The effects of reduced gluten barley diet on humoral and cell-mediated systemic immune responses of gluten-sensitive rhesus macaques

Abstract

Celiac disease (CD) affects approximately 1% of the general population while an estimated additional 6% suffers from a recently characterized, rapidly emerging, similar disease, referred to as non-celiac gluten sensitivity (NCGS). The only effective treatment of CD and NCGS requires removal of gluten sources from the diet. Since required adherence to a gluten-free diet (GFD) is difficult to accomplish, efforts to develop alternative treatments have been intensifying in recent years. In this study, the non-human primate model of CD/NCGS, e.g., gluten-sensitive rhesus macaque, was utilized with the objective to evaluate the treatment potential of reduced gluten cereals using a reduced gluten (RG; 1% of normal gluten) barley mutant as a model. Conventional and RG barleys were used for the formulation of experimental chows and fed to gluten-sensitive (GS) and control macaques to determine if RG barley causes a remission of dietary gluten-induced clinical and immune responses in GS macaques. The impacts of the RG barley diet were compared with the impacts of the conventional barley-containing chow and the GFD. Although remission of the anti-gliadin antibody (AGA) serum responses and an improvement of clinical diarrhea were noted after switching the conventional to the RG barley diet, production of inflammatory cytokines, e.g., interferon-gamma (IFN-γ), tumor necrosis factor (TNF) and interleukin-8 (IL-8) by peripheral CD4+ T helper lymphocytes, persisted during the RG chow treatment and were partially abolished only upon re-administration of the GFD. It was concluded that the RG barley diet might be used for the partial improvement of gluten-induced disease but its therapeutic value still requires upgrading-by co-administration of additional treatments.

Figure 1

The kinetics of serum AGA antibody levels in three control (KC73, KD17 and KD82) and three GS (KF97, JR67 and KG49) macaques during the periods of (1) immunological remission e.g., GFD; (2) conventional barley diet; (3) RG barley diet and (4) GFD. Vertical, dotted lines indicate borders between different diets. Only the GS animals but not the healthy controls seroconverted to dietary gluten. The two GS macaques (KF97 and JR67) responded to its presence (AGA increase) as well as withdrawal (AGA decrease). KG49 macaque remained with elevated AGAs throughout the entire period of experiment regardless of dietary changes. AGA-ELISA cut-off = 40 Units.

Figure 2

The effects of conventional and RG barley diets on small intestinal tissue architecture of control and GS macaques (H & E staining, jejunum, 100×). After being on conventional barley diet for 64 days, jejunum of control macaque (KD17) looks normal, unaffected, with only minimal lacteal dilatation (A) while GS macaque (KF97) jejunum is showing shortened villi e.g., villous atrophy (B). Multifocally, KF97’s lamina propria is moderately expanded by lymphoid aggregates. The lacteals are mildly dilated (lymphangiectasia). The submucosa is expanded by edema containing infiltrates of small number of neutrophils, eosinophils, lymphocytes and plasma cells (B). Follow-up treatment of KF97 with RG barley diet for 56 days resulted in an improvement: Jejunum became close to normal, with only moderate lymphangiectasia (C).

Figure 3

The production of inflammatory (IFN-γ, IL-8 and TNF) and anti-inflammatory (IL-10) cytokines by peripheral T helper (CD3+CD4+), cytotoxic T (CD3+CD8+) and B (CD3-CD20+) lymphocytes from control and GS macaques is shown at four selected time points. While only the minimal cytokine production by CD4+ T helper cells was measured at the stage of immunological remission induced by GFD (A), the use of conventional barley diet was characterized with significantly increased cytokine production: Day 64 is shown (B) with differentially increased cytokine production (*). An introduction of RG barley diet and its continuous administration for two months led only to transient decrease of TNF production by CD3+CD8+ T and CD20+ B cells in all animals (C). Upon reintroduction of GFD for one month, a further but still transient decrease of inflammatory cytokine-producing cells was noted (D) indicating the necessity of more sustained treatment.

Figure 4

The proportions of CD152 (marker of T cell inhibition) expression by peripheral CD3+CD4+ T cells from control (C = blue columns) and gluten-sensitive (GS = red columns) macaques were evaluated. Six selected time points are shown including the GFD day 28 (X axis value 1); conventional barley diet days 14 (2) and 43 (3); RG barley diet days 28 (4) and 42 (5); followed again by GFD day 13 (6). While only the minimal expression of CD152 was measured during the first GFD period, an introduction of conventional barley diet led to an increased expression of CD152 by day 14, and further increase by day 43 in both groups. Replacement of conventional barley with RG barley diet did result in transiently lower % of CD3+CD4+CD152+ T cells. No significant differences in CD152 expression were observed between the two groups.