Neuropsychiatric systemic lupus erythematosus: pathogenesis and biomarkers.

REVIEWS Neuropsychiatric systemic lupus erythematosus: pathogenesis and biomarkers Hélène Jeltsch-David and Sylviane Muller Abstract | Systemic lupus erythematosus (SLE) is a complex clinical syndrome, elements of which remain poorly understood. Although recognized over 140 years ago when Kaposi recorded the systemic nature and manifestations of the disease, CNS involvement represents one of the least understood aspects of SLE. This knowledge gap remains despite the fact that up to 75% of adults and children with SLE will, at some point over the course of the disease and to different extents, experience the various disabling effects of neuropsychiatric SLE (NPSLE). Indeed, after decades of research, our understanding of the underlying pathophysiology of NPSLE, in particular, remains limited. Numerous factors contribute to the immune dysfunction that occurs in SLE, including genetic, environmental and hormonal influences, and the contributory or predisposing components that lead to neurological tropism of disease in some patients have not been clearly demonstrated. Features of NPSLE pathogenesis that might be directly linked to clinical manifestations have been identified; however, the complexity and variety of NPSLE symptoms and the clinical overlap with other psychiatric disorders continue to make accurate diagnosis difficult and time-consuming. Thus, efforts to define biomarkers of NPSLE are needed to improve prediction of disease outcomes and guide treatment. In this article, we review the manifestation and pathogenesis of NPSLE, focusing on the features that might aid identification of potential biomarkers. Jeltsch-David, H. & Muller, S. Nat. Rev. Neurol. advance online publication 9 September 2014; doi:10.1038/nrneurol.2014.148

Introduction

Centre National de la Recherche Scientifique (CNRS), Immunopathologie et Chimie Thérapeutique/ Laboratory of Excellence Medalis, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67000 Strasbourg, France (H.J.‑D., S.M.).

Systemic lupus erythematosus (SLE) is a chronic, sys temic, relapsing–remitting autoimmune inflamma tory disease, which can also involve the CNS. Given the considerable heterogeneity in the manifestation of the disease within the patient population, SLE has been called “the disease with a thousand faces.”1 The disease is characterized by loss of immune tolerance, production of autoantibodies that target self antigens, and occurrence of immune complexes that can deposit in tissues and promote systemic inflammation. Although the aetiological causes of SLE and a cure for the disease remain elusive, important progress has been made towards elucidating the numerous intricately linked genetic and nongenetic factors that are involved in the triggering, flaring and amplification of disease activity.2–5 Over the past few decades, as the longevity of patients with SLE has increased, clinicians have become more aware of the late sequelae of the disease;6 nowadays, we realize that an important proportion of the patient population develop neurological and psychiatric (neuro psychiatric) symptoms, which cover the whole spectrum of psychiatric dysfunction (including cognitive changes, mood and anxiety disorders, acute confusional state, and psychosis; Table 1). The manifestations of neuro psychiatric SLE (NPSLE) are associated with different

Correspondence to: S.M. s.muller@ ibmc-cnrs.unistra.fr

Competing interests S.M. is a consultant for ImmuPharma. H.J.‑D. declares no competing interests.

degrees of morbidity, vary in presentation (having vari able onset and duration, for example) and severity between patients, and are often difficult to distinguish from other neuropsychiatric conditions with different aetiologies. Whether the CNS itself is a primary target of autoimmune dysfunction in SLE or whether neuropsychi atric symptoms are secondary manifestations of wide spread organ dysfunction is currently unclear. Although a variety of clinical, laboratory and radiographic findings have been reported to be potentially useful for differen tial diagnosis of neuropsychiatric conditions associated with SLE, no ‘gold standard’ approach is available at present. Thus, efforts to identify biomarkers that facili tate the development of an accurate and reliable diag nostic test, and novel specific therapeutic agents, are of utmost importance. Indeed, we hypothesize that as our understanding of the pathogenic mechanisms underlying NPSLE expands, both specifically designed and adapted diagnostic tools will become available, and therapeutic agents selectively targeting specific pathways involved in pathogenesis will be generated to slow or halt the development of this debilitating form of SLE. In this Review, we provide an overview of the mani festations and pathogenesis of NPSLE—aspects of the disease that might inform the development of muchneeded biomarkers. We also briefly discuss the classi fication and treatment of NPSLE, and identify facets of the disease that should be the subjects of future research.

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REVIEWS Key points ■■ Neuropsychiatric symptoms can be an important complication of systemic lupus erythematosus (SLE); among patients with neuropsychiatric SLE (NPSLE), neuropsychiatric manifestations contribute considerably to morbidity and mortality ■■ Clinical manifestations of NPSLE are wide-ranging, and can involve the CNS and PNS; thus, no single diagnostic test for NPSLE exists, and diagnosis remains mainly an exercise of exclusion ■■ Both the profile and severity of neuropsychiatric impairment in patients with NPSLE fluctuate over time, independently of disease activity ■■ Disruption of blood–brain barrier integrity, which enables diffusion of small molecules and cytokines into the cerebrospinal fluid, is considered a pivotal component of NPSLE development ■■ Consensus case-definition criteria for NPSLE-associated central and peripheral neurological syndromes represent an important development that has improved the diagnosis and management of NPSLE; however, some manifestations are still misdiagnosed ■■ The rationale for understanding the pathophysiology of NPSLE is the potential to develop biomarkers and selective therapies targeting the identified pathogenetic processes, thereby ultimately improving the management of the disease

Table 1 | Neuropsychiatric syndromes associated with SLE Neurological manifestations

ACR criteria (1999)28

Revision of the ACR criteria by Ainiala et al. (2001)163*

Syndromes associated with the CNS‡

Cerebrovascular disease Seizures Myelopathy Aseptic meningitis Movement disorder Demyelinating syndrome Cognitive dysfunction Psychosis Acute confusional state Headache Mood disorder Anxiety disorder

Cerebrovascular disease Seizures Myelopathy Aseptic meningitis Movement disorder Demyelinating syndrome Cognitive dysfunction (moderate or severe) Psychosis Acute confusional state Severe depression

Syndromes associated with the PNS

Cranial neuropathy Mononeuropathy Acute inflammatory demyelinating polyradiculoneuropathy Myasthenia gravis Plexopathy Autonomic neuropathy Polyneuropathy

Cranial neuropathy Mononeuropathy, single or multiplex Acute inflammatory demyelinating polyradiculoneuropathy Myasthenia gravis Plexopathy Autonomic disorder Polyneuropathy (with electroneuromyographic confirmation)

*In this cross-sectional validation study, the 1999 ACR criteria28 were shown to have a specificity of only 46%; however, exclusion of the syndromes without evidence for neuronal damage (headache, mild cognitive dysfunction, and mild mood and anxiety disorders), as well as polyneuropathy without electrophysiological confirmation, halved the frequency of NPSLE diagnosis and increased the specificity of the 1999 ACR criteria to 91%.163 ‡Cerebrovascular disease, seizures, myelopathy, aseptic meningitis, movement disorder and demyelinating syndrome are considered as focal neurological manifestations, whereas mood and anxiety disorders, cognitive dysfunction, psychosis, acute confusional state and headache are considered as diffuse psychiatric or neuropsychological manifestations. Abbreviations: ACR, American College of Rheumatology; SLE, systemic lupus erythematosus.

Epidemiology and genetics of NPSLE

Epidemiological studies have highlighted important differences in the prevalence and characteristics of SLE according to sex, ethnicity, age, and geographical loca tion.7,8 A marked female predominance of the disease is well established,9 with a peak incidence rate among women during childbearing years, which suggests a hormonal component to the disease.10 Hormones might constitute an endogenous ‘milieu’ that promotes the development of the disease in susceptible individuals. Changes in concentrations of sex steroids, coupled with currently undiscovered environmental factors, might

also underlie disease flares and could potentially explain the ‘waxing and waning’ nature of the disease. Non-white individuals, including persons of African descent, Asian people, and members of American and Australian aboriginal populations, are more commonly affected by SLE, including NPSLE, than are white indi viduals. 11 CNS involvement, which can be severe in SLE, substantially affects the patient’s quality of life and is associated with poor prognosis.12 Accurate data on survival rates in populations of patients with NPSLE are scarce and conflicting, ranging from no increased mortality to highly increased mortality rates compared with the general population.13 Furthermore, in a context in which diagnosis of NPSLE remains challenging—no single diagnostic test is available for NPSLE and differen tial diagnosis remains mainly an exercise of exclusion— the reported frequency of neurological involvement varies greatly among studies, ranging from 12% to 95% of patients with SLE.14–17 Many factors potentially con tribute to this lack of consistency, including variations in study design (prospective versus retrospective), study methodology (follow-up duration and types of neuro psychiatric manifestations considered), and patient selection criteria (ethnic, demographic and clinical differences such as variations in disease duration and activity), as well as the rarity of some neuropsychiatric syndromes. A 3‑year prospective study that followed 370 patients with SLE with no previous history of CNS involvement found that when major CNS manifesta tions (strokes, seizures, myelopathy, aseptic meningi tis, chorea, demyelinating syndrome, psychosis, and acute confusional state) were included, and minor or nonspecific symptoms (headaches, subclinical cogni tive dysfunction, and mild depression) were discarded, the prevalence of CNS involvement was only 4.3% and the incidence was 7.8 events per 100 person-years.18 Neuropsychiatric manifestations are at least as common in children with SLE as they are in adults,19 but paediatric NPSLE is not c onsidered herein. Intense research effort—rarely focused specifically on NPSLE—has been devoted to genetic studies that aimed to identify determinants of disease susceptibility in patients with SLE. Although most investigations were underpowered to detect distinct genotype–phenotype relationships, some evidence suggests an increased abundance of genetic susceptibility factors in patients with NPSLE compared with patients who have lupus without neurological features.2 In particular, mutations in TREX1, which encodes three-prime repair exonucle ase 1 (also known as DNase III), have been identified in patients with NPSLE,20 and polymorphisms in this gene are associated with manifestations of CNS involvement, such as seizures.21 Interestingly, Trex1-deficient mice develop lethal autoimmunity and increased levels of type 1 interferon, which is relevant to SLE pathogenesis.22

NPSLE classification criteria

CNS involvement in SLE is considered primary when directly linked to disease activity and second ary when related to other causes, such as medications,

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Figure 1 | SLE-associated neurological features. MRI scans represent three main categories of severe CNS involvement: ischaemic stroke, epileptic seizures, and acute myelitis. a | 26-year-old woman with recently diagnosed SLE, who was positive for antiphospholipid antibodies and had acute ischaemic stroke. She presented with right hemiparesis, and MRI revealed an infarct in the hind limb of the left internal capsule (arrow). b | 17-year-old woman with known SLE and CNS vasculitis. She presented with grand mal epileptic seizures, ultimately leading to status epilepticus, and had high SLE disease activity. MRI revealed multiple bilateral white matter lesions and diffuse angiogenic oedema, suggesting acute vasculitis. c | 35-year-old woman with known SLE, acute myelitis, and positivity for neuromyelitis opticaassociated IgG autoantibodies, who presented with reduced muscle strength and sensibility in the right half of her body. MRI revealed a small intramedullary demyelinating lesion in the spinal cord (C6–C7 level; arrow). Abbreviation: SLE, systemic lupus erythematosus. Courtesy of Athanasios G. Tzioufas.

infections and metabolic abnormalities.23–25 Whether primary or secondary, CNS involvement has been rec ognized in patients with SLE for more than 100 years.26 Prior to 1999, several classifications had been proposed to describe the diverse clinical presentations of CNS involvement in SLE,27 but the terminologies used were a source of confusion, and discrepancies existed among recommended methods of evaluation. In 1999, however, The American College of Rheumatology (ACR) harmo nized case definition in NPSLE by describing 12 CNS syndromes and seven syndromes of the PNS that are compatible with the disease (Table 1),28 thus facilitating comparison of results between studies. The CNS symptoms, as defined by the ACR, are common in patients with NPSLE, accounting for around 93% of cases.29 These symptoms can be further catego rized as either focal neurological manifestations (cerebro vascular disease, seizures, myelopathy, aseptic meningitis, movement disorder, and demyelinating syndrome) or diffuse psychiatric/neuropsychological syndromes (cog nitive dysfunction, mood and anxiety disorders, psy chosis, acute confusional state, and headache), largely depending on the anatomical sites of the CNS pathology. PNS conditions comprise the remaining 7% of NPSLE cases, and include cranial neuropathy, polyneuropathy, mononeuropathy, acute inflammatory demyelinating polyradiculoneuropathy (Guillain–Barré syndrome), myasthenia gravis, plexopathy, and autonomic disorders (Table 1). PNS symptoms are beyond the scope of this article and, therefore, will not be discussed further.30

Neuropsychiatric manifestations of SLE

Focal neurological symptoms in SLE are often second ary to vascular events caused by antiphospholipid (aPL)

autoantibodies, including lupus anticoagulant, anticardio lipin and anti‑β2‑glycoprotein‑1 (β2GP1) antibodies.31 Such manifestations are typically acute in onset, refrac tory to therapy, and can be associated with structural abnormalities at autopsy. By contrast, the pathogenic mechanisms underlying diffuse CNS symptoms are less well defined. These manifestations are insidious, develop slowly over time and independently of disease activity, are often short-lived and reversible with therapy, and are not usually associated with structural pathology. The CNS symptoms that can be observed in patients with NPSLE are discussed in more detail in the sections that follow; these brief descriptions illustrate the complexity of NPSLE. In addition, we provide representative MRI images, which highlight neuroimaging findings associated with three major categories of these manifestations: cerebrovascular disease, epileptic seizures, and acute myelitis (Figure 1).

Cerebrovascular disease Cerebrovascular disease occurs commonly in the context of high disease activity and/or disease-related tissue injury, and can result in chronic cerebrovascular disease, stroke, transient ischaemic attack (Figure 1a), myelopathy, movement disorders, and seizures.30 In NPSLE, thrombo sis driven by aPL antibodies is the most accepted aetiol ogy for cerebrovascular disease, and robust risk factors include chronic and high disease activity, high cumulative corticosteroid dose, persistently positivity for aPL anti bodies at moderate-to-high titres, heart valve disease, and systemic hypertension.32,33 Seizures Seizures are a frequent neurological complication in SLE, occuring in 20–25% of patients (compared with 0.5–1.0% in the general population). They are often among the earliest manifestations of CNS involvement,34 and some times occur several years prior to other SLE-related symptoms, leading to erroneous diagnosis of such events as isolated epileptic seizures.35 Seizures are ususally of the primary generalized type, but partial episodes also occur (Figure 1b). Cerebral atrophy, a common finding in patients with SLE, might predispose to seizures.36 Cerebrospinal fluid (CSF) pleocytosis is common, sug gesting that a low-grade lupus-related encephalopathy may be a possible underlying process. Myelopathy Myelopathy in NPSLE presents as rapidly evolving transverse myelitis (Figure 1c). Ischaemic–thrombotic and inflammatory myelopathy might be observed, and patients can present with signs of grey matter dysfunc tion (flaccidity and hyporeflexia, for example) or white matter dysfunction (such as spasticity and hyper reflexia);37 white matter dysfunction is associated with neuromyelitis optica and positivity for aPL antibodies (lupus anticoagulant). Aseptic meningitis Acute, chronic or recurrent aseptic meningitis is a rare manifestation of SLE and requires confirmation by CSF



REVIEWS analysis, which should reveal increased lymphocyte and leukocyte counts. It usually occurs early in the course of the disease and may herald the onset of more-significant neurological phenomena, such as transverse myelitis, and can be complicated by ischaemic strokes.38 Aseptic meningitis may arise as a result of treatment with NSAIDs.

Movement disorders Movement disorders resulting from SLE can present as chorea (cogwheel rigidity, coarse tremors, lack of facial expression, or akinesia), and can also occur in juven ile SLE.39 Several case reports suggest the presence of circulating aPL antibodies in patients with SLE who exhibit movement disorders;40 however, these manifes tations are rare, accounting for less than 2% of NPSLE cases, and the infrequency of this syndrome precludes extensive research. Demyelinating syndrome Demyelinating syndrome can produce clinical fea tures and imaging characteristics that closely resem ble those seen in multiple sclerosis. This syndrome is rare, being reported in less than 4% of patients with NPSLE,41 and of unknown cause. It has been described as a combination of multiple sclerosis symptoms and SLE antibodies. Spastic paraplegia is the most common manifestation. Anticardiolipin and antimyelin antibodies may contribute to the pathogenesis of neural lesions in this syndrome.42

quality of life, and are probably associated, to some extent, with physical disability and perceived lack of control over the disease.48 Nevertheless, some patients with SLE might also present with ‘organic’ forms of depression caused by autoimmune lesions in the CNS. For example, anti- ribosomal P protein antibodies were demonstrated to be associated with both lupus psychosis and severe depres sion.49,50 Interestingly, in MRLlpr/lpr mice, which naturally develop an SLE-like disease, elevated titres of several types of autoantibodies—targeting nuclear antigens, cardiolipin, ribosomal P proteins, and the N‑methyl‑d‑aspartate (NMDA) receptor—closely correlated with depressive symptoms, which were often observed prior to the onset of involvement in other organs (for example, nephritis, arthritis, lymphadenopathy).51,52

Lupus psychosis Lupus psychosis is an uncommon SLE-associated event that is characterized by hallucinations and delusions. Patients who experience this rare complication gener ally present early in the course of the systemic disease,53 and most episodes resolve within 2–4 weeks.30 Psychosis is usually ascribed to damage mediated by dysimmun ity, although metabolic disturbances or medications may be involved. Schizophrenia and substance abuse must be ruled out.54 Although anti-ribosomal P protein antibodies have been associated with lupus psychosis in prospective studies,55,56 a meta-analysis reported that this biomarker had limited diagnostic accuracy.57

Cognitive dysfunction Mild-to-moderate cognitive dysfunction is common among patients with SLE; up to 80% are reported to be affected, depending on the cohort studied, the definitions of impairment, and the neuropsychological assessments used to measure cognition.14,43 Severe cognitive dysfunc tion develops in a considerably smaller proportion of patients, typically 3–5%.30 The cognitive domains affected most frequently are visual and verbal memory, attention, executive function, and psychomotor speed.44 Cognitive impairment can occur in the absence of either serological disease activity or other systemic disease manifestations, and fluctuates over the course of the disease indepen dently of depression or anxiety.45 Furthermore, symptoms can be compounded by several confounding influences related to systemic disease that affect cognition (such as fatigue and pain), by medications, and by other associated pathological states, such as aPL syndrome.46

Acute confusional state Acute confusional state has been reported in 4–7% of patients with SLE.58 This syndrome is characterized by acute-onset fluctuations in the level of consciousness, which can progress to coma, reduced ability to focus attention, mood disturbances, and impaired cognition. SLE-related acute confusional state must be differenti ated from other causes, which include certain infections and metabolic abnormalities.30

Mood and anxiety disorders Mood and anxiety disorders associated with SLE include major depressive-like episodes; mood disorders with depressive, manic or mixed features; prominent anxiety; panic disorders or attack obsession; and compulsion. They are common in patients with NPSLE, depression being the predominant psychiatric manifestation, with rates reach ing 54% of patients. These affective disorders, which might also contribute to the worsening of other neuropsychiatric symptoms,47 are difficult to separate from the stress of suf fering from a chronic disease and its detrimental effect on

Patient evaluation

Headache Headache is frequently reported by patients with SLE, but its association with the disease remains controver sial and a subject of debate.59–61 Thus, although head ache might be a component of SLE disease activity in individual patients, the majority of headaches occurring in patients with SLE are more likely to have an aetiology unrelated to the systemic disease. When evaluating patients with SLE who present with neuropsychiatric symptoms, careful assessment is neces sary to exclude other potential causes, and requires a rational holistic approach (Figure 2).24,62 Both anatomi cal and functional neuroimaging techniques (Table 2) are recommended for identifying CNS abnormalities (Figure 1).23,63,64 Neurophysiological studies are par ticularly valuable in the diagnosis of subsets of NPSLE syndromes. For example, an EEG can enable the identi fication of patients presenting with seizures who are at



REVIEWS Neuropsychiatric manifestations

Suspicion of NPSLE

Detailed medical history Timing of clinical events (neuropsychiatric and others) Review of medication use

Treatment of aggravating factors Infections, hypertension, metabolic disturbances

Exclusion of infections Leukocytes counts and CSF analysis Urine culture Exclusion of hormonal/metabolic dysfunctions Exclusion of other systemic diseases Exclusion of any medication-related adverse-events

Laboratory investigation for disease activity Analyses of urine sediment, complement, CSF, and erythrocyte sedimentation rate Immunoserological analysis Autoantibody (dsDNA antibodies, aPL antibodies, anti-ribosomal P protein antibodies) and proinflammatory cytokine profiling

Neurological and rheumatological clinical assessment

Neuroimaging investigation Structural imaging of the brain (CT, MRI, MTI, DTI, DWI) and functional brain imaging (fMRI, PET, SPECT, MRS, MRA) Electrophysiological investigation

Neuropsychological and psychiatric evaluation

Other

NPSLE

Figure 2 | Rational approach to evaluation and differential diagnosis of NPSLE. Patients presenting with neuropsychiatric symptoms suspected to be SLE-related require careful assessment to exclude other potential causes. First, one must determine—usually through a process of exclusion—whether the neuropsychiatric manifestations can be attributed convincingly to SLE. This approach requires detailed assessment of the medical history and thorough clinical evaluation. Laboratory analyses, including serological assays for immunological factors, are a key part of this process. Various circulating autoantibodies are associated with NPSLE; assessment of serum aPL antibody levels probably has the greatest diagnostic potential. Anatomical and functional brain imaging, electrophysiological studies, and objective cognitive assessment can also facilitate differential diagnosis and evaluation of the nature and extent of neuropsychiatric disease. Abbreviations: aPL, antiphospholipid; CSF, cerebrospinal fluid; dsDNA, double-stranded DNA; DTI, diffusion tensor imaging; DWI, diffusion-weighted imaging; fMRI, functional MRI; MRA, magnetic resonance angiography; MRS, magnetic resonance spectroscopy; MTI, magnetization transfer imaging; NPSLE, neuropsychiatric SLE; SLE, systemic lupus erythematosus; SPECT, single-photon emission CT.

high risk of recurrence of this symptom;30 this modal ity can also be used to exclude the presence of seizure disorder in patients who present with acute confusional state. In patients with PNS symptoms, diagnosis of plexo pathy, neuromuscular disorders, and different types of neuropathy (mononeuropathy versus polyneuropathy; demyelinating versus axonal) is facilitated by nerve con duction studies and electromyography. Finally, cognitive testing can help evaluate cerebral involvement, and is of particular importance considering that cognitive dysfunc tion is the most prevalent neuropsychiatric manifestation

in patients with SLE.63 More-detailed discussion of the evaluation of patients with SLE and suspected neuro logical involvement is beyond the scope of this article, and has recently been extensively reviewed.24,62

NPSLE pathogenesis Implications for biomarker identification Identification of targets for selective therapeutic strat egies is an obvious rationale for elucidation of the patho genetic mechanisms of SLE-associated neuropsychiatric disease. To this end, increased interest is being devoted to the identification of biomarkers that are integral to the pathogenesis of the disease and, therefore, correlate closely with disease activity and outcomes—as opposed to opportunistic, nonspecific markers of inflammation or injury. However, few robust biomarkers of NPSLE that fulfil this requirement have been validated to date. Thus, further delineation of the pathogenetic pathways underlying NPSLE is required to identify biomarkers of the disease that not only inform diagnosis, prognostica tion and treatment, but might also be important targets for therapy. The pathogenesis of NPSLE is particularly complex and the precise mechanisms remain elusive, in some cases provoking controversy.24,65 No single pathogenic pathway is likely to account for the variety of neuropsychiatric symptoms reported among patients with SLE. However, complex, interrelated mechanisms, including blood– brain barrier (BBB) dysfunction, vascular occlusion, neuroendocrine–immune imbalance, tissue and neuronal damage mediated by autoantibodies and proinflammatory cytokines (IL‑1, IL‑6, IL‑8, IL‑17, tumour necrosis factor [TNF], colony-stimulating and macrophage-stimulating factors), as well as direct neuronal cell death (see below) are undoubtedly implicated (Figure 3).15,43,66,67 Of the panel of autoantibodies that have been exam ined, those targeting phospholipids, ribosomal P pep tides, glial fibrillary acidic protein (GFAP), the NMDA receptor, microtubule-associated protein 2 (MAP‑2), and matrix metalloproteinase 9 (MMP‑9) have emerged as possible biomarkers (Table 3), as levels of these immunoglobulins have been shown to be increased in serum from patients with NPSLE;68–71 however, incon clusive data have been reported, notably in the case of antibodies directed at neuronal proteins and ribosomal P peptides.56 This uncertainty might be the result of the inherent complexity of NPSLE pathogenesis, which is reflected in variable disease activity and symptoms that wax and wane. Other potential biomarkers have been associated with NPSLE (Table 3), and might help clinicians to confirm or rule out SLE as the underlying cause of neuropsychiatric events (Figure 3). However, general SLE-related disease activity, previous or concurrent neuropsychiatric events, and persistent positivity for aPL antibodies at moderateto-high titres have been shown to be the most informa tive indicators of NPSLE.30 Elements of the proposed pathogenic mechanisms of NPSLE that are relevant to biomarker discovery in particular are described in more detail in the sections that follow.



REVIEWS Table 2 | Anatomical and functional neuroimaging techniques commonly used in evaluation of NPSLE Technique

Data examined

Most prevalent findings in NPSLE

Comments

CT166,167

Cross-sectional radiographic images of the brain

Cerebral atrophy; acute intracranial haemorrhage; oedema

Poor specificity for features of NPSLE

MRI25,100,136,168–172

Signals related to the properties of hydrogen nuclei (protons) within water and fats in biological tissue, including the brain

Focal lesions in subcortical white matter; cortical atrophy; diffuse cortical grey matter changes; reduced brain and corpus callosum volumes

Noninvasive; more sensitive than CT; currently considered as the imaging modality of choice for anatomical neuroimaging, but has poor sensitivity and specificity for features of NPSLE

Magnetization transfer imaging (MTI)173–177

Magnetization transfer between hydrogen nuclei in water with restricted and unrestricted motion (for example, between water molecules in white matter and CSF)

Abnormalities of the brain parenchyma, and atrophy (oedema, demyelination)

Quantitative MRI technique; more sensitive than standard MRI, and greater sensitivity in chronic NPSLE than in active or acute disease; enables quantification of structural damage

Diffusion tensor imaging (DTI)178–180

Signals indicative of restricted diffusion of water in tissue, enabling in vivo mapping of brain microstructure

Degradation of white matter fibre integrity due to microstructural pathology related to neuronal loss and mild hypoperfusion

Noninvasive MRI technique; enables visualization of early changes in normal-appearing grey and white matter

fMRI181–183

Changes in local brain deoxyhaemoglobin levels, reflecting neuronal activity and thereby providing an indirect measure of brain function

Activation in cerebral areas serving working memory, executive function, and attention

Noninvasive; analysis of brain activation patterns associated with specific cognitive tasks; enables study of the neuropathological basis for cognitive dysfunction

PET43,167,184–188

Brain glucose uptake (as a correlate of brain function) or oxygen consumption

Parieto-occipital hypometabolism revealing impending tissue loss and atrophy

Sensitive method for functional assessment of tissues; findings could potentially be predictive of considerable clinical deterioration; limited applicability; unsuitable for routine clinical use due to considerable expense, large dose of radiation and requirement for a cyclotron to produce the relevant isotopes

Single-photon emission CT (SPECT)184,188–191

Regional cerebral blood flow and metabolism

Widespread small areas of hypoperfusion in parietal, frontal and temporal lobes

Functional imaging method that is more sensitive than fMRI; surrogate measure of tissue integrity; practical limitations prevent routine clinical use

Magnetic resonance spectroscopy (MRS)126,175,192–196

Levels of metabolites such as NAA (indicative of neuronal and axonal density), choline (representative of myelin content and reflects membrane turnover) and creatine (reference metabolite); data are expressed either as ratios or absolute concentrations

Neuronal/axonal damage and demyelination indicated by reduced levels of NAA or decreased NAA:creatine ratio; inflammation and disease activity reflected by elevated levels of choline; these findings are often evident even in white and grey matter that appear normal on conventional MRI

More sensitive than MRI; reflects the integrity of neuronal cells

Abbreviations: CSF, cerebrospinal fluid; fMRI, functional MRI; NAA, N‑acetylaspartate; NPSLE, neuropsychiatric systemic lupus erythematosus.

Blood–brain barrier dysfunction Normally, the brain is immunologically privileged and is sheltered from substances in the circulation by the BBB. This specialized vascular system limits the entry of soluble molecules and cells into the brain parenchyma, and incorporates specific transport mechanisms to regu late the uptake and efflux of substances into and out of the brain.72 Evidence suggests that a certain degree of BBB disruption or leakage, such that immunoglobulins and other large molecules, and immune cells can gain access to brain tissue, is required to permit immune responses in the brain. Indeed, evidence for BBB disruption in neurodegenerative diseases,73 as well as in SLE-related neuropathology,72,74 is accumulating. However, the mecha nism by which self-reactive autoantibodies pass through the BBB to affect neurological functions remains an issue that is poorly understood.75,76

Analyses of CSF from patients with NPSLE have repeatedly demonstrated increased levels of immuno globulins in general (as well as specific types of anti body), proinflammatory cytokines, and albumin, indicative of increased BBB permeability.77–79 Similarly, in BXSB and MRLlpr/lpr lupus-prone mice, an increased IgG index and elevated albumin concentrations have been reported in CSF,80–82 and these CSF markers increase concomitantly with neurodegeneration in periventricu lar areas and correlate positively with disease activity.83 Several experimental findings, such as IgG infiltration into the brain parenchyma, might account for a definite loss of BBB integrity observed in the MRLlpr/lpr model. In line with this observation, it has also been suggested that the complement system (in particular, C5a/C5aR) has a key role in disruption of BBB integrity through differ ent cascades of events leading to increased generation of



REVIEWS

Genetic factors (Mutation in TREX1)

Neuroendocrine factors Hormonal influence (sex hormones as modulators of immune system, antibody production) Activation of the HPA axis (by cytokines and via CRH) Synthesis of glucocorticoids and other neuropeptides (association with hippocampal atrophy, impaired memory and learning)

Environmental factors (Infection, UV light, stress, smoking)

Vascular mechanisms (occlusion, microangiopathy and haemorrhage) Antiphospholipid antibodies (lupus anticoagulant, anticardiolipin, anti-β2GP1 antibodies) Immune complexes

Neuroinflammatory mechanisms BBB dysfunction Cytokines/chemokines (potentially produced by astrocytes and microglia) Autoantibodies (anti-ribosomal P protein, anti-NR2 antibodies)

Pathological processes Widespread microvascular infarcts Ischaemic and thrombotic brain infarction and microhaemorrhage Cortical atrophy Perivascular lymphocytic infiltrates Endothelial cell proliferation, promoting the clotting cascade Production of inflammatory and vasoconstrictive mediators True vasculitis (rare)

Pathological processes Direct interaction of inflammatory mediators with, and subsequent activation of, endothelial cells in the brain Complement activation Direct CNS tissue injury (by excitatory amino acid toxicity, oxidative stress, and PAI-1 or MMP activity) Activation of microglial cells Neuronal cell death by apoptosis

Neuropsychiatric manifestations of SLE

Focal neuropsychiatric manifestations Acute onset Can be associated to structural abnormalities at autopsy Examples include: cerebrovascular disease, seizures, myelopathy, aseptic meningitis, movement disorders, demyelinating syndrome

Diffuse neuropsychiatric manifestations More insidious than focal lesions; develop slowly and independently of disease activity, without associated structural pathology Examples include: cognitive dysfunction, mood and anxiety disorders, psychosis, acute confusional state

Figure 3 | Potential pathogenic mechanisms in NPSLE. Factors implicated in, and relative contribution to, the disease might vary between individuals, by stage of development of the autoimmune process, and by pathoanatomical localization. Genetic, hormonal and environmental factors probably all contribute to immune dysfunction. Vascular mechanisms contribute to pathogenesis of neuropsychiatric disorders classically distinguished as ‘focal’. Vascular syndromes involve autoantibodies and cytokines, and therapeutic approaches might involve depletion or inactivation of autoantibodies, interference with the clotting cascade, or inhibition of endothelial cell activation. In neuroinflammatory syndromes, BBB disruption, possibly resulting from infection or hypertension, might permit entry of inflammatory mediators into the CNS. These inflammatory mediators contribute to the pathogenesis of the classic ‘diffuse’ disorders; therapeutic approaches should focus on protecting BBB integrity and inactivating inflammatory initiators before they gain access to vulnerable CNS components. Abbreviations: β2GP1, β2-glycoprotein 1; BBB, blood–brain barrier; CRH, corticotropin-releasing hormone; HPA, hypothalamic– pituitary–adrenal; MMP, matrix metalloproteinase; NPSLE, neuropsychiatric systemic lupus erythematosus; NR2, N-methyl-d-aspartate receptor subtype 2; PAI‑1, plasminogen activator inhibitor 1; UV, ultraviolet.

inducible nitric oxide (NO) synthase (iNOS) and reactive oxygen species, and actin reorganization. These results indicate that inhibition of C5aR is a potential treatment strategy for SLE.84 The process leading to neurological dysfunction in SLE probably involves abnormal endothelial cell– immune cell interactions that permit access of immune cells and, therefore, immune mediators to the CNS. This mechanism might be mediated by proinflammatory cytokines or autoantibodies that upregulate the expres sion of adhesion proteins, such as intercellular adhesion

molecule 1 (ICAM‑1) and vascular cell adhesion mol ecule 1 (VCAM‑1), thus facilitating leukocyte entry into the CNS.85 In turn, immune-mediated injury to the brain tissues might lead to neurological disease. Although these findings were mostly reported in MRLlpr/lpr lupusprone mice, they are consistent with clinical evidence obtained in patients with SLE, in whom infiltration of autoantibodies and immune cells into the brain has been observed.86 Serum levels of soluble ICAM‑1 have been shown to correlate with disease activity and to normalize with remission,85,87,88 thus strengthening the hypothesis that endothelial cell activation and subsequent compro mise of BBB integrity might be an essential r equisite for SLE-related disease activity in the brain. Importantly, clinical evidence does not unequivocally support the relationship between serum expression of brain-reactive antibodies and neuropsychiatric mani festations,89–91 raising the question of whether autoanti bodies to brain tissues are also produced intrathecally in NPSLE. Of note, an elevated CSF IgG index (CSF IgG:serum IgG ratio) has been attributed to increased intrathecal antibody synthesis. CSF levels of autoantibod ies have been shown to correlate more closely with psy chiatric manifestations and brain injury in patients with NPSLE than do serum autoantibody levels;92–96 the occur rence of autoantibodies in the CSF might be due to passive transfer from the circulation through a disrupted BBB, or to intrathecal production. Similarly, in MRLlpr/lpr mice, high levels of brain-reactive antibodies in the CSF correlated positively with increased immobility in the forced-swim test, and CSF IgG antibodies bound directly to brain regions implicated in neurogenesis, memory formation and emotional reactivity.97 However, further investigation is required—ideally using affinity-purified antibodies that recognize specific antigens—to define the possible direct pathological effects resulting from the binding of these antibodies to brain tissues; the poly clonal immunoglobulin species evaluated in the studies probably target diverse epitopes, preventing delineation of their role in NPSLE pathogenesis. Further confir mation that some of these CSF antibodies are indeed produced intrathecally, rather than being derived from serum, would also contribute in a decisive way to our understanding of the pathophysiological events associated with this specific autoimmunity.

Cerebrovascular mechanisms Autopsy studies have suggested that vasculopathy is involved in CNS damage in patients with NPSLE; multi focal microinfarcts, small-vessel noninflammatory vas culopathy and occlusion, intranial embolism, cortical atrophy, and microhaemorrhages were the most common pathological findings.67,98 aPL antibodies (see below) are likely to be associated with these conditions—principally with focal manifestations of NPSLE (seizures, strokes)— as are complement proteins.99,100 Microvasculopathy was initially attributed to deposition of immune complexes but is now suspected to arise from complement activa tion.101 In the brain, microhaemorrhages, infarcts and focal injury seem to be caused by impaired blood flow



REVIEWS Table 3 | Potential biomarkers associated with NPSLE* Biomarker

Location

Prevalence in patients with (NP)SLE

Chemokine receptors and cell types

Association with disease activity or phenotype

Level of evidence/strength of associations

Phospholipids (β2-glycoprotein 1 and cardiolipin)63,66,71,197

Serum and CSF

~45%

NA

Focal neuropsychiatric diseases: cerebrovascular disease, seizures, chorea Diffuse neuropsychiatric diseases: cognitive dysfunction, psychosis, depression, headache

Controversial However, these antibodies seem to be the laboratory parameter most frequently relied on in the diagnosis of NPSLE and in decisions regarding treatment

Ribosomal P protein49,52,55–57,66,71,95,113

Serum and CSF

6–46%

NA

Elevated titres detected during active SLE; may be particularly associated with episodes of psychosis and severe depression

Controversial Measurements in CSF may be more accurate for diagnosis than measurements in serum Not helpful in differentiating among various disease phenotypes

NMDA receptors, specifically NMDA receptor subtype 2 (NR2; also known as GluN2)66,71,92,93,116,119,124,126,198,199

Serum and CSF

30–40%

NA

Diffuse neuropsychiatric manifestations such as depression, cognitive dysfunction

Controversial

Microtubule-associated protein 2 (MAP‑2)130

Serum

17%

NA

Seizures, chorea, psychosis, headache, sensory neuropathy

Strong association (76.5%)

U1 ribonucleoprotein (U1RNP)79,200

Serum and CSF

NR

NA

NPSLE in general

64.3% sensitivity and 92.9% specificity for central NPSLE

Glial fibrillary acidic protein (GFAP)71,201

Serum

NR

NA

Neuropsychiatric manifestations of organic/major type

Significant positive association with neuropsychiatric manifestations

IL‑170

CSF

NR

NA

NPSLE in general

Elevated levels

IL‑293,147

CSF

NR

NA

NPSLE in general

Controversial

70,142,143,146–148

IL‑6

CSF

NR

NA

NPSLE in general

Consistently high levels Among reported cytokines, IL‑6 has been shown to have the strongest positive association with NPSLE; significant correlation with levels of neuronal degradation product (neurofilament)

IL‑870,142,143,146,147

CSF

NR

NA

NPSLE in general

Consistently high levels

IL‑1070,147,156

Serum and CSF

NR

NA

NPSLE in general

Consistently high levels; correlation with disease activity

IFN‑α70,202

Serum and CSF

NR

NA

NPSLE in general


IFN‑γ70,147,156

Serum and CSF

NR

NA

NPSLE in general


TNF70,147,156

Serum and CSF

NR

NA

NPSLE in general

Controversial

TNF-related weak inducer of apoptosis (TWEAK; also known as TNF ligand superfamily member 12A)76,150

CSF

NR

NA

Profound depression-like behaviour, cognitive impairment, anhedonia

Association with decreased brain expression of RANTES

IL‑8 (also known as CXC chemokine ligand 8 [CXCL8])70,147,149,202

CSF

NR

CXCR1 on monocytes and neutrophils

NPSLE in general

Increased levels

Monocyte chemotactic protein 1 (MCP‑1; also known as CC chemokine ligand 2 [CCL2])70,147,200,202

CSF

NR

CCR2 on memory T cells, monocytes and immature dendritic cells

NPSLE in general

Increased levels

Autoantibodies (by target)

Cytokines

Chemokines



REVIEWS Table 3 (cont.) | Potential biomarkers associated with NPSLE* Biomarker

Location

Prevalence in patients with (NP)SLE

Chemokine receptors and cell types

Association with disease activity or phenotype

Level of evidence/strength of associations

IFN‑γ-inducible protein‑10 (IP‑10; also known as CXC chemokine ligand 10 [CXCL10])70,147,202

CSF

NR

CXCR3 on TH1, mast and mesangial cells

NPSLE in general

Increased levels

RANTES (also known as CC chemokine ligand 5 [CCL5])70,147,149

CSF

NR

CCR1 on T cells, monocytes, eosinophils and basophils CCR3 on TH2 cells, mast cells, eosinophils and basophils

NPSLE in general

Increased levels

Fractalkine (also known as CX3C chemokine ligand 1 [CX3CL1])70

CSF

NR

CX3CR1d on macrophages and endothelial cells

NPSLE in general

Controversial

Plasminogen activator inhibitor 1 (PAI‑1)143

CSF

NR

NA

NPSLE in general

Strong association with CSF markers of inflammation (IL‑6 and IL‑8) and neuronal and astrocytic damage (GFAP)

Matrix metalloproteinase‑9 (MMP9)141,142

Serum and CSF

NR

NA

NPSLE in general, and particularly cognitive impairment

Significant association with CSF markers of inflammation and MRI findings

Others

*A biomarker is a measurable element that enables diagnosis of a disease or indicates disease severity (disease state). The list of markers presented here, which is not exhaustive, includes potential biomarkers of NPSLE as well as molecular elements that have been linked to NPSLE pathogenesis but are not exclusively found in this form of SLE. As indicated, in some cases contradictory reports failed to confirm any association with NPSLE. Abbreviations: CSF, cerebrospinal fluid; NMDA, N‑methyl‑d-aspartate; NPSLE, neuropsychiatric SLE; NA, not applicable; NR, not reported; SLE, systemic lupus erythematosus; TH, T helper; TNF, tumour necrosis factor.

secondary to thrombi or emboli.102,103 However, micro infarcts are unlikely to account for all the functional impairments seen in NPSLE.

Serum and CSF autoantibody-mediated effects Appreciation of the neuroactivatory and/or neurotoxic role and biochemical identity of brain-reactive auto antibodies in the pathogenesis of various psychiatric syndromes is increasing. 104–106 Indeed, various auto antibodies have been implicated in NPSLE manifesta tions (Table 3); however, as mentioned previously, the clinical relevance of a large proportion of these bio markers has not been consistently demonstrated across studies. Furthermore, no autoantibody species targeting any brain-specific or systemic antigen has been linked to a particular neuropsychiatric manifestation of SLE. The autoantibodies potentially associated with NPSLE have been extensively reviewed elsewhere.66,71,105 Antiphospholipid antibodies The aPL antibody family targets proteins that interact with anionic phospholipids present in the plasma mem brane and regulate the blood clotting cascade; thereby, aPL antibodies alter the activation of procoagulants, promoting thrombosis and cerebral infarcts.107 aPL anti bodies, particularly lupus anticoagulant, anticardiolipin and anti‑β2GP1 antibodies, are by far the most widely investigated autoantibodies in NPSLE.108,109 Owing to their diverse effects on platelets, coagulation proteins and endothelial cells, certain subtypes of aPL antibodies have been considered not only as serological biomark ers, but also as potential direct contributors to the devel opment of thrombosis and other NPSLE manifestations.

There is abundant research on the association of these antibodies with both focal and diffuse neurological manifestations in NPSLE,23,110,111 and they seem to be closely associated with focal cerebrov ascular symp toms.24 However, the results of studies attempting to correlate these antibodies with cognitive impairment— a diffuse neuropsychiatric symptom—are inconsist ent; importantly, aPL antibodies are not present in all patients who experience cognitive decline 38,112 and, therefore, their association with c ognitive dysfunction remains to be clearly established. Anti-ribosomal P protein antibodies Anti-ribosomal P protein antibodies occur in up to 46% of patients with SLE.113 These antibodies target epitopes located in the C‑terminal end of three highly conserved phosphorylated proteins, P0, P1 and P2, which are present in the 60S subunit of ribosomes.114 A possible clinical association between elevated serum or CSF titres of anti-ribosomal P protein antibodies and lupus psychosis remains a matter of debate.49,56,95 The prevailing conclusion at present is that these antibodies display limited diagnostic value in NPSLE and, accord ing to the results of an international meta-analysis of 14 studies,57 are not helpful for differentiating various disease phenotypes (such as psychosis, mood disorder, and other diffuse or focal manifestations). Nevertheless, in a study in healthy mice, anti-ribosomal P protein antibodies were shown to recognize neurons in the hippocampus, cingulate and primary olfactory piriform cortex in immunostaining experiments, and induced a long-term increase in depressive-like behaviour when administered intracerebroventricularly.52



REVIEWS

Frequency of autoantibody positivity (%)

100

80

*

**

PARP-1

PARP F2

60

40

20

0 dsDNA

Chromatin

HSP70

Ubiquitin

Autoantigen Patients Patients Patients Patients

with neuropsychiatric symptoms (n = 19) with renal symptoms (n = 32) with neuropsychiatric and renal symptoms (n = 17) without renal or neuropsychiatric symptoms (n = 17)

Figure 4 | Immune response to molecules related to cell stress and repair in NPSLE. Graph presents data on frequency of seropositivity for IgG autoantibodies targeting various factors associated with cell stress and repair in patients with and without neuropsychiatric and/or renal features of SLE.130 Antibodies targeting PARP‑1 and, in particular, a PARP‑1-derived peptide (PARP F2) were markedly more common in patients without neuropsychological and renal symptoms; the difference was statistically significant between this group and patients with neuropsychiatric manifestations (*P = 0.01529; **P = 0.0036). One interpretation of these findings is that autoantibodies targeting PARP‑1 are decreased in patients with neuropsychiatric symptoms, as they are sequestered from the serum by widespread engagement of PARP‑1 and deposition in tissues where this protein is expressed, such as the brain. This scenario implicates anti-PARP‑1 antibodies in NPSLE pathogenesis. Abbreviations: dsDNA, double-stranded DNA; HSP70, heat shock 70 kDa protein; NPSLE, neuropsychiatric SLE; PARP‑1, poly (ADP-ribose) polymerase 1; SLE, systemic lupus erythematosus.

The neuropathogenic potential of anti-ribosomal P protein antibodies was also demonstrated in an inde pendent study, which showed that these antibodies, derived from patients with SLE, induced a rapid and sustained increase in calcium influx and subsequent apoptosis in rat neurons that expressed a cell-surface protein termed p331; thus, p331 was designated as a new neuronal surface P‑antigen.115 Death of these neurons, which were found in specific cerebral regions involved in memory, emotional behaviour and other higher brain functions in rats, could potentially account for a broad range of deficits observed in patients with NPSLE. Anti-glutamate receptor antibodies Anti-glutamate receptor antibodies have been described as anti-double-stranded-DNA (dsDNA) antibodies that cross-react with NMDA receptors.116–119 NMDA recep tors are widely distributed throughout the brain, local ized within glutamatergic synapses, with a particularly high density observed in the amygdala and hippo campus;120 of note, these two cerebral subregions are implicated in cognitive functions such as emotional pro cesses and memory, respectively.121,122 The pathogenicity of CSF IgG antibodies to the NMDA receptor subunit 2 (NR2; also known as GluN2) has been demonstrated in independent studies, both in vitro and in vivo.116,118,123,124 Interestingly, no neuronal damage was observed if

the BBB remained intact, whereas several pathologi cal alterations were detectable when the BBB was dis rupted. 118,123,124 Although these data provide strong evidence of a relationship between anti‑NR2/dsDNA antibodies and NPSLE, this association remains a matter of controversy. Numerous studies91,119,125,126 have attempted to correlate seropositivity for anti‑NR2/dsDNA antibodies—which occurs in 30–40% of patients with SLE117—with clinical aspects of NPSLE, yet robust evidence of such an associ ation has not been presented. On the other hand, clinical studies that explored the presence of anti‑NR2/dsDNA antibodies in the CSF demonstrated a correlation with diffuse CNS manifestations of NPSLE.92,96,127 In contrast to other autoantibody types, such as anti-ribosomal P protein and anticardiolipin antibodies, anti‑NR2/dsDNA antibodies also distinguish patients with SLE and central, diffuse CNS manifestations from patients with PNS involvement or no neuropsychiatric manifestations.93 These data emphasize the need to consider CNS and PNS manifestations—and potentially also diffuse and focal CNS symptoms—separately when studying NPSLE, as the pathogenic factors and mechanisms underlying these disease phenotypes are probably distinct. A remaining question is how do anti‑NR2/dsDNA antibodies gain access to the brain in patients with SLE: by direct intrathecal antibody production or through increased permeability of the BBB?128 Of note, endothelial cells in the brain express NMDA receptors, and binding of glutamate to these receptors has been shown to result in loss of BBB integrity.129 Therefore, antib odies that recognize NR2 might promote their own transport into the brain by acting as agonists or co-agonists for NMDA receptors.75 Anti-NR2/dsDNA antibodies from patients with SLE have also been shown to bind to human umbili cal vein endothelial cells and upregulate endot helial expression of ICAM‑1 and VCAM‑1, as well as the pro duction of IL‑6 and IL‑8.127 Activation of BBB endothelial cells by anti-NR2/dsDNA antibodies in the same manner might cause inflammation, BBB disruption, and entry of antibodies into the CSF in patients with SLE. Other autoantibodies Among the autoantibodies that have been closely associ ated with NPSLE, antibodies targeting MAP‑2, a cellu lar protein essential to cytoskeletal integrity and found almost exclusively in neurons, demand further consider ation.130 Furthermore, anti‑U1RNP antibodies might represent a clinically important CSF biomarker that is even more specific for NPSLE than anti-ribosomal P protein or anti-NR2/dsDNA antibodies.79 Data obtained in our laboratory indicate that positiv ity for IgG antibodies that recognize poly (ADP‑ribose) polymerase 1 (PARP‑1) is significantly less frequently observed in patients with NPSLE than in patients with SLE who have no renal or neurological features (P = 0.01529; Figure 4). The results observed with recombinant full-length PARP‑1 protein were confirmed using PARP F2, a peptide encompassing only the second zinc finger F2 region of the protein, which harbours a



REVIEWS dominant antigenic epitope of PARP‑1.131,132 Even if these findings do not directly help in the ongoing quest to identify NPSLE biomarkers, they open up new avenues of investigation regarding the possible pathophysiologi cal mechanisms operating in NPSLE—in particular, the role of autoimmune reactivity to PARP‑1 and the pos sible influence of PARP‑1 autoantibodies on repair of single-stranded DNA breaks, a process in which PARP‑1 is implicated. Strikingly, Parp1‑knockout mice exhibit behavioural alterations affecting food entrainment of peripheral cir cadian clocks,133 as well as long-term (but not short-term) memory formation.134 Indeed, PARP‑1 has been found to be highly expressed in the hippocampus of adult mice,135 a compartment that is the prominent target of cortico steroids in the brain, and can be atrophic in patients with NPSLE and in lupus-prone mice. 136,137 Bearing these findings in mind, the fact that the frequency of anti-PARP‑1 autoantibody seropositivity was reduced in patients with NPSLE might indicate that the auto antibodies have gained access to their target throughout the body, including the brain (considering that the BBB is altered in NPSLE), and are deposited in and largely confined to immune complexes. Interestingly, a similar effect has been demonstrated for certain antibodies associated with kidney pathology in patients with SLE who develop typical lupus glomerulonephritis, with high levels of anti-dsDNA antibodies being observed prior to kidney involvement, but decreasing sharply at onset of serious renal manifestations.138 Identification and fine characterization of autoanti bodies that are relevant to the pathogenesis of NPSLE remains an area of active investigation and a subject of intense debate. Many of the observations reported here, though clinically important, relate to associ ations only,68,139,140 rather than confirmed mechanistic involvement of particular autoantibodies in NPSLE pathogenesis. Numerous questions remain unanswered regarding the origin and pharmacokinetics of such anti bodies, their capacity to target precise brain epitopes, and the molecular basis of their proposed pathogenic prop erties. Obtaining information to answer these questions is, therefore, central to the development of improved strategies for effective treatment of NPSLE.

Potential non-antibody biomarkers of NPSLE Other possible intrathecal markers of NPSLE include plasminogen activator inhibitor 1 (PAI‑1) and MMP‑9 (Table 3). The primary function of MMP‑9 is to enhance T‑cell migration through connective tissues. Two independent studies reported that levels of MMP‑9 in serum141 and in CSF142 were markedly higher in patients with NPSLE than in patients without neuropsychiat ric symptoms. Furthermore, MMP‑9 levels correlated positively with both the volume of MRI-detected brain lesion141,142 and intrathecal levels of the proinflamma tory cytokines IL‑6 and IL‑8.142 Similarly, intrathecal levels of PAI‑1 were substantially increased in patients with NPSLE compared with SLE patients who did not exhibit overt neuropsychiatric involvement. These levels

also correlated with CSF levels of IL‑6 and IL‑8, and were associated with the presence of neuronal damage markers and pathologically elevated GFAP levels in CSF.143

Cytokine and chemokine effects Cytokines and chemokines are known to promote intra thecal antibody production, recruit immune cells and modulate neurotransmitter release.70,144 In SLE, these factors modify the homeostatic immune regulatory mechanisms and participate in local inflammation pro cesses that ultimately lead to tissue destruction and, in the context of BBB disruption, influence the hypothalamus.67 Levels of a variety of cytokines can be elevated in the serum and CSF of patients with NPSLE, and levels of certain cytokines, such as IL‑6 and IL‑10, have been shown to cor relate with disease activity, assessed using various meas ures including anti-dsDNA antibody levels and the SLE disease activity index (SLEDAI) score.145 Some cytokines have also been associated with specific clinical manifesta tions and MRI findings; for example, CSF levels of IL‑6 and IL‑8 were elevated in patients with NPSLE, compared with SLE patients without CNS involvement.146 On the basis of the reported data on cytokines, CSF IL‑6 seems to have the most robust positive association with NPSLE, and levels of this cytokine are increased147,148 even in the absence of BBB alteration.94 Thus, the CSF level of IL‑6, and possibly also IL‑8, might represent a biomarker that holds promise in facilitating the diagnosis of NPSLE.146,149 Studies in both patients with SLE and lupus-prone mice have pointed to a possible role for TNF-related weak inducer of apoptosis (TWEAK) in NPSLE patho genesis.76,150 Through activation of its cognate recep tor fibroblast growth factor-inducible immediate-early response protein 14 (Fn14; also known as TNF recep tor superfamily member 12A), TWEAK triggers the assembly of receptor-associated signalling complex, which in turn mediates the activation of cellular prolif eration, angiogenesis, inflammation and apoptosis.151 Increased cerebral expression of TWEAK and Fn14 was observed in wild-type MRLlpr/lpr mice, as compared with MRLlpr/lpr mice deficient for Fn14.150 Behaviourally, wildtype MRLlpr/lpr mice displayed profound depression-like behaviour, impaired cognitive function and anhedonia consistent with NPSLE symptoms in this lupus-prone strain, and these neuropsychiatric manifestations were ameliorated in Fn14-deficient mice.150 No statistically significant differences were found between wild-type and Fn14-deficient MRLlpr/lpr mice with regard to serum levels of autoantibodies associated with neuropsychi atric disease (anticardiolipin, anti-ribosomal P protein and anti-NR2/dsDNA antibodies);150 however, MRLlpr/lpr mice lacking Fn14, which had greater BBB integrity than wild-type mice of the same strain, showed markedly decreased brain expression of CC chemokine ligand 5 (CCL5; also known as RANTES) and other proinflam matory mediators.150 The mechanism by which Fn14 deficiency ameliorates neuropsychiatric disease might, therefore, involve decreased cerebral expression of CCL5 and other proinflammatory substances. Together, these results indicate a role for the TWEAK–Fn14 pathway in



REVIEWS the pathogenesis of NPSLE, and suggest not only that TWEAK might represent an informative biomarker in NPSLE, but also that this ligand–receptor pair could represent a potential therapeutic target in NPSLE.151 In patients with SLE, CSF levels of B‑cell-activating factor (BAFF; also known as TNF ligand superfamily member 13B)—another cytokine that, like TWEAK, belongs to the TNF ligand family—were shown to be increased more than 200-fold compared with control samples from healthy individuals. 152 CSF levels of a related cytokine that is also essential for B‑cell survival and function, a proliferation-inducing ligand (APRIL; known as TNF ligand superfamily member 13), were also elevated 20-fold in patients with SLE.152 APRIL levels—but not BAFF levels—were further augmented in the CSF of patients with NPSLE compared with SLE patients without CNS involvement. CSF levels of BAFF and APRIL did not correlate with serum levels of these cytokines,152 and independent studies concluded that serum levels of BAFF and APRIL did not correlate with disease activity.152,153 No information seems to be avail able regarding CSF levels of BAFF and APRIL in relation to disease activity. Nevertheless, BAFF and/or APRIL might be important targets for therapy in NPSLE. Indeed, atacicept, a human recombinant fusion protein that com prises the ligand-binding portion of TACI, the receptor for both BAFF and APRIL, has already entered clinical trials in various diseases, including SLE. However, after the failure of a randomized, placebo-controlled, doubleblind phase II trial of this agent in multiple sclerosis, further studies are needed before evaluating strategies targeting BAFF and APRIL in NPSLE. Several cytokines and chemokines also seem to be upregulated during active disease and downregulated after treatment in patients with NPSLE. 154 Further investigation of the role of these immune modulators in the pathogenesis of NPSLE, and their biomarker and therapeutic potential, are required.

Direct neuronal cell injury and death Stress factors, including those associated with inflam mation, can directly affect neural tissues. Production of NO in the brain, which typically occurs at low levels under the control of neuronal NO synthase, substantially increases during stress via iNOS. iNOS expression is con trolled by inflammatory cytokines and can also be upreg ulated by complement components, immune complexes and aPL antibodies.67 Some cytokines are potent indu cers of iNOS in cerebral cell types (such as human astro cytes),155 and might, therefore, trigger the production of toxic NO metabolites within the CNS. This pathway has been reported to be active in patients with NPSLE;156 patients with NPSLE had high numbers of peripheral blood cells expressing TNF mRNA, which closely cor related with increased levels of NO metabolites in the CSF, and with disease severity.156 These findings suggest that peripheral TNF production leads to NO produc tion in the CNS and the development of clinical NPSLE. Importantly, when produced in large quantities, NO provokes neuronal cell death by various mechanisms,

in particular, direct nitrosylation of NMDA receptors.67 Levels of MMP‑9, which is ubiquitously secreted by immune cells and endothelial cells, are elevated in patients with NPSLE as compared with SLE patients without CNS involvement. Furthermore, the MMP-9 level correlates with SLE-related injury and lesion volume, as measured by brain MRI;141 thus, this proteo lytic enzyme might also mediate direct neuronal damage in NPSLE. Further elucidation of these pathways might clarify the potential of such NPSLE-associated factors as informative biomarkers and therapeutic targets.

Treatment of patients with NPSLE

Recommendations for the management of NPSLE were published in 2010 by the European League Against Rheumatism (EULAR).30 Since then, several comprehen sive reviews on this subject have been published,15,23,24,140 which provide numerous details on the management of NPSLE that are beyond the scope of this Review. The EULAR recommendations indicate that it is imperative to quickly identify and manage any aggravating factors for neuropsychiatric symptoms, including metabolic abnormalities, as well as predisposing medications such as corticosteroids,30 which can have considerable CNS toxicity. In addition, provision of symptomatic treatment for the NPSLE manifestations is essential.30 To date, the treatment of NPSLE remains relatively empirical, and is characterized by a multifocal approach (Figure 2) that should be tailored to address the presenting symptoms in individual patients. Medications that can be used in the management of NPSLE include NSAIDs for symptomatic relief, anticoagulants for thrombotic disease, and immuno suppressive agents (corticosteroids, cyclophosphamide, azathioprine, methotrexate and mycophenolate mofetil) for severe inflammation (Box 1). B‑cell-depletion thera pies have been shown to have efficacy in the treatment of NPSLE symptoms, including in the setting of disease that is refractory to conventional immunosuppression. Although the data come from uncontrolled studies, the results are encouraging, with over 80% of patients with NPSLE that is refractory to conventional treatments demonstrating at least a partial clinical response.157 Various other treatment modalities that have been applied in patients with drug-refractory NPSLE include plasmapheresis, intrathecal administration of metho trexate, dexamethasone, intravenous immunoglobulin, and haematopoietic stem cell transplantation. Mild forms of NPSLE might only necessitate symptomatic treatment; for example, adjunctive symptomatic treat ment can complement conventional corticosteroid and immunosuppressive therapies by targeting mood disorders (which generally improve after antidepres sant therapy with selective serotonin reuptake inhibi tors such as fluoxetine), psychosis (with antipsychotic agents), cognitive impairment (using cognitive rehab ilitation programmes and psychological group inter vention), seizures (with anticonvulsants), and headaches (with analgesics). As these treatments are all associated



REVIEWS Box 1 | Treatment approaches in NPSLE After diagnosis of NPSLE and treatment of any aggravating factors, such as infections, metabolic abnormalities and hypertension, the patient can be provided with therapies targeting the neuropsychiatric manifestions of the disease and/or the underlying systemic disease.15,30,58,140,203 Approaches to the management of patients with NPSLE are detailed below.*

Symptomatic treatment of neuropsychiatric manifestations ■■ NSAIDs for symptomatic relief ■■ Cerebrovascular disease: attempt to control cardiovascular risk factors (lifestyle changes including sleep hygiene, encouragement to quit smoking, relaxation, regular exercise programmes); anticoagulants in patients with APS; corticosteroids (prednisone, methylprednisolone) and/or immunosuppressants for generalized SLE disease activity ■■ Seizures: anticonvulsants, antiepileptic agents; aspirin or other anticoagulants in patients with APS; corticosteroids and/or immunosuppressants for generalized SLE disease activity ■■ Myelopathy: corticosteroids and/or immunosuppressants ■■ Movement disorder: corticosteroids; aspirin or anticoagulants in patients with APS ■■ Cognitive dysfunction: nonpharmacological approaches (cognitive rehabilitation programmes, psychological group intervention); control disease activity, for example, using corticosteroids; aspirin or anticoagulants in patients with APS ■■ Depression: anxiolytic agents; antidepressants ■■ Psychosis: exclude secondary causes before considering antipsychotic agents; corticosteroids and/or immunosuppressants to control generalized SLEassociated disease activity ■■ Acute confusional state: corticosteroids and/or immunosuppressants ■■ Headache: aspirin or anticoagulants in patients with APS; corticosteroids (in refractory or severe cases) Immunosuppression ■■ For patients with severe inflammation: corticosteroids (prednisone, methylprednisolone); cyclophosphamide; azathioprine; methotrexate; mycophenolate mofetil; hydroxychloroquine ■■ In refractory cases: B‑cell depletion; B‑cell-directed cytokines; plasmapheresis; intravenous immunoglobulin; rituximab (anti-CD20 monoclonal antibody); haematopoietic stem cell transplantation; intrathecal administration of methotrexate or dexamethasone Anticoagulation therapies ■■ For patients with thrombotic disease: anticoagulants and/or antiplatelets (aspirin, heparin, warfarin) *Most, but not all, of the possible neuropsychiatric manifestions of NPSLE are considered. Abbreviations: APS, antiphospholipid syndrome; NPSLE, neuropsychiatric SLE; SLE, systemic lupus erythematosus.

with adverse effects ranging in severity, some of which can be life-threatening, efforts should be made to identify and treat any secondary causes of CNS dysfunction, such as infection, increased intracranial pressure, or primary psychiatric disorders (Figure 2). In general, prompt initi ation of immunosuppressive and symptomatic treatment can result in improved long-term outcomes, at least for certain manifestations (such as psychosis).158,159 With regard to emerging therapeutic strategies in SLE, few studies have examined the efficacy of such approaches in subgroups of patients with SLE and neuropsychiatric manifestations. This research gap is attributable to the fact that the NPSLE patient sub group is heterogeneous and remains difficult to accu rately define; moreover, tools (assessment instruments and informative biomarkers) that enable accurate and robust recording of neuropsychiatric outcomes are not widely available in evaluation centres. These limitations, as well as ethical and health concerns (these patients are affected by one of the most severe forms of the disease,

and some have behavourial or emotional disorders), restrict the inclusion of such patients in interventional clinical trials. Thus, to date, new drugs have not been studied in patients with severe active NPSLE involving the CNS. For example, belimumab, a human monoclonal antibody antagonist of BAFF, is now approved for the treatment of SLE; however, the lack of evidence, spe cifically in patients with neuropsychiatric symptoms, essentially precludes recommendation of this drug for the treatment of NPSLE. Other therapies with limited or no data specifically obtained in patients with NPSLE include: antibody therapies targeting BAFF or other B‑cell markers, such as CD22 (epratuzumab) and CD20 (rituximab);157 anti bodies against cytokines or cytokine receptors, such as IL‑6, IFN‑α, IFN‑α receptor and TWEAK; medi ators of lymphocyte activation (for example, abata cept, a CTLA‑4–Ig fusion protein that binds to CD80 and CD86);160 and non-antibody agents, such as the T‑cell-modulator peptide P140.161 In addition to these medications, nonpharmacological approaches might be important in the management of patients with SLE who present with psychiatric disorders and impaired attention, concentration and memory.162

Current limitations in NPSLE

In the light of the considerations described above, we must acknowledge that limitations remain concerning the description and classification of the complex sub group of patients with SLE who present with neurologi cal disease. According to case definitions, the prevalence of neuropsychiatric events among patients with SLE is often overestimated because the diagnosis of several manifestations depends on subjective complaints that are also commonly reported in the general population. Thus, many of the classically reported neuropsychiatric manifestations of SLE might not be directly attributable to SLE,163 and other possible causes must be investi gated and excluded before concluding that a patient has NPSLE (Figure 2).164 Furthermore, the definitions of neuropsychiatric symptoms used in the published studies are not ideal. As stated by some researchers,65,165 NPSLE symptoms should be recategorized according to a pathoanatomical localization of the disease (Figure 3); that is, the exact nature of the syndrome should be defined according to the anatomical location of the insult (for example, vascular, CNS or PNS). Another source of potential bias in the currently available evi dence stems from the fact that the majority of published studies investigated neuropsychiatric manifestations in the active phase of SLE, and others reported both overt and subclinical neuropsychiatric manifestations in the absence of markers of systemic SLE disease activity. Evaluative modalities with diagnostic potential are limited in NPSLE; although a multiplicity of labora tory, neuroimaging and psychological tests can assist diagnosis, no single test with high sensitivity and high specificity is available for NPSLE. The diagnostic value of MRI remains limited by the lack of specific ity of this technique (Table 2): similar abnormalities can be



REVIEWS detected in patients without neuropsychiatric mani festations, patients with diffuse neuropsychiatric manifestations (nonspecific abnormalities), and even in healthy individuals.

Future research

Many questions remain unanswered in NPSLE. For example, can neuropsychiatric symptoms in SLE be attributed to specific autoantibodies? What are the anti genic specificities of brain-reactive autoantibodies? How do autoantibodies modulate cerebral functions (assuming that they do)? What part does the genetic and hormo nal component of SLE play in the response of neurons to antibody-mediated effects? What are the possible causes of severe damage of the endothelial cells of BBB that lead to dysfunction of this specialized endothelium? To answer all of these questions, future studies must further elucidate the pathogenic mechanisms underlying NPSLE, and cor relate these mechanisms with specific NPSLE phenotypes. Such information would provide justification for devoting substantial effort to identifying reliable biomarkers that are specific for SLE-associated neuropsychiatric disease, with the aim of developing accurate tests that enable early diagnosis. This issue, which is central to the manage ment of NPSLE, can potentially be realized using highthroughput screening approaches based on pathological and healthy tissues. Some progress has to be made concerning the devel opment and implementation of advanced functional neuroimaging techniques capable of identifying the underlying pathological mechanism of NPSLE (throm botic and inflammatory; Table 2). Moreover, it can be hypothesized that repeated neuroimaging examinations and follow-up serological assessment (to differentiate active and chronic stages of the disease, or the reversibil ity of lesions or manifestations) will contribute consider ably to the treatment decision-making process. However, continued development of imaging techniques, as well as laboratory and psychological tests, that can identify and assess key biomarkers to inform diagnosis and treatment will be important. The development and characterization of animal models, in which controlled immune mechanisms induce specific cognitive and behavioural deficits, is another important area of research. Such models could poten tially be based on active immunization with peptides that 1.

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Esdaile, J. M. Lupus. The disease with a thousand faces [French]. Union Med. Can. 120, 357–358 (1991). Koga, M. et al. Cumulative association of eight susceptibility genes with systemic lupus erythematosus in a Japanese female population. J. Hum. Genet. 56, 503–507 (2011). Lundström, E. et al. HLA-DRB1*04/*13 alleles are associated with vascular disease and antiphospholipid antibodies in systemic lupus erythematosus. Ann. Rheum. Dis. 72, 1018–1025 (2013). Rubtsov, A. V., Rubtsova, K., Kappler, J. W. & Marrack, P. Genetic and hormonal factors in female-biased autoimmunity. Autoimmun. Rev. 9, 494–498 (2010).

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mimic epitopes on brain antigens, passive brain-reactive autoantibody transfer, or intracerebroventricular admin istration of antibodies in order to confirm their causal relationship with neurological manifestations. In addition, on the basis of the association of SLE with common neuropsychiatric syndromes, future work should be aimed at determining whether any conditions that are currently diagnosed as primary psychiatric disorder actu ally represent a definable subset of treatable autoimmune syndromes. Such investigations into the pathogenesis of psychiatric disorders could also be of mutual benefit in NPSLE, owing to overlapping mechanisms.

Conclusions

At least five million people worldwide have some form of SLE, and it is estimated that up to 75% of these patients will experience neuropsychiatric manifestations at some point in the course of the disease.43,45 Neuropsychiatric involvement represents one of the main causes of morbid ity and mortality in SLE, and this complication is certainly the least well-understood aspect of the disease. Clinical, serological and neuroimaging tests should be interpreted in combination to correctly diagnose or exclude NPSLE on an individual-patient basis. Appropriate evaluation and accurate classification of neuropsychiatric manifes tations is also an important aspect of NPSLE treatment and research; however, the multifactorial pathogenesis of NPSLE and its polymorphic phenotype continues to considerably complicate the identification of pertinent biomarkers, research into novel therapies, and the imple mentation of well-established treatments. Thus, substan tial efforts are needed to increase our understanding and, ultimately, improve the management of NPSLE. Review criteria Articles for citation were selected by searching PubMed for English-language publications, using the terms “neuropsychiatric systemic lupus erythematosus”, “systemic lupus erythematosus”, “classification”, “brain”, “cerebral”, “central nervous system”, “pathogenesis”, “epidemiology”, “autoantibodies”, “antiphospholipid”, “NMDA”, “blood–brain barrier”, “neuroimaging” and “treatment” in various combinations. Additional references were identified through the references cited in the publications identified, as well as from the authors’ personal reference libraries.

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