Original articleBiomarkers of inflammation and epithelial barrier function in multiple sclerosis
Introduction
Multiple sclerosis (MS) is a chronic immune-mediated disease of the central nervous system (CNS) leading to demyelination and axonal loss. The aetiology is still not fully understood, but peripherally activated immune cells are involved in the initiation of pathogenic inflammation in the CNS. (Hemmer et al., 2015) Currently, detailed clinical examination and imaging analysis are used to diagnose and monitor the disease. (Compston and Coles, 2008) Moreover, biomarkers in the cerebrospinal fluid (CSF), such as oligoclonal bands, are widely used supporting the diagnosis of MS. There is; however, a lack of validated blood biomarkers in MS.
One of the most studied biomarkers in MS is neurofilament light chain (NfL). Neurofilaments are structural components of myelinated axons. When axons are injured, neurofilaments are released and can be measured in CSF and blood. Most studies on NfL have been done on CSF, but sensitive techniques such as the single-molecule array (SIMOA) technology enables quantification of NfL at the very low concentrations in serum or plasma. (Rissin et al., 2010) NfL levels increase during MS relapses and when active contrast-enhancing lesions are present on magnetic resonance imaging (MRI). Further, when treated with disease-modifying therapies (DMTs), the concentration decreases. (Disanto et al., 2017) However, the relation between NfL and blood biomarkers of systemic inflammation has received little attention.
Recent data suggest a potential involvement of mucosal epithelial barriers in relation to peripheral immune activation in metabolic, infectious and inflammatory diseases, including MS. (König et al., 2016, Wittekindt, 2017, Buscarinu et al., 2018) Both the pulmonary and the intestinal epithelial barriers inhibit passage of harmful substances and pathogens into the submucosa and bloodstream. In case of a disruption of these barriers the permeability increases, which may cause excessive activation of the immune system. The intestinal tract is the largest body surface where external stimuli interacts with the internal; in addition, a substantial amount of our immune cells resides in the intestinal tract. Studies on intestinal permeability with lactulose/mannitol ratio tests have shown that MS patients have increased intestinal permeability compared with healthy controls (HCs). (Buscarinu et al., 2017) Pulmonary epithelial barrier damage and subsequent immune activation is hypothesized as being one of the underlying causes of the increased risk of MS due to cigarette smoking. (Rosso and Chitnis, 2020) Blood biomarkers of epithelial barrier function have, however, not been widely studied in MS. To address this, we conducted an exploratory study measuring the concentration of a comprehensive panel of blood-based biomarkers of inflammation and epithelial barrier disruption in serum or plasma samples from newly diagnosed, untreated RRMS patients and HCs. Next, we performed a validation in a second independent cohort to reproduce our findings.
Section snippets
Exploratory cohort
Forty-nine patients with RRMS and 58 HCs were included between 2013 and 2015 at our centre. At baseline 29 patients were treatment naïve and 20 patients untreated. Untreated RRMS was defined as no DMT for at least 3 months prior inclusion. Inclusion criteria were newly diagnosed (<5 years), untreated RRMS or clinically isolated syndrome (CIS) according the McDonald criteria 2010, and HCs not fulfilling exclusion criteria, aged 18 to 60 years. Exclusion criteria were other autoimmune disorders,
Results
Demographics and clinical characteristics of all participants are presented in table 1A and 1B. Age, sex, body mass index (BMI) and smoking did not differ between MS and HCs. NfL levels were significantly higher in MS patients compared with HCs in the exploratory cohort (median 8.26 pg/ml vs 4.90 pg/ml, p<0.001: Table 1A) and validation cohort (median 12.0 pg/ml vs. 6.02 pg/ml, p<0.001: Table 1B). A list of all measured molecules in MS and HCs in the exploratory cohort is presented in table 2.
Discussion
In this study we measured biomarkers of inflammation and epithelial barrier function in serum or plasma of RRMS patients and HCs using electrochemiluminescence, ELISA and SIMOA assays, in separate exploratory and validation cohorts. The main findings were increased serum concentration of calprotectin in MS patients compared with HCs, and calprotectin's association with disease activity.
Unexpectedly, the measured biomarkers of epithelial barrier function zonulin, uteroglobin and SP-D did not
CRediT authorship contribution statement
A. Olsson: Conceptualization, Methodology, Data curation, Formal analysis, Writing - original draft. S. Gustavsen: Data curation, Writing - review & editing. I. Chenoufi Hasselbalch: Data curation, Writing - review & editing. A.R. Langkilde: Data curation, Writing - review & editing. F. Sellebjerg: Conceptualization, Methodology, Supervision, Writing - review & editing. A.B. Oturai: Conceptualization, Methodology, Supervision, Writing - review & editing. H. Bach Søndergaard: Conceptualization,
Declaration of Competing Interest
Anna Olsson has received support for congress participation from Roche. Stefan Gustavsen has received support for congress participation from Merck. Ida Chenoufi Hasselbalch has nothing to declare. Annika R. Langkilde has nothing to declare. Finn Sellebjerg has served on scientific advisory boards, been on the steering committees of clinical trials, served as a consultant, received support for congress participation, received speaker honoraria, or received research support for his laboratory
Acknowledgements
We wish to thank Ulla Abildtrup, Linda Wolter Christensen and Birgit Højgaard Kassow for their excellent technical assistance. We thank the Karolinska Institute in Stockholm for permission to translate and use the Genetic and Environment in Multiple Sclerosis (GEMS) questionnaire.
Funding
This research was supported by the Danish Multiple Sclerosis Society [A-38469 and A35043], Overlæge Johan Boserup og Lise Boserups Legat [J.nr. 20795-24], Grosserer A.V. Lykfeldt og Hustrus Legat, Trigon fonden, Civilingeniør Bent Bøgh og hustru Inge Bøghs fond, Carl og Ellen Hertz Legat [J.nr. 7179-2], Direktør Emil C. Hertz og hustru Inger Hertz' Fond, Civilingeniør Frode V. Nyegaard og Hustrus Fond, Th. Maigaards Eftf. Fru Lily Benthine Lunds Fond, Jascha Fonden, Torben og Alice Frimodts
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