ABSTRACT
Introduction: Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterised by itching, erythema, and epidermal barrier dysfunction. The pathogenesis of AD is complex and multifactorial; however,mast cell (MC) activation has been reported to be one of the crucial mechanisms in the pathogenesis of AD. The MC receptor Mas related G protein-coupled receptor-X2 (MRGPRX2) has been identified as a prominent alternative receptor to the IgE receptor in causing MC activation and the subsequent release of inflammatory mediators. The current study aimed to evaluate the therapeutic effect of a novel small molecule MRGPRX2 antagonist GE1111 in AD using in vitro and in vivo approaches. Methods: We developed an in vitro cell culture disease model by using LAD-2 MC, HaCaT keratinocytes and RAW 264.7 macrophage cell lines. We challenged keratinocytes and macrophage cells with CST-14 treated MC supernatant in the presence and absence of GE1111 and measured the expression of tight junction protein claudin 1, inflammatory cytokines and macrophage phagocytosis activity through immunohistochemistry, western blotting, RT-qPCR and fluorescence imaging techniques. In addition to this, we developed a DFNB-induced AD model in mice and evaluated the protective effect and underlying mechanism of GE1111. Results and Discussion: Our in vitro findings demonstrated a potential therapeutic effect of GE1111, which inhibits the expression of TSLP, IL-13, MCP-1, TNF-a, and IL-1ß in MC and keratinocytes. In addition to this, GE1111 was able to preserve the expression of claudin 1 in keratinocytes and the phagocytotic activity of macrophage cells. The in vivo results demonstrated that GE1111 treatment significantly reduced phenotypic changes associated with AD (skin thickening, scaling, erythema and epidermal thickness). Furthermore, immunohistochemical analysis demonstrated that GE1111 treatment preserved the expression of the tight junction protein Involucrin and reduced the expression of the inflammatory mediator periostin in the mouse model of AD. These findings were supported by gene and protein expression analysis, where GE1111 treatment reduced the expression of TSLP, IL-13, and IL-1ß, as well as downstream signalling pathways of MRGPRX2 in AD skin lesions. In conclusion, our findings provide compelling in vitro and in vivo evidence supporting the contribution of MRGPRX2-MC interaction with keratinocytes and macrophages in the pathogenesis of AD.
Subject(s)
Cytokines , Dermatitis, Atopic , Disease Models, Animal , Keratinocytes , Receptors, G-Protein-Coupled , Receptors, Neuropeptide , Skin , Animals , Dermatitis, Atopic/drug therapy , Dermatitis, Atopic/immunology , Mice , Cytokines/metabolism , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/metabolism , Humans , Receptors, Neuropeptide/antagonists & inhibitors , Receptors, Neuropeptide/metabolism , Skin/pathology , Skin/drug effects , Skin/metabolism , Skin/immunology , Keratinocytes/drug effects , Keratinocytes/metabolism , HaCaT Cells , Macrophages/immunology , Macrophages/metabolism , Macrophages/drug effects , Mast Cells/drug effects , Mast Cells/immunology , Mast Cells/metabolism , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/metabolism , RAW 264.7 Cells , Inflammation Mediators/metabolismABSTRACT
This study presents a dataset of audio-visual soundscape recordings at 62 different locations in Singapore, initially made as full-length recordings over spans of 9-38 min. For consistency and reduction in listener fatigue in future subjective studies, one-minute excerpts were cropped from the full-length recordings. An automated method using pre-trained models for Pleasantness and Eventfulness (according to ISO 12913) in a modified partitioning around medoids algorithm was employed to generate the set of excerpts by balancing the need to encompass the perceptual space with uniformity in distribution. A validation study on the method confirmed its adherence to the intended design.
Subject(s)
Auditory Perception , Singapore , Humans , Auditory Perception/physiology , Algorithms , SoundABSTRACT
Background Lipoprotein lipase (LPL)-derived fatty acid is a major source of energy for cardiac contraction. Synthesized in cardiomyocytes, LPL requires translocation to the vascular lumen for hydrolysis of lipoprotein triglyceride, an action mediated by endothelial cell (EC) release of heparanase. We determined whether flow-mediated biophysical forces can cause ECs to secrete heparanase and thus regulate cardiac metabolism. Methods and Results Isolated hearts were retrogradely perfused. Confluent rat aortic ECs were exposed to laminar flow using an orbital shaker. Cathepsin L activity was determined using gelatin-zymography. Diabetes was induced in rats with streptozotocin. Despite the abundance of enzymatically active heparanase in the heart, it was the enzymatically inactive, latent heparanase that was exceptionally responsive to flow-induced release. EC exposed to orbital rotation exhibited a similar pattern of heparanase secretion, an effect that was reproduced by activation of the mechanosensor, Piezo1. The laminar flow-mediated release of heparanase from EC required activation of both the purinergic receptor and protein kinase D, a kinase that assists in vesicular transport of proteins. Heparanase influenced cardiac metabolism by increasing cardiomyocyte LPL displacement along with subsequent replenishment. The flow-induced heparanase secretion was augmented following diabetes and could explain the increased heparin-releasable pool of LPL at the coronary lumen in these diabetic hearts. Conclusions ECs sense fluid shear-stress and communicate this information to subjacent cardiomyocytes with the help of heparanase. This flow-induced mechanosensing and its dynamic control of cardiac metabolism to generate ATP, using LPL-derived fatty acid, is exquisitely adapted to respond to disease conditions, like diabetes.
