Martine Heyman

Celiac disease (CD) is an enteropathy resulting from an abnormal immune response to gluten-derived peptides in genetically susceptible individuals. This immune response is initiated by intestinal transport of intact peptide 31-49 (p31-49) and 33-mer gliadin peptides through an unknown mechanism. We show that the transferrin receptor CD71 is responsible for apical to basal retrotranscytosis of gliadin peptides, a process during which p31-49 and 33-mer peptides are protected from degradation. In patients with active CD, CD71 is overexpressed in the intestinal epithelium and colocalizes with immunoglobulin (Ig) A. Intestinal transport of intact p31-49 and 33-mer peptides was blocked by polymeric and secretory IgA (SIgA) and by soluble CD71 receptors, pointing to a role of SIgA–gliadin complexes in this abnormal intestinal transport. This retrotranscytosis of SIgA–gliadin complexes may promote the entry of harmful gliadin peptides into the intestinal mucosa, thereby triggering an immune response and perpetuating intestinal inflammation. Our findings strongly implicate CD71 in the pathogenesis of CD.

Objectives: Little information is available on the properties of fermented milk formula intended to healthy infants. This study analyzes the effect of long‐term ingestion of a heat‐treated, fermented milk formula on the development of oral tolerance or systemic immune response to soluble antigens in mice.Materials and Methods: The C3H/HeN mice, fed with a heat‐treated fermented (Bifidobacterium breve C50 and Streptococcus thermophilus 065) infant formula (htFF) or a matched control diet (control), were immunized with ovalbumin (OVA) with or without gavage of 20 mg OVA to induce tolerance or immunity, respectively. Systemic and local anti‐OVA immune responses and intestinal barrier function were measured after 5 to 6 weeks.Results: Oral tolerance to OVA developed similarly in htFF‐ and control‐fed mice, attested to by the downregulation of OVA‐specific immunoglobulin (Ig) G and IgE after oral OVA administration. In contrast, immunization with OVA led to significantly higher titers in htFF‐fed mice than in control‐fed mice (log2 IgG titers, 16.45 ± 1.24 and 15.46 ± 0.79, respectively; P = 0.012). Jejunal interferon gamma, interleukin 12p40 and interleukin 10 expressions were significantly higher in tolerized mice fed with htFF compared with those fed with the control diet. Mucosal to serosal intact horseradish peroxidase fluxes were lower in htFF‐fed mice than in control‐fed mice (39 ± 8 and 118 ± 38 ng/h.cm², respectively; P < 0.0001), indicating that the htFF diet reinforces intestinal barrier capacity to macromolecules.Conclusions: In mice, htFF strengthens intestinal barrier and enhances systemic immune responses to antigens without interfering with the development of oral tolerance, suggesting a potential beneficial effect in host defence and vaccination.

Intestinal permeability tests used in the diagnosis of allergic diseases in response to oral food challenge have led to the conclusion that constitutive defects of the intestinal barrier are not the primary cause of allergic diseases. However, perturbation of environmental factors (infection, stress), by increasing intestinal permeability and enhancing danger signals, may favour food allergy in susceptible individuals. The mechanisms of enhanced permeability to specific and bystander antigens have been delineated as well as the molecular events involved in the sequential phases of allergic reactions. Intestinal absorption of food antigens and immune responses are mutually dependent, and luminal (environmental) and serosal (intrinsic) factors synergize to maintain a self-perpetuating cycle in which antigens penetrate the mucosa and induce allergic inflammation.

Background: Probiotic bacteria have a beneficial effect on intestinal inflammation. In this study, we have examined the effect of lactic acid and commensal Gram positive (+) bacteria conditioned media (CM) on tumour necrosis factor α (TNF-α) release and the mechanisms involved. Methods: Lipopolysaccharide (LPS) induced TNF-α secretion by peripheral blood mononuclear cells or the THP-1 cell line was monitored in the presence or absence of bacteria CM obtained from two probiotic strains, Bifidobacterium breve ( Bb ) and Streptococcus thermophilus ( St ), and three commensal bacterial strains ( Bifidobacterium bifidum , Ruminococcus gnavus , and unidentified Streptococcus ). Bb and St bacteria CM were allowed to cross filter grown intestinal epithelial cell monolayers (HT29-19A) to assess intestinal transport of active bacterial products. These products were characterised and their effect on LPS binding to THP-1 cells and nuclear factor κB (NFκB) activation assessed. Results: Dose dependent inhibition of LPS induced TNF-α secretion was noted for both probiotic bacteria CM (64% and 71% inhibition for Bb and St , respectively) and to a lesser extent commensal bacteria CM (21–32% inhibition). Active products from Bb and St were resistant to digestive enzymes and had a molecular mass Bb and St CM. Conclusion: B breve and S thermophilus release metabolites exerting an anti-TNF-α effect capable of crossing the intestinal barrier. Commensal bacteria also display a TNF-α inhibitory capacity but to a lesser extent. These results underline the beneficial effect of commensal bacteria in intestinal homeostasis and may explain the role of some probiotic bacteria in alleviating digestive inflammation.

Background: Intestinal epithelial cells secrete exosome-like vesicles. The aim of this study was to characterise murine intestinal epithelial exosomes and to analyse their capacity to inform the immune system in vivo in mice. Methods: Epithelial exosomes were obtained from the murine epithelial cell line MODE K incubated in the presence or absence of interferon γ (IFN-γ) together with pepsin/trypsin ovalbumin hydrolysate (hOVA) to mimic luminal digestion. Exosomes isolated from MODE K conditioned media (EXO-hOVA and EXO-hOVA-IFN) were characterised by western blot, peptide mapping, and mass spectrometry. They were injected intraperitoneally to C3H/HeN mice to test their immunocompetence. Results: MODE K epithelial exosomes displayed major histocompatibility complex (MHC) class I and class II (upregulated by IFN-γ) molecules and tetraspan proteins (CD9, CD81, CD82) potentially involved in the binding to target cells. A33 antigen, an Ig-like molecule highly specific for intestinal epithelial cells, was enriched in exosomes and was also found in mice mesenteric lymph nodes, suggesting exosome migration towards the gut associated lymphoid tissues. Intraperitoneal injection of EXO-hOVA or EXO-hOVA-IFN did not induce humoral or cellular tolerance to OVA in mice. In contrast, exosomes obtained after incubation with IFN-γ (EXO-hOVA-IFN), bearing abundant MHC class II/OVA complexes, induced a specific humoral immune response. Conclusions: Epithelial exosomes are antigen presenting vesicles bearing MHC class II/peptide complexes that prime for an immunogenic rather than tolerogenic response in the context of a systemic challenge. In the intestine, both the mucosal microenvironment and local effector cells are probably key players in determining the outcome of the immune response to exosome derived epitopes.

Intestinal epithelial cells (IEC) are located at the strategic interface between the external environment and the most extensive lymphoid compartment in the body. Besides their central role in the absorption of nutrients, they also provide sample information to the immune system on soluble or particulate antigens present in the intestinal lumen. Like professional antigen-presenting cells, IEC have recently been shown to secrete 30- to 90-nm diameter vesicles named exosomes from their apical and basolateral surfaces. These vesicles carry molecules that are implicated in adhesion and antigen presentation, such as major histocompatibility complex (MHC) class I molecules, MHC class II molecules, CD63, CD26/dipeptidyl-peptidase IV, tetraspan proteins, and A33 antigen. IEC exosomes therefore, constitute a link by which IEC may influence antigen presentation in the mucosal or systemic immune system independent of direct cellular contact with effector cells.

The intestinal epithelium is a selective barrier where incompletely-digested food antigens are transmitted to the immune system. Food antigens are often the starting point of intestinal diseases such as food allergy or coeliac disease. The intestinal epithelial cells (IEC) take up and process food antigens mainly by fluid-phase transcytosis involving two functional pathways, one minor direct pathway without degradation and another major lysosomal degradative pathway. Among the peptidic metabolites generated during transepithelial transport of luminal antigens, some have a molecular mass compatible with a binding to restriction (major histocompatibility complex; MHC) molecules; the latter can be up regulated on enterocytes, especially in inflammatory conditions. Indeed, interferon-gamma not only increases the paracellular absorption of antigens, but also their transcytosis across epithelial cells. It has been reported that enterocytes may even directly present peptidic epitopes to underlying T-cells. As a new potential way of transmitting peptidic information to the local or systemic immune system, the secretion by IEC of antigen-presenting vesicles called exosomes and bearing MHC-peptide complexes has recently been proposed. Many other factors such as nutritional or environmental factors can also influence the properties of the epithelial barrier and the outcome of the immune response to lumen antigens.

Contradictory results have been reported on the use of goats' milk in cows' milk allergy. In this study the hypothesis was tested, using a guinea pig model of cows' milk allergy, that these discrepancies could be due to the high genetic polymorphism of goats' milk proteins. Forty guinea pigs were fed over a 20 d period with pelleted diets containing one of the following: soyabean proteins (group S), cows' milk proteins (group CM), goats' milk proteins with high (group GM1) or low (group GM2) α_s1-casein content. Parenteral sensitization to GM1 and GM2 proteins was also assessed. The sensitization was measured (1) by systemic IgG1 antibodies directed against bovine or caprine β-lactoglobulin (β-lg), α-lactalbumin (α-la) and whole caseins, and (2) by intestinal anaphylaxis measured in vitro in Ussing chambers, by the rise in short-circuit current (ΔIsc) in response to milk proteins. Guinea pigs fed on CM and GM1 developed high titres (> 1500) of anti-β-lg IgG1, with an important cross reactivity between goat and cow β-lg. However, in guinea pigs fed on GM2, anti-goat β-lg IgG1 antibodies were significantly decreased compared with GM1 guinea pigs (mean IgG1 titres were 546 and 2046 respectively), and the intestinal anaphylaxis was significantly decreased (3·5±4·5 μA/cm²) compared with that observed in GM1 guinea pigs (8·3±7·6 μA/cm²). Animals receiving GM1 or GM2 proteins via the parenteral route developed a marked sensitization. These results suggest that the discrepancies observed in the use of goats' milk in cows' milk allergy could be due, at least in part, to the high genetic polymorphism of goats' milk proteins.