Clustering analysis of HRCT parameters measured using a texture-based automated system: relationship with clinical outcomes of IPF.

PURPOSE: The extent of honeycombing and reticulation predict the clinical prognosis of IPF. Emphysema, consolidation, and ground glass opacity are visible in HRCT scans. To date, there have been few comprehensive studies that have used these parameters. We conducted automated quantitative analysis to identify predictive parameters for clinical outcomes and then grouped the subjects accordingly. METHODS: CT images were obtained while patients held their breath at full inspiration. Parameters were analyzed using an automated lung texture quantification system. Cluster analysis was conducted on 159 IPF patients and clinical profiles were compared between clusters in terms of survival. RESULTS: Kaplan-Meier analysis revealed that survival rates declined as fibrosis, reticulation, honeycombing, consolidation, and emphysema scores increased. Cox regression analysis revealed that reticulation had the most significant impact on survival rate, followed by honeycombing, consolidation, and emphysema scores. Hierarchical and K-means cluster analyses revealed 3 clusters. Cluster 1 (n = 126) with the lowest values for all parameters had the longest survival duration, and relatively-well preserved FVC and DLCO. Cluster 2 (n = 15) with high reticulation and consolidation scores had the lowest FVC and DLCO values with a predominance of female, while cluster 3 (n = 18) with high honeycombing and emphysema scores predominantly consisted of male smokers. Kaplan-Meier analysis revealed that cluster 2 had the lowest survival rate, followed by cluster 3 and cluster 1. CONCLUSION: Automated quantitative CT analysis provides valuable information for predicting clinical outcomes, and clustering based on these parameters may help identify the high-risk group for management.

S100 Calcium-Binding Protein A9, a Potential Novel Diagnostic Biomarker for Idiopathic Pulmonary Fibrosis.

BACKGROUND: Neutrophilic inflammation is a characteristic feature of idiopathic pulmonary fibrosis (IPF). S100 calcium-binding protein A9 (S100A9) is a neutrophil-derived protein involved in the development of neutrophil-related chronic inflammatory disorders. However, the role of S100A9 in IPF remains unclear. METHODS: We used enzyme-linked immunosorbent assays to measure S100A9 levels in bronchoalveolar lavage fluid (BALF) and serum obtained from healthy controls (HCs) and patients with IPF, non-specific interstitial pneumonia, hypersensitivity pneumonitis, and sarcoidosis. RESULTS: Compared with HCs, BALF S100A9 levels were significantly higher in IPF patients (P < 0.001), patients with hypersensitivity pneumonitis (P = 0.043), and patients with nonspecific interstitial pneumonia (P < 0.001). The S100A9 level in BALF of 0.093 ng/mL could distinguish IPF patients from HCs, with a specificity of 78.8% and a sensitivity of 81.6%. Similarly, the S100A9 level in BALF of 0.239 ng/mL had a specificity of 64.7% and a sensitivity of 66.7% for distinguishing IPF patients from patients with other interstitial lung diseases. Additionally, BALF S100A9 levels were significantly correlated with neutrophil counts (r = 0.356, P < 0.001) in BALF. IPF patients with S100A9 levels in BALF > 0.533 ng/mL had lower survival rates, compared with patients who had levels ≤ 0.553 ng/mL (n = 49; hazard ratio [HR], 3.62; P = 0.021). Combination analysis revealed that IPF patients with S100A9 levels in BALF> 0.553 ng/mL or neutrophil percentages > 49.1% (n = 43) had significantly lower survival rates than patients with S100A9 levels in BALF ≤ 0.553 ng/mL and neutrophil percentages ≤ 49.1% (n = 41) (HR, 3.91; P = 0.014). Additionally, patients with serum S100A9 levels > 0.077 ng/mL (n = 29) had significantly lower survival rates than patients with levels ≤ 0.077 ng/mL (n = 53, HR, 2.52; P = 0.013). S100A9 was expressed on neutrophils and macrophages in BALF from IPF patients as well as α-smooth muscle actin positive cells in the lung tissues. CONCLUSION: S100A9 is involved in the development and progression of IPF. Moreover, S100A9 levels in BALF and serum may be surrogate markers for IPF diagnosis and survival prediction, particularly when analyzed in combination with neutrophil percentages.

Intranasal administration of nucleus-deliverable GATA3-TMD alleviates the symptoms of allergic asthma.

Although the T helper 2 (Th2) subset is a critical player in the humoral immune response to extracellular parasites and suppression of Th1-mediated inflammation, Th2 cells have been implicated in allergic inflammatory diseases such as asthma, allergic rhinitis, and atopic dermatitis. GATA binding protein 3 (GATA3) is a primary transcription factor that mediates Th2 differentiation and secretion of Th2 cytokines, including IL-4, IL-5, and IL-13. Here, a nucleus-deliverable form of GATA3-transcription modulation domain (TMD) (ndG3-TMD) was generated using Hph-1 human protein transduction domain (PTD) to modulate the transcriptional function of endogenous GATA3 without genetic manipulation. ndG3-TMD was shown to be efficiently delivered into the cell nucleus quickly without affecting cell viability or intracellular signaling events for T cell activation. ndG3-TMD exhibited a specific inhibitory function for the endogenous GATA3-mediated transcription, such as Th2 cell differentiation and Th2-type cytokine production. Intranasal administration of ndG3-TMD significantly alleviated airway hyperresponsiveness, infiltration of immune cells, and serum IgE level in an OVA-induced mouse model of asthma. Also, Th2 cytokine secretion by the splenocytes isolated from the ndG3-TMD-treated mice substantially decreased. Our results suggest that ndG3-TMD can be a new therapeutic reagent to suppress Th2-mediated allergic diseases through intranasal delivery.

The Role of Erythrocyte Membrane Protein Band 4.1-like 3 in Idiopathic Pulmonary Fibrosis.

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-ß-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.

Association of ATP8B3 gene polymorphisms with aspirin-exacerbated respiratory disease in asthmatics.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD), an asthma phenotype, often presents with severe manifestations and it remains widely underdiagnosed because of insufficient awareness of the relationship between the ingestion of nonsteroidal anti-inflammatory drugs, including acetylsalicylic acid (ASA), and asthma exacerbation. Our previous genome-wide association study demonstrated an association between a single nucleotide polymorphism (SNP) of the ATP8B3 gene and the risk of AERD. This study examined AERD-related SNPs of the ATP8B3 gene in a large population. METHODS: Twenty-five SNPs of ATP8B3 were genotyped with the GoldenGate assay using VeraCode microbeads in 141 asthmatics with AERD and 995 Aspirin-tolerant asthma (ATA). The genotype distribution was analyzed using logistic regression models. The declines in forced expiratory volume in 1 second (FEV1)following an ASA challenge were compared among the genotypes and haplotypes using a type III generalized linear model. RESULTS: The minor allele frequencies (MAFs) of rs10421558 A>G in the 5'UTR and rs10403288 G>A in the intron were significantly lower in the AERD than the ATA [34.0% vs. 43.8%, OR = 0.66 (0.62-0.92), Pcorr = 0.03 and 28.4% vs. 35.4%, OR = 0.62 (0.59-0.89), Pcorr = 0.016, respectively]. BL1ht5 was significantly higher in the AERD [7.6% vs. 1.6%, OR = 12.23 (0.2-0.51), P = 4.7 × 10 -4 , Pcorr = 0.001]. Among them, rs10421558 A>G and BL1ht5 were associated with the percent decline in FEV1 on the oral ASA challenge test. CONCLUSION: The minor allele of rs10421558 A>G in the 5'UTR may protect against the development of AERD via the increased production of ATP8B3.

Genome-wide DNA methylation profile of peripheral blood lymphocytes from subjects with nonsteroidal anti-inflammatory drug-induced respiratory diseases.

BACKGROUND: Significant changes in CpG methylation have been identified in nasal polyps, which are the main targets of nonsteroidal anti-inflammatory drug-exacerbated respiratory disease (NERD); however, these polyps are composed of various cellular components. In the present study, whole-genome CpG methylation in peripheral blood lymphocytes (PBLs) was analyzed to define the epigenetic changes in lymphocytes, which are the primary immune cells involved in NERD. MATERIALS AND METHODS: Genomic DNA from peripheral blood mononuclear cells from 27 NERD and 24 aspirin-tolerant asthma (ATA) was subjected to bisulfate conversion and a methylation array. Quantitative CpG methylation, the ß-values as a quantitative measure of DNA methylation, in lymphocytes were calculated after adjustments for cellular composition. RESULTS: Fifty-six hypermethylated and three hypomethylated differentially methylated CpGs (DMCs) in PBLs in the NERD compared with ATA. The top 10 CpG loci predicted the methylation risk score, with a positive predictive value of 91.3%, a negative predictive value of 81.5% and an accuracy of 84.3%. As demonstrated in the nasal polyps, 30 DMCs were predicted to bind to the following 10 transcription factors, ranked in descending order: AP-2alphaA, TFII-1, STAT4, FOXP3, GR, c-Est-1, E2F-1, XBP1, ENKTF-1 and NF-1. Gene ontology analysis identified 13 categories such as regulation of T-helper 17 cell differentiation, including SMAD7 and NFKBIZ. PBLs in NERD contained no DMCs in genes associated with the prostaglandin and leukotriene pathways, which were found in ATA. CONCLUSION: PBLs in NERD form a unique pattern of DNA CpG methylation, and the combined analysis may provide predictive values for NERD.

Multicenter Prospective Observational Study to Evaluate the Therapeutic Effect and Safety of a Combination of Montelukast and Levocetirizine for Allergic Rhinitis when Administered to Patients with Allergic Rhinitis and Asthma.

INTRODUCTION: Allergic rhinitis and asthma share a common inflammatory mechanism and are closely related, recognized as "one airway disease." Thus, the guidelines recommend allergic rhinitis and asthma be treated together, and leukotriene antagonists and antihistamines have been administered simultaneously; however, there are few reports of the use of combination drugs so far. METHODS: The aim of the study was to evaluate the treatment effects and adverse events of Monterizine® (a combination of montelukast and levocetirizine); a total of 2,254 patients with perennial allergic rhinitis and asthma were prospectively enrolled from 60 hospitals nationwide in Korea. They were followed up for 3 (Period 1) or 6 months (Period 2). Total nasal symptom score (TNSS), satisfaction, and safety data were collected and compared to baseline. RESULTS: TNSS scores were analyzed for 2,254 subjects. At Period 1 (n = 2,024) and 2 (n = 1,861), the scores decreased significantly from baseline (-1.20 ± 2.49 and -1.63 ± 2.78, p < 0.001). The mean quality of life (QoL) was significantly improved at Period 1 and 2 relative to baseline (-3.75 ± 6.58, -4.83 ± 7.11, both p < 0.0001). There were no serious adverse drug reactions, but there were some minor reactions including nasopharyngitis (2.92%), rhinitis (0.37%), and somnolence (0.34%). CONCLUSIONS: TNSS score and QoL were significantly improved by 3-6 months' treatment with Monterizine without significant adverse reactions. These results indicate that Monterizine, as a combination drug, is effective and safe for improving nasal symptoms and quality of life in patients with allergic rhinitis who also have asthma.

Association of genetic variants of oxidative stress responsive kinase 1 (OXSR1) with asthma exacerbations in non-smoking asthmatics.

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.

Characteristics of Specialist-Diagnosed Asthma-COPD Overlap in Severe Asthma: Observations from the Korean Severe Asthma Registry (KoSAR).

BACKGROUND: While the clinical characteristics and outcomes of asthma-chronic obstructive pulmonary disease (COPD) overlap (ACO) have been frequently compared with those of COPD or asthma, the prevalence and features of ACO in patients with severe asthma are unclear. OBJECTIVES: Evaluation of the prevalence and clinical features of ACO using the Korean severe asthma registry. METHODS: At the time of registration, ACO was determined in patients with severe asthma by attending specialists. Patients were classified into ACO and non-ACO groups, and the demographic and clinical characteristics of these two groups were compared. RESULTS: Of 482 patients with severe asthma, 23.7% had ACO. Patients in the ACO group were more likely to be male (P < .001), older (P < .001), and ex- or current smokers (P < .001) compared with those in the non-ACO group. Patients in the ACO group had lower mean forced expiratory volume in 1 second (P < .001) and blood eosinophil percentage (P = .006), but higher blood neutrophil percentage (P = .027) than those in the non-ACO group. The ACO group used more inhaled long-acting muscarinic antagonist (P < .001), methylxanthine (P = .001), or sustained systemic corticosteroid (P = .002). In addition, unscheduled emergency department visits due to exacerbation were more frequent in the ACO group (P = .006). CONCLUSION: Among patients with severe asthma, those with ACO were older, predominantly male, and were more likely to have a smoking history than those with asthma only. Patients with ACO used more systemic corticosteroid and had more frequent exacerbations related to emergency department visits than those with severe asthma only.

Erratum to "Effects of Ammonium Chloride on Ozone-induced Airway Inflammation: the Role of Slc26a4 in the Lungs of Mice".

This corrects the article on p. e272 in vol. 35, PMID: 32808511.

Airway G-CSF identifies neutrophilic inflammation and contributes to asthma progression.

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.

Effects of air pollution on moderate and severe asthma exacerbations.

Background: Few studies have evaluated the impact of air pollution levels on the severity of exacerbations. Thus, we compared the relative risks posed by air pollutant levels on moderate and severe exacerbations.Methods: Exacerbation episodes of 618 from 143 adult asthmatics were retrospectively collected between 2005 and 2015 in a tertiary hospital of Korea. Air pollution GPS data for the location closest to each patient's home were obtained from the national ambient monitoring station. The relative impacts of air pollutants on asthma exacerbations were evaluated via a time-trend controlled symmetrical, bidirectional, case-crossover design using conditional logistic regression models on the day of the exacerbation (T-0) and up to 3 days before the exacerbation (T-1-T-3).Results: Overall asthma exacerbation were associated with O3 levels in summer and winter (OR: 1.012[1.003-1.02] and 1.009[1.003-1.016]), SO2 levels in spring and summer (OR: 1.009[1-1.018] and 1.02[1.006-1.035]) and NO2 levels in winter (OR: 1.007[1.003-1.011]). Analyses of the temporal relationship between O3 concentrations and exacerbations demonstrated that 63.2% of episodes in the summer occurred when the O3 concentrations on T-1 were significantly higher than those on control days, while 51% of exacerbation episodes in the winter occurred. Severe and moderate exacerbations were similarly associated with O3 levels in winter (OR: 1.012 [1.003-1.02] vs. 1.01 [0.999-1.021], p > 0.05) and in summer (OR: 1.006 [1.002-1.009] vs. 1.009 [1.003-1.016], p > 0.05).Conclusions: Asthma exacerbations may be associated with the seasonal elevation of O3, SO2 and NO2 levels in summer and winter with the similar relative risk between moderate and severe exacerbations.

Effects of Ammonium Chloride on Ozone-induced Airway Inflammation: the Role of Slc26a4 in the Lungs of Mice.

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-1ß, 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-1ß, 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.

Associations between TMEM196 polymorphisms and NSAID-exacerbated respiratory disease in asthma.

BACKGROUND: We previously found differences in the minor allele frequency (MAF) of single-nucleotide polymorphisms (SNPs) in transmembrane protein 196 (TMEM196) between 995 patients with aspirin-tolerant asthma (ATA) and 141 asthmatic patients with NSAID-exacerbated respiratory disease (NERD). In this study, we statistically analyzed the distributions of the genotypes and haplotypes of these SNPs to determine the exact association between TMEM196 genetic variants and the risk for NERD. MATERIALS AND METHODS: Lewontin's D' and r values were used to measure linkage disequilibrium between the biallelic loci having MAFs more than 0.05, and haplotypes were inferred using the PHASE algorithm (version 2.0). The genotype distribution was analyzed by logistic regression models using age of onset, smoking status (nonsmoker=0, ex-smoker=1, smoker=2), and BMI as covariates. Regression analysis of the association between SNPs and the risk of NERD was analyzed using SPSS version 12.0 and PLINK version 1.9. RESULTS: The MAF of rs9886152 C>T was significantly lower in NERD than in ATA [24.8 vs. 34.0%, odds ratio=0.64 (0.48-0.85), P=2.07×10, Pcorr=0.048]. The rate of the rs9886152 C>T minor allele was significantly lower in NERD than in ATA [44.0 vs. 56.4% in the codominant model, P=0.002, Pcorr=0.049, odds ratio=0.64 (0.48-0.85)]. An additional three SNPs (rs9639334 A>G, rs9638765 A>G, and rs2097811 G>A) showed similar associations with the risk of NERD. NERD patients had lower frequencies of the rs9639334 A>G minor allele (51.1 vs. 64.4%, P=0.002, Pcorr=0.043), rs9638765 A>G (49.7 vs. 64.2%, P=0.001, Pcorr=0.017), and rs2097811 G>A (51.1 vs. 64.5%, P=0.002, Pcorr=0.04) compared with ATA patients. Patients homozygous for the minor alleles of the four SNPs showed significantly less of an aspirin-induced decrease in forced expiratory volume in one second compared with those homozygous for the common alleles (P=0.003-0.012). CONCLUSION: The minor alleles of the four SNPs in TMEM196 may exert a protective effect against the development of NERD and may be useful genetic markers to predict the risk of NERD.

Involvement of Alveolar Epithelial Cell Necroptosis in Idiopathic Pulmonary Fibrosis Pathogenesis.

Alveolar epithelial cell (AEC) injury leading to cell death is involved in the process of fibrosis development during idiopathic pulmonary fibrosis (IPF). Among regulated/programmed cell death, the excessive apoptosis of AECs has been widely implicated in IPF pathogenesis. Necroptosis is a type of regulated/programmed necrosis. A multiprotein complex composed of receptor-interacting protein kinase (RIPK)-1 and -3 plays a key regulatory role in initiating necroptosis. Although necroptosis participates in disease pathogeneses through the release of damage-associated molecular patterns, its association with IPF progression remains elusive. In this study, we attempted to illuminate the involvement of RIPK3-regulated necroptosis in IPF pathogenesis. IPF lung tissues were used to detect necroptosis, and the role of RIPK3 was determined using cell culturing models of AECs. Lung fibrosis models of bleomycin (BLM) treatment were also used. RIPK3 expression levels were increased in IPF lungs, and both apoptosis and necroptosis were detected mainly in AECs. Necrostatin-1 and RIPK3 knockout experiments in AECs revealed the participation of necroptosis in BLM and hydrogen peroxide-induced cell death. BLM treatment induced RIPK3 expression in AECs and increased high-mobility group box 1 and IL-1ß levels in mouse lungs. The efficient attenuation of BLM-induced lung inflammation and fibrosis was determined in RIPK3 knockout mice and by necrostatin-1 with a concomitant reduction in high-mobility group box 1 and IL-1ß. RIPK3-regulated necroptosis in AECs is involved in the mechanism of lung fibrosis development through the release of damage-associated molecular patterns as part of the pathogenic sequence of IPF.

Annexin A1 in plasma from patients with bronchial asthma: its association with lung function.

BACKGROUND: Annexin-A1 (ANXA1) is a glucocorticoid-induced protein with multiple actions in the regulation of inflammatory cell activation. The anti-inflammatory protein ANXA1 and its N-formyl peptide receptor 2 (FPR2) have protective effects on organ fibrosis. However, the exact role of ANXA1 in asthma remains to be determined. The aim of this study was to identify the role of ANXA1 in bronchial asthma. METHODS: In mice sensitized and challenged with ovalbumin (OVA-OVA mice) and mice sensitized with saline and challenged with air (control mice), we investigated the potential links between ANXA1 levels and bronchial asthma using ELISA, immunoblotting, and immunohistochemical staining. Moreover, we also determined ANXA1 levels in blood from 50 asthmatic patients (stable and exacerbated states). RESULTS: ANXA1 protein levels in lung tissue and bronchoalveolar lavage fluid were significantly higher in OVA-OVA mice compared with control mice. FPR2 protein levels in lung tissue were significantly higher in OVA-OVA mice compared with control mice. Plasma ANXA1 levels were increased in asthmatic patients compared with healthy controls. Plasma ANXA1 levels were significantly lower in exacerbated patients compared with stable patients with bronchial asthma (p < 0.05). The plasma ANXA1 levels in controlled asthmatic patients were correlated with forced expiratory volume in 1 s (FEV1) (r = - 0.191, p = 0.033) and FEV1/forced vital capacity (FVC) (r = -0.202, p = 0.024). CONCLUSION: These results suggest that ANXA1 may be a potential marker and therapeutic target for asthma.

Impact of ozone on claudins and tight junctions in the lungs.

Claudins (CLDNs) are a major transmembrane protein component of tight junctions (TJs) in endothelia and epithelia. CLDNs are not only essential for sustaining the role of TJs in cell permeability but are also vital for cell signaling through protein-protein interactions. Ozone induces oxidative stress and lung inflammation in humans and experimental models, but the impact of ozone on claudins remains poorly understood. This study was to determine the expression of TJ proteins, such as claudin 3, 4, 5, and 14 following ozone exposure. Mice were exposed to 0.1, 1, or 2 ppm of ozone or ambient air for 6 h for 3 days. The impact of ozone on CLDNs, Nrf2, Keap1, and reactive oxygen species (ROS) were estimated using immunoblotting, immunohistochemical staining, confocal imaging, and ELISA analysis in mice and bronchial epithelial cells. Mice exposed to ozone experienced increased airway inflammatory cell infiltration and bronchial hyper-responsiveness compared to control mice. Additionally, CLDN3, CLDN4, ROS, Nrf2, and Keap1 protein expression increased, and lung CLDN14 protein expression decreased, in mice exposed to ozone compared with control mice. These results indicate that CLDNs are involved in airway inflammation following ozone exposure, suggesting that ozone affects TJ proteins through oxidative mechanisms.

Role of S100A9 in the development of neutrophilic inflammation in asthmatics and in a murine model.

S100A9 is an endogenous danger signal that promotes and exacerbates the neutrophilic inflammatory response. To investigate the role of S100A9 in neutrophilic asthma, S100A9 levels were measured in sputum from 101 steroid-naïve asthmatics using an ELISA kit and the levels were significantly correlated with percentages of neutrophils in sputum. Intranasal administration of recombinant S100A9 markedly increased neutrophil numbers at 8h and 24h later with concomitant elevation of IL-1ß, IL-17, and IFN-γ levels. Treatment with an anti-S100A9 antibody restored the increased numbers of neutrophils and the increased airway resistance in OVA/CFA mice toward the levels of sham-treated mice. Concomitantly, the S100A9 and neutrophil elastase double positive cells were markedly reduced with attenuation of IL-1ß, IL-17, and IFN-γ levels by the treatment with the anti-S100A9 antibody. Our data support a role of S100A9 to initiate and amplify the neutrophilic inflammation in asthma, possibly via inducing IL-1ß, IL-17 and IFN-γ.

Gene profile of fibroblasts identify relation of CCL8 with idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is characterized by the complex interaction of cells involved in chronic inflammation and fibrosis. Global gene expression of a homogenous cell population will identify novel candidate genes. METHODS: Gene expression of fibroblasts derived from lung tissues (8 IPF and 4 controls) was profiled, and ontology and functional pathway were analyzed in the genes exhibiting >2 absolute fold changes with p-values < 0.05. CCL8 mRNA and protein levels were quantified using real-time PCR and ELISA. CCL8 localization was evaluated by immunofluorescence staining. RESULTS: One hundred seventy eight genes differentially expressed and 15 genes exhibited >10-fold change. Among them, 13 were novel in relation with IPF. CCL8 expression was 22.8-fold higher in IPF fibroblasts. The levels of CCL8 mRNA and protein were 3 and 9-fold higher in 14 IPF fibroblasts than those in 10 control fibroblasts by real-time PCR and ELISA (p = 0.022 and p = 0.026, respectively). The CCL8 concentrations in BAL fluid was significantly higher in 86 patients with IPF than those in 41 controls, and other interstitial lung diseases including non-specific interstitial pneumonia (n = 22), hypersensitivity pneumonitis (n = 20) and sarcoidosis (n = 19) (p < 0.005, respectively). Cut-off values of 2.29 pg/mL and 0.43 pg/mL possessed 80.2 and 70.7% accuracy for the discrimination of IPF from NC and the other lung diseases, respectively. IPF subjects with CCL8 levels >28.61 pg/mL showed shorter survival compared to those with lower levels (p = 0.012). CCL8 was expressed by α-SMA-positive cells in the interstitium of IPF. CONCLUSIONS: Transcriptome analysis identified several novel IPF-related genes. Among them, CCL8 is a candidate molecule for the differential diagnosis and prediction of survival.

Factors affecting treatment outcome in patients with idiopathic nonspecific interstitial pneumonia: a nationwide cohort study.

BACKGROUND: The effects of corticosteroid-based therapy in patients with idiopathic nonspecific interstitial pneumonia (iNSIP), and factors affecting treatment outcome, are not fully understood. We aimed to investigate the long-term treatment response and factors affecting the treatment outcome in iNSIP patients from a multi-center study in Korea. METHODS: The Korean interstitial lung disease (ILD) Study Group surveyed ILD patients from 2003 to 2007. Patients were divided into two groups to compare the treatment response: response group (forced vital capacity (FVC) improves ≥10% after 1 year) and non-response group (FVC <10%). Factors affecting treatment response were evaluated by multivariate logistic regression analysis. RESULTS: A total of 261 patients with iNSIP were enrolled, and 95 patients were followed-up for more than 1 year. Corticosteroid treatment was performed in 86 patients. The treatment group showed a significant improvement in lung function after 1-year: FVC, 10.0%; forced expiratory volume (FEV1), 9.8%; diffusing capacity of the lung for carbon monoxide (DLco), 8.4% (p < 0.001). Sero-negative anti-nuclear antibody (ANA) was significantly related with lung function improvement. Sero-positivity ANA was significantly lower in the response group (p = 0.013), compared to that in the non-response group. A shorter duration of respiratory symptoms at diagnosis was significantly associated with a good response to treatment (p = 0.018). CONCLUSION: Treatment with corticosteroids and/or immunosuppressants improved lung function in iNSIP patients, which was more pronounced in sero-negative ANA and shorter symptom duration patients. These findings suggest that early treatment should be considered in iNSIP patients, even in an early disease stage.