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Adaptive immune response in the intestinal mucosa of microscopic colitis patients
Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Microscopic colitis (MC) is a chronic diarrhoeal disease of unknown aetiology, comprising collagenous colitis (CC) and lymphocytic colitis (LC). The nature of the adaptive local immune responses in the mucosa of MC patients is however far from elucidated. The present study investigates phenotypic and functional characteristics of the adaptive local immune responses in the colonic mucosa of these patients.

Our immunohistochemistry and flow cytometry studies (Paper I & II) demonstrated increased frequencies of CD8+ T cells in the colonic epithelium and lamina propria of both LC and CC patients compared to controls, whereas the frequencies of CD4+ T cells were unaltered or reduced. Our flow cytometry data revealed increased local activation of both CD4+ and CD8+ T cells in the lamina propria as well as the intraepithelial compartment of CC and LC patients compared to controls, demonstrated as increased proportions of these cells expressing the active/memory marker CD45RO and the proliferation marker Ki67.

Analysis of recent thymic emigrants by measuring T cell receptor excision circle (TREC) levels in the colonic mucosa of CC and LC patients revealed reduced TRECs levels in these patients compared to controls (Paper III). These results suggests that the observed increased numbers of T cells in the mucosa of CC and LC patients is due to the expansion of local resident T cells rather than direct recruitment of recent thymic emigrants to the mucosa.

Molecular analysis of T helper (Th) cell and cytotoxic T lymphocyte (Tc) mucosal cytokines at messenger and protein levels in the colonic biopsies from CC and LC patients demonstrated a mixed Th17/Tc17 and Th1/Tc1 mucosal cytokine profile and revealed significant differences in the mucosal cytokine levels in CC and LC patients compared to controls (Paper IV).

Finally, we have set up an in vitro model to investigate how the colonic milieu affects the activation and differentiation of T lymphocytes (Paper V). Our preliminary data indicate increased production of both pro inflammatory and antiinflammatory cytokines by peripheral blood T cells in the presence of soluble factors from the inflamed colonic mucosa of CC patients compared to controls.

Place, publisher, year, edition, pages
Örebro: Örebro universitet , 2013. , p. 83
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 84
Keywords [en]
Microscopic colitis, collagenous colitis, lymphocytic colitis, intraepithelial lymphocytes, lamina propria lymphocytes, T cell receptor excision circle, T helper cells, cytotoxic T lymphocyte and mucosal cytokines
National Category
Medical and Health Sciences
Research subject
Biomedicine
Identifiers
URN: urn:nbn:se:oru:diva-27894ISBN: 978-91-7668-929-5 (print)OAI: oai:DiVA.org:oru-27894DiVA, id: diva2:610490
Public defence
2013-05-24, Hörsal P2, Prismahuset, Örebro universitet, Fakultetsgatan 1, Örebro, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2013-03-18 Created: 2013-03-11 Last updated: 2017-10-17Bibliographically approved
List of papers
1. Immunohistochemical characterization of lymphocytes in microscopic colitis
Open this publication in new window or tab >>Immunohistochemical characterization of lymphocytes in microscopic colitis
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2013 (English)In: Journal of Crohn's and Colitis, ISSN 1197-4982, Vol. 7, no 10, p. e434-e442Article in journal (Refereed) Published
Abstract [en]

Background and Aims: Microscopic colitis (MC), encompassing the subgroups collagenous colitis (CC) and lymphocytic colitis (LC), is characterized by macroscopically normal or near-normal colonic mucosa, and an increased number of intraepithelial lymphocytes (IELs) and mononuclear cell infiltration in the underlying lamina propria (LP), in addition to an increased collagen layer in CC. This study aimed to characterize the inflammatory cells involved in mucosal inflammation, using immunohistochemistry.

Methods Paraffin-embedded biopsies from 23 untreated patients with MC (CC = 13, LC = 10) and 17 controls were stained with antibodies against CD3, CD4, CD8, CD20, CD30, Foxp3, CD45RO and Ki67. Computerized image analysis was used to calculate areas of stained lymphocytes in the surface and crypt epithelia as well as in the LP.

Results In CC and LC, an increase of predominantly CD8+ lymphocytes was seen in both the epithelium and the lamina propria, whereas a decreased amount of CD4+ lymphocytes was found in the lamina propria. CD45RO+ and Foxp3+ cells were more abundant in all areas in both patient groups compared to controls, as were CD20+ areas, although more scarce. Ki67+ areas were only more abundant in the epithelium, whereas CD30+ areas were more abundant in the lamina propria of both patient groups compared to controls.

Conclusions This study confirms an increased amount of CD8+ lymphocytes in the epithelium. Lymphocytic proliferation and activation markers were more abundant, whereas a decreased amount of CD4+ lymphocytes was seen in the LP. Further studies are needed to reveal the underlying mechanism(s).

Place, publisher, year, edition, pages
Elsevier, 2013
Keywords
Collagenous colitis, Lymphocytic colitis, Microscopic colitis, Lymphocytes, Immunohistochemistry
National Category
Biomedical Laboratory Science/Technology
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-30117 (URN)10.1016/j.crohns.2013.02.007 (DOI)23523417 (PubMedID)2-s2.0-84884146798 (Scopus ID)
Available from: 2013-08-02 Created: 2013-08-02 Last updated: 2018-05-19Bibliographically approved
2. Microscopic colitis patients have increased frequencies of Ki67+proliferating and CD45RO+ active/memory CD8+ and CD4+8mucosal T cells
Open this publication in new window or tab >>Microscopic colitis patients have increased frequencies of Ki67+proliferating and CD45RO+ active/memory CD8+ and CD4+8mucosal T cells
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2013 (English)In: Journal of Crohn's & Colitis, ISSN 1873-9946, E-ISSN 1876-4479, Vol. 7, no 9, p. 694-705Article in journal (Refereed) Published
Abstract [en]

Background: Collagenous colitis (CC) and lymphocytic colitis (LC) are chronic inflammatory bowel disorders of unknown etiology. This study investigated phenotypic characteristics of the mucosal lymphocytes in CC and LC.

Methods: Lamina propria and intraepithelial lymphocytes (LPLs, IELs) isolated from mucosal biopsies from CC (n = 7), LC (n = 6), as well as LC or CC patients in histopathological remission, (LC-HR) (n = 6) and CC-HR (n = 4) and non-inflamed controls (n = 10) were phenotypically characterized by four-color flow cytometry.

Results: The proportions of CD8+ IELs were increased in CC and LC (p < 0.01) compared to controls. Increased proportions of CD45RO+CD8+ IELs and LPLs were observed in LC and even more in CC patients (p < 0.01). Both CC (p < 0.05) and LC patients had elevated proportions of CD4+8+ IELs and LPLs compared to controls. The proportions of CD45RO+ cells were increased in CD4+8+ IELs and LPLs (p < 0.05) in CC and LC patients compared to controls. Both CC (p < 0.05) and LC patients had higher proportions of Ki67+CD8+ IELs and LPLs compared to controls.

In contrast, decreased proportions of CD4+ LPLs were observed in CC and LC as well as CD4+ IELs in LC compared to controls. Increased proportions of Ki67+CD4+ IELs and LPLs (p < 0.05) were observed in CC and LC patients. CC-HR but not LC-HR patients demonstrated normalized proportions of both IELs and LPLs compared to CC and LC patients respectively.

Conclusion: LC and CC patients have differences in mucosal lymphocyte subsets, with increased proportions of Ki67+ and CD45RO+ CD8+ and CD4+8+ mucosal T cells.

Place, publisher, year, edition, pages
Oxford, United Kingdom: Oxford University Press, 2013
Keywords
Collagenous colitis, Lymphocytic colitis, Flow cytometry, Lamina propria lymphocytes, Intraepithelial lymphocytes
National Category
Medical and Health Sciences Gastroenterology and Hepatology
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-25588 (URN)10.1016/j.crohns.2012.08.014 (DOI)000323995900002 ()22995775 (PubMedID)
Available from: 2012-08-30 Created: 2012-08-30 Last updated: 2017-12-07Bibliographically approved
3. Reduced T cell receptor excision circle (TREC) levels in the colonic mucosa of microscopic colitis patients indicate local proliferation rather than homing of peripheral lymphocytes to the inflamed mucosa
Open this publication in new window or tab >>Reduced T cell receptor excision circle (TREC) levels in the colonic mucosa of microscopic colitis patients indicate local proliferation rather than homing of peripheral lymphocytes to the inflamed mucosa
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Aims: Dysregulated T cell responses in the intestine may lead to chronic bowel inflammation such as collagenous colitis (CC) and lymphocytic colitis (LC), together known as microscopic colitis (MC). Having demonstrated increased local T cell responses in the intestinal mucosa of MC patients, we investigated the recent thymic emigrants by measuring T cell receptor excision circle (TREC) levels in the colonic mucosa of CC and LC patients.

Methods: Mucosal biopsies from CC (n=8), LC (n=5), and CC or LC patients in histopathological remission, (CC-HR, n=3), (LC-HR, n=6), non-inflamed diarrhoea patients (n=17) and controls (n=10) were analysed for TRECs expression by real time PCR.

Results: The median TREC levels were lower in both CC and LC patients as well as in LCHR patients compared to controls. In contrast to MC patients, non-inflamed diarrhoea patients presented with enhanced TREC levels compared to controls. None of the recorded differences did however reach statistical significance. No differences were observed in median TREC levels in either CC-HR or LC-HR patients compared to active CC and LC patients. A trend towards increased relative expression of CD3 was noted in all MC subgroups examined; and reached statistical significance in LC patients compared to controls. LC patients had ignificantly increased CD3 mRNA levels also compared to CC, CC-HR, LC-HR and non-inflamed iarrhoea patients.

Conclusions: Reduced TRECs level in the colonic mucosa, together with our previously demonstrated enhanced expression of Ki67+ T cells, suggest local expansion of resident T lymphocytes in the inflamed mucosa of MC patients.

Keywords
Microscopic colitis, collagenous colitis, lymphocytic colitis, T cells, T cell receptor excision circles (TRECs)
National Category
Biomedical Laboratory Science/Technology
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-30120 (URN)
Available from: 2013-08-05 Created: 2013-08-05 Last updated: 2017-10-17Bibliographically approved
4. Microscopic colitis patients demonstrate a mixed Th17/Tc17 and Th1/Tc1 mucosal cytokine profile
Open this publication in new window or tab >>Microscopic colitis patients demonstrate a mixed Th17/Tc17 and Th1/Tc1 mucosal cytokine profile
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2013 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 55, no 3-4, p. 355-364Article in journal (Refereed) Published
Abstract [en]

Background:

Microscopic colitis (MC) is a chronic inflammatory bowel disorder of unknown aetiology comprising collagenous colitis (CC) and lymphocytic colitis (LC). Data on the local cytokine profile in MC is limited. This study investigated the T helper (Th) cell and cytotoxic T lymphocyte (CTL) mucosal cytokine profile at messenger and protein levels in MC patients.

Methods:

Mucosal biopsies from CC (n = 10), LC (n = 5), and CC or LC patients in histopathological remission (CC-HR, n = 4), (LC-HR, n = 6), ulcerative colitis (UC, n = 3) and controls (n = 10) were analysed by real-time PCR and Luminex for expression/production of IL-1 beta, -4, -5, -6, -10, -12, -17, -21, -22, -23, IFN-gamma, TNF-alpha, T-bet and RORC2.

Results:

Mucosal mRNA but not protein levels of IFN-gamma and IL-12 were significantly up regulated in CC, LC as well as UC patients compared to controls. Transcription of the Th1 transcription factor T-bet was significantly enhanced in CC but not LC patients. mRNA levels for IL-17A, IL-21, IL-22 and IL-6 were significantly up regulated in CC and LC patients compared to controls, albeit less than in UC patients. Significantly enhanced IL-21 protein levels were noted in both CC and LC patients. IL-6 protein and IL-1 beta mRNA levels were increased in CC and UC but not LC patients. Increased mucosal mRNA levels of IFN-gamma, IL-21 and IL-22 were correlated with higher clinical activity, recorded as the number of bowel movements per day, in MC patients.

Although at lower magnitude, IL-23A mRNA was upregulated in CC and LC, whereas TNF-alpha protein was increased in CC, LC as well as in UC patients.

Neither mRNA nor protein levels of IL-4, IL-5 or IL-10 were significantly changed in any of the colitis groups. LC-HR and especially CC-HR patients had normalized mRNA and protein levels of the above cytokines compared to LC and CC patients. No significant differences were found between LC and CC in cytokine expression/production.

Conclusion:

LC and CC patients demonstrate a mixed Th17/Tc17 and Th1/Tc1 mucosal cytokine profile.

Keywords
T cells, Mucosal cytokines, Microscopic colitis, Collagenous colitis, Lymphocytic colitis
National Category
Immunology in the medical area
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-29849 (URN)10.1016/j.molimm.2013.03.007 (DOI)000319540200020 ()
Available from: 2013-06-28 Created: 2013-06-28 Last updated: 2018-05-19Bibliographically approved
5. An in vitro model for analysis of the impact of the colonic milieu in collagenous colitis patients on peripheral T lymphocyte activation and differentiation
Open this publication in new window or tab >>An in vitro model for analysis of the impact of the colonic milieu in collagenous colitis patients on peripheral T lymphocyte activation and differentiation
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Soluble factors released by intestinal mucosal cells contribute to immune homeostasis in the gut. This is the first study to investigate the role of soluble factors from the intestinal mucosa of collagenous colitis (CC) patients in the regulation of effector T cells using a novel system that mimics the in vivo exposure of newly recruited peripheral blood T cells to soluble factors derived from the colonic milieu of normal individuals and inflamed CC patient mucosa.

Methods: Denuded biopsies (DNB) and isolated lamina propria mononuclear cells (LPMCs) from mucosal biopsies from CC patients and non-inflamed controls were cultured to collect conditioned medium (CM). Enriched peripheral blood CD4+ T cells from healthy donors were polyclonally activated in the absence or presence of CM from CC patients and controls. Proliferation, as well as secretion of IL-1β IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF-α was analysed the latter with Luminex® analysis.

Results: Peripheral CD4+ T cells exposed to CM from the colonic mucosa demonstrated reduced proliferation. This inhibition was less pronounced with DNB-CM derived from CC patients compared to non-inflamed control mucosa. In contrast, LPMC-CM from non-inflamed controls inhibited T-cell proliferation less than LPMC-CM from CC patients. Both DNB-CM and LPMC-CM from CC patients induced more or less increased production of the proinflammatory cytokines IFN-γ, IL-17A, IL-6 and TNF-α as well as the anti-inflammatory cytokines IL-4 and IL-10 from peripheral CD4+ T cells compared to non-inflamed controls. In contrast, IL-1β production by peripheral T cells showed mixed results – it was either increased or reduced in the presence of both DNB and LPMC-CM from CC patients compared to noninflamed controls with different blood donors and different concentrations.

Conclusion: Our preliminary data indicates reduced inhibition of proliferation of peripheral CD4+ T cells in the presence of mucosa-derived soluble factors from CC patients compared to controls. In addition, increased production of both inflammatory and anti-inflammatory cytokines by peripheral CD4+ T cells was recorded in the presence of soluble factors from the colonic mucosa of CC patients compared to controls. This model can be valuable in evaluating the effect(s) of existing and new drugs on T cell differentiation in the intestinal mucosa.

National Category
Biomedical Laboratory Science/Technology
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-30121 (URN)
Available from: 2013-08-05 Created: 2013-08-05 Last updated: 2017-10-17Bibliographically approved

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