Abstract

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.

Notes

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Graduation Date

2022

Semester

Spring

Advisor

Naser, Saleh

Degree

Master of Science (M.S.)

College

College of Medicine

Department

Burnett School of Biomedical Sciences

Degree Program

Biotechnology

Format

application/pdf

Identifier

CFE0009017; DP0026350

URL

https://purls.library.ucf.edu/go/DP0026350

Language

English

Release Date

May 2023

Length of Campus-only Access

1 year

Access Status

Masters Thesis (Campus-only Access)

Restricted to the UCF community until May 2023; it will then be open access.

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