Arash Zandian

Anoctamin 2 identified as an autoimmune target in multiple sclerosis

Significance Despite the growing evidence that autoantibodies are team players in the pathogenesis of multiple sclerosis (MS), the target autoantigens are yet to be identified. In this work, we mined the autoantibody repertoire within MS by screening more than 2,000 plasma samples from patients with MS and controls and identified increased autoantibody reactivity against an ion-channel protein called “anoctamin 2” (ANO2). This finding points toward an ANO2 autoimmune sub-phenotype in MS and might contribute to the development of clinical algorithms to characterize a subgroup of MS patients. Multiple sclerosis (MS) is the most common chronic inflammatory disease of the central nervous system and also is regarded as an autoimmune condition. However, the antigenic targets of the autoimmune response in MS have not yet been deciphered. In an effort to mine the autoantibody repertoire within MS, we profiled 2,169 plasma samples from MS cases and population-based controls using bead arrays built with 384 human protein fragments selected from an initial screening with 11,520 antigens. Our data revealed prominently increased autoantibody reactivity against the chloride-channel protein anoctamin 2 (ANO2) in MS cases compared with controls. This finding was corroborated in independent assays with alternative protein constructs and by epitope mapping with peptides covering the identified region of ANO2. Additionally, we found a strong interaction between the presence of ANO2 autoantibodies and the HLA complex MS-associated DRB1*15 allele, reinforcing a potential role for ANO2 autoreactivity in MS etiopathogenesis. Furthermore, immunofluorescence analysis in human MS brain tissue showed ANO2 expression as small cellular aggregates near and inside MS lesions. Thus this study represents one of the largest efforts to characterize the autoantibody repertoire within MS. The findings presented here demonstrate that an ANO2 autoimmune subphenotype may exist in MS and lay the groundwork for further studies focusing on the pathogenic role of ANO2 autoantibodies in MS.

Whole-Proteome Peptide Microarrays for Profiling Autoantibody Repertoires within Multiple Sclerosis and Narcolepsy

The underlying molecular mechanisms of autoimmune diseases are poorly understood. To unravel the autoimmune processes across diseases, comprehensive and unbiased analyses of proteins targets recognized by the adaptive immune system are needed. Here we present an approach starting from high-density peptide arrays to characterize autoantibody repertoires and to identify new autoantigens. A set of ten plasma and serum samples from subjects with multiple sclerosis, narcolepsy, and without any disease diagnosis were profiled on a peptide array representing the whole proteome, hosting 2.2 million 12-mer peptides with a six amino acid lateral shift. On the basis of the IgG reactivities found on these whole-proteome peptide microarrays, a set of 23 samples was then studied on a targeted array with 174 000 12-mer peptides of single amino acid lateral shift. Finally, verification of IgG reactivities was conducted with a larger sample set (n = 448) using the bead-based peptide microarrays. The presented workflow employed three different peptide microarray formats to discover and resolve the epitopes of human autoantibodies and revealed two potentially new autoantigens: MAP3K7 in multiple sclerosis and NRXN1 in narcolepsy. The presented strategy provides insights into antibody repertoire reactivity at a peptide level and may accelerate the discovery and validation of autoantigens in human diseases.

Dysregulations in circulating sphingolipids associate with disease activity indices in female patients with systemic lupus erythematosus: a cross-sectional study

Objective The objective of this study was to investigate the association of clinical and renal disease activity with circulating sphingolipids in patients with systemic lupus erythematosus. Methods We used liquid chromatography tandem mass spectrometry to measure the levels of 27 sphingolipids in plasma from 107 female systemic lupus erythematosus patients and 23 controls selected using a design of experiment approach. We investigated the associations between sphingolipids and two disease activity indices, the Systemic Lupus Activity Measurement and the Systemic Lupus Erythematosus Disease Activity Index. Damage was scored according to the Systemic Lupus International Collaborating Clinics damage index. Renal activity was evaluated with the British Island Lupus Activity Group index. The effects of immunosuppressive treatment on sphingolipid levels were evaluated before and after treatment in 22 female systemic lupus erythematosus patients with active disease. Results Circulating sphingolipids from the ceramide and hexosylceramide families were increased, and sphingoid bases were decreased, in systemic lupus erythematosus patients compared to controls. The ratio of C16:0-ceramide to sphingosine-1-phosphate was the best discriminator between patients and controls, with an area under the receiver-operating curve of 0.77. The C16:0-ceramide to sphingosine-1-phosphate ratio was associated with ongoing disease activity according to the Systemic Lupus Activity Measurement and the Systemic Lupus Erythematosus Disease Activity Index, but not with accumulated damage according to the Systemic Lupus International Collaborating Clinics Damage Index. Levels of C16:0- and C24:1-hexosylceramides were able to discriminate patients with current versus inactive/no renal involvement. All dysregulated sphingolipids were normalized after immunosuppressive treatment. Conclusion We provide evidence that sphingolipids are dysregulated in systemic lupus erythematosus and associated with disease activity. This study demonstrates the utility of simultaneously targeting multiple components of a pathway to establish disease associations.

Untargeted screening for novel autoantibodies with prognostic value in first-episode psychosis

Immunological and inflammatory reactions have been suggested to have a role in the development of schizophrenia, a hypothesis that has recently been supported by genetic data. The aim of our study was to perform an unbiased search for autoantibodies in patients with a first psychotic episode, and to explore the association between any seroreactivity and the development of a Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) disorder characterized by chronic or relapsing psychotic symptoms. We collected plasma samples from 53 patients when they were treated for their first-episode psychosis, and 41 non-psychotic controls, after which the patients were followed for a mean duration of 7 years. Thirty patients were diagnosed with schizophrenia, delusional disorder, schizoaffective disorder, bipolar disorder or a long-term unspecified nonorganic psychosis during follow-up, whereas 23 patients achieved complete remission. At the end of follow-up, plasma samples were analyzed for IgG reactivity to 2304 fragments of human proteins using a multiplexed affinity proteomic technique. Eight patient samples showed autoreactivity to the N-terminal fragment of the PAGE (P antigen) protein family (PAGE2B/PAGE2/PAGE5), whereas no such autoreactivity was seen among the controls. PAGE autoreactivity was associated with a significantly increased risk of being diagnosed with schizophrenia during follow-up (odds ratio 6.7, relative risk 4.6). An immunohistochemistry analysis using antisera raised against the N-terminal fragment stained an unknown extracellular target in human cortical brain tissue. Our findings suggest that autoreactivity to the N-terminal portion of the PAGE protein family is associated with schizophrenia in a subset of patients with first-episode psychosis.

Antibodies in children with malaria to PfEMP1, RIFIN and SURFIN expressed at the Plasmodium falciparum parasitized red blood cell surface

Naturally acquired antibodies to proteins expressed on the Plasmodium falciparum parasitized red blood cell (pRBC) surface steer the course of a malaria infection by reducing sequestration and stimulating phagocytosis of pRBC. Here we have studied a selection of proteins representing three different parasite gene families employing a well-characterized parasite with a severe malaria phenotype (FCR3S1.2). The presence of naturally acquired antibodies, impact on rosetting rate, surface reactivity and opsonization for phagocytosis in relation to different blood groups of the ABO system were assessed in a set of sera from children with mild or complicated malaria from an endemic area. We show that the naturally acquired immune responses, developed during malaria natural infection, have limited access to the pRBCs inside a blood group A rosette. The data also indicate that SURFIN4.2 may have a function at the pRBC surface, particularly during rosette formation, this role however needs to be further validated. Our results also indicate epitopes differentially recognized by rosette-disrupting antibodies on a peptide array. Antibodies towards parasite-derived proteins such as PfEMP1, RIFIN and SURFIN in combination with host factors, essentially the ABO blood group of a malaria patient, are suggested to determine the outcome of a malaria infection.

SURGE complex of Plasmodium falciparum in the rhoptry-neck (SURFIN4.2-RON4-GLURP) contributes to merozoite invasion

Plasmodium falciparum invasion into red blood cells (RBCs) is a complex process engaging proteins on the merozoite surface and those contained and sequentially released from the apical organelles (micronemes and rhoptries). Fundamental to invasion is the formation of a moving junction (MJ), a region of close apposition of the merozoite and the RBC plasma membranes, through which the merozoite draws itself before settling into a newly formed parasitophorous vacuole (PV). SURFIN4.2 was identified at the surface of the parasitized RBCs (pRBCs) but was also found apically associated with the merozoite. Using antibodies against the N-terminus of the protein we show the presence of SURFIN4.2 in the neck of the rhoptries, its secretion into the PV and shedding into the culture supernatant upon schizont rupture. Using immunoprecipitation followed by mass spectrometry we describe here a novel protein complex we have named SURGE where SURFIN4.2 forms interacts with the rhoptry neck protein 4 (RON4) and the Glutamate Rich Protein (GLURP). The N-terminal cysteine-rich–domain (CRD) of SURFIN4.2 mediates binding to the RBC membrane and its interaction with RON4 suggests its involvement in the contact between the merozoite apex and the RBC at the MJ. Supporting this suggestion, we also found that polyclonal antibodies to the extracellular domain (including the CRD) of SURFIN4.2 partially inhibit merozoite invasion. We propose that the formation of the SURGE complex participates in the establishment of parasite infection within the PV and the RBCs.

Epitopes of anti-RIFIN antibodies and characterization of rif -expressing Plasmodium falciparum parasites by RNA sequencing

Variable surface antigens of Plasmodium falciparum have been a major research focus since they facilitate parasite sequestration and give rise to deadly malaria complications. Coupled with its potential use as a vaccine candidate, the recent suggestion that the repetitive interspersed families of polypeptides (RIFINs) mediate blood group A rosetting and influence blood group distribution has raised the research profile of these adhesins. Nevertheless, detailed investigations into the functions of this highly diverse multigene family remain hampered by the limited number of validated reagents. In this study, we assess the specificities of three promising polyclonal anti-RIFIN antibodies that were IgG-purified from sera of immunized animals. Their epitope regions were mapped using a 175,000-peptide microarray holding overlapping peptides of the P. falciparum variable surface antigens. Through immunoblotting and immunofluorescence imaging, we show that different antibodies give varying results in different applications/assays. Finally, we authenticate the antibody-based detection of RIFINs in two previously uncharacterized non-rosetting parasite lines by identifying the dominant rif transcripts using RNA sequencing.

05.01 Protein profiling in plasma reveals molecular subgroups in systemic lupus erythematosus

Objective Systemic Lupus Erythematosus (SLE) is a heterogeneous systemic autoimmune disease that is currently lacking specific diagnostic biomarkers. The diversity within the patients might obstruct clinical trials and could reflect differences in underlying pathogenesis. Our objective was to identify protein profiles that could be used for diagnosis and to identify molecular subgroups within SLE for patient stratification subjected to different treatment. Method In a cross-sectional study we performed protein profiling of 695 plasma samples from SLE patients and matched controls. This was achieved by utilising an antibody suspension bead array targeting 367 proteins. T-test and ROC analysis was performed to identify differences in the protein profiles between SLE and controls. Unsupervised K-means clustering was performed to identify data-driven SLE subgroups. Results We report that the novel proteins MMP1, SELE, and S100A12 can distinguish between SLE patients and controls with an area under curve of 0.80 in a ROC analysis. In addition, 28 proteins were found to show differences (corrected p-value<0.05) between SLE patients and controls. By unsupervised clustering we identified an IRF5, NOS3 and CLDN8-driven subgroup, an ARID2 and SELE-driven subgroup and one subgroup characterised by low SLC22A2 levels. Conclusion We have identified potential biomarkers of SLE that may be used to improve the diagnosis of SLE patients. Our suggested panel of biomarkers needs to be validated in an additional SLE cohort and also in relation to other systemic autoimmune diseases before it can be used as a diagnostic test. We have also identified subgroups characterised by different molecular patterns, indicating underlying pathogenic differences. These patient groups might benefit from different treatment strategies. Our work adds new information to today’s view of classifying the heterogeneous subgroups within SLE and the importance of personalised medicine.

A6.15 Characterisation of systemic lupus erythematosus subgroups with features of antiphospholipid or sjögrens´s syndrome utilising affinity proteomics

Background and objectives Systemic Lupus Erythematosus (SLE) is an autoimmune disease that covers a wide range of phenotypes, from subtle symptoms to life-threatening conditions. The heterogeneous presentation of SLE is a major obstacle in clinical trials and the lack of biomarkers also hampers accurate diagnosis and choice of treatment. In this study we explored biochemical pathways in two suggested subgroups of SLE utilising affinity proteomics in order to characterise subgroups and identify biomarkers for personalised medicine. Materials and methods We have utilised Karolinska SLE cohort consisting of 320 well-characterised SLE patients and 320 individually matched population based controls in a cross-sectional study. SLE subgroups were defined only based on patients´ autoantibody profile: an Antiphospholipid Syndrome-like (APS-like, n = 55) and a Sjögrens Syndrome-like (SS-like, n = 58) subgroup. Based on literature and clinical knowledge we have made a targeted selection of 281 proteins targeted by in total 367 antibodies from the Human Protein Atlas. Antibodies were covalently coupled to colour-coded magnetic beads. EDTA-plasma samples were labelled with biotin and screened in a 384 bead array format. The read-out was made by adding a streptavidin fluorophore and using the Luminex-technology. Results We identified differences in protein profiles comparing APS-like and SS-like SLE subgroups: The top most significantly different proteins were Integrin beta 1 (p = 9.8e-8, Log2 fold change=1.5), renin (p = 2.7e-5, Log2 fold change=0.5), glutamic-oxaloacetic transaminase 1 (p = 2.9e-5, Log2 fold change=0.5) and Apolipoprotein M (p = 3.4e-5, Log2 fold change=0.3) that all were increased in SS-like SLE, while Apolipoprotein H (β2-GPI, p = 6.6e-5, Log2 fold change=-0.3) were increased in the APS-like subgroup. Conclusions The two suggested SLE subgroups were defined exclusively on autoantibody profile as the APS-like and the SS-like subgroups. Our results demonstrate that the two subgroups differ in their protein profiles and that these observations indicate underlying pathogenic differences between SS-like and APS-like SLE. A common aetiology of the presence of β2-GPI in both APS and in APS-like SLE is suggested. Therefore, we believe that stratification of SLE patients should be further explored towards personalised medicine.