Author ORCID Identifier
https://orcid.org/0000-0002-9209-9559
Defense Date
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Microbiology & Immunology
First Advisor
John Ryan
Second Advisor
Aron Lichtman
Third Advisor
David Straus
Fourth Advisor
Rebecca Martin
Fifth Advisor
Eddie Wook-Jin Chae
Abstract
Allergic diseases such as asthma involve complex inflammatory pathways, with mast cells playing a central role in mediating allergic responses through degranulation and cytokine release that regulate the immune environment. Current therapies often provide suboptimal control, highlighting the need for novel or repurposed treatments. This dissertation investigates the potential of fluoxetine, a selective serotonin reuptake inhibitor, and simvastatin, a widely prescribed cholesterol lowering drug, as modulators of mast cell function in allergic inflammation through inhibition of molecular pathways involved in IL-33 and IgE-mediated signaling. Prior data from our lab testing the effects of fluoxetine on IgE-driven mast cell function indicated a role for the purinergic receptor, P2X3. Purinergic receptors are ATP-gated cation channels most often associated with nerve function but are also expressed on the surface of mast cells. IgE cross-linkage elicits a rapid release of ATP from mast cells, suggesting that ATP-P2X3 receptor signaling may potentiate IgE-driven responses. We hypothesized that fluoxetine inhibits mast cell function by binding to the ATP pocket on P2X receptors via its strongly negative trifluoromethyl (CF3) group. We show that genetic deletion of P2X3 had no effect on the function of fluoxetine, indicating that fluoxetine does not inhibit IgE-mediated mast cell function through P2X3 interactions. Fluoxetine inhibition did present with a sexually dimorphic phenotype in a model of IgE-induced passive systemic anaphylaxis (PSA), in which the drug reduced hypothermia only in females. Therefore, identifying the fluoxetine target could aid in our fundamental understanding of why some allergic diseases show female dominance. Despite finding that P2X3-/- mast cells remained fluoxetine-responsive, we uncovered interesting and unexpected effects of P2X receptors in this study. We found that P2X2 and P2X3 do play a minor role in IgE-mediated mast cell function, in a female-dominant manner. Genetic deletion of the P2X3 receptor reduced hypothermia during IgE-mediated PSA in a 50/50 split, and only in females. Further, while P2X3-/- mast cells had no change in IgE responses, deleting both P2X2 and P2X3 inhibited IgE-induced cytokine production only in female mast cells in vitro. These data support the hypothesis that P2X2 and 3 must have redundant and female-dominant roles in IgE-mediated mast cell function. Further investigation is needed. Our lab has previously shown that cholesterol-lowering statin drugs can suppress IgE-mediated mast cell function by inhibiting protein isoprenylation, which uses cholesterol pathway intermediates. In addition to IgE, mast cells are also activated by the alarmin IL-33, released by epithelial cells after contact with cellular stressors. We hypothesized that IL-33-mediated mast cell function can be inhibited by disrupting isoprenylation via statins or the dual isoprenyl transferase inhibitor, FGTI-2734. We show that simvastatin and FGTI-2734 suppress IL-33-mediated cytokine mRNA and protein production in mouse mast cells and eosinophils. Investigating the signaling mechanisms revealed that simvastatin and FGTI-2734 did not suppress IL-33-induced phosphorylation of JNK, ERK, P38, or NFκB, and did not change expression of the IL-33 receptor, ST2. In an in vivo model of peritonitis, FGTI-2734 suppressed IL-33-mediated neutrophil and eosinophil influx. Simvastatin, however, did not suppress peritonitis. Lastly, we saw modest inhibitory effects of simvastatin or FGTI-2734 on IL-33- mediated cytokine production by human mast cells in preliminary studies. These findings indicate that targeting isoprenylation via statins or selective isoprenyl transferase inhibitors is a viable target in IL-33-mediated mast cell function. Further investigation into the mechanism of action is required.
Rights
© Jason R. Burchett
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
12-9-2024