Background:
Disruption of the blood brain barrier (BBB) is believed to play a critical role in the
pathogenesis of multiple sclerosis (MS). Cramer et al. (2014) found increased BBB
permeability in MS patients compared to healthy controls (HC) with values being higher in
cases with recent relapses (1). Furthermore, BBB permeability can contribute to predicting
the conversion of optic neuritis (ON) to MS (2). In the same study, significant correlations
were found between BBB permeability, leukocyte counts and levels of the chemokine CXCL10 in
cerebrospinal fluid (CSF). Chemokines are believed to contribute to MS pathogenesis by
attracting leukocyte populations to the CNS.
Sørensen et al. (1999) found elevated levels of the cytokines CXCL9, CXCL10 and CCL5 together
with their receptors CXCR3 and CCR5 on leukocytes in CSF from MS patients during attacks (3).
CXCR3 is expressed in human enterocytes, endothelial cells in the BBB and a variety of immune
cells. Elevated expression of CXCR3 has also been measured on T cells from MS patients
compared to HC, and in patients during relapses compared to remission (4). Exposure of
intestinal epithelial cell lines to gliadin has been shown to activate the chemokine receptor
CXCR3 and hereby leading to release of zonulin (5). Ex vivo experiments illustrated that
zonulin, which has been identified as pre-haptoglobin 2, leads to a time- and dose dependent,
reversible reduction of the transepithelial electrical resistance (TEER) of murine small
intestinal mucosa (6). The effects of zonulin on gut permeability are assumed to be due to
disruption of tight junctional integrity.
A pilot study
- (7) showed a higher proportion of individuals with increased intestinal
permeability among patients with MS compared to sex-matched HC.
A randomized clinical trial
showed beneficial effects of a gluten-free diet (GFD) on annual relapse rate, lesional
activity and expanded disability status scale (EDSS) in patients with relapsing-remitting MS
(RRMS), when compared to a regular diet (8). Additionally, findings of significantly higher
titers of IgA against gliadin, gluten and casein in MS patients compared to HC, could
indicate a role of nutritional factors in MS (8).
The role of gluten in the pathogenesis of celiac disease (CD) is established. In CD dietary
intake of gluten leads to the development of autoantibodies against the enzyme
transglutaminase 2 (TG2). White matter autopsies have shown TG2 immunoreactivity in
astrocytes from activated MS lesions (9). Furthermore, treatment with TG2 inhibitors led to
attenuation of demyelination and clinical deficits in rats with chronic-relapsing
experimental autoimmune encephalomyelitis (EAE) (10). A GFD has also been documented to
protect against type 1 diabetes (T1D) in mice (11) and prolong the asymptomatic period after
diagnosis of T1D in humans (12).
Lastly, gliadin fragments have been documented to stimulate the production of TNF-α and IL-8
in human monocytes directly (13). TNF-α has also been shown to have a regulatory effect on
zonulin and to increase the permeability of the endothelial and epithelial cell layers (14),
which could be an indirect mechanism through which gluten can induce alterations of gut and
possibly also BBB permeability. A high gluten intake could thereby not only increase the
influx of antigens, but also activate pathways of the innate immune system. This can
theoretically contribute to establishing an inflammatory milieu and thereby facilitate the
activation of autoreactive T cells. It could be hypothesized that the role of gluten in
autoimmunity is not restricted to gluten itself being recognized as an antigen by people with
specific HLA genotypes, but gluten might also increase the risk of losing tolerance against
other antigens.
Purpose:
The study aims to investigate whether a GFD can contribute to decreasing BBB permeability in
patients with ON, other clinically isolated syndrome (CIS) and MS. Furthermore, the study
seeks to identify possible effects of a GFD on markers of systemic as well as CNS
inflammation. Lastly, gut permeability is measured in order to examine whether there are any
correlations between the permeability of the gut and the BBB as well as the inflammatory
state in the intestine and CNS. Evaluating the effects of gluten intake on disease
progression could contribute to identifying crucial mechanisms that underlie the pathogenesis
of CIS and MS, directing future research and designing new, optimized treatment protocols.
Hypothesis:
Gluten may increase intestinal permeability and thereby elevate antigen influx possibly by
activating CXCR3 on enterocytes. Increased gut permeability, gut inflammation and absorption
of larger gliadin molecules may be associated with increased permeability of the BBB.
Activation of BBB endothelial cells may lead to loss of tight junctional integrity, but also
increased integrin expression and thereby enhanced extravasation of immune cells into the
CNS.
Expected research value:
The study can contribute to a better understanding of the pathologies of ON and MS and
provide a basis for the development of new treatment protocols. Conducting evidence-based
research regarding the possible effects of gluten intake on the progression of autoimmune
diseases will reduce misinformation as well as shed light on practical challenges associated
with the implementation of dietary interventions for treatment purposes. The study is
approved of the Scientific Ethics Committee.
Methods:
The trial is a clinically controlled, open label, intervention study including 40 patients
with newly diagnosed CIS or MS. Patients will be divided into an intervention group and a
control group, each of 20 patients. The intervention group will abstain from gluten for six
months, while patients in the control group will retain their usual dietary habits. Before
and after the intervention period, a variety of measurements will be conducted. These
include:
- - Anthropometric measurements: conducted to monitor possible effects of the GFD on
participants´ body composition.
- - Diet and lifestyle surveys: used to monitor participants´ dietary habits, smoking
status, exercise, physical activity, sleeping pattern and quality of life.
- - Clinical measures: Relapses, expanded disability status scale (EDSS), MS diagnosis,
disease modifying treatment, T2 lesions count and lesion load.
Treatment status and
recent relapses will be used as covariates in the logistic regression analysis.
- - Dynamic contrast-enhanced MRI: used to measure the permeability of the BBB in several
tissue subtypes (normal appearing white matter and grey matter and MS lesions) as
segmented on high resolution T2 FLAIR and 3D T1 weighted images.
The number of new or
enhancing lesions is being monitored.
- - Intestinal permeability test: conducted to measure intestinal permeability and
absorption capacity.
- - Lumbar puncture: used to determine disease activity in the CNS.
Analyses include
pre-haptoglobin 2, oligoclonal bands, TNF-α, IL-1β, IL-8, INF-γ, CXCL9, CXCL10, CCL5,
immune cell counts, expression of CXCR3 on immune cells.
- - Blood samples: used to determine the grade of inflammation outside the CNS and diagnose
celiac disease.
Analyses include S100β, pre-haptoglobin 2, anti-gliadin antibodies, CRP,
TNF-α, IL-1β, INF-γ, immune cell counts, expression of CXCR3 on immune cells, endotoxin,
intestinal fatty acid binding protein, 25-(OH)-cholecalciferol.
- - Fecal samples: used to measure gut microbiota profiling and short chain fatty acids.
BBB and intestinal permeability will not only be measured through an MRI scan and an
intestinal permeability test respectively, but also using the biomarker zonulin. Of notice is
that our measurements of zonulin in CSF (for BBB permeability) and serum (for intestinal
permeability) will be carried out in collaboration with Assistant Professor Dr. Ian Galea,
who has established a unique analytical method, based on western blotting using a
non-commercially, proprietary antibody manufactured by Biorad, for this purpose. Every other
assay used in the scientific community measures both haptoglobin and zonulin, whereas our
assay measures only zonulin and is therefore characterized by increased accuracy.
Expected results:
A gluten-free diet is expected to alleviate CIS symptoms, improve quality of life and reduce
the risk of CIS progressing into MS. Mechanistically this is expected to be manifested
through a normalized BBB and intestinal permeability, decreased lesional activity and by
dampening the activity of a previously overactive immune system. If our hypothesis is proven
to be correct, MS and CIS patients should be informed about the results of the study and the
option of following a GFD could potentially become part of the standardized patient treatment
in hospitals.
Publication of results:
Results will be published independent of study outcome. Positive, negative as well as
inconclusive results will be published. Results are expected to be published in peer-reviewed
international journals.
