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The imbalance between pathogenic and protective T cell subsets is a cardinal feature of autoimmune disorders such as multiple sclerosis (MS). Emerging evidence indicates that endogenous and dietary-induced changes in fatty acid metabolism have a major impact on both T cell fate and autoimmunity. To date, however, the molecular mechanisms that underlie the impact of fatty acid metabolism on T cell physiology and autoimmunity remain poorly understood. Here, we report that stearoyl-CoA desaturase-1 (SCD1), an enzyme essential for the desaturation of fatty acids and highly regulated by dietary factors, acts as an endogenous brake on regulatory T-cell (Treg) differentiation and augments autoimmunity in an animal model of MS in a T cell-dependent manner. Guided by RNA sequencing and lipidomics analysis, we found that the absence of Scd1 in T cells promotes the hydrolysis of triglycerides and phosphatidylcholine through adipose triglyceride lipase (ATGL). ATGL-dependent release of docosahexaenoic acid enhanced Treg differentiation by activating the nuclear receptor peroxisome proliferator-activated receptor gamma. Our findings identify fatty acid desaturation by SCD1 as an essential determinant of Treg differentiation and autoimmunity, with potentially broad implications for the development of novel therapeutic strategies and dietary interventions for autoimmune disorders such as MS. The work has been supported by the Flemish Fund for Scientific Research (FWO Vlaanderen; 12J9116N, 12JG119N, 12U7718N, 1S15519N, and G099618N), the Belgian Charcot Foundation (FCS-2016-EG7, R-8676, and R-6832), the Interreg V‐A EMR program (EURLIPIDS, EMR23), and the special research fund UHasselt (BOF). JMN is supported by a National Institutes of Health Grant (R01 DK062388). MK was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (640116) and by a SALK grant from the government of Flanders and by an Odysseus grant of the Research Foundation Flanders, Belgium (FWO). We thank MP Tulleners for excellent technical assistance. We would also like to thank other members of the Biomedical Research Institute (Hasselt University) for providing feedback and suggestions during the preparation of the manuscript.

The initiation and progression of autoimmune disorders such as multiple sclerosis (MS) is linked to aberrant cholesterol metabolism and overt inflammation. Liver X receptors (LXR) are nuclear receptors that function at the crossroads of cholesterol metabolism and immunity, and their activation is considered a promising therapeutic strategy to attenuate autoimmunity. However, despite clear functional heterogeneity and cell-specific expression profiles, the impact of the individual LXR isoforms on autoimmunity remains poorly understood. Here, we show that LXR alpha and LXR beta have an opposite impact on immune cell function and disease severity in the experimental autoimmune encephalomyelitis model, an experimental MS model. While Lxr alpha deficiency aggravated disease pathology and severity, absence of Lxr beta was protective. Guided by flow cytometry and by using cell-specific knockout models, reduced disease severity in Lxr beta-deficient mice was primarily attributed to changes in peripheral T cell physiology and occurred independent from alterations in microglia function. Collectively, our findings indicate that LXR isoforms play functionally non-redundant roles in autoimmunity, potentially having broad implications for the development of LXR-based therapeutic strategies aimed at dampening autoimmunity and neuroinflammation.

The Multiple Sclerosis Data Alliance (MSDA), a global multi-stakeholder collaboration, is working to accelerate research insights for innovative care and treatment for people with multiple sclerosis (MS) through better use of real-world data (RWD). Despite the increasing reliance on RWD, challenges and limitations complicate the generation, collection, and use of these data. MSDA aims to tackle sociological and technical challenges arising with scaling up RWD, specifically focused on MS data. MSDA envisions a patient-centred data ecosystem in which all stakeholders contribute and use big data to co-create the innovations needed to advance timely treatment and care of people with MS. We would like to thank the sponsors of the MS Data Alliance. The MSDA receives income from a range of corporate sponsors, recently including: Biogen, BristolMyersSquibb (formerly Celgene), Canopy Growth Corporation, Genzyme, Icometrix, Janssen, Merck, Mylan, Novartis, QMENTA, Quanterix, Roche. We would like to thank the European MS Platform and the University MS Center of Hasselt University for founding the initiative. Next to this, we would like to thank the other core partners for their continued support: the European Charcot Foundation, The European Institute for Innovation through Health Data, the University of Gottingen and KULeuven. A special thanks to our pioneer registries: the UK MS registry and the German MS registry. Peeters, LM (corresponding author), Agoralaan C Bis, B-3590 Diepenbeek, Belgium. Liesbet.peeters@uhasselt.be

Cytotoxic CD4+ T cells (CD4 CTL) are terminally differentiated T helper cells that contribute to autoimmune diseases, such as multiple sclerosis. We developed a novel triple co-culture transwell assay to study mutual interactions between CD4 CTL, conventional TH cells, and regulatory T cells (Tregs) simultaneously. We show that, while CD4 CTL are resistant to suppression by Tregs in vitro, the conditioned medium of CD4 CTL accentuates the suppressive phenotype of Tregs by upregulating IL-10, Granzyme B, CTLA-4, and PD-1. We demonstrate that CD4 CTL conditioned medium skews memory TH cells to a TH17 phenotype, suggesting that the CD4 CTL induce bystander polarization. In our triple co-culture assay, the CD4 CTL secretome promotes the proliferation of TH cells, even in the presence of Tregs. However, when cell−cell contact is established between CD4 CTL and TH cells, the proliferation of TH cells is no longer increased and Treg-mediated suppression is restored. Taken together, our results suggest that when TH cells acquire cytotoxic properties, these Treg-resistant CD4 CTL affect the proliferation and phenotype of conventional TH cells in their vicinity. By creating such a pro-inflammatory microenvironment, CD4 CTL may favor their own persistence and expansion, and that of other potentially pathogenic TH cells, thereby contributing to pathogenic responses in autoimmune disorders.

Macrophages play a crucial role during the pathogenesis of multiple sclerosis (MS), a neuroinflammatory autoimmune disorder of the central nervous system. Important regulators of the metabolic and inflammatory phenotype of macrophages are liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs). Previously, it has been reported that PPAR&gamma, expression is decreased in peripheral blood mononuclear cells of MS patients. The goal of the present study was to determine to what extent PPAR&gamma, as well as the closely related nuclear receptors PPAR&alpha, and &beta, and LXR&alpha, are differentially expressed in monocytes from MS patients and how this change in expression affects the function of monocyte-derived macrophages. We demonstrate that monocytes of relapsing-remitting MS patients display a marked decrease in PPAR&gamma, expression, while the expression of PPAR&alpha, /&beta, is not altered. Interestingly, exposure of monocyte-derived macrophages from healthy donors to MS-associated proinflammatory cytokines mimicked this reduction in PPAR&gamma, expression. While a reduced PPAR&gamma, expression did not affect the inflammatory and phagocytic properties of myelin-loaded macrophages, it did impact myelin processing by increasing the intracellular cholesterol load of myelin-phagocytosing macrophages. Collectively, our findings indicate that an inflammation-induced reduction in PPAR&gamma, expression promotes myelin-induced foam cell formation in macrophages in MS.

Background and aims: Alemtuzumab is a humanized monoclonal antibody that depletes CD52-bearing B and T lymphocytes. Clinical trials defined that systemic administration of alemtuzumab reduces disease severity in the relapsing–remitting phase of multiple sclerosis (MS). However, its efficacy in progressive MS patients is limited, which may reflect the inability of alemtuzumab to cross the reconstituted BBB in these patients. Objective: to study whether central nervous system (CNS) delivery of anti-CD52 antibodies reduces disease severity and the neuroinflammatory burden in the experimental autoimmune encephalomyelitis (EAE) model. Methods: Anti-CD52 antibodies were administered intrathecally during the acute and chronic phases of EAE. Flow cytometry and immunohistochemistry were utilized to define immunological and pathological parameters. Results: We show that subcutaneously administrated anti-CD52 antibodies completely abolish EAE disease severity. CNS delivery of anti-CD52 antibodies during both the acute and chronic phases of EAE moderately reduces disease severity and the neuroinflammatory burden. Our findings further suggest that CNS delivery of anti-CD52 antibodies impacts both the peripheral and CNS immune cell compartments in the EAE model but not in healthy mice. Conclusion: Collectively, our findings highlight the therapeutic potential of CNS delivery of alemtuzumab for the treatment of progressive as well as early MS.

The aim of this study is to examine whether the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), can generate dendritic cells (DCs) with a stable tolerogenic phenotype to counteract autoimmune responses in an animal model of multiple sclerosis. We investigated if the tolerogenic potency of DCs could be increased by continuous treatment during in vitro differentiation toward DCs compared to standard 24-h in vitro treatment of already terminally differentiated DCs. We show that in vitro treatment with SAHA reduces the generation of new CD11c+ DCs out of mouse bone marrow. SAHA-generated DCs show reduced antigen-presenting function as evidenced by a reduction in myelin endocytosis, a decreased MHC II expression, and a failure to upregulate costimulatory molecules upon LPS challenge. In addition, SAHA-generated DCs display a reduction in proinflammatory cytokines and molecules involved in apoptosis induction, inflammatory migration, and TLR signaling, and they are less immunostimulatory compared to untreated DCs. We demonstrated that the underlying mechanism involves a diminished STAT1 phosphorylation and was independent of STAT6 activation. Although in vitro results were promising, SAHA-generated DCs were not able to alleviate the development of experimental autoimmune encephalomyelitis in mice. In vitro washout experiments demonstrated that the tolerogenic phenotype of SAHA-treated DCs is reversible. Taken together, while SAHA potently boosts tolerogenic properties in DCs during the differentiation process in vitro, SAHA-generated DCs were unable to reduce autoimmunity in vivo. Our results imply that caution needs to be taken when developing DC-based therapies to induce tolerance in the context of autoimmune disease.

Mesenchymal stromal cells (MSCs) are multipotent stem cells with immunosuppressive and trophic support functions. While MSCs from different sources frequently display a similar appearance in culture, they often show differences in their surface marker and gene expression profiles. Although bone marrow is considered the ‘‘gold standard’’ tissue to isolate classical MSCs (BM-MSC), MSC-like cells are currently also derived from more easily accessible extra-embryonic tissues such as the umbilical cord. In this study, we defined the best way to isolate MSCs from the Wharton’s jelly of the human umbilical cord (WJ-MSC) and assessed the mesenchymal and immunological phenotype of BM-MSC and WJ-MSC. Moreover, the gene expression profile of established WJ-MSC cultures was compared to two different bone marrow-derived stem cell populations (BM-MSC and multipotent adult progenitor cells or MAPC). We observed that explant culturing of Wharton’s jelly matrix is superior to collagenase tissue digestion for obtaining mesenchymal-like cells, with explant isolated cells displaying increased expansion potential. While being phenotypically similar to adult MSCs, WJ-MSC show a different gene expression profile. Gene ontology analysis revealed that genes associated with cell adhesion, proliferation, and immune system functioning are enriched in WJ-MSC. In vivo transplantation confirms their immune modulatory effect on T cells, similar to BMMSC and MAPC. Furthermore, WJ-MSC intrinsically overexpress genes involved in neurotrophic support and their secretome induces neuronal maturation of SH-SY5Y neuroblastoma cells to a greater extent than BM-MSC. This signature makes WJ-MSC an attractive candidate for cell-based therapy in neurodegenerative and immune-mediated central nervous system disorders such as multiple sclerosis, Parkinson’s disease, or amyotrophic lateral sclerosis. This research was promoted by grants from the IWT (agentschap voor Innovatie door Wetenschap en Technology), Alma-in-Silico (EMR INT4.-1.3.-2008-03/003), the VIB (Vlaams Instituut voor Biotechnologie), the transnational University Limburg, Hasselt University, and Limburg Sterk Merk. Pascal Gervois is supported by grant number 12 U7718 N of the Fonds Wetenschappelijk Onderzoek Vlaanderen.

Umbilical cord matrix or Wharton?s jelly-derived stromal cells (WJ-MSCs) are an easily accessible source of mesenchymal-like stem cells. Recent studies describe a hypo-immunogenic phenotype, multipotent differentiation potential and trophic support function for WJ-MSCs, with variable clinical benefit in degenerative disease models such as stroke, myocardial infarction and Parkinson?s disease. It remains unclear whether WJ-MSCs have therapeutic value for multiple sclerosis (MS), where autoimmune-mediated demyelination and neurodegeneration need to be halted. In this study, we investigated whether WJ-MSCs possess the required properties to effectively and durably reverse these pathological hallmarks, and whether they survive in an inflammatory environment after transplantation. WJ-MSCs displayed a lowly immunogenic phenotype and showed intrinsic expression of neurotrophic factors and a variety of anti-inflammatory molecules. Furthermore, they dose-dependently suppressed proliferation of activated T cells using contact-dependent and paracrine mechanisms. Indoleamine 2,3-dioxygenase 1 was identified as one of the main effector molecules responsible for the observed T cell suppression. The immune-modulatory phenotype of WJ-MSCs was further enhanced after pro-inflammatory cytokine treatment in vitro (licensing). In addition to their effect on adaptive immunity, WJ-MSCs interfered with dendritic cell differentiation and maturation, thus directly affecting antigen presentation and therefore T cell priming. Systemically infused WJ-MSCs potently but transiently ameliorated experimental autoimmune encephalomyelitis (EAE), an animal model for MS, when injected at onset or during chronic disease. This protective effect was paralleled with a reduction in autoantigen-induced T cell proliferation, confirming their immune-modulatory activity in vivo. Surprisingly, in vitro licensed WJ-MSCs did not ameliorate EAE, indicative of a fast rejection as a result of enhanced immunogenicity. Collectively, we show that WJ-MSCs have trophic support properties and effectively modulate immune cell functioning both in vitro and in the EAE model, suggesting WJ-MSC may hold promise for MS therapy. Future research is needed to optimize survival of stem cells and enhance clinical durability. We thank Ms. Katrien Wauterickx and Ms. Christel Bocken (Hasselt University, Biomedical Research institute) for their technical assistance with the cell cultures and animal experiments. In addition, we gratefitlly thank Ms. Wai Ping Lee and Dr Nathalie Cools (Antwerp University) for their suggestions regarding the dendritic cell cultures. This research was supported by the agency for Innovation through Science and Technology (agentschap voor Innovatie door Wetenschap en Technology -IWT), the Research Foundation-Flanders (Fonds Wetenschappelijk Onderzoek Vlaanderen - FWO), Transnational University Limburg, Hasselt University, and Limburg Sterk Merk. R.D. benefits from a PhD scholarship of the IWT. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Authors' contributions: Designed the experiments: R.D., N.H.; performed the experiments and analyzed the data: R.D., M.V; contributed reagents, materials, or analysis tools: J.B., S.R., K.T., and W.G.; wrote the paper: R.D.; revised the manuscript: J.B., PS., J.H., W.G., and N.H.; all authors read and approved the final manuscript. The authors declare no conflicts of interest.

Multiple sclerosis (MS) is a chronic disabling disease of the central nervous system (CNS), in which destruction of myelin sheaths leads to disturbed axonal conduction. Available MS therapies modulate the immune response, but are unable to prevent neurological decline. Therefore, great efforts are made to develop therapies that limit demyelination and axonal degeneration. Oncostatin M (OSM), a member of the interleukin (IL)-6 cytokine family, is produced in demyelinating lesions of MS patients and stimulates neuronal survival. In this study, we reveal that the OSM receptor (OSMR) was robustly upregulated on microglia/macrophages and astrocytes in the cuprizone-induced demyelination model. While OSMR deficiency led to aggravated demyelination, CNS-targeted OSM treatment largely prevented demyelination. OSM treatment increased IL-4 expression and induced polarization of myeloid cells towards an anti-inflammatory M2 phenotype in vivo. This study reveals a previously uncharacterized and protective role for OSM during demyelination, and indicates that OSM is a promising therapeutic candidate to limit CNS damage in demyelinating diseases including MS. GLIA 2015;63:1729–1737

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), for which current treatments are unable to prevent disease progression. Based on its neuroprotective and neuroregenerating properties, leukemia inhibitory factor (LIF), a member of the interleukin-6 (IL-6) cytokine family, is proposed as a novel candidate for MS therapy. However, its effect on the autoimmune response remains unclear. In this study, we determined how LIF modulates T cell responses that play a crucial role in the pathogenesis of MS. We demonstrate that expression of the LIF receptor was strongly increased on immune cells of MS patients. LIF treatment potently boosted the number of regulatory T cells (Tregs) in CD4(+) T cells isolated from healthy controls and MS patients with low serum levels of IL-6. Moreover, IL-6 signaling was reduced in the donors that responded to LIF treatment in vitro. Our data together with previous findings revealing that IL-6 inhibits Treg development, suggest an opposing function of LIF and IL-6. In a preclinical animal model of MS we shifted the LIF/IL-6 balance in favor of LIF by CNS-targeted overexpression. This increased the number of Tregs in the CNS during active autoimmune responses and reduced disease symptoms. In conclusion, our data show that LIF downregulates the autoimmune response by enhancing Treg numbers, providing further impetus for the use of LIF as a novel treatment for MS and other autoimmune diseases.

Multiple sclerosis (MS) is the leading cause of chronic neurological disability in young adults. The clinical disease course of MS varies greatly between individuals, with some patients progressing much more rapidly than others, making prognosis almost impossible. We previously discovered that cytotoxic CD4+ Tcells (CD4+ CTL), identified by the loss of CD28, are able to migrate to sites of inflammation and that they contribute to tissue damage. Furthermore, in an animal model for MS, we showed that these cells are correlated with inflammation, demyelination, and disability. Therefore, we hypothesize that CD4+ CTL drive progression of MS and have prognostic value. To support this hypothesis, we investigated whether CD4+ CTL are correlated with worse clinical outcome and evaluated the prognostic value of these cells in MS. To this end, the percentage of CD4+ CD28null T cells was measured in the blood of 176 patients with relapsing-remitting MS (=baseline). Multimodal evoked potentials (EP) combining information on motoric, visual, and somatosensoric EP, as well as Kurtzke expanded disability status scale (EDSS) were used as outcome measurements at baseline and after 3 and 5 years. The baseline CD4+ CD28null T cell percentage is associated with EP (P = 0.003, R-2 = 0.28), indicating a link between these cells and disease severity. In addition, the baseline CD4+ CD28null T cell percentage has a prognostic value since it is associated with EP after 3 years (P = 0.005, R-2 = 0.29) and with EP and EDSS after 5 years (P = 0.008, R-2 = 0.42 and P = 0.003, R-2 = 0.27). To the best of our knowledge, this study provides the first direct link between the presence of CD4+ CTL and MS disease severity, as well as its prognostic value. Therefore, we further elaborate on two important research perspectives: 1 degrees investigating strategies to block or reverse pathways in the formation of these cells resulting in new treatments that slow down MS disease progression, 2 degrees including immunophenotyping in prediction modeling studies to aim for personalized medicine. This work was supported by the Scientific Research Flanders (FWO, no G021412N). LP is supported by Interuniversity Attraction Pole (IUAP, no IAP VII/39) from the Belspo Agency. BB is an ECTRIMS postdoctoral fellow.

Macrophages and microglia are key effector cells in immune-mediated neuroinflammatory disorders. Driving myeloid cells towards an anti-inflammatory, tissue repair-promoting phenotype is considered a promising strategy to halt neuroinflammation and promote central nervous system (CNS) repair. In this study, we defined the impact of multipotent adult progenitor cells (MAPC), a stem cell population sharing common mesodermal origin with mesenchymal stem cells (MSCs), on the phenotype of macrophages and the reciprocal interactions between these two cell types. We show that MAPC suppress the secretion of tumor necrosis factor alpha (TNF-alpha) by inflammatory macrophages partially through a cyclooxygenase 2- (COX-2-) dependent mechanism. In turn, we demonstrate that inflammatory macrophages trigger the immunomodulatory properties of MAPC, including an increased expression of immunomodulatory mediators (e.g., inducible nitric oxide synthase (iNOS) and COX-2), chemokines, and chemokine receptors. Macrophage-primed MAPC secrete soluble factors that suppress TNF-alpha release by macrophages. Moreover, the MAPC secretome suppresses the antigen-specific proliferation of autoreactive T cells and the T cell stimulatory capacity of macrophages. Finally, MAPC increase their motility towards secreted factors of activated macrophages. Collectively, these in vitro findings reveal intimate reciprocal interactions between MAPC and inflammatory macrophages, which are of importance in the design of MAPC-based therapeutic strategies for neuroinflammatory disorders in which myeloid cells play a crucial role. The current study was financially supported by the Belgian Charcot Foundation.

Lipoproteins modulate innate and adaptive immune responses. In the chronic inflammatory disease multiple sclerosis (MS), reports on lipoprotein level alterations are inconsistent and it is unclear whether lipoprotein function is affected. Using nuclear magnetic resonance (NMR) spectroscopy, we analysed the lipoprotein profile of relapsing-remitting (RR) MS patients, progressive MS patients and healthy controls (HC). We observed smaller LDL in RRMS patients compared to healthy controls and to progressive MS patients. Furthermore, low-BMI (BMI

Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR-MG) is considered as a prototypic autoimmune disease. The thymus is important in the pathophysiology of the disease since thymus hyperplasia is a characteristic of early-onset AChR-MG and patients often improve after thymectomy. We hypothesized that thymic B cell and antibody repertoires of AChR-MG patients differ intrinsically from those of control individuals. Using immortalization with Epstein Barr Virus and Toll-like receptor 9 activation, we isolated and characterized monoclonal B cell lines from 5 MG patients and 8 controls. Only 2 of 570 immortalized B cell clones from MG patients produced antibodies against the AChR (both clones were from the same patient), suggesting that AChR-specific B cells are not enriched in the thymus. Surprisingly, many B cell lines from both AChR-MG and control thymus samples displayed reactivity against striated muscle proteins. Striational antibodies were produced by 15% of B cell clones from AChR-MG versus 6% in control thymus. The IgV(H) gene sequence analysis showed remarkable similarities, concerning V-H family gene distribution, mutation frequency and CDR3 composition, between B cells of AChR-MG patients and controls. MG patients showed clear evidence of clonal B cell expansion in contrast to controls. In this latter aspect, MG resembles multiple sclerosis and clinically isolated syndrome, but differs from systemic lupus erythemitosus. Our results support an antigen driven immune response in the MG thymus, but the paucity of AChR-specific B cells, in combination with the observed polyclonal expansions suggest a more diverse immune response than expected. (C) 2013 Elsevier Ltd. All rights reserved.

Along with microglia and monocyte-derived macrophages, macrophages in the perivascular space, choroid plexus, and meninges are the principal effector cells in neuroinflammatory and neurodegenerative disorders. These phagocytes are highly heterogeneous cells displaying spatial- and temporal-dependent identities in the healthy, injured, and inflamed CNS. In the last decade, researchers have debated on whether phagocytes subtypes and phenotypes are pathogenic or protective in CNS pathologies. In the context of this dichotomy, we summarize and discuss the current knowledge on the spatiotemporal physiology of macrophage subsets and microglia in the healthy and diseased CNS, and elaborate on factors regulating their behavior. In addition, the impact of macrophages present in lymphoid organs on CNS pathologies is defined. The prime focus of this review is on multiple sclerosis (MS), which is characterized by inflammation, demyelination, neurodegeneration, and CNS repair, and in which microglia and macrophages have been extensively scrutinized. On one hand, microglia and macrophages promote neuroinflammatory and neurodegenerative events in MS by releasing inflammatory mediators and stimulating leukocyte activity and infiltration into the CNS. On the other hand, microglia and macrophages assist in CNS repair through the production of neurotrophic factors and clearance of inhibitory myelin debris. Finally, we define how microglia and macrophage physiology can be harnessed for new therapeutics aimed at suppressing neuroinflammatory and cytodegenerative events, as well as promoting CNS repair. We conclude that microglia and macrophages are highly dynamic cells displaying disease stage and location-specific fates in neurological disorders. Changing the physiology of divergent phagocyte subsets at particular disease stages holds promise for future therapeutics for CNS pathologies.

Through their tumor-promoting and/or tumor-suppressive properties, cytokines can influence progression of cancer. We systematically reviewed the current literature on the prognostic value of the circulating cytokines IL-1α/β, IL-6, IL-8, IL-10, IL-12, TNF-α, TGF-β and IFN-γ to predict overall and disease-free survival in any type of cancer patients. PubMed was systematically searched and based on eligibility assessment using our five criteria of the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) checklist, six unique studies were reviewed. Elevated IL-6 and IL-10 levels seem independently associated with worse prognosis in terms of overall and disease-free survival. The prognostic value of IL-1α/β, IL-8, IL-12, TNF-α, TGF-β and IFN-γ could not be demonstrated. The small number of selected studies underlines the need for large well-designed prospective studies, using the REMARK checklist as a guideline, to determine which cytokines have prognostic value on survival in cancer patients.

The population of elderly individuals with rheumatoid arthritis (RA) is expanding, due mainly to increasing life expectancy. A variety of theories have been proposed to explain the ageing process, including accumulation of DNA damage and resultant changes in biological processes. Such changes can influence the development and/or course of disease. Furthermore, alterations in biological function determine the biological age-as opposed to chronological age-of an individual, which strongly influences their ability to cope with disease. Moreover, comorbidities are more frequent in elderly individuals. Together, these factors complicate treatment of disease and necessitate careful patient management. Indeed, although evidence from clinical trials suggests that DMARDs and biologic agents have good efficacy and are well tolerated in elderly patients with RA, such individuals are often undertreated and inadequately managed. Unfortunately, insufficient data are available for the development of evidence-based guidelines for this population, as elderly patients are often excluded from clinical trials owing to age restrictions or comorbidities. Thus, additional clinical studies in elderly patients are warranted, with treatment regimens tailored according to vitality or frailty parameters. This Review focuses on the pathophysiological aspects of ageing and their implications for the management of RA in elderly patients.

Epidemiological studies suggest a positive correlation between the incidence and severity of multiple sclerosis (MS) and the intake of fatty acids. It remains to be clarified whether high fat diet (HFD) indeed can exacerbate the disease pathology associated with MS and what the underlying mechanisms are. In this study, we determined the influence of HFD on the severity and pathology of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Mice were fed either normal diet (ND) or HFD and subsequently induced with EAE. Immunohistochemical staining and real-time PCR were used to determine immune cell infiltration and inflammatory mediators in the central nervous system (CNS). Our data show that HFD increases immune cell infiltration and inflammatory mediator production in the CNS and thereby aggravates EAE. Moreover, our data demonstrate that activation of the renin angiotensin system (RAS) is associated with the HFD-mediated effects on EAE severity. These results show that HFD exacerbates an autoreactive immune response within the CNS. This indicates that diets containing excess fat have a significant influence on neuroinflammation in EAE, which may have important implications for the treatment and prevention of neuroinflammatory disorders.

Transcriptomic and proteomic analyses of multiple sclerosis (MS) lesions indicate alterations in the gamma-aminobutyric acid (GABA) inhibitory system, suggesting its involvement in the disease process. To further elucidate the role of GABA in central nervous system (CNS) inflammation in vivo, the chronic myelin oligodendrocyte glycoprotein (MOG)(35-55) experimental autoimmune encephalomyelitis (EAE) model was used. Daily GABA injections (200mg/kg) from day 3 onwards significantly augmented disease severity, which was associated with increased CNS mRNA expression levels of tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6. GABA-treated mice showed enhanced MOG-dependent proliferation and were skewed towards a T helper 1 phenotype. Moreover, in vitro, the lipopolysaccharide (LPS)-induced increase in interleukin (IL)-6 production by macrophages was enhanced at low GABA concentrations (0.03-0.3mM). In sharp contrast to exogenous GABA administration, endogenous GABA increment by systemic treatment with the GABA-transaminase inhibitor vigabatrin (250mg/kg) had prophylactic as well as therapeutic potential in EAE. Together, these results indicate an immune amplifying role of GABA in neuroinflammatory diseases like MS.

Antibody-independent B cell functions play an important role in multiple sclerosis (MS) pathogenesis. In this study, B cell antigen presentation and costimulation in MS were studied. Peripheral blood B cells of MS patients showed increased expression of costimulatory CD86 and CD80 molecules compared with healthy controls (HC). In MS cerebrospinal fluid (CSF), 12-fold and 2-fold increases in CD86(+) and CD80 B cells, respectively, were evidenced compared with peripheral blood. Further, B cells from MS patients induced proinflammatory T cells in response to myelin basic protein (MBP). Immunomodulatory treatment restored B cell costimulatory molecule expression and caused significantly reduced B cell induced T cell responses. Together, these results demonstrate the potential of B cells from MS patients to induce autoreactive proinflammatory T cell responses. Immunomodulatory therapy abrogated this effect, emphasizing the importance of B cell antigen presentation and costimulation in MS pathology. (C) 2016 Elsevier Inc. All rights reserved. This work was supported by Hasselt University and the Belgian Charcot Foundation. J. Fraussen is a postdoctoral fellow of the Fund for Scientific Research (FWO), Flanders.

Objectives. Despite recent progress in biomarker discovery for RA diagnostics, still over one-third of RA patients-and even more in early disease-present without RF or ACPA. The aim of this study was to confirm the presence of previously identified autoantibodies to novel Hasselt University (UH) peptides in early and seronegative RA. Methods. Screening for antibodies against novel UH peptides UH-RA.1, UH-RA.9, UH-RA.14 and UH-RA.21, was performed in two large independent cohorts. Peptide ELISAs were developed to screen for the presence of antibodies to UH-RA peptides. First, 292 RA patients (including 39 early patients), 90 rheumatic and 97 healthy controls from UH were studied. Antibody reactivity to two peptides (UH-RA. 1 and UH-RA. 21) was also evaluated in 600 RA patients, 309 patients with undifferentiated arthritis and 157 rheumatic controls from the Leiden Early Arthritis Clinic cohort. Results. In both cohorts, 38% of RA patients were seronegative for RF and ACPA. Testing for autoantibodies to UH-RA. 1 and UH-RA. 21 reduced the serological gap from 38% to 29% in the UH cohort (P = 0.03) and from 38% to 32% in the Leiden Early Arthritis Clinic cohort (P = 0.01). Furthermore, 19-33% of early RA patients carried antibodies to these peptides. Specificities in rheumatic controls ranged from 82 to 96%. Whereas antibodies against UH-RA. 1 were related to remission, anti-UH-RA. 21 antibodies were associated with inflammation, joint erosion and higher tender and swollen joint counts. Conclusion. This study validates the presence of antibody reactivity to novel UH-RA peptides in seronegative and early RA. This might reinforce current diagnostics and improve early diagnosis and intervention in RA.

In situ detection of MSCs remains difficult and warrants additional methods to aid with their characterization in vivo. Two-photon confocal laser scanning microscopy (TPM) and second harmonic generation (SHG) could fill this gap. Both techniques enable the detection of cells and extracellular structures, based on intrinsic properties of the specific tissue and intracellular molecules under optical irradiation. TPM imaging and SHG imaging have been used for label-free monitoring of stem cells differentiation, assessment of their behavior in biocompatible scaffolds, and even cell tracking in vivo. In this study, we show that TPM and SHG can accurately depict the umbilical cord architecture and visualize individual cells both in situ and during culture initiation, without the use of exogenously applied labels. In combination with nuclear DNA staining, we observed a variance in fluorescent intensity in the vessel walls. In addition, antibody staining showed differences in Oct4, aSMA, vimentin, and ALDH1A1 expression in situ, indicating functional differences among the umbilical cord cell populations. In future research, marker-free imaging can be of great added value to the current antigen-based staining methods for describing tissue structures and for the identification of progenitor cells in their tissue of origin. The authors would like to thank Ms. Katrien Wauterickx, Dr. Petra Hilkens, and Ms. Jeanine Santermans for assistance with the immunohistochemical staining and paraffin embedding of the umbilical cord tissues. This research was financially supported by the agency for Innovation through Science and Technology (Agentschap voor Innovatie door Wetenschap en Technologie, IWT), the Research Foundation-Flanders (Fonds Wetenschappelijk Onderzoek Vlaanderen, FWO), the Transnational University Limburg, Hasselt University, and the Belgian Charcot Foundation. The Province of Limburg (Belgium) is acknowledged for financial support within the tUL IMPULS FASE II program, allowing for the upgrading of the laser source used in this work.

Aging of the immune system contributes to the increased morbidity and mortality of the elderly population and may occur prematurely in patients with immune disorders. One of the main characteristics of immunosenescence is the expansion of CD4(+)CD28(-) T cells in the blood. These cells are effector memory T cells with cytotoxic capacity, and have been recently described to have pathogenic potential in a variety of immune disorders. Interestingly, CD4(+)CD28(-) T cells have now been found to infiltrate target tissues of patients with multiple sclerosis, rheumatoid arthritis, myopathies, acute coronary syndromes, and other immune-related diseases. In this review, we discuss potential factors and mechanisms that may induce the expansion of these cells, as well as their putative pathogenic mechanisms in immune disorders.

Phage display is a high-throughput technology used to identify ligands for a given target. A drawback of the approach is the absence of PTMs in phage-displayed peptides. The applicability of phage display could be broadened considerably by the implementation of PTMs in this system. The aim of this study was to investigate the possible application of citrullination, a PTM of an arginine into a citrulline amino acid, in filamentous (M13) and lytic (T7) phage display. After in vitro citrullination of T7 and M13 phages, citrullination was confirmed and the infectivity of both citrullinated and non-citrullinated phage was compared by titer determination. We demonstrated the successful in vitro citrullination of T7 and M13 phage-displayed peptides. This in vitro modification did not affect the viability or infectivity of the T7 virions, a necessary prerequisite for the implementation of this approach in T7 phage display. For M13 phage, however, the infecting phage titer decreased five-fold upon citrullination, limiting the use of this modification in M13 phage display. In conclusion, in vitro citrullination can be applied in T7 phage display giving rise to a high-throughput and sensitive approach to identify citrulline-containing ligands by the use of the strengths of phage display technology.

Approximately one-third of rheumatoid arthritis (RA) patients are seronegative for the 2 serological RA markers, rheumatoid factor (RF) and antibodies against cyclic citrullinated peptides (ACCP). Moreover, the sensitivities of both markers are lower in the diagnostically important early disease phase. The aim of this study was to identify additional autoantibody markers for early RA and for RF-negative, ACCP-negative (seronegative) RA. We screened an RA synovium cDNA phage display library with autoantibodies in plasma from 10 early (symptoms of maximum 1 year) and 10 seronegative (RF-negative, ACCP-negative) RA patients with validation in 72 additional RA patients and 121 controls (38 healthy controls, 43 patients with other inflammatory rheumatic diseases, 20 osteoarthritis patients and 20 subjects with mechanical joint complaints). Fourteen novel autoantibodies were identified that showed a 54% sensitivity and 90% specificity for RA. For 11 of these autoantibodies, an exclusive presence was demonstrated in RA patients (100% specificity, 37% sensitivity) as compared to controls. All early RA patients were positive for at least one of the identified autoantibodies and antibody-positivity was associated with a shorter disease duration (P = 0.0087). 52% of RA patients who initially tested negative for RF and ACCP, tested positive for at least one of the 14 novel autoantibodies, resulting in a 19% increase in sensitivity compared to current serological testing. Moreover, 5 identified autoantibodies were detected more frequently in seronegative RA patients, indicating that these autoantibodies constitute novel candidate markers for this RA subtype. We demonstrated that the targets of 3 of these 5 autoantibodies had an increased expression in RA synovial tissue compared to control synovial tissue, pointing towards a biological rationale for these auto antibody targets in RA. In conclusion, we identified novel candidate autoantibody markers for RA that can be detected in early and seronegative RA patients indicating the potential added value for RA diagnostics.

We have developed a B cell immortalization method for low B cell numbers per well using simultaneous B cell stimulation by CpG2006 and B cell infection by Epstein-Barr virus (EBV), followed by an additional CpG2006 and interleukin-2 (IL-2) stimulus. Using this method, immunoglobulin G (IgG)-producing immortalized B cell lines were generated from peripheral blood IgG(+)CD22(+) B cells with an efficiency of up to 83%. Antibody can already be obtained from the culture supernatant after 3-4 weeks. Moreover, clonality analysis demonstrated monoclonality in 87% of the resulting immortalized B cell lines. Given the high immortalization efficiency and monoclonality rate, evidence is provided that no further sub-cloning is necessary. An important application of this B cell immortalization method is the characterization of (autoreactive) antibodies from patients with autoimmune disease. This could eventually lead to the identification of new autoantigens, disease markers or targets for therapy.

Otosclerosis is a common form of hearing loss, characterized by disordered bone remodeling in the otic capsule. Within the otosclerotic foci, several immunocompetent cells and immune-modulating factors can be found. Different etiological theories involving the immune system have been suggested. However, a genetic component is clearly present. In large otosclerosis families, seven autosomal-dominant loci have been found, but none of the disease-causing genes has been identified. This study focused on the exploration of the second otosclerosis locus on chromosome 7q34-36 (OTSC2), holding the T-cell receptor beta locus (TRB locus). A significantly lower T-cell receptor-beta (TCR-beta) mRNA expression and percentage of blood circulating TCR-alphabeta(+) T cells was detected in OTSC2 patients compared with controls and patients with the complex form of the disease. Further analysis illustrated more significant disturbances in specific T-cell subsets, including an increased CD28(null) cell population, suggesting a disturbed T-cell development and ageing in OTSC2 patients. These disturbances could be associated with otosclerotic bone remodeling, given the known effects of immunocompetent cells on bone physiology. These data implicate the TRB locus as the causative gene in the OTSC2 region and represent an important finding in the elucidation of the disease pathology.Genes and Immunity advance online publication, 25 February 2010; doi:10.1038/gene.2010.3.

The receptor for the homeostatic T cell cytokine interleukin-7 (IL-7Ralpha) has recently shown genetic association to multiple sclerosis (MS). To investigate the functional contribution of IL-7Ralpha polymorphisms to the pathogenesis of MS, we correlated the IL-7Ralpha haplotypes with different T cell parameters in a group of MS patients and healthy controls. We show that carriers of one of the four IL-7Ralpha haplotypes (Hap4) show a higher expression of IL-7Ralpha (CD127) on their CD4(+) T cells, compared with noncarriers (P=0.04). Moreover, Hap4 carriers possess higher frequencies of recent thymic emigrants (RTEs, CD31(+)) in both the regulatory T cell (Treg; P=0.007) and conventional T cell (Tconv) population (P=0.0001). This effect is most pronounced within the MS population (Treg, P=0.0077; Tconv, P=0.0007), whereas in healthy controls significance was only reached for Tconv (P=0.043; Treg, P=0.11). Because previous studies showed a decreased RTE-Treg frequency in MS patients compared to healthy subjects, we here conclude that this decrease is localized within the MS population of non-Hap4 carriers. In conclusion, our findings suggest that IL-7Ralpha polymorphisms can influence T cell development and homeostasis, and thereby contribute to the altered immune regulation associated with disease development in patients with MS.Genes and Immunity advance online publication, 14 January 2010; doi:10.1038/gene.2009.106.

Myasthenia Gravis (MG) is an antibody-mediated autoimmune disorder affecting the postsynaptic membrane of the neuromuscular junction (NMJ). MG is characterized by an impaired signal transmission between the motor neuron and the skeletal muscle cell, caused by auto-antibodies directed against NMJ proteins. The auto-antibodies target the nicotinic acetylcholine receptor (nAChR) in about 90% of MG patients. In approximately 5% of MG patients, the muscle specific kinase (MuSK) is the auto-antigen. In the remaining 5% of MG patients, however, antibodies against the nAChR or MuSK are not detectable (idiopathic MG, iMG). Although only the anti-nAChR and anti-MuSK auto-antibodies have been demonstrated to be pathogenic, several other antibodies recognizing self-antigens can also be found in MG patients. Various auto-antibodies associated with thymic abnormalities have been reported, as well as many non-MG-specific auto-antibodies. However, their contribution to the cause, pathology and severity of the disease is still poorly understood. Here, we comprehensively review the reported auto-antibodies in MG patients and discuss their role in the pathology of this autoimmune disease.

A major focus in rheumatoid arthritis (RA) research is the identification of the antigens that are targeted by the joint-directed autoimmune response. B cells and associated autoantibodies have been studied in RA to identify the antigenic targets and to discover RA-associated autoantibodies which can be used as disease markers. This research indicated the heterogeneity of the autoantibody profile in RA and the large overlap in antibody specificities with other rheumatic diseases pointing toward the need for multiplexing to identify an RA-associated autoantibody profile. The discovery of antibodies directed against cyclic citrullinated peptides (ACPA) has led to great advances in RA research. This finding generated novel autoantigen suspects in ACPA-positive RA patients, which comprise approximately two-thirds of the entire RA population, namely citrullinated peptides and/or proteins. One-third of the RA patients, however, do not show ACPA, and it is now postulated that ACPA-positive and ACPA-negative RA are two different disease entities with different genetic associations, pathogenesis, and etiology. The analysis of autoantibodies in ACPA-negative RA could provide insight into the identity of antigenic targets and markers for this disease subtype. We report here the optimization of an unbiased, high-throughput autoantibody profiling procedure based on cDNA phage display for the detection of novel autoantibody targets in ACPA-negative RA. The discovery of specific autoantibodies in this RA subtype could lead to great advances in the diagnosis of these patients and could provide clues regarding disease etiology and pathogenesis of ACPA-negative RA.

The immune system has developed several checkpoints to prevent the induction of autoimmune diseases like multiple sclerosis and rheumatoid arthritis. Autoreactive T cells are negatively selected in the thymus but can escape this selection procedure. Peripherally, they are further controlled by homeostatic mechanisms and regulatory T cells (Tregs). Genetic and exogenous factors as well as a disbalanced functioning of Tregs may all influence these checkpoints and contribute to the breakdown of tolerance against ‘self’. Re-establishment of tolerance can be achieved by different vaccination strategies, which are divided into three major groups based on the composition of the vaccine: DNA vaccination, peptide-based-vaccination and T cell vaccination. This review summarizes the mechanisms, clinical trials and pros and cons of these different vaccination strategies.

While it is widely accepted that myelin reactive T cells are key players in the immunopathogenesis of multiple sclerosis (MS), the initial triggers that turn on these self-reactive T-cells remain to be determined. One mechanism, which is already text book knowledge for a decade, is molecular mimicry by which viral or microbial antigens are able to cross-activate T cells specific for myelin epitopes, the major target in MS pathology. Although proof of concept for this principle was given in animal model studies, evidence for such a mechanism in MS is limited. In this issue, Zhang et al. demonstrate an increased frequency of T cells that cross-react with a wide variety of antigens, a phenomenon termed as T cell degeneracy. While the role of these degenerate T cells in MS remains to be determined the authors now provide new elegant tools to study this T cell population, thus providing a starting point to better understand their function in MS.

Leukemia inhibitory factor (LIF) promotes the survival of oligodendrocytes (OLG) both in vitro and in an animal model of multiple sclerosis. Here, we show that LIF protects mature rat OLG cultures selectively against the combined insult of the proinflammatory cytokines interferon-gamma and tumor necrosis factor-alpha, but it does not protect against oxidative stress nor against staurosporine induced apoptosis. We further demonstrate that LIF activates the janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) and the phosphatidylinositol 3 kinase/Akt pathway in mature OLG. We show that LIF protection is independent of suppressors of cytokine signaling and Bcl-2 mRNA expression levels. To gain further insight into the protective mechanism, a quantitative proteomic approach (DIGE) was applied to identify differentially expressed proteins in LIF-treated OLG. Our results indicate that LIF induces a shift in the cellular machinery toward a prosurvival execution program, illustrated by an enhanced expression of isoforms of the antiapoptotic molecule 14-3-3. These data provide further insight into the mechanisms of LIF-mediated protection of mature OLGs.

Complex autoimmune diseases are chronic conditions initiated by a loss of immunological tolerance to self-antigens. Despite many years of intense investigation, the etiopathogenic mechanisms initiating these chronic inflammatory conditions remain undefined. The relatively high prevalence and the clustering of multiple autoimmune diseases within individuals or within families suggest that rather common immune mechanisms may facilitate the development of autoimmune diseases. However, specific genetic or environmental factors seem to be necessary for triggering disease. Clearly, immune mechanisms, which are intended to protect from disease, fail or become dysregulatcd. In this review, we discuss the mechanisms of immune homeostasis, immune regulation, and immunosenescence. We focus on the merit, consequences, and/or shortcomings of these mechanisms and speculate on their possible involvement in autoimmune-disease development.

We applied a cDNA phage display method called serological antigen selection (SAS) to identify immunogenic targets that evoke an autoantibody response in the serum of multiple sclerosis (MS) patients. This method involves the display of a cDNA expression library, in this study a MS brain library, on filamentous phage and subsequent selection using patient immunoglobulin G (IgG). To apply the SAS technology for autoantibodies in the serum of MS patients, an optimization was necessary to deplete cDNA products that encode IgG fragments derived from B cells present in the MS brain plaques. We describe a differential screening procedure in which positive selection rounds on MS serum and negative selection rounds on healthy control serum were alternated to optimize the selection procedure. As a result, a substantial decrease of IgG-displaying phage clones was observed after each negative selection round, thereby preventing an overgrowth of IgG-displaying phage clones. Our depletion strategy was therefore successful in preventing the enrichment of IgG-displaying phage clones. This approach will facilitate the identification of possible MS-related antigens.