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Isolated anti-Ro52 identifies a severe subset of Sjögren’s syndrome patients

ABSTRACT: Serum autoantibodies targeting the SSA/Ro proteins are a key component of the classification criteria for the diagnosis of Sjögren’s syndrome (SS). Most patient serum reacts with both Ro60 and Ro52 proteins. Here we compare the molecular and clinical characteristics of patients diagnosed with SS with anti-Ro52 in the presence or absence of anti-Ro60/La autoantibodies. Patients in the SS biobank at Westmead Hospital that were positive for anti-Ro52 were included and stratified based on the absence (isolated) or presence (combined) of anti-Ro60/La, measured by line immunoassay. We examined clinical associations and the serological and molecular characteristics of anti-Ro52 using ELISA and mass spectrometry in serological groups. A total of 123 SS patients were included for study. SS patients with isolated anti-Ro52 (12%) identified a severe serological subset characterised by higher disease activity, vasculitis, pulmonary involvement, rheumatoid factor (RhF) and cryoglobulinaemia. Anti-Ro52 IgG titre was correlated with RhF. Serum antibodies reacting with Ro52 in the isolated anti-Ro52 subset displayed less isotype switching, less immunoglobulin variable region subfamily usage and a lower degree of somatic hypermutation than the combined anti-Ro52 subset. In our cohort of SS patients, isolated anti-Ro52 represents a severe subset of SS, and is independently associated with the presence of cryoglobulinaemia. We therefore provide clinical relevance to the stratification of SS patients by their sero-reactivities. It is possible that the autoantibody patterns may be immunological epiphenomena of the underlying disease process, and further work is required to unearth the mechanisms of the differential clinical phenotypes.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Serum

DISEASE(S): Sjogren's Syndrome

SUBMITTER: Jing Jing Wang

LAB HEAD: Jing Jing Wang

PROVIDER: PXD038765 | Pride | 2023-03-14

REPOSITORIES: Pride

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Project description:Introduction: Pediatric systemic lupus erythematosus (pSLE) patients often initially present with more active and severe disease than adults, including a higher frequency of lupus nephritis. Specific autoantibodies, including anti-C1q, anti-DNA and anti-alpha-actinin, have been associated with kidney involvement in SLE, and DNA antibodies are capable of initiating early stage lupus nephritis in severe combined immunodeficiency (SCID) mice. Over 100 different autoantibodies have been described in SLE patients, highlighting the need for comprehensive autoantibody profiling. Knowledge of the antibodies associated with pSLE and proliferative nephritis will increase the understanding of SLE pathogenesis, and may aid in monitoring patients for renal flare. Methods: We used autoantigen microarrays composed of 140 recombinant or purified antigens to compare the serum autoantibody profiles of new-onset pSLE patients (n=45) to healthy controls (n=17). We also compared pSLE patients with biopsy-confirmed class III or IV proliferative nephritis (n=23) and without significant renal involvement (n=18). We performed ELISA with selected autoantigens to validate the microarray findings. We created a multiple logistic regression model, based on the ELISA and clinical information, to predict whether a patient had proliferative nephritis, and used a validation cohort (n=23) and longitudinal samples (88 patient visits) to test its accuracy. Results: Fifty autoantibodies were at significantly higher levels in the sera of pSLE patients compared to healthy controls, including anti-B-cell activating factor (BAFF). High levels of anti-BAFF were associated with active disease. Thirteen serum autoantibodies were present at significantly higher levels in pSLE patients with proliferative nephritis than those without, and we confirmed five autoantigens (dsDNA, C1q, collagens IV & X and aggrecan) by ELISA. Our model, based on ELISA measurements and clinical variables, correctly identified patients with proliferative nephritis with 91% accuracy. Conclusions: Autoantigen microarrays are an ideal platform for identifying autoantibodies associated with both pSLE and specific clinical manifestations of pSLE. Using multiple regression analysis to integrate autoantibody and clinical data permits accurate prediction of clinical manifestations with complex etiologies in pSLE.

Project description:We previously reported the establishment of a rabbit (Oryctolagus cuniculus) model of Systemic Lupus Erythematosus (SLE) in which peptide immunization led to lupus-like autoantibody production including anti-Sm, -RNP, -SS-A, -SS-B and -dsDNA. Some neurological symptoms in form of seizures and nystagmus were observed. The animals used in the previous and in the present study were from a National Institute of Allergy and Infectious Diseases colony of rabbits that were pedigreed, immunoglobulin allotype-defined but not inbred. Their genetic heterogeneity may correspond to that found among patients of a given ethnicity. We extended the information about this rabbit model of SLE by microarray based expression profiling. We first demonstrated that human expression arrays could be used with rabbit RNA to yield information on molecular pathways. We then designed a study evaluating gene expression profiles in 8 groups of control and SLE rabbits (46 rabbits in total). Genes significantly upregulated in SLE rabbits were associated with NK cytotoxicity, antigen presentation, leukocyte migration, cytokine activity, protein kinases, RNA spliceosomal ribonucleoproteins, intracellular signaling cascades, and glutamate receptor activity. These results link increased immune activation with up-regulation of components associated with neurological and anti-RNP responses, demonstrating the utility of the rabbit SLE model to uncover biological pathways related to SLE-induced clinical symptoms, including Neuropsychiatric Lupus. Our finding of distinct gene expression patterns in rabbits that made anti-dsDNA compared to those that only made other anti-nuclear antibodies should be further investigated in subsets of SLE patients with different autoantibody profiles. An important goal of biomedical research is to translate basic findings into clinical applications. Models in inbred mice that spontaneously develop SLE, along with various mutant, transgenic and knockout models have documented a variety of genetic defects leading to SLE, but from the clinical perspective, the degree to which these findings using the inbred or homogeneous artificially mutated strains apply to individuals in heterogeneous outbred human populations is open to question. Given that there is still no cure available for SLE, it is important that we continue to explore possibilities using new animal models of SLE including neuropsychiatric lupus (NPSLE). The gene expression study reported here was conducted to expand our understanding of SLE and in particular NPSLE using our rabbit model. We reported earlier that immunization of rabbits with the SM- or GR-MAP peptides led to development of anti-nuclear autoantibodies, including anti-dsDNA, as well as neurological symptoms in the form of seizures and nystagmus in some rabbits. After establishing that it was possible to use the Affymetrix U95 human microarray for the rabbit gene expression studies, through comparative hybridization of identically prepared cRNA from human and rabbit PWBC, the human microarray was used due to lack of rabbit-specific microarrays. We describe unique gene expression changes associated with lupus like serological patterns in immunized rabbits. Our results also demonstrate that caution must be applied when choosing the structure of the carrier Multiple Antigen Peptide (MAP-peptide) for immunization. We discovered that using MAP-4 rather than MAP-8 significantly altered patterns of immune response and gene expression. Currently, microarrays specific for study of gene expression profiles are not available for rabbits. Therefore, we first conducted studies that compared identically prepared rabbit and human cRNA binding to the Affymetrix U95 microarray available for human gene expression analyses. It was determined that the human microarray could be used with rabbit cRNA to yield information on genetic pathways activated and/or suppressed in autoantibody-producing immunized rabbits. In the current report, gene expression profiles of a total of 46 rabbits, from 4 generations within a pedigreed group of control and immunized rabbits, were obtained and analyzed.

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	JW20_E08.mzid.gz	Mzid
	JW20_E08.raw	Raw
	JW20_E08_pSS_B007_2016_anti-Ro52_neat_50kd_T.mgf	Mgf
	JW20_E09.mzid.gz	Mzid
	JW20_E09.raw	Raw

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Isolated anti-Ro52 identifies a severe subset of Sjögren’s syndrome patients

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