Rohit Gaurav

Clinical view on the importance of dendritic cells in asthma

Allergic asthma is characterized by airway hyperresponsiveness and inflammation and may lead to airway remodeling in uncontrolled cases. Genetic predisposition to an atopic phenotype plays a major component in the pathophysiology of asthma. However, with tremendous role of epigenetic factors and environmental stimuli in precipitating an immune response, the underlying pathophysiological mechanisms are complicated. Dendritic cells are principal antigen-presenting cells and initiators of the immune response in allergic asthma. Their phenotype, guided by multiple factors may dictate the immune reaction to an allergic or tolerogenic response. Involvement of the local cytokine milieu, microbiome and interplay between immune cells add dimension to the fate of immune response. In addition to allergen exposure, these factors modulate DC phenotype and function. In this article, integration of many factors and pathways associated with the recruitment and activation of DCs in the pathophysiology of allergic asthma is presented in a clinical and translational manner.

Superoxide Dismutase 3 R213G Single-Nucleotide Polymorphism Blocks Murine Bleomycin-Induced Fibrosis and Promotes Resolution of Inflammation

&NA; Loss of extracellular superoxide dismutase 3 (SOD3) contributes to inflammatory and fibrotic lung diseases. The human SOD3 R213G polymorphism decreases matrix binding, redistributing SOD3 from the lung to extracellular fluids, and protects against LPS‐induced alveolar inflammation. We used R213G mice expressing a naturally occurring single‐nucleotide polymorphism, rs1799895, within the heparin‐binding domain of SOD3, which results in an amino acid substitution at position 213 to test the hypothesis that the redistribution of SOD3 into the extracellular fluids would impart protection against bleomycin‐induced lung fibrosis and secondary pulmonary hypertension (PH). In R213G mice, SOD3 content and activity was increased in extracellular fluids and decreased in lung at baseline, with greater increases in bronchoalveolar lavage fluid (BALF) SOD3 compared with wild‐type mice 3 days after bleomycin. R213G mice developed less fibrosis based on pulmonary mechanics, fibrosis scoring, collagen quantification, and gene expression at 21 days, and less PH by right ventricular systolic pressure and pulmonary arteriole medial wall thickening at 28 days. In wild‐type mice, macrophages, lymphocytes, neutrophils, proinflammatory cytokines, and protein increased in BALF on Day 7 and/or 21. In R213G mice, total BALF cell counts increased on Day 7 but resolved by 21 days. At 1 or 3 days, BALF pro‐ and antiinflammatory cytokines and BALF protein were higher in R213G mice, resolving by 21 days. We conclude that the redistribution of SOD3 as a result of the R213G single‐nucleotide polymorphism protects mice from bleomycin‐induced fibrosis and secondary PH by improved resolution of alveolar inflammation.

Successful Transfection of Genes Using AAV-2/9 Vector in Swine Coronary and Peripheral Arteries

BACKGROUND Gene therapy has attracted attention for its potential to treat several cardiovascular diseases. The use of adeno-associated viral (AAV) vectors to facilitate therapeutic gene transfer to suppress intimal hyperplasia is a promising concept. The objective of this study was to analyze the in vivo transduction of a novel recombinant AAV-2/9 vector with SM22α promoter, containing β-galactosidase gene (LacZ) or green fluorescent protein (GFP) as reporter genes, to the medial layer smooth muscle cells (SMCs) of swine coronary and peripheral arteries. METHODS The AAV-2/9 vector containing SM22α (1 × 10(13) pfu) were administered into carotid/femoral/coronary arteries of domestic swine using irrigating balloon catheter-based gene delivery. Following gene transfer, cryosections of arteries were processed for X-Gal and GFP analysis. Fluorescence microscopy and Western blotting were done to analyze the GFP expression in the SMCs. RESULTS LacZ mRNA expression was visualized in the medial layer 7 d after vector administration. The GFP expression was detected at day 7 and lasted for at least 2 mo showing the longer-lasting expression of the AAV-2/9 vector. Control arteries did not show any expression of GFP or LacZ. There was no significant effect of AAV-2/9 viral transduction on serum amylase, fibrinogen, and serum CRP levels. CONCLUSION These finding support the use of AAV-2/9 as a vector to effectively transduce a gene in SMCs of coronary and peripheral arteries without causing inflammation.

The R213G polymorphism in SOD3 protects against allergic airway inflammation

Oxidative stress is important in the pathogenesis of allergic asthma. Extracellular superoxide dismutase (EC-SOD; SOD3) is the major antioxidant in lungs, but its role in allergic asthma is unknown. Here we report that asthmatics have increased SOD3 transcript levels in sputum and that a single nucleotide polymorphism (SNP) in SOD3 (R213G; rs1799895) changes lung distribution of EC-SOD, and decreases likelihood of asthma-related symptoms. Knockin mice analogous to the human R213G SNP had lower airway hyperresponsiveness, inflammation, and mucus hypersecretion with decreased interleukin-33 (IL-33) in bronchoalveolar lavage fluid and reduced type II innate lymphoid cells (ILC2s) in lungs. SOD mimetic (Mn (III) tetrakis (N-ethylpyridinium-2-yl) porphyrin) attenuated Alternaria-induced expression of IL-33 and IL-8 release in BEAS-2B cells. These results suggest that R213G SNP potentially benefits its carriers by resulting in high EC-SOD in airway-lining fluid, which ameliorates allergic airway inflammation by dampening the innate immune response, including IL-33/ST2-mediated changes in ILC2s.

Chloride Channel 3 Channels in the Activation and Migration of Human Blood Eosinophils in Allergic Asthma

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is responsible for respiratory burst in immune cells. Chloride channel 3 (CLC3) has been linked to the respiratory burst in eosinophils and neutrophils. The effect of cytokines and the involvement of CLC3 in the regulation of NADPH-dependent oxidative stress and on cytokine-mediated migration of eosinophils are not known. Human peripheral blood eosinophils were isolated from healthy individuals and from individuals with asthma by negative selection. Real-time PCR was used to detect the expression of NADPH oxidases in eosinophils. Intracellular reactive oxygen species (ROS) measurement was done with flow cytometry. Superoxide generation was measured with transforming growth factor (TGF)-β, eotaxin, and CLC3 blockers. CLC3 dependence of eosinophils in TGF-β- and eotaxin-induced migration was also examined. The messenger RNA (mRNA) transcripts of NADPH oxidase (NOX) 2, dual oxidase (DUOX) 1, and DUOX2 were detected in blood eosinophils, with very low expression of NOX1, NOX3, and NOX5 and no NOX4 mRNA. The level of NOX2 mRNA transcripts increased with disease severity in the eosinophils of subjects with asthma compared with healthy nonatopic volunteers. Change in granularity and size in eosinophils, but no change in intracellular ROS, was observed with phorbol myristate acetate (PMA). PMA, TGF-β, and eotaxin used the CLC3-dependent pathway to increase superoxide radicals. TGF-β and eotaxin induced CLC3-dependent chemotaxis of eosinophils. These findings support the requirement of CLC3 in the activation and migration of human blood eosinophils and may provide a potential novel therapeutic target to regulate eosinophil hyperactivity in allergic airway inflammation in asthma.

Novel CLC3 transcript variants in blood eosinophils and increased CLC3 expression in nasal lavage and blood eosinophils of asthmatics

Eosinophilia is a characteristic feature of allergic airway inflammation and remodeling. Chloride channel‐3 (CLC3) in eosinophils has been associated with superoxide generation and respiratory burst. The CLC3 gene may produce multiple transcript variants through alternative splicing. However, the presence of CLC3 variants in human eosinophils is unknown. We examined the expression of CLC3 transcript variants in peripheral blood eosinophils of allergic asthmatics and healthy individuals. Potential of these obligatory dimers to form homo‐ or hetero‐dimers was examined in HEK293 cells co‐transfected with CLC3b‐GFP and CLC3e‐RFP. Eosinophils were isolated from peripheral blood by negative selection. Expression of CLC3 and CLC3 transcript variants was examined by qPCR, Western blot, and immunofluorescence. Confocal micrographs were analyzed with Image J software. Higher levels of novel transcript variants of CLC3 (CLC3b and CLC3e) were found in peripheral blood eosinophils of asthmatics compared to healthy non‐atopic subjects. We also found higher CLC3 protein expression in the blood and nasal lavage eosinophils of asthmatics than healthy subjects. Both membranous and intracellular CLC3 expression were observed. Also, we found the presence of both homodimers and heterodimers of CLC3b‐GFP and CLC3e‐RFP in HEK293 cells. Higher and differential expression of novel CLC3 transcript variants in mild‐to‐moderate and moderate‐to‐severe asthmatic eosinophils suggest their critical role in allergic asthma. Membranous and intracellular (granular) expression of CLC3 in nasal lavage and peripheral blood eosinophils suggest their involvement in the activation and migration of eosinophils in allergic asthma. Moreover, homo‐ and hetero‐dimerization of these transcript variants may change the channel properties to exhibit these states. Presence of CLC3 variants may serve as a biomarker in allergic asthma and additional knowledge of interaction between CLC3 transcript variants and their specific role in the activation and migration of eosinophils will allow to explore novel therapeutic approach in allergic asthma.