Electronic Screening for Adolescent Risk Behaviors in the Emergency Department: A Randomized Controlled Trial.

INTRODUCTION: In this study we aimed to assess the impact of an electronic health assessment with individualized feedback for risk behaviors in adolescents seeking care in a pediatric emergency department (ED). METHODS: We conducted a randomized control trial using a tablet-based screening program with a study population of adolescents in a busy pediatric ED. The intervention group received the screening program with individualized feedback. The control group received the screening program without feedback. All participants received one-day and three-month follow-up surveys to assess behaviors and attitudes toward health behaviors. RESULTS: A total of 296 subjects were enrolled and randomized. There was no difference in changes in risky behaviors between the control and experimental groups. A higher proportion of participants in the intervention groups reported that the screener changed the way they thought about their health at one-day follow-up (27.0%, 36/133) compared to the control group (15.5%, 20/129, P = .02). CONCLUSION: This study successfully tested a multivariable electronic health screener in a real-world setting of a busy pediatric ED. The tool did not significantly change risky health behaviors in the adolescent population screened. However, our finding that the intervention changed adolescents' perceptions of their health opens a door to the continued development of electronic interventions to screen for and target risk behaviors in adolescents in the ED setting.

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.

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.

TRIM68 negatively regulates IFN-ß production by degrading TRK fused gene, a novel driver of IFN-ß downstream of anti-viral detection systems.

In recent years members of the tripartite motif-containing (TRIM) family of E3 ubiquitin ligases have been shown to both positively and negatively regulate viral defence and as such are emerging as compelling targets for modulating the anti-viral immune response. In this study we identify TRIM68, a close homologue of TRIM21, as a novel regulator of Toll-like receptor (TLR)- and RIG-I-like receptor (RLR)-driven type I IFN production. Proteomic analysis of TRIM68-containing complexes identified TRK-fused gene (TFG) as a potential TRIM68 target. Overexpression of TRIM68 and TFG confirmed their ability to associate, with TLR3 stimulation appearing to enhance the interaction. TFG is a known activator of NF-κB via its ability to interact with inhibitor of NF-κB kinase subunit gamma (IKK-γ) and TRAF family member-associated NF-κB activator (TANK). Our data identifies a novel role for TFG as a positive regulator of type I IFN production and suggests that TRIM68 targets TFG for lysosomal degradation, thus turning off TFG-mediated IFN-ß production. Knockdown of TRIM68 in primary human monocytes resulted in enhanced levels of type I IFN and TFG following poly(I:C) treatment. Thus TRIM68 targets TFG, a novel regulator of IFN production, and in doing so turns off and limits type I IFN production in response to anti-viral detection systems.

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.

Btk regulates macrophage polarization in response to lipopolysaccharide.

Bacterial Lipopolysaccharide (LPS) is a strong inducer of inflammation and does so by inducing polarization of macrophages to the classic inflammatory M1 population. Given the role of Btk as a critical signal transducer downstream of TLR4, we investigated its role in M1/M2 induction. In Btk deficient (Btk (-\-)) mice we observed markedly reduced recruitment of M1 macrophages following intraperitoneal administration of LPS. Ex vivo analysis demonstrated an impaired ability of Btk(-/-) macrophages to polarize into M1 macrophages, instead showing enhanced induction of immunosuppressive M2-associated markers in response to M1 polarizing stimuli, a finding accompanied by reduced phosphorylation of STAT1 and enhanced STAT6 phosphorylation. In addition to STAT activation, M1 and M2 polarizing signals modulate the expression of inflammatory genes via differential activation of transcription factors and regulatory proteins, including NF-κB and SHIP1. In keeping with a critical role for Btk in macrophage polarization, we observed reduced levels of NF-κB p65 and Akt phosphorylation, as well as reduced induction of the M1 associated marker iNOS in Btk(-/-) macrophages in response to M1 polarizing stimuli. Additionally enhanced expression of SHIP1, a key negative regulator of macrophage polarisation, was observed in Btk(-/-) macrophages in response to M2 polarizing stimuli. Employing classic models of allergic M2 inflammation, treatment of Btk (-/-) mice with either Schistosoma mansoni eggs or chitin resulted in increased recruitment of M2 macrophages and induction of M2-associated genes. This demonstrates an enhanced M2 skew in the absence of Btk, thus promoting the development of allergic inflammation.

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.

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.

Suppressors of cytokine signaling 2 and 3 diametrically control macrophage polarization.

Suppressors of cytokine signaling (SOCS) are important regulators of lipopolysaccharide (LPS) and cytokine responses but their role in macrophage polarization is unknown. We have shown here that myeloid-restricted Socs3 deletion (Socs3(Lyz2cre)) resulted in resistance to LPS-induced endotoxic shock, whereas Socs2(-/-) mice were highly susceptible. We observed striking bias toward M2-like macrophages in Socs3(Lyz2cre) mice, whereas the M1-like population was enriched in Socs2(-/-) mice. Adoptive transfer experiments showed that responses to endotoxic shock and polymicrobial sepsis were transferable and macrophage dependent. Critically, this dichotomous response was associated with enhanced regulatory T (Treg) cell recruitment by Socs3(Lyz2cre) cells, whereas Treg cell recruitment was absent in the presence of Socs2(-/-) macrophages. In addition, altered polarization coincided with enhanced interferon-gamma (IFN-γ)-induced signal transducer and activator of transcription-1 (STAT1) activation in Socs2(-/-) macrophages and enhanced interleukin-4 (IL-4) plus IL-13-induced STAT6 phosphorylation in Socs3(Lyz2cre) macrophages. SOCS, therefore, are essential controllers of macrophage polarization, regulating inflammatory responses.

Genetics of SLE: functional relevance for monocytes/macrophages in disease.

Genetic studies in the last 5 years have greatly facilitated our understanding of how the dysregulation of diverse components of the innate immune system contributes to pathophysiology of SLE. A role for macrophages in the pathogenesis of SLE was first proposed as early as the 1980s following the discovery that SLE macrophages were defective in their ability to clear apoptotic cell debris, thus prolonging exposure of potential autoantigens to the adaptive immune response. More recently, there is an emerging appreciation of the contribution both monocytes and macrophages play in orchestrating immune responses with perturbations in their activation or regulation leading to immune dysregulation. This paper will focus on understanding the relevance of genes identified as being associated with innate immune function of monocytes and macrophages and development of SLE, particularly with respect to their role in (1) immune complex (IC) recognition and clearance, (2) nucleic acid recognition via toll-like receptors (TLRs) and downstream signalling, and (3) interferon signalling. Particular attention will be paid to the functional consequences these genetic associations have for disease susceptibility or pathogenesis.

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.