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Defensins and cytokines in inflammatory bowel disease
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ulcerative colitis (UC) and Crohn’s disease (CD) constitute the two major inflammatory bowel diseases in man. Both are serious chronic illnesses of the intestine with severe debilitating effects. The etiology of the diseases is unknown, but involvement of both adaptive and innate immune reactions seems to be major factors in the pathogenesis. In this thesis the roles of key molecules of the adaptive immunity, i.e. interleukin-2 (IL-2), and innate immunity, i.e. β-defensins, were studied both in human inflammation of the large intestine and in mouse colitis models.

β-defensins are small endogenous peptides with antimicrobial activity. Previous studies showed that expression of human β-defensin-2 (hBD-2), hBD-3, and hBD-4 is induced in colonic epithelial cells of UC patients. Here we demonstrate that cells expressing these three β-defensins are present also in the colonic lamina propria of UC patients and less frequently in CD patients, and controls. These cells were identified as mature plasma cells by the highly specific CD138 marker, by their prominent IgA or IgG expression, and by their ultra structural characteristics. Immunoelectron microscopy analysis of the hBD-2 peptide demonstrated synthesis and transport for secretion. Defensin producing plasma cells were 2-3 times more abundant in UC colon than in control and CD colon. Additionally, hBD-2 mRNA expression was demonstrated in 3 out of 4 well-characterized plasma cell lines.

Defensin expression was studied in large intestinal mucosa before and after onset of colitis in two colitis models - the IL-2 KO mouse and the dextran sulphate sodium (DSS) induced colitis mouse. Mouse β-defensin-3 (mBD-3) and mBD-4 mRNA was expressed in colonic epithelial cells of homozygous IL-2 KO (IL-2-/-) mice with established colitis (15 weeks old) and at significantly higher levels than in apparently healthy wild type mice, heterozygous (IL-2+/-) mice, and 5 weeks old IL-2-/- mice. Similarly mBD-3 was expressed in epithelial cells of DSS treated mice with chronic colitis but not in DSS treated mice with acute inflammation. Cells expressing mBD-3 mRNA were seen also in colonic lamina propria of diseased animals. Thus, expression of β-defensins in the colonic epithelium seems to be a consequence of the chronic inflammation.

IL-2+/- mice have reduced levels of IL-2 in the intestinal mucosa but are clinically healthy. IL-2+/- mice showed markedly reduced susceptibility to DSS induced colitis. This was associated with a significantly reduced infiltration of both CD4+ and CD8+ T cells in the colonic mucosa and lower expression levels of the cytokines IL-2, IL-4, and IL-10 in colonic T cells compared to DSS treated wild type mice. These results suggest that reduced level of IL-2 leads to attenuated activation and function of colonic T cells in turn causing a milder colitis in response to DSS challenge.

Interestingly, IL-2+/- mice had a reduced frequency of regulatory T cells (CD4+CD25+) in both small and large intestine compared to wild type mice. As the small intestine of IL-2-/- and IL-2+/- mice appear normal, small intestinal T cells have never been critically analyzed in these mice. The cytokine profile of small intestinal T cells in IL-2-/- and IL-2+/- mice was changed compared to wild type control mice (IL-2+/+) with significantly elevated expression levels of IL-10 and IL-4. DSS treatment of IL-2+/- mice caused a marked reduction in cytokine expression levels in small intestinal T-cells. These results suggest that lack of IL-2 and even the partial decrease seen in IL-2+/- mice influence T cell function locally in the intestinal mucosa and cause a skewed cytokine milieu also in the small intestine despite its normal histology.

In this thesis we demonstrate the pivotal importance of different T-cell subsets in intestinal inflammation. The upregulation of intestinal antimicrobial peptides seems to be a consequence of chronic inflammation in an effort to minimize intestinal damage.

Place, publisher, year, edition, pages
Umeå: Medicin , 2007. , 58 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1129
Research subject
Immunology
Identifiers
URN: urn:nbn:se:umu:diva-1377ISBN: 978-91-7264-421-2 (print)OAI: oai:DiVA.org:umu-1377DiVA: diva2:140806
Public defence
2007-10-19, Astrid Fagreussalen A103, NUS, Umeå, 09:00 (English)
Opponent
Available from: 2007-09-28 Created: 2007-09-28 Last updated: 2011-04-04Bibliographically approved
List of papers
1. Beta-defensin production by human colonic plasma cells: a new look at plasma cells in ulcerative colitis
Open this publication in new window or tab >>Beta-defensin production by human colonic plasma cells: a new look at plasma cells in ulcerative colitis
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2007 (English)In: Inflammatory Bowel Diseases, ISSN 1078-0998, E-ISSN 1536-4844, Vol. 13, no 7, 847-855 p.Article in journal (Refereed) Published
Abstract [en]

Background: Previously, we showed that colonic epithelium of ulcerative colitis (UC) patients expresses increased levels of mRNA for 3 antimicrobial peptides, human P-defensin 2 (hBD-2), hBD-3, and hBD-4 compared to controls. Methods: Human colon mucosa was analyzed using double immunofluorescence staining, in situ hybridization, iumn,moelectron microscopy, and quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) with specific antibodies and probes in the respective assays. Results: We demonstrate that lamina propria in colon from UC patients, Crohn's colitis patients, and controls contain cells that express hBD-2. These cells were identified as mature plasma cells by the highly specific CD 138 marker, by their prominent IgA or IgG expression, and by their ultrastructural characteristics. By immuno-electron microscopy it was furthermore shown that the hBD-2 peptide was expressed in rough endoplasmic reticulum, the Golgi complex, and cytoplasmic vesicles, reflecting consecutive steps of synthesis and transport for secretion. Plasma cells were 2-3 times more abundant in UC colon than in control colon and Crohn's colitis. Moreover, plasma cells in UC colon expressed hBD-3 and hBD-4 mRNA. Additionally, hBD-2 mRNA expression was demonstrated in 3 out of 4 well-characterized plasma cell lines. Conclusions: Mature colonic plasma cells can express multiple beta-defensins. In UC, defensin production by plasma cell, is probably clinically relevant since plasma cells accumulate in large numbers between the distorted crypts and muscularis mucosae, first focally than diffusely, so as to protect against microbial attack.

Keyword
Crohn's colitis, intestine, innate immunity, ultrastructure
National Category
Gastroenterology and Hepatology
Identifiers
urn:nbn:se:umu:diva-2595 (URN)10.1002/ibd.20141 (DOI)000247784800004 ()17387677 (PubMedID)
Available from: 2007-09-28 Created: 2007-09-28 Last updated: 2017-12-14Bibliographically approved
2. Chronic colitis induces expression of β-defensins in murine intestinal epithelial cells
Open this publication in new window or tab >>Chronic colitis induces expression of β-defensins in murine intestinal epithelial cells
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2011 (English)In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 163, no 1, 123-130 p.Article in journal (Refereed) Published
Abstract [en]

Anti-microbial peptides are important effectors in innate immunity. In the gut they defend against pathogens, shape the commensal microbiota and probably control intestinal homeostasis. Ulcerative colitis (UC), but not Crohn's disease, shows increased expression of inducible β-defensins (hBD-2, hBD-3 and hBD-4) in colonic epithelial cells. Does inducible defensin production precede the chronic intestinal inflammation characteristic of UC, or is it a consequence of the T cell-driven chronic inflammation? The aim was to analyse defensin mRNA and protein expression in colonic epithelial cells in two colitis mouse models resembling UC, the interleukin (IL)-2(-/-) mouse and the dextran sulphate sodium (DSS)-induced colitis mouse. Defensin mRNA was assayed by in situ hybridization and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Defensin peptide was assayed by immunohistochemistry. Mouse β-defensin 3 (mBD-3, orthologue to hBD-2) was up-regulated strongly in colonic epithelium of 15-week-old IL-2(-/-) mice and DSS-induced colitis mice with chronic bowel inflammation, but not in apparently healthy IL-2(-/-) 5-week-old mice, IL-2(+/-) 15-week-old mice or in acute stage DSS mice. Up-regulation was seen both at the mRNA- and at the protein level (only mBD-3 investigated). IL-17, but not several other cytokines, including interferon (IFN)-γ, induced mBD-3 mRNA expression in mouse colon carcinoma cells. The mRNA expression level of the constitutively expressed α-defensin, cryptdin-4, was up-regulated marginally in acute stage DSS-colitis mice and in IL-2(-/-) mice before signs of colitis. Inducible β-defensin expression in colonic epithelium is the consequence of the chronic bowel inflammation caused by activated T cells releasing cytokines including IL-17.

Keyword
acute and chronic DSS-induced colitis;anti-microbial peptides;colonic epithelium;IL-2−/− mice;interleukin-17
National Category
Immunology
Identifiers
urn:nbn:se:umu:diva-2596 (URN)10.1111/j.1365-2249.2010.04282.x (DOI)
Available from: 2007-09-28 Created: 2007-09-28 Last updated: 2017-12-14Bibliographically approved
3. Reduced susceptibility to dextran sulphate sodium-induced colitis in the interleukin-2 heterozygous (IL-2) mouse.
Open this publication in new window or tab >>Reduced susceptibility to dextran sulphate sodium-induced colitis in the interleukin-2 heterozygous (IL-2) mouse.
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2005 (English)In: Immunology, ISSN 0019-2805, E-ISSN 1365-2567, Vol. 114, no 4, 554-564 p.Article in journal (Refereed) Published
Keyword
Animals, CD4-Positive T-Lymphocytes/immunology, Cell Proliferation, Colitis/*immunology/pathology, Colon/pathology, Cytokines/immunology, Dextran Sulfate, Disease Susceptibility, Female, Flow Cytometry, Heterozygote, Interleukin-2/*genetics, Intestinal Mucosa/*immunology, Lymphocyte Activation, Male, Mice, Mice; Inbred C57BL, Mice; Mutant Strains, Receptors; Interleukin-2/immunology, Reverse Transcriptase Polymerase Chain Reaction, T-Lymphocytes/*immunology
National Category
Immunology
Identifiers
urn:nbn:se:umu:diva-15032 (URN)10.1111/j.1365-2567.2005.02123.x (DOI)15804292 (PubMedID)
Available from: 2007-06-25 Created: 2007-06-25 Last updated: 2017-12-14Bibliographically approved
4. IL-2 levels influence the function of small intestinal T cells: a study in interleukin-2 (IL-2) deficient mice
Open this publication in new window or tab >>IL-2 levels influence the function of small intestinal T cells: a study in interleukin-2 (IL-2) deficient mice
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(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-2598 (URN)
Available from: 2007-09-28 Created: 2007-09-28 Last updated: 2011-04-04Bibliographically approved

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