Gliadin Nanoparticles Induce Immune Tolerance to Gliadin in Mouse Models of Celiac Disease (1)
The tolerogenic potential of antigen loaded, negatively charged nanoparticles (referred to as Tolerogenic Immune Modifying Nanoparticles, TIMP) in murine models of multiple sclerosis (MS), transplantation, airway allergy, and type 1 diabetes (T1D). Studies using nanoparticles or apoptotic cells have shown that treatment resulted in the upregulation of inhibitory ligands on macrophages, such as PD-L1, and the production of regulatory cytokines, such as interleukin 10 (IL10) and transforming growth factor beta (TGFB), in the spleen.
Gliadin delayed-type hypersensitivity (DTH) model
Female C57BL/6 mice were immunized subcutaneously with 100μl of an emulsion containing 200μg of M. tuberculosis H37Ra and 100μg of gliadin, distributed over three sites on the flank. Gliadin was reconstituted from lyophilized powder in 50mM acetic acid (5 mg/ml). This gliadin solution was then further diluted for use in PBS. Mice were treated intravenously with different nanoparticle preparations, on days -7, 0 or 7, as indicated. On day 14 after priming, mice were bled, and tested for delayed type hypersensitivity (DTH).
In a gliadin DTH mouse model, intravenous treatment on days 0 and 7 with two injections of 2.5 mg/mouse PLGA-PEMA-GLIA following gliadin/CFA priming was associated with significant decreases in ear swelling when compared to control animals receiving either PLGA-PVA-GLIA, or PLGA-PEMA-OVA. The intravenous injection of 25 μg soluble (free) gliadin alone did not show any efficacy in reducing ear swelling in the gliadin DTH model.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot confirmed the presence of gliadin α/β, γ and ω proteins in TIMP-GLIA. Scanning electron microscopy of TIMP-GLIA suspensions confirmed the size and a spherical morphology with a smooth surface.
The spleens of animals that had received two infusions of TIMP-GLIA had significantly reduced numbers of proliferating, interferon gamma (IFNG)-producing CD4+ effector T cells in the spleen, as determined by intracellular cytokine staining. The decrease in effector T cells observed in TIMP-GLIA treated animals correlated with a dose-dependent reduction in T cell proliferation in splenocyte cultures when restimulated with gliadin. This phenotype appeared to result in reduced T cell help, required for anti-gliadin antibody production, as treatment with 2.5mg/mouse TIMP-GLIA resulted in significant reduction in circulating levels of gliadin-specific IgG. Doses of 0.25-2.5 mg/mouse (12.5-125 mg/kg) were associated with significant decreases in ear swelling when compared to controls. The most robust decrease in DTH was observed in animals receiving 2.5 mg/mouse. TIMP-GLIA administration after immunization was associated with a reduction in circulating gliadin specific IgG.
Treatment of HLA-DQ8 mice with 2.5 mg/mouse (125 mg/kg) TIMP-GLIA or TIMP-OVA control on days -11 and -3 prior to immunization with gliadin/CFA, did not appear to alter the levels of circulating anti-gliadin IgG1, but reduced the amount of T-helper 1 (Th1) cell-associated, complement-fixing anti-gliadin IgG2c, compared to TIMP-OVA control-treated mice. The identities of these 15 genes, and their expression levels between samples from different groups, revealed long-term changes induced by TIMP-GLIA treatment in pathways of APC function (Tspan13, Cd83, Nrp2), B cell activation and differentiation (Cd79a, Ms4a1/Cd20, Ms4a4c), MHC II peptide loading (H2-DM, H2-O) and T cell cytokine secretion (Il17a, Il17f; gene expression heatmap).
The GLUTEN positive control group gained less body weight until week 4, and subsequently lost weight more rapidly, when compared to the GFD control. However, mice in the TIMP-GLIA group were protected from weight loss over the entire study period, similar to gluten free diet control animals. Duodenal sections that exemplify histological duodenitis severity scores of normal, mild, moderate or severe duodenitis are shown. Treatment with TIMP-GLIA reduced secretion of pro-inflammatory IFNG, IL17, and IL2 cytokines by spleen cells restimulated with gliadin, compared to GLUTEN positive control or TIMP-LYS treatment control, but not of regulatory cytokine IL10.
The gliadin plasma concentration after injection of TIMP-GLIA peaked at 1h, rapidly declined until 4h, and had returned to basline at 24h after injection, as determined by ELISA.
A result of the protein solubilization procedure in acetic acid, gliadin encapsulated in TIMP-GLIA is already partially deamidated. Processing of TIMP-GLIA by APC, equipped with enzymes that can further deamidate glutamine residues of gliadin peptides, most importantly tissue transglutaminase (TG2), may lead to the presentation of both native and deamidated gliadin peptides.