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.

Keywords: Crohn's disease; TACE (TNF-α converting enzyme); ectodomain shedding; iRHOM 1/2; mycobacteria paratuberculosis; rhomboid proteins.

Figure 1

TGFβRII shedding in clinical plasma samples obtained from healthy control subjects, MAP-negative CD patients, and MAP-positive CD patients (N= 40 per group). The Kolmogorov–Smirnov normality test was used to test the normal distribution for all values and a two-way analysis of variance (ANOVA) was used to assess significance among experiments, which was followed by a Bonferroni correction test. All experiments were performed in triplicates. *P-value < 0.05 compared to healthy controls. ^#P-value < 0.05 compared to healthy controls and MAP-negative CD patients.

Figure 2

TGFβRII shedding in bacteria infected THP-1 derived macrophages. The Kolmogorov–Smirnov normality test was used to test the normal distribution for all values and a two-way analysis of variance (ANOVA) was used to assess significance among experiments, which was followed by a Bonferroni correction test. All experiments were performed in triplicates. *Indicates P-value of <0.05.

Figure 3

The (A) expression and (B) production of TGF-β ligand in bacteria infected THP-1 derived macrophages. The Kolmogorov–Smirnov normality test was used to test the normal distribution for all values and a two-way analysis of variance (ANOVA) was used to assess significance among experiments, which was followed by a Bonferroni correction test. All experiments were performed in triplicates. *Indicates P-value of less than 0.05.

Figure 4

The impact of an iRHOM 1/2 siRNA mock transfection of the expression of (A) TNF-α and (B) TACE. The Kolmogorov–Smirnov normality test was used to test the normal distribution for all values and a two-way analysis of variance (ANOVA) was used to assess significance among experiments, which was followed by a Bonferroni correction test. All experiments were performed in triplicates. The difference in gene expression among the control and treated groups was not significant (NS).

Figure 5

The total concentration of TACE within bacteria infected cells (A) and the impact of TACE regression on the concentration of (B) transmembrane TACE levels and (C) TGFβRII shedding. The Kolmogorov–Smirnov normality test was used to test the normal distribution for all values and a two-way analysis of variance (ANOVA) was used to assess significance among experiments, which was followed by a Bonferroni correction test. All experiments were performed in triplicates. *Indicates P-value of less than 0.05.

Figure 6

The impact of TACE regression on the downstream phosphorylation of Smad3. The Kolmogorov–Smirnov normality test was used to test the normal distribution for all values and a two-way analysis of variance (ANOVA) was used to assess significance among experiments, which was followed by a Bonferroni correction test. All experiments were performed in triplicates. *Indicates P-value of less than 0.05.

Figure 7

The impact of TACE regression on the expression of (A) TNFα, (B) IL-1β, (C) IL-6, and (D) IL-10. The Kolmogorov–Smirnov normality test was used to test the normal distribution for all values and a two-way analysis of variance (ANOVA) was used to assess significance among experiments, which was followed by a Bonferroni correction test. All experiments were performed in triplicates. *Indicates P-value of less than 0.05.

Figure 8

How regressing TACE trafficking affects (A) the expression of NADPH oxidase-1 (NOX-1) and (B) the resulting ratio of NADP⁺/NADPH in co-cultured Caco-2 monolayers. The Kolmogorov–Smirnov normality test was used to test the normal distribution for all values and a two-way analysis of variance (ANOVA) was used to assess significance among experiments, which was followed by a Bonferroni correction test. All experiments were performed in triplicates. *Indicates P-value of less than 0.05.

Figure 9

The impact of TACE regression on oxidative stress experienced by co-cultured Caco-2 monolayers. (A) The total nuclei are stained with DAPI blue and the DHE positive cells are stained in red; the merged cells are presented in pink. (B) The quantitative corrected DHE fluorescence integrated density from Caco-2 cells co-cultured with control and treated THP-1 macrophages. The Kolmogorov–Smirnov normality test was used to test the normal distribution for all values and a two-way analysis of variance (ANOVA) was used to assess significance among experiments, which was followed by a Bonferroni correction test. All experiments were performed in triplicates. *Indicates P-value of less than 0.05. All DHE fluorescence staining experiments were performed in triplicates.

Figure 10

The impact of TACE regression on the (A) expression and (B) production of plasminogen activator inhibitor-1 (PAI-1). The Kolmogorov–Smirnov normality test was used to test the normal distribution for all values and a two-way analysis of variance (ANOVA) was used to assess significance among experiments, which was followed by a Bonferroni correction test. All experiments were performed in triplicates. *Indicates P-value of less than 0.05.

Figure 11

Inhibitory rhomboid proteins (iRHOM1/2) regulate TNF-α converting enzyme (TACE) membrane trafficking. Macrophages recognize M. paratuberculosis ManLAM through toll-like 2 receptors (TLR-2) and external mannose receptors (EMR). Upon binding, the p38 MAPK pathway is activated, which facilitates TACE trafficking from the ER to the cell surface via iRHOM1/2 regulation. Increasing TACE activity cleaves transmembranous TNF-α and induces TGFβRII shedding, which exacerbates inflammation and attenuates TGF-β signaling pathway. Silencing iRHOM1/2 downregulates TACE trafficking and maturation, which consequently decreases TNF-α cleavage and restores TGF-β signaling pathway.