A Novel Pool of Microparticle Cholesterol Is Elevated in Rheumatoid Arthritis but Not in Systemic Lupus Erythematosus Patients.

Microparticles are sub-micron, membrane-bound particles released from virtually all cells and which are present in the circulation. In several autoimmune disorders their amount and composition in the circulation is altered. Microparticle surface protein expression has been explored as a differentiating tool in autoimmune disorders where the clinical pictures can overlap. Here, we examine the utility of a novel lipid-based marker-microparticle cholesterol, present in all microparticles regardless of cellular origin-to distinguish between rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We first isolated a series of microparticle containing lipoprotein deficient fractions from patient and control plasma. There were no significant differences in the size, structure or protein content of microparticles isolated from each group. Compared to controls, both patient groups contained significantly greater amounts of platelet and endothelial cell-derived microparticles. The cholesterol content of microparticle fractions isolated from RA patients was significantly greater than those from either SLE patients or healthy controls. Our data indicate that circulating non-lipoprotein microparticle cholesterol, which may account for 1-2% of measured cholesterol in patient samples, may represent a novel differentiator of disease, which is independent of cellular origin.

MicroRNA-302d targets IRF9 to regulate the IFN-induced gene expression in SLE.

Systemic lupus erythematosus (SLE) is a complex disease targeting multiple organs as a result of overactivation of the type I interferon (IFN) system, a feature currently being targeted by multiple biologic therapies against IFN-α. We have identified an estrogen-regulated microRNA, miR-302d, whose expression is decreased in SLE patient monocytes and identify its target as interferon regulatory factor (IRF)-9, a critical component of the transcriptional complex that regulates expression of interferon-stimulated genes (ISGs). In keeping with the reduced expression of miR-302d in SLE patient monocytes, IRF9 levels were increased, as was expression of a number of ISGs including MX1 and OAS1. In vivo evaluation revealed that miR-302d protects against pristane-induced inflammation in mice by targeting IRF9 and hence ISG expression. Importantly, patients with enhanced disease activity have markedly reduced expression of miR-302d and enhanced IRF9 and ISG expression, with miR-302d negatively correlating with IFN score. Together these findings identify miR-302d as a key regulator of type I IFN driven gene expression via its ability to target IRF9 and regulate ISG expression, underscoring the importance of non-coding RNA in regulating the IFN pathway in SLE.

The association of cytokines with disease activity and damage scores in systemic lupus erythematosus patients.

OBJECTIVE: The aim of this study was to explore the role of cytokines in the pathogenesis of SLE in a genetically homogeneous Caucasian SLE patient population. METHODS: Serum levels of the following cytokines were determined by ELISA in SLE patients (diagnosed as per ACR diagnostic criteria): IL-1ß, IL-10, IL-12p70 and TNF-α. Demographic data, disease activity as per the SLEDAI and damage scores (SLICC) at the 5-year follow-up were calculated. RESULTS: Enhanced production of TNF-α, IL-1 and IL-10 were observed in SLE patients compared with controls. A strong positive correlation was seen between levels of IL-12p70 and IL-10. In addition, IL-10, TNF-α and IL-1 demonstrated a significant relationship with disease activity. Interestingly, elevated levels of IL-10 were observed in SLE patients with CNS involvement while patients with elevated levels of TNF-α were more likely to have renal involvement and sustain damage over the follow-up period. Additionally, the ratio of all cytokines assayed to IL-12p70 levels were significantly higher in SLE patients when compared with controls, with an association seen between damage accrual and the IL-1ß/IL-12p70 ratio (r = 0.431, P = 0.003), IL-10/IL-12p70 ratio (r = 0.351, P = 0.018) and TNF-α/IL-12p70 ratio (r = 0.33, P = 0.028). When the respective ratios were analysed for organ-specific disease, significant differences were observed for the IL-1ß/IL-12p70 ratio (0.79 vs 0.47, P = 0.036), IL-10/IL-12p70 ratio (4.29 vs 1.87, P = 0.018) and TNF-α/IL-12p70 ratio (7.49 vs 5.21, P = 0.018) with respect to renal involvement. CONCLUSION: Increased levels of a number of immunomodulatory cytokines relative to IL-12p70 in this Caucasian SLE patient population are seen in patients with renal involvement and are associated with increased accrual of damage at the 5-year follow-up.

Elevated B lymphocyte stimulator levels are associated with increased damage in an Irish systemic lupus erythematosus cohort.

OBJECTIVE: The overall aim of this study is to identify clinical and serological features that are associated with B lymphocyte stimulator (BLyS) elevation in a homogeneous Caucasian SLE population and thereby identify patients who are most likely to benefit from BLyS blockade. METHODS: Patients with SLE (as per ACR criteria) were recruited. Clinical history, disease activity measures and laboratory measures of disease were recorded. BLyS levels were determined by ELISA. RESULTS: BLyS elevation was defined as being higher than the 95th percentile of BLyS levels measured in controls. Patients were divided into two groups: those with elevated BLyS levels (group 1, n = 23) and those with normal BLyS levels (group 2, n = 22). Elevated BLyS levels were significantly associated with patients of younger age and shorter disease duration. In keeping with previous reports, patients with elevated BLyS levels had more active disease (SLEDAI 5.1 vs 0.86, P < 0.001); however, our analysis also demonstrates that BLyS elevation was significantly associated with increased organ damage at 5-year follow-up [Systemic Lupus International Collaborating Clinics/ACR Damage Index (SLICC/ACR DI) 0.53 vs 0.13, P = 0.012]. Furthermore, the presence of Sm autoantibody significantly predicted elevated BLyS levels in a Caucasian population. BLyS levels were significantly higher in those with musculoskeletal involvement, malar rash, renal disease and evidence of immunological activity. CONCLUSION: BLyS blockade may be most beneficial if introduced early in the course of disease in young Caucasian patients presenting with renal, musculoskeletal and skin disease in an effort to reduce long-term damage.

Enhanced interferon regulatory factor 3 binding to the interleukin-23p19 promoter correlates with enhanced interleukin-23 expression in systemic lupus erythematosus.

OBJECTIVE: To examine the role of interferon regulatory factor 3 (IRF-3) in the regulation of interleukin-23 (IL-23) production in patients with systemic lupus erythematosus (SLE). METHODS: Bone marrow-derived macrophages were isolated from both wild-type and IRF3(-/-) C57BL/6 mice. These cells were stimulated with the Toll-like receptor 3 (TLR-3) agonist poly(I-C), and IL-23p19 cytokine levels were analyzed by enzyme-linked immunosorbent assay. IRF-3 binding to the IL-23p19 gene promoter region in monocytes from patients with SLE and healthy control subjects was analyzed by chromatin immunoprecipitation (ChIP) assay. Luciferase reporter gene assays were performed to identify key drivers of IL-23p19 promoter activity. TANK-binding kinase 1 (TBK-1) protein levels were determined by Western blotting. RESULTS: ChIP assays demonstrated that IRF-3 was stably bound to the human IL-23p19 promoter in monocytes; this association increased following TLR-3 stimulation. Patients with SLE demonstrated increased levels of IRF-3 bound to the IL-23p19 promoter compared with control subjects, which correlated with enhanced IL-23p19 production in monocytes from patients with SLE. Investigations of the TLR-3-driven responses in monocytes from patients with SLE revealed that TBK-1, which is critical for regulating IRF-3 activity, was hyperactivated in both resting and TLR-3-stimulated cells. CONCLUSION: Our results demonstrate for the first time that patients with SLE display enhanced IL-23p19 expression as a result of hyperactivation of TBK-1, resulting in increased binding of IRF-3 to the promoter. These findings provide novel insights into the molecular pathogenesis of SLE and the potential role for TLR-3 in driving this response.

Photoprotective behaviour and sunscreen use: impact on vitamin D levels in cutaneous lupus erythematosus.

BACKGROUND: Sun exposure of the skin, independent of dietary sources, may provide sufficient vitamin D in healthy individuals. A recent study of patients with cutaneous lupus erythematosus concluded that over 70% of them restrict their sun exposure. METHODS: We recruited 52 patients with biopsy-proven cutaneous lupus erythematosus to establish whether they are deficient in 25-hydroxyvitamin D [25(OH)D]. We measured their serum 25(OH)D levels during summer months, investigated the effects of several variables on 25(OH)D levels and assessed the role of vitamin D supplementation. RESULTS: An overall mean 25(OH)D level of 63.03 (+/-23.3) nmol/l was obtained. Significantly low values (<25 nmol/l) were recorded in two (3.8%) patients and concentrations below 75 nmol/l were found in 34 (65.4%) patients. 25(OH)D levels were significantly lower among sun avoiders and daily sunscreen users, while significantly higher values were found among those who took cholecalciferol (vitamin D3) supplements. Low values were recorded among those with renal disease despite supplementation with vitamin D3 in some cases. CONCLUSIONS: We suggest that patients with cutaneous lupus erythematous have suboptimal 25(OH)D levels, which are significantly raised by the addition of at least 400 IU/day of cholecalciferol. We recommend supplementation with an active vitamin D analogue in collaboration with a consultant nephrologist, for the subgroup of patients with renal disease.

IL-16/miR-125a axis controls neutrophil recruitment in pristane-induced lung inflammation.

Severe lung inflammation and alveolar hemorrhage can be life-threatening in systemic lupus erythematosus (SLE) patients if not treated early and aggressively. Neutrophil influx is the driver key of this pathology, but little is known regarding the molecular events regulating this recruitment. Here, we uncover a role for IL-16/mir-125a in this pathology and show not only that IL-16 is a target for miR-125a but that reduced miR-125a expression in SLE patients associates with lung involvement. Furthermore, in the pristane model of acute "SLE-like" lung inflammation and alveolar hemorrhage, we observed reduced pulmonary miR-125a and enhanced IL-16 expression. Neutrophil infiltration was markedly reduced in the peritoneal lavage of pristane-treated IL-16-deficient mice and elevated following i.n. delivery of IL-16. Moreover, a miR-125a mimic reduced pristane-induced IL-16 expression and neutrophil recruitment and rescued lung pathology. Mechanistically, IL-16 acts directly on the pulmonary epithelium and markedly enhances neutrophil chemoattractant expression both in vitro and in vivo, while the miR-125a mimic can prevent this. Our results reveal a role for miR-125a/IL-16 in regulating lung inflammation and suggest this axis may be a therapeutic target for management of acute lung injury in SLE.

Estrogen receptor α regulates tripartite motif-containing protein 21 expression, contributing to dysregulated cytokine production in systemic lupus erythematosus.

OBJECTIVE: To examine the role of 17ß-estradiol in the regulation of the autoantigen tripartite motif-containing protein 21 (TRIM-21) in patients with systemic lupus erythematosus (SLE). METHODS: Monocytes isolated from healthy control subjects and patients with SLE were stimulated with 17ß-estradiol and/or the estrogen receptor α (ERα) antagonist methyl-piperidino-pyrazole dihydrochloride. TRIM-21, ERα, and CREMα expression was determined by real-time polymerase chain reaction (PCR) analysis. MatInspector software was used to identify putative binding sites within the TRIM-21 promoter. ERα binding to the TRIM-21 gene promoter region in monocytes was analyzed by chromatin immunoprecipitation (ChIP) assay. TRIM-21 and interferon regulatory factor 3 protein levels were analyzed by Western blotting. RESULTS: Real-time PCR analysis demonstrated a role of estrogen in the regulation of TRIM-21 expression in monocytes, which correlated positively with ERα gene expression in patients with SLE. Investigations into the human TRIM-21 promoter revealed the presence of an estrogen response element, with ChIP assays confirming ERα binding to this site. Studies into estrogen-induced TRIM-21 expression revealed a hyperresponsiveness of SLE patients to 17ß-estradiol, which led to the enhanced levels of TRIM-21 observed in these individuals. CONCLUSION: Our results demonstrate a role of estrogen in the regulation of TRIM-21 expression through an ERα-dependent mechanism, a pathway that we observed to be overactive in SLE patients. Treatment of monocytes with an ERα antagonist abrogated estrogen-induced TRIM-21 expression and, as a consequence, decreased the expression of interleukin-23. These findings identify TRIM-21 as a novel ERα-regulated gene and provide novel insights into the link between estrogen and the molecular pathogenesis of SLE.

Meticillin-sensitive Staphylococcus aureus costochondritis in a healthy man.

BACKGROUND: A 54-year-old previously healthy white man presented to hospital with fever, right parasternal pain and swelling over the right second and third costochondral joints. The symptoms had developed 1 week earlier. INVESTIGATIONS: Physical examination, white blood cell count, erythrocyte sedimentation rate, C-reactive protein level, blood and urine culture, plain radiography and CT of the chest, (99m)Tc bone scintigraphy, ultrasound-guided needle aspiration of soft tissue mass, Gram staining and culture of aspirated fluid. DIAGNOSIS: Meticillin-sensitive Staphylococcus aureus costochondritis. MANAGEMENT: CT revealed a 2 x 5 cm soft tissue mass at the posterior aspect of the right second and third costochondral joints. The fluid aspirated contained Gram-positive cocci, and culture revealed the presence of meticillin-sensitive S. aureus. The patient received a 6-week course of flucloxacillin (2 g by intravenous injection every 6 h for 2 weeks, then 1 g orally every 6 h for 4 weeks). He responded well to treatment, and was discharged from hospital.