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The role of infections in Behçet disease and neuro-Behçet syndrome Monica Marta1, Ernestina Santos2,5, Ester Coutinho2,6, Ana Martins Silva2,4,5, João Correia4, Carlos Vasconcelos4 e Gavin Giovannoni1 1. Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, 4 Newark Street, London E1 2AT, UK 2. Neurology Department, Centro Hospitalar do Porto, Largo Prof. Abel Salazar, 4099-001 Porto, Portugal 3. Unidade de Imunologia Clínica, Centro Hospitalar do Porto, Largo Prof. Abel Salazar, 4099001 Porto, Portugal 4. Unidade Multidisciplinar de Investigação Biomédica, Instituto de Ciências Biomédicas de Abel Salazar, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal 5. Present address: Nuffield Department of Clinical Neurosciences. Level 6, West Wing, John Radcliffe Hospital, Oxford OX3 9DU, UK Corresponding author: Monica Marta, MD, PhD [email protected] Neuroimmunology Unit, Neuroscience and Trauma Centre Blizard Institute, Queen Mary University London 4 Newark Street, Whitechapel London E1 2AT Phone no: +44 20 78822677 Fax no: +44 20 78822180 Ernestina Santos: [email protected] Ester Coutinho: [email protected] Ana Martins Silva: [email protected] João Correia: [email protected] Carlos Vasconcelos: [email protected] Gavin Giovannoni: [email protected] Abstract Infections are considered an environmental trigger for exacerbations of immune-mediated diseases. We aimed to establish if common viral infections could be identified as a precipitant of Behçet disease (BD) with or without neurological involvement and to assess the variability of the immune response to common viruses. We also investigated whether cytokines and chemokines could be markers of neurological involvement. Finally, we explored if anti-basal ganglia antibodies (ABGA) would be associated with neurological involvement in BD. Our study included 14 individuals with BD with neurological involvement (neuroBehçet syndrome - NBS), 16 individuals with BD without neurological involvement and 18 healthy controls (HC). Overall we found a tendency for increased levels of anti-viral IgG antibody levels in BD, more evident in NBS patients versus HC. Epstein-Barr viral-capsid antigen IgG titres were increased in NBS patients versus other BD patients (p=0.032). Anti-measles antibody titres induced by vaccination were similarly elevated. ABGA were not detected in the serum of our cohort. Raised levels of serum IL-8 in some BD patients did not reflect clinical activity or severity. In conclusion, there was evidence for a polyclonal immune activation rather than a specific virus effect in the sera of individuals with BD or NBS. Keywords: Behçet, infections, EBV, IL-8 Abbreviations: BD: Behçet disease; NBS: neuroBehçet syndrome; CSF: cerebro-spinal fluid; MS: multiple sclerosis; ABGA: anti-basal ganglia antibodies; CNS: central nervous system; BBB: blood-brain barrier; EBV: Epstein-Barr; VZV: varicella zoster; CMV: cytomegalovirus; HSV-1: herpes simplex type 1; ASO: antistreptolysin O; VCA: viral capsid antigen; EBNA-1: EBV Nuclear Antigen-1 1. Introduction NeuroBehçet syndrome (NBS) is defined as the presence of neurological symptoms associated with signs and/or with imaging and/or abnormal cerebro-spinal fluid (CSF) in an individual who fulfils the Diagnostic Criteria for Behçet Disease (BD) 1, after exclusion of other possible causes 2 . Neurological involvement ranges from 3.2 to 49% in BD series and may present as headache (the most frequent initial neurological symptom), meningoencephalitis, multiple sclerosis (MS)– like illness, stroke, pseudotumor cerebri, organic confusional syndrome or a combination of the above 3. Neurologic involvement is one of the most serious complications, leading to severe disability and ensuing high fatality rate. BD is a multisystem vasculitis that affects small vessels causing focal or multifocal intraparenchymal disease in the central nervous system (CNS) 4,5; it also affects the peripheral nerves in a distal and symmetrical way or as mononeuritis multiplex. Frequently, BD involves large vessels presenting with thrombosis of the major cerebral venous sinuses 6. BD patients have a preferential genetic background with a significant association with HLA-B51 (B101) and the MICA6 allele in most populations. But apart from this genetic susceptibility, the aetiology remains unknown 7. An important role is recognized for infections acting as environmental triggers in the induction or promotion of immune-mediated diseases in genetically predisposed individuals 8. Further, infections and stress may lead to disruption of the blood-brain barrier (BBB) and lead to neurological involvement 9,10. Common virus such as Epstein-Barr (EBV), varicella zoster (VZV), cytomegalovirus (CMV), herpes simplex type 1 (HSV-1) or measles cause infections that range from clinically asymptomatic to life threatening, mostly when they occur in healthy infants or in immunocompromised individuals respectively 11. The indirect effects of the viral infection, either due to the long-term presence of latent virus or to the large proportion of the immune system they engage, are associated with autoimmunity through mechanisms like molecular mimicry, polyclonal activation or superantigens. These are the mechanisms that in some cases link infections to autoimmunity, and that counter aspects of the hygiene hypothesis 12. Several studies have associated BD with infections, in particular streptococcal infections but also altered microbiota of the gut 13. Streptococcal infection is usually confirmed by culture or by demonstrating a raised antistreptolysin O (ASO) or anti-DNAse B antibody titre. Further, acquired hypersensitivity to streptococcal antigens plays an important role in the aetiopathology of BD. Persistently high ASO titers in BD patients could indicate that streptococcal infections such as pharyngitis are related to BD symptoms like erythema nodosum-like lesions 14. Group A beta-hemolytic streptococcal infections cause immune-mediated delayed rheumatic fever in a proportion of individuals and Sydenham's chorea is part of that phenotype. A type of anti-neuronal antibodies, anti-basal ganglia antibodies (ABGA), are associated with a wide spectrum of post-streptococcal neuropsychiatric disorders 15. The possible aberrant immune response may be triggered by streptococcal infection and the auto-antibodies cross-react with human brain tissue and some individuals have dopamine-2 receptor antibodies 16,17. The overall aim of the study was to identify an infection that would be associated with BD with or without neurological involvement. In particular, we looked at (a) common viral infections as possible precipitants or (b) a variation in the immune response to a common viral agent, (c) cytokines and chemokines that may play a role or be a marker of permeability of the BBB and finally at (d) the possibility that streptococcal infection could elicit the presence of ABGA, which have been associated with neurological syndromes. 2. Methods 2.1 Sample collection and ethical approval The study was approved by the Ethics Committee of Hospital Santo Antonio, Centro Hospitalar do Porto, Portugal. Every participant signed an Informed Consent Form. Clinical and epidemiological data of individuals with BD only and NBD followed in the Clinical Immunology Unit and Neuroinflammation Clinic was retrospectively compiled. There was an independent healthy control group. 2.2 Measurement of serum immunoglobulin G (IgG) antibodies to EBV (EBNA1 and VCA), VZV, CMV, measles and HSV-1 Cross-sectional measurement of serum immunoglobulin G (IgG) antibodies to EBV (EBNA1: Epstein–Barr virus nuclear antigen and VCA: viral capsid antigen), VZV, CMV, measles and HSV-1 was done with commercial enzyme linked immunoassays. Control and standard samples (supplied) and samples diluted 1:100 (1:400 for EBNA1 and 1:1000 for VCA) were added to the microtest strips pre-coated with the antigen (supplied) and incubated for one hour at 37C in moist chamber and washed thoroughly; the anti-human-IgG-alkaline phosphatase-conjugate solution was then added, incubated for 30 minutes in the same conditions and washed thoroughly; the p-nitrophenyl phosphate substrate solution was then added and incubated for up to 30 minutes and finally HCl was used to stop the reaction. The optical density of the plate was read at 405nm. 2.3 Measurement of serum cytokines Cross-sectional measurement of 11 serum cytokines: IL (interleukin)-2, IFN-gamma, IL-12p70 and TNF-beta (type-1 cytokines); IL -4, IL-5, IL-6, IL-10 and IL-13 (type-2 cytokines), IL-1b, IL-8 and TNF with a flow cytometry multiplex platform (Human Th1/Th2 11plex FlowCytomix Multiplex, Bender MedSystems, eBioscience, Austria) 18 according to suppliers instructions. In brief, the bead mixture and the biotin-conjugate mixture were added to the samples, standards and blank tubes, mixed and incubated at RT for one hour protected from the light. Buffer was added to all tubes and spun then the supernatant was discarded leaving 100μL. Streptavidin-PE solution was added to all tubes, mixed and incubated protected from the light for one hour. Buffer was added, spun and the supernatant was discarded twice. It was then analysed in the flow cytometer. The bead mixture included beads of two different sizes (4 and 5 μm) and each had 9 or 11 populations internally dyed with different intensities of a fluorescent dye for individual detection and coated with antibodies specifically reacting with the analyte. 2.4 Detection of ABGA in the sera Detection of ABGA in the sera by Western blotting was described elsewhere 19. In brief, human basal ganglia with no pathology were physically disrupted and homogenized with protein extractor and protease inhibitors (Sigma-Aldrich Co., UK). The protein fraction was then collected and stored at −80 °C until required. The homogenate and a molecular weight ladder were loaded onto a Nu-Page gel (Invitrogen, Life Technologies Ltd, UK) and electrophoresis was run at 200V, 120mA and 25W for about 1 hour. The proteins were blotted onto nitrocellulose membranes (Bio-Rad Laboratories, Inc., UK) and blocked with 2% milk proteins for 2 hours. Samples were diluted at 1:300, applied to the blot with the help of a sample manifold saving one well for the mix of control antibodies. The membranes were incubated overnight at 4 °C and after ten wash cycles and incubated for 2 hours with rabbit anti–human IgG, FAB fragments conjugated with horseradish peroxide diluted at 1:1,000 (Dako, Cambridge, UK). After washing, the substrate 4-chloro-1-naphthol (Sigma-Aldrich Co., UK) was added and the blot was allowed to develop for 20 minutes. 2.5 Statistical analyses Mann-Whitney without corrections for multi-testing, were done with GraphPad Prism 5. 3. Results 3.1 Characteristics of the patients and clinical manifestation of BD and NBS The group studied originated from the same Clinical Immunology Unit and Neuroimmunology Clinic and included 14 individuals with BD and neurological involvement (NBD) and 16 individuals with BD. The clinical and radiological characteristics of the NBD patients were detailed in a previous publication 20. The neurological syndrome included cognitive impairment, visual defects, hemiparesis (2 patients), aseptic meningitis, psychosis, meningoencephalitis involving brain stem (2 patients) or hemispheres (2 patients), cerebellar syndrome (2 patients), cerebral venous thrombosis (CVT) and vasculitic polyneuropathy. All individuals except the one with cerebro-venous thrombosis had multiple small hyperintense lesions on T2/FLAIR predominantly in the meso-diencephalic and ponto-bulbar regions. Serum samples were collected in a cross-sectional way, independent of the activity of disease or treatment and a healthy control group from relatives and friends of patients attending the Neuroimmunology Clinic was matched for sex and area of origin. The BD clinical syndrome and the treatments used at time of sample collection are described in Table 1. 3.2 Immune response to common viral infections The IgG antibodies to EBV, VZV, measles, HSV-1 and CMV were measured in a serum sample from all thirty individuals with BD with and without neurological involvement and in eighteen healthy controls. Comparisons were made between the titres in healthy controls (HC) and all the individuals with BD and between the individuals with and without neurological involvement. 3.3 EBV serology The prevalence of latent infection with EBV in the general population is 85-90% and increases with age. Elevated titres of EBNA1 IgG are associated with an increased risk for MS. Only one person in the healthy control group was negative for EBV infection (negative EBNA1 and VCA serology). There was a non-significant increased titre of EBNA1 in NBS patients compared to BD patients, but this was not apparent when all individuals with BD were compared with HC. The titres of VCA IgG were increased in NBS patients when compared to BD patients (p=0.032) but the difference was not significant when all individuals with BD were compared to HC (Fig1). VZV serology Varicella zoster is another common herpes family infection, which has a much lower prevalence in the Portuguese population. In countries like the USA where the vaccination recommendation is for universal coverage, the results would differ. The titres of VZV IgG were increased in NBS when compared to BD patients (p= 0.0323) and in individuals with BD when compared with HC (not significant) (Fig 2). 3.4 Measles serology The majority individuals tested were under universal vaccination coverage for measles (in Portugal since 1974). There were six individuals with BD who had titres below the positive threshold for measles, but, according to their age all of them had been vaccinated. The sera IgG titres showed a tendency to be higher in NBD patients when compared with BD patients and in all individuals with BD when compared with HC (not significant) (Fig 3). 3.5 HSV-1 serology HSV-1 persists in neural ganglia in a latent state after the initial primary infection. Different stimuli cause the virus to reactivate and cause new lesions in the respective dermatome. The percentage of people who are positive for HSV-1 infection as measured by IgG generally increases with age and is higher in lower socioeconomic strata. The titres of HSV-1 IgG had a tendency to be increased in NBS patients when compared to BD patients and in BD patients when compared with HC (not significant) (Fig 4). 3.6 Cytokines in sera of patients with BD with and without neurological involvement and HC Our results showed that IL-8, IL-2 and IL-10 were the only detectable serum cytokines in any patient when we tested for IL-2, IFN-gamma, IL-12p70, TNF-beta, IL-4, IL-5, IL-6, IL-10, IL13, IL-1b, IL-8 and TNF-alpha with the multiplex platform used. IL-8 was detected in the 11 of 17 BD patients and 7 of 13 NBS patients. The levels of IL8 had a tendency to be higher in all individuals with BD than in NBS patients. The two NBS patients with the highest IL-8 levels also had detectable IL-2. There was no correlation between activity of disease and IL-8 levels (Fig 5). IL-10 was detected in only two BD patients without neurological manifestations. Both patients were on colchicine and had moderately severe disease; there was no correlation of IL-10 with the levels of IL-8, which were undetectable in one patient and high in another. 3.7 Detection of anti-basal ganglia antibodies in sera of patients with NBS BD has been associated with Streptococcal colonization and infections and post-streptococcal ABGA are associated with many neuropsychiatric manifestations. We investigated whether BD neurological involvement was associated with the presence of anti-neuronal antibodies, in particular ABGA. We tested for presence of anti-aldolase-C (neuron-specific), anti-gammaenolase, anti-pyruvate kinase antibodies in sera of all individuals with BD with the routinely used western blot and did not find a specific band. 3. Discussion There is a continuing debate whether specific autoimmune diseases are triggered by infections. In particular for diseases that do not fulfill the strict criteria for autoimmunity (characterized antibody mediated diseases), the presence of infectious particles can trigger self-immune recognition as a bystander phenomenon. Polyclonal activation, molecular mimicry, superantigens and bystander activation are all mechanisms that have an effect and are independent of a particular agent. We chose to test EBV, CMV, VZV and HSV-1 IgG titres as they are neurotropic, often found in a latent state after acute infection and have previously been associated with other autoimmune diseases 11,21,22. On the other hand, measles can rarely cause subacute sclerosing panencephalitis, a progressive infection caused by persistent mutated virus, but is part of the routine infant vaccination program in most countries, and so most people have antibodies to the vaccine. The increased levels of VCA IgG indicate that the immune responses to the main protein that characterize the lytic phase of EBV infection may be associated with NBS. This will need to be confirmed, but it is an interesting observation as EBNA1 IgG levels, rather than VCA, are associated with an increased risk for MS 23–25. Overall, for all tested viral infections, there was a tendency towards higher IgG levels in people with BD than HC. Similarly, the titres of antimeasles IgG detected were also increased. They probably represent the post immune response childhood vaccination and not exposure to or response to infection, which makes us believe in a plasmocyte polyclonal activation rather than a specific increased exposure or response to viral infections. Nevertheless, it was interesting that the patient with most severe residual neurological disability had increased IgG levels for all viruses except for measles. It is possible that IgG responses are dampened by the use of steroids but the differences between BD and NBS patients could not be accounted for. None of our patients were in flare and there is no accurate measure of clinical activity or severity between BD and NBS patients, hence no correlation can be made. Viral infections are good candidates for environmental promoters of autoimmunity 26,27. The number of individuals in the groups would need to be increased to reach statistical significance. Streptococcal infection is a well-known cause of autoimmune disease: a delayed postinfectious inflammatory disease that affects the heart muscle and valves, large joints, subcutaneous tissue and brain. Acute rheumatic fever and Sydenham’s chorea are well established but PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections), which presents with tics and/or obsessive compulsive behavior soon after infections, has yet to be widely accepted as an specific disease entity. Streptococci are more frequently isolated from the oral cavity of people with BD, who also have more frequent streptococcal infections. Initially, S sanguis was the identified strain but others have been isolated 28,29. We hypothesized that if BD is triggered by a streptococcal infection, anti-neuronal antibodies could mediate some neurological involvement and/or constitute a biomarker to help clinical therapeutic decisions. ABGA WB recognises four protein bands: the 40kDa antigen is aldolase-C (neuronspecific), the 45 kDa is gamma-enolase, the 60 kDa is pyruvate kinase and the 98 kDA that possibly is a dimmer of gamma- and alpha-enolase. The negative findings can either be because of the presence of ABGA in higher detectable quantity in the CSF rather than sera unlike other diseases, or because the individuals were not in acute stage and the ABGA had lower levels. ABGA are associated with typical phenotype that includes hyperkinetic movement disorders with a relatively characteristic neuropsychiatric syndrome 19. The NBS patients in this study did not present with this phenotype. In systemic inflammatory diseases potentially driven by antibodies, disruption of the BBB by cytokines or chemokines is needed for access of inflammatory cells and antibodies to the brain. The mechanism that increases the permeability of the BBB may also be related to cellular entry; peripheral blood phenotyping was not performed in this study. Autoantibodies, binding molecules exposed on the surface of neurons, lead to a neurotoxic effect 1,30. Increased levels of TNF, IL-6 31, IL-18 32 and IL-8 have been reported in cultures of monocytes from people with BD 33,34. IL-8 in particular attracts and activates neutrophils and other leukocytes and is thought to mediate endothelial damage in BD. The association between IL-8 and activity of BD has not been tested in a NBD cohort 35. Surprisingly, serum TNF-alpha or IL-6 were not detected in serum. We would expect increased levels of TNF-alpha as infliximab and other TNFalpha inhibitors have proven to be effective in severe BD 36–38. Probably TNF-alpha does not act at a systemic level, but at cellular level, and the titres detected in serum do not correlate with clinical disease. In conclusion, our results do not identify increased IL-8 levels as markers for neurological involvement. Further, our analysis of the clinical picture at the time of sample collection did not demonstrate a correlation between IL-8 levels and clinical severity. It is likely though the activity of BD was not overt or the treatment has an effect in the symptoms but not in this inflammatory mediator. Alternative facilitators of BBB permeability are anti-endothelial cell antibodies. These may activate the endothelium inducing the synthesis of pro-inflammatory cytokines and chemokines and expression of adhesion molecules, leading to increased permeability of the BBB 39. Ideally, our study would have collected serum and CSF samples at the presentation of neurological manifestations and in a prospective way. The numbers in our cohort are limited and make it difficult to achieve sufficient power for robust statistical analyses. The wide number of virus and possibly bacteria that have not been tested is large and the causal infectious etiology may yet to be identified 40. Overall we found a tendency for increased levels of anti-viral IgG antibody levels in BD and more so in NBS patients than in HC. The observation that also anti-measles antibodies elicited by vaccination where elevated in a similar fashion argues for a polyclonal activation rather than a specific viral effect. ABGA were not detected in the serum of our cohort. The high levels of serum IL-8 detected in some of the individuals did not reflect clinical activity or severity and were not increased in NBS patients. Conflict of Interest: “The authors declare(s) that there are no conflicts of interest regarding the data and publication of this paper.” Acknowledgements: We thank Dr Andrew Church for technical help with the ABGA assay. References 1. Criteria for diagnosis of Behçet’s disease. International Study Group for Behçet's Disease. Lancet. 1990;335(8697):1078-80. Available at: http://www.ncbi.nlm.nih.gov/pubmed/1970380. Accessed July 5, 2011. 2. Al-Araji A, Kidd DP. Neuro-Behçet’s disease: epidemiology, clinical characteristics, and management. 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Infectome: a platform to trace infectious triggers of autoimmunity. Autoimmun Rev. 2013;12(7):726-40. doi:10.1016/j.autrev.2012.12.005. Figure 1. EBV IgG antibodies against EBNA1 and VCA. There was no significant difference in the number of positive results or levels of IgG levels between BD patients, NBS patients or HC. The top graphs show the levels of IgG in individuals with Behçet with (NB; n=14) and without (B; n=16) neurological features. The bottom graphs show the levels of IgG in all Behçet patients (BD) compared with healthy controls (HC; n=18). The titres of VCA IgG were increased in NBS when compared to BD patients (p=0.032), but this was not significant after multi-testing. Figure 2. VZV IgG antibodies. There was no difference between BD only, NBS patients or HC. The graph on the left shows the levels of IgG in Behçet patients with (NB; n=14) and without (B; n=16) neurological features. The graph on the right shows the levels of IgG in all Behçet patients (BD) compared with healthy controls (HC; n=18). Figure 3. Measles IgG antibodies. There was no difference between BD only, NBS patients or HC. The graph on the left shows the levels of IgG in Behçet patients with (NB; n=14) and without (B; n=16) neurological features. The graph on the right shows the levels of IgG in all Behçet patients (BD) compared with healthy controls (HC; n=18). Figure 4. HSV-1 IgG antibodies. There was no difference between BD only, NBS patients or HC. The graph on the left shows the levels of IgG in Behçet patients with (NB; n=14) and without (B; n=16) neurological features. The graph on the right shows the levels of IgG in all Behçet patients (BD) compared with healthy controls (HC; n=18). Figure 5. IL-8 levels in Behçet’s patients with (NB; n=14) and without (B; n=16) neurological involvement. U/mL Figure 2. VZV IgG antibodies. BD NB HC U/mL U/mL 10 3000 00 60 00 70 00 0 20 4000 0 60 0 8000 00 U/mL 80 12 0 0 16 0 00 VZV 40 0 B 50 0 HC 0 25 0 U/mL 10 0 80 20 0 NB 20 0 U/mL 10 0 50 0 Behcet Behcet B 0 0 10 0 20 0 30 0 40 0 50 0 80 0 90 10 0 00 11 0 12 0 0 13 0 0 14 0 00 15 00 16 00 17 00 18 00 Behcet EBNA1 VCA B NB EBNA1 VCA BD BD HC Figure 1. EBV IgG antibodies against EBNA1 and VCA. VZV Measles Measles BD NBS HC U/mL 10 00 0 15 00 0 0 50 00 U/mL 75 0 10 0 00 0 15 00 0 50 00 25 00 0 Behcet BD Figure 3. Measles IgG antibodies. HSV1 HSV1 BD NBS HC Figure 4. HSV-1 IgG antibodies. U/mL 40 5000 10 00 00 0 30 00 20 00 0 10 00 U/mL 10 0 15 00 00 0 60 00 40 00 20 00 0 Behcet BD IL-8 Behcet B NB 0 0 20 0 40 00 6 U/mL 0 80 00 00 10 20 Figure 5. IL-8 levels in Behçet’s patients with (NB; n=14) and without (B; n=16) neurological involvement. Table 1. Clinical characteristics of people with BD with and without neurological involvement and medication at time of blood collection Sex NBS BD 10 females 4 males 10 females 6 males Age BD diagnosis 34 for females, 31 for males Neurological involvement 4.5 years (from -4 to 18 years) orals ulcers (OU) only (4) BD clinical syndrome 32 for females , 29 for males oral ulcers only (4) OU and erythema nodosum (1) OU and OU + arthralgia (1) vaginal ulcers (1) arthritis only (1) cyclosporin only (1), steroids only (1), colchicin only (6) Drugs at time of Colchicin (C) + clopidogrel (1) steroids only (3) (2.5 – 30mg blood collection C + steroids (2), C + sulfasalazine (1) prednisolone) talidomide (1), aspirin (1) Colchicin and prednisolone (4) Highlights polyclonal immune activation in individuals with BD or NBS raised sera IL-8 levels in individuals with BD or NBS even under treatment no specific virus effect no differences in sera IgG or cytokines values for individuals with BD or NBS