Improved xenobiotic metabolism and reduced susceptibility to cancer in gluten-sensitive macaques upon introduction of a gluten-free diet

Abstract

Background: A non-human primate (NHP) model of gluten sensitivity was employed to study the gene perturbations associated with dietary gluten changes in small intestinal tissues from gluten-sensitive rhesus macaques (Macaca mulatta).

Methodology: Stages of remission and relapse were accomplished in gluten-sensitive animals by administration of gluten-free (GFD) and gluten-containing (GD) diets, as described previously. Pin-head-sized biopsies, obtained non-invasively by pediatric endoscope from duodenum while on GFD or GD, were used for preparation of total RNA and gene profiling, using the commercial Rhesus Macaque Microarray (Agilent Technologies),targeting expression of over 20,000 genes.

Principal findings: When compared with normal healthy control, gluten-sensitive macaques showed differential gene expressions induced by GD. While observed gene perturbations were classified into one of 12 overlapping categories--cancer, metabolism, digestive tract function, immune response, cell growth, signal transduction, autoimmunity, detoxification of xenobiotics, apoptosis, actin-collagen deposition, neuronal and unknown function--this study focused on cancer-related gene networks such as cytochrome P450 family (detoxification function) and actin-collagen-matrix metalloproteinases (MMP) genes.

Conclusions/significance: A loss of detoxification function paralleled with necessity to metabolize carcinogens was revealed in gluten-sensitive animals while on GD. An increase in cancer-promoting factors and a simultaneous decrease in cancer-preventing factors associated with altered expression of actin-collagen-MMP gene network were noted. In addition, gluten-sensitive macaques showed reduced number of differentially expressed genes including the cancer-associated ones upon withdrawal of dietary gluten. Taken together, these findings indicate potentially expanded utility of gluten-sensitive rhesus macaques in cancer research.

Figure 1. AGA and anti-TG2 antibodies in plasma.

Plasma antibody responses to gliadin and TG2 were measured in four gluten-sensitive (FH45, FB97, DJ68 and DE87) and four normal healthy control (HK31, HT24, HD60 and IA24) rhesus macaques at the time of remission and/or relapse. Graph depicts fold-increases of antibodies over the baseline upon introduction of dietary gluten. Significant (p<0.05) increases are indicated (*).

Figure 2. Differential gene expression.

A commercial rhesus-specific gene array was used to measure the differential gene expression in eight duodenum samples from four selected study macaques: Normal healthy control (HK31), andFH45, FB97 and DJ68 gluten sensitive macaques at the stages of remission and/or relapse. Absolute numbers of perturbed genes out of ∼20,000 tested per each sample are shown.

Figure 3. FH45 macaque.

Selected three categories of perturbed genes in duodenum of FH45 gluten-sensitive macaque (normalized against normal, healthy control macaque HK31) while on gluten diet. Different color shades correspond with four categories of perturbed genes. Similar patterns of gene perturbation were identified also in FB97 and DJ68 gluten-sensitive macaques (not shown).

Figure 4. Categories of differentially expressed genes in gluten-sensitive macaques during the relapse stage.

1 – cancer, 2 – metabolism, 3 – digestive tract function, 4 – immune response, 5 – cell growth, 6 – signal transduction, 7 – autoimmunity, allergy, 8 – detoxification of xenobiotics, 9 – apoptosis, 10 – not known function, 11 – actin-collagen-MMP network, and 12 – neuronal function. Relative distributions of up-regulated gene categories are shown in panels A (FH45), B (FB97) and C (DJ68) while down-regulated genes are shown in panels D (FH45), E (FB97) and F (DJ68).

Figure 5. Dietary gluten withdrawal and differential gene expression.

Dietary gluten withdrawal impacts relative distribution of perturbed gene categories in gluten-sensitive macaques. In three selected categories [1 (cancer), 8 (detoxification of xenobiotics), and 11 (actin-collagen-MMP network)], these changes included decreased number of “cancer-promoting” events such as down-regulation of category 8 genes and up-regulation of category 11 genes. Data generated with FB97 and DJ68 macaques during the stage of remission and relapse are shown. FH45 macaque had only few gene perturbations during the stage of remission while on gluten-free diet, thus relative gene counts are not shown for this animal.

Figure 6. Down-regulation of cytochrome P450 family gene network.

Introduction of dietary gluten (relapse stage) down-regulates several of the genes in P450 network while dietary gluten withdrawal (remission stage) normalizes its expression towards baseline level: Expression in FH45 gluten-sensitive macaque shows 5 out of 6 perturbed genes being more down-regulated during the relapse stage than during the remission stage. Red signifies up-regulation of a gene while green signifies down-regulation, with the standard level of expression determined by HK31 normal healthy control macaque. Intensity of color represents the magnitude of change in expression. The solid lines mean that the gene's protein product directly affects the target gene's protein levels, whereas dotted lines mean that the gene's protein product indirectly alters the target gene's protein levels through the involvement of an intermediate gene.

Figure 7. Perturbations in the actin-collagen-MMP gene network.

Introduction of dietary gluten (relapse stage) up-regulates most of the actin and collagen genes while MMP3 and MMP9 are down-regulated. Data generated with FB97 gluten-sensitive macaque during the stage of remission and relapse are shown. Red signifies up-regulation of a gene while green signifies down-regulation, with the standard level of expression determined by HK31 normal healthy control macaque. Intensity of color represents the magnitude of change in expression. The solid lines mean that the gene's protein product directly affects the target gene's protein levels, where as dotted lines mean that the gene's protein product indirectly alters the target gene's protein levels through the involvement of an intermediate gene.

Figure 8. DJ68 and FB97 macaques.

Selected categories (a. Celiac Disease, b. general gluten sensitivity or inflammation and c. other disease) of upregulated genes in duodenum of DJ68 and FB97 gluten-sensitive macaques (normalized against normal, healthy control macaque HK31) while on gluten-containing diet. Different color shades correspond with different categories of perturbed genes. ^aTGM2 = tissue transglutaminase 2 functions as celiac disease autoantigen; ^bIL17B = IL-17 cytokine reported to be produced in celiacs but not in other gluten-sensitive patients; ^cMICB = NKG2D ligand suggested to mediate the transformation of TCR-dependent CD8+ CTLs to TCR-independent (NK-like) effector IELs in celiac patients; ^dIFNg = interferon gamma is produced by T cells and promotes gluten peptide flux across intestinal epithelium; ^eITGAE i.e. CD103 antigen was suggested to mediate the celiac-specific autoimmune response in the small intestine.