The Role of SAHA for Preventing IL-13-Induced Colon Epithelial Cell Dysfunction

Abstract

IL-13 has been implicated as an effector cytokine in the pathophysiology of ulcerative colitis (UC) through increasing colon epithelial cell apoptosis and expression of the pore-forming tight junction protein claudin-2. Binding of IL-13 to its receptor leads to activation of STAT6. The STAT6 phosphorylation status in patients with UC, however, is unknown, as is the effect of STAT6 inhibition on colonic epithelium exposed to IL-13. Suberoylanilide hydroxamic acid (SAHA) is a FDA-approved histone deacetylase inhibitor that inhibits constitutive STAT6 activation in lymphoma cell lines. Therefore, the aim of this study was to determine if STAT6 phosphorylation is increased in patients with UC, and if inhibition of STAT6 signaling with SAHA attenuates IL-13-induced apoptosis and claudin-2 expression. To assess IL-13 signaling, immunohistochemical staining for phosphorylated (p) STAT6 was performed on colonic tissue from pediatric subjects with UC (early UC, n= 10) and Crohn's disease (CD, n= 10) at diagnosis, colectomy tissue from adults with UC (late UC, n= 5) and controls (n= 10). A pathologist blinded to the diagnosis scored histological sections on a 0-4 scale for pSTAT6. In cell culture, human colon HT-29 cells were pre-treated with SAHA (1 or 5 μM) or vehicle control prior to treatment with IL-13 (10 ng/ml). Western blot analysis was performed on cellular lysates for pSTAT6 and claudin-2 with results quantified by densitometry. Apoptosis was determined by flow cytometry of Annexin V-PE-stained cells and by immunodetection of cleaved caspase-3. Epithelial pSTAT6 staining was increased in UC with median score of 0 in control subjects, 2 in early UC (vs. control P= 0.004), 4 in late UC (P= 0.014), and 0 in CD. In HT-29 cell culture, SAHA inhibited IL-13-induced pSTAT6 in a dose-dependent fashion with a 57% reduction of pSTAT6 induction at 5 μM (P < 0.01). IL-13 induced a 2-fold increase in claudin-2 expression (p < 0.05), and SAHA reduced IL-13-induced claudin-2 expression by 70% to baseline levels (p<0.05). By Annexin V staining, IL-13 increased apoptosis 2.7-fold (3.7 ±1.6% vs. 10.0±1.4% Annexin V+, P<0.01). SAHA alone did not increase apoptosis and reduced IL-13-induced apoptosis in a dose-dependent manner with complete suppression to baseline at 5 μM (IL-13 + SAHA 1 μM 7.5±0.8%, + 5 μM 4.8±0.9, Ptrend< 0.001). Similarly, by immunodetection of cleaved caspase-3, SAHA reduced IL-13 induced apoptosis by 57% at 1 μM and 94% at 5 μM (P<0.001 for each compared to IL-13 alone). Increased STAT6 phosphorylation in the colonic epithelium of pediatric subjects at presentation with UC supports an effector role of IL-13 early in the disease. Treatment of colonic epithelial cells with SAHA, a drug that inhibits IL-13-induced STAT6 activation, reduces IL-13-induced apoptosis and claudin-2 expression, suggesting that STAT6 may be a novel therapeutic target for UC.