Supplementation of Reduced Gluten Barley Diet with Oral Prolyl Endopeptidase Effectively Abrogates Enteropathy-Associated Changes in Gluten-Sensitive Macaques

Abstract

Celiac disease (CD) is an autoimmune disorder that affects approximately three million people in the United States. Furthermore, non-celiac gluten sensitivity (NCGS) affects an estimated additional 6% of the population, e.g., 20 million in the U.S. The only effective treatment of CD and NCGS requires complete removal of gluten sources from the diet. While required adherence to a gluten-free diet (GFD) is extremely difficult to accomplish, efforts to develop additional supportive treatments are needed. To facilitate these efforts, we developed a gluten-sensitive (GS) rhesus macaque model to study the effects of novel therapies. Recently reported results from phase one of this project suggest that partial improvement-but not remission-of gluten-induced disease can be accomplished by 100-fold reduction of dietary gluten, i.e., 200 ppm-by replacement of conventional dietary sources of gluten with a mutant, reduced gluten (RG) barley (lys3a)-derived source. The main focus of this (phase two) study was to determine if the inflammatory effects of the residual gluten in lys3a mutant barley grain could be further reduced by oral supplementation with a prolylendopeptidase (PE). Results reveal that PE supplementation of RG barley diet induces more complete immunological, histopathological and clinical remission than RG barley diet alone. The combined effects of RG barley diet and PE supplementation resulted in a further decrease of inflammatory mediators IFN-γ and TNF secretion by peripheral lymphocytes, as well as decreased plasma anti-gliadin and anti-intestinal tissue transglutaminase (TG2) antibodies, diminished active caspase production in small intestinal mucosa, and eliminated clinical diarrhea-all comparable with a gluten-free diet induced remission. In summary, the beneficial results of a combined RG barley and PE administration in GS macaques may warrant the investigation of similar synergistic approaches.

Keywords: IL-15; celiac; gluten; gluten-free; gluten-sensitive enteropathy; glutenase; oral supplement; protease; rhesus macaque.

Figure 1

Anti-gliadin antibodies (AGA) and anti-intestinal tissue transglutaminase (TG2) plasma antibodies in four gluten-sensitive (GS) rhesus macaques (A–D). Gluten-modified diets (GD, GFD, Bomi + B, RGB and RGB + PE) that were used to feed the macaques are indicated. Individual time points represent two-week intervals. Blue dashed line represents AGA baseline, i.e., 25 ELISA units while red dashed line represents TG2 antibody baseline, i.e., 40 units. Values elevated above these lines were significantly greater (p < 0.05) than values generated with plasmas from healthy, normal macaques.

Figure 2

Clinical diarrhea scores generated with GS macaques fed gluten-modified diets (GFD, Bomi + B, RGB and RGB + PE) are shown.

Figure 3

H & E staining of jejunum from a GS macaque while on GFD reveals normal-range intestinal architecture, magnification 10× (A); Four-color confocal microscopy of jejunum from another animal on GFD (B) shows undisrupted continuity of villin (green) and tight junction protein ZO-1 staining (red). Abundant IgA-positive B cells are seen in the subepithelium (blue). Gray = nuclear DNA.

Figure 4

The combination of cytokeratin 1 (red = epithelial cells), active caspase (green = cells undergoing apoptosis) and diamino-2-phenylindole (DAPI, blue = nuclear DNA) antibodies was used to examine the diet-induced changes within intestinal mucosa. Jejunum from juvenile GS macaques on gluten-free diet (GFD) shows prominent red staining corresponding to epithelial cells with very few of the green cells (A); Jejunum from macaque on Bomi + B diet shows an increased number of green/apoptotic cells positive for active caspase inside the lamina propria (LP) (B); Control jejunum tissue from an adult macaque with gluten-sensitive enteropathy (GSE) exhibits high % of epithelial apoptotic cells (C); Charts reflecting the proportions (%) of apoptotic cells inside the epithelium (D) and LP (E) of GS macaques fed dietary gluten-modified diets in this study plus the control GSE macaques fed regular monkey (gluten-containing) chow are shown.

Figure 5

Jejunum biopsies collected from GS macaques on Bomi + B diet show an increased IL-15 staining, indicated by arrows (A,B) while lower intensity of such signal was detected in tissues stained with unrelated, isotype-matched antibodies or in healthy control biopsies (C,D), magnification 40×.

Figure 6

Proportions of pro- and anti-inflammatory mediator expressions by peripheral blood mononuclear cells (PBMCs) following the administration of gluten-modified diets are shown. Significant differences in IFN-γ production between the GFD vs. Bomi + B fed macaques (p = 0.006) were found (A). Bomi + B vs. RGB or RGB + PE fed macaques also differed significantly (p < 0.05) in IFN-γ production (A); A similar but more robust difference (p < 0.0001) between the Bomi + B fed macaques and other diets was found in the case of TNF production by CD20 + B cells (B); Non-significant trends for differences were found in the case of TNF production by CD4 + T cells (C) while an increase in expression of anti-inflammatory CTLA-4 (CD152) by CD4 + T cells (p < 0.05) was observed after the macaques were placed on RGB + PE diet (D). Time intervals between indicated measurements correspond to four weeks.