004, Pcorr = 0.039). The frequent exacerbators had a significantly higher frequency of the common allele of rs1384006 C > T than did the infrequent exacerbators (74.4% vs. 55.2%, P = 0.004, Pcorr = 0.038). Conclusion The common allele of rs1384006 C > T of OXSR1 was associated with the asthma exacerbation rate and a higher risk of being a frequent exacerbator, indicating that non-smoking asthmatics who carry common alleles may be vulnerable to asthma exacerbations.

Background This study aimed to investigate the association between e-cigarette (EC) use and development of acute severe pneumonia in the Korean population using a national database. Methods We conducted a retrospective analysis using linkage of data between the Korean National Health and Nutrition Examination Survey (KNHANES) and the National Health Insurance Service (NHIS) administrative claims database. The primary endpoint of this study was development of severe pneumonia requiring hospital admission according to EC use during the study period. The secondary endpoints were in-hospital mortality, intensive care unit (ICU) admission, ventilator care, and days of hospital stay. Results The final analysis included 28,950 individuals, of which 578 (2.0%) were EC users. EC users were younger and more often male than non-EC users. The EC users showed higher level of education and household income and had fewer comorbidities. Severe pneumonia was noted in 37 of 28,372 non-EC users (0.13%), but there were no occurrences of severe pneumonia in EC users. The incidence of pneumonia occurrence was not different between the two groups (P = 1.000). Conclusions Since e-cigarette or vaping use-associated lung injury (EVALI) is most likely included in acute severe pneumonia occurring within 3 months of EC use, it is considered that there might be no EVALI patients in Korea during the investigation period. A large-scale, prospective study is necessary to evaluate the association between EC use and acute lung injury., Graphical Abstract

Background Cancer stem-like cells (CSCs) play a pivotal role in lung tumor formation and progression. Nerve injury-induced protein 1 (Ninjurin1, Ninj1) has been implicated in lung cancer; however, the pathological role of Ninj1 in the context of lung tumorigenesis remains largely unknown. Methods The role of Ninj1 in the survival of non-small cell lung cancer (NSCLC) CSCs within microenvironments exhibiting hazardous conditions was assessed by utilizing patient tissues and transgenic mouse models where Ninj1 repression and oncogenic KrasG12D/+ or carcinogen-induced genetic changes were induced in putative pulmonary stem cells (SCs). Additionally, NSCLC cell lines and primary cultures of patient-derived tumors, particularly Ninj1high and Ninj1low subpopulations and those with gain- or loss-of-Ninj1 expression, and also publicly available data were all used to assess the role of Ninj1 in lung tumorigenesis. Results Ninj1 expression is elevated in various human NSCLC cell lines and tumors, and elevated expression of this protein can serve as a biomarker for poor prognosis in patients with NSCLC. Elevated Ninj1 expression in pulmonary SCs with oncogenic changes promotes lung tumor growth in mice. Ninj1high subpopulations within NSCLC cell lines, patient-derived tumors, and NSCLC cells with gain-of-Ninj1 expression exhibited CSC-associated phenotypes and significantly enhanced survival capacities in vitro and in vivo in the presence of various cell death inducers. Mechanistically, Ninj1 forms an assembly with lipoprotein receptor-related protein 6 (LRP6) through its extracellular N-terminal domain and recruits Frizzled2 (FZD2) and various downstream signaling mediators, ultimately resulting in transcriptional upregulation of target genes of the LRP6/β-catenin signaling pathway. Conclusions Ninj1 may act as a driver of lung tumor formation and progression by protecting NSCLC CSCs from hostile microenvironments through ligand-independent activation of LRP6/β-catenin signaling.

Shinhee Park,1 Pureun-Haneul Lee,2 Ae Rin Baek,1 Jong Sook Park,1 Junehyuk Lee,1 Sung-Woo Park,1 Do Jin Kim,1 An-Soo Jang1 1Division of Allergy and Respiratory Medicine, Department of Internal Medicine, Soonchunhyang University Bucheon Hospital, Bucheon, Republic of Korea; 2Department of Interdisciplinary Program in Biomedical Science Major, Soonchunhyang Graduate School, Soonchunhyang University Bucheon Hospital, Bucheon, Republic of KoreaCorrespondence: An-Soo JangDivision of Allergy and Respiratory Medicine, Department of Internal Medicine, Soonchunhyang University Bucheon Hospital, 170 Jomaru-ro, Bucheon, 14584, Republic of KoreaTel +82 32 621 5143Fax +82 32 621 6950Email jas877@schmc.ac.krPurpose: Chronic obstructive pulmonary disease (COPD) imposes a major healthcare burden. A tight junction protein, claudin-4 (CLDN4), may play a protective role in acute lung injury, but its role in COPD is unclear. To investigate the relationship between CLDN4 and COPD, we evaluated the association of CLDN4 with the clinical parameters of COPD, including exacerbations.Patients and Methods: We analyzed a cohort of 30 patients with COPD and 25 healthy controls and evaluated their clinical parameters, including lung function. The plasma CLDN4 level in stable and exacerbated COPD was measured.Results: The COPD patients were all males and predominantly smokers; their initial lung function was poorer than the healthy controls. The mean CLDN4 plasma level was 0.0219 ± 0.0205 ng/mg in the control group, 0.0086 ± 0.0158 ng/mg in the stable COPD group (COPD-ST) and 0.0917 ± 0.0871 ng/mg in the exacerbated COPD (COPD-EXA) group. The plasma CLDN4 level was significantly lower in the COPD-ST than the control group, but was significantly elevated in the COPD-EXA group. The plasma CLDN4 level was inversely correlated with forced vital capacity and forced expiratory volume in 1 second in the COPD-EXA group (r=0.506, P=0.001 and r=0.527, P< 0.001, respectively).Conclusion: The plasma CLDN4 level is closely correlated with COPD exacerbations and decreased lung function. This suggests that CLDN4 has potential as a severity marker for COPD.Keywords: claudin-4, COPD, lung function

Toluene diisocyanate (TDI) causes occupational asthma by generating oxidative stress, leading to tissue injury and inflammation. Glutathione transferases (GSTs) are detoxifying enzymes that eliminate oxidative stress. We examined whether the genotypes of theThe study population consisted of 26 asthmatics with a positive response to the TDI challenge (TDI-PA) and 27 asthmatics with negative responses (TDI-NA).TheThe

Background This pilot study aimed to investigate the current status of e-cigarettes (ECs) use patterns among patients with chronic airway disease or chronic respiratory symptoms and the effects of ECs use on respiratory and mental health. Methods A cross-sectional survey was conducted at the outpatient clinic of eight teaching hospitals in South Korea between November 2019 and December 2019. All adult ECs users (19 years and above) who visited the outpatient clinic as a patient with chronic airway disease or chronic respiratory symptoms were eligible to participate in this study. Results A total of 51 subjects responded to the survey. Most of the participants were male (92.2%) and the mean age was 41.8 years. Dominant airway diseases were asthma and chronic obstructive pulmonary disease. Most of the subjects had a history of cigarette smoking and 19 subjects were dual users of current cigarettes and ECs. Most of the subjects started ECs use due to health-related reasons. When comparing exclusive ECs users and dual users, St. George's respiraory questionaire (SGRQ) scores, the proportion of cases with moderate to severe depressive symptoms, and average Fagerstrom Test for Nicotine Dependence scores for ECs were higher in dual users than exclusive ECs users (mean 4.64 vs. 2.38, p=0.006), respectively. Conclusion Most of the subjects started ECs use due to health concerns, but dual users have more respiratory symptoms and higher nicotine dependence in this pilot study. One hypothesis that comes from these results is that greater nicotine dependence may influence behaviours, habits and views about ECs. These preliminary observations need confirmation in a large cohort.

Quinoline-3-carboxamides have been used to treat autoimmune/inflammatory diseases in humans because they inhibit the functions of S100 calcium-binding protein A9 (S100A9), which participates in the development of neutrophilic inflammation in asthmatics and in an animal model of neutrophilic asthma. However, the therapeutic effects of these chemicals have not been evaluated in asthma. The purpose of this study was to evaluate the effect of paquinimod, one of the quinoline-3-carboxamides, on a murine model of neutrophilic asthma.Paquinimod was orally administered to 6-week-old C57BL/6 mice sensitized and challenged with ovalbumin (OVA)/complete Freund's adjuvant (CFA) and OVA. Lung inflammation and remodeling were evaluated using bronchoalveolar lavage (BAL) and histologic findings including goblet cell count. S100A9, caspase-1, IL-1Paquinimod restored the enhancement of airway resistance and the increases in numbers of neutrophils and macrophages of BAL fluids and those of goblet cells in OVA/CFA mice toward the levels of sham-treated mice in a dose-dependent manner (0.1, 1, 10, and 25 mg/kg/day, p.o.). Concomitantly, p20 activated caspase-1, IL-1These data indicate that paquinimod effectively inhibits neutrophilic inflammation and remodeling in the murine model of neutrophilic asthma, possibly via downregulation of IL-17, IFN

Exacerbation of asthma is affected by genetic and environmental factors, but little is known about genetic differences according to smoking status. We evaluated genetic factors associated with asthma exacerbations in smokers and non-smokers, and identified the underlying mechanisms via a genome-wide association study (GWAS) and gene-level analyses according to smoking status.A GWAS on the annual frequency of asthma exacerbations was performed in 420 non-smoking and 188 smoking patients with asthma. Gene-wise associations were analyzed by Multi-marker Analysis of GenoMic Annotation (MAGMA); Gene Ontology analysis was also performed.In the non-smoker group, 189 genes showed significant associations with the annual frequency of exacerbations (permutated P 0.001). The top 10 genes were F5, KLRC1, TAFA2, AIRE, IER3IP1, CHMP2A, IL31RA, ZNF497, DNMT3L, and MYT1L (permutated P = 1.0 × 10Our findings not only suggest novel genetic candidates for predicting asthma exacerbations, but also that asthma treatment strategies should take into account smoking behavior.

Background. Quinoline-3-carboxamides have been used to treat autoimmune/inflammatory diseases in humans because they inhibit the functions of S100 calcium-binding protein A9 (S100A9), which participates in the development of neutrophilic inflammation in asthmatics and in an animal model of neutrophilic asthma. However, the therapeutic effects of these chemicals have not been evaluated in asthma. The purpose of this study was to evaluate the effect of paquinimod, one of the quinoline-3-carboxamides, on a murine model of neutrophilic asthma. Methods. Paquinimod was orally administered to 6-week-old C57BL/6 mice sensitized and challenged with ovalbumin (OVA)/complete Freund’s adjuvant (CFA) and OVA. Lung inflammation and remodeling were evaluated using bronchoalveolar lavage (BAL) and histologic findings including goblet cell count. S100A9, caspase-1, IL-1β, MPO, IL-17, IFN-γ, and TNF-α were measured in lung lysates using western blotting. Results. Paquinimod restored the enhancement of airway resistance and the increases in numbers of neutrophils and macrophages of BAL fluids and those of goblet cells in OVA/CFA mice toward the levels of sham-treated mice in a dose-dependent manner (0.1, 1, 10, and 25 mg/kg/day, p.o.). Concomitantly, p20 activated caspase-1, IL-1β, IL-17, TNF-α, and IFN-γ levels were markedly attenuated. Conclusion. These data indicate that paquinimod effectively inhibits neutrophilic inflammation and remodeling in the murine model of neutrophilic asthma, possibly via downregulation of IL-17, IFN-γ, and IL-1β.

Background: Neutrophilic inflammation is a predominant characteristic of idiopathic pulmonary fibrosis (IPF). S100 Calcium-Binding Protein A9 (S100A9) is a neutrophil-derived protein and is involved in the development of neutrophil-related chronic inflammatory disorders. However, the role of S100A9 in IPF has not been evaluated.Methods : S100A9 concentrations were measured by ELISA in the BAL fluid obtained from NCs (n = 33) and patients with IPF (n = 87), NSIP (n = 22), HP (n = 19), or sarcoidosis (n = 10).Results: The S100A9 levels in BALF were significantly higher in patients with IPF than in those with NC (0.4 [0.18–0.9] vs. 0 [0–0.5] ng/mL, p < 0.001), HP (0.19 [0.07–0.33] ng/mL, p = 0.043), or sarcoidosis (0.06 [0–0.11] ng/mL, p < 0.001) patients. A S100A9 level of 0.093 ng/mL had discriminating powers of 78.79% for specificity and 81.61% for sensitivity between IPF patients and NCs. S100A9 levels were also correlated with neutrophil numbers (r = 0.356, p = 0.0007) and S100A9 was expressed on neutrophils and macrophages in the BALF of IPF patients. Patients with S100A9 levels above 0.5535 ng/mL or a neutrophil percentage above 49.09% (n = 43) had significantly lower survival rates than those with S100A9 levels at or below 0.5535 ng/mL and a neutrophil percentage at or below 49.09% (n = 41) (HR, 9.28; p = 0.0004).Conclusion: S100A9 may participate in the development and progression of IPF. The levels of S100A9 in BALF may be a surrogate marker for diagnosing IPF and predicting its prognosis.

Background. Fibroblast dysfunction is the main pathogenic mechanism underpinning idiopathic pulmonary fibrosis (IPF). Potassium voltage-gated channel subfamily J member 2 (KCNJ2) plays critical roles in the proliferation of myofibroblasts and in the development of cardiac fibrosis. Objectives. This study aimed to evaluate the role of KCNJ2 in IPF. Methods. KCNJ2 mRNA expression was measured using real-time PCR in fibroblasts from IPF patients and normal controls (NCs). Protein concentrations were measured by ELISA in bronchoalveolar lavage (BAL) fluid obtained from NCs (n = 30), IPF (n = 84), nonspecific interstitial pneumonia (NSIP; n = 9), hypersensitivity pneumonitis (HP; n = 8), and sarcoidosis (n = 10). Results. KCNJ2 mRNA levels were significantly higher in fibroblasts from IPF (n = 14) than those from NCs (n = 10, p<0.001). KCNJ2 protein levels in BAL fluid were significantly higher in IPF (6.587 [1.441–26.01] ng/mL) than in NCs (0.084 [0.00–0.260] ng/mL, p < 0.001), NSIP (0.301 [0.070–5.059] ng/mL, p = 0.006), HP (0.365 [0.000–3.407] ng/mL, p = 0.02), and sarcoidosis (0.170 [0.057–1.179] ng/mL, p = 0.001). Receiver operating characteristic curves showed a clear difference between the IPF and NCs according to the KCNJ2 protein level (area under the curve = 0.893). The KCNJ2 protein cutoff level determined from the curves (0.636 ng/mL) showed a 90.0% specificity and 83.3% sensitivity in distinguishing IPF from NCs. Conclusion. KCNJ2 may participate in the development of IPF, and its protein level may be a candidate diagnostic and therapeutic molecule for IPF.

Background Exposure to ozone (O3) induces neutrophilic inflammation and goblet cell hyperplasia in humans and experimental animals. Because the solute carrier family 26-member 4 (Slc26a4; pendrin) gene induces mucin production and intraluminal acidification in the airways, it was hypothesized to be a key molecule in O3-induced airway injury. Thus, we evaluated the role of Slc26a4 and the protective effects of ammonium chloride (NH4Cl) in O3-induced airway injury in mice. Methods Six-week-old female BALB/c mice were exposed to filtered air or O3 for 21 days (2 ppm for 3 hr/day). NH4Cl (0, 0.1, 1, and 10 mM) was administered intratracheally into the airways. Airway resistance was measured using a flexiVent system, and bronchoalveolar lavage fluid (BALF) cells were differentially counted. Slc26a4 and Muc5ac proteins and mRNA were measured via western blotting, real-time polymerase chain reaction, and immunostaining. Tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-17, IL-1β, and caspase-1 were analyzed via western blotting. Results The levels Slc26a4 protein and mRNA significantly increased in lung tissues from Day 7 to Day 21 of O3 exposure, with concomitant increases in lung resistance, numbers of goblet cells in lung tissues, and inflammatory cells and thiocyanate (SCN−) levels in BALF in a time-dependent manner. Treatment with NH4Cl significantly reduced these changes to levels similar to those of sham-treated mice, with a concomitant reduction of Slc26a4 proteins in lung lysates and SCN− levels in BALF. Slc26a4 protein was co-expressed with muc5ac protein in the bronchial epithelium, as indicated by immunofluorescence staining. NH4Cl treatment also significantly attenuated the O3-induced increases in IFN-γ, TNF-α, IL-17, IL-1β, and p20-activated caspase-1. Conclusion Slc26a4 may be involved in O3-induced inflammatory and epithelial changes in the airways via activation of the inflammasome and the induction of IL-17 and IFN-γ. NH4Cl shows a potential as a therapeutic agent for controlling O3-induced airway inflammation and epithelial damage by modulating Slc26a4 expression., Graphical Abstract

Background/aims The methacholine bronchial provocation test (MBPT) is used to detect and quantify airway hyper-responsiveness (AHR). Since improvements in the severity of asthma are associated with improvements in AHR, clinical studies of asthma therapies routinely use the change of airway responsiveness as an objective outcome. The aim of this study was to assess the relationship between serial MBPT and clinical profiles in patients with asthma. Methods A total of 323 asthma patients were included in this study. The MBPT was performed on all patients beginning at their initial diagnosis until asthma was considered controlled based on the Global Initiative for Asthma guidelines. A responder was defined by a decrease in AHR while all other patients were considered non-responders. Results A total of 213 patients (66%) were responders, while 110 patients (34%) were non-responders. The responder group had a lower initial PC20 (provocative concentration of methacholine required to decrease the forced expiratory volume in 1 second by 20%) and longer duration compared to the non-responder group. Members of the responder group also had superior qualities of life, compared to members of the non-responder group. Whole blood cell counts were not related to differences in PC20; however, eosinophil concentration was. No differences in sex, age, body mass index, smoking history, serum immunoglobulin E, or frequency of acute exacerbation were observed between responders and non-responders. Conclusions The initial PC20, the duration of asthma, eosinophil concentrations, and quality-of-life may be useful variables to identify improvements in AHR in asthma patients.

Background We previously reported that the ILVBL gene on chromosome 19p13.1 was associated with the risk for aspirin-exacerbated respiratory disease (AERD) and the percent decline of forced expired volume in one second (FEV1) after an oral aspirin challenge test. In this study, we confirmed the association between polymorphisms and haplotypes of the ILVBL gene and the risk for AERD and its phenotype. Methods We recruited 141 AERD and 995 aspirin-tolerant asthmatic (ATA) subjects. All study subjects underwent an oral aspirin challenge (OAC). Nine single nucleotide polymorphisms (SNPs) with minor allele frequencies above 0.05, which were present in the region from 2 kb upstream to 0.5 kb downstream of ILVBL in Asian populations, were selected and genotyped. Results In an allelic association analysis, seven of nine SNPs were significantly associated with the risk for AERD after correction for multiple comparisons. In a codominant model, the five SNPs making up block2 (rs2240299, rs7507755, rs1468198, rs2074261, and rs13301) showed significant associations with the risk for AERD (corrected P = 0.001–0.004, OR = 0.59–0.64). Rs1468198 was also significantly associated with the percent decline in FEV1 in OAC tests after correction for multiple comparisons in the codominant model (corrected P = 0.033), but the other four SNPs in hapblock2 were not. Conclusion To the best of our knowledge, this is the first report of an association between SNPs on ILVBL and AERD. SNPs on ILVBL could be promising genetic markers of this condition. Electronic supplementary material The online version of this article (10.1186/s12890-017-0556-6) contains supplementary material, which is available to authorized users.

Background Interleukin-32(IL-32)γ is a pro-inflammatory cytokine involved in the development and severity of chronic inflammatory diseases, but its role in asthma is unclear. Objective This study was conducted to evaluate the relationship of IL-32γ levels in sputum with the severity of asthma. Methods IL-32γ levels in the supernatant of induced sputum obtained from 89 patients with stable asthma were measured using a sandwich enzyme-linked immunosorbent assay (ELISA). The relationships between sputum IL-32γ levels and baseline forced expiratory volume in 1 s (FEV 1 % pred.), inflammatory cell profiles in sputum, and annual frequency of asthma exacerbation were determined. Results IL-32γ was detected in the sputum of 25 of 89 (28.1%) asthma patients, and the levels of sputum were negatively correlated with FEV 1 % pred. (ρ = −0.312, p = 0.003). The annual exacerbation rate was significantly higher in this group than in the IL-32-negative group (n = 64) (p = 0.03). Sputum IL-32γ levels correlated well with the annual exacerbation rate (ρ = 0.261, p = 0.014), but there were no differences in the inflammatory cell profiles in the induced sputum of IL-32-positive and IL-32-negative patients. Conclusion The level of IL-32γ in induced sputum may be associated with asthma severity and related with higher risk of asthma exacerbation.

Background Aspirin-exacerbated respiratory diseases (AERD) are caused by ingestion of non-steroidal anti-inflammatory drugs and are characterized by acute bronchospasms and marked infiltration of eosinophils, the latter being attributable to altered synthesis of cysteinyl leukotrienes (LT) and prostaglandins (PG). Recently, the innate Th2 response is revealed to induce eosinophil infiltration in allergic inflammation, however the role of the innate Th2 response has not been studies in AERD. Thus, we evaluated the relationship between the innate Th2 cytokines including IL-25, thymic stromal lymphopoietin (TSLP) and IL-33 and the development of AERD. Methods and materials Plasma IL-25, IL-33, and TSLP levels were measured before and after aspirin challenge in subjects with AERD (n = 25) and aspirin-tolerant asthma (ATA, n = 25) by enzyme-linked immunosorbent assay (ELISA). Pre and post-aspirin challenge levels of LTC4 and PGD2 were measured using ELISA. Results Basal plasma IL-25 levels were significantly higher in AERD group than in normal controls and in ATA group (p = 0.025 and 0.031, respectively). IL-33 and TSLP levels were comparable in the AERD and ATA groups. After the aspirin challenge, the IL-25 levels were markedly decreased in the ATA group (p = 0.024), while not changed in the AERD group. The post-challenge IL-25 levels of all asthmatic subjects were significantly correlated with aspirin challenge - induced declines in FEV1 (r = 0.357, p = 0.011), but not with basal and post challenge LTC4 and PGD2 levels. Conclusions IL-25 is associated with bronchospasm after aspirin challenge, possibly via mechanisms other than altered LTC4 and PGD2 production.

PURPOSE Different characteristics of airway microbiome in asthmatics may lead to differential immune responses, which in turn cause eosinophilic or neutrophilic airway inflammation. However, the relationships among these factors have yet to be fully elucidated. METHODS Microbes in induced sputum samples were subjected to sequence analysis of 16S rRNA. Airway inflammatory phenotypes were defined as neutrophils (>60%) and eosinophils (>3%), and inflammation endotypes were defined by levels of T helper (Th) 1 (interferon-γ), Th2 (interleukin [IL]-5 and IL-13), Th-17 (IL-17), and innate Th2 (IL-25, IL-33, and thymic stromal lymphopoietin) cytokines, inflammasomes (IL-1β), epithelial activation markers (granulocyte-macrophage colony-stimulating factor and IL-8), and Inflammation (IL-6 and tumor necrosis factor-α) cytokines in sputum supernatants was assessed by enzyme-linked immunosorbent assay. RESULTS The numbers of operational taxonomic units were significantly higher in the mixed (n = 21) and neutrophilic (n = 23) inflammation groups than in the paucigranulocytic inflammation group (n = 19; p < 0.05). At the species level, Granulicatella adiacens, Streptococcus parasanguinis, Streptococcus pneumoniae, Veillonella rogosae, Haemophilus parainfluenzae, and Neisseria perflava levels were significantly higher in the eosinophilic inflammation group (n = 20), whereas JYGU_s levels were significantly higher in the neutrophilic inflammation group compared to the other subtypes (p < 0.05). Additionally, IL-5 and IL-13 concentrations were correlated with the percentage of eosinophils (p < 0.05) and IL-13 levels were positively correlated with the read counts of Porphyromonas pasteri and V. rogosae (p < 0.05). IL-1β concentrations were correlated with the percentage of neutrophils (p < 0.05). had a tendency to be positively correlated with the read count of JYGU_s (p = 0.095), and was negatively correlated with that of S. pneumoniae (p < 0.05). CONCLUSIONS Difference of microbial patterns in airways may induce distinctive endotypes of asthma, which is responsible for the neutrophilic or eosinophilic inflammation in asthma.

Stratification of asthmatic patients based on relevant biomarkers enables the prediction of responsiveness against immune-targeted therapies in patients with asthma. Individualised therapy in patients with eosinophilic asthma has yielded improved clinical outcomes; similar approaches in patients with neutrophilic asthma have yet to be developed. We determined whether colony-stimulating factors (CSFs) in the airway reflect the inflammatory phenotypes of asthma and contribute to disease progression of neutrophilic asthma.We analysed three different mouse models of asthma and assessed cytokine profiles in sputum from human patients with asthma stratified according to inflammatory phenotype. In addition, we evaluated the therapeutic efficacy of various cytokine blockades in a mouse model of neutrophilic asthma.Among the CSFs, airway granulocyte CSF (G-CSF) contributes to airway neutrophilia by promoting neutrophil development in bone marrow and thereby distinguishes neutrophilic inflammation from eosinophilic inflammation in mouse models of asthma. G-CSF is produced by concurrent stimulation of the lung epithelium with interleukin (IL)-17A and tumour necrosis factor (TNF)-α; therefore, dual blockade of upstream stimuli using monoclonal antibodies or genetic deficiency of the cytokines in IL-17A×TNF-α double-knockout mice reduced the serum level of G-CSF, leading to alleviation of neutrophilic inflammation in the airway. In humans, the sputum level of G-CSF can be used to stratify patients with asthma with neutrophil-dominated inflammation.Our results indicated that myelopoiesis-promoting G-CSF and cytokines as the upstream inducing factors are potential diagnostic and therapeutic targets in patients with neutrophilic asthma.

Fibroblast dysfunction is the main pathogenic mechanism of idiopathic pulmonary fibrosis (IPF). S100 calcium-binding protein A4 (S100A4) plays critical roles in the proliferation of fibroblasts and in the development of pulmonary, hepatic, and renal fibrosis. However, the clinical implications of S100A4 in IPF have not been evaluated.The S100A4 mRNA and protein levels were measured by real-time PCR and immunoblotting in fibroblasts from IPF patients and controls. The S100A4 level was measured by enzyme-linked immunosorbent assay in bronchoalveolar lavage fluid (BALF) from the normal controls (NCs; n = 33) and from patients with IPF (n = 87), non-specific interstitial pneumonia (NSIP; n = 22), hypersensitivity pneumonitis (HP; n = 19), and sarcoidosis (n = 9). S100A4 localization was evaluated by immunofluorescence staining.The S100A4 mRNA and protein levels were significantly higher in fibroblasts from IPF patients (n = 14) than in those from controls (n = 10, p 0.001). The S100A4 protein level in BALF was significantly higher in the IPF (89.25 [49.92-203.02 pg/mL]), NSIP (74.53 [41.88-131.45 pg/mL]), HP (222.36 [104.92-436.92 pg/mL]) and sarcoidosis (101.62 [59.36-300.62 pg/mL]) patients than in the NCs (7.57 [1.31-14.04 pg/mL], p 0.01, respectively). Cutoff S100A4 levels of 18.85 and 28.88 pg/mL had 87.4% and 87.8% accuracy, respectively, for discriminating IPF and other lung diseases from NCs.S100A4 is expressed by α-SMA-positive cells in the interstitium of the IPF patients. S100A4 may participate in the development of IPF, and its protein level may be a candidate diagnostic and therapeutic marker for IPF.