Although treatment of Crohn's disease (CD) with TNFa monoclonal antibodies is beneficial, many patients stop responding over time and become at risk of acquiring infections. This study investigated an alternative approach to attenuate the release of excess TNFα by silencing iRHOMs 1/2 with the intention of restoring TGFβ-mediated Immunosuppression through Mitigation of TNFα converting enzyme (TACE) Trafficking. TACE, a transmembrane metalloprotease involved in signaling of receptors/cytokines/growth factors/adhesion proteins, is upregulated in CD, resulting in a marked increase of membranous TNFα cleavage and inflammation. Initially, we measured TGFβRII shedding in plasma from CD patients and healthy controls (N=40 per group). We also measured the expression and production of TGFβ ligand, TGFβRII shedding, TNFα, IL-6, IL-1β, IL-10, and global versus membranous TACE production in CD-like macrophages during infection. Furthermore, we transfected CD-like macrophages with iRHOM siRNA during infection and measured the phosphorylation of Smad3, and the expression and production of key cytokines, and plasminogen activator inhibitor-1 (PAI-1). The impact of TNFα release and TGFβRII shedding in co-cultured Caco-2 monolayers was measured via Dihydroethidium (DHE) staining, NOX-1 gene expression analysis, and NADP/NADPH quantification. TGFβRII shedding was significantly higher in CD plasma compared to healthy controls [515.52 ± 54.23 pg/mL vs 310.81 ± 43.16 pg/mL, respectively]. Plasma from CD patients infected with Mycobacterium avium subspecies paratuberculosis (MAP) had significantly more TGFβRII shedding (601.83 ± 49.56 pg/mL) than MAP-negative CD samples (430.37 ± 45.73 pg/mL). TACE production, TGFβ expression and production, and TGFβRII shedding were also higher in MAP-infected macrophages compared to the controls. Following iRHOMs siRNA transfection, TACE expression, production, and membrane localization were significantly decreased. There was significant decrease in the expression and production of TGFβRII shedding, TNFα, IL-6, and IL-1β, and PAI-1; and an increase in IL-10 and Smad3 phosphorylation. Overall, there was decrease in inflammation and Caco-2 oxidative stress. The data clearly demonstrated that silencing iRHOMs 1 and 2 has significantly reduced TACE membrane trafficking, restored TGFβ-mediated immunosuppression, reduced the inflammatory response, and ultimately reversed tissue damage. The study provides a framework for new and safer therapeutic options to treat autoimmune disease including CD and Rheumatoid Arthritis.
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Master of Science (M.S.)
College of Medicine
Burnett School of Biomedical Sciences
Length of Campus-only Access
Masters Thesis (Open Access)
Louis, Taylor, "Attenuation of Excess TNFα Release in Crohn's Disease by Silencing of iRHOMs 1/2 and the Restoration of TGFβ-mediated Immunosuppression Through the Modulation of TACE Trafficking" (2022). Electronic Theses and Dissertations, 2020-. 1046.