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OBJECTIVE To evaluate the impact of early treatment and IL1RN genetic variants on the response to anakinra in systemic juvenile idiopathic arthritis (JIA). METHODS Response to anakinra was defined as achievement of clinically inactive disease (CID) at 6 months without glucocorticoid treatment. Demographic, clinical, and laboratory characteristics of 56 patients were evaluated in univariate and multivariate analyses as predictors of response to treatment. Six single-nucleotide polymorphisms (SNPs) in the IL1RN gene, previously demonstrated to be associated with a poor response to anakinra, were genotyped by quantitative polymerase chain reaction (qPCR) or Sanger sequencing. Haplotype mapping was performed with Haploview software. IL1RN messenger RNA (mRNA) expression in whole blood from patients, prior to anakinra treatment initiation, was assessed by qPCR. RESULTS After 6 months of anakinra treatment, 73.2% of patients met the criteria for CID without receiving glucocorticoids. In the univariate analysis, the variable most strongly related to the response was disease duration from onset to initiation of anakinra treatment, with an optimal cutoff at 3 months (area under the curve 84.1%). Patients who started anakinra treatment ≥3 months after disease onset had an 8-fold higher risk of nonresponse at 6 months of treatment. We confirmed that the 6 IL1RN SNPs were inherited as a common haplotype. We found that homozygosity for ≥1 high-expression SNP correlated with higher IL1RN mRNA levels and was associated with a 6-fold higher risk of nonresponse, independent of disease duration. CONCLUSION Our findings on patients with systemic JIA confirm the important role of early interleukin-1 inhibition and suggest that genetic IL1RN variants predict nonresponse to therapy with anakinra.

Haploinsufficiency of A20 (HA20) is an inflammatory disease caused by mutations in the

ObjectiveTo investigate the activation of the IFNγ signaling pathway in monocytes of patients with secondary hemophagocytic lymphohistiocytosis (sHLH)/macrophage activation syndrome (MAS) and to evaluate whether levels of phosphorylated STAT1 represent a biomarker for the identification of patients at early stages of the disease.MethodsFresh whole blood samples from pediatric patients with active sHLH/MAS, not receiving (n=10) and receiving glucocorticoids (n=14) at time of sampling, were prospectively collected. As disease control groups, patients with active systemic juvenile idiopathic arthritis (sJIA) without MAS, patients with sHLH/MAS in remission and patients with other rheumatic diseases were also sampled. Whole blood cells were left unstimulated or stimulated with increasing concentrations of IFNγ for 10 minutes and the intracellular Tyrosine (701)-phosphorylated STAT1 (pSTAT1) levels were evaluated in monocytes by flow cytometry.ResultsMonocytes from untreated sHLH/MAS patients showed significantly higher basal levels of pSTAT1 compared to those observed in monocytes from glucocorticoid-treated sHLH/MAS patients and from all the other disease controls. In addition, a significant increase in responsiveness to IFNγ, as assessed by increased levels of pSTAT1 following ex vivo stimulation, was observed in monocytes from untreated sHLH/MAS patients. pSTAT1 levels in monocytes distinguished patients with sHLH/MAS not treated with glucocorticoids from patients with active sJIA or with other rheumatic diseases [AUC, 0.93; 95% confidence interval 0.85-1.00, pIFNG mRNA levels in whole blood cells, circulating IFNγ levels and pSTAT1 levels in sHLH/MAS monocytes were found.ConclusionOur data demonstrating higher basal levels of pSTAT1 as well as a hyperreactivity to IFNγ stimulation in monocytes from patients with sHLH/MAS point to perturbations in the activation of downstream IFNγ signaling pathway as a contributor to the hyperinflammation occurring in these patients. Finally, the observation that glucocorticoids affect pSTAT1 levels in vivo, makes it difficult to consider the measurement of pSTAT1 levels as a biomarker to identify patients at early stages of sHLH/MAS in clinical practice.

We describe a 2 year old boy with two previously undescribed frameshift mutations in the interferon (IFN)α/β receptor 2 (IFNAR2) gene presenting with hemophagocytic lymphohistiocytosis (HLH) following measles-mumps-rubella vaccination. Functional analyses show the absence of response to type I IFN in the patient’s cells, as revealed by the lack of phosphorylation of STAT1 and the lack of induction of interferon-stimulated genes upon ex vivo stimulation with IFNα. HLH has been reported in patients with inborn errors of type I IFN-mediated immune responses following vaccination with live-attenuated viruses. The relation between HLH and defective type I IFN-mediated responses is unclear. We show that in patient’s natural killer (NK) cells stimulated with IFNα the expected increase in degranulation and inhibition of IFNγ production were affected. These data support a role for NK cell function dysregulation and lack of inhibition of IFNγ production as contributors to the development of HLH in patients with impaired type I IFN signaling.

Revealing the interactions of long noncoding RNAs (LncRNAs) with specific genomic regions is of basic importance to explore the mechanisms by which they regulate gene expression. Chromatin oligo-affinity precipitation (ChOP) technique was the first method developed to analyze the association of LncRNAs with genomic regions in the chromatin context. The first step of the procedure is cell cross-linking, aimed at stabilizing the RNA-protein-DNA complexes. Next, after chromatin fragmentation, the RNA complexes are pulled down through hybridization with antisense oligonucleotides tagged with biotin and purification with anti-biotin antibody. After extensive wash, the RNA-interacting chromatin is eluted by RNase treatment. Subsequent protein elimination and DNA purification allow to retrieve DNA fragments for following analyses such as qPCR or sequencing.In the present chapter, we describe the ChOP protocol, as used in our laboratory for investigating the interaction of the LncRNA Kcnq1ot1 with chromatin at specific regulatory regions of the Cdkn1c locus.

Revealing the interactions of long noncoding RNAs (LncRNAs) with specific genomic regions is of basic importance to explore the mechanisms by which they regulate gene expression. Chromatin oligo-affinity precipitation (ChOP) technique was the first method developed to analyze the association of LncRNAs with genomic regions in the chromatin context. The first step of the procedure is cell cross-linking, aimed at stabilizing the RNA-protein-DNA complexes. Next, after chromatin fragmentation, the RNA complexes are pulled down through hybridization with antisense oligonucleotides tagged with biotin and purification with anti-biotin antibody. After extensive wash, the RNA-interacting chromatin is eluted by RNase treatment. Subsequent protein elimination and DNA purification allow to retrieve DNA fragments for following analyses such as qPCR or sequencing.In the present chapter, we describe the ChOP protocol, as used in our laboratory for investigating the interaction of the LncRNA Kcnq1ot1 with chromatin at specific regulatory regions of the Cdkn1c locus.

Nod-like Receptor Pyrin domain containing proteins (NLRPs) expressed by resident renal cells may contribute to the pathogenesis of multiple renal diseases. Cystinosis is a genetic disorder that affects kidney and particularly proximal tubular epithelial cells (PTEC). Here, we investigated the expression of NLRP family members in human control and cystinotic conditionally immortalized PTEC. Among all the NLRPs tested, we found that NLRP2 is highly expressed in cystinostic PTEC, but not in PTEC from healthy subjects. The NLRP2 overexpression was confirmed in primary PTEC and in kidney biopsies from cystinotic patients. In order to elucidate the role of NLRP2 in PTEC, we stably transfected control PTEC with an NLRP2-containing plasmid. We showed that NLRP2 markedly increases the production of several NF-κB regulated cytokines and chemokines. Accordingly, we demonstrated that NLRP2 interacts with IKKa and positively regulates the DNA-binding activity of p50 and p65 NF-κB, by modulating the p65 NF-κB phosphorylation status in Serine 536. Transcriptome analysis revealed that NLRP2 also upregulates the expression of profibrotic mediators and reduces that of several interferon-inducible genes. Finally, NLRP2 overexpression decreased the apoptotic cell rate. Consistently, silencing of NLRP2 by small-interfering RNA in cystinotic PTEC resulted in a significant decrease in cytokine and chemokine production as well as in an increase in the apoptosis rate. Altogether, our data reveals a previously unrecognized role for NLRP2 in regulating proinflammatory, profibrotic and antiapoptotic responses in PTEC, through NF-κB activation. Moreover, our findings unveil a novel potential mechanism involving NLRP2 overexpression in the pathogenesis of cystinosis. ispartof: FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY vol:7 ispartof: location:Switzerland status: published

The myogenic factor MyoD regulates skeletal muscle differentiation by interacting with a variety of chromatin-modifying complexes. Although MyoD can induce and maintain chromatin accessibility at its target genes, its binding and trans-activation ability can be limited by some types of not fully characterized epigenetic constraints. In this work we analysed the role of PARP1 in regulating MyoD-dependent gene expression. PARP1 is a chromatin-associated enzyme, playing a well recognized role in DNA repair and that is implicated in transcriptional regulation. PARP1 affects gene expression through multiple mechanisms, often involving the Poly(ADP-ribosyl)ation of chromatin proteins. In line with PARP1 down-regulation during differentiation, we observed that PARP1 depletion boosts the up-regulation of MyoD targets, such as p57, myogenin, Mef2C and p21, while its re-expression reverts this effect. We also found that PARP1 interacts with some MyoD-binding regions and that its presence, independently of the enzymatic activity, interferes with MyoD recruitment and gene induction. We finally suggest a relationship between the binding of PARP1 and the loss of the activating histone modification H3K4me3 at MyoD-binding regions. This work highlights not only a novel player in the epigenetic control of myogenesis, but also a repressive and catalytic-independent mechanisms by which PARP1 regulates transcription.

Background: Systemic juvenile idiopathic arthritis (sJIA) is a polygenic autoinflammatory disease. Innate immune mechanisms appear to play a central role in the pathogenesis of the disease. Nevertheless, a better understanding of the pathophysiology of sJIA is still needed to identify patients responsive to IL-1 or IL-6 targeted therapies. Objectives: In this study, we evaluated the production of IL-1β, IL-6 and TNF-α by fresh whole blood cells isolated from sJIA patients in disease remission, after stimulation with the TLR-4 ligand lipopolysaccharide (LPS), and we investigated whether sJIA patients that respond or not-respond to treatment with the IL-1 receptor antagonist anakinra show a different response. Methods: We collected fresh whole blood samples from sJIA patients during clinical inactive disease (inactive sJIA, n=19) and sJIA patients during active disease (active sJIA n=4). Active and inactive disease was defined at time of sampling. As controls, fresh whole blood samples from healthy subjects (HS, n=10) were used. Whole blood cells were left unstimulated or stimulated with 10ug/mL of LPS for 24 hours. Cytokine levels (IL-1β, IL-6 and TNF-α) released in the supernatants were measured by ELISA. Response to anakinra was defined as achievement of clinical inactive disease off glucocorticoids at 6 months after initiation of anakinra treatment. Results: We found that LPS-stimulated cells from inactive sJIA patients released significantly higher amounts of all the inflammatory cytokines tested, compared to HS (p Conclusion: Our preliminary results show a dysregulated production of inflammatory cytokines by whole blood cells from sJIA patients in remission disease, when stimulated with a TLR-4 agonist. Disclosure of Interests: : Antonia Pascarella: None declared, Manuela Pardeo: None declared, Ivan Caiello: None declared, Claudia Bracaglia: None declared, Marianna Rossi: None declared, Giulia Marucci: None declared, Emanuela Sacco: None declared, Fabrizio De Benedetti Grant/research support from: Abbvie, SOBI, Novimmune, Roche, Novartis, Sanofi, Pfizer, Giusi Prencipe: None declared

Aim of this study was to investigate the activation of the IFNγ pathway in the affected liver and in the blood of patients with secondary hemophagocytic lymphohistiocytosis (sHLH). To this purpose, the mRNA expression levels of IFNG and IFNγ-inducible genes as well as Tyrosine (701)-phosphorylated signal transducer and activator of transcription 1 (STAT1) protein levels were evaluated in the liver and in peripheral blood mononuclear cells (PBMCs) of three patients with sHLH with predominant liver involvement. The mRNA expression levels of IFNG and IFNγ-inducible genes were markedly higher in patient livers compared to control livers and to one disease control liver. Conversely, slight differences in the expression levels of Type I IFN-inducible genes and other classical inflammatory cytokine genes were found. Further supporting the activation of the IFNγ pathway, higher protein levels of phosphorylated and total STAT1 were detected in patient livers compared to control livers. When the expression of the same genes analysed in liver tissues was evaluated in PBMCs collected from 2 out of 3 patients before the liver biopsy, we found that mRNA levels of IFNγ-inducible genes were markedly increased. Accordingly, high circulating levels of IFNγ-inducible CXCL9 were observed in patients. Altogether, these data demonstrate the selective and marked up-regulation of the IFNγ pathway in the liver tissue and blood of patients with active sHLH. Finally, we show that measurement of circulating CXCL9 levels and evaluation of IFNγ-inducible gene expression levels in PBMCs may represent a new valid tool to better identify patients with suspected HLH with predominant liver involvement.

p57kip2 is the most complex member of the CIP/KIP family of cyclin-dependent kinase inhibitors and plays a fundamental role in regulating cell cycle and differentiation during mammalian development. Consistently with a key role for p57kip2 in the spatial and temporal control of cell proliferation, its expression is fine-tuned by multiple regulatory mechanisms, resulting in a tissue-, developmental phase- and cell type-specific pattern. Moreover, p57kip2 is an imprinted gene, further supporting the importance of its proper expression dosage. Importantly, misregulation of p57kip2 expression has been associated, more frequently than mutations in its coding region, to human growth disorders, such as Beckwith-Wiedemann and Silver-Russell syndromes, as well as to the onset of several types of cancers. This review will summarize the molecular mechanisms regulating p57kip2 transcription during differentiation and development, their relationship with the imprinting control and their alterations in growth-related diseases and cancer. Particular attention will be given to the role of epigenetic mechanisms, involving DNA methylation, histone modifications, long-range chromatin interactions and non-coding RNAs in modulating and integrating the functions of cis-regulatory elements and trans-acting factors.

The cdk inhibitor p57kip2, encoded by the Cdkn1c gene, plays a critical role in mammalian development and in the differentiation of several tissues. Cdkn1c protein levels are carefully regulated via imprinting and other epigenetic mechanisms affecting both the promoter and distant regulatory elements, which restrict its expression to particular developmental phases or specific cell types. Inappropriate activation of these regulatory mechanisms leads to Cdkn1c silencing, causing growth disorders and cancer. We have previously reported that, in skeletal muscle cells, induction of Cdkn1c expression requires the binding of the bHLH myogenic factor MyoD to a long-distance regulatory element within the imprinting control region KvDMR1. Interestingly, MyoD binding to KvDMR1 is prevented in myogenic cell types refractory to the induction of Cdkn1c. In the present work, we took advantage of this model system to investigate the epigenetic determinants of the differential interaction of MyoD with KvDMR1. We show that treatment with the DNA demethylating agent 5-azacytidine restores the binding of MyoD to KvDMR1 in cells unresponsive to Cdkn1c induction. This, in turn, promotes the release of a repressive chromatin loop between KvDMR1 and Cdkn1c promoter and, thus, the upregulation of the gene. Analysis of the chromatin status of Cdkn1c promoter and KvDMR1 in unresponsive compared to responsive cell types showed that their differential responsiveness to the MyoD-dependent induction of the gene does not involve just their methylation status but, rather, the differential H3 lysine 9 dimethylation at KvDMR1. Finally, we report that the same histone modification also marks the KvDMR1 region of human cancer cells in which Cdkn1c is silenced. On the basis of these results, we suggest that the epigenetic status of KvDMR1 represents a critical determinant of the cell type-restricted expression of Cdkn1c and, possibly, of its aberrant silencing in some pathological conditions.