While Th2-driven inflammation is a distinguishing feature of UC (3
), no currently approved therapies for its treatment specifically target Th2 lymphocytes, cytokines, or transcription factors associated with a Th2 immune response. Since IL-13 is a key Th2 cytokine in the pathogenesis of UC(4
), we sought to evaluate whether activation of STAT6, a transcription factor downstream of IL-13 signaling, is altered in UC, and whether STAT6 inhibition limits the effects of IL-13 on colon epithelial cells. To our knowledge, this study is the first demonstration of increased pSTAT6 in the epithelium of subjects with new-onset ulcerative colitis. Furthermore, we demonstrate that SAHA, a compound that inhibits constitutive STAT6 activation in lymphoma cell lines,(12
) inhibits IL-13-induced apoptosis, claudin-2 expression, and barrier dysfunction in colon epithelial cells.
Fuss and colleagues were the first to report the importance of IL-13 in UC by demonstrating that this cytokine is abundantly secreted by lamina propria lymphocytes from patients with advanced disease.(4
) The potential importance of IL-13 in the pathogenesis of UC is underscored by the finding that neutralization of IL-13 prevents oxazalone-induced colitis, a mouse model with similar features to human UC.(19
) These seminal studies, however, do not provide in situ
evidence that the colon epithelium is actually exposed to IL-13 in patients with UC. In fact, other groups examining cytokine levels from tissue homogenates or supernatants from organ culture have reported down regulation of IL-13 in UC.(20
) Our finding of increased pSTAT6 in the colonic epithelium of pediatric subjects with UC is evidence for IL-13-induced signaling and consistent with the notion that the colonic epithelium in UC is exposed to increased IL-13. Although IL-4 is known to also signal through STAT6, many investigators using various methods have demonstrated low or normal levels of IL-4 in patients with both UC and CD. (3
) While prior studies investigating IL-13 in UC used colectomy tissue from patients with severe or established UC, our findings are from tissues of pediatric patients at their diagnostic colonoscopies, which suggests a role for Th2 cytokine signaling in the early pathogenesis of UC.
We found that a subset of 4 patients with CD had increased epithelial pSTAT6 staining. Interestingly, 2 of these patients had strictly colonic involvement (without perianal disease or granulomas). The remainder of CD patients, with none to minimal epithelial pSTAT6, had both small bowel and colonic involvement. One possible explanation is the CD patients with only colonic involvement were misdiagnosed and truly had ulcerative colitis. Since the tissue specimens were obtained from a pathology repository, we did not have access to the entire detailed medical record to determine the clinical criteria on which each patient was diagnosed. However, in our practice, in the absence of granulomas, small bowel involvement, or perianal disease, patients would have to display clearly distinguishing signs of Crohn’s disease such as discrete apthous or linear ulceration, or skip lesions to be diagnosed with CD (as opposed to IBD-unspecified or UC). Alternatively, since CD is a phenotypically heterogeneous disorder, we can speculate that this finding could represent overlap in the pathogenesis of UC and a specific colonic subtype of CD. Interestingly, perinuclear antineutrophil cytoplasmic antibodies (pANCA) are another biomarker generally more specific for ulcerative colitis which, when present in patients with CD, are associated with a colonic phenotype and UC-like features.(27
) A larger prospective study of STAT6 signaling in the mucosa of patients with Crohn’s disease is needed to test this hypothesis.
Given our finding of increased pSTAT6 in UC and the established role of IL-13 in the disease, we hypothesize that STAT6 is a potential target against which to develop future UC therapies. We show that the two known mechanisms by which IL-13 directly increases colon epithelial permeability, induction of apoptosis and induction of claudin-2 expression(5
), are STAT6 dependent. Our results support the findings of Madden et al
who, using a STAT6 knockout mouse, demonstrated that IL-13-induced increases in mucosal permeability are STAT6-dependent.(29
) In contrast, Capons et al
found that in T84 cells, IL-13 regulation of epithelial permeability was not STAT6-dependent, but rather mediated by phosphoinositide 3-kinase signaling.(30
) There are several explanations for these conflicting results including the use of different cell lines and model systems, and different approaches to reduce or eliminate STAT6 signaling; in the current report we used siRNA, whereas Madden et al
used a knockout mouse, and Capons et al
used transcription factor decoys. Recently, STAT proteins have been shown to have cellular roles other than as transcription factors.(31
) Therefore, transcription factor decoys might not mitigate all the relevant cellular effects of STAT6 regulation of epithelial permeability.
The HDAC inhibitor SAHA has been shown to inhibit constitutive STAT6 activation in several lymphoma cell lines(12
), and we demonstrate that SAHA also inhibits IL-13-induced STAT6 activation in HT-29 colon epithelial cells. Furthermore, SAHA prevented IL-13-induced apoptosis without altering baseline apoptosis. SAHA and other HDAC inhibitors induce cell cycle arrest and apoptosis in many transformed cell lines while sparing normal cells.(33
) While HT29 cells are a transformed cell line, they are resistant to HDAC-inhibitor-induced apoptosis(36
), which more closely models the response of non-transformed intestinal epithelial cells in this regard.(37
Our finding that SAHA inhibits IL-13-induced claudin-2 expression in HT-29 cells is in line with those of Weber et al
who demonstrated the same results in T84 cells.(17
) Weber et al
also demonstrated that IL-13-induced reductions in TER are dependent on the induction of claudin-2. Here we add to these findings by demonstrating that SAHA alone is capable of abrogating the detrimental effects of IL-13 on TER.
While the mechanism underlying blockade of STAT6 activation by SAHA remains an important area for further investigation, the findings presented here suggest avenues for future investigation. SAHA inhibited both IL-13- and IL-4-induced STAT6 activation, suggesting that SAHA acts on elements common to the signal transduction cascade of both cytokines. SAHA did not affect expression of either the IL-4Rα subunit, which is a component of both the IL-4 receptor and IL-13Rα1 heterodimers, or the IL-13Rα1 subunit, which is only a part of the IL-13Rα1 heterodimer. Therefore, SAHA may act on IL-4Rα activation (as opposed to expression) or on components of both the IL-13 and IL-4 signaling cascade downstream of the receptors.
Both STAT1 and STAT3 are directly acetylated, and thus regulated by HDACs.(38
) In the case of STAT1, acetylating leads to recruitment of tyrosine phosphotases, rendering the protein resistant to persistent phosphorylation. Furthermore, treatment of human embryonic kidney cells with HDAC inhibitors prevents interferon alpha-induced STAT1 phosphorylation.(39
) Future studies will be needed to determine if direct acetylating of STAT6 regulates phosphorylation by a similar mechanism.
Our results suggest that the use of SAHA or other drugs that inhibit STAT6 activation may be a potential treatment strategy for ulcerative colitis. HDAC inhibitors have already shown potential in mouse models of Th1-mediated colitis, ostensibly due to their effects on T-cell function. (40
) SAHA and another HDAC inhibitor, trichostatin A (TSA), ameliorated dextran sodium sulfate-induced colitis and a T-cell transfer model of colitis in association with decreases in pro-inflammatory cytokine production and augmentation of the number of regulatory T-cells, and their function. Here we add to these findings by demonstrating a protective effect of SAHA and inhibition of STAT6 on a cell culture model of colon epithelium exposed to IL-13, a key cytokine involved in Th2-mediated colitis.
In conclusion, we demonstrate increased epithelial pSTAT6 in pediatric subjects newly diagnosed with ulcerative colitis. We further find that IL-13-induced human intestinal epithelial cell apoptosis and claudin-2 expression are STAT6 dependent. Lastly, SAHA protects colon epithelial cells from IL-13-induced apoptosis and epithelial barrier dysfunction, likely through inhibition of STAT6 activation. Thus, the role of SAHA, or other means of STAT6 inhibition as a novel Th2-inflammation-targeted therapy for UC warrants further investigation.