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Aberrant extrathymic T cell receptor gene rearrangement in the small intestinal mucosa: a risk factor for coeliac disease?
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. (Hammarström)
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. (Hammarström)
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2009 (English)In: Gut, ISSN 0017-5749, E-ISSN 1468-3288, Vol. 58, no 2, 189-195 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Coeliac disease is a small intestine enteropathy caused by permanent intolerance to wheat gluten. Gluten intake by patients with coeliac disease provokes a strong reaction by intestinal intraepithelial lymphocytes (IELs), which normalises on a gluten-free diet. AIM: To investigate whether impaired extrathymic T cell maturation and/or secondary T cell receptor (TCR) gene recombination in IELs are features of coeliac disease which could contribute to the failure of establishing tolerance to gluten.

METHODS: Expression levels of the four splice-forms of recombination activating gene-1 (RAG1) mRNA and preT alpha-chain (preTalpha) mRNA were determined in IEL-subsets of children with coeliac disease and controls. Frequencies of RAG1 expressing IELs were determined by immunomorphometry.

RESULTS: In controls, the RAG1-1A/2 splice-form selectively expressed outside the thymus, was dominant and expressed in both mature (TCR(+)) and immature (CD2(+)CD7(+)TCR(-)) IELs ( approximately 8 mRNA copies/18S rRNA U). PreTalpha was expressed almost exclusively in CD2(+)CD7(+)TCR(-) IELs ( approximately 40 mRNA copies/18S rRNA U). By contrast, RAG1 and preTalpha mRNA levels were low in patients with coeliac disease compared to controls, both with active disease and with inactive, symptom-free disease on a gluten-free diet (p values <0.01 for mature and <0.05 for immature IELs). Similarly, the frequencies of RAG1+ IELs were significantly lower in patients with coeliac disease compared to controls (p<0.001).

CONCLUSIONS: Patients with coeliac disease appear to have an impaired capacity for extrathymic TCR gene rearrangement. This is an inherent feature, which probably plays a pivotal role in the failure to efficiently downregulate the T cell response to gluten.

Place, publisher, year, edition, pages
2009. Vol. 58, no 2, 189-195 p.
National Category
Immunology
Identifiers
URN: urn:nbn:se:umu:diva-21080DOI: 10.1136/gut.2007.125526PubMedID: 18299319OAI: oai:DiVA.org:umu-21080DiVA: diva2:210796
Available from: 2009-04-06 Created: 2009-04-02 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Innate and adaptive immunity in childhood celiac disease
Open this publication in new window or tab >>Innate and adaptive immunity in childhood celiac disease
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Celiac disease (CD) is an inflammatory small-bowel enteropathy caused by a permanent intolerance to wheat gluten and related proteins in rye and barley. Even though the disease originate from the small intestine the clinical symptoms varies in affected individuals and are often different in small children compared to adolescents and adults. Susceptibility to develop the disease is strongly associated with certain genetic factors i.e. HLADQ2/DQ8 but it is undoubtedly that additional inherited and environmental factors are involved. As specific T lymphocyte reactions are central in the pathogenesis of CD, six key cytokine messenger RNA levels in intestinal intraepithelial and lamina propria T lymphocytes (IEL, LPL), retrieved from small intestinal biopsies, were determined by using quantitative real-time reverse transcription polymerase chain reaction (RTPCR). Levels of cytokines, small secreted proteins which mediate and regulate immunity, in children with active disease were compared with that of treated children and controls. Interferon (IFN)-γ and interleukin (IL)-10 were also determined at the protein level by immunohistochemistry. Active celiac disease was characterized by distortions in cytokine expression, with highly significant increases of IFN-γ and IL-10 but no concomitant increases in tumor necrosis factor α (TNF-α), transforming growth factor β1 (TGFβ1), or IL-2 and no induction of IL-4. A marked shift of IFN-γ and IL-10 production from LPLs to IELs was characteristic of active celiac disease, and as many as one fourth of the IELs expressed IFN-γ. IELs in treated, symptom-free celiac patients still had increased IFN-γ levels compared with controls. In CD, gluten intake seems to cause an overreaction in IELs, with uncontrolled production of IFN-γ and IL-10 which may cause both recruitment of more IELs and a leaky epithelium, leading to a vicious circle with amplified immune activity and establishment of the intestinal lesion. In order to determine different IEL subsets contribution of the produced cytokines, γδIELs, CD4+αβIELs, and CD8+αβIELs as well as CD94+CD8+αβIELs and CD94CD8+αβIELs of children with active CD and children with no food-intolerance were analyzed for cytokine mRNA expression levels by RT-PCR. In active CD, CD8+αβIELs had the highest expression levels of IFN-γ- and IL-10 mRNA and constituted the cellular source for almost all IFN-γ and a large fraction of the IL-10. Expression levels of these two cytokines correlated and were higher in CD94-CD8+αβIELs than CD94+CD8+αβIELs CD4+αβIELs had the highest expression levels of TNF-α and despite the small number of this cell subset they contributed with half of the small amounts of this cytokine. Interestingly, TNF-α levels correlated with IL-10 in CD4+αβIELs. γδIELs had the lowest expression levels of IFN-γ, TNF-α, IL-10, and TGF-β1. Essentially no IL-2 mRNA was detected in the three IEL subpopulations. “Classical” CD8+CD94-αβT cells in the epithelial compartment are responsible for most of the excessive production of proinflammatory IFN-γ. The question whether an impaired extrathymic T cell maturation and/or capacity for secondary T cell receptor (TCR) gene recombination in iIELs is a contributing factor to CD was addressed. Expression levels of recombination activating gene-1 (RAG1) and the pre T α-chain (preTα) mRNAs were determined in IEL T cell lineage subsets of children with CD and controls. In controls, RAG1 was expressed in both mature (TCRγδ+ and TCRαβ+) and immature (CD2+CD7+TCR-) IELs while preTα was expressed preferentially in immature IELs. The RAG1 splice form selectively expressed outside thymus (RAG1 1A/2) as well as preTα were significantly decreased in CD patients both in active and inactive disease suggesting a deteriorated capacity of de novo TCR gene rearrangement in local T cell development and / or of secondary TCR gene rearrangement during editing or antigen-driven revision. This may lead to an imbalance between thymus- and gut derived T lymphocytes in the intestinal mucosa with consequent inefficient regulation of T cell responses against food antigens. Innate or nonspecific immunity is the first line, immediate defense against pathogens mediated by the epithelial cells in the intestine (IECs). As certain adaptive immune reaction in CD mimics that of intestinal infections, aberrant innate immune reaction could be a contributing factor to CD. Therefore jejunal biopsies were screened for bacteria and the innate immune status of the epithelium was investigated. Bacteria were freqently (40%) associated with the mucosa of children with active but also treated disease (20%) compared to controls (2%). Lack of antimicrobial factors such as mucins, proteins forming protective biofilm on the IECs, defensins and lysozym, peptides and enzymes with antibacterial effects, could not explain the presence of bacteria. If anything, mucin-2 (MUC2), α-defensins, HD-5, HD-6, and lysozyme mRNA levels were increased in epithelial cells in active CD, returning to normal levels in treated CD. Their expression levels correlated to the IFN-γ mRNA levels in IELs. Analysis of beta defensins, hBD-1 and hBD-2 as well as carcinoembryonic antigen (CEA) cell adhesion molecule 1a (CECAM1a), glycoproteins in the glycocalyx with ability to bind micro organisms, were not affected by the disease. Lectin staining by histochemistry revealed that goblet cells were stained by UEA1 in CD both active and treated but not in controls. The opposite pattern was seen for the lectin PNA where staining was seen in controls in the glycocalyx layer but not in CD. Thus altered glycocalyx/mucous layer may promote bacterial adhesion in CD.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2006. 69 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1054
National Category
Immunology in the medical area
Research subject
Immunology
Identifiers
urn:nbn:se:umu:diva-874 (URN)91-7264-162-2 (ISBN)
Public defence
2006-10-06, Astrid Fagreussalen, 6A, NUS, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2006-09-25 Created: 2006-09-25 Last updated: 2013-03-25Bibliographically approved
2. Immune response of the small intestinal mucosa in children with celiac disease: impact of two environmental factors, resident microbiota and oats
Open this publication in new window or tab >>Immune response of the small intestinal mucosa in children with celiac disease: impact of two environmental factors, resident microbiota and oats
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Celiac disease (CD) is an immune-mediated enteropathy caused by permanent intolerance to dietary gliadin in wheat gluten and related prolamines in barley and rye. The pathogenesis of CD is still unknown and several different environmental factors have been associated with CD, such as dysbiosis of the microflora. In this translational study we investigated the immune status and the interplay of T-cells and Tregs in the mucosa of children with CD and controls, as well as the immune status in treated CD patients, provoked by either dietary oats, CD associated bacteria or gluten.

The major findings in the studies were: First, indicators of extrathymic T-cells maturation (ETCM), i.e., the RAG1 enzyme required for recombination of the T cell receptor (TCR) genes and the preTα-surrogate chain in the immature TCR, were both expressed at lower levels in CD patients compared to controls. In addition, IELs expressing RAG1 were less abundant in CD patients compared to controls. The levels of these two indicators stayed low in treated CD patients as well, suggesting that impaired capacity of ETCM is an inherent feature of CD patients. Second, IL-17A, a cytokine involved in both inflammation and anti-bacterial responses was increased in active CD. The major cellular source was CD8+IELs. Furthermore, ex vivo challenge of biopsies from treated CD patients with gluten and with CD-associated bacteria induced an IL-17A response. The CD-associated bacteria also influenced the magnitude of the IL-17A response to gluten. Third, we investigated the effect of dietary oats on local immune status in the intestinal mucosa by comparing CD patients receiving GFD with and without oats. 22 different mRNAs for immunity effector molecules and tight junction proteins were analyzed. We found that expression of two down-regulatory cytokines, two activating NK-receptors and the tight-junction protein claudin-4 normalized in patients on a standard GFD while they did not normalize in patients on a GFD with oats. Fourth, we analyzed the expression level of mRNAs for chemokines, cytotoxic effector molecules, NK-receptors and their ligands in IELs and epithelial cells. Expression levels of several of these genes follow disease activity, suggesting massive recruitment of immune cells by both cell types accompanied by increased IEL-mediated cytotoxicity in the epithelium of inflamed mucosa.

In this thesis we have identified three potential risk factors for development of CD: 1) an inherent lower level of ETCM in the small intestinal mucosa than in controls. This could lead to decreased generation of regulatory T cells and less capacity to tolerate gluten and adapt to the local milieu in the mucosa. 2) Dysbiosis of the resident microbiota with increased IL-17A production that could promote local inflammation and immune cell infiltration as well as antibacterial reactions. 3) Dietary oats may provoke a local immune response in a sub-population of CD patients. These patients should probably avoid oats in their GFD but larger studies are needed.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2013. 69 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1576
Keyword
Celiac disease, Oats, Microflora, Immune response
National Category
Immunology in the medical area
Research subject
Immunology
Identifiers
urn:nbn:se:umu:diva-71030 (URN)978-91-7459-663-2 (ISBN)978-91-7459-664-9 (ISBN)
Public defence
2013-06-13, E04, byggnad 6E, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2013-05-23 Created: 2013-05-16 Last updated: 2013-05-23Bibliographically approved

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