Pretreatment with Yeast-Derived Complex Dietary Polysaccharides Suppresses Gut Inflammation, Alters the Microbiota Composition, and Increases Immune Regulatory Short-Chain Fatty Acid Production in C57BL/6 Mice.

BACKGROUND: ß-Glucans (BGs), a group of complex dietary polysaccharides (CDPs), are available as dietary supplements. However, the effects of orally administered highly purified BGs on gut inflammation are largely unknown. OBJECTIVES: The aim of this study was to investigate the impact of orally administering highly purified, yeast-derived BG (YBG; ß-1,3/1,6-d-glucan) on susceptibility to colitis. METHODS: Eight-week-old C57BL/6 (B6) mice were used in a series of experiments. Experiment (Expt) 1: male and female mice were treated every day, for 40 d, with saline (control) or 250 µg YBG, followed by 2.5% (wt:vol) dextran sulfate sodium (DSS) in drinking water during days 30-35; and colitis severity and intestinal immune phenotype were determined. Expt 2: female B6 mice were treated with saline or YBG for 30 d and intestinal immune phenotype, gut microbiota composition, and fecal SCFA concentrations were determined. Expt 3: female B6 mice were treated as in Expt 2, given drinking water with or without antibiotics [Abx; ampicillin (1 g/L), vancomycin (0.5 g/L), neomycin (1 g/L), and metronidazole (1 g/L)] during days 16-30, and gut immune phenotype and fecal SCFA concentrations were determined. Expt 4: female B6 Foxp3-green fluorescent protein (-GFP) reporter mice were treated as in Expt 3, and intestinal T-regulatory cell (Treg) frequencies and immune phenotypes were determined. Expt 5: female mice were treated as in Expt 1, given drinking water with or without antibiotics during days 16-40, and colitis severity and intestinal cytokine production were determined. RESULTS: Compared with controls, the YBG group in Expt 1 exhibited suppressive effects on features of colitis, such as loss of body weight (by 47%; P < 0.001), shortening of colon (by 24%; P = 0.016), and histopathology severity score (by 45%; P = 0.01). The YBG group of Expt 2 showed a shift in the abundance of gut microbiota towards Bacteroides (by 16%; P = 0.049) and Verrucomicrobia (mean ± SD: control = 7.8 ± 0.44 vs. YBG = 21.0 ± 9.6%) and a reduction in Firmicutes (by 66%; P < 0.001). The YBG group also showed significantly higher concentrations of fecal SCFAs such as acetic (by 37%; P = 0.016), propionic (by 47%; P = 0.026), and butyric (by 57%; P = 0.013) acids. Compared with controls, the YBG group of Expt 2 showed higher frequencies of Tregs (by 32%; P = 0.043) in the gut mucosa. Depletion of gut microbiota in the YBG group of mice caused diminished fecal SCFA concentrations (Expt 3) and intestinal Treg frequencies (Expt 4). Compared with the YBG group, the YBG-(Abx) group of Expt 5 showed aggravated colitis features including loss of body weight (by >100%; P < 0.01) and colonic inflammation score (by 42%; P = 0.04). CONCLUSIONS: Studies using B6 mice show that dietary BGs are beneficial for promoting intestinal health when the gut microbiota is intact. However, these CDPs may produce adverse effects if gut microbiota is compromised.

Strategies for Mast Cell Inhibition in Food Allergy.

Mast cells are tissue resident allergic effector cells that drive IgE-mediated food allergies. There are several steps leading to mast cell activation in the context of allergic disease that can be targeted to prevent mast cell activation and degranulation. These include blocking IgE-FcεRI crosslinking and type 2 cytokine receptor activation; modulating cell-surface neural chemical receptors; stabilizing mast cell membranes to prevent co-localization of activating receptors; impeding intracellular signaling; and engaging cell surface inhibitory receptors. This review highlights several ITIM-containing inhibitory mast cell surface receptors that could serve as pharmaceutical targets to prevent mast cell activation and degranulation in the context of food allergy. When activated, these ITIM-containing inhibitory receptors recruit the phosphatases SHP-1, SHP-2, and/or SHIP to dephosphorylate the tyrosine kinases responsible for activation signals downstream of the IgE-FcεRI complex. We describe several members of the Ig and Ig-like inhibitory receptor and C-type lectin inhibitory receptor superfamilies. Fundamental studies exploring the behavior of these receptors within the context of experimental food allergy models are needed. A deeper understanding of how these receptors modulate mast cell-driven food allergic responses will shape future strategies to harness these inhibitory receptors to treat food allergy.

Blocking antibodies induced by peanut oral and sublingual immunotherapy suppress basophil activation and are associated with sustained unresponsiveness.

BACKGROUND: Oral and sublingual immunotherapies for peanut allergy have demonstrated efficacy in small clinical trials; however, mechanisms and biomarkers correlating with clinical outcomes remain elusive. Previous studies have demonstrated a role for IgG in post-OIT plasma in the suppression of IgE-mediated mast cell reactions. OBJECTIVE: The aim of this study was to characterize the role that peanut oral and sublingual immunotherapy-induced plasma factors play in the inhibition of ex vivo basophil activation and whether inhibitory activity is associated with clinical outcomes. METHODS: Plasma samples from subjects on placebo, peanut oral immunotherapy (OIT) or peanut sublingual immunotherapy (SLIT), and IgG-depleted plasma or the IgG fraction were incubated with sensitized basophils, and the inhibition of basophil activation following stimulation with peanut extract was measured. Basophil inhibition results were compared between the two routes of immunotherapy, time on treatment and clinical outcomes. RESULTS: Plasma from subjects after 12 months of active peanut OIT, but not placebo, inhibits basophil activation ex vivo. Depletion of IgG abrogated the blocking effect of OIT plasma, while the IgG fraction substantially blocked basophil activation. Basophils are inhibited to a similar extent by undiluted OIT and SLIT plasma; however, diluted OIT plasma from the time of desensitization challenge inhibited basophils more than diluted SLIT plasma from time of desensitization challenge. Plasma from subjects who experienced sustained unresponsiveness following OIT inhibited basophils to a greater extent than plasma from subjects who were desensitized, but this was not true for SLIT. CONCLUSIONS AND CLINICAL RELEVANCE: Peanut immunotherapy induces IgG-dependent functional changes in plasma that are associated with OIT but not SLIT clinical outcomes. Understanding the mechanisms of peanut OIT and SLIT may help derive informative biomarkers.

Novel peanut-specific human IgE monoclonal antibodies enable screens for inhibitors of the effector phase in food allergy.

Background: 10% of US residents have food allergies, including 2% with peanut allergy. Mast cell mediators released during the allergy effector phase drive allergic reactions. Therefore, targeting sensitized mast cells may prevent food allergy symptoms. Objective: We used novel, human, allergen-specific, IgE monoclonal antibodies (mAbs) created using human hybridoma techniques to design an in vitro system to evaluate potential therapeutics targeting sensitized effector cells. Methods: Two human IgE mAbs specific for peanut, generated through human hybridoma techniques, were used to sensitize rat basophilic leukemia (RBL) SX-38 cells expressing the human IgE receptor (FcϵRI). Beta-hexosaminidase release (a marker of degranulation), cytokine production, and phosphorylation of signal transduction proteins downstream of FcϵRI were measured after stimulation with peanut. Degranulation was also measured after engaging inhibitory receptors CD300a and Siglec-8. Results: Peanut-specific human IgE mAbs bound FcϵRI, triggering degranulation after stimulation with peanut in RBL SX-38 cells. Sensitized RBL SX-38 cells stimulated with peanut increased levels of phosphorylated SYK and ERK, signal transduction proteins downstream of FcϵRI. Engaging inhibitory cell surface receptors CD300a or Siglec-8 blunted peanut-specific activation. Conclusion: Allergen-specific human IgE mAbs, expressed from human hybridomas and specific for a clinically relevant food allergen, passively sensitize allergy effector cells central to the in vitro models of the effector phase of food allergy. Peanut reproducibly activates and induces degranulation of RBL SX-38 cells sensitized with peanut-specific human IgE mAbs. This system provides a unique screening tool to assess the efficacy of therapeutics that target allergy effector cells and inhibit food allergen-induced effector cell activation.