Effect of anti-gliadin IgY antibody on epithelial intestinal integrity and inflammatory response induced by gliadin

Gujral et al. BMC Immunology (2015) 16:41 DOI 10.1186/s12865-015-0104-1 RESEARCH ARTICLE Open Access Effect of anti-gliadin IgY antibody on epithelial intestinal integrity and inflammatory response induced by gliadin Naiyana Gujral1, Ju Won Suh2* and Hoon H. Sunwoo1* Abstract Background: Pepsin-trypsin resistant gliadin (PT-gliadin) promotes intestinal tissue inflammation and increases paracellular permeability of immunogenic gliadin peptides into the lamina propria. This leads to the complications seen in the pathogenesis of celiac disease (CD). In this study, specific anti-gliadin IgY antibody was produced and evaluated for its efficacy on gliadin induced intestinal integrity impairment and proinflammatory effects on intestinal epithelial (Caco-2) cell culture model for CD. Methods: Caco-2 (passages 20-24) monolayers were subjected to 7 experimental conditions (n=3 each): phosphatebufferedsaline (PBS; control), pancreatic digested-casein (PD-casein; negative control), PT-gliadin (positive control), nonspecific IgY with PT-gliadin, and anti-wheat gliadin IgY with PT-gliadin at a ratio of 1:6,000, 1:3,000 and 1:1,500. Caco-2 monolayers were then evaluated for effects of gliadin and/or anti-wheat gliadin IgY after 24 h exposure. Enzyme-linked immunosorbent assay (ELISA) was used to quantify anti-inflammatory markers (TNF-α and IL-1β) 5 days after cells were exposed to PT-gliadin and/or anti-wheat gliadin IgY. Results: Among other conditions, anti-wheat gliadin IgY at a ratio of 1:3,000 (anti-gliadin IgY: PT-gliadin) significantlyprevented gliadin toxicity on Caco-2 by maintaining intestinal integrity, inhibiting phenol red permeation, and inhibiting gliadin absorption and production of proinflammatory cytokines (TNF-α and IL-1β) as compared to PT-gliadin stimulated cultures (P < 0.05). Conclusion: The anti-wheat gliadin IgY antibody produced in this study has proved to inhibit absorption of gliadin and gliadin-induced inflammatory response in Caco2 cell culture model of CD. Anti-gliadin IgY, therefore has potential to be used as an oral passive antibody therapy to treat CD. Keywords: Celiac disease, Gliadin, Immunoglobulin Y, Intestinal integrity, Cytokines Background Celiac disease is one of the most common autoimmune diseases, occurring in 1 out of 100–300 people worldwide [1]. CD is driven by an abnormal immune response to the ingestion of gluten in genetically (HLA DQ2/ DQ8) predisposed individuals. Among these gluten proteins, gliadin is exceptionally resistant to enzymatic degradation due to its high proline and glutamine content [2]. The PT-gliadin can cross the membrane of * Correspondence: ; 2 Center for Nutraceutical and Pharmaceutical Materials, Myongji University, Cheoin-gu, Yongin, Gyeonggi-Do 449-728, Korea 1 3142G Katz Group Centre for Pharmacy & Health Research, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 11361 – 87 Ave, Edmonton, AB T6G 2E1, Canada enterocytes and provoke damage in a variety of ways. Firstly, the binding of gliadin to the CXCR3 receptor results in the increased release of the protein zonulin, which can lead to impaired mucosal integrity. Gliadin can then enter enterocytes by transcytosis or retrotranscytosis via secretory IgA through the transferrin receptor CD71 [3]. The release of p31–43/49 peptides triggers the innate immune response [4], localizes to endocytic vesicles which leads to the production of inflammatory cytokines, such as IL-1β and TNF-α, and impaired mucosal integrity [5]. P31-43 interferes with the endocytic pathway by causing delay maturation of early endosomes to late endosomes, thus affecting various metabolic pathways and cellular functions [6, 7]. The 33-mer (p56- © 2015 Gujral et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gujral et al. BMC Immunology (2015) 16:41 89) gliadin can also penetrate into the lamina propria, triggering the T-helper cell mediated adaptive immune response [8] that contributes to the ongoing inflammation in the small intestine of CD patients [9]. Currently, there is no pharmacological therapy available for CD patients. Only strict adherence to a GFD helps to alleviate symptoms. Finding a more convenient, safe, and cost-effective way of relieving symptoms would contribute greatly to the quality of life for these patients, and one avenue of study includes how to prevent triggering the immune system upon the ingestion of gluten. Potential therapeutic options include the hydrolysis of toxic gliadin by exogenous enzymes [10], the modification of gliadin-derived peptide pattern by Bifidobacteria [8], the prevention of gliadin absorption by polymeric binders [11], the inhibition of tight junction opening by zonulin antagonists [12], the blockage of selective deamidation of specific glutamine residues by tissue tranglutaminase inhibitors [13], the restoration of immune tolerance towards gluten by vaccines [14], the modulation of immune response to gliadin by NKG2D/MICA blockers [15] and the neutralization of gliadin in vivo by IgY antibody [16]. Among these, oral passive immunotherapy may be the best avenue to pursue simply by virtue of its advantages, including safety, reduced cost, ease of administration, and potential to treat localized conditions in the gastrointestinal tract (GIT) [17]. Chicken egg yolk immunoglobulin (IgY) is ideal for passive immunotherapy, as it may be readily obtained in large quantities from egg yolk. Compared to the traditional method of harvesting mammalian antibodies, IgY purification is more cost-effective, convenient, and hygienic. IgY antibodies have already been shown to be effective in neutralizing disease-causing pathogens such as Rotavirus [18], E. coli O157:H7 [19], Salmonella enteritis [20], Clostridium perfringens [21]. Despite this, there is only limited information available that describes the use of IgY antibodies in neutralizing toxic gliadin in an intestinal epithelium culture system. The Caco-2 cell line has been used in several studies as an ex vivo model of CD intestinal epithelia for initial testing of novel CD treatment options [22–24]. In this study, Caco-2 cell cultures were used to evaluate the effectiveness of anti-gliadin IgY in inhibiting gliadin induced impaired intestinal integrity, gliadin absorption and the inflammatory response induced by gliadin. The objectives of this study are to produce anti-gliadin IgY antibodies by immunizing chickens with gliadin, to purify the resultant IgY antibodies by gel chromatography, and to characterize its reactivity to gliadin by western blot and ELISA t (...truncated)