Angela Midgley

Urinary Monocyte Chemoattractant protein 1 and Alpha-1-acid glycoprotein as biomarkers of renal disease activity in Juvenile-onset Systemic Lupus Erythematosus

A higher proportion of patients with juvenile-onset systemic lupus erythematosus (JSLE) will have renal involvement compared with adult-onset disease, some progressing to renal failure in adulthood. Histological examination is the gold standard for diagnosing lupus nephritis (LN), but its invasive nature limits routine use. Using cross-sectional cohort analysis, we aimed to determine whether urinary concentrations of monocyte chemoattractant protein-1 (MCP1), alpha-1-acid glycoprotein (AGP) and interferon-inducible protein 10 (IP10) are biomarkers of active LN. Sixty JSLE patients recruited to the UK JSLE Cohort Study were categorized according to the British Isles Lupus Assessment Group (BILAG) activity index. Patients with active renal JSLE (n = 8; renal BILAG score A, B) had significantly higher urinary MCP1 concentrations than patients with inactive renal disease (n = 52; renal BILAG score C, D, E; 582 pg/mg creatinine [Cr], 207 pg/mg Cr; p = 0.018) or healthy controls (n = 23; 117 pg/mg Cr; p = 0.005). Urinary AGP concentration was significantly elevated in patients with active renal disease compared with inactive renal disease (1517 ng/mg Cr, 485 ng/mg Cr; p = 0.027) or healthy controls (313 ng/mg Cr; p = 0.013). Urinary IP10 concentration was not significantly different between groups, but did strongly correlate with uMCP and uAGP levels (rho = 0.38, p = 0.009; rho = 0.33, p = 0.021). Urinary MCP1 and AGP are biomarkers of LN, providing insight into its pathophysiology. Longitudinal studies are warranted.

New insights into the pathogenesis and management of lupus in children

Systemic lupus erythematosus (SLE) is the archetypal systemic autoimmune disease, characterised by inflammation causing a wide spectrum of major clinical manifestations that may affect any organ. Childhood-onset SLE (cSLE) is more severe with greater damage and drug burden than adult-onset SLE. Understanding the pathogenesis of cSLE is a key step in directing medical management. The dysregulated immune system, that in health is usually vital in protecting the body from infection, contributes significantly to the disease process. Improved knowledge of disease mechanism will help to identify potential targets for novel agents and the identification of new biomarkers of disease activity. This review will present current knowledge of the innate and adaptive immune responses in cSLE and the optimal patient management that aims to control the disease. Innate immune dysregulation includes the overexpression of interferon-α, dendritic cell activation, neutrophil extracellular traps and phagocyte abnormalities. The classical adaptive immune system is over activated in lupus with excessive autoantibody production due to abnormalities in B and T cell regulation. Novel biologic medications are being developed to specifically target these areas with the ultimate aim of improving the long-term outlook and quality of life for children living with Lupus.

Increased expression of low density granulocytes in juvenile-onset systemic lupus erythematosus patients correlates with disease activity

Neutrophils are implicated in a wide range of non-infectious inflammatory conditions. A subset of neutrophils in the peripheral circulation of systemic lupus erythematosus (SLE) patients has been described and termed low density granulocytes (LDGs). This study investigates the expression of LDG in juvenile-onset SLE (JSLE) patients compared to controls, and any correlations with disease activity. Neutrophils and LDGs were isolated from JSLE (n = 13) and paediatric non-inflammatory control patients (n = 12). Cell populations were assessed and compared using flow cytometry and morphological analysis. Standard clinical data, which included disease activity markers/scores, were collected for each patient. Significantly increased LDG expression (%mean ± SEM, range) was observed in JSLE patients (10.4 ± 3.26, 3.41–36.3) compared to controls (2.4 ± 0.44, 0.36–5.27; p = 0.005). A statistically significant positive correlation was observed between LDG expression and the British Isles Lupus Activity Group (correlation coefficient 0.685; p = 0.010) and SLE Disease Activity Index (correlation coefficient 0.567; p = 0.043) and the biomarker of dsDNA-antibodies (correlation coefficient 0.590; p = 0.043). Here we observe increased expression in LDGs in JSLE patients, which correlate with dsDNA antibody concentration and scores of disease activity. These correlations indicate that the increased LDG expression observed in this study may have a potential role in the pathogenesis of JSLE, and may be a useful biomarker.

Expression of Toll-like receptors and their detection of nuclear self-antigen leading to immune activation in JSLE

OBJECTIVES Toll-like receptors (TLRs) essential in the functioning of the immune system have been implicated in the development of autoimmunity. TLR3, 7, 8 and 9 are capable of recognizing nucleic autoantigens typical of SLE. Their expression correlates positively with disease activity in adult-onset SLE. This study aimed to determine the role of TLRs in JSLE and whether apoptotic neutrophils are a source of nuclear autoantigen being detected through TLR3, 7, 8 and 9, leading to an inflammatory response. METHODS TLR3, 7, 8 and 9 mRNA and protein expression were measured in peripheral blood mononuclear cells (PBMCs) in JSLE patients compared with JIA and non-inflammatory controls. Activation of the TLRs by JSLE serum-induced apoptotic neutrophils was detected by measuring IFN-α mRNA and protein expression, and confirmed using myeloid differentiation factor 88 (MyD88) and TIR domain-containing adapter-inducing IFN-β (TRIF) inhibitors. RESULTS JSLE patients have increased TLR3, 8 and 9 mRNA and protein expression compared with controls (P < 0.05). Incubation of PBMCs with apoptotic neutrophils demonstrated a dose-response relationship for IFN-α mRNA expression. Inhibition of TLR signalling by blocking MyD88 and TRIF signalling decreased IFN-α mRNA expression in PBMCs incubated with apoptotic neutrophils (P < 0.05). CONCLUSIONS This study demonstrated significantly increased TLR expression in JSLE compared with controls. Our data indicate that apoptotic neutrophils trigger TLR activation through their presentation of autoantigens. The role of TLRs in this inflammatory response was demonstrated by a dose-response relationship to apoptotic neutrophil concentration and confirmed by a decrease in IFN-α production after inhibition of TLR signalling.

Increased soluble phagocytic receptors sMer, sTyro3 and sAxl and reduced phagocytosis in Juvenile-onset Systemic Lupus Erythematosus

BackgroundThe TAM-receptor tyrosine kinase family, Tyro3, Axl and Mer are key to apoptotic cell clearance. Reduced phagocytic clearance in systemic lupus erythematosus (SLE) leads to prolonged exposure of nuclear autoantigen to the immune system. Here we measure the levels of TAM receptors and the phagocytic capacity of monocytes and macrophages in juvenile-onset SLE (JSLE).MethodMer protein was measured on monocytes from JSLE, healthy control and JIA patients. JSLE, healthy control and JIA patients’ plasma were analysed for soluble Mer (sMer), soluble Tyro3 (sTyro) and soluble Axl (sAxl). A phagocytosis assay measured the effect of JSLE serum on phagocytic potential of JSLE and control monocytes to engulf E. Coli bacteria and healthy macrophages to engulf apoptotic neutrophils.ResultsMer receptor expression was significantly decreased on JSLE monocytes compared to healthy controls. Plasma sMer, sTyro and sAxl were significantly increased in JSLE patients compared to controls (p < 0.05). Adult healthy control macrophages had significantly decreased phagocytosis of E. Coli and apoptotic neutrophils in the presence of 10% JSLE serum compared to control serum (p < 0.05).ConclusionJSLE patients have a decreased phagocytosis due to both serum and cell-derived factors. Significantly increased levels of sMer, sTyro3 and sAxl may be important factors contributing to the deficit in phagocytosis ability.

Autoantibodies in juvenile-onset myositis: Their diagnostic value and associated clinical phenotype in a large UK cohort

Objectives Juvenile myositis is a rare and heterogeneous disease. Diagnosis is often difficult but early treatment is important in reducing the risk of associated morbidity and poor outcomes. Myositis specific autoantibodies have been described in both juvenile and adult patients with myositis and can be helpful in dividing patients into clinically homogenous groups. We aimed to explore the utility of myositis specific autoantibodies as diagnostic and prognostic biomarkers in patients with juvenile-onset disease. Methods Using radio-labelled immunoprecipitation and previously validated ELISAs we examined the presence of myositis specific autoantibodies in 380 patients with juvenile-onset myositis in addition to, 318 patients with juvenile idiopathic arthritis, 21 patients with juvenile-onset SLE, 27 patients with muscular dystrophies, and 48 healthy children. Results An autoantibody was identified in 60% of juvenile-onset myositis patients. Myositis specific autoantibodies (49% patients) were exclusively found in patients with myositis and with the exception of one case were mutually exclusive and not found in conjunction with another autoantibody. Autoantibody subtypes were associated with age at disease onset, key clinical disease features and treatment received. Conclusions In juvenile patients the identification of a myositis specific autoantibody is highly suggestive of myositis. Autoantibodies can be identified in the majority of affected children and provide useful prognostic information. There is evidence of a differential treatment approach and patients with anti-TIF1γ autoantibodies are significantly more likely to receive aggressive treatment with IV cyclophosphamide and/or biologic drugs, clear trends are also visible in other autoantibody subgroups.

Cellular localization of nuclear antigen during neutrophil apoptosis: mechanism for autoantigen exposure?

Juvenile-onset systemic lupus erythematosus (JSLE) is a multisystem autoimmune disease characterized by hyperactive B-cells producing auto-antibodies directed against nuclear antigens. A potential source of these antigenic components is apoptotic cells. We have previously demonstrated increased dysregulated neutrophil apoptosis in JSLE patients. Here we investigate autoantigen expression on JSLE neutrophils during apoptosis. Neutrophils from non-inflammatory controls and JSLE patients were incubated with JSLE and control serum. Apoptosis and dsDNA expression was measured using flow cytometry and confocal microscopy. Increased neutrophil apoptosis and dsDNA expression was observed in JSLE and control neutrophils incubated with JSLE serum. During neutrophil apoptosis nuclear material was exposed on the cell surface rather than within the cell as seen with viable neutrophils. The increased neutrophil apoptosis induced by JSLE compared with control serum resulted in increased surface expression of nuclear antigens. This may provide an additional mechanism leading to the generation of autoantibodies in JSLE.

Differential expression of factors involved in the intrinsic and extrinsic apoptotic pathways in juvenile systemic lupus erythematosus

Dysregulated neutrophil apoptosis may result in the development of autoimmune disease by contributing to nuclear autoantigen exposure, leading to autoantibody generation and a breakdown in immune tolerance. It has previously been shown that neutrophil apoptosis is increased in juvenile-onset systemic lupus erythematosus (JSLE). This study aims to investigate the pathways involved in JSLE serum-induced apoptosis. Caspases 3, 7–9, IAP1/2, XIAP and FADD mRNA levels and TRAIL R2, BID/tBID, caspase 8 and 9 protein expression were measured in neutrophils from JSLE patients (n = 14) and controls (n = 10). The mRNA levels of caspases 7–9 were significantly higher in JSLE neutrophils than in controls, whereas the mRNA levels of IAP1, IAP2 and XIAP were decreased (p < 0.05). A decrease in neutrophil apoptosis induced by JSLE serum was observed in the presence of caspase 8 and 9 inhibitors (p < 0.05), and the activity of caspases 8 and 9 increased over time. tBID protein expression increased following incubation with JSLE serum. These data focus specifically on the expression and activity of the main caspases in the intrinsic and extrinsic apoptotic pathways. Increased expression of factors involved in the downstream signalling of the extrinsic apoptotic pathway indicates a prominent involvement of this pathway in JSLE serum-induced apoptosis.

Increased concentration of plasma TNFR1 and TNFR2 in paediatric lupus nephritis

Background Juvenile-onset systemic lupus erythematous (JSLE) is a debilitating condition that frequently involves the kidneys (lupus nephritis; LN). Tumour necrosis factor alpha (TNF-α), an important pro-inflammatory cytokine, is expressed locally in the kidney and correlates with LN disease activity. The aim of this study was to ascertain whether soluble receptors for TNF-α (sTNFR1/sTNFR2) are significantly increased in children with LN. Methods Plasma samples were collected from JSLE patients at routine review. Concentrations of sTNFR1 and sTNFR2 were measured (median; interquartile range, IQR) using enzyme-linked immunosorbent assay (ELISA) in 25 JSLE patients (seven LN) and 20 healthy controls (HCs). Results sTNFR2 concentration was significantly increased in JSLE (5149 pg/dl, 3413–8561) compared to HCs (3858 pg/dl, 2254–5165; p = 0.049). sTNFR1 concentration was significantly increased in active LN (n = 7, 1765 pg/dl, IQR 1133–4167) compared to inactive LN (n = 18, 1104 pg/dl, 886–1272; p = 0.018). There was a non-significant increase in sTNFR2 concentration in active LN (9829 pg/dl, 3298–21271) compared to inactive LN (4595 pg/dl, 3345–6993; p = 0.146). sTNFR1 concentration correlated moderately with sTNFR2 (r = 0.66, p < 0.001). sTNFR2 demonstrated strong positive correlations with ESR (r = 0.941, p < 0.01) and anti-dsDNA antibodies (r = 0.998, p = 0.041). Both receptors also positively correlated with creatinine (TNFR1 r = 0.81, p < 0.001; TNFR2 r = 0.50, p = 0.015) and urinary albumin creatinine ratio (TNFR1 r = 0.64, p < 0.01; TNFR2 r = 0.63, p < 0.01). Conclusions These data indicate that sTNFR1 and sTNFR2 concentrations are elevated in LN and may reflect renal activity. These results provide basis for further investigation into the pathological pathways underlying LN.

THU0373 Demonstration of an “Excellent” Biomarker Panel for Identifying Active Lupus Nephritis in Children

Background Lupus nephritis (LN) is a serious manifestation of Juvenile-onset Systemic Lupus Erythematosus (JSLE), affecting up to 80% of patients [1]. Conventional markers of JSLE disease activity fail to adequately predict impending LN flares. Renal histology is the gold standard for diagnosing and predicting renal prognosis in LN, and is rarely repeated for monitoring purposes due to its invasive nature. Single novel urine biomarkers are good at predicting and detecting LN flares [2], but to date, no individual urine biomarkers have achieved an “excellent” predictive value (area under the curve (AUC) >0.9). Objectives To assess if combining novel and traditional biomarkers can result in an excellent biomarker panel for identifying active LN. Methods Participants of the UK JSLE cohort study were aged less than 16 years at the time of diagnosis. Disease activity data was collected using the paediatric British Isles Lupus Assessment Group (pBILAG2004) score. Patients were cross-sectionally categorised as active LN (pBILAG2004 renal domain score A or B plus previous histological confirmation of LN), in-active LN (pBILAG2004 renal domain score D or E) or healthy controls (HCs). Novel urinary biomarkers; vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein 1 (MCP-1), lipocalin like prostaglandin D synthase (LPGDS), transferrin, ceruloplasmin and alpha-1-acid glycoprotein (AGP) were quantified by enzyme-linked immunosorbent assays. Neutrophil gelatinase associated lipocalin (NGAL) was measured using the Abbot Architect assay. Binary logistic regression modeling and receiver-operating curve (ROC curve) analysis assessed combinations of novel and traditional biomarkers. The study had full ethical approval in place. Results 61 JSLE patients and 19 HCs were recruited. 15 (25%) JSLE patients had active LN and 46 (75%) had in-active LN. Urinary AGP, ceruloplasmin, VCAM-1, MCP-1, LPGDS and transferrin levels were significantly increased in active LN (all p≤0.01). Urinary NGAL levels did not differ between patient groups (p=0.245). AGP was the best single biomarker differentiating active and inactive LN (good AUC 0.890, p≤0.001). Combining novel biomarkers improved the identification of active LN (optimal combination; AGP, ceruloplasmin, LPGDS, transferrin, excellent AUC 0.923, p<0.001). Additional improvement was seen with the addition of dsDNA to this combination (excellent AUC 0.933, p≤0.001). Conclusions A combination of novel urinary and traditional biomarkers produces an excellent “biomarker panel” for active LN identification on a cross sectional basis. It is anticipated that biomarker led monitoring will improve long-term renal outcomes in JSLE patients in the future. References Watson L, Leone V, Pilkington C, et al. Disease activity, severity, and damage in the UK Juvenile-Onset Systemic Lupus Erythematosus Cohort. Arth Rheum 2012;64(7):2356-65. Watson L, Tullus K, Pilkington C, et al. Urine biomarkers for monitoring juvenile lupus nephritis: a prospective longitudinal study. Ped Nephrol 2013;29(3):397-405. Disclosure of Interest None declared