Group III secreted phospholipase A2 -driven lysophospholipid pathway protects against allergic asthma.

Asthma is a chronic inflammatory disease of the airways characterized by recurrent episodes of airway obstruction, hyperresponsiveness, remodeling, and eosinophilia. Phospholipase A2 s (PLA2 s), which release fatty acids and lysophospholipids from membrane phospholipids, have been implicated in exacerbating asthma by generating pro-asthmatic lipid mediators, but an understanding of the association between individual PLA2 subtypes and asthma is still incomplete. Here, we show that group III-secreted PLA2 (sPLA2 -III) plays an ameliorating, rather than aggravating, role in asthma pathology. In both mouse and human lungs, sPLA2 -III was expressed in bronchial epithelial cells and decreased during the asthmatic response. In an ovalbumin (OVA)-induced asthma model, Pla2g3-/- mice exhibited enhanced airway hyperresponsiveness, eosinophilia, OVA-specific IgE production, and type 2 cytokine expression as compared to Pla2g3+/+ mice. Lipidomics analysis showed that the pulmonary levels of several lysophospholipids, including lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidic acid (LPA), were decreased in OVA-challenged Pla2g3-/- mice relative to Pla2g3+/+ mice. LPA receptor 2 (LPA2 ) agonists suppressed thymic stromal lymphopoietin (TSLP) expression in bronchial epithelial cells and reversed airway hyperresponsiveness and eosinophilia in Pla2g3-/- mice, suggesting that sPLA2 -III negatively regulates allergen-induced asthma at least by producing LPA. Thus, the activation of the sPLA2 -III-LPA pathway may be a new therapeutic target for allergic asthma.

Identification of distinct N-glycosylation patterns on extracellular vesicles from small-cell and non-small-cell lung cancer cells.

Asparagine-linked glycosylation (N-glycosylation) of proteins in the cancer secretome has been gaining increasing attention as a potential biomarker for cancer detection and diagnosis. Small extracellular vesicles (sEVs) constitute a large part of the cancer secretome, yet little is known about whether their N-glycosylation status reflects known cancer characteristics. Here, we investigated the N-glycosylation of sEVs released from small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC) cells. We found that the N-glycans of SCLC-sEVs were characterized by the presence of structural units also found in the brain N-glycome, while NSCLC-sEVs were dominated by typical lung-type N-glycans with NSCLC-associated core fucosylation. In addition, lectin-assisted N-glycoproteomics of SCLC-sEVs and NSCLC-sEVs revealed that integrin αV was commonly expressed in sEVs of both cancer cell types, while the epithelium-specific integrin α6ß4 heterodimer was selectively expressed in NSCLC-sEVs. Importantly, N-glycomics of the immunopurified integrin α6 from NSCLC-sEVs identified NSCLC-type N-glycans on this integrin subunit. Thus, we conclude that protein N-glycosylation in lung cancer sEVs may potentially reflect the histology of lung cancers.

Long-acting muscarinic antagonist regulates group 2 innate lymphoid cell-dependent airway eosinophilic inflammation.

BACKGROUND: Tiotropium bromide, a long-acting muscarinic antagonist, reduces the frequency of exacerbation in patients with moderate to severe asthma, but its underlying mechanism is not clear. Asthma exacerbations are associated with exposure to external stimuli, and group 2 innate lymphoid cells (ILC2s) are considered to be involved in the pathophysiology of asthma exacerbation. We investigated whether tiotropium modulates airway inflammation through ILC2 functions. METHODS: Mice were administered papain intranasally to induce innate-type airway inflammation with or without tiotropium pretreatment, and bronchoalveolar lavage fluids (BALF) and lung tissues were collected. Lung-derived ILC2s and bone marrow-derived basophils were stimulated in vitro with IL-33 in the presence or absence of tiotropium. Muscarinic M3 receptor (M3R) expression on immune cells was assessed by RNA sequence. RESULTS: Papain induced airway eosinophilic inflammation, and tiotropium reduced the numbers of eosinophils in BALF. The concentrations of IL-4, IL-5, and IL-13, and the numbers of ILC2s in BALF were also reduced by tiotropium treatment. However, tiotropium did not affect IL-33-induced IL-5 and IL-13 production from ILC2s, suggesting that tiotropium regulates ILC2s indirectly. Gene-expression analysis showed that basophils predominantly expressed M3R mRNA among murine immune cells. Tiotropium reduced IL-4 production from basophils derived from mouse bone marrow and human basophils after stimulation with IL-33. CONCLUSIONS: These findings suggest that tiotropium attenuates ILC2-dependent airway inflammation by suppressing IL-4 production from basophils and, subsequently, regulating ILC2 activation. The inhibitory effects of long-acting muscarinic antagonists on the innate response may contribute to reducing asthma exacerbation.

Cytomegalovirus cavitary pneumonia in a human immunodeficiency virus-infected patient.

Cavitary lung lesions are uncommon radiological findings in cytomegalovirus pneumonia, and tissue biopsy is rarely performed for diagnosis. A 67-year-old man presented with a wet cough. Extensive white moss in the oral cavity was found on physical examination, and chest computed tomography revealed an approximately 4 cm cavitary lesion in the upper lobe of the right lung. Blood tests showed a critically low CD4+ T lymphocyte count and positivity for human immunodeficiency virus type 1 antibodies. A transbronchial biopsy of the cavitary lung lesion was performed, and inclusion bodies in the nuclei of enlarged alveolar epithelial cells were seen in the histopathological findings. Immunohistochemistry staining for cytomegalovirus was positive, and cytomegalovirus pneumonia was diagnosed. Ganciclovir treatment was initiated, and the symptoms and imaging findings resolved. Cytomegalovirus pneumonia can present as cavitary lung lesions in patients with acquired immunodeficiency syndrome, and a transbronchial biopsy is essentially useful for a definitive diagnosis.

Glycometabolic Regulation of the Biogenesis of Small Extracellular Vesicles.

The biogenesis of small extracellular vesicles (sEVs) is regulated by multiple molecular machineries generating considerably heterogeneous vesicle populations, including exosomes and non-exosomal vesicles, with distinct cargo molecules. However, the role of carbohydrate metabolism in generating such vesicle heterogeneity remains largely elusive. Here, we discover that 2-deoxyglucose (2-DG), a well-known glycolysis inhibitor, suppresses the secretion of non-exosomal vesicles by impairing asparagine-linked glycosylation (N-glycosylation) in mouse melanoma cells. Mechanistically, 2-DG is metabolically incorporated into N-glycan precursors, causing precursor degradation and partial hypoglycosylation. N-glycosylation blockade by Stt3a silencing is sufficient to inhibit non-exosomal vesicle secretion. In contrast, N-glycosylation blockade barely influences exosomal secretion of tetraspanin proteins. Functionally, N-glycosylation at specific sites of the hepatocyte growth factor receptor, a cargo protein of non-exosomal vesicles, facilitates its sorting into vesicles. These results uncover a link between N-glycosylation and unconventional vesicle secretion and suggest that N-glycosylation facilitates sEV biogenesis through cargo protein sorting.

N-glycome inheritance from cells to extracellular vesicles in B16 melanomas.

We investigated the correlation between metastatic behaviors of tumor cells and asparagine-linked glycosylation (N-glycosylation) of tumor-derived extracellular vesicles (EVs). Three mouse melanoma B16 variants with distinct metastatic potentials show similar gene expression levels and enzymatic activities of glycosyltransferases involved in N-glycosylation. All melanoma variants and EVs have nearly identical profiles of de-sialylated N-glycans. The major de-sialylated N-glycan structures of cells and EVs are core-fucosylated, tetra-antennary N-glycans with ß1,6-N-acetylglucosamine branches. A few N-glycans are extended by N-acetyllactosamine repeats. Sialylation of these N-glycans may generate cell-type-specific N-glycomes on EVs. Taken together, melanoma-derived EVs show high expression of tumor-associated N-glycans, and the core structure profile is inherited during multiple selection cycles of B16 melanomas and from tumor cells to EVs.

Generation of the heterogeneity of extracellular vesicles by membrane organization and sorting machineries.

BACKGROUND: Cells secrete heterogeneous populations of extracellular vesicles (EVs) via unknown mechanisms. EV biogenesis has been postulated to involve lipid-protein clusters, also known as membrane microdomains. METHODS: Membrane properties and heterogeneity of melanoma-derived EVs were analyzed by a detergent solubilization assay, sucrose density gradient ultracentrifugation and immunoprecipitation. EV secretion was modulated by RNA interference and pharmacological treatments. RESULTS: We identified two EV membranes (low-density exosomal detergent-insoluble membranes [EV-DIMs]; EV detergent-soluble membranes [EV-DSMs]) and discovered an abundant, novel type of high-density EV-DIMs. The high-density EV-DIMs accumulated the microdomain-resident protein flotillin-1, as well as a disintegrin and metalloproteinase domain containing protein 10 (Adam10), the hepatocyte growth factor receptor Met and its proteolytic fragments. Low-density EV-DIMs also contained flotillin-1. EV-DSMs were enriched with tetraspanin CD81, melanogenic enzymes and proteolytic fragments of Adam10. Intact and fragmented forms of Adam10, which resided in distinct membrane types, were secreted by different EVs. The fragmented form of Met was associated with DIMs much more efficiently than the intact form and they were secreted by distinct EVs. We identified that the endosomal sorting complexes required for transport machinery was indispensable for EV secretion of both mature and fragmented forms of Adam10 and Met. CONCLUSION: The findings of this study reveal the role of the interplay between membrane organization and sorting machineries in generating the heterogeneity of EVs. GENERAL SIGNIFICANCE: This study provides novel insights into important aspects of EV biogenesis.

IL-13 enhances mesenchymal transition of pulmonary artery endothelial cells via down-regulation of miR-424/503 in vitro.

Pulmonary arterial hypertension (PAH) has a major effect on life expectancy with functional degeneracy of the lungs and right heart. Interleukin-13 (IL-13), one of the type 2 cytokines mainly associated with allergic diseases, has recently been reported to be associated with Schistosomiasis-associated PAH which shares pathological features with other forms of PAH, such as idiopathic PAH and connective tissue disease-associated PAH. But a direct pathological role of IL-13 in the development of PAH has not been explored. We examined the effects of recombinant human IL-13 on the function of primary human pulmonary artery endothelial cells (HPAECs) to examine how IL-13 influences exacerbation of PAH. IL-13 increased the expression of Rictor, which is a key molecule of mammalian target of rapamycin complex 2. Treatment of IL-13 induced HPAEC migration via Rictor. Rictor was directly regulated by both miR-424 and 503 (miR-424/503). Therefore, IL-13 increases Rictor level by regulating miR-424/503, causing the increase of HPAEC migration. Since enhancement of HPAEC migration in the lung is thought to be associated with PAH, these data suggest that IL-13 takes some roles in exacerbating PAH.