Plasma interferon-alpha protein levels during pregnancy are associated with lower birth weight in systemic lupus erythematosus.

OBJECTIVES: Adverse pregnancy outcomes are more common in women with systemic lupus erythematosus (SLE) compared with healthy women, but we lack prognostic biomarkers. Plasma interferon alpha (IFNα) protein levels are elevated in a subgroup of pregnant women with SLE, but whether this is associated with pregnancy outcomes is unknown. We investigated the relationship between IFNα, adverse pregnancy outcomes and the presence of autoantibodies in SLE pregnancy. METHODS: We followed 76 women with SLE prospectively. Protein levels of IFNα were quantified in plasma collected in the 2nd and 3rd trimester with single-molecule array. Positivity for antinuclear and antiphospholipid antibodies was assessed during late pregnancy with multiplexed bead assay. Clinical outcomes included the adverse pregnancy outcomes small for gestational age (SGA), preterm birth, and preeclampsia. RESULTS: During SLE pregnancy, women with SGA infants compared with those without had higher levels of plasma IFNα protein, and IFNα positivity was associated with lower birth weight of the infant. Preterm birth was associated with autoantibodies against chromatin. IFNα protein levels associated positively with autoantibodies against chromatin, Smith/ribonucleoprotein (SmRNP) and RNP, but negatively with phospholipid antibodies. CONCLUSION: Elevated IFNα protein in plasma of women with SLE is a potential risk factor for lower birth weight of their infants. The association between IFNα and lower birth weight warrants further investigation regarding the pathophysiological role of IFNα during SLE pregnancy.

The serological immunogenicity of the third and fourth doses of COVID-19 vaccine in patients with inflammatory rheumatic diseases on different biologic or targeted DMARDs: a Swedish nationwide study (COVID-19-REUMA).

Studies investigating the immunogenicity of additional COVID-19 vaccine doses in immunosuppressed patients with inflammatory rheumatic diseases (IRD) are still limited. The objective was to explore the antibody response including response to omicron virus subvariants (sBA.1 and sBS.2) after third and fourth COVID-19 vaccine doses in Swedish IRD patients treated with immunomodulating drugs compared to controls. Antibody levels to spike wild-type antigens (full-length protein and S1) and the omicron variants sBA.1 and sBA.2 (full-length proteins) were measured. A positive response was defined as having antibody levels over cut-off or ≥fourfold increase in post-vaccination levels for both antigens. Patients with arthritis, vasculitis, and other autoimmune diseases (n = 414), and controls (n = 61) receiving biologic/targeted synthetic disease-modifying anti-rheumatic drugs (DMARDs) with or without conventional synthetic DMARDs participated. Of these, blood samples were available for 370 patients and 52 controls after three doses, and 65 patients and 15 controls after four doses. Treatment groups after three vaccine doses were rituximab (n = 133), abatacept (n = 22), IL6r inhibitors (n = 71), JAnus Kinase inhibitors (JAK-inhibitors) (n = 56), tumor necrosis factor inhibitor (TNF-inhibitors) (n = 61), IL12/23/17 inhibitors (n = 27), and controls (n = 52). The percentage of responders after three and four vaccine doses was lower in rituximab-treated patients (59% and 57%) compared to controls (100%) (P < 0.001). After three doses, the percentage of responders in all other groups was 100%, including response to omicron sBA.1 and sBA.2. In rituximab-treated patients, higher baseline immunoglobulin G (IgG) and longer time-period between rituximab and vaccination predicted better response. In this Swedish nationwide study including IRD patients three and four COVID-19 vaccine doses were immunogenic in patients treated with IL6r inhibitors, TNF-inhibitors, JAK-inhibitors, and IL12/23/17-inhibitors but not in rituximab. As >50% of rituximab patients responded to vaccines including omicron subvariants, these patients should be prioritized for additional vaccine doses. IMPORTANCE: Results from this study provide further evidence that additional doses of COVID-19 vaccines are immunogenic and result in satisfactory antibody response in a majority of patients with inflammatory rheumatic diseases (IRD) receiving potent immunomodulating treatments such as biological or targeted disease-modifying anti-rheumatic drugs (DMARDs) given as monotherapy or combined with traditional DMARDs. We observed that rituximab treatment, both as monotherapy and combined with csDMARDs, impaired antibody response, and only roughly 50% of patients developed a satisfactory antibody response including response to omicron subvariants after the third vaccine. In addition, higher IgG levels at the last rituximab course before the third vaccine dose and a longer time after the last rituximab treatment increased the chance of a satisfactory antibody response. These results indicate that rituximab-treated patients should be prioritized for additional vaccine doses. CLINICAL TRIALS: EudraCT (European Union Drug Regulating Authorities Clinical Trials Database) with number 2021-000880-63.

Low CD4 + T cell count is related to specific anti-nuclear antibodies, IFNα protein positivity and disease activity in systemic lupus erythematosus pregnancy.

BACKGROUND: Lymphopenia, autoantibodies and activation of the type I interferon (IFN) system are common features in systemic lupus erythematosus (SLE). We speculate whether lymphocyte subset counts are affected by pregnancy and if they relate to autoantibody profiles and/or IFNα protein in SLE pregnancy. METHODS: Repeated blood samples were collected during pregnancy from 80 women with SLE and 51 healthy controls (HC). Late postpartum samples were obtained from 19 of the women with SLE. Counts of CD4 + and CD8 + T cells, B cells and NK cells were measured by flow cytometry. Positivity for anti-nuclear antibodies (ANA) fine specificities (double-stranded DNA [dsDNA], Smith [Sm], ribonucleoprotein [RNP], chromatin, Sjögren's syndrome antigen A [SSA] and B [SSB]) and anti-phospholipid antibodies (cardiolipin [CL] and ß2 glycoprotein I [ß2GPI]) was assessed with multiplexed bead assay. IFNα protein concentration was quantified with Single molecule array (Simoa) immune assay. Clinical data were retrieved from medical records. RESULTS: Women with SLE had lower counts of all lymphocyte subsets compared to HC throughout pregnancy, but counts did not differ during pregnancy compared to postpartum. Principal component analysis revealed that low lymphocyte subset counts differentially related to autoantibody profiles, cluster one (anti-dsDNA/anti-Sm/anti-RNP/anti-Sm/RNP/anti-chromatin), cluster two (anti-SSA/anti-SSB) and cluster three (anti-CL/anti-ß2GPI), IFNα protein levels and disease activity. CD4 + T cell counts were lower in women positive to all ANA fine specificities in cluster one compared to those who were negative, and B cell numbers were lower in women positive for anti-dsDNA and anti-Sm compared to negative women. Moreover, CD4 + T cell and B cell counts were lower in women with moderate/high compared to no/low disease activity, and CD4 + T cell count was lower in IFNα protein positive relative to negative women. Finally, CD4 + T cell count was unrelated to treatment. CONCLUSION: Lymphocyte subset counts are lower in SLE compared to healthy pregnancies, which seems to be a feature of the disease per se and not affected by pregnancy. Our results also indicate that low lymphocyte subset counts relate differentially to autoantibody profiles, IFNα protein levels and disease activity, which could be due to divergent disease pathways.

A survey of ficolin-3 activity in Systemic Lupus Erythematosus reveals a link to hematological disease manifestations and autoantibody profile.

The complement system plays a central role in the pathogenesis of Systemic Lupus Erythematosus (SLE), but most studies have focused on the classical pathway. Ficolin-3 is the main initiator of the lectin pathway of complement in humans, but its role in systemic autoimmune disease has not been conclusively determined. Here, we combined biochemical and genetic approaches to assess the contribution of ficolin-3 to SLE risk and disease manifestations. Ficolin-3 activity was measured by a functional assay in serum or plasma samples from Swedish SLE patients (n = 786) and controls matched for age and sex (n = 566). Genetic variants in an extended 300 kb genomic region spanning the FCN3 locus were analyzed for their association with ficolin-3 activity and SLE manifestations in a Swedish multicenter cohort (n = 985). Patients with ficolin-3 activity in the highest tertile showed a strong enrichment in an SLE cluster defined by anti-Sm/DNA/nucleosome antibodies (OR 3.0, p < 0.001) and had increased rates of hematological disease (OR 1.4, p = 0.078) and lymphopenia (OR = 1.6, p = 0.039). Genetic variants associated with low ficolin-3 activity mapped to an extended haplotype in high linkage disequilibrium upstream of the FCN3 gene. Patients carrying the lead genetic variant associated with low ficolin-3 activity had a lower frequency of hematological disease (OR 0.67, p = 0.018) and lymphopenia (OR 0.63, p = 0.031) and fewer autoantibodies (p = 0.0019). Loss-of-function variants in the FCN3 gene were not associated with SLE, but four (0.5 %) SLE patients developed acquired ficolin-3 deficiency where ficolin-3 activity in serum was depleted following diagnosis of SLE. Taken together, our results provide genetic and biochemical evidence that implicate the lectin pathway in hematological SLE manifestations. We also identify lectin pathway activation through ficolin-3 as a factor that contributes to the autoantibody response in SLE.

MRI BrainAGE demonstrates increased brain aging in systemic lupus erythematosus patients.

Introduction: Systemic lupus erythematosus (SLE) is an autoimmune connective tissue disease affecting multiple organs in the human body, including the central nervous system. Recently, an artificial intelligence method called BrainAGE (Brain Age Gap Estimation), defined as predicted age minus chronological age, has been developed to measure the deviation of brain aging from a healthy population using MRI. Our aim was to evaluate brain aging in SLE patients using a deep-learning BrainAGE model. Methods: Seventy female patients with a clinical diagnosis of SLE and 24 healthy age-matched control females, were included in this post-hoc analysis of prospectively acquired data. All subjects had previously undergone a 3 T MRI acquisition, a neuropsychological evaluation and a measurement of neurofilament light protein in plasma (NfL). A BrainAGE model with a 3D convolutional neural network architecture, pre-trained on the 3D-T1 images of 1,295 healthy female subjects to predict their chronological age, was applied on the images of SLE patients and controls in order to compute the BrainAGE. SLE patients were divided into 2 groups according to the BrainAGE distribution (high vs. low BrainAGE). Results: BrainAGE z-score was significantly higher in SLE patients than in controls (+0.6 [±1.1] vs. 0 [±1.0], p = 0.02). In SLE patients, high BrainAGE was associated with longer reaction times (p = 0.02), lower psychomotor speed (p = 0.001) and cognitive flexibility (p = 0.04), as well as with higher NfL after adjusting for age (p = 0.001). Conclusion: Using a deep-learning BrainAGE model, we provide evidence of increased brain aging in SLE patients, which reflected neuronal damage and cognitive impairment.

Serologic immunogenicity and safety of Herpes Zoster subunit vaccine in patients with Rheumatoid Arthritis receiving Janus Kinase inhibitors.

OBJECTIVE: Patients with rheumatoid arthritis (RA) treated with Janus Kinase inhibitors (JAKi) are at increased risk of Herpes Zoster (HZ). The objective of this study was to investigate serological immunogenicity and safety of the HZ subunit (HZ/su) vaccine in RA patients treated with JAKi, for which little is known. METHODS: RA patients treated with JAKi (n = 82) at the Department of Rheumatology, Skåne University Hospital, Sweden, and healthy controls (n = 51) received two doses of the HZ/su vaccine (Shingrix). Vaccine-specific antibody responses were analysed using indirect enzyme-linked immunosorbent assay (ELISA). Post-vaccination antibody levels were compared between patients and controls using analysis of covariance. Potential predictors for vaccine response were investigated using a multivariable linear regression analysis. Self-reported adverse events (AEs) and changes in RA disease activity were analysed. RESULTS: Following vaccination, vaccine-specific antibody levels increased significantly in both patients and controls (p< 0.0001). 80.5% of patients and 98.0% of controls achieved a ≥ 4-fold increase in antibody levels. Post-vaccination antibody levels were lower in patients than controls (ratio 0.44, 95% CI 0.31-0.63), and lower in patients receiving JAKi+Methotrexate than JAKi monotherapy (ratio 0.43, 95% CI 0.24-0.79). AEs, mostly mild/moderate, were common. One patient developed HZ and six patients (6.5%) had increased RA disease activity following vaccination. CONCLUSION: The HZ/su vaccine was serologically immunogenic in most RA patients treated with JAKi. Moreover, the vaccine had an acceptable safety profile. These results support recommendations for usage of the HZ/su vaccine in this vulnerable population. TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, NCT03886038.

B cell polygenic risk scores associate with anti-dsDNA antibodies and nephritis in systemic lupus erythematosus.

OBJECTIVE: B cell function and autoantibodies are important in SLE pathogenesis. In this work, we aimed to investigate the impact of cumulative SLE B cell genetics on SLE subphenotype and autoantibody profile. METHODS: Female patients with SLE (n=1248) and healthy controls (n=400) were genotyped using Illumina's Global Screening Array. Two polygenic risk scores (PRSs), one representing B cell genes and the other B cell activation genes, were calculated for each individual using risk loci for SLE in genes assigned to B cell-related pathways according to the Kyoto Encyclopedia of Genes and Genomes, Gene Ontology and Reactome Databases. RESULTS: Double-stranded DNA (dsDNA) antibodies were more prevalent among patients with a high compared with a low SLE B cell PRS (OR 1.47 (1.07 to 2.01), p=0.018), and effect sizes were augmented in patients with human leucocyte antigen (HLA) risk haplotypes HLA-DRB1*03:01 and HLA-DRB1*15:01 (DRB1*03/15 -/- (OR 0.99 (0.56 to 1.77), p=0.98; DRB1*03/15 +/- or -/+ (OR 1.64 (1.06 to 2.54), p=0.028; and DRB1*03/15 +/+ (OR 4.47 (1.21 to 16.47), p=0.024). Further, a high compared with a low B cell PRS was associated with low complement levels in DRB1*03/15 +/+ patients (OR 3.92 (1.22 to 12.64), p=0.022). The prevalence of lupus nephritis (LN) was higher in patients with a B cell activation PRS above the third quartile compared with patients below (OR 1.32 (1.00 to 1.74), p=0.048). CONCLUSIONS: High genetic burden related to B cell function is associated with dsDNA antibody development and LN. Assessing B cell PRSs may be important in order to determine immunological pathways influencing SLE and to predict clinical phenotype.

Synovial monocytes contribute to chronic inflammation in childhood-onset arthritis via IL-6/STAT signalling and cell-cell interactions.

Introduction: Monocytes are key effector cells in inflammatory processes. We and others have previously shown that synovial monocytes in childhood-onset arthritis are activated. However, very little is known about how they contribute to disease and attain their pathological features. Therefore, we set out to investigate the functional alterations of synovial monocytes in childhood-onset arthritis, how they acquire this phenotype, and whether these mechanisms could be used to tailorize treatment. Methods: The function of synovial monocytes was analysed by assays believed to reflect key pathological events, such as T-cell activation-, efferocytosis- and cytokine production assays using flow cytometry in untreated oligoarticular juvenile idiopathic arthritis (oJIA) patients (n=33). The effect of synovial fluid on healthy monocytes was investigated through mass spectrometry and functional assays. To characterize pathways induced by synovial fluid, we utilized broad-spectrum phosphorylation assays and flow cytometry, as well as inhibitors to block specific pathways. Additional effects on monocytes were studied through co-cultures with fibroblast-like synoviocytes or migration in transwell systems. Results: Synovial monocytes display functional alterations with inflammatory and regulatory features, e.g., increased ability to induce T-cell activation, resistance to cytokine production following activation with LPS and increased efferocytosis. In vitro, synovial fluid from patients induced the regulatory features in healthy monocytes, such as resistance to cytokine production and increased efferocytosis. IL-6/JAK/STAT signalling was identified as the main pathway induced by synovial fluid, which also was responsible for a majority of the induced features. The magnitude of synovial IL-6 driven activation in monocytes was reflected in circulating cytokine levels, reflecting two groups of low vs. high local and systemic inflammation. Remaining features, such as an increased ability to induce T-cell activation and markers of antigen presentation, could be induced by cell-cell interactions, specifically via co-culture with fibroblast-like synoviocytes. Conclusions: Synovial monocytes in childhood-onset arthritis are functionally affected and contribute to chronic inflammation, e.g., via promoting adaptive immune responses. These data support a role of monocytes in the pathogenesis of oJIA and highlight a group of patients more likely to benefit from targeting the IL-6/JAK/STAT axis to restore synovial homeostasis.

Low-density granulocytes are related to shorter pregnancy duration but not to interferon alpha protein blood levels in systemic lupus erythematosus.

BACKGROUND: An increased risk of pregnancy complications is seen in women with systemic lupus erythematosus (SLE), but the specific immunopathological drivers are still unclear. Hallmarks of SLE are granulocyte activation, type I interferon (IFN) overproduction, and autoantibodies. Here we examined whether low-density granulocytes (LDG) and granulocyte activation increase during pregnancy, and related the results to IFNα protein levels, autoantibody profile, and gestational age at birth. METHODS: Repeated blood samples were collected during pregnancy in trimesters one, two, and three from 69 women with SLE and 27 healthy pregnant women (HC). Nineteen of the SLE women were also sampled late postpartum. LDG proportions and granulocyte activation (CD62L shedding) were measured by flow cytometry. Plasma IFNα protein concentrations were quantified by single molecule array (Simoa) immune assay. Clinical data were obtained from medical records. RESULTS: Women with SLE had higher LDG proportions and increased IFNα protein levels compared to HC throughout pregnancy, but neither LDG fractions nor IFNα levels differed during pregnancy compared to postpartum in SLE. Granulocyte activation status was higher in SLE relative to HC pregnancies, and it was increased during pregnancy compared to after pregnancy in SLE. Higher LDG proportions in SLE were associated with antiphospholipid positivity but not to IFNα protein levels. Finally, higher LDG proportions in trimester three correlated independently with lower gestational age at birth in SLE. CONCLUSION: Our results suggest that SLE pregnancy results in increased peripheral granulocyte priming, and that higher LDG proportions late in pregnancy are related to shorter pregnancy duration but not to IFNα blood levels in SLE.

Argonaute, Vault, and Ribosomal Proteins Targeted by Autoantibodies in Systemic Lupus Erythematosus.

OBJECTIVE: To expand, in an unbiased manner, our knowledge of autoantigens and autoantibodies in patients with systemic lupus erythematosus (SLE) and evaluate their associations with serological and clinical variables. METHODS: Human proteome arrays (> 21,000 proteins) were screened with serum from patients with SLE (n = 12) and healthy controls (n = 6) for IgG and IgA binding. Top hits were validated with 2 cohorts of patients with SLE (cohort 1, n = 49; cohort 2, n = 46) and other rheumatic diseases by ELISA. Clinical associations of the autoantibodies were tested. RESULTS: Ro60 was the top hit in the screen, and the 10 following proteins included 2 additional known SLE autoantigens plus 8 novel autoantigens involved in microRNA processing (Argonaute protein 1 [AGO1], AGO2, and AGO3), ribosomes (ribosomal protein lateral stalk subunit P2 and ovarian tumor deubiquitinase 5 [OTUD5]), RNA transport by the vault (major vault protein), and the immune proteasome (proteasome activator complex subunit 3). Patient serum contained IgG reactive with these proteins and IgA against the AGO proteins. Using the 95th percentile of healthy donor reactivity, 5-43% were positive for the novel antigens, with OTUD5 and AGO1 showing the highest percentages of positivity. Autoantibodies against AGO1 proteins were more prevalent in patients with oral ulcers in a statistically significant manner. IgG autoantibodies against AGO proteins were also seen in other rheumatic diseases. CONCLUSION: We discovered new autoantigens existing in cytosolic macromolecular protein assemblies containing RNA (except the proteasome) in cells. A more comprehensive list of autoantigens will allow for a better analysis of how proteins are targeted by the autoimmune response. Future research will also reveal whether specific autoantibodies have utility in the diagnosis or management of SLE.

The impact of immunomodulating treatment on the immunogenicity of COVID-19 vaccines in patients with immune-mediated inflammatory rheumatic diseases compared to healthy controls. A Swedish nationwide study (COVID19-REUMA).

OBJECTIVES: To elucidate antibody responses after the second and third dose of COVID-19 vaccine in patients with inflammatory rheumatic diseases (IRD) treated with biologic/targeted disease modifying anti-rheumatic drugs (b/ts DMARDs). METHODS: Antibody levels to antigens representing spike full length protein and spike S1 were measured before vaccination, 2-12 weeks after the second dose, before and after the third dose using multiplex bead-based serology assay. Positive antibody response was defined as antibody levels over cut off (seropositivity) in seronegative individuals or ≥ 4-fold increase in antibodies in individuals seropositive for both spike proteins. RESULTS: Patients (n = 414) receiving b/ts DMARDs (283 had arthritis, 75 systemic vasculitis and 56 other autoimmune diseases) and controls (n = 61) from five Swedish regions participated. Treatments groups were: rituximab (n = 145); abatacept (n = 22); Interleukin 6 receptor inhibitors [IL6i (n = 79)]; JAnus Kinase Inhibitors [JAKi (n = 58)], Tumour Necrosis Factor inhibitor [TNFi (n = 68)] and Interleukin12/23/17 inhibitors [IL12/23/17i (n = 42)]. Percentage of patients with positive antibody response after two doses was significantly lower in rituximab (33,8%) and abatacept (40,9%) (p < 0,001) but not in IL12/23/17i, TNFi or JAKi groups compared to controls (80,3%). Higher age, rituximab treatment and shorter time between last rituximab course and vaccination predicted impaired antibody response. Antibody levels collected 21-40 weeks after second dose decreased significantly (IL6i: p = 0,02; other groups: p < 0,001) compared to levels at 2-12 week but most participants remained seropositive. Proportion of patients with positive antibody response increased after third dose but was still significantly lower in rituximab (p < 0,001). CONCLUSIONS: Older individuals and patients on maintenance rituximab have an impaired response after two doses of COVID-19 vaccine which improves if the time between last rituximab course and vaccination extends and also after an additional vaccine dose. Rituximab patients should be prioritized for booster vaccine doses. TNFi, JAKi and IL12/23/17i does not diminished humoral response to primary and an additional vaccination.

Role of Mannose-binding Lectin and Association with Microbial Sensitization in a Cohort of Patients with Atopic Dermatitis.

Atopic dermatitis is a relapsing inflammatory skin condition, in which bacteria, fungi and viruses may colonize the skin and aggravate the condition. Mannose-binding lectin is part of the innate immune system. Polymorphism in the mannose-binding lectin gene can result in deficiency of mannose-binding lectin, which may affect defence against microbes. The aim of this study was to investigate whether polymorphisms in the mannose-binding lectin gene affect the extent of sensitization to common skin microbes, the skin barrier function, or the severity of the disease in a cohort of patients with atopic dermatitis. Genetic testing of mannose-binding lectin polymorphism was performed in 60 patients with atopic dermatitis. The disease severity, skin barrier function, and serum levels of specific immunoglobulin E against skin microbes were measured. In patients with low mannose-binding lectin genotype (group 1) 6 of 8 (75%) were sensitized to Candida albicans, compared to 14 of 22 (63.6%) patients with intermediate mannose-binding genotype  (group 2) and 10 of 30 (33.3%) patients with high mannose-binding genotype (group 3). Group 1 (low mannose-binding lectin) was more likely to be sensitized to Candida albicans compared with group 3 (high mannose-binding lectin) (odds ratio 6.34, p-value 0.045). In this cohort of patients with atopic dermatitis, mannose-binding lectin deficiency was associated with increased sensitization to Candida albicans.

Tract-based white matter hyperintensity patterns in patients with systemic lupus erythematosus using an unsupervised machine learning approach.

Currently, little is known about the spatial distribution of white matter hyperintensities (WMH) in the brain of patients with Systemic Lupus erythematosus (SLE). Previous lesion markers, such as number and volume, ignore the strategic location of WMH. The goal of this work was to develop a fully-automated method to identify predominant patterns of WMH across WM tracts based on cluster analysis. A total of 221 SLE patients with and without neuropsychiatric symptoms from two different sites were included in this study. WMH segmentations and lesion locations were acquired automatically. Cluster analysis was performed on the WMH distribution in 20 WM tracts. Our pipeline identified five distinct clusters with predominant involvement of the forceps major, forceps minor, as well as right and left anterior thalamic radiations and the right inferior fronto-occipital fasciculus. The patterns of the affected WM tracts were consistent over the SLE subtypes and sites. Our approach revealed distinct and robust tract-based WMH patterns within SLE patients. This method could provide a basis, to link the location of WMH with clinical symptoms. Furthermore, it could be used for other diseases characterized by presence of WMH to investigate both the clinical relevance of WMH and underlying pathomechanism in the brain.

Plasma and cerebrospinal fluid neurofilament light concentrations reflect neuronal damage in systemic lupus Erythematosus.

BACKGROUND: Neuronal damage in systemic lupus erythematosus (SLE) is common, but the extent and mechanisms are unclear. Neurofilament light (NfL) concentrations rise in plasma and cerebrospinal fluid (CSF) during neuronal damage in various neurological disorders. In this cross-sectional study, plasma and CSF concentrations of NfL were explored as a marker of neuronal damage in SLE. METHODS: Seventy-two consecutive SLE out-patients and 26 healthy controls, all female, aged < 55 years, underwent magnetic resonance imaging (MRI) and neurocognitive testing. NfL concentrations in plasma from all individuals and in CSF from 32 patients were measured with single-molecule array technology. Patients were assessed by a rheumatologist and neurologist to define neuropsychiatric involvement (NPSLE) according to three attribution models: SLICC A, SLICC B and ACR. RESULTS: Plasma and CSF NfL concentrations correlated strongly (r = 0.72, p < 0.001). Both NPSLE and non-NPSLE patients in all attribution models had higher plasma NfL concentrations compared with healthy controls (log-NfL, pg/ml, mean (SD); healthy controls (0.71 (0.17)); SLICC A model: NPSLE (0.87 (0.13), p = 0.003), non-NPSLE (0.83 (0.18), p = 0.005); SLICC B model: NPSLE (0.87 (0.14), p = 0.001), non-NPSLE (0.83 (0.18), p = 0.008); ACR model: NPSLE (0.86 (0.16), p < 0.001), non-NPSLE (0.81 (0.17), p = 0.044)). Plasma and CSF NfL concentrations did not differ between NPSLE and non-NPSLE patients. Higher plasma NfL concentrations correlated with larger CSF volumes on MRI (r = 0.34, p = 0.005), and was associated with poorer cognitive performance in the domains of simple attention, psychomotor speed and verbal memory. SLICC/ACR-Damage Index ≥1 was independently associated with higher plasma NfL concentrations (ß = 0.074, p = 0.038). Higher plasma creatinine concentrations, anti-dsDNA-positivity, low complement C3 levels, or a history of renal involvement were associated with higher plasma NfL concentrations (ß = 0.003, p = 0.009; ß = 0.072, p = 0.031; ß = 0.077, p = 0.027; ß = 0.069, p = 0.047, respectively). CONCLUSIONS: Higher plasma NfL concentrations in NPSLE and non-NPSLE patients may indicate a higher degree of neuronal damage in SLE in general, corresponding to cognitive impairment and organ damage development. Furthermore, our results may indicate a higher degree of neuronal breakdown in patients with active SLE, also without overt clinical symptoms. NfL may serve as an indicator of neuronal damage in SLE in further studies.

Autoantibodies associated with systemic sclerosis in three autoimmune diseases imprinted by type I interferon gene dysregulation: a comparison across SLE, primary Sjögren's syndrome and systemic sclerosis.

OBJECTIVE: SLE, primary Sjögren's syndrome (pSS) and systemic sclerosis (SSc) are heterogeneous autoimmune diseases with a dysregulated type I interferon (IFN) system. The diseases often show overlapping clinical manifestations, which may result in diagnostic challenges. We asked to which extent SSc-associated autoantibodies are present in SLE and pSS, and whether these link to serum IFN-α, clinical phenotypes and sex. Samples with clinical data from patients with SSc and healthy blood donors (HBDs) served as controls. Finally, the diagnostic performance of SSc-associated autoantibodies was evaluated. METHODS: Samples from well-characterised subjects with SLE (n=510), pSS (n=116), SSc (n=57) and HBDs (n=236) were analysed using a commercially available immunoassay (EuroLine Systemic Sclerosis Profile (IgG)). IFN-α was quantified by ELISA. Self-reported data on Raynaud's phenomenon (RP) were available. RESULTS: With exceptions for anti-Ro52/SSA and anti-Th/To, SSc-associated autoantibodies were more frequent in SSc than in SLE, pSS and HBDs regardless of sex. IFN-α levels correlated with the number of positive SSc-associated autoantibodies (r=0.29, p<0.0001) and associated with Ro52/SSA positivity (p<0.0001). By using data from SLE, SSc and HBDs, RP was significantly associated with topoisomerase I, centromere protein (CENP)-B, RNA polymerase III 11 kDa, RNA polymerase III 155 kDa and PM-Scl100 whereas Ro52/SSA associated inversely with RP. In SLE, CENP-A was associated with immunological disorder, CENP-B with serositis and Ku with lupus nephritis. By combining analysis of ANA (immunofluorescence) with SSc-associated autoantibodies, the diagnostic sensitivity reached 98% and the specificity 33%. CONCLUSIONS: The 13 specificities included in the EuroLine immunoassay are commonly detected in SSc, but they are also frequent among individuals with other diseases imprinted by type I IFNs. These findings are valuable when interpreting serological data on patients with suspected SSc, especially as patients may present with disease manifestations overlapping different rheumatological diseases. In SLE, we observed associations between manifestations and SSc-associated autoantibodies which have not previously been reported.

Mer-tyrosine kinase: a novel susceptibility gene for SLE related end-stage renal disease.

OBJECTIVE: Lupus nephritis (LN) is a common and severe manifestation of SLE. The genetic risk for nephritis and progression to end-stage renal disease (ESRD) in patients with LN remains unclear. Herein, we aimed to identify novel genetic associations with LN, focusing on subphenotypes and ESRD. METHODS: We analysed genomic data on 958 patients with SLE (discovery cohort: LN=338) with targeted sequencing data from 1832 immunological pathway genes. We used an independent multiethnic cohort comprising 1226 patients with SLE (LN=603) as a replication dataset. Detailed functional annotation and functional epigenomic enrichment analyses were applied to predict functional effects of the candidate variants. RESULTS: A genetic variant (rs56097910) within the MERTK gene was associated with ESRD in both cohorts, meta-analysis OR=5.4 (2.8 to 10.6); p=1.0×10-6. We observed decreased methylation levels in peripheral blood cells from SLE patients with ESRD, compared with patients without renal SLE (p=2.7×10-4), at one CpG site (cg16333401) in close vicinity to the transcription start site of MERTK and located in a DNAse hypersensitivity region in T and B cells. Rs56097910 is linked to altered MERTK expression in kidney tissue in public eQTL databases. Two loci were replicated for association with proliferative LN: PRDM1 (rs6924535, pmeta=1.6×10-5, OR=0.58) and APOA1BP (NAXE) (rs942960, pmeta=1.2×10-5, OR=2.64). CONCLUSION: We identified a novel genetic risk locus, MERTK, associated with SLE-ESRD using the data from two large SLE cohorts. Through DNA methylation analysis and functional annotation, we showed that the risk could be mediated through regulation of gene expression. Our results suggest that variants in the MERTK gene are important for the risk of developing SLE-ESRD and suggest a role for PRDM1 and APOA1BP in proliferative LN.

Serum S100A8/A9 concentrations are associated with neuropsychiatric involvement in systemic lupus erythematosus: a cross-sectional study.

BACKGROUND: Neuropsychiatric (NP) involvement and fatigue are major problems in systemic lupus erythematosus (SLE). S100A8/A9 is a marker of inflammation and responds to therapy in SLE patients. S100A8/A9 has an immunopathogenic role in various neurological diseases. We investigated S100A8/A9 in relation to NP-involvement and fatigue in SLE. METHODS: 72 consecutive SLE outpatients at a tertiary centre and 26 healthy controls were included in this cross-sectional study. NPSLE was determined by specialists in rheumatology and neurology and defined according to three attribution models: "ACR", "SLICC A" and "SLICC B". Cerebral MRI was assessed by a neuroradiologist and neurocognitive testing by a neuropsychologist. The individuals were assessed by scores of pain (VAS), fatigue (VAS and FSS), and depression (MADRS-S). Concentrations of S100A8/A9 in serum and cerebrospinal fluid were measured with ELISA. Statistical calculations were performed using non-parametric methods. RESULTS: Serum concentrations of S100A8/A9 were higher in SLE patients compared with controls (medians 1230 ng/ml; 790 ng/ml, p = 0.023). The concentrations were higher in NPSLE patients compared with non-NPSLE patients when applying the SLICC A and ACR models, but not significant when applying the SLICC B model (medians 1400 ng/ml; 920 ng/ml, p = 0.011; 1560 ng/ml; 1090 ng/ml, p = 0.050; 1460 ng/ml; 1090 ng/ml, p = 0.083, respectively). No differences of CSF S100A8/A9 concentrations were observed between NPSLE and non-NPSLE patients. SLE patients with depression or cognitive dysfunction as an ACR NPSLE manifestation had higher serum S100A8/A9 concentrations than non-NPSLE patients (median 1460 ng/ml, p = 0.007 and 1380 ng/ml, p = 0.013, respectively). Higher serum S100A8/A9 correlated with higher VAS fatigue (r = 0.31; p = 0.008) and VAS pain (r = 0.27, p = 0.021) in SLE patients. Serum S100A8/A9 was not independently associated with NPSLE when adjusting for scores of fatigue (FSS) and pain (VAS) (OR 1.86, 95% CI 0.93-3.73, p = 0.08). CONCLUSIONS: Serum S100A8/A9 concentrations may be associated with NPSLE and fatigue. S100A8/A9 may be of interest in evaluating NPSLE, although further investigations are needed.

Strong Association of Combined Genetic Deficiencies in the Classical Complement Pathway With Risk of Systemic Lupus Erythematosus and Primary Sjögren's Syndrome.

OBJECTIVE: Complete genetic deficiency of the complement component C2 is a strong risk factor for monogenic systemic lupus erythematosus (SLE), but whether heterozygous C2 deficiency adds to the risk of SLE or primary Sjögren's syndrome (SS) has not been studied systematically. This study was undertaken to investigate potential associations of heterozygous C2 deficiency and C4 copy number variation with clinical manifestations in patients with SLE and patients with primary SS. METHODS: The presence of the common 28-bp C2 deletion rs9332736 and C4 copy number variation was examined in Scandinavian patients who had received a diagnosis of SLE (n = 958) or primary SS (n = 911) and in 2,262 healthy controls through the use of DNA sequencing. The concentration of complement proteins in plasma and classical complement function were analyzed in a subgroup of SLE patients. RESULTS: Heterozygous C2 deficiency-when present in combination with a low C4A copy number-substantially increased the risk of SLE (odds ratio [OR] 10.2 [95% confidence interval (95% CI) 3.5-37.0]) and the risk of primary SS (OR 13.0 [95% CI 4.5-48.4]) when compared to individuals with 2 C4A copies and normal C2. For patients heterozygous for rs9332736 with 1 C4A copy, the median age at diagnosis was 7 years earlier in patients with SLE and 12 years earlier in patients with primary SS when compared to patients with normal C2. Reduced C2 levels in plasma (P = 2 × 10-9 ) and impaired function of the classical complement pathway (P = 0.03) were detected in SLE patients with heterozygous C2 deficiency. Finally, in a primary SS patient homozygous for C2 deficiency, we observed low levels of anti-Scl-70, which suggests a risk of developing systemic sclerosis or potential overlap between primary SS and other systemic autoimmune diseases. CONCLUSION: We demonstrate that a genetic pattern involving partial deficiencies of C2 and C4A in the classical complement pathway is a strong risk factor for SLE and for primary SS. Our results emphasize the central role of the complement system in the pathogenesis of both SLE and primary SS.

Enrichment of Complement, Immunoglobulins, and Autoantibody Targets in the Proteome of Platelets from Patients with Systemic Lupus Erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a complex disease characterized by autoimmunity toward apoptotic cells, excessive amounts of circulating immune complexes, and complement activation. A decreased platelet size has been observed in SLE and their nonhemostatic functions may play an active role in the disease. The main objective of this study was to find clues that could explain their decreased size and functional role, analyzing the entire platelet proteome. METHODS: Platelets were isolated from 23 patients with SLE. The five individuals with the highest and lowest average platelet forward scatter were selected for further analysis. Platelet protein content was analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS) and compared with platelets from five healthy controls. Data are available via ProteomeXchange with identifier PXD031202. RESULTS: Out of 2,572 proteins identified, 396 had significantly different levels (ANOVA q-value ≤ 0.01). Forty proteins, including immunoglobulin-, complement- and phosphatidylserine-binding proteins had higher abundance in platelets from SLE patients, largely independent of size (fold difference of ≥1.5 and a t-test p-value of ≤0.05 as cut-off). Functional characterization revealed increased degranulation and skewed hemostatic balance in platelets from SLE patients. In the SLE proteome, immunoglobulin proteins were negatively correlated to serum complement C3 and C4 and the highest relative levels were detected in platelets of normal size. CONCLUSION: Platelets from SLE patients shared a specific protein profile, including immunoglobulins, complement proteins, and autoantigens, largely independent of the platelet size and in agreement with an integrated role for platelets in SLE.

Complement C4 Copy Number Variation is Linked to SSA/Ro and SSB/La Autoantibodies in Systemic Inflammatory Autoimmune Diseases.

OBJECTIVE: Copy number variation of the C4 complement components, C4A and C4B, has been associated with systemic inflammatory autoimmune diseases. This study was undertaken to investigate whether C4 copy number variation is connected to the autoimmune repertoire in systemic lupus erythematosus (SLE), primary Sjögren's syndrome (SS), or myositis. METHODS: Using targeted DNA sequencing, we determined the copy number and genetic variants of C4 in 2,290 well-characterized Scandinavian patients with SLE, primary SS, or myositis and 1,251 healthy controls. RESULTS: A prominent relationship was observed between C4A copy number and the presence of SSA/SSB autoantibodies, which was shared between the 3 diseases. The strongest association was detected in patients with autoantibodies against both SSA and SSB and 0 C4A copies when compared to healthy controls (odds ratio [OR] 18.0 [95% confidence interval (95% CI) 10.2-33.3]), whereas a weaker association was seen in patients without SSA/SSB autoantibodies (OR 3.1 [95% CI 1.7-5.5]). The copy number of C4 correlated positively with C4 plasma levels. Further, a common loss-of-function variant in C4A leading to reduced plasma C4 was more prevalent in SLE patients with a low copy number of C4A. Functionally, we showed that absence of C4A reduced the individuals' capacity to deposit C4b on immune complexes. CONCLUSION: We show that a low C4A copy number is more strongly associated with the autoantibody repertoire than with the clinically defined disease entities. These findings may have implications for understanding the etiopathogenetic mechanisms of systemic inflammatory autoimmune diseases and for patient stratification when taking the genetic profile into account.