Short-term intermittent cigarette smoke exposure enhances alveolar type 2 cell stemness via fatty acid oxidation.

BACKGROUND: Cigarette smoke (CS) is associated with chronic obstructive pulmonary disease (COPD) and cancer. However, the underlying pathological mechanisms are not well understood. We recently reported that mice exposed to long-term intermittent CS for 3 months developed more severe emphysema and higher incidence of adenocarcinoma than mice exposed to long-term continuous CS for 3 months and long-term continuous CS exposure activated alveolar stem cell proliferation. However, the influence of variations in the CS exposure pattern in alveolar stem cell in unknown. Here, we exposed mice to 3 weeks of continuous or intermittent CS to identify whether different CS exposure patterns would result in differential effects on stem cells and the mechanisms underlying these potential differences. METHODS: Female mice expressing GFP in alveolar type 2 (AT2) cells, which are stem cells of the alveolar compartment, were exposed to mainstream CS via nasal inhalation. AT2 cells were collected based on their GFP expression by flow cytometry and co-cultured with fibroblasts in stem cell 3D organoid/colony-forming assays. We compared gene expression profiles of continuous and intermittent CS-exposed AT2 cells using microarray analysis and performed a functional assessment of a differentially expressed gene to confirm its involvement in the process using activator and inhibitor studies. RESULTS: AT2 cells sorted from intermittent CS-exposed mice formed significantly more colonies compared to those from continuous CS-exposed mice, and both CS-exposed groups formed significantly more colonies when compared to air-exposed cells. Comparative microarray analysis revealed the upregulation of genes related to fatty acid oxidation (FAO) pathways in AT2 cells from intermittent CS-exposed mice. Treatment of intermittent CS-exposed mice with etomoxir, an inhibitor of the FAO regulator Cpt1a, for 5 weeks resulted in a significant suppression of the efficiency of AT2 cell colony formation. In vitro treatment of naïve AT2 cells with a FAO activator and inhibitor further confirmed the relationship between FAO and AT2 stem cell function. CONCLUSIONS: Alveolar stem cell function was more strongly activated by intermittent CS exposure than by continuous CS exposure. We provide evidence that AT2 stem cells respond to intermittent CS exposure by activating stem cell proliferation via the activation of FAO.

TLR7 Agonist Suppresses Group 2 Innate Lymphoid Cell-mediated Inflammation via IL-27-Producing Interstitial Macrophages.

Group 2 innate lymphoid cells (ILC2s) play an important role in the pathophysiology of asthma via the robust production of type 2 cytokines. Recent studies have demonstrated that TLR7 (Toll-like receptor 7) signaling skews toward a type 1 inflammatory response in asthma, which may lead to the development of novel treatment strategies. However, the effect of TLR7 signaling on ILC2-dependent nonallergic eosinophilic inflammation remains unclear. In this study, we investigated the effects of R848, a TLR7 agonist, in a mouse model of IL-33-induced eosinophilic airway inflammation. Intranasal administration of R848 decreased infiltration of airway eosinophils and ILC2s, mucus production in epithelial cells, and type 2 cytokine production. Flow cytometric analysis identified an increased number of interstitial macrophages (IMs) expressing a high level of TLR7 in the lung upon IL-33 stimulation. IL-33-induced IMs also expressed high levels of alternatively activated (M2)-type genes and chemokines (CCL17 and CCL24). However, R848 stimulation modified these gene expressions and elicited the production of IL-27. Coculture experiments revealed that IL-33-induced IMs directly suppressed ILC2 activation in response to R848. In addition, the inhibitory effects of R848 on ILC2-induced type 2 inflammation were defective in WSX-1-deficient mice lacking the IL-27 receptor. Taken together, these findings indicate that R848 stimulates IL-33-induced IMs to suppress ILC2-mediated type 2 airway inflammation via IL-27. These findings highlight the therapeutic potential of TLR7 agonists and/or IL-27 cascades in nonallergic asthma.

ADAM10 partially protects mice against influenza pneumonia by suppressing specific myeloid cell population.

The influenza virus infection poses a serious health threat worldwide. Myeloid cells play pivotal roles in regulating innate and adaptive immune defense. A disintegrin and metalloproteinase (ADAM) family of proteins contributes to various immune responses; however, the role of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) in influenza virus infection remains largely unknown. Herein, we investigated its role, focusing on myeloid cells, during influenza virus infection in mice. ADAM10 gene (Adam10)flox/flox/Lyz2-Cre (Adam10ΔLyz2) and control Adam10flox/flox mice were intranasally infected with 200 plaque-forming units of influenza virus A/H1N1/PR8/34. Adam10ΔLyz2 mice exhibited a significantly higher mortality rate, stronger lung inflammation, and a higher virus titer in the lungs than control mice. Macrophages and inflammatory cytokines, such as TNF-α, IL-1ß, and CCL2, were increased in bronchoalveolar lavage fluid from Adam10ΔLyz2 mice following infection. CD11b+Ly6G-F4/80+ myeloid cells, which had an inflammatory monocyte/macrophage-like phenotype, were significantly increased in the lungs of Adam10ΔLyz2 mice. Adoptive transfer experiments suggested that these cells likely contributed to the poorer prognosis in Adam10ΔLyz2 mice. Seven days after infection, CD11b+Ly6G-F4/80+ lung cells exhibited significantly higher arginase-1 expression levels in Adam10ΔLyz2 mice than in control mice, whereas an arginase-1 inhibitor improved the prognosis of Adam10ΔLyz2 mice. Enhanced granulocyte-macrophage colony-stimulating factor (GM-CSF)/GM-CSF receptor signaling likely contributed to this process. Collectively, these results indicate that myeloid ADAM10 protects against influenza virus pneumonia and may be a promising therapeutic target.

ADAM17 protects against elastase-induced emphysema by suppressing CD62L+ leukocyte infiltration in mice.

Pulmonary emphysema is a major manifestation of chronic obstructive pulmonary disease and is associated with chronic pulmonary inflammation caused by cigarette smoking, with contributions from immune cells such as neutrophils, macrophages, and lymphocytes. Although matrix metalloproteinases are well known to contribute to emphysema progression, the role of a disintegrin and metalloproteinase (ADAM) family proteins, other major metalloproteinases, in disease pathogenesis is largely unknown. ADAM17 is a major sheddase that cleaves various cell surface proteins, including CD62L, an adhesion molecule that plays a critical role in promoting the migration of immune cells to the site of inflammation. In the present study, we aimed to investigate the potential role of ADAM17 and CD62L in the development of elastase-induced emphysema. Control and Adam17flox/flox/Mx1-Cre (Adam17ΔMx1) mice (8-10 wk old) were intratracheally injected with 5 units of porcine pancreas elastase and monitored for 35 days after injection. Lung alveolar destruction was evaluated by analyzing the mean linear intercepts of lung tissue specimens and by histopathological examination. Mean linear intercepts data indicated that the degree of elastase-induced emphysema was significantly more severe in Adam17ΔMx1 mice. Furthermore, flow cytometry showed that CD62L+ neutrophil, CD62L+ macrophage, and CD62L+ B lymphocyte numbers were significantly increased in Adam17ΔMx1 mice. Moreover, the pharmacological depletion of CD62L+ cells with a CD62L-neutralizing antibody ameliorated the extent of emphysema in Adam17ΔMx1 mice. Collectively, these results suggest that ADAM17 possibly suppresses the progression of emphysema by proteolytically processing CD62L in immune cells and that ADAM17 and CD62L could be novel therapeutic targets for treating pulmonary emphysema.

Effect of FGF/FGFR pathway blocking on lung adenocarcinoma and its cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) are known to promote tumourigenesis through various mechanisms. Fibroblast growth factor (FGF)/FGF receptor (FGFR)-dependent lung cancers have been described. We have developed a mouse model of lung adenocarcinoma that was constructed through the induction of Fgf9 overexpression in type 2 alveolar cells. The expression of Fgf9 in adult lungs resulted in the rapid development of multiple adenocarcinoma-like tumour nodules. Here, we have characterised the contribution of CAFs and the Fgf/Fgfr signalling pathway in maintaining the lung tumours initiated by Fgf9 overexpression. We found that CAF-secreted Fgf2 contributes to tumour cell growth. CAFs overexpressed Tgfb, Mmp7, Fgf9, and Fgf2; synthesised more collagen, and secreted inflammatory cell-recruiting cytokines. CAFs also enhanced the conversion of tumour-associated macrophages (TAMs) to the tumour-supportive M2 phenotype but did not influence angiogenesis. In vivo inhibition of Fgfrs during early lung tumour development resulted in significantly smaller and fewer tumour nodules, whereas inhibition in established lung tumours caused a significant reduction in tumour size and number. Fgfr inhibition also influenced tumour stromal cells, as it significantly abolished TAM recruitment and reduced tumour vascularity. However, the withdrawal of the inhibitor caused a significant recurrence/regrowth of Fgf/Fgfr-independent lung tumours. These recurrent tumours did not possess a higher proliferative or propagative potential. Our results provide evidence that fibroblasts associated with the Fgf9-induced lung adenocarcinoma provide multiple means of support to the tumour. Although the Fgfr blocker significantly suppressed the tumour and its stromal cells, it was not sufficient to completely eliminate the tumour, probably due to the emergence of alternative (resistance/maintenance) mechanism(s). This model represents an excellent tool to further study the complex interactions between CAFs, their related chemokines, and the progression of lung adenocarcinoma; it also provides further evidence to support the need for a combinatorial strategy to treat lung cancer. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Deficiency of CRTH2, a Prostaglandin D2 Receptor, Aggravates Bleomycin-induced Pulmonary Inflammation and Fibrosis.

Chemoattractant receptor homologous with T-helper cell type 2 cells (CRTH2), a receptor for prostaglandin D2, is preferentially expressed on T-helper cell type 2 lymphocytes, group 2 innate lymphoid cells, eosinophils, and basophils, and elicits the production of type 2 cytokines, including profibrotic IL-13. We hypothesized that lack of CRTH2 might protect against fibrotic lung disease, and we tested this hypothesis using a bleomycin-induced lung inflammation and fibrosis model in CRTH2-deficient (CRTH2-/-) or wild-type BALB/c mice. Compared with wild-type mice, CRTH2-/- mice treated with bleomycin exhibited significantly higher mortality, enhanced accumulation of inflammatory cells 14-21 days after bleomycin injection, reduced pulmonary compliance, and increased levels of collagen and total protein in the lungs. These phenotypes were associated with decreased levels of IFN-γ, IL-6, IL-10, and IL-17A in BAL fluid. Adoptive transfer of splenocytes from wild-type, but not CRTH2-/-, mice 2 days before injection of bleomycin resolved the sustained inflammation as well as the increased collagen and protein accumulation in the lungs of CRTH2-/- mice. We consider that the disease model is driven by γδT cells that express CRTH2; thus, the adoptive transfer of γδT cells could ameliorate bleomycin-induced alveolar inflammation and fibrosis.

Intermittent Exposure to Cigarette Smoke Increases Lung Tumors and the Severity of Emphysema More than Continuous Exposure.

Lung cancer and chronic obstructive pulmonary disease are leading causes of morbidity and mortality worldwide, and cigarette smoking is a main risk factor for both. The presence of emphysema, an irreversible lung disease, further raises the risk of lung cancer in patients with chronic obstructive pulmonary disease. The mechanisms involved in smoke-induced tumorigenesis and emphysema are not fully understood, attributable to a lack of appropriate animal models. Here, we optimized a model of cigarette smoke (CS)-induced lung cancer and emphysema in A/J mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a potent carcinogen. We investigated whether variations in CS exposure patterns with the same total amount and duration of exposure affect tumorigenesis and/or development of emphysema. Continuous CS exposure for 3 months significantly suppressed 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced development of adenomas and adenocarcinomas; however, emphysema independently developed during this period. Surprisingly, intermittent CS exposure increased the severity of emphysema and resulted in a higher incidence of adenocarcinomas. Furthermore, intermittent CS exposure elicited a marked increase in M2-polarized macrophages within and near the developed tumors. By employing a CS exposure protocol with repeated cycles of cessation and relapse, we provide evidence that intermittent CS exposure enhances tumorigenesis and emphysema progression more than that of continuous CS exposure.

Effects of the common polymorphism in the human aldehyde dehydrogenase 2 (ALDH2) gene on the lung.

BACKGROUND: Aldehyde dehydrogenases (ALDHs) play a major role in detoxification of aldehydes. High expression of ALDHs is a marker for stem cells of many organs including the lungs. A common polymorphism in ALDH2 gene (ALDH2*2) results in inactivation of the enzyme and is associated with alcohol flushing syndrome and increased risk for cardiovascular and Alzheimer's diseases and some cancers. The effect of this ALDH2 polymorphism on the lung and its stem cells has not been thoroughly examined. METHODS: We examined the association between the ALDH2*2 allele and lung function parameters in a population of healthy individuals. We also examined its association with the incidence of asthma and COPD in patient cohorts. We used the in vitro colony forming assay to detect the effect of the polymorphism on lung epithelial stem cells from both primary human surgical samples and Aldh2*2 transgenic (Tg) and Aldh2 -/- mice. Response to acute and chronic lung injuries was compared between wild type (WT), Aldh2*2 Tg and Aldh2 -/- mice. RESULTS: In humans, the ALDH2*2 allele was associated with lower FEV1/FVC in the general population, but not with the development of asthma or COPD. Both the bronchial and lung epithelium carrying the ALDH2*2 allele showed a tendency for lower colony forming efficiency (CFE) compared to ALDH2 allele. In mice, the tracheal epithelial thickness, nuclear density, and number of basal stem cells were significantly lower in Aldh2 -/- and Aldh2*2 Tg adult mice than in WT. Electron microscopy showed significantly increased number of morphologically abnormal mitochondria in the trachea of Aldh2 -/- mice. Aldh2 -/- tracheal and lung cells showed higher ROS levels and fewer functional mitochondria than those from WT mice. No significant differences were detected when tracheal and lung epithelial stem cells were examined for their in vitro CFE. When exposed to chronic cigarette smoke, Aldh2*2 Tg mice were resistant to emphysema development, whereas influenza infection caused more epithelial damage in Aldh2 -/- mice than in WT mice. CONCLUSIONS: ALDH2 polymorphism has several subtle effects on the lungs, some of which are similar to changes observed during normal aging, suggesting a "premature lung aging" effect.

Distinct effects of endogenous interleukin-23 on eosinophilic airway inflammation in response to different antigens.

BACKGROUND: The role of interleukin (IL)-23 in asthma pathophysiology is still controversial. We examined its role in allergic airway inflammation in response to two distinct antigens using IL-23-deficient mice. METHODS: Allergic airway inflammation was evaluated in wild-type and IL-23p19(-/-) mice. Mice were sensitized to ovalbumin (OVA) or house dust mite (HDM) by intraperitoneal injection of antigen and their airways were then exposed to the same antigen. Levels of antigen-specific immunoglobulins in serum as well as cytokines in bronchoalveolar or peritoneal lavage fluid and lung tissue were determined by enzyme-linked immunosorbent assay and/or quantitative polymerase chain reaction. RESULTS: Deficiency of IL-23p19 decreased eosinophils and Th2 cytokines in bronchoalveolar lavage fluid (BALF) of OVA-treated mice, while it increased BALF eosinophils of HDM-treated mice. Peritoneal injection of OVA with alum, but not of HDM, induced local synthesis of IL-6, IL-10, and IL-23. Systemic production of antigen-specific IgG1 was partially dependent on IL-23. In contrast, airway exposure to HDM, but not to OVA, induced IL-23p19 mRNA expression in the lungs. In IL-23p19-deficient mice, HDM-exposed lungs did not exhibit the induction of IL-17A, which negatively regulates eosinophilic inflammation. CONCLUSIONS: Different antigens induced IL-23 at different part of the body in our similar asthma models. Endogenous IL-23 production at the site of antigen sensitization facilitates type-2 immune responses, whereas IL-23 production and subsequent IL-17A synthesis in the airways suppresses allergic inflammation.

CRTH2 is a critical regulator of neutrophil migration and resistance to polymicrobial sepsis.

Although arachidonic acid cascade has been shown to be involved in sepsis, little is known about the role of PGD(2) and its newly found receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), on the septic response. Severe sepsis is associated with the failure of neutrophil migration. To investigate whether CRTH2 influences neutrophil recruitment and the lethality during sepsis, sepsis was induced by cecal ligation and puncture (CLP) surgery in mice. CRTH2 knockout (CRTH2(-/-)) mice were highly resistant to CLP-induced sepsis, which was associated with lower bacterial load and lower production of TNF-α, IL-6, and CCL3. IL-10, an anti-inflammatory cytokine, was higher in CRTH2(-/-) mice, blunting CLP-induced lethality in CRTH2(-/-) mice. Neutrophil accumulation in the peritoneum was more pronounced after CLP in CRTH2(-/-) mice, which was associated with higher CXCR2 levels in circulating neutrophils. Furthermore, sepsis caused a decrease in the level of acetylation of histone H3, an activation mark, at the CXCR2 promoter in wild-type neutrophils, suggesting that CXCR2 expression levels are epigenetically regulated. Finally, both pharmacological depletion of neutrophils and inhibition of CXCR2 abrogated the survival benefit in CRTH2(-/-) mice. These results demonstrate that genetic ablation of CRTH2 improved impaired neutrophil migration and survival during severe sepsis, which was mechanistically associated with epigenetic-mediated CXCR2 expression. Thus, CRTH2 is a potential therapeutic target for polymicrobial sepsis.

Dual role of interleukin-23 in epicutaneously-sensitized asthma in mice.

BACKGROUND: Interleukin (IL)-23/Th17 axis plays an important role in the pathophysiology of asthma and eczema, however, there are some conflicting data about the effects of this system on allergic airway inflammation. In the present study, we aim to dissect the spatiotemporal differences in the roles of IL-23 in an epicutaneously-sensitized asthma model of mice. METHODS: C57BL/6 mice were sensitized to ovalbumin (OVA) by patch application on the skin, followed by airway exposure to aerosolized OVA. During sensitization and/or challenge phase, either a specific neutralizing antibody (Ab) against IL-23 or control IgG was injected intraperitoneally. On days 1 and 8 after the final OVA exposure, airway inflammation and responsiveness to methacholine, immunoglobulin levels in serum, and cytokine release from splenocytes were evaluated. Skin Il23a mRNA levels were evaluated with quantitative RT-PCR. RESULTS: Patch application time-dependently increased the expression of Il23a mRNA expression in the skin. Treatment with the anti-IL-23 Ab during sensitization phase alone significantly reduced the number of eosinophils in bronchoalveolar lavage fluids and peribronchial spaces after allergen challenge compared with treatment with control IgG. Anti-IL-23 Ab also reduced serum levels of OVA-specific IgG1. In contrast, treatment with the anti-IL-23 Ab during the challenge phase alone rather exacerbated airway hyperresponsiveness to methacholine with little effects on airway eosinophilia or serum IgG1 levels. CONCLUSIONS: IL-23 expressed in the skin during the sensitization phase plays an essential role in the development of allergic phenotypes, whereas IL-23 in the airways during the challenge phase suppresses airway hyperresponsiveness.

Induction of mucin and MUC5AC expression by the protease activity of Aspergillus fumigatus in airway epithelial cells.

Allergic bronchopulmonary mycosis, characterized by excessive mucus secretion, airflow limitation, bronchiectasis, and peripheral blood eosinophilia, is predominantly caused by a fungal pathogen, Aspergillus fumigatus. Using DNA microarray analysis of NCI-H292 cells, a human bronchial epithelial cell line, stimulated with fungal extracts from A. fumigatus, Alternaria alternata, or Penicillium notatum, we identified a mucin-related MUC5AC as one of the genes, the expression of which was selectively induced by A. fumigatus. Quantitative RT-PCR, ELISA, and histochemical analyses confirmed an induction of mucin and MUC5AC expression by A. fumigatus extracts or the culture supernatant of live microorganisms in NCI-H292 cells and primary cultures of airway epithelial cells. The expression of MUC5AC induced by A. fumigatus extracts diminished in the presence of neutralizing Abs or of inhibitors of the epidermal growth factor receptor or its ligand, TGF-α. We also found that A. fumigatus extracts activated the TNF-α-converting enzyme (TACE), critical for the cleavage of membrane-bound pro-TGF-α, and its inhibition with low-molecular weight inhibitors or small interfering RNA suppressed the expression of MUC5AC. The protease activity of A. fumigatus extracts was greater than that of other fungal extracts, and treatment with a serine protease inhibitor, but not with a cysteine protease inhibitor, eliminated its ability to activate TACE or induce the expression of MUC5AC mRNA in NCI-H292. In conclusion, the prominent serine protease activity of A. fumigatus, which caused the overproduction of mucus by the bronchial epithelium via the activation of the TACE/TGF-α/epidermal growth factor receptor pathway, may be a pathogenetic mechanism of allergic bronchopulmonary mycosis.

Dexamethasone restores TNFα-induced epithelial barrier dysfunction in primary rat alveolar epithelial cells.

Alveolar barrier dysfunction is one of the major pathophysiological changes in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). In ALI/ARDS, tumor necrosis factor-alpha (TNFα) disrupts the barriers of alveolar epithelium and endothelium. Glucocorticoids (GCs) exert anti-inflammatory effects and ameliorate pulmonary edema in ALI/ARDS. However, the involvement of GCs in the restoration of alveolar epithelial barrier dysfunction has not been extensively studied. Here, we elucidated that dexamethasone (Dex) restored TNFα-induced alveolar epithelial barrier dysfunction in vitro using primary rat alveolar epithelial cells isolated from Sprague-Dawley rats. Moreover, Dex promoted the alveolar epithelial cell barrier integrity by initiating GC receptor-mediated signaling via the downregulation of myosin light chain kinase (MLCK) expression and the dephosphorylation of myosin light chain (MLC) 2. Further investigation revealed that Dex enhanced the expression of zonula occludens-1 (ZO-1), a tight junction-related protein, at intercellular junction sites. These findings suggest that GCs strengthen the integrity of the alveolar epithelial barrier in ALI/ARDS via the GR-MLCK-pMLC2 axis.

Lysophosphatidylcholine Acyltransferase 1 Deficiency Promotes Pulmonary Emphysema via Apoptosis of Alveolar Epithelial Cells.

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.

Role of prostaglandin D2 receptor CRTH2 in sustained eosinophil accumulation in the airways of mice with chronic asthma.

The prostaglandin D(2) (PGD(2))/CRTH2 pathway is important for eosinophil trafficking in vitro; however, genetic deficiency of CRTH2 does not suppress in vivo eosinophilic airway inflammation in acute models of asthma, and the role of CRTH2 in the pathogenesis of asthma is still ambiguous. Therefore, in the present study we explored whether the PGD(2)/CRTH2 pathway could affect the phenotypes of chronic asthma. Either CRTH2-deficient (CRTH2-/-) or wild-type mice were sensitized and exposed to ovalbumin (OVA) for 3 days (acute model) or 6 weeks (chronic model). While the magnitude of the acute eosinophilic inflammation was equivalent between CRTH2-/- and wild-type mice, the number of inflammatory cells and eosinophils in bronchoalveolar lavage fluid after chronic OVA exposure was significantly reduced in CRTH2-/- mice (18.0 ± 2.6 × 10(4) cells and 2.0 ± 0.5 × 10(4) cells) compared to wild-type mice (27.9 ± 2.5 × 10(4) cells and 6.8 ± 1.1 × 10(4) cells, p < 0.001). On the contrary, no difference was observed between CRTH2-/- and wild-type mice in terms of airway hyperresponsiveness or remodeling (goblet cell hyperplasia) in the chronic model of asthma. In conclusion, CRTH2 that mediates PGD(2) activity is essential for sustained eosinophilic inflammation in the airways, and its antagonists could exert an anti-inflammatory effect in chronic asthma.

Effect of High Fat Diet on the Severity and Repair of Lung Fibrosis in Mice.

Lung fibrosis is a progressive fatal disease, and the underlying mechanisms remain unclear. These involve a combination of altered fibroblasts, excessive accumulation of extracellular matrix, inflammation, and aberrant activation of epithelial cells. Previously, we showed that high-fat diet (HFD) induces lung inflammation, aberrant activation of stem cells, and lung mitochondria impairment. Therefore, we hypothesized that HFD-induced changes would influence lung fibrosis. Mice were fed standard diet (SD) or HFD, administered bleomycin, then examined for fibrosis severity and the start of repair 3 weeks after injury, and for fibrosis repair/resolution 6-9 weeks after injury. At 3 weeks, no significant differences in inflammation and fibrosis severity were observed between SD- and HFD-fed mice. However, infiltration of alveolar type (AT)-2 cells and bronchioalveolar stem cells (BASCs) into the fibrotic areas (the start of repair) was impaired in HFD-fed mice. At 6 weeks, SD-fed mice showed near-complete resolution/repair of fibrosis and inflammation, while HFD-fed mice still showed residual fibrosis and inflammation. Infiltration of the fibrotic areas with AT2 cells was observed, but very few BASCs were detectable. At 9 weeks, mice from both groups showed complete resolution/repair of fibrosis and inflammation, indicating that HFD induced delayed, rather than failed, resolution of fibrosis and alveolar repair. To further confirm the direct role of enhanced fatty-acid oxidation (FAO) in delayed resolution/repair, we administered etomoxir, a FAO inhibitor, to HFD-fed mice for 3-6 weeks after bleomycin injury. Inhibition of FAO abolished the HFD-induced delay in alveolar repair and fibrosis resolution at both time points. In conclusion, after a fibrosis-inducing injury, HFD slows resolution of fibrosis/inflammation and delays alveolar repair by slowing the contribution of AT2 stem cells and abolishing the contribution of BASCs in the repair process. FAO activation appears to be involved in this delay mechanism; thus, inhibiting FAO may be useful in the treatment of lung injury and fibrosis.

Regulation of peripheral Th/Treg differentiation and suppression of airway inflammation by Nr4a transcription factors.

Helper T (Th) and regulatory T (Treg) cell differentiation programs promote the eradication of pathogens, while minimizing adverse immune reactions. Here, we found that Nr4a family of nuclear receptors supports Treg cell induction and represses Th1 and Th2 cell differentiation from naive CD4+ T cells. Nr4a factors are transiently induced in CD4+ T cells immediately after antigen stimulation, thereby mediating epigenetic changes. In differentiating Treg cells, Nr4a factors mainly upregulated the early responsive genes in the Treg cell-specifying gene set, either directly or in cooperation with Ets family transcription factors. In contrast, Nr4a factors repressed AP-1 activity by interrupting a positive feedback loop for Batf factor expression, thus suppressing Th2 cell-associated genes. In an allergic airway inflammation model, Nr4a factors suppressed the pathogenesis, mediating oral tolerance. Lastly, pharmacological activation of an engineered Nr4a molecule prevented allergic airway inflammation, indicating that Nr4a factors may be novel therapeutic targets for inflammatory diseases.

Strain-specific phenotypes of airway inflammation and bronchial hyperresponsiveness induced by epicutaneous allergen sensitization in BALB/c and C57BL/6 mice.

BACKGROUND: Allergen sensitization through a disrupted skin barrier appears to play a prominent role in the development of atopic diseases, including allergic asthma. The role of the genetic background in immunological and physiological phenotypes induced by epicutaneous sensitization is undetermined. METHODS: BALB/c and C57BL/6 mice were sensitized either epicutaneously by patch application of ovalbumin (OVA) or systemically by intraperitoneal injection of OVA with alum before exposure to aerosolized OVA. The concentrations of OVA-specific immunoglobulin in serum and cytokines in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay. The severity of airway inflammation was evaluated by cell counts in BALF, and bronchial responsiveness to methacholine was measured by the flexiVent system. RESULTS: The production of OVA-specific IgG1 and IgE was greater in the epicutaneously sensitized BALB/c than C57BL/6 mice. In contrast, both eosinophilic airway inflammation and bronchial responsiveness to methacholine were more prominent in the C57BL/6 than in the BALB/c mice. The concentrations of interleukin-4 increased significantly in the BALF from C57BL/6 mice only. No between-strain differences were observed after intraperitoneal sensitization. CONCLUSIONS: The C57BL/6 mouse is a more appropriate model than the BALB/c mouse to study the relationship between skin barrier dysfunction and the pathogenesis of allergic asthma.

Optimizing the in vitro colony-forming assay for more efficient delineation of the interaction between lung epithelial stem cells and their niche.

The use of in vitro 3D organoid/colony forming assay (CFA); which mimics the in vivo environment have provided insight into the mechanisms by which lung stem cells maintain and repair the lung. In recent years, the use of CFA has markedly expanded. However, variations among laboratories in lung cell isolation methods, media used, type, origin, and processing methods of mesenchymal cells used as feeders for the epithelial colonies, and terms utilized to describe and quantify the growing colonies, have caused difficulty in reproducing results among different labs. In this study, we compared several previously described methods for lung cell isolation and culture media, to identify their influence on retrieved cells and growing colonies. We also characterized the effect of freeze/thaw, and propagation of fibroblasts on their ability to support epithelial colonies. Importantly, we suggested markers to identify fibroblast subtypes that offer the best support to alveolar stem cell proliferation. Then, we used our optimized assay to confirm the in vitro identity of recently described epithelial progenitors. We also tested the effect of hyperoxia on lung stem cells, and examined the expression of the receptors for the SARS-COV-2 virus's entry into epithelial cells, on our organoids. In summary, our findings facilitate CFA standardization, help understand how niche cell variations influence growing colonies, and confirm some of the recently described lung stem cells.

Calorie restriction enhances adult mouse lung stem cells function and reverses several ageing-induced changes.

Ageing is associated with decreased lung function and an increased incidence of lung infections. Several studies have suggested that long-term calorie restriction (CR) promotes health and longevity and results in the reduced risk of several diseases. The effect of CR is thought to be through improving the function of tissue stem cells. Stem cell function is known to decline with ageing. In this study, we examined the effects of ageing on lung epithelial and stem cells and the effect of CR on young and old lungs. We found that ageing results in a decrease in tracheal basal stem cells. CR induced an increase in basal stem cells in both young and old mice. In addition, ageing induced lung inflammation, and CR tended to reduce baseline lung inflammatory cell infiltration in young mice and significantly reduced ageing-induced lung inflammation. Furthermore, ageing reduced the number and function of mitochondria in lung and increased the level of mitochondrial reactive oxygen species. CR increased the number and function of mitochondria both in young and old mice. Moreover, ageing reduced lung stem cell colony-forming efficiency (CFE), and CR increased the CFE in both young and old mice. Finally, CR improved epithelial cell survival in injured lungs of young mice. In conclusion, ageing causes several structural and functional changes/impairments in lung epithelial cells. CR induces several potentially beneficial changes in lung epithelial cells, even when it is initiated at an older age, including reversal of some ageing-induced changes.