Asthma Sample Collection Protocol: Defining the Role of Apolipoprotein Pathways in Asthma

National Heart, Lung, And Blood Institute

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Abstract

The progress achieved under this protocol is summarized as follows: 1. The structural cell populations obtained by this protocol have been utilized to characterize the expression of ARTS-1 in lung cell populations. Bronchial epithelial cells that have been obtained by bronchial brushings have been utilized to demonstrate co- localization of membrane-associated ARTS-1 and TNFR1 in the apical cell membrane of ciliated bronchial epithelial cells. 2. Bronchoalveolar lavage fluid obtained by this protocol has been utilized to identify exosome-like vesicle release as a new mechanism by which soluble cytokine receptors can be generated, independent of ectodomain cleavage by receptor sheddases. The majority of soluble type I TNF receptor (TNFR1) in bronchoalveolar lavage fluid was found to be full-length, 55-kDa, exosome-associated TNFR1, whereas the 28-kDa cleaved TNFR1 ectodomain represented only a minor fraction. Thus, release of exosome-associated TNFR1 (i.e., eTNFR1) represents an important alternative mechanism for the release of soluble TNF receptors. 3. BAL fluid from asthmatic patients has been utilized in studies investigating the ADP-ribosyltransferase-specific modification of human neutrophil peptide-1. 4. Expression of the VLDL receptor was demonstrated for the first time on circulating CD11c+/CD14-/HLA-DR+ dendritic cells. This confirmed our result in a murine model that the VLDL receptor is expressed by dendritic cells and attenuates house dust mite-induced airway inflammation by suppressing dendritic cell-mediated adaptive immune responses. 5. BAL fluid cells have been used to show that expression of CD163 is reduced on alveolar macrophages from asthmatics as compared to normal subjects. 6. Induced sputum cells have been used to develop components of a nanoscale microfluidic flow cytometer that will liquefy sputum samples for point-of-care inflammatory phenotyping of asthmatic patients. 7. Myeloid dendritic cell subsets from patients with eosinophil-high asthma were shown to have lower levels of LRP-1 expression than those from healthy nonasthmatic subjects and that a negative correlation exists between LRP-1 expression by myeloid dendritic cell subsets and peripheral blood eosinophil counts in asthmatic patients. 8. HDL particles were shown to be negatively correlated, whereas serum triglycerides were shown to be positively correlated, with blood eosinophils in atopic asthmatics. This supports the concept that serum levels of HDL and triglycerides may be linked to systemic type 2 inflammation in atopic asthma. (J Lipid Research 2017 Aug; 58(8): 1713-1721. PMID: 28655726.) 9. Human peripheral blood myeloid DC subsets from patients with eosinophilic asthma have lower LRP-1 expression than cells from healthy nonasthmatic subjects. (JACI 2018 Oct; 142(4):1066-1079.e6. DOI: 10.1016/j.jaci.2017.10.044. PMID: 29274414.) 10. Peripheral blood mononuclear cells from asthmatics were used to show that prolonged fasting blunts the NLRP3 inflammasome and Th2 cell activation in steroid-naive asthmatics as well as diminishes airway epithelial cell cytokine production. This identifies a potential role for nutrient level-dependent regulation of inflammation in asthma. (J Immunol 2018 Sep 1; 201(5):1382-1388. PMID 30021766). 11. Bronchoalveolar lavage macrophages from asthmatics were used to show that APOE can function as an endogenous, concentration-dependent pulmonary danger signal that primes and activates the NLPR3 inflammasome to secrete IL-1. This might represent a mechanism through which APOE amplifies pulmonary inflammatory responses when concentrations in the lung are increased to greater than normal levels, which can occur during viral exacerbations of HDM-induced asthma characterized by neutrophilic airway inflammation. (J Allergy Clinical Immunology 2019; 144(2):426-441.e3. DOI: https://doi.org/10.1016/jaci.2019.02.027). 12. RNA sequencing of human asthmatic bronchial brushing cells stimulated with APOE identified increased expression of mRNA transcripts encoding multiple proinflammatory genes, including CXCL5 (C-X-C motif chemokine ligand 5), an epithelial-derived chemokine that promotes neutrophil activation and chemotaxis. We subsequently characterized the APOE signaling pathway that induces CXCL5 secretion by human asthmatic small airway epithelial cells (SAECs). We showed that elevated levels of APOE in the airway may activate a TLR4/TAK1/IK/NF-B/TPL2/JNK signaling pathway that induces CXCL5 secretion by human asthmatic SAECs. These findings identify new roles for TLR4 and TPL2 in APOE-mediated proinflammatory responses in asthma. (Am J Resp Cell Mol Biol 2020; 63 (2): 185 - 197.)