Date of Award

Summer 2019

Document Type

Open Access Dissertation

Department

Biomedical Science

First Advisor

Gregorio Gomez

Second Advisor

Jason Kubinak

Abstract

Mast cells (MCs) are well known for their implications in allergic reactions. They are also known to have multiple functions in the innate and adaptive immune system. Their activation plays an essential role in many aspects of physiological and pathological conditions. Allergies are considered chronic conditions that affect more than 60 million people in the U.S. These diseases are driven by the activation of MCs in response to IgE-mediated antigen, rendering these cells as targets for the management of allergies and asthma. Therefore, this research considers the understanding of their activation and the regulation of their response to be an important step for the management of allergies. Previous studies have shown the role of adenosine in the modulation of the activation of mast cells through its interaction with its receptors in allergic asthma. In this study, we investigated the role of the adenosine receptors, and particularly the A2A subtype, on the regulation of allergic mediators from human skin mast cells. We demonstrated that A2A signals inhibit FcɛRI–induced proinflammatory cytokines via cAMP mechanism. However, the A2A receptor has no effects on FcɛRI-induced degranulation or PGD2 production. We also showed that FcɛRI signaling plays a significant role in the modulation of the expression and the function of adenosine receptors on mast cells. We found that sub-threshold stimulation of FcɛRI led to up-regulation of the A2A, and to down-regulation of the A3 receptors at the mRNA and protein levels. Additionally, we observed that up-regulation of the A2A receptors by sub-threshold of FcɛRI led to be more pronounced inhibition of TNF by adenosine, which shifts mast cells into anti-inflammatory phenotype.

Next, we explored the role of miR-155 in MCs function. MiRNAs have been reported to regulate different genes involved in MCs activation, and miRNAs impact the function of MCs in various allergic reactions.

Research indicates miR-155 plays a key regulatory role in the pathogenesis of allergy. In this project, miR-155 expression was induced following IgE-receptor crosslinking on human skin mast cells as well as mast cells derived from bone mouse bone marrow (BMMCs). MiR-155 had no effect on IgE-dependent degranulation or leukotriene C4 secretion. Accordingly, arachidonate 5-lipoxygenase (ALOX5) expression was similar in WT and miR-155 KO BMMCs.

In contrast, FcɛRI-induced COX-2 expression was significantly diminished in the absence of miR-155, suggesting that miR-155 plays a critical role in prostaglandin D2 biosynthesis. In addition, FcɛRI-induced TNF, IL-6, and IL-13 was significantly diminished in miR-155 KO BMMCs compared to WT. Interestingly, the amount of these cytokines from miR-155 KO BMMCs increased compared to WT following LPS treatment. The phosphorylation of AKT was significantly decreased in miR-155 KO compared with WT following FcɛRI crosslinking, whereas p38, and p42/p44 phosphorylation were the same in both types of mast cells. Collectively, these data demonstrate that miR-155 has both a positive and a negative regulatory action on the mast cell mediator release.

Recently, many studies have revealed that the natural polyphenol Resveratrol exhibits different biological and pharmacological properties, including anti- allergic effect. In the current study, experiments were designed to study the role of miRNAs in Resveratrol-mediated inhibition COX-2 expression in the activated mast cells. We showed that Resveratrol inhibited FcɛRI-induced COX-2 production in WT BMMCs and failed to inhibit COX-2 expression in miR-155 KO BMMCs. MiRNA array analysis and Ingenuity Pathway Analysis (IPA) revealed an altered miRNAs profile following Resveratrol treatment. One of the miRNAs that was significantly downregulated after Resveratrol treatment was miR-155. We then validated the miRNA array and IPA analysis by qRT-PCR. According to our results, there was a positive correlation between miR-155 and COX-2 expression in activated SMCs, in which both were downregulated after RSV treatment. However, the ATF3 expression was increased, which suggests that miR-155 could be the target of COX-2 expression. Collectively, Resveratrol inhibits FcɛRI-induced COX-2 expression through inhibition miR-155 in mast cells. Therefore targeting miR-155- mediated inhibition of COX-2 by Resveratrol may serve as a new approach for the treatment of the allergic condition.

Rights

© 2019, Zahraa Abdulmohsin Mohammed

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