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Immunological Mechanisms and Natalizumab Treatment in Multiple Sclerosis: Studies on lymphocytes, inflammatory markers and magnetic resonance spectroscopy
Linköping University, Department of Clinical and Experimental Medicine, Neurology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Local Health Care Services in Central Östergötland, Department of Neurology.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: Multiple sclerosis (MS) is a chronic inflammatory, demyelinating disease of the central nervous system (CNS), and a frequent cause of neurological disability among young adults. In addition to focal inflammatory demyelinated lesions, diffuse white matter pathology as well as a neurodegenerative component with accumulating axonal damage and gliosis have been demonstrated and contribute to MS disease characteristics. The inflammatory component is considered autoimmune and mediated by auto-reactive T lymphocytes together with other cell populations of the immune system and their respective products like cytokines and chemokines. Treatment with natalizumab, a monoclonal antibody directed against the α4β1-integrin (VLA-4), reduces migration of potential disease-promoting cells to the CNS. The efficacy of natalizumab in reducing relapses and MRI activity is evident, however associated effects on the immune response and the neurodegenerative component in MS are not clear.

Methods: In total 72 MS patients were included, distributed among paper I-IV. We investigated effects associated with one-year natalizumab treatment in 31 MS patients regarding cytokine and chemokine levels in CSF and blood using multiplex bead assay analyses (paper I), as well as treatment effects on blood lymphocyte composition in 40 patients using flow cytometry, including functional assays of lymphocyte activation (paper II). Normal appearing white matter (NAWM) metabolite concentrations were assessed with proton magnetic resonance spectroscopy (1H-MRS) in 27 MS patients before and after one year of treatment (paper III). We also evaluated the balance between circulating T helper (Th) subsets in 33 MS patients using gene expression analyses of the CD4+ T cell related transcription factors in whole blood (paper IV).

Results: One-year natalizumab treatment was associated with a marked decline in pro-inflammatory cytokines (IL-1β and IL-6) and chemokines (CXCL8, CXCL9, CXCL10 and CXCL11) intrathecally. Circulating plasma levels of some cytokines (GM-CSF, TNF, IL-6 and IL-10) also decreased after treatment. Natalizumab treatment was further associated with an increase in lymphocyte numbers of major populations in blood (total lymphocytes, T cells, T helper cells, cytotoxic T cells, NK cells and B cells). In addition, T cell responsiveness to recall antigens and mitogens was restored after treatment. As to 1H-MRS metabolite concentrations in NAWM, no change in levels were detected post-to pretreatment on a group level. However, correlation analyses between one-year change in metabolite levels (total creatine and total choline) and levels of pro-inflammatory IL-1β and CXCL8 showed a pattern of high magnitude correlation coefficients (r=0.43-0.67). Gene expression analyses demonstrated a systemically reduced expression of transcription factors related to immunoregulatory T cell populations (regulatory T cells and Th2) in relapsing MS compared with controls.

Conclusions: Our findings support that an important mode of action of natalizumab is reducing lymphocyte extravasation, although cell-signalling effects through VLA-4 also may be operative. Correlation analyses between changes 1H-MRS metabolite concentrations and inflammatory markers possibly point towards an association between intrathecal inflammation and gliosis development in NAWM. Finally, gene expression analyses indicate a systemic defect at the mRNA level in relapsing MS, involving downregulation of beneficial CD4+ phenotypes.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. , 83 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1332
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-84274ISBN: 978-91-7519-787-6 (print)OAI: oai:DiVA.org:liu-84274DiVA: diva2:558428
Public defence
2012-11-02, Berzeliussalen, Campus US, Linköpings universitet, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2012-10-24Bibliographically approved
List of papers
1. Natalizumab treatment in multiple sclerosis: marked decline of chemokines and cytokines in cerebrospinal fluid
Open this publication in new window or tab >>Natalizumab treatment in multiple sclerosis: marked decline of chemokines and cytokines in cerebrospinal fluid
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2010 (English)In: Multiple Sclerosis, ISSN 1352-4585, E-ISSN 1477-0970, Vol. 16, no 2, 208-217 p.Article in journal (Refereed) Published
Abstract [en]

Natalizumab exerts impressive therapeutic effects in patients with multiple sclerosis (MS). The proposed main mode of action is reducing transmigration of leukocytes into the CNS, but other immunological effects may also be operative. Cytokines and chemokines are involved in the regulation of inflammatory responses and may reflect the disease process in MS. The objective of this study was to evaluate the effects of natalizumab treatment on cytokine and chemokine profiles systemically and intrathecally in multiple sclerosis. We used luminex to analyse a panel of cytokines (IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma, GM-CSF) and chemokines (CXCL9, CXCL10, CXCL11, CCL17, CCL22) in blood and cerebrospinal fluid (CSF) from 31 patients with relapsing MS before and after one year of natalizumab treatment. There was a marked decline in CSF levels of cytokines and chemokines, thus including pro-inflammatory cytokines (IL-1 beta, IL-6 and IL-8) as well as chemokines associated with both Th1 (CXCL9, CXCL10, CXCL11) and Th2 (CCL22). Circulating plasma levels of some cytokines (GM-CSF, TNF-alpha, IL-6 and IL-10) also decreased after one year of treatment. This is the first study to show that natalizumab treatment is associated with a global decline in cytokine and chemokine levels at a protein level. This finding was most pronounced in CSF, in line with the reduced transmigration of cells into CNS, whereas reduction in plasma levels indicates other possible mechanisms of natalizumab treatment.

Keyword
cerebrospinal fluid, chemokines, cytokines, luminex, multiple sclerosis, natalizumab, peripheral blood
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-54061 (URN)10.1177/1352458509355068 (DOI)000274326500010 ()
Note

Original Publication: Johan Mellergård, Måns Edström, Magnus Vrethem, Jan Ernerudh and Charlotte Dahle, Natalizumab treatment in multiple sclerosis: marked decline of chemokines and cytokines in cerebrospinal fluid, 2010, MULTIPLE SCLEROSIS, (16), 2, 208-217. http://dx.doi.org/10.1177/1352458509355068 Copyright: SAGE Publications http://www.uk.sagepub.com/

Available from: 2010-02-22 Created: 2010-02-22 Last updated: 2017-12-12
2. An Increase in B cell and Cytotoxic NK cell Proportions and Increased T cell Responsiveness in Blood of Natalizumab-treated Multiple Sclerosis Patients
Open this publication in new window or tab >>An Increase in B cell and Cytotoxic NK cell Proportions and Increased T cell Responsiveness in Blood of Natalizumab-treated Multiple Sclerosis Patients
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 12, e81685Article in journal (Refereed) Published
Abstract [en]

Background

Changes in the peripheral blood lymphocyte composition probably both mediate and reflect the effects of natalizumab treatment in multiple sclerosis, with implications for treatment benefits and risks.

Objectives

To assess changes in circulating lymphocyte subpopulation compositions and T-cell responses during natalizumab treatment.

Material and methods

A broad panel of markers for blood lymphocyte populations, including states of activation and co-stimulation as well as T-cell responses to recall antigens and mitogens, was assessed by flow cytometry in 40 patients with relapsing multiple sclerosis before and after one-year natalizumab treatment.

Results

Absolute numbers of all major populations of lymphocytes increased after treatment, most markedly for NK- and B-cells. The fraction of both memory and presumed regulatory B-cell subsets increased, as did CD3-CD56dim cytotoxic NK-cells, whereas CD3-CD56bright regulatory NK-cells decreased. Treatment was also associated with a restored T-cell responsiveness to recall antigens and mitogens.

Conclusions

Our data confirms that natalizumab treatment increases the number of lymphocytes in blood, likely mirroring the expression of VLA-4 being highest on NK- and B-cells. This supports reduction of lymphocyte extravasation as a main mode of action, although the differential composition of lymphocyte subpopulations suggests cell-signalling effects may also be operative. The systemic increase in T-cell responsiveness reflects the increase in numbers, and while augmenting anti-infectious responses systemically, localized responses become correspondingly decreased.

Place, publisher, year, edition, pages
San Francisco, USA: Public Library of Science, 2013
Keyword
Multiple sclerosis, natalizumab, flow cytometry, T-cells, NK-cells, B-cells, lymphocyte proliferation
National Category
Neurology Immunology in the medical area
Identifiers
urn:nbn:se:liu:diva-84268 (URN)10.1371/journal.pone.0081685 (DOI)000327944500088 ()24312575 (PubMedID)2-s2.0-84891420120 (Scopus ID)
Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2017-12-07Bibliographically approved
3. Association between Change in Normal Appearing White Matter Metabolites and Intrathecal Inflammation in Natalizumab-Treated Multiple Sclerosis
Open this publication in new window or tab >>Association between Change in Normal Appearing White Matter Metabolites and Intrathecal Inflammation in Natalizumab-Treated Multiple Sclerosis
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 9, e44739- p.Article in journal (Refereed) Published
Abstract [en]

Background: Multiple sclerosis (MS) is associated not only with focal inflammatory lesions but also diffuse pathology in the central nervous system (CNS). Since there is no firm association between the amount of focal inflammatory lesions and disease severity, diffuse pathology in normal appearing white matter (NAWM) may be crucial for disease progression. Immunomodulating treatments for MS reduce the number of focal lesions, but possible effects on diffuse white matter pathology are less studied. Furthermore, it is not known whether intrathecal levels of inflammatory or neurodegenerative markers are associated with development of pathology in NAWM.

Methods: Quantitative proton magnetic resonance spectroscopy (1H-MRS) was used to investigate NAWM in 27 patients with relapsing MS before and after one year of treatment with natalizumab as well as NAWM in 20 healthy controls at baseline. Changes in 1H-MRS metabolite concentrations during treatment were also correlated with a panel of intrathecal markers of inflammation and neurodegeneration in 24 of these 27 patients.

Results: The group levels of 1H-MRS metabolite concentrations were unchanged pre-to posttreatment, but a pattern of high magnitude correlation coefficients (r = 0.43–0.67, p<0.0005–0.03) were found between changes in individual metabolite concentrations (total creatine and total choline) and levels of pro-inflammatory markers (IL-1β and CXCL8).

Conclusions: Despite a clinical improvement and a global decrease in levels of inflammatory markers in cerebrospinal fluid during treatment, high levels of pro-inflammatory CXCL8 and IL-1β were associated with an increase in 1H-MRS metabolites indicative of continued gliosis development and membrane turnover in NAWM.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84270 (URN)10.1371/journal.pone.0044739 (DOI)000309742800016 ()
Note

funding agencies|Swedish Society of Neurologically Disabled||Swedish Society of Medicine||National Research Council (VR/NT)||University Hospital of Linkoping||County Council of Ostergotland||Teva||Biogen Idec||

Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2017-12-07
4. Transcriptional characteristics of CD4+ T cells in multiple sclerosis: relative lack of suppressive populations in blood
Open this publication in new window or tab >>Transcriptional characteristics of CD4+ T cells in multiple sclerosis: relative lack of suppressive populations in blood
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2011 (English)In: Multiple Sclerosis, ISSN 1352-4585, E-ISSN 1477-0970, Vol. 17, no 1, 57-66 p.Article in journal (Refereed) Published
Abstract [en]

Background:Multiple sclerosis (MS) is hypothetically caused by autoreactive Th1 and Th17 cells, whereas Th2 and regulatory T cells may confer protection. The development of Th subpopulations is dependant on the expression of lineage-specific transcription factors.

Objective:The aim of this study was to assess the balance of CD4+T cell populations in relapsing-remitting MS.

Methods:Blood mRNA expression of TBX21, GATA3, RORC, FOXP3 and EBI3 was assessed in 33 patients with relapsing-remitting MS and 20 healthy controls. In addition, flow cytometry was performed to assess T lymphocyte numbers.

Results:In relapsing-remitting MS, diminished expression of FOXP3 (Treg) was found (p < 0.05), despite normal numbers of CD4+CD25hiTreg. Immunoregulatory EBI3 and Th2-associated GATA3 ([a-z]+) was also decreased in MS (p < 0.005 and p < 0.05, respectively). Expression of TBX21 (Th1) and RORC (Th17) did not differ between patients and controls. Similar changes were observed when analysing beta-interferon treated (n = 12) or untreated (n = 21) patients. Analysis of transcription factor ratios, comparing TBX21/GATA3 and RORC/FOXP3, revealed an increase in the RORC/FOXP3 ratio in patients with relapsing-remitting MS (p < 0.005).

Conclusion:Our findings indicate systemic defects at the mRNA level, involving downregulation of beneficial CD4+phenotypes. This might play a role in disease development by permitting activation of harmful T cell populations.

Place, publisher, year, edition, pages
Sage Publications, 2011
Keyword
EBI3, FOXP3, multiple sclerosis, RORC, T cells, transcription factors
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-64758 (URN)10.1177/1352458510381256 (DOI)000285867200006 ()20847001 (PubMedID)
Available from: 2011-02-04 Created: 2011-02-04 Last updated: 2017-12-11

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