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The inhalation challenge test is considered to be the item for diagnosis of hypersensitivity pneumonitis (HP) and identifying the causative antigen in patients with fibrotic HP. However, the inhalation challenge test is not widely used.To evaluate the values of the inhalation challenge test by comparing with serum immunoglobulin (Ig)G test.This was a single-center, retrospective study. The patients with fibrotic HP were diagnosed pathologically by surgical lung biopsy or transbronchial lung cryobiopsy and were assumed to have bird-related fibrotic HP if they had a history of obvious avian exposure.On the basis of pathologic findings and history of avian exposure, 43 of 86 patients were diagnosed with having bird-related fibrotic HP. In 43 patients with bird-related fibrotic HP, 15 (35%) were positive for anti-bird IgG antibody and 36 (84%) were positive for the inhalation challenge test; in addition, the specificity of the inhalation challenge test was 67%. Patients with both positive results from inhalation challenge test and anti-bird IgG antibodies had a 2.7% decline in annual forced vital capacity (FVC) before the inhalation (P = .02). In patients with positive result from inhalation challenge test and negative result from anti-bird IgG antibodies, the annual FVC decreased by 5.8% (P = .03). FVC was not consistent in patients with positive result from the anti-bird IgG antibodies.The inhalation challenge test for bird-related fibrotic HP was more sensitive than the anti-bird IgG antibodies. Furthermore, the inhalation challenge test could select patients with similar disease progression.

Chronic obstructive pulmonary disease (COPD) is primarily caused by inhalation of cigarette smoke and is the third leading cause of death worldwide. Pulmonary surfactant, a complex of phospholipids and proteins, plays an essential role in respiration by reducing the surface tension in the alveoli. Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is an enzyme that catalyzes the biosynthesis of surfactant lipids and is expressed in type 2 alveolar epithelial cells. Its dysfunction is suggested to be involved in various lung diseases; however, the relationship between LPCAT1 and COPD remains unclear. To investigate the role of LPCAT1 in the pathology of COPD, we analyzed an elastase-induced emphysema model using Lpcat1 knockout (KO) mice. In Lpcat1 KO mice, elastase-induced emphysema was significantly exacerbated with increased apoptotic cells, which was not ameliorated by supplementation with dipalmitoylphosphatidylcholine, which is a major component of the surfactant synthesized by LPCAT1. We subsequently evaluated the effects of cigarette smoking on primary human type 2 alveolar epithelial cells (hAEC2s) and found that cigarette smoke extract (CSE) downregulated the expression of Lpcat1. Furthermore, RNA sequencing analysis revealed that the apoptosis pathway was significantly enriched in CSE-treated primary hAEC2s. Finally, we downregulated the expression of Lpcat1 using small interfering RNA, which resulted in enhanced CSE-induced apoptosis in A549 cells. Taken together, cigarette smoke-induced downregulation of LPCAT1 can promote the exacerbation of pulmonary emphysema by increasing the susceptibility of alveolar epithelial cells to apoptosis, thereby suggesting that Lpcat1 is a novel therapeutic target for irreversible emphysema.

Background Chronic hypersensitivity pneumonitis (CHP) remains a diagnostic challenge. The process of collecting and extracting serum and droppings from causative animals for the inhalation challenge test is complicated and the risk of inducing disease progression exists. Objective To investigate the utility and safety of an inhalation challenge test using pigeon eggs. Methods Pigeon eggs were pasteurized and mixed with a saline solution to produce an inhalation fluid. An inhalation challenge test was conducted on 19 patients with bird-related CHP and 17 patients with interstitial lung disease other than bird-related CHP. To identify antigens in pigeon eggs, the antigen-antibody responses of the pigeon eggs and serum from patients were evaluated using Western blotting. Results The mean changes in C-reactive protein, alveolar-arterial oxygen difference, erythrocyte sedimentation rate, and lactate dehydrogenase significantly increased by 0.32 mg/dL (P = .014), 7.8 Torr (P = .002), 1.4 mm/h (P = .012), and 5.4 U/mL (P = .0019), respectively, in bird-related CHP group compared to the control 24 hours after the inhalation challenge test. Furthermore, within 24 hours of the inhalation test, the mean forced vital capacity decreased by 2.3% in the bird-related CHP group compared with a decline of 0.05% in the control group (P = .035). Serum collected from seven bird-related CHP patients who underwent the inhalation challenge test and reacted to antigens with molecular weights of 37-75 KDa, and these molecular weights were consistent with egg albumin and globulin. Conclusion Since a mild response was observed after the inhalation challenge test using pigeon eggs, this test was an obvious candidate for diagnosing bird-related CHP.

Despite signs of infection, the involvement of the oral cavity in COVID-19 is poorly understood. To address this, single-cell RNA sequencing data-sets were integrated from human minor salivary glands and gingiva to identify 11 epithelial, 7 mesenchymal, and 15 immune cell clusters. Analysis of SARS-CoV-2 viral entry factor expression showed enrichment in epithelia including the ducts and acini of the salivary glands and the suprabasal cells of the mucosae. COVID-19 autopsy tissues confirmed in vivo SARS-CoV-2 infection in the salivary glands and mucosa. Saliva from SARS-CoV-2-infected individuals harbored epithelial cells exhibitingACE2expression and SARS-CoV-2 RNA. Matched nasopharyngeal and saliva samples found distinct viral shedding dynamics and viral burden in saliva correlated with COVID-19 symptoms including taste loss. Upon recovery, this cohort exhibited salivary antibodies against SARS-CoV-2 proteins. Collectively, the oral cavity represents a robust site for COVID-19 infection and implicates saliva in viral transmission.

TitleA mouse model of asthma­–chronic obstructive pulmonary disease overlap induced by intratracheal papain

Background: Chronic Obstructive Pulmonary Disease (COPD) is a systemic inflammatory disease, and often causes weight loss, which is considered as a poor prognostic factor. A Japanese herbal medicine, Hochu-ekki-to (TJ-41), was reported to prevent weight loss in a small study of COPD patients, while its biological mechanism is unknown. Objective: The objective of our study was to evaluate the role of TJ-41 using mouse lung emphysema model, and to investigate its biological mechanism in vivo and in vitro. Methods: We used lung epithelial specific Taz conditional knockout mice (Taz CKO mice) as emphysema model mice, and fed a diet containing 2% TJ-41 or a control diet for 12 weeks. During administration, body weights were measured. After seeding, Taz CKO mice and control mice were subjected to BALF analysis, and lung mRNA expressions of inflammatory cytokines were analysed by RT-PCR. Results: After treatment, Taz CKO mice showed higher body weights in TJ-41 group compared to control diet group. BALF analysis showed decreased number of inflammatory cells in TJ-41 group, and among inflammatory cytokines, IL-8 expression was also significantly decreased. Conclusion: Our results suggest the possibility that TJ-41 attenuates lung inflammation, and thereby prevents weight loss of COPD patients. Among inflammatory cytokines, IL-8 is downregulated by administration of TJ-41, which coincided with decreased number of inflammatory cells in BALF in TJ-41 group. Our study sheds light on the biological mechanism of TJ-41 in lung emphysema, and suggests its potential for anti-inflammatory therapy for COPD patients.

Backgrounds: Ｃhronic obstructive pulmonary disease (COPD) is a pulmonary disease with permanent airflow obstruction and emphysema, and acute exacerbation of COPD is often induced by infection often resulting in a poor prognosis. Reactive oxygen species (ROS), producted in mitochondria, has been reported to accelerate the establishment of COPD. Sirtuin3(SIRT3) is the mitochondrial NAD(+)-dependent deacetylase, which regulates ROS formation and proinflammatory responses. It is also reported to be related with lifespan longevity. Aims: The aim of our study was to investigate the role of SIRT3 in COPD exacerbation mouse model generated by porcine pancreatic elastase(PPE) and lipopolysaccharide(LPS). Methods: PPE followed by LPS was administered intratracheally to wild-type (WT) or SIRT3 overexpressing transgenic(SIRT3 OE), SIRT3 knockout (SIRT3 KO) male mice. The next day (early phase) and 4weeks later (late phase) after LPS administration, BALF analysis and lung function test was conducted. Results: In early phase, the BALF cell count of SIRT3 OE mice was significantly increased compared to control WT mice, whereas that of SIRT3 KO mice was significantly decreased. In late phase, the lung compliance was increased in SIRT3 OE mice compared to WT mice, and decreased in SIRT3 KO mice. Conclusions: Our study showed that SIRT3 surpressed acute lung injury and emphysematous worsening in COPD model mice. SIRT3 would serve as a new therapeutic target to COPD and COPD exacerbation.

Background and Aim: Recently, it has been recognized that asthma and COPD can coexist as asthma-COPD overlap (ACO). Due to the difference of clinical course, it is thought the pathogenesis of ACO is different from that of asthma or COPD; however, its animal models have not yet been established. In animal experiments, papain (a cysteine protease enzyme present in papaya) is known to cause asthmatic response by daily inhalation, and lung emphysema by intermittent inhalation. Here we developed ACO mice model by weekly intratracheal administrations of papain, and we examined whether the mice model well reflects the features of ACO, comparing its features with elastase induced COPD model. Methods: PBS, papain, or porcine pancreatic elastase (PPE) was intratracheally administered to female C57BL/6N mice (5-6 weeks old) on days 0, 7, 14, 21, while PPE on day 0 only. The mice were sacrificed on day 25. Lung mechanics, airway responsiveness to methacholine, bronchoalveolar lavage fluid (BALF), lung tissue, and mRNA in lung homogenates were analyzed. Results: In lung mechanics tests, both papain and PPE group showed significantly higher compliance and inspiratory capacity than PBS group, while the airway responsiveness to methacholine was significantly higher in papain group than in other groups. Papain increased eosinophils in BALF. Both papain and PPE induced alveolar air-space enlargement. In addition, type 2 inflammatory cytokines such as IL-13 and IL-25 were significantly higher in papain group than in other groups. Conclusions: Weekly papain administrations induced ACO like features in mice. Clarification of the underlying pathogenesis and development of novel treatment might be expected using this single-agent simple model.

Coronavirus infection causes diffuse alveolar damage leading to acute respiratory distress syndrome. The absence of ex vivo models of human alveolar epithelium is hindering an understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here, we report a feeder-free, scalable, chemically defined, and modular alveolosphere culture system for the propagation and differentiation of human alveolar type 2 cells/pneumocytes derived from primary lung tissue. Cultured pneumocytes express the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor angiotensin-converting enzyme receptor type-2 (ACE2) and can be infected with virus. Transcriptome and histological analysis of infected alveolospheres mirror features of COVID-19 lungs, including emergence of interferon (IFN)-mediated inflammatory responses, loss of surfactant proteins, and apoptosis. Treatment of alveolospheres with IFNs recapitulates features of virus infection, including cell death. In contrast, alveolospheres pretreated with low-dose IFNs show a reduction in viral replication, suggesting the prophylactic effectiveness of IFNs against SARS-CoV-2. Human stem cell-based alveolospheres, thus, provide novel insights into COVID-19 pathogenesis and can serve as a model for understanding human respiratory diseases., Graphical Abstract, Highlights • Stroma-free long-term expansion and differentiation of adult human lung stem cells • AT2 response to SARS-CoV-2 infection mirrors features of COVID-19 lungs • Infected AT2s upregulate IFNs and apoptotic pathways and decrease surfactants • Low-dose IFN pre-treatment blocks SARS-CoV-2 replication in alveolospheres, Tata and colleagues report defined conditions for long-term expansion and differentiation of adult human primary alveolar stem cells. Cultured AT2s are conducive to SARS-CoV-2 infection and elicit transcriptome-wide changes that mirror COVID-19 histopathology, including upregulation of inflammatory responses, cell death, and downregulation of surfactant expression, leading to pneumocyte dysfunction.

Background: Asthma is a chronic airway inflammatory disease characterized by airway remodeling, in which the bronchial smooth muscle (BSM) cells play an important role. Periostin, a biomarker that reflects Th2-driven inflammatory diseases such as asthma, may play an important role in the asthmatic airway. Although periostin is mainly produced in airway epithelial cells and fibroblasts after interleukin (IL)-13 stimulation, whether BSM cells produce periostin remains unclear. Therefore, we investigated periostin production in BSM cells and the mechanisms involved. Methods: Human BSM cells were cultured, and the effect of IL-13 stimulation on periostin production was evaluated using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). We evaluated the phosphorylation of signal transducer and activator of transcription factor 6 (STAT6), extracellular signal-regulated kinase (ERK)1/2, and Akt after IL-13 stimulation. Furthermore, using ELISA, we evaluated the influence of several phosphorylation inhibitors on periostin production. Results: Periostin mRNA expression increased in a dose- and time-dependent manner after IL-13 stimulation; periostin production was induced 24 and 48 h after stimulation. IL-13 stimulation induced the phosphorylation of STAT6, ERK1/2, and Akt. IL-13-induced periostin production was attenuated by inhibiting STAT6 phosphorylation and strongly suppressed by inhibiting mitogen-activated protein kinase kinase 1/2 phosphorylation or phosphatidylinositol 3-kinase (PI3K) phosphorylation. Conclusions: BSM cells produced periostin after IL-13 stimulation, via the JAK/STAT6, ERK1/2, and PI3K/Akt pathways. Understanding the mechanism of periostin production in BSM cells may help to clarify asthma pathogenesis.

Background Transfusion-related acute lung injury (TRALI) is a life-threatening complication of blood transfusion. Antibodies against human leukocyte antigens in donors' plasma are the major causes of TRALI. Several animal models of TRALI have been developed, and the mechanism underlying TRALI development has been extensively investigated using rodent models. Although sheep models of nonimmune TRALI have been developed, large-animal models of antibody-mediated TRALI are not yet available. Study Design and Methods To develop a swine model of TRALI, male Clawn strain miniature pigs were used. A monoclonal antibody (MoAb) against swine leukocyte antigens (SLAs) Class I (4G8, 0.3 or 1.0 mg/kg body weight [BW]) and a control antibody (1.0 mg/kg BW) were injected into the peripheral vein after priming with or without 1 μg/kg BW lipopolysaccharide (LPS; n = 3 each). Lung injury was assessed using PaO2/FiO2 (P/F) ratio and by chest X-ray imaging. Histopathologic analysis was also conducted. Results Lung injury could be induced by injecting 4G8 at an amount of 1.0 mg/kg BW, after LPS. The P/F ratio 90 minutes after the administration of 4G8 significantly decreased (p

Lung cancer is the most common cause of cancer-related death worldwide. Stromal cancer-associated fibroblasts (CAFs) play crucial roles in carcinogenesis, proliferation, invasion, and metastasis of non-small cell lung carcinoma, and targeting of CAFs could be a novel strategy for cancer treatment. However, the characteristics of human CAFs still remain to be better defined. In this study, we established patient-matched CAFs and normal fibroblasts (NFs), from tumoral and non-tumoral portions of resected lung tissue from lung cancer patients. CAFs showed higher α-smooth muscle actin (α-SMA) expression than NFs, and CAFs clearly enhanced collagen gel contraction. Furthermore, we employed three-dimensional co-culture assay with A549 lung cancer cells, where CAFs were more potent in inducing collagen gel contraction. Hematoxylin and eosin staining of co-cultured collagen gel revealed that CAFs had the potential to increase invasion of A549 cells compared to NFs. These observations provide evidence that lung CAFs have the tumor-promoting capacity distinct from NFs.

Background and Purpose Bronchial asthma (BA) is a chronic airway disease characterized by airway hyperresponsiveness and remodeling, which are intimately linked to chronic airway inflammation. Reactive oxygen species (ROS) such as hydrogen peroxide are generated by inflammatory cells that are involved in the pathogenesis of BA. However, the role of ROS in the management of BA patients is not yet clear. We attempted to determine the role of ROS as a biomarker in the clinical setting of BA. Subjects and Methods We enrolled patients with BA from 2013 through 2015 and studied the degrees of asthma control, anti-asthma treatment, pulmonary function test results, fractional exhaled nitric oxide (FeNO), serum reactive oxygen metabolite (ROM) levels, and serum levels of interleukin (IL)-6 and IL-8. Results We recruited 110 patients with BA. Serum ROM levels correlated with white blood cell (WBC) count (rs = 0.273, p = 0.004), neutrophil count (rs = 0.235, p = 0.014), CRP (rs = 0.403, p < 0.001), and IL-6 (rs = 0.339, p < 0.001). Serum ROM levels and IL-8 and CRP levels negatively correlated with %FEV1 (rs = -0.240, p = 0.012, rs = -0.362, p < 0.001, rs = -0.197, p = 0.039, respectively). Serum ROM levels were significantly higher in patients who experienced severe exacerbation within 3 months than in patients who did not (339 [302–381] vs. 376 [352–414] CARR U, p < 0.025). Receiver-operating characteristics analysis showed that ROM levels correlated significantly with the occurrence of severe exacerbation (area under the curve: 0.699, 95% CI: 0.597–0.801, p = 0.025). Conclusions Serum levels of ROM were significantly associated with the degrees of airway obstruction, WBC counts, neutrophil counts, IL-6, and severe exacerbations. This biomarker may be useful in predicting severe exacerbations of BA.

Fibrosis is an abnormal response to organ injury, characterized by accumulation of activated fibroblasts at the sites of injury. Fibroblasts arise from several sources, including resident fibroblasts and circulating fibrocytes that infiltrate organ tissue. Recently, epithelial-mesenchymal transition (EMT) has been recognized as a source of mesenchymal cells. EMT is induced by various growth factors, such as transforming growth factor (TGF)-β1, and enhanced by inflammatory cytokines. Recently the tumor necrosis factor superfamily member LIGHT has been implicated in the pathogenesis of inflammatory disease and airway remodeling in severe asthma. We hypothesized that LIGHT might contribute to the pathogenesis of airway fibrosis via enhancement of EMT. Therefore, we investigated LIGHT's ability to induce EMT. A549 cells were stimulated with LIGHT, TGF-β1 or both for 48h. To estimate EMT, we evaluated the expression of epithelial and mesenchymal markers using immunocytochemistry, Western blotting and quantitative RT-PCR. Signaling pathways for EMT were characterized by Western analysis to detect phosphorylation of Erk1/2 and smad2. LIGHT enhanced TGF-β1-induced EMT both morphologically, by suppressing E-cadherin and enhancing vimentin, and functionally, by enhancing cell contractility. Additionally, LIGHT induced EMT without TGF-β1. Evaluation of the mechanism showed that LIGHT did not induce TGF-β1 production or affect the smad-snai1 pathway. Inhibition of Erk1/2 phosphorylation reduced LIGHT-induced EMT, indicating the Erk1/2 pathway to be a key pathway in LIGHT-induced EMT. In summary, LIGHT enhanced TGF-β1-induced EMT but also induced EMT via the Erk1/2 pathway by itself, without TGF-β1 signaling. LIGHT may contribute to the pathogenesis of airway fibrosis through enhancement of EMT.

Chronic inflammatory airway diseases, such as bronchial asthma and chronic obstructive pulmonary disease, are common respiratory disorders worldwide. Exacerbations of these diseases are frequent and worsen patients' respiratory condition and overall health. However, the mechanisms of exacerbation have not been fully elucidated. Recently, it was reported that interleukin (IL)-17A might play an important role in neutrophilic inflammation, which is characteristic of such exacerbations, through increased production of neutrophil chemoattractants. Therefore, we hypothesized that IL-17A was involved in the pathogenesis of acute exacerbation, due to viral infection in chronic inflammatory airway diseases. In this study, we assessed chemokine production by bronchial epithelial cells and investigated the underlying mechanisms. Comprehensive chemokine analysis showed that, compared with poly(I:C) alone, co-stimulation of BEAS-2B cells with IL-17A and poly(I:C) strongly induced production of such neutrophil chemoattractants as CXC chemokine ligand (CXCL)8, growth-related oncogene (GRO), and CXCL1. Co-stimulation synergistically induced CXCL8 and CXCL1 mRNA and protein production by BEAS-2B cells and normal human bronchial epithelial cells. Poly(I:C) induced chemokine expression by BEAS-2B cells mainly via Toll-like receptor 3/TIR-domain-containing adapter-inducing interferon-β-mediated signals. The co-stimulation with IL-17A and poly(I:C) markedly activated the p38 and extracellular-signal-regulated kinase 1/2 pathway, compared with poly(I:C), although there was little change in nuclear factor-κB translocation into the nucleus or the transcriptional activities of nuclear factor-κB and activator protein 1. IL-17A promoted stabilization of CXCL8 mRNA in BEAS-2B cells treated with poly(I:C). In conclusion, IL-17A appears to be involved in the pathogenesis of chronic inflammatory airway disease exacerbation, due to viral infection by promoting release of neutrophil chemoattractants from bronchial epithelial cells.

Asthma-related mortality has been decreasing due to inhaled corticosteroid use, but severe asthma remains a major clinical problem. One characteristic of severe asthma is resistance to steroid therapy, which is related to neutrophilic inflammation. Recently, the tumor necrosis factor superfamily member (TNFSF) 14/LIGHT has been recognized as a key mediator in severe asthmatic airway inflammation. However, the profiles and intracellular mechanisms of cytokine/chemokine production induced in cells by LIGHT are poorly understood. We aimed to elucidate the molecular mechanism of LIGHT-induced cytokine/chemokine production by bronchial epithelial cells. Human bronchial epithelial cells express lymphotoxin β receptor (LTβR), but not herpesvirus entry mediator, which are receptors for LIGHT. LIGHT induced various cytokines/chemokines, such as interleukin (IL)-6, oncostatin M, monocyte chemotactic protein-1, growth-regulated protein α and IL-8. Specific siRNA for LTβR attenuated IL-6 and IL-8 production by BEAS-2B and normal human bronchial epithelial cells. LIGHT activated intracellular signaling, such as mitogen-activated protein kinase and nuclear factor-κB (NF-κB) signaling. LIGHT also induced luciferase activity of NF-κB response element, but not of activator protein-1 or serum response element. Specific inhibitors of phosphorylation of extracellular signal-regulated kinase (Erk) and that of inhibitor κB attenuated IL-8 production, suggesting that LIGHT-LTβR signaling induces IL-8 production via the Erk and NF-κB pathways. LIGHT, via LTβR signaling, may contribute to exacerbation of airway neutrophilic inflammation through cytokine and chemokine production by bronchial epithelial cells.