Juvenile Neuropsychiatric Systemic Lupus Erythematosus: Identification of Novel Central Neuroinflammation Biomarkers

Patients identified through the French database of rare diseases who met the 2019 European League Against Rheumatism (EULAR) ACR classification criteria for SLE with onset < 16 years (j-SLE) followed at a tertiary reference center for autoimmune diseases in children, Robert-Debré hospital, Paris, France, from January 2017 to March 2022, were included. These patients were investigated according to the same routine-care procedure over the entire period: suspected cases of j-NPSLE were evaluated by a multidisciplinary pediatric team (rheumatologist, psychiatrist, psychologist, and neurologist) and longitudinally assessed during their follow-up. Standardized criteria (1999 ACR nomenclature, DSM V-Diagnostic and Statistical Manual of Mental Disorders) and multidisciplinary consensus were combined to attribute NP features to j-SLE. Isolated headaches were excluded. CSF neopterin and IFN-α were not considered for classification and only analyzed after group attribution. The study protocol followed ethics guidelines (Robert-Debré hospital ethical committee, authorization N˚ 2020–478). Data processing was approved by the AP-HP (Public Hospitals of Paris) data protection office (registration number N°20200227113056). All samples were collected with informed consent. The study was approved by the Comité de protection des personnes Ile de France II and the French advisory committee on data processing in medical research (ID-RCB: 2014-A01017-40).

Demographic information, past medical history, clinical features and laboratory findings, treatments, outcome, brain computed tomography scans, magnetic resonance imaging (MRI), and electroencephalogram (EEG) were recorded. All patients with j-NPSLE underwent brain MRI with non-contrast–enhanced MR angiography (TOF, time-of-flight) using either a 1.5 or 3 T MRI scanner. Global disease activity and cumulative organ damage were respectively quantified by the SLE Disease Activity Index (SLEDAI) and the Systemic Lupus International Collaborating Clinics (SLICC)/ACR damage index. Active NPSLE status was attributed with ACR and DSM V criteria and multidisciplinary expertise. Inactive NPSLE status was defined as the resolution of acute clinical signs which had led to the diagnosis of j-NPSLE.

Lumbar puncture analysis included cytology, protein, bacteriology, oligoclonal bands, CNS auto antibodies, neopterin, and IFN-α. CNS auto antibodies (including anti-N-methyl-D-aspartate receptor) were assessed by immunohistochemistry on rat brains slices. Neopterin was determined by liquid chromatography coupled with tandem mass spectrometry. IFN-α was quantified by different methods in serum and CSF: biological titration of antiviral activity [27, 28], and with a single-molecule array (Simoa) digital ELISA (enzyme-linked immunosorbent assay) using a monoclonal antibody pair isolated from patients with APECED with high specificity for all IFN-α subtypes (pan-IFN-α assay) as previously described [19]. Interferon stimulated gene (ISG) expression was assessed in whole blood and an ‘ISG score’ derived as previously described [29].

Medians and interquartile ranges were used for quantitative variable description. Qualitative variables were described as number of patients (n) and percentages (%). Fisher’s exact test was used for categorical data. Given the small sample size, continuous data were compared using Mann–Whitney’s test. A paired data test was not performed in the principal analysis for neopterin and pan-IFN-α in j-NPSLE because of the consequences in terms of data loss. A p-value below 0.05 was considered statistically significant. Analyses were performed with GraphPad PRISM (version 9).

Fifty-one j-SLE patients were included, 20 of whom were diagnosed with j-NPSLE with CNS involvement (39%) and shown in a flow chart (Figure S1). Median follow-up of all j-SLE patients was 34 months [20–59]. Patient demographic and clinical characteristics are summarized in Tables 1, S1, and S2. Of the 20 j-NPSLE patients, 13 demonstrated NP features at SLE onset (65%). The seven other patients received a diagnosis of j-NPSLE later in the course of their disease (median time: 15 months after SLE diagnosis, [2-36]). SLEDAI score at the time of CSF assessment did not differ significantly between both groups (j-NPSLE vs j-SLE-controls). On the contrary, SLICC/ACR damage score was significantly higher in the j-NPSLE group (Table 1: median = 1 [0;1] vs 0 [0;0], p = 0.0065; number of patients with a score ≥ 1 n = 11 [61%] vs 0; p = 0.0074) at last follow-up evaluation (median time follow-up from acute NP episode to last follow-up evaluation: 24 months [16;37] for j-NPSLE vs 14 months [12;29] for j-SLE-controls, p = 0.2627). We collected 38 CSF samples from 28 individuals, comprising 20 j-NPSLE patients with CNS involvement (19 and 11 samples during active and inactive disease respectively) and 8 controls (j-SLE where NPSLE was discarded). The following assessments were available: CSF IFN activity in 18 active and 9 inactive j-NPSLE and 8 controls, blood IFN activity in 19 active and 9 inactive j-NPSLE and 7 controls, CSF pan-IFN-α measured by Simoa in 9 active and 5 inactive j-NPSLE patients and 3 controls, serum pan-IFN-α measured by Simoa in 8 active and 5 inactive j-NPSLE and 3 controls, CSF neopterin in 18 active and 11 inactive j-NPSLE and 7 controls, 17 ISG scores in peripheral blood in 5 active and 7 inactive j-NPSLE and 5 controls, taken concomitant to CSF sampling (Figs. 1 and S1; Tables 1 and S3).

Electroencephalogram (EEG) nonspecific features were reported in 11/14 investigated j-NPSLE patients (79%): diffuse slow activity (n = 5), diffuse micro-voltage (n = 3), encephalopathy with disturbance in maintaining vigilance (n = 1), focal slow waves (n = 3), focal spikes (n = 2), and diffuse spikes (n = 1). CSF analysis was performed at first neuropsychiatric manifestation during the study period in all j-NPSLE patients (Table S3) except one due to profound thrombocytopenia (19/20). Moderate hyperproteinorachia (n = 3; 16%), associated with an increased white blood cell count (> 5 cells/mm³, n = 2; 11%), and isolated pleocytosis (n = 1; 5%) were the only abnormalities recorded. Oligoclonal bands were noted in 4 of 15 patients tested (27%). We observed positivity of CNS autoantibodies in none of the 12 patients tested. Brain MRI was performed in all j-NPSLE patients, and abnormal nonspecific morphological features were reported in 19 of 20 patients (95%): a mild enlargement of supratentorial anterior subarachnoid spaces associated with slight widening of sulci was seen in most patients (74%, n = 14), often after the use of corticosteroid treatment (64%, n = 9) and weight loss (64%, n = 9). White matter hyperintensities (WMH) on T2 or FLAIR were observed in 13 patients, without any correlation between their number or size and clinical features. Only one patient displayed ischemic brain lesions on MRI, after severe myocarditis and low brain blood-flow, without any feature of MRI inflammation.

Only six j-NPSLE patients and three non-j-NPSLE patients had not received any immunosuppressive treatment the month before CSF assessment. Neopterin levels were significantly higher in active versus inactive j-NPSLE and non-NPSLE patients respectively (Fig. 1A, p = 0.0015, p = 0.0008, n = 36 samples of 26 patients assessed). ISG expression (IFN signature) was assessed in active j-NPSLE, inactive j-NPSLE, and j-SLE controls in 5, 7, and 5 patients, respectively, and median of IFN signature was not significantly different between active j-NPSLE patients and active j-SLE controls (median: 13 (n = 5) vs 19 (n = 5), p = 0.8968, data not shown). CSF IFN activity was assessed in active j-NPSLE, inactive j-NPSLE, and j-SLE controls in 18, 9, and 8 patients, respectively, as negative in 83% (n = 15/18) of active j-NPSLE (negative in all inactive j-NPSLE and j-SLE controls) and not statistically different (active vs inactive j-NPSLE p = 0.4554 and active j-NPSLE vs j-SLE controls p = 0.5292, data not shown). In contrast, levels of pan-IFN-α in the CSF measured using the more sensitive Simoa digital ELISA were significantly higher in active versus inactive j-NPSLE (Fig. 1B, p = 0.0010, n = 14 samples of 9 patients assessed). CSF neopterin and pan-IFN-α levels correlated strongly (Fig. 1C, R = 0.8323, p < 0.0001, n = 23 paired samples). On the contrary, levels of pan-IFN-α in the serum measured by Simoa were not significantly different in active versus inactive j-NPSLE, nor j-SLE controls (Fig. 1D, p = 0.0932 and p = 0.7758 respectively; n = 16 samples of 12 patients assessed). Interferon activity was higher in serum than in CSF for all j-NPSLE patients, except for one, who is currently being assessed for a monogenic cause of SLE by whole-genome sequencing. All serum IFN-α concentrations measured with the Simoa pan-IFN-α assay were higher than those in the CSF in j-NPSLE patients, except for one patient who had also a diagnosis of AGS by homozygous mutation c.529G > A, p.Ala177Thr in RNASEH2B (Fig. 2B). The analysis did not differ statistically, if groups were analyzed without the AGS patient associated to acute j-NPSLE episode (CSF neopterin j-NPSLE active vs inactive p = 0.0033; CSF neopterin controls vs j-NPSLE active p = 0.0011; CSF pan-IFN-α j-NPSLE active vs inactive p = 0.0040; CSF pan-IFN-α controls vs active j-NPSLE p =  0.1939). A decrease of CSF IFN-α concentrations, measured with the Simoa pan-IFN-α assay (n = 5), and CSF neopterin (n = 10) upon immunosuppressive treatment was observed in all patients tested, correlating with improvement of SLE and j-NPSLE features (Figs. 1, 2A, and S2).