Abstract
Numerous inflammatory skin disorders display a high prevalence of itch. The Mas-related G protein coupled receptor X2 (MRGPRX2) has been shown to modulate itch by inducing non-IgE-mediated mast cell degranulation and the release of endogenous inducers of pruritus. Various substances collectively known as basic secretagogues, which include inflammatory peptides and certain drugs, can trigger MRGPRX2 and thereby induce pseudo-allergic reactions characterized by histamine and protease release as well as inflammation. Here, we investigated the capacity of an immunomodulatory single-stranded oligonucleotide (ssON) to modulate IgE-independent mast cell degranulation and, more specifically, its ability to inhibit the basic secretagogues compound 48/80 (C48/80)-and LL-37 in vitro and in vivo. We examined the effect of ssON on MRGPRX2 activation in vitro by measuring degranulation in a human mast cell line (LAD2) and calcium influx in MRGPRX2-transfected HEK293 cells. To determine the effect of ssON on itch, we performed behavioral studies in established mouse models and collected skin biopsies for histological analysis. Additionally, with the use of a rosacea mouse model and RT-qPCR, we investigated the effect on ssON on LL-37-induced inflammation. We reveal that both mast cell degranulation and calcium influx in MRGPRX2 transfected HEK293 cells, induced by the antimicrobial peptide LL-37 and the basic secretagogue C48/80, are effectively inhibited by ssON in a dose-dependent manner. Further, ssON demonstrates a capability to inhibit LL-37 and C48/80 activation in vivo in two mouse models. We show that intradermal injection of ssON in mice is able to block itch induced via C48/80 in a dose-dependent manner. Histological staining revealed that ssON inhibits acute mast cell degranulation in murine skin treated with C48/80. Lastly, we show that ssON treatment ameliorates LL-37-induced inflammation in a rosacea mouse model. Since there is a need for new therapeutics targeting non-IgE-mediated activation of mast cells, ssON could be used as a prospective drug candidate to resolve itch and inflammation in certain dermatoses.
Highlights
Numerous inflammatory skin disorders exhibit an increased or dysregulated expression of antimicrobial peptides, which is often associated with pro-inflammatory, innate immune responses [1]
To determine the effect of single-stranded oligonucleotide (ssON) on IgEindependent degranulation, we treated a human mast cell (MC) line, LAD2, with a variety of degranulating compounds, with or without ssON (Figure 1). These compounds included: compound 48/80 (C48/80), a potent, synthetic MC degranulator extensively used in field; LL-37, a naturally occurring antimicrobial peptide of high interest due its potential involvement in a variety of skin disease including rosacea and psoriasis; substance P (SP), a neuropeptide associated with itch and inflammation; and A23187, a calcium ionophore
We further found that ssON has the capacity to inhibit certain endocytic uptake routes utilized by ligands destined for Toll-like receptor 3 (TLR3)/4 and 7 expressing endosomes, thereby preventing TLR signaling in human monocyte-derived dendritic cells and peripheral blood mononuclear cells [22]
Summary
Numerous inflammatory skin disorders exhibit an increased or dysregulated expression of antimicrobial peptides, which is often associated with pro-inflammatory, innate immune responses [1]. An elevated presence of LL-37 has been reported in the lesional skin of rosacea patients but has been linked to the disease pathology and induction of inflammation in the skin of mice [4]. Itch, or pruritus, presents as a dominant symptom in a multitude of cutaneous disorders [5, 6], and elicits the desire or reflex to scratch, which often exacerbates the disease [7]. The differential induction of itch is due to a vast and diverse assortment of pruritogens, which are substances that induce itch-mediated signaling by interacting with cellular detectors [i.e., G proteincoupled receptors (GPCR), ion channels] expressed mainly on nerve fibers [6]. Histamine, which is one of the most well-characterized endogenous pruritogens [10], is predominantly stored in mast cell (MC) secretory vesicles
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