Attenuation of Excess TNF-α Release in Crohn’s Disease by Silencing of iRHOMs 1/2 and the Restoration of TGF-β Mediated Immunosuppression Through Modulation of TACE Trafficking

Abstract

TNFα 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 TNFα secretion and inflammation. Although treatment of CD with TNFα monoclonal antibodies is beneficial, many patients are at risk for acquiring opportunistic infections, and the treatment efficacy of TNFα monoclonal antibodies typically decreases over time. This study investigated an alternative approach for mitigating TNFα release by knocking down TACE membrane translocation in macrophages via inhibitory rhomboid proteins 1 and 2 (iRHOMs 1/2) siRNA treatment. First we measured TGFβRII shedding in ex vivo plasma samples collected from CD patients and healthy control subjects (N=40 per group). Then, we measured TGFβRII shedding and the expression and production of TGFβ ligand, TNFα, IL-6, IL-1β, 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 TGFβRII 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 TGFβRII 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; TGFβ ligand expression and production; and TGFβRII 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 TGFβRII 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 TNFα and restores the immunosuppressive capabilities of TGFβ 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.

Attenuation of Chronic Inflammation in Intestinal Organoids with Graphene Oxide-Mediated Tumor Necrosis Factor-α_Small Interfering RNA Delivery

“…In the context of chronic or acute inflammation due to injury or infection, cells in the affected tissue produce elevated and high levels of TNF-α cytokine, triggering the production of other inflammatory cytokines, thereby intensifying the inflammatory response. ,, Inhibition of TNF-α has been shown to restore immunosuppressive capabilities, reduce the release of other cytokines, such as IL-1β and IL-6, and attenuate inflammatory damage in IBD. ,, Thus, we further validated gene silencing by evaluating secreted cytokine levels, revealing significant downregulations of TNF-α and its downstream effectors, IL-1β and IL-6 (as shown in Figure B-D), with smGO exhibiting the most pronounced reduction, while bmGO-mediated transfection had no effects compared to the controls. Similar downregulation of TNF-α (4-fold) and IL-6 (3-fold) cytokines has been reported for LPS-induced mouse macrophages upon transfection with TNF-α_siRNA .…”

Mycobacterium avium paratuberculosis Infection Suppresses Vitamin D Activation and Cathelicidin Production in Macrophages through Modulation of the TLR2-Dependent p38/MAPK-CYP27B1-VDR-CAMP Axis

“…We prepared the knockdown transfection mix as described earlier by Vaccaro et al, 2022 [4], and Louis et al, 2022 [22]. Briefly, 5 nmol of Silencer™ pre-designed siRNA (siRNA ID: 4392420) specific to TLR2 was diluted in 50 µL nuclease-free water.…”

“…Caco-2 monolayers were grown at a density of 5 × 10 5 cells/2 mL in Falcon 8-well chambered cell culture slides (ThermoFisher, Waltham, MA, USA) as described earlier [22]. DHE fluorescence staining was performed on the monolayers following 24 h of treatment with supernatant from MAP-infected THP-1 macrophages.…”

“…Slides were examined under an Amscope IN480TC-FL-MF603 fluorescence microscope, where blue staining represents nuclei and red indicates oxidative stress. Images were captured on 100× magnification power and analysis was conducted by measuring the average integrated density using NIH Image J 1.39o software as described previously [22]. Merged images of DAPI and DHE stains were generated by the same software.…”

The dual role of interleukin-6 in Crohn’s disease pathophysiology

“…TACE is also involved in the shedding of many inflammatory cytokines and their receptors including IL-6R, which mediates the trans-signaling on gp130-expressing cells (51). TACE membrane trafficking increases in macrophages during Mycobacterium avium paratuberculosis (MAP) infection and in IBD, which results in an upregulation of proinflammatory cytokine release (51,52). iRhom1 and iRhom2 are to a large extent redundant and must be abolished together to achieve an effect on TACE membrane trafficking (51).…”