Regulation of allergic immune responses by IKKb in intestinal epithelial cells : Effect on allergic inflammation at distant mucosal sites

La régulation de l'homéostasie intestinale est de la plus haute importance en raison de la constante exposition de l'intestin aux antigènes alimentaires et à la flore commensale. La perturbation de la flore intestinale est souvent associée à diverses maladies telles que l'allergie, l'obésité et certaines maladies inflammatoires. La plupart des individus sont tolérant aux antigènes alimentaires et ne développe pas de réponse immunitaire sauf en cas de prédisposition génétique ou d'exposition à un environnement défavorable. La réponse allergique se caractérise par la production d'IgE stimulé par les lymphocytes Th2. Les symptômes allergiques sont très variés et affectent plusieurs parties de l'organisme. La plupart des travaux de recherche se sont focalisé jusqu'à présent sur le rôle des cellules de l'immunité adaptative dans le développement de l'allergie en sous-estimant le rôle majeur des cellules épithéliales et des cellules de l'immunité innée. L'objectif de ce projet est de comprendre comment les cellules épithéliales intestinales modulent la réponse immunitaire à distance vers la muqueuse pulmonaire ou la peau après stimulation allergique. L'ingestion de l'antigène associé à l'adjuvant de la toxine cholérique permet d'étudier la réponse allergique chez l'animal. Nous avons démontré sur ce modèle animal que l'absence de la kinase inhibitrice IKKb dans la voie de signalisation du facteur de transcription NF-kB altère la composition de la flore intestinal d'une part et transforme la réponse immunitaire inflammatoire au niveau pulmonaire et de la peau grâce à la présence d'IgA et de lymphocyte Th17 d'autre part. En adéquation avec les observations cliniques rapportées chez les patients allergiques (allergies alimentaires, asthme, dermatite atopique), nos résultats identifient IKKb dans la cellule épithéliale intestinale comme cible potentielle pour traiter les allergies alimentaires. De futurs efforts devront être faits pour développer de nouvelles stratégies thérapeutiques qui considèrent la muqueuse intestinale, la production d'IgA et l'importance des bactéries commensales dans le traitement des allergies.Immune homeostasis is of paramount importance in the gastrointestinal tract, which is constantly exposed to ingested antigens and commensal microbiota. The gut microbiota can be perturbed by endogenous or exogenous factors and it is now established that microbial dysbiosis is associated with allergy, obesity, and inflammatory diseases. Ingestion of food antigens generally fails to promote brisk immune responses but rather results in a state of immune tolerance. However, aberrant immune responses can develop in individuals with a genetic predisposition. Food allergies are generally regarded as pathologic responses to food antigens mediated by excessive Th2 responses and antigen-specific IgE antibody responses. Clinical manifestations of food allergies are very broad and symptoms can affect different organs. While past research on allergy focused on the role of cells and molecules involved in adaptive immunity, epithelial cells lining the sites of antigen entry and innate immune responses have recently emerged as important players in allergy. This project was undertaken to understand the mechanisms employed by intestinal epithelial cells (IECs) to shape immune responses to allergens and influence allergic manifestations in distant mucosal sites such as the airways or the skin. Oral administration of food antigen with cholera toxin as adjuvant in experimental animals is a well-accepted model to study allergic sensitization to food antigens. Using this model, we show that a localized impairment of the canonical NF-κB pathway through deletion of IkB kinase (IKKβ) in IECs alters the gut microbiota during oral allergic sensitization and regulates the magnitude of allergic inflammatory responses at distant sites of the airway and the skin through enhancement of IgA Abs and Th17 responses. Consistent with the clinical observations linking atopic diseases (food allergy, allergic asthma, atopic dermatitis), our results identify IKKβ in IECs as a potential therapeutic target for treatment of food allergies and subsequent disease. They also suggest that future efforts for controlling allergic responses in the airways and the skin could include strategies that use the gut microbiota and promote IgA Ab responses and prevent IL-17 responses

Gender does not alter the pattern of antigen-specific serum Ab responses to ingested antigens.

<p>(<b>A</b>) Male (black) and female (white) mice were sensitized on days 0 and 7 by intraperitoneal injection of ovalumin (0.1 mg) and cholera toxin (1 μg) (<b>B</b>) Mice were sensitized by oral administration of ovalbumin (1 mg) and cholera toxin (10 μg). Blood was collected on day 14. OVA-specific IgE, IgA isotypes and IgG subclasses (i.e., IgG1, IgG2a/c, IgG2b and IgG3) were measured by ELISA. The results are expressed as the log₂ titers ± one SD from three experiments with five mice per group.</p

Airway hyper-responsiveness to nasal challenge and lung inflammation in male and female IKKβ^ΔMye mice sensitized by the oral or parenteral route.

<p>The IKKβ^ΔMye mice were sensitized on days 0 and 7 either by oral administration of ovalbumin (1000 μg) and cholera toxin (10 μg) or intraperitoneal injection of ovalbumin (100 μg) and cholera toxin (1 µ g). (<b>A</b>) Mice were exposed to increasing amounts of methacholine and lung resistance was measured. The results are expressed as mean resistance ± one SD (five mice per group). (<b>B</b>) Hematoxylin and eosin staining of lung sections from male and female IKKβ^ΔMye mice sensitized by intraperitoneal injection (magnification×200). The results are representative of two experiments with four mice per group. (<b>C</b>) Inflammation scores after nasal antigen challenge of male and female IKKβ^ΔMye mice sensitized by intraperitoneal injection (four mice per group).</p

Gender influences lung inflammation after nasal antigen challenge in mice sensitized by the intraperitoneal route.

<p>Mice were sensitized on days 0 and 7 either by oral administration of ovalbumin (1000 μg) and cholera toxin (10 μg) or intraperitoneal injection of ovalbumin (100 μg) and cholera toxin (1 μg). Nasal challenges were performed on days 15, 16 and 19 and lungs were collected on day 20. (<b>A</b>) Timeline of experiments showing the days of intervention. (<b>B</b>) Hematoxylin and eosin staining of lung sections from C57BL/6 mice (magnification×200). Black arrows denote areas of inflammation. The results are representative of three experiments with five mice per group. (<b>C</b>) Pictures of BAL in mice immunized ip or orally with OVA and nasally challenged (×600). Black arrows denote eosinophils. (<b>D</b>) Percentage of immune cell populations in BALF in mice sensitized orally or intraperitoneally and nasally challenged. Results are expressed as mean ± SD of 5 mice per group. (*, p<0.05).</p

Airway hyper-responsiveness and cytokine responses to nasal challenge of male and female mice sensitized by the oral or parenteral route.

<p>Mice were sensitized on days 0 and 7 either by oral administration of ovalbumin (1000 μg) and cholera toxin (10 μg) or intraperitoneal injection of ovalbumin (100 μg) and cholera toxin (1 μg). (<b>A</b>) Mice were exposed to increasing amounts of methacholine and lung resistance was measured. (<b>B</b>) Mice were exposed to increasing amount of methacholine and lung compliance was measured. (<b>C</b>) Real-time RT-PCR analysis of cytokine, chemokine and eosinophil peroxidase mRNA responses in lung tissues after nasal antigen challenge in mice immunized ip with OVA and CT. (<b>D</b>) Cytokine, chemokine and eosinophil peroxidase mRNA responses in lung tissues after nasal antigen challenge in mice immunized orally with OVA and CT. Results are expressed in Relative Copy Number (1/2∧ΔCt *100*1000) as mean ± SD of three separate experiments, with 5 mice per group. (* p<0.05).</p

IKKβ in myeloid cells regulates male and female responses to intraperitoneal sensitization to allergen.

<p>(<b>A</b>) Cytokine responses in spleen tissues after oral or intraperitoneal sensitization. Spleens were collected 16 hours after intraperitoneal or oral sensitization and cytokine mRNA responses were determined by Real-time RT-PCR. (<b>B</b>) IKKβ^ΔMye mice were sensitized on days 0 and 7 by intraperitoneal injection of ovalbumin (100 μg) and cholera toxin (1 μg) or by oral administration of ovalbumin (1 mg) and cholera toxin (10 μg). Blood was collected on day 14. OVA-specific IgE, IgA isotypes and IgG subclass were measured by ELISA. The results are expressed as the log₂ titers ± one SD of two experiments with five mice per group.</p

Cry Protein Crystals: A Novel Platform for Protein Delivery

<div><p>Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium <i>Bacillus thuringiensis</i>. We demonstrate that fusion of the <i>cry3Aa </i>gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines <i>in vitro</i>, and can be delivered to mice <i>in vivo</i> via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer’s patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.</p></div

Fluorescence and TEM images of Cry3Aa-GFP and Cry3Aa-mCherry crystals uptaken into cells.

<p>Macrophage cells (RAW264.7) were incubated with Cry3Aa-GFP crystals for different time periods. Fluorescence images at (a) 15-min, demonstrating fast initial uptake and at (b) 1-h, where the green fluorescence intensity reached its maximum value. Note the punctate fluorescence pattern, which supports uptake of the whole crystal. (c) Red fluorescence image of primary mouse fibroblasts after 1.5 h incubation with Cry3Aa-mCherry protein crystals is not as intense as seen with macrophages. TEM images of (d) untreated RAW264.7 cells, and (e) cells treated with Cry3Aa-GFP. The Cry3Aa-GFP treated cells exhibit a distinct cytoplasmic particulate not observed in the control cells. (f) Enlarged TEM image of the cytoplasmic particulate observed in the Cry3Aa-GFP treated macrophages highlighting the likely crystal degradation in the cytoplasm.</p