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Toll-like receptors, Wnt receptors and Calcium-activated Chloride/Potassium Channels as promising immunomodulators of allergic airway inflammation, airway hyper-responsiveness and asthma: translational research impact

Saumya Pandey (M.Sc., Ph.D.)

Department of Clinical Research, Indira IVF Hospital, Udaipur, India

Introduction: Allergic airway inflammation/airway hyper-responsiveness and asthma have emerged as major public health challenges in United States of America/Asia-Pacific region. Targeting Toll-like receptor (TLR), Wnt-Frizzled receptor signaling and Calcium-activated-Chloride (ClCa)/Potassium (IKCa3.1) Channels in unraveling the cellular/molecular/genetic basis of susceptibility to inflammatory diseases in specific human patient population subset(s) is emerging as an attractive immunotherapeutic pharmacological strategy in management/prevention of asthma in the Covid-19 pandemic era. 

Objectives: My exploratory study aimed to investigate the role of TLRs, Wnt-Fzd receptors and ClCa/IKCa3.1 ion-channels in human airway smooth muscle cells, bronchial epithelial cells (ASMCs/NHBEC/BEAS-2B from ATCC), and eosinophils-derived from asthma patients of North American ethnicity.

Methods: Whole cell patch-clamp electrophysiological-recordings with stringent pH/ osmolality-checks were conducted for ClCa/IKCa3.1 ion-channel physiology and outwardly-/inwardly-rectifying currents in cultured cells (passages: P2-P5) grown on sterile cover-slips. RNA and Protein were extracted from ASMCs/NHBECs/BEAS-2B/eosinophils using Trizol and RIPA methods. Borosilicate patch-pipettes were fabricated using Sutter instrument and resistance was checked alongwith bore-diameter prior to filling bath- and pipette-solutions; micromanipulators were adjusted for gigaseal-recordings. The study was approved by Institutional Review Board. 

Results: Cell-viability assays (MTT) demonstrated >80% viability of ASMCs/NHBECs/ BEAS-2B cells and asthma patients’-isolated eosinophils. Mean age of American patients (N=7; White N=2/African-American N=4/Caucasian N=1) was 47.0 years (S.D±5.0 years). Receptor/ion channel physiology studies demonstrated the expression of TLR2/4, Wnt2/4, Fzd2 and intermediate conductance IKCa3.1/ClCa mRNA transcripts; beta-actin/GAPDH were used as internal controls. Patch-clamp electrophysiology recordings detected Chloride/Potassium channel current-spikes in cultured cells in presence of intracellular Calcium, and DIDS-Chloride channel inhibitor. My preliminary data implicates the public health impact of TLR2/4-Wnt2/4-Fzd2/ClCa-KCa regulatory networks-mediated immunomodulation in AHR and asthma management in North American cohort. Future pharmacogenetics-based epidemiology/association-studies with larger sample-size and subgroup-stratification for Covid-19 positivity status and Covid-19 relapse/recurrence rates are warranted for development of cost-effective predictive biomarkers for AHR and asthma susceptible populations of diverse ethnicities. 

Acknowledgement: Dr. Pandey acknowledges the fellowship/fund support from Department of Biomedical Sciences, School of Medicine, Creighton University, Omaha, Nebraska, USA-National Institutes of Health, Bethesda, Maryland, USA during her tenure.