Larazotide acetate regulates epithelial tight junctions in vitro and in vivo
Highlights
► Larazotide acetate (LA) inhibits gliadin-induced actin and ZO-1 redistribution. ► LA inhibits cytokine-induced permeability. ► LA inhibits gliadin translocation across Caco-2 cell monolayers. ► LA preserves tight junction structure and function in gluten-sensitive mice. ► LA is a novel compound with therapeutic value in celiac disease management.
Introduction
Multicellular organisms can survive only if they establish a distinct internal environment, separating “self” from the “non-self” environment. The mucosal surfaces of the genitourinary, gastrointestinal, and respiratory tracts have epithelial barriers that are “sealed” by tight junctions (TJs) [31], [40]. TJs are located at the apico-lateral borders of adjoining epithelial cells, regulating the patency of the paracellular space and limiting the bidirectional diffusion of particles, water, and solutes across mucosal surfaces. TJs consist of over 50 proteins, including the transmembrane proteins junctional adhesion molecule (JAM), occludin, and claudin, and the cytoplasmic scaffolding proteins zonula occludens (ZO)-1, ZO-2, and ZO-3 [1], [16].
In the absence of inflammation or epithelial disruption, the functional state of the TJ determines paracellular permeability and polarity [33]. TJs are highly dynamic, opening and closing in response to cytoskeletal reorganization that occurs upon exposure to external antigens, such as gliadin in celiac disease [30], and internal inflammatory cytokines, such as TNF-α [31]. Increased exposure of the submucosa to external antigens is putatively caused by “barrier dysfunction” in the small bowel, and “intestinal leak” is associated with numerous intestinal and extra-intestinal autoimmune conditions, including celiac disease, inflammatory bowel disease, type 1 diabetes, multiple sclerosis, primary biliary cirrhosis, autoimmune hepatitis, and systemic sclerosis [14], [15], [42].
Celiac disease, an autoimmune disorder, is triggered by gluten/gliadin or gliadin fragments in individuals with the HLA-DQ2/DQ8 allele that facilitates presentation of gliadin peptides to T cells [20]. Gliadin induces a loss of barrier function and stimulates innate and adaptive immune responses, followed by intestinal damage [29]. Gliadin also disrupts TJ integrity by altering actin and ZO-1 distribution in intestinal epithelial cells [9], [12], [30]. Alterations in ZO-1 phosphorylation and expression have also been reported in response to gliadin and in celiac disease mucosa, respectively [7], [30].
Protease-resistant gliadin peptides are transported, by as yet unknown mechanisms, from the lumen of the gut to the lamina propria, where tissue transglutaminase catalyzes the conversion of glutamine in certain peptides to glutamate. Deamidated versions of the gliadin peptides cause immune activation and initiate an inflammatory cascade with the production of IFNγ and other cytokines [17]. One of the “toxic” gliadin peptides, P31–43 (13-mer), also induces intestinal permeability and stimulates cytokine and chemokine production by macrophages in vitro [39]. Inflammatory cytokines have been shown to enhance the permeability of the gut epithelium [2], [8], which could lead to the translocation of more gliadin. Currently, a gluten-free diet is the only management that helps to alleviate the symptoms of celiac disease [10]. Therefore, there is a great need for novel therapeutic approaches that target the earliest event at the mucosal surface to inhibit barrier dysfunction, and hence prevent immune activation.
We have previously shown that AT-1002, a synthetic peptide comprising the first 6 amino acids of the active fragment of zonula occludens toxin (ZOT) from Vibrio cholerae, lowers transepithelial electrical resistance (TEER) and increases TJ permeability and alters ZO-1 and actin distribution in Caco-2 cells [19]. As mentioned previously, gliadin also elicits similar responses in intestinal epithelial cells. It is also well established that cytokines decrease TEER and induce paracellular permeability in epithelial cells [32], [45]. We have developed an 8-mer peptide and TJ regulator larazotide acetate that has therapeutic value for celiac disease owing to its ability to inhibit early mucosal events that lead to barrier dysfunction and immune activation [26]. Furthermore, ZOT-induced immune responses are inhibited by intranasal administration of larazotide acetate [22], and oral larazotide acetate reduces the incidence of type I diabetes in BB Wor/DP rats [46]. In this study, we examined the mechanism by which larazotide acetate inhibits TJ opening caused by AT-1002, gliadin, and cytokines. We studied effects on TJ and cytoskeletal organization in vitro, and examined whether the “epithelial leak” induced by various stimuli was inhibited by larazotide acetate. Finally, we examined whether these in vitro effects of larazotide acetate on TJs could be translated in vivo to a gluten-sensitive transgenic HLA-HCD4/DQ8 mouse model developed at the Mayo clinic [5]. In this mouse model, gluten sensitization induces changes in intestinal permeability and innate immune cell infiltration making it an excellent model to test potential therapeutics for gluten-induced effects [25], [28].
Section snippets
Reagents
The permeability inducer AT-1002 and the 13-mer gliadin peptide AT-4067 were synthesized using F-moc solid phase chemistry as described previously [19]. The final products were isolated as TFA salts in a lyophilized form (>95% purity by HPLC/MS).
Larazotide acetate (AT-1001) was synthesized using solution phase synthesis [9], and the identity of the compound was confirmed by LC/MS. The final product was isolated as an acetate salt in a lyophilized form (>99% purity by HPLC/MS).
Pepsin-trypsin
Effects of larazotide acetate on AT-1002-induced TJ dysfunction in Caco-2 cells
We have previously shown that AT-1002 induces TJ barrier dysfunction in human-derived Caco-2 cells [19]. To determine the effects of larazotide acetate on AT-1002-induced barrier dysfunction, we measured the mean LY passage across Caco-2 cell monolayers following exposure to AT-1002 in the presence or absence of larazotide acetate (Fig. 1). AT-1002 caused a substantial increase in LY passage, which was inhibited by larazotide acetate in a dose-dependent manner. Larazotide acetate at 15 and 12.5
Discussion
“Barrier dysfunction” and “intestinal leak” are associated with numerous intestinal and extra-intestinal autoimmune conditions, and barrier function is a new target for drug development, drug delivery, and adjuvant development. In this study, we examined the effects of larazotide acetate, an 8-mer peptide and TJ regulator [10], [26], on TJ disruption caused by AT-1002, gliadin, and proinflammatory cytokines.
We have previously shown that AT-1002, a ZOT-derived peptide, disrupts TJs, causes actin
Acknowledgments
This work was partially supported by grants from the Canadian Association of Gastroenterology (CAG)/Canadian Institute of Health Research (CIHR) (GN2-114709), the Canadian Celiac Association New Investigator Award (to E. Verdu), and ALBA Therapeutics. E. Verdu holds a McMaster University Dep. of Medicine Internal Career Research Award. Joseph Murray was supported by NIH grant DK 70031. We are grateful to Drs. Alessio Fasano, Linda Arterburn, and Francisco Leon for critical reading of the
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