Inflammatory bowel diseases (IBDs) are chronic disorders affecting the gastrointestinal (GI) tract in humans. The etiology of IBD is not completely understood; however, the pathogenesis is associated with contributions from genetic predisposition, the mucosal immune system, and exposure to environmental factors.1 Genome-wide association studies for IBD susceptibility loci have revealed the importance of epithelial barrier function and innate and adaptive immunity in disease pathogenesis.1 A key factor in the development of mucosal inflammation associated with IBD is disturbed intestinal epithelial barrier function and increased intestinal permeability. Decreased barrier function has been shown to positively correlate with mucosal inflammation in Crohn's disease (CD) and ulcerative colitis (UC) patients,2 and increased epithelial permeability precedes clinical relapse.3, 4 Additionally, first-degree relatives of CD patients who are at risk of developing disease often have increased intestinal epithelial permeability relative to the general population.5 In animal models of IBD, increased epithelial paracellular permeability can precede chronic mucosal inflammation,6 and in addition, altered epithelial barrier function has been associated with the subsequent development of colitis.7
The integrity of mucosal epithelial barrier function is preserved by the intestinal epithelium, which regulates the trafficking of macromolecules between the environment and the host and serves as a central coordinator of communication between the immune system and the external environment.8 The intestinal epithelium is comprised of apical and subapical junctional complexes, which seal the paracellular space and regulate mucosal barrier permeability. While substantial progress has been achieved in understanding structural changes associated with barrier disruption and reassembly at the molecular level, regulatory pathways that dynamically control intestinal barrier homeostasis remain less well understood. Recently, the type II transmembrane serine protease, matriptase (also known as ST14, MT-SP1, TADG-15, epithin, and SNC199), was identified as a factor critical for maintenance of epithelial barrier homeostasis. Matriptase is localized to apical junctional complexes and on the basolateral surfaces of polarized intestinal epithelium and demonstrates potent epithelial barrier protective properties.10, 11 Initial analyses of mice with complete deficiency in the matriptase gene (St14) uncovered a critical function for matriptase in skin development and function.12 Matriptase knockout mice die shortly after birth, due to a severe dehydration caused by impaired epidermal barrier. Conditional knockout of matriptase from the murine GI tract under the direction of a Villin-Cre promoter results in persistent diarrhea, inflammation, edema, gross disruption of colonic architecture, and general loss of mucosal barrier function that leads to rapid tissue degeneration and death within a few weeks after weaning, although at birth the microarchitecture of the GI tract appears structurally and morphologically intact.10 Constitutive expression of matriptase is required throughout adulthood to maintain intestinal barrier integrity, since inducible ablation of matriptase from the GI tract of adult mice also results in loss of mucosal barrier function and rapid tissue degeneration.10
The importance of intestinal epithelial barrier function to the etiology of IBD and the functional link between matriptase and paracellular permeability in the GI tract prompted us to investigate the role of matriptase in IBD. Here we report that matriptase expression is dramatically suppressed in colonic mucosa during human and experimental IBD. St14 hypomorphic mice, which express minimal levels of matriptase and are born free of intestinal disease,11, 13 are more susceptible to dextran sodium sulfate (DSS)-induced colitis, with the normally transient DSS-induced intestinal injury converted to a severe, persistent colitis, with reduced mouse survival. These data demonstrate a critical role for matriptase in the restoration of barrier function following injury of the GI tract, and suggest that matriptase dysregulation may contribute to the pathogenesis of IBD.
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- MATERIALS AND METHODS
- Supporting Information
Compromised epithelial barrier function is considered an important pathophysiologic basis for IBD. The increased permeability enhances the transport of microbial flora and other antigenic material from the intestinal lumen into the submucosa, resulting in a recurring cycle of inflammation and mucosal injury.29 A consequence of excessive activation of the immune system can be the weakening of countering mechanisms critically required to restore normal homeostasis. Here we show that matriptase, an enzyme critical for maintenance of epithelial homeostasis, is a key factor in the restoration and maintenance of intestinal barrier function after injury, and that its downregulation by inflammatory cytokines generated by activation of the immune system contributes to perpetuation of disease severity and impaired mucosal recovery.
Mouse models of IBD have shown that a compromised epithelium is sufficient to cause intestinal inflammation, and that resident microbial flora is necessary for colitis induction.33 An initiating event, whether genetic, immune, or environmental, that enhances barrier permeability can initiate activation of the innate immune system given the exceptional challenge provided by the dense microbial flora unique to the gut. Indeed, complete genetic deficiency of matriptase abrogates epithelial barrier function resulting in acute and severe inflammation along with massive tissue destruction.10 Although the minimal levels of matriptase expressed by St14 hypomorphic mice weaken barrier function, they do not result in spontaneous disease in the absence of an initiating trigger. Thus, the ST14 hypomorph represents a pathologically relevant model of disease susceptibility in patients who have increased intestinal epithelial permeability and are at risk of developing disease.
In the DSS model of colitis, an initiating trigger, e.g., exposure to DSS, causes damage to the epithelial layer and initiates activation of innate immune responses.25 Persistent inflammation associated with DSS injury in the ST14 hypomorphic mice was not caused by a more robust inflammatory response but was related to the inability to recover barrier function after the DSS insult was removed. Matriptase downregulation occurs secondary to the inflammatory response and, in control mice, restoration of normal matriptase levels during the recovery phase promotes colitis resolution. In St14 hypomorphic mice the inability to restore normal matriptase levels prevents gut barrier recovery and perpetuates the destructive inflammation. These data highlight the critical role of host barrier protective mechanisms in the resolution of colitis, which can become suppressed by excessive activation of the immune system that drives tissue damage.
The specific molecular pathways initiated by matriptase activity in the intestinal epithelium are incompletely understood.11 Matriptase colocalizes with E-cadherin to apical junctional complexes and evidence suggests that it is required for interepithelial junction formation.10, 11, 34 Matriptase has also been implicated in the control of epithelial-cell turnover by regulating cell-cell and/or cell-substratum adhesions,35 and in the removal of aged epithelial cells in the small intestine through detachment from the basement membrane component laminin.36 Substrates that are targeted by matriptase proteolytic activities in other cell systems,37 pro-urokinase-type plasminogen activator (pro-uPA) and protease activated receptor-2 (PAR-2) do not appear to mediate matriptase intestinal barrier protective activities, since mice deficient in uPA or PAR-2 do not show similar susceptibility to DSS-induced colitis (Fig. S6). Altered pericellular activation of HGF may be involved in this phenotype since matriptase is an activator of pro-HGF38 and HGF/c-met signaling is important for repair of injured mucosa.39, 40 Interestingly, conditional knockout of HAI-1 in the intestine of mice was shown recently to enhance susceptibility to DSS-induced colitis, possibly caused in part by deregulated matriptase activity.41
Increased inflammatory cytokine production is associated with mucosal inflammation, and studies in vitro and in animal models demonstrate that inflammatory cytokines associated with IBD cause tight junction barrier dysfunction.42 Matriptase is downregulated in inflamed intestinal epithelia of IBD patients (Fig 1A), and by the inflammatory cytokines IL-13 and IL-4 in in vitro cultures (Fig. 7A). IL-13 enhances barrier permeability in part by stimulating the synthesis of claudin-220, 26, 43 and we have shown that matriptase deficiency results in increased claudin-2 expression in both Caco-2 monolayers and in St14 hypomorph intestinal tissues.11 Endogenous mechanisms that regulate matriptase expression and activation in the gut are not currently known. Nonetheless, in vitro data suggest that restoration of matriptase expression and activity could interrupt the cytokine-mediated inflammatory cycle to promote epithelial repair and disease resolution.
While dysregulated inflammation induced by immunogen exposure is considered a major cause of mucosal tissue damage and injury in IBD, the present findings highlight the critical importance of host barrier repair mechanisms for restriction of the offending insult and its destructive consequences. Intestinal permeability has been shown to predict and possibly cause relapse in patients suffering from IBD.3, 44 A better understanding of the role of matriptase in controlling mucosal homeostasis by regulating the integrity and permeability of epithelial barrier function will be important since matriptase-based therapeutic strategies could have application for preventing, treating, or altering the natural progression of IBD.