Germ-Free Mice Exhibit Mast Cells With Impaired Functionality and Gut Homing and Do Not Develop Food Allergy

Background: Mucosal mast cells (MC) are key players in IgE-mediated food allergy (FA). The evidence on the interaction between gut microbiota, MC and susceptibility to FA is contradictory.Objective: We tested the hypothesis that commensal bacteria are essential for MC migration to the gut and their maturation impacting the susceptibility to FA.Methods: The development and severity of FA symptoms was studied in sensitized germ-free (GF), conventional (CV), and mice mono-colonized with L. plantarum WCFS1 or co-housed with CV mice. MC were phenotypically and functionally characterized.Results: Systemic sensitization and oral challenge of GF mice with ovalbumin led to increased levels of specific IgE in serum compared to CV mice. Remarkably, despite the high levels of sensitization, GF mice did not develop diarrhea or anaphylactic hypothermia, common symptoms of FA. In the gut, GF mice expressed low levels of the MC tissue-homing markers CXCL1 and CXCL2, and harbored fewer MC which exhibited lower levels of MC protease-1 after challenge. Additionally, MC in GF mice were less mature as confirmed by flow-cytometry and their functionality was impaired as shown by reduced edema formation after injection of degranulation-provoking compound 48/80. Co-housing of GF mice with CV mice fully restored their susceptibility to develop FA. However, this did not occur when mice were mono-colonized with L. plantarum.Conclusion: Our results demonstrate that microbiota-induced maturation and gut-homing of MC is a critical step for the development of symptoms of experimental FA. This new mechanistic insight into microbiota-MC-FA axis can be exploited in the prevention and treatment of FA in humans

Heat-Induced Structural Changes Affect OVA-Antigen Processing and Reduce Allergic Response in Mouse Model of Food Allergy

BACKGROUND AND AIMS: The egg protein ovalbumin (OVA) belongs to six most frequent food allergens. We investigated how thermal processing influences its ability to induce allergic symptoms and immune responses in mouse model of food allergy. METHODOLOGY/PRINCIPAL FINDINGS: Effect of increased temperature (70°C and 95°C) on OVA secondary structure was characterized by circular dichroism and by the kinetics of pepsin digestion with subsequent HPLC. BALB/c mice were sensitized intraperitoneally and challenged with repeated gavages of OVA or OVA heated to 70°C (h-OVA). Levels of allergen-specific serum antibodies were determined by ELISA (IgA and IgGs) or by β-hexosaminidase release test (IgE). Specific activities of digestive enzymes were determined in brush border membrane vesicles of jejunal enterocytes. Cytokine production and changes in regulatory T cells in mesenteric lymph nodes and spleen were assessed by ELISA and FACS. Heating of OVA to 70°C caused mild irreversible changes in secondary structure compared to boiling to 95°C (b-OVA), but both OVA treatments led to markedly different digestion kinetics and Tregs induction ability in vitro, compared to native OVA. Heating of OVA significantly decreased clinical symptoms (allergic diarrhea) and immune allergic response on the level of IgE, IL-4, IL-5, IL-13. Furthermore, h-OVA induced lower activities of serum mast cell protease-1 and enterocyte brush border membrane alkaline phosphatase as compared to native OVA. On the other hand h-OVA stimulated higher IgG2a in sera and IFN-γ secretion by splenocytes. CONCLUSIONS: Minor irreversible changes in OVA secondary structure caused by thermal processing changes both its digestion and antigenic epitopes formation, which leads to activation of different T cell subpopulations, induces shift towards Th1 response and ultimately reduces its allergenicity

Protective Effect of Vegan Microbiota on Liver Steatosis Is Conveyed by Dietary Fiber: Implications for Fecal Microbiota Transfer Therapy

Fecal microbiota transfer may serve as a therapeutic tool for treating obesity and related disorders but currently, there is no consensus regarding the optimal donor characteristics. We studied how microbiota from vegan donors, who exhibit a low incidence of non-communicable diseases, impact on metabolic effects of an obesogenic diet and the potential role of dietary inulin in mediating these effects. Ex-germ-free animals were colonized with human vegan microbiota and fed a standard or Western-type diet (WD) with or without inulin supplementation. Despite the colonization with vegan microbiota, WD induced excessive weight gain, impaired glucose metabolism, insulin resistance, and liver steatosis. However, supplementation with inulin reversed steatosis and improved glucose homeostasis. In contrast, inulin did not affect WD-induced metabolic changes in non-humanized conventional mice. In vegan microbiota-colonized mice, inulin supplementation resulted in a significant change in gut microbiota composition and its metabolic performance, inducing the shift from proteolytic towards saccharolytic fermentation (decrease of sulfur-containing compounds, increase of SCFA). We found that (i) vegan microbiota alone does not protect against adverse effects of WD; and (ii) supplementation with inulin reversed steatosis and normalized glucose metabolism. This phenomenon is associated with the shift in microbiota composition and accentuation of saccharolytic fermentation at the expense of proteolytic fermentation

Numbers of Tregs in splenocytes and mesenteric lymph nodes of OVA treated mice.

<p>Typical plots depicting numbers of Tregs in mouse splenocytes (a) and mesenteric lymph node (b) in gated CD3+CD4+CD8– T helper cells after feeding with OVA, h-OVA or PBS, respectively. Numbers in upper quadrants shows proportions (mean ± SD) of either CD25–Foxp3+ or CD25+Foxp3+ Th cells out of all cells. Representative data from one out of three independent experiments. *P≤0.05.</p

Number of Tregs in spleen cell suspensions co-cultured <i>in vitro</i> with OVA digests.

<p>The percentage of Tregs in cell suspension isolated from spleens of non-stimulated (naïve) BALB/c mice cultured <i>in vitro</i> for 48 hours with undigested (0′) and after 20 (20′) and 40 minutes (40′) peptic digest of OVA (white bars), h-OVA (black bars) or b-OVA (grey bars). The data represent the percentage of CD4+Foxp3+ cells out of all live cells as measured by FACS. Representative data from one out of three independent experiments are shown. Data are represented as mean ± SEM.</p

Cytokine production after <i>in vitro</i> restimulation with OVA.

<p>The cytokine production from mesenteric lymph nodes (a) and splenocytes (b) of BALB/c mice fed with OVA (white bars) or h-OVA (black bars) and stimulated <i>in vitro</i> with appropriate allergen. Cytokine levels are expressed after subtraction of base line levels of unstimulated lymph node cells or splenocytes. Data shown are mean values ± SEM (n = 4–7 mice/group), representative data from one out of three independent experiments. *P≤0.05, **P≤0.01, ***P≤0.001, n.d. =  not detectable.</p

Decreased mast cell protease induction by heated-OVA.

<p>Heated OVA (h-OVA, black bar) induced significantly lower amounts of mast cell protease (MMCP-1), the marker of mast cell activation, compared to mice fed with native OVA (white bar). Data are represented as mean ± SEM (n = 10 mice/group), representative data from one out of three independent experiments. *P≤0.05, **P≤0.01, ***P≤0.001.</p

RP-HPLC separation profile of native-OVA and heated-OVA peptic digests.

<p>RP-HPLC separation profile monitored at 280 nm corresponds to OVA and OVA heated at 70°C (h)-OVA or boiled at 95°C (b)-OVA undigested (0′) and after 20 (20′) and 40 minutes (40′) of digestion by pepsin. RT – retention time.</p