Babak Noamani

A discrete cluster of urinary biomarkers discriminates between active systemic lupus erythematosus patients with and without glomerulonephritis

BackgroundManagement of lupus nephritis (LN) would be greatly aided by the discovery of biomarkers that accurately reflect changes in disease activity. Here, we used a proteomics approach to identify potential urinary biomarkers associated with LN.MethodsUrine was obtained from 60 LN patients with paired renal biopsies, 25 active non-LN SLE patients, and 24 healthy controls. Using Luminex, 128 analytes were quantified and normalized to urinary creatinine levels. Data were analyzed by linear modeling and non-parametric statistics, with corrections for multiple comparisons. A second cohort of 33 active LN, 16 active non-LN, and 30 remission LN SLE patients was used to validate the results.ResultsForty-four analytes were identified that were significantly increased in active LN as compared to active non-LN. This included a number of unique proteins (e.g., TIMP-1, PAI-1, PF4, vWF, and IL-15) as well as known candidate LN biomarkers (e.g., adiponectin, sVCAM-1, and IL-6), that differed markedly (>4-fold) between active LN and non-LN, all of which were confirmed in the validation cohort and normalized in remission LN patients. These proteins demonstrated an enhanced ability to discriminate between active LN and non-LN patients over several previously reported biomarkers. Ten proteins were found to significantly correlate with the activity score on renal biopsy, eight of which strongly discriminated between active proliferative and non-proliferative/chronic renal lesions.ConclusionsA number of promising urinary biomarkers that correlate with the presence of active renal disease and/or renal biopsy changes were identified and appear to outperform many of the existing proposed biomarkers.

T Cell and Dendritic Cell Abnormalities Synergize to Expand Pro-Inflammatory T Cell Subsets Leading to Fatal Autoimmunity in B6.NZBc1 Lupus-Prone Mice

We have previously shown that B6 congenic mice with a New Zealand Black chromosome 1 (c1) 96-100 cM interval produce anti-nuclear Abs and that at least two additional genetic loci are required to convert this subclinical disease to fatal glomerulonephritis in mice with a c1 70-100 cM interval (c1(70-100)). Here we show that the number of T follicular helper and IL-21-, IFN-γ-, and IL-17-secreting CD4+ T cells parallels disease severity and the number of susceptibility loci in these mice. Immunization of pre-autoimmune mice with OVA recapitulated these differences. Differentiation of naïve T cells in-vitro under polarizing conditions and in-vivo following adoptive transfer of OVA-specific TCR transgenic cells into c1(70-100) or B6 recipient mice, revealed T cell functional defects leading to increased differentiation of IFN-γ- and IL-17-producing cells in the 96-100 cM and 88-96 cM intervals, respectively. However, in-vivo enhanced differentiation of pro-inflammatory T cell subsets was predominantly restricted to c1(70-100) recipient mice, which demonstrated altered dendritic cell function, with increased production of IL-6 and IL-12. The data provide support for the role of pro-inflammatory T cells in the conversion of subclinical disease to fatal autoimmunity and highlight the importance of synergistic interactions between individual susceptibility loci in this process.

Lack of Interferon and Proinflammatory Cyto/chemokines in Serologically Active Clinically Quiescent Systemic Lupus Erythematosus

Objective. Serologically active clinically quiescent (SACQ) patients with systemic lupus erythematosus (SLE) remain clinically quiescent for prolonged periods despite anti-dsDNA antibodies and/or low complements, indicating the presence of immune complexes. The immune mechanisms leading to this quiescence are unknown. However, in addition to activating complement, immune complex uptake by various cells leads to the production of interferon (IFN)-α and other proinflammatory factors that are also involved in tissue damage. Here we investigate whether production of these factors is reduced in SACQ patients. Methods. The levels of 5 IFN-induced genes and 19 cyto/chemokines were measured in SACQ patients and were compared with those in serologically and clinically active (SACA) and serologically and clinically quiescent (SQCQ) patients. SACQ and SQCQ were defined as ≥ 2 years without clinical activity, with/without persistent serologic activity, respectively, and off corticosteroids/immunosuppressives. SACA was defined as disease activity compelling immunosuppression. Levels of OAS1, IFIT1, MX1, LY6E, and ISG15 were measured by quantitative real-time polymerase chain reaction (PCR) and a composite score (IFN-5) derived from this. Plasma cyto/chemokines were measured by Luminex assay. Nonparametric univariate and logistic regression analyses were conducted. Results. There were no differences in gene expression or cyto/chemokine levels between SACQ and SQCQ patients. The SACQ IFN-5 score was significantly lower than that of SACA (p = 0.003) and was driven by SACQ status, not by autoantibody profile or disease duration. Levels of granulocyte-macrophage colony-stimulating factor, interleukin (IL) 6, IL-10, IFN-γ-inducible protein 10, monocyte chemoattractant protein 1, and tumor necrosis factor-α were significantly lower in SACQ than SACA. Conclusion. The levels of proinflammatory factors in SACQ mirror those of SQCQ patients, indicating reduced production of these factors despite the presence of immune complexes.

Identification of a neutrophil-related gene expression signature that is enriched in adult systemic lupus erythematosus patients with active nephritis: Clinical/pathologic associations and etiologic mechanisms

Both a lack of biomarkers and relatively ineffective treatments constitute impediments to management of lupus nephritis (LN). Here we used gene expression microarrays to contrast the transcriptomic profiles of active SLE patients with and without LN to identify potential biomarkers for this condition. RNA isolated from whole peripheral blood of active SLE patients was used for transcriptomic profiling and the data analyzed by linear modeling, with corrections for multiple testing. Results were validated in a second cohort of SLE patients, using NanoString technology. The majority of genes demonstrating altered transcript abundance between patients with and without LN were neutrophil-related. Findings in the validation cohort confirmed this observation and showed that levels of RNA abundance in renal remission were similar to active patients without LN. In secondary analyses, RNA abundance correlated with disease activity, hematuria and proteinuria, but not renal biopsy changes. As abundance levels of the individual transcripts correlated strongly with each other, a composite neutrophil score was generated by summing all levels before examining additional correlations. There was a modest correlation between the neutrophil score and the blood neutrophil count, which was largely driven by the dose of glucocorticosteroids and not the proportion of low density and/or activated neutrophils. Analysis of longitudinal data revealed no correlation between baseline neutrophil score or changes over the first year of follow-up with subsequent renal flare or treatment outcomes, respectively. The findings argue that although the neutrophil score is associated with LN, its clinical utility as a biomarker may be limited.

Presence of an interferon signature in individuals who are anti-nuclear antibody positive lacking a systemic autoimmune rheumatic disease diagnosis

BackgroundElevated levels of type I interferons (IFNs) are a characteristic feature of the systemic autoimmune rheumatic diseases (SARDs) and are thought to play an important pathogenic role. However, it is unknown whether these elevations are seen in anti-nuclear antibody–positive (ANA+) individuals who lack sufficient criteria for a SARD diagnosis. We examined IFN-induced gene expression in asymptomatic ANA+ individuals and patients with undifferentiated connective tissue disease (UCTD) to address this question.MethodsHealthy ANA− control subjects and ANA+ titre (≥1:160 by immunofluorescence) participants meeting no criteria, meeting at least one criterion (UCTD) or meeting SARD classification criteria were recruited. Whole peripheral blood IFN-induced and BAFF gene expression were quantified using NanoString technology. The normalized levels of five IFN-induced genes were summed to produce an IFN5 score.ResultsThe mean IFN5 scores were increased in all ANA+ participant subsets as compared with healthy control subjects. We found that 36.8% of asymptomatic ANA+ and 50% of UCTD participants had IFN5 scores >2 SD above the mean for healthy control subjects. In all ANA+ subsets, the IFN5 score correlated with the presence of anti-Ro/La antibodies. In the asymptomatic ANA+ subset, this score also correlated with the ANA titre, whereas in the other ANA+ subsets, it correlated with the number of different ANA specificities. Development of new SARD criteria was seen in individuals with normal and high IFN5 scores.ConclusionsAn IFN signature is seen in a significant proportion of ANA+ individuals and appears to be associated with ANA titre and type of autoantibodies, rather than with the presence or development of clinical SARD symptoms.

AI-08 B cell phenotypic changes in anti-nuclear antibody positive individuals prior to the onset of systemic autoimmune rheumatic disease

Background Patients with systemic autoimmune rheumatic diseases (SARD) often have a prolonged pre-clinical phase during which they are anti-nuclear antibody (ANA)+ but lack clinical symptoms. Here we sought to determine whether ANA+ individuals who lack sufficient symptoms for a SARD diagnosis share the B cell phenotypic changes seen in SARD. Materials and methods Healthy controls (HC) and ANA+ individuals who: 1) lacked clinical symptoms of SARD (ANS); 2) had at least one clinical symptom of SARD (UCTD); or 3) had recently diagnosed steroid and immunosuppressive naïve SARD (SLE, SS, SSc, MCTD, DM) were recruited. PBMCs were stained with various combinations of fluorescently labelled antibodies and analysed by flow cytometry. Anti-nuclear antibodies were measured through the hospital laboratory. Whole blood IFN signature and BAFF RNA levels were measured by NanoString. Results B cell phenotypes were examined for 32 HC, 38 ANS, 28 UCTD, and 59 early SARD patients. Patients with early SARD had a number of changes in their naïve and memory B cell subsets including: increased proportions of mature naïve (SSc) and T1T2 cells (SLE and SS), and decreased proportions of switched memory cells (all SARD). Similar decreases in the proportion of switched memory B cells were seen in ANS and UCTD patients, and as seen for the SARD patients, these cells were activated with elevated levels of CD86 as compared to HC. Significantly increased activation of the CD27-IgD- memory compartment was also seen in ANS, UCTD, SLE and SjD patients. Although significantly increased proportions of plasmablasts and/or CD138+ plasma cells were seen in early SARD patients, these were not seen in ANS and UCTD patients. Nevertheless, in pre-SARD individuals (ANS + UCTD) there was a significant positive correlation between the size of these cell subsets and ANA titer as well as the number of different anti-nuclear antibody specificities. As observed for early SARD patients, there was a trend to increased BAFF levels as compared to HC in pre-SARD individuals, which achieved statistical significance in UCTD patients. However, there was no association between the levels of BAFF and any of the B cell phenotypes, whereas the IFN signature was positively associated with the proportion of T1T2 cells. Conclusions B cell phenotypic abnormalities precede the onset of clinical disease in ANA+ individuals and have a pattern suggesting ongoing activation through T-B collaboration.

FRI0307 Changes in Urinary Biomarker Levels Can Predict Treatment Responses in Lupus Nephritis

Background The relapsing and remitting nature of lupus nephritis (LN) poses a challenge to clinicians who must balance the risk of long term kidney damage with the side effects of treatment. Management of this condition would be greatly aided by the identification of biomarkers that accurately reflect and predict treatment responses. We previously identified and validated a panel of urinary biomarkers that are specifically elevated in SLE patients with active LN as compared to active patients without LN or LN patients in remission. Objectives To determine whether changes in the levels of these urinary biomarkers predict treatment responses. Methods 21 SLE patients with biopsy-proven LN were followed longitudinally for a minimum of 2 years after treatment. Levels of 15 urinary biomarkers including Clusterin, Cystatin C, NGAL, PF4, vWF,sVCAM-1, GM-CSF, GRO, IL-15, IL-6, MCP-1, Adiponectin, PAI-1, MMP-7, and TIMP-1, were measured by Luminex. Patients were classified as having a complete response (n=12), partial response (n=4), or treatment (Tx) failure (n=5) at 2 years following initiation of treatment, based upon previously established criteria. Urinary biomarker levels were considered abnormal if they were >2 SD above the mean for 24 healthy controls. Data were analyzed using non-parametric statistics. Results At 3–6 months following treatment, the changes in biomarker levels from the first visit were not significantly different between complete responders and partial responders or Tx failures. However, at 1 year (11–16 months) following treatment, 5 urinary biomarkers, sVCAM-1, Adiponectin, IL-15, vWF, and MCP-1, demonstrated significantly different changes from baseline in complete responders as compared to Tx failures, with the majority of responders demonstrating improvement and the majority of Tx failures demonstrating worsening. As urinary biomarker levels not corrected for urinary osmolality correlated best with clinical outcomes, subsequent analyses were done with the uncorrected values. Normalization of urinary Adiponectin by 11–16 months was the best predictor of a complete response, with 11 of 12 patients who normalized being complete responders and 1 a partial responder. Similar but slightly less discriminative results were obtained for the other 4 urinary biomarkers. Conversely, the presence of an abnormal urinary vWF at 11–16 months was the strongest predictor of an adverse outcome with 4 of 6 patients with abnormal levels being Tx failures. Notably, all patients that were Tx failures that had normal levels of urinary biomarkers at 11–16 months subsequently developed abnormal levels, whereas patients who were complete responders eventually normalized the majority of these 5 biomarkers. Partial responders demonstrated normalization with delayed kinetics. Conclusions Measurement of urinary biomarkers can provide valuable insight into treatment responses in lupus nephritis. Disclosure of Interest None declared

THU0265 B Cell Phenotypic Changes in anti-Nuclear Antibody Positive Individuals Prior To The Onset of Systemic Autoimmune Rheumatic Disease

Background Patients with systemic autoimmune rheumatic diseases (SARD) often have a prolonged pre-clinical phase during which they are anti-nuclear antibody (ANA)+ but lack clinical symptoms. It has been proposed that progression from asymptomatic autoimmunity to clinical disease is accompanied by immunologic changes that could be used as predictors of disease development. Previous studies indicate that a number of B cell phenotypic changes are seen in SARD patients including changes in the proportions of various naïve and memory B cell subsets, increased B cell activation and elevated levels of plasmablasts/plasma cells. Objectives To determine whether ANA+ individuals who lack sufficient symptoms for a SARD diagnosis have B cell phenotypic changes similar to those seen in SARD. Methods Healthy controls (HC) and ANA+ individuals who: 1) lacked clinical symptoms of SARD (ANS); 2) had at least one clinical symptom of SARD (UCTD); or 3) had a recently diagnosed steroid and immunosuppressive naïve SARD (SLE, SS, SSc, MCTD, DM) were recruited. PBMCs were isolated over a Ficoll gradient, stained with various combinations of fluorescently labeled antibodies and analyzed by flow cytometry. Anti-nuclear antibodies were measured through the hospital laboratory. Whole blood IFN signature and BAFF RNA levels were measured by NanoString. Results B cell phenotypes were examined for 32 HC, 38 ANS, 28 UCTD, and 59 early SARD (24 SS, 26 SSc, 6 SLE, 2 MCTD, 1 DM) patients. Patients with early SARD had a number of changes in their naïve and memory B cell subsets, as previously reported for patients with established disease, including: increased proportions of mature naïve B cells (SSc); increased proportions of T1T2 cells (SLE and SS); and trends to decreased proportions of switched memory B cells (CD27+IgD–) in all SARD. Similar decreases in the proportion of switched memory B cells were seen in ANS and UCTD patients, and as seen for the SARD patients, these cells were activated with elevated levels of CD86 as compared to HC. Significantly increased activation of the CD27–IgD– memory compartment was also seen in ANS, UCTD, SLE and SjD patients. Although significantly increased proportions of plasmablasts and/or CD138+ plasma cells were seen in all SARD patients (except those with SSc), these were not seen in ANS and UCTD patients. Nevertheless, in pre-SARD individuals (ANS + UCTD) there was a significant positive correlation between the size of these cell subsets, as well as the proportion of T1T2 cells, and ANA titer and the number of different anti-nuclear antibodies specificities. As observed for early SARD patients, there was a trend to increased BAFF levels as compared to HC in pre-SARD individuals, which achieved statistical significance in UCTD patients. However, there was no association between the levels of BAFF and any of the B cell phenotypes, whereas the IFN signature was positively associated with the proportion of T1T2 cells. Conclusions B cell phenotypic abnormalities precede the onset of clinical disease in ANA+ individuals and have a pattern suggesting ongoing activation through T-B collaboration. Disclosure of Interest None declared

FRI0004 A Progressive Stepwise Accrual of T Cell Abnormalities Marks the Transition from Benign to Symptomatic Autoimmunity

Background The systemic autoimmune rheumatic diseases (SARD; Systemic Lupus Erythematosus, Rheumatoid Arthritis, Sjogrens Disease, Systemic Sclerosis) are proposed to have a prolonged period of pre-clinical autoimmunity culminating in clinical disease. Evidence from disease-specific studies (e.g., SLE, RA) suggests that the pre-clinical phase is marked by the accrual of immunological abnormalities such as pathogenic auto-antibodies (auto-Ab). Little is known about the cellular derangements that accompany the transition from benign to pathological autoimmunity. Abnormalities in T cell subsets including invariant NKT (iNKT) and T follicular helper (TFH) cells are implicated in the development of systemic autoimmunity. Both a decrease in iNKT cells and an increase in TFH cells are found in patients with established SARD. Objectives To determine if T cell abnormalities associated with SARD are present in pre-clinical autoimmunity. Methods Patients (n=87) who were ANA+ (titer ≥1:160) and healthy controls (HC, n=37) were recruited. Patients were stratified into clinical subsets: (1) no defining SARD symptoms (n=24); (2) undifferentiated connective tissue disease (UCTD, n=17), one or more SARD defining symptom; and (3) early SARD (n=46), fulfilling ACR criteria for a SARD diagnosis. Patients were immunosuppressive and steroid naïve. PBMCs were isolated over a Ficoll gradient, stained with combinations of fluorescently labeled antibodies and analyzed by flow cytometry. ANA titer and auto-Ab profile were determined. Results A statistically significant decrease in the proportion of iNKT cells (CD3+Vα24Jα18 TCR+) was found for all ANA+ patients relative to HC (p=0.0001). Similar results were found for each patient subset when compared to HC; ANA+ asymptomatic (p=0.001), UCTD (p=0.02) and SARD (p=0.001), with no differences amongst groups. The proportion of TFH (CD4+CXCR5highPD-1high) cells was significantly elevated (p=0.01) in patients versus HC. While the proportion of TFH cells was similar between asymptomatic ANA+ patients and HC, there was a significant expansion of TFH in UCTD as compared to both groups (p=0.02 and p=0.04, respectively). SARD patients had a non-significant trend to increased proportions of TFH as compared to UCTD. Patients (n=50) with higher ANA titers (≥1:640) had a significant increase (p=001) in TFH when compared to individuals (n=13) with lower ANA levels (1:160). A positive correlation (p<0.0001) between the proportion of TFH and the number of auto-Abs within the patient population was found. No significant differences were noted in the iNKT or TFH cell proportions between SARD patients stratified by specific disease diagnosis. Conclusions A decrease in the iNKT cell subset is present at the earliest phase of pre-clinical autoimmunity (asymptomatic ANA+) suggesting that loss of this T cell subset contributes to a breach of tolerance to nuclear antigens. In contrast, increases in the TFH compartment appear to parallel the onset of clinical symptoms and accrual of auto-Abs. These results suggest that an incremental development of T cell abnormalities marks the progression towards clinically significant autoimmunity. Disclosure of Interest None declared

OP0078 Presence of an Interferon Signature in Anti-Nuclear Antibody Positive Individuals Prior to the Onset of Systemic Autoimmune Rheumatic Disease

Background Patients with systemic autoimmune rheumatic diseases (SARD) often have a prolonged pre-clinical phase during which they are anti-nuclear antibody (ANA)+ but lack clinical symptoms. It has been proposed that progression from asymptomatic autoimmunity to clinical disease is accompanied by immunologic changes that could be used as predictors of disease development. Elevated levels of interferon (IFN)-induced gene expression, termed the IFN signature, are found in several SARD conditions, and IFNs appear to play an important role in disease pathogenesis. Objectives To determine whether ANA+ individuals who lack sufficient symptoms for a SARD diagnosis share the IFN-signature. Methods ANA+ individuals who: 1) lacked clinical symptoms of SARD (ANS); 2) had a least one clinical symptom of SARD (Undifferentiated Connective Tissue Disease, UCTD); or 3) had a recently diagnosed SARD (Systemic Lupus Erythematosus, SLE; Sjogrens Disease, SjD; Scleroderma, SSc; Mixed Connective Tissue Disease, MCTD; Dermatomyositis, DM) were recruited from clinics at UHN/MSH hospitals. None of the patients were on corticosteroids or DMARDs with the exception of hydroxychloroquine. Healthy controls (HC) were also recruited. RNA was prepared from blood archived in Tempus tubes. Expression of 5 IFN-induced genes was quantified by Nanostring, normalized to expression of housekeeping genes, and summed to generate an IFN5 score. ANAs and levels of specific autoantibodies were measured by the hospital laboratory. Results To date we have measured the IFN signature on 95 individuals (21 HC, 21 ANS, 16 UCTD, 22 SjD, 7 SSc, 6 SLE, 1 MCTD, 1 DM). There was a trend to higher mean IFN score in all groups as compared to HC (mean ± SD: HC 7,071±6,321; ANS 27,245±36,037; UCTD 27,624±24,827; SSc 34,940±40,940; SjD 61,877±33,404; SLE 62,769±50,233; MCTD/DM 97,716±24,973), which achieved statistical significance for ANS, UCTD, SjD, and SLE (corrected p=0.044, 0.003, <0.0001, 0.0075, respectively). Using a cutoff of 2 SD above the mean of HC as indicative of an elevated IFN5 score; 8/21 ANS, 8/16 UCTD, 3/7 SSc, 18/22 SjD, 5/6 SLE, and 2/2 MCTD/DM participants had elevated IFN levels. Marked elevations of the IFN5 score were seen in a subset of ANS and UCTD participants, which could not be attributed to recent infection. Although there was a significant correlation between the ANA titer (p=0.002) and IFN5 score for all ANA+ individuals, this was not seen in the ANS or UCTD subsets of this population. However the IFN5 score was positively correlated with the number of different ANA specificities present in the UCTD subset (p=0.048) and all ANA+ individuals (p<0.0001). Within the ANS subset, there was a strong correlation between the presence of anti-Ro/La antibodies with 6/8 IFN5 high as compared to 1/13 IFN low individuals being antibody positive (p=0.003). Conclusions An IFN signature is seen in a subset of ANA+ individuals prior to a confirmed diagnosis of SARD and appears to correlate with the type and number of specific ANAs rather than onset of clinical disease. Disclosure of Interest None declared