TNFalpha converting enzyme (TACE) is a transmembrane metalloprotease that sheds an assortment of signaling receptors, cytokines, growth factors, and pro-inflammatory mediators. In Crohn's disease (CD), TACE activity is upregulated, resulting in a marked increase of TNFalpha secretion and inflammation. Although treatment of CD with TNFalpha monoclonal antibodies is beneficial, many patients are at risk for acquiring opportunistic infections, and the treatment efficacy of TNFalpha monoclonal antibodies typically decreases over time. This study investigated an alternative approach for mitigating TNFalpha release by knocking down TACE membrane translocation in macrophages via inhibitory rhomboid proteins 1 and 2 (iRHOMs 1/2) siRNA treatment. First we measured TGFbetaRII shedding in ex vivo plasma samples collected from CD patients and healthy control subjects (N=40 per group). Then, we measured TGFbetaRII shedding and the expression and production of TGFbeta ligand, TNFalpha, IL-6, IL-1beta, IL-10, and total versus membranous TACE in vitro with THP-1 derived macrophage infected with Mycobacterium avium subspecies paratuberculosis (MAP), a highly studied CD-related pathogen. We determined that TGFbetaRII shedding was significantly higher in CD patients compared to healthy controls [515.52 +/- 54.23 pg/mL vs 310.81 +/- 43.16 pg/mL, respectively], and MAP-infected CD plasma samples had significantly more TGFbetaRII shedding (601.83 +/- 49.56 pg/mL) than MAP-negative CD samples (430.37 +/- 45.73 pg/mL). Moreover, we also determined that TACE production; TGFbeta ligand expression and production; and TGFbetaRII shedding were also higher in MAP-infected THP-1 macrophages. Nevertheless, once we transfected the MAP infected macrophages with iRHOM siRNA, TACE production and membrane localization were significantly decreased, resulting in a significant decrease in TGFbetaRII shedding; an increase in Smad3 phosphorylation; a decrease in the expression and production of pro-inflammatory cytokines; and a decrease in the expression and production of stricture-associated factor, plasminogen activator inhibitor-1 (PAI-1). Our data clearly demonstrates that the regression of TACE trafficking, via iRHOM 1/2 silencing, significantly reduces the release of TNFalpha and restores the immunosuppressive capabilities of TGFbeta signaling, which ultimately reverses inflammatory tissue damage. Accordingly, this study may provide a framework for the creation of newer, safer therapeutic options designed to treat inflammatory autoimmune diseases such as CD and rheumatoid arthritis.