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Innate immunity of human intestinal epithelium in childhood celiac disease: influences from celiac disease associated bacteria and dietary oats
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Marie-Louise Hammarström)ORCID iD: 0000-0003-4172-549X
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background & Aims: Celiac disease (CD) is a chronic inflammatory small-bowel enteropathy caused by permanent intolerance to gliadin in wheat gluten, and related proteins in ray and barley. It is disputed whether CD patients tolerate oats. The only treatment of CD is lifelong gluten-free diet (GFD). Only individuals that carry the HLA-DQ2 and/or DQ8 alleles, and eat gluten can develop CD. Dysbiosis in the gut microbiota is a suggested risk factor for CD. T cells in small intestinal mucosa, including intraepithelial lymphocytes (IELs), are known to be important in the pathogenesis of CD. In contrast, the role of intestinal epithelial cells (IECs) is poorly understood. In this thesis we investigated the role of IECs in the immune pathology of CD from duodenal mucosa of children with CD, clinical controls and treated CD. We also investigated the role of CD associated bacteria and oats supplemented GFD on the mucosal immune system.

Results: A new CD-associated bacterium, Prevotella jejuni, was isolated and characterized. It is a saccharolytic and proteolytic anaerobe. More than 25 defense-related genes, including IRF1, SPINK4, ITLN1, OAS2, CIITA, HLA-DMB, HLA-DOB, PSMB9, TAP1, BTN3A1, and CX3CL1, were upregulated in IECs in active CD. In two in vitro models for intestinal epithelium, small intestine enteroids and T84 polarized tight monolayers, we showed that 70% of these genes were upregulated by interferon (IFN)-γ via the IRF1 pathway. IRF1 was also upregulated by the CD-associated bacteria P. jejuni and Actinomyces gravenitzii. IECs expressed the NLRP6/8 inflammasome yielding CASP1 and biologically active interleukin (IL)-18, which induces IFN-γ in IELs. P. jejuni bound the intestinal epithelial cell lines T84, Caco2, HT29, and INT407, while Lachnoanaerobaculum umeaense preferentially bound Caco2. P. jejuni caused decreased transepithelial resistance over tight monolayers, while L. umeaense caused an increase. P. jejuni upregulated mRNAs for the detoxification molecules CYP1A1, CYP1A2, CYP1B1, and TIPARP, the chemokines CX3CL1, CXCL1, and CXCL10, the sialyltranserase ST3GAL4, and the inflammation promoting protein S100A3 in tight monolayers. L. umeaense upregulated the chemokines CCL20 and CXCL10, and down-regulated TLR2. In a randomized, double-blinded intervention trial comparing two study-groups, standard GFD and oat-containing GFD, we found that mRNAs for several immune effector molecules and tight junction proteins were only reduced in patients receiving GFD, but not in a substantial fraction of patients on GFD with oats. The down-regulatory cytokines IL-10 and TGF-β1, the cytotoxicity-activating NK-receptors NKG2C and NKG2E, and the tight junction protein claudin-4 remained elevated in the study group on GFD with oats.

Conclusions: IECs are far from inactive in CD. A key factor in the epithelial reaction in CD appears to be over-expression of IRF1 in IECs. Dual activation of IRF1 and IRF1-regulated genes, both directly by P. jejuni and indirectly by IFN-γ via the IL-18-inflammasome, would drastically enhance the inflammatory response and lead to the pathological situation seen in active CD. P. jejuni harms the intestinal epithelium, i.e., it is a likely risk factor for CD, while L. umeaense strengthen barrier function and local immunity, possibly acting as a protective. A fraction of CD patients should avoid oats in the diet.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2017. , 75 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1924
Keyword [en]
Celiac disease, dietary oats, gut microbiota, intestinal epithelium, IEL, IFN-γ, IRF1, IL-18, CXC3L1, inflammasome, permeability, Prevotella jejuni, Lachnoanaerobaculum umeaense
National Category
Immunology in the medical area
Research subject
Immunology
Identifiers
URN: urn:nbn:se:umu:diva-140691ISBN: 978-91-7601-768-5 (print)OAI: oai:DiVA.org:umu-140691DiVA: diva2:1152533
Public defence
2017-11-23, A5_R0, Analysvägen 3, Umeå, 09:00 (English)
Opponent
Supervisors
Projects
Doctoral thesis
Available from: 2017-11-02 Created: 2017-10-25 Last updated: 2017-10-31Bibliographically approved
List of papers
1. Noncontaminated dietary oats may hamper normalization of the intestinal immune status in childhood celiac disease
Open this publication in new window or tab >>Noncontaminated dietary oats may hamper normalization of the intestinal immune status in childhood celiac disease
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2014 (English)In: Clinical and Translational Gastroenterology, ISSN 2155-384X, E-ISSN 2155-384X, Vol. 5, e58Article in journal (Refereed) Published
Abstract [en]

OBJECTIVES: Life-long, strict gluten-free diet (GFD) is the only treatment for celiac disease (CD). Because there is still uncertainty regarding the safety of oats for CD patients, the aim was to investigate whether dietary oats influence the immune status of their intestinal mucosa.

METHODS: Paired small intestinal biopsies, before and after >11 months on a GFD, were collected from children with CD who were enrolled in a randomized, double-blind intervention trial to either of two diets: standard GFD (GFD-std; n=13) and noncontaminated oat-containing GFD (GFD-oats; n=15). Expression levels of mRNAs for 22 different immune effector molecules and tight junction proteins were determined by quantitative reverse transcriptase (RT)-PCR.

RESULTS: The number of mRNAs that remained elevated was higher in the GFD-oats group (P=0.05). In particular, mRNAs for the regulatory T cell (Treg) signature molecules interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1), the cytotoxicity-activating natural killer (NK) receptors KLRC2/NKG2C and KLRC3/NKG2E, and the tight junction protein claudin-4 remained elevated. Between the two groups, most significant differences were seen for claudin-4 (P=0.003) and KLRC3/NKG2E (P=0.04).

CONCLUSIONS: A substantial fraction of pediatric CD patients seem to not tolerate oats. In these patients, dietary oats influence the immune status of the intestinal mucosa with an mRNA profile suggesting presence of activated cytotoxic lymphocytes and Tregs and a stressed epithelium with affected tight junctions. Assessment of changes in levels of mRNA for claudin-4 and KLC3/NKG2E from onset to after a year on oats containing GFD shows promise to identify these CD patients.

National Category
Basic Medicine Pediatrics
Research subject
Immunology
Identifiers
urn:nbn:se:umu:diva-93052 (URN)10.1038/ctg.2014.9 (DOI)000355530800002 ()24964993 (PubMedID)2-s2.0-84903278030 (Scopus ID)
Available from: 2014-09-11 Created: 2014-09-11 Last updated: 2017-10-31Bibliographically approved
2. Prevotella jejuni sp nov., isolated from the small intestine of a child with coeliac disease
Open this publication in new window or tab >>Prevotella jejuni sp nov., isolated from the small intestine of a child with coeliac disease
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2013 (English)In: International Journal of Systematic and Evolutionary Microbiology, ISSN 1466-5026, E-ISSN 1466-5034, Vol. 63, no 11, 4218-4223 p.Article in journal (Refereed) Published
Abstract [en]

Five obligately anaerobic, Gram-stain-negative, saccharolytic and proteolytic, non-spore-forming bacilli (strains CD3 :27, CD3 :28(T), CD3 :33, CD3 :32 and CD3 :34) are described. All five strains were isolated from the small intestine of a female child with coeliac disease. Cells of the five strains were short rods or coccoid cells with longer filamentous forms seen sporadically. The organisms produced acetic acid and succinic acid as major metabolic end products. Phylogenetic analysis based on comparative 16S rRNA gene sequence analysis revealed close relationships between CD3 : 27, CD3 :28(T) and CD3 :33, between CD3 :32 and Prevotella histicola CCUG 55407(T), and between CD3 :34 and Prevotella melaninogenica CCUG 4944B(T). Strains CD3 : 27, CD3 :28(T) and CD3 :33 were clearly different from all recognized species within the genus Prevotella and related most closely to but distinct from P. melaninogenica. Based on 16S rRNA, RNA polymerase) beta-subunit (rpoB) and 60 kDa chaperonin protein subunit (cpn60) gene sequencing, and phenotypic, chemical and biochemical properties, strains CD3 :27, CD3 :28(T) and CD3 :33 are considered to represent a novel species within the genus Prevotella, for which the name Prevotella jejuni sp. nov. is proposed. Strain CD3 : 28(T) (=CCUG 60371(T)=DSM 26989(T)) is the type strain of the proposed novel species. All five strains were able to form homologous aggregates, in which tube-like structures were connecting individual bacteria cells. The five strains were able to bind to human intestinal carcinoma cell lines at 37 degrees C.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-84790 (URN)10.1099/ijs.0.052647-0 (DOI)000328666600045 ()
Available from: 2014-01-28 Created: 2014-01-20 Last updated: 2017-10-31Bibliographically approved
3. Influences of celiac disease associated bacteria on functions of intestinal epithelium: an in vitro study
Open this publication in new window or tab >>Influences of celiac disease associated bacteria on functions of intestinal epithelium: an in vitro study
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(English)Manuscript (preprint) (Other academic)
National Category
Immunology
Identifiers
urn:nbn:se:umu:diva-141364 (URN)
Available from: 2017-10-31 Created: 2017-10-31 Last updated: 2017-10-31
4. Immunopathology of childhood celiac disease: Key role of intestinal epithelial cells
Open this publication in new window or tab >>Immunopathology of childhood celiac disease: Key role of intestinal epithelial cells
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2017 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 9, e0185025Article in journal (Refereed) Published
Abstract [en]

BACKGROUND & AIMS: Celiac disease is a chronic inflammatory disease of the small intestine mucosa due to permanent intolerance to dietary gluten. The aim was to elucidate the role of small intestinal epithelial cells in the immunopathology of celiac disease in particular the influence of celiac disease-associated bacteria.

METHODS: Duodenal biopsies were collected from children with active celiac disease, treated celiac disease, and clinical controls. Intestinal epithelial cells were purified and analyzed for gene expression changes at the mRNA and protein levels. Two in vitro models for human intestinal epithelium, small intestinal enteroids and polarized tight monolayers, were utilized to assess how interferon-γ, interleukin-17A, celiac disease-associated bacteria and gluten influence intestinal epithelial cells.

RESULTS: More than 25 defense-related genes, including IRF1, SPINK4, ITLN1, OAS2, CIITA, HLA-DMB, HLA-DOB, PSMB9, TAP1, BTN3A1, and CX3CL1, were significantly upregulated in intestinal epithelial cells at active celiac disease. Of these genes, 70% were upregulated by interferon-γ via the IRF1 pathway. Most interestingly, IRF1 was also upregulated by celiac disease-associated bacteria. The NLRP6/8 inflammasome yielding CASP1 and biologically active interleukin-18, which induces interferon-γ in intraepithelial lymphocytes, was expressed in intestinal epithelial cells.

CONCLUSION: A key factor in the epithelial reaction in celiac disease appears to be over-expression of IRF1 that could be inherent and/or due to presence of undesirable microbes that act directly on IRF1. Dual activation of IRF1 and IRF1-regulated genes, both directly and via the interleukin-18 dependent inflammasome would drastically enhance the inflammatory response and lead to the pathological situation seen in active celiac disease.

National Category
Immunology
Identifiers
urn:nbn:se:umu:diva-139860 (URN)10.1371/journal.pone.0185025 (DOI)000411339900076 ()28934294 (PubMedID)
Available from: 2017-09-25 Created: 2017-09-25 Last updated: 2017-11-16Bibliographically approved

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