Gut Mucosal Antibody Responses and Implications for Food Allergy

The gastrointestinal mucosa is a critical environmental interface where plasma cells and B cells are exposed to orally-ingested antigens such as food allergen proteins. It is unclear how the development of B cells and plasma cells in the gastrointestinal mucosa differs between healthy humans and those with food allergy, and how B cells contribute to, or are affected by, the breakdown of oral tolerance. In particular, the antibody gene repertoires associated with symptomatic allergy have only begun to be characterized in full molecular detail. Here, we review literature concerning B cells and plasma cells in the gastrointestinal system in the context of food allergy, with a focus on human studies

Local immune response to food antigens drives meal-induced abdominal pain

Up to 20% of people worldwide develop gastrointestinal symptoms following a meal1, leading to decreased quality of life, substantial morbidity and high medical costs. Although the interest of both the scientific and lay communities in this issue has increased markedly in recent years, with the worldwide introduction of gluten-free and other diets, the underlying mechanisms of food-induced abdominal complaints remain largely unknown. Here we show that a bacterial infection and bacterial toxins can trigger an immune response that leads to the production of dietary-antigen-specific IgE antibodies in mice, which are limited to the intestine. Following subsequent oral ingestion of the respective dietary antigen, an IgE- and mast-cell-dependent mechanism induced increased visceral pain. This aberrant pain signalling resulted from histamine receptor H1-mediated sensitization of visceral afferents. Moreover, injection of food antigens (gluten, wheat, soy and milk) into the rectosigmoid mucosa of patients with irritable bowel syndrome induced local oedema and mast cell activation. Our results identify and characterize a peripheral mechanism that underlies food-induced abdominal pain, thereby creating new possibilities for the treatment of irritable bowel syndrome and related abdominal pain disorders

Transcriptional program of ciliated epithelial cells reveals new cilium and centrosome components and links to human disease.

Defects in the centrosome and cilium are associated with a set of human diseases having diverse phenotypes. To further characterize the components that define the function of these organelles we determined the transcriptional profile of multiciliated tracheal epithelial cells. Cultures of mouse tracheal epithelial cells undergoing differentiation in vitro were derived from mice expressing GFP from the ciliated-cell specific FOXJ1 promoter (FOXJ1:GFP). The transcriptional profile of ciliating GFP+ cells from these cultures was defined at an early and a late time point during differentiation and was refined by subtraction of the profile of the non-ciliated GFP- cells. We identified 649 genes upregulated early, when most cells were forming basal bodies, and 73 genes genes upregulated late, when most cells were fully ciliated. Most, but not all, of known centrosome proteins are transcriptionally upregulated early, particularly Plk4, a master regulator of centriole formation. We found that three genes associated with human disease states, Mdm1, Mlf1, and Dyx1c1, are upregulated during ciliogenesis and localize to centrioles and cilia. This transcriptome for mammalian multiciliated epithelial cells identifies new candidate centrosome and cilia proteins, highlights similarities between components of motile and primary cilia, and identifies new links between cilia proteins and human disease

Differential regulation of disease genes associated with motile and nonmotile ciliopathies.

<p>Heat maps showing the expression of genes associated with motile (top) and nonmotile (middle) ciliopathies, as well as genes linked to Bardet-Biedl syndrome (bottom), a model ciliopathy.</p

Identification of a novel centrosome component.

<p>NIH/3T3 cells were transfected with a plasmid encoding TTC12-GFP. TTC12-GFP localizes to the centrosome as shown by overlap with gamma-tubulin staining. Green, TTC12-GFP. Red, gamma-tubulin.</p

Genes encoding centrosome proteins are differentially regulated during basal body formation.

<p>(A) Upregulation of regulatory and structural proteins required for centriole duplication in cycling cells and (B) heat map of putative and known centrosomal components. Genes are shown in rows and replicate arrays are shown in columns. Three biological replicates were performed for ALI+4, ALI+12 and GFP-. In addition, two technical replicates were performed for two of the ALI+4 biological replicates (columns 1,2 and 4,5) for a total of five columns. Data were zero-transformed against non-ciliated (GFP-) cells. The scale indicates the fold change in expression (log₂). Grey boxes represent gene spots that failed to pass quality control filters for the indicated array.</p

Functional mapping of candidate centrosome/cilia proteins and identification of novel centrosome components.

<p>Candidate genes were organized by known physical and genetic interactions mined from Mitocheck, the Human Protein Reference Database and BioGrid. Clusters containing genes associated with cell cycle control (A), hedgehog signaling (B) and the PCM1 complex (C) are shown.</p

Microarray analysis of ciliating mouse tracheal epithelial cells. (

<p>A) Immunofluorescence staining showing representative images of GFP expression and ciliogenesis in <i>FOXJ1:GFP</i> MTEC cultures before establishment of the air-liquid interface (pre-ALI), at 4 days after ALI (ALI+4) and at 12 days after ALI (ALI+12). MTEC cultures were stained with antibodies against GFP, against glutamylated tubulin to mark cilia and basal bodies, and with DAPI to mark nuclei. GFP is expressed from the <i>FOXJ1</i> promoter early during ciliogenesis, but is not expressed pre-ALI. At ALI+4, most <i>FOXJ1:GFP+</i> cells are undergoing centriole formation but have not formed cilia. At ALI+12, most <i>FOXJ1:GFP+</i> cells are forming cilia or have completed ciliogenesis. Scale bars, 5 µm. (B) FACS analysis of cells dissociated from a trachea from a wild type mouse (left panel) and a trachea from a <i>FOXJ1:GFP</i>-expressing mouse (right panel). The red and blue rectangles are representative gates used to sort GFP+ and GFP- populations, respectively. (C) Sorted cell populations were stained with DAPI (blue) and combined acetylated alpha-tubulin and gamma-tubulin antibodies (green) to detect cilia and basal bodies. After sorting, 90–95% of cells in the GFP+ population at ALI+12 had observable cilia and or amplified basal bodies, whereas <1% of cells in the GFP- population stained positive for these markers, although some had a single primary cilium (arrowhead).</p

Identification of new links between centrosomes, cilia and human disease.

<p>(A, B) NIH/3T3 cells were transfected with a plasmid encoding MDM1-GFP. MDM1-GFP localizes to centrosomes (A) and the primary cilium (B). NIH/3T3 cells and MTECs were stained for MLF1 with an anti-MLF1 antibody. MLF1 localizes to the primary cilium in NIH/3T3 cells (C) and motile cilia in MTECs (D). Insets show higher magnification of merged images, slightly offset as indicated by circles in upper left. Scale bars, 5 µm. Green, GFP (A, B); MLF1 (C, D). Red, gamma-tubulin (A); acetylated tubulin (B); polaris (C, D).</p

Subtractive analysis of MTEC microarray data.

<p>(A) Experimental flowchart detailing microarray analysis. (B) The number of differentially expressed genes in ciliated MTECs at adj. p-values <0.05, <0.01 and <0.001 before subtraction (non-subtracted) and after subtraction. The non-subtracted gene list is the transcriptional profile of GFP+ cells compared to a universal mouse reference RNA. The subtracted gene list was derived analytically by comparing the transcriptional profile of GFP+ cells to that of GFP- cells from MTEC cultures. The number of genes that show 2-fold or greater differential expression and have adjusted p-values of 0.05, 0.01 and 0.001 are shown before and after subtractive analysis. After subtraction, 649 genes were significantly upregulated at ALI+4 with respect to GFP- cells, and 73 genes at ALI+12. 143 genes were downregulated at ALI+4 and none at ALI+12 (adj. p<0.05). (C) The overlap of significantly upregulated and downregulated genes (adj. p<0.05) from ALI+4, ALI+12 and GFP- pools decreases after subtraction. The GFP- group in both panels represents genes that are differentially expressed in non-ciliated cells relative to the universal reference RNA. M = log₂(fold change).</p