Dimitrios T. Boumpas

Enhanced activity of NLRP3 inflammasome in peripheral blood cells of patients with active rheumatoid arthritis

IntroductionInterleukin-1β (IL-1β) is a major inflammatory cytokine, produced predominantly by innate immune cells through NLRP3-inflammasome activation. Both intrinsic and extrinsic danger signals may activate NLRP3. Genetic variations in NLRP3-inflammasome components have been reported to influence rheumatoid arthritis (RA) susceptibility and severity. We sought to assess the activity of NLRP3-inflammasome in patients with active RA compared to healthy individuals.MethodIntracellular protein expression of NLRP3, ASC, pro- and active caspase-1, pro- and active IL-1β was assessed by immunoblotting both at baseline and upon inflammasome activation. NLRP3 function (IL-1β secretion) was assessed upon priming of TLR2 (Pam(3)CysSK(4), TLR3 (poly(I:C)) or TLR4 (LPS) and ATP sequential treatment. We used caspase inhibitors (casp-1, 3/7 and 8) to assess their contribution to IL-1β maturation. All experiments were performed in whole blood cells.ResultsActive RA patients (n = 11) expressed higher basal intracellular levels of NLRP3 (p < 0.008), ASC (p < 0.003), active caspase-1 (p < 0.02) and pro-IL-1β (p < 0.001). Upon priming with TLR4 (LPS) and ATP, RA-derived cell extracts (n = 7) displayed increased expression of NLRP3 (p < 0.01) and active caspase-1 (p < 0.001). Secreted IL-1β in culture supernatants from whole blood cells activated with TLR4 (LPS) or TLR3 agonist (poly(I:C)) plus ATP was higher in RA patients (n = 20) versus controls (n = 18) (p < 0.02 for both). Caspase-1 inhibition significantly reduced IL-1β secretion induced by all stimuli, whereas caspase-8 inhibition affected only TLR4 and TLR3 cell priming.ConclusionPatients with active RA have increased expression of NLRP3 and NLRP3-mediated IL-1β secretion in whole blood cells upon stimulation via TLR3 and TLR4 but not TLR2. In these patients, IL-1β secretion seems to be predominately driven by caspase-1 and caspase-8. Targeting NLRP3 or downstream caspases may be of benefit in suppressing IL-1β production in RA.

Lupus nephritis management guidelines compared

In the past years, many (randomized) trials have been performed comparing the treatment strategies for lupus nephritis. In 2012, these data were incorporated in six different guidelines for treating lupus nephritis. These guidelines are European, American and internationally based, with one separate guideline for children. They offer information on different aspects of the management of lupus nephritis including induction and maintenance treatment of the different histological classes, adjunctive treatment, monitoring of the patient, definitions of response and relapse, indications for (repeat) renal biopsy, and additional challenges such as the presence of vascular complications, the pregnant SLE patient, treatment in children and adolescents and considerations about end-stage renal disease and transplantation. In this review, we summarize the guidelines, determine the common ground between them, highlight the differences and discuss recent literature.

Genetic variation near IRF8 is associated with serologic and cytokine profiles in systemic lupus erythematosus and multiple sclerosis

Alleles of interferon (IFN) regulatory factor 8 (IRF8) are associated with susceptibility to both systemic lupus erythematosus (SLE) and multiple sclerosis (MS). Although high-type I IFN is thought to be causal in SLE, type I IFN is used as a therapy in MS. We investigated whether IRF8 alleles were associated with type I IFN levels or serologic profiles in SLE and MS. Alleles that have been previously associated with SLE or MS were genotyped in SLE and MS patients. The MS-associated rs17445836G allele was associated with anti-double-stranded DNA (dsDNA) autoantibodies in SLE patients (meta-analysis odds ratio=1.92). The same allele was associated with decreased serum IFN activity in SLE patients with anti-dsDNA antibodies, and with decreased type I IFN-induced gene expression in peripheral blood mononuclear cell from anti-dsDNA-negative SLE patients. In secondary progressive MS patients, rs17445836G was associated with decreased serum type I IFN. Rs17445836G was associated with increased IRF8 expression in SLE patient B cells. In summary, IRF8 rs17445836G is associated with human autoimmune disease characterized by low-type I IFN levels, and this may have pharmacogenetic relevance as type I IFN is modulated in SLE and MS. The association with autoantibodies and increased IRF8 expression in B cells supports a role for rs17445836G in humoral tolerance.

High-density lipoprotein attenuates Th1 and Th17 autoimmune responses by modulating dendritic cell maturation and function

Aberrant levels and function of the potent anti-inflammatory high-density lipoprotein (HDL) and accelerated atherosclerosis have been reported in patients with autoimmune inflammatory diseases. Whether HDL affects the development of an autoimmune response remains elusive. In this study, we used apolipoprotein A-I–deficient (apoA-I−/−) mice, characterized by diminished circulating HDL levels, to delineate the role of HDL in autoimmunity. ApoA-I−/− mice exhibited increased severity of Ag-induced arthritis compared with wild-type mice, and this was associated with elevated Th1 and Th17 cell reactivity in the draining lymph nodes. Furthermore, reconstituted HDL (rHDL) attenuated IFN-γ and IL-17 secretion by Ag-specific T cells upon stimulation of draining lymph nodes in vitro. The suppressive effects of rHDL were mediated through modulation of dendritic cell (DC) function. Specifically, rHDL-treated DCs demonstrated an immature phenotype characterized by downregulated costimulatory molecules, the release of low amounts of proinflammatory cytokines, and failure to promote T cell proliferation in vitro. The mechanism of action involved the inhibition of NF-κB nuclear translocation and the decrease of Myd88 mRNA levels by rHDL. Finally, modulation of DC function by rHDL was critically dependent on the presence of scavenger receptor class B type I and ATP Binding Cassette Transporter A1, but not the ATP Binding Cassette Transporter G1. These findings reveal a novel role of HDL in the regulation of adaptive inflammatory responses through suppression of DC function that could be exploited therapeutically in autoimmune inflammatory diseases.

Coexistence of systemic lupus erythematosus and multiple sclerosis: prevalence, clinical characteristics, and natural history.

OBJECTIVES The coexistence of systemic lupus erythematosus (SLE) and multiple sclerosis (MS) in the same individual has rarely been described. Our objective was to report on the prevalence, clinical characteristics, and prognosis of cases fulfilling the criteria for both SLE and MS. METHODS We utilized existing patient cohorts from the Departments of Rheumatology and Neurology, University of Crete, and screened patients diagnosed with either SLE (n = 728) or MS (n = 819) for features of both diseases. The clinical, laboratory, and neuroimaging findings were assessed. RESULTS We identified nine patients who fulfilled the diagnostic criteria for both SLE and MS, corresponding to a prevalence rate of 1.0-1.2% in each cohort. All patients were women, with an average age at SLE diagnosis of 42.1 years (range: 34-56 years). The diagnosis of SLE preceded the development of MS in five patients, with a time lag ≤ 5 years in four of them. Initial presentation of MS included spinal symptoms in seven patients. All patients had features of mild SLE with predominantly cutaneous, mucosal, and musculoskeletal manifestations. Accordingly, therapeutic decisions were mainly guided by the severity of the neurological syndrome. During the median follow-up of 4 years (range: 1-10 years), three patients remained stable and the remaining experienced gradual deterioration in their neurological status. SLE remained quiescent in all patients while on standard immunomodulatory MS therapy. CONCLUSIONS Occurrence of both diseases in the same individual is rare, corroborating data that suggest distinct molecular signatures. SLE and MS coexistence was not associated with a severe phenotype for either entity.

Differential Expression of miR-4520a Associated With Pyrin Mutations in Familial Mediterranean Fever (FMF)

Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute, and self‐limiting attacks of fever. Mutations in MEFV gene encoding pyrin account for FMF, but the high number of heterozygote patients with typical symptoms of the disease has driven a number of alternative aetiopathogenic hypotheses. The MEFV gene was knocked down in human myelomonocytic cells that express endogenous pyrin to identify deregulated microRNAs (miRNAs). Microarray analyses revealed 29 significantly differentially expressed miRNAs implicated in pathways associated with cellular integrity and survival. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that miR‐4520a is predicted to target genes implicated in autophagy through regulation of RHEB/mTOR signaling. Differential expression levels of RHEB were confirmed by luciferase reporter gene assays providing further evidence that is directly targeted by miR‐4520a. Although the relative expression levels of miR‐4520a were variable among FMF patients, the statistical expression of miR‐4520a was different between FMF mutation carriers and controls (P = 0.0061), indicating an association between miR‐4520a expression and MEFV mutations. Comparison between FMF patients bearing the M694V mutation, associated with severe disease, and healthy controls showed a significant increase in miR‐4520a expression levels (P = 0.00545). These data suggest that RHEB, the main activator of mTOR signaling, is a valid target of miR‐4520a with the relative expression levels of the latter being significantly deregulated in FMF patients and highly dependent on the presence of pyrin mutations, especially of the M694V type. These results suggest a role of deregulated autophagy in the pathogenesis of FMF. J. Cell. Physiol. 232: 1326–1336, 2017.

Differential Expression of miR-4520a Associated with Pyrin Mutations Suggesting a Role of Autophagy in Familial Mediterranean Fever (FMF).

Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute, and self‐limiting attacks of fever. Mutations in MEFV gene encoding pyrin account for FMF, but the high number of heterozygote patients with typical symptoms of the disease has driven a number of alternative aetiopathogenic hypotheses. The MEFV gene was knocked down in human myelomonocytic cells that express endogenous pyrin to identify deregulated microRNAs (miRNAs). Microarray analyses revealed 29 significantly differentially expressed miRNAs implicated in pathways associated with cellular integrity and survival. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that miR‐4520a is predicted to target genes implicated in autophagy through regulation of RHEB/mTOR signaling. Differential expression levels of RHEB were confirmed by luciferase reporter gene assays providing further evidence that is directly targeted by miR‐4520a. Although the relative expression levels of miR‐4520a were variable among FMF patients, the statistical expression of miR‐4520a was different between FMF mutation carriers and controls (P = 0.0061), indicating an association between miR‐4520a expression and MEFV mutations. Comparison between FMF patients bearing the M694V mutation, associated with severe disease, and healthy controls showed a significant increase in miR‐4520a expression levels (P = 0.00545). These data suggest that RHEB, the main activator of mTOR signaling, is a valid target of miR‐4520a with the relative expression levels of the latter being significantly deregulated in FMF patients and highly dependent on the presence of pyrin mutations, especially of the M694V type. These results suggest a role of deregulated autophagy in the pathogenesis of FMF. J. Cell. Physiol. 232: 1326–1336, 2017.

Dysregulated production of interleukin-1β upon activation of the NLRP3 inflammasome in patients with familial Mediterranean fever.

Familial Mediterranean fever (FMF) is caused by mutations in pyrin, a protein expressed in innate immune cells that interacts with caspase-1 and other inflammasome components to regulate interleukin (IL)-1β maturation. Since NLRP3 inflammasome represents major source of IL-1β, we studied its protein expression and function in FMF. We isolated peripheral white blood cells (WBCs) from 20 symptoms-free FMF patients and 21 healthy individuals. Intracellular protein expression of NLRP3, caspase-1, IL-1β at baseline and after LPS/ATP sequential treatment for NLRP3 activation was assessed by immunoblotting. Secreted IL-1β was quantified by ELISA. THP-1 cells were transfected with wild-type or mutant pyrin and IL-1β secretion was measured. FMF WBCs exhibited lower NLRP3 and active caspase-1 protein expression compared to healthy individuals, and LPS/ATP treatment resulted in significantly lower intracellular IL-1β levels in FMF patients. Likewise, LPS/ATP induced caspase-1-dependent IL-1β release at significantly lower amounts in the FMF group (1182±192 versus 2134±245pg/mL in controls, p=0.004). Consistently, THP-1 cells transfected with FMF-associated M694V mutant pyrin displayed lower LPS/ATP-induced IL-1β compared with wild-type pyrin-transfected cells. FMF WBCs demonstrate reduced NLRP3-mediated IL-1β production. Additional studies are needed to define whether this finding represents a compensatory mechanism to control inflammation or is directly linked to disease pathogenesis.

A8.28 Reconstituted high density lipoprotein (RHDL) modulates TH1 and TH17 immune responses and pro-inflammatory cytokine production in a murine model of rheumatoid arthritis

Background and Objectives High density lipoprotein (HDL) has a variety of functions which confer protection from cardiovascular and other human diseases. Increased cardiovascular risk is observed in humans with autoimmune disorders such as rheumatoid arthritis (RA), which is associated with either low levels or dysfunctional HDL. Recent evidence suggests a role of HDL in modulating both innate and adaptive immune responses. We sought to investigate the role of rHDL during the development of an autoimmune response in the antigen-induced arthritis (AIA) mouse model. Materials and Methods C57BL/6 mice were subcutaneously immunised with ovalbumin (OVA) in complete Freund’s adjuvant (CFA) and the inguinal lymph nodes (LN) were excised 9 days after the antigenic challenge. The LN cells were cultured in vitro in the presence of varying concentrations of reconstituted HDL (rHDL) in the presence or absence of OVA and OVA-specific immune responses were measured. To assess the effect of HDL on dendritic cell activation and maturation, mouse bone marrow was cultured with GM-CSF to generate dendritic cells (BM-DCs), which were collected, cultured and treated with LPS in the presence or absence of rHDL. Results Presence of rHDL significantly inhibited the proliferation of OVA-primed LN cells in vitro, in a dose-dependent manner, as indicated by IL-2 measurement. Suppressed proliferation accompanied by reduced levels of IFN-γ and IL-17 in culture supernatants indicating that rHDL modulates the induction of Th1 and Th17 effector cells. Finally, rHDL-treated LPS-stimulated BM-DCs secreted significantly lower amounts of pro-inflammatory cytokines such as IL-6 and IL-8 whereas secretion of TNF-α was not affected. Conclusions rHDL exerts a direct immunomodulatory function on T cells in vitro by suppressing their proliferation and the induction of Th1 and Th17 effector cells. Furthermore, rHDL modulate the secretion of pro-inflammatory cytokines by DCs. Ongoing work is focused on the delineation of the mechanism involved in the rHDL-mediated suppression of the immune response both in vitro and in vivo. These data identify rHDL as an important player in the homeostatic regulation of the inflammatory response and a potential therapeutic target for chronic inflammatory diseases.

THU0467 Silencing of the MEFV Gene in Familial Mediterranean Fever and Transcriptomic Analysis in Human Monocytes: Evidence for the Involvement of Pyrin in Innate Immune Responses and Autophagy

Background Mutations in MEFV gene encoding pyrin [1] account for Familial Mediterranean fever FMF, a recessively inherited disease but the high number of heterozygote patients is puzzling and requires additional investigation [2,3]. Objectives Elucidation of the pathophysiology of the disease, through the identification of novel genes and miRNAs that interact with or regulate the MEFV gene. Methods Silencing of MEFV gene by AccellsiRNAs was performed in THP-1 cell line that expresses endogenous pyrin. Global changes in mRNA and microRNA expression were assessed by GeneChip HuGene-1.0-ST-v1 and GeneChipmiRNA 3.0 Array, respectively. Putative target genes of the differentially regulated miRNAs were identified by using appropriate algorithms (TargetScan, PicTar). Functional classifications and biological processes of differentially expressed mRNAs and miRNA target genes were assigned according to Gene Ontology (GO) and the updated Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. mRNA and miRNA target genetic or protein interactions were constructed using Cytoscape 3.0.2 (Plugins GeneMania and BioGrid). Results We achieved 62% knockdown of the MEFV mRNA as measured by Taqman PCR, following siRNA-mediated knockdown of the respective gene.Global transcriptome analysis revealed 89 differentially expressed genes and 25 deregulated miRNAs involved in inflammatory-mediated responses, cell cycle, cytoskeleton/microtubule dynamics, intracellular trafficking and survival. Specifically, miR-4520a and miR-19b demonstrate altered expression levels during TLR-mediated signaling, which corroborate the role of pyrin in the regulation of inflammatory responses. Interestingly, the aforementioned deregulated miRNAs are also shown to play a significant role in intracellular membrane trafficking and subsequently autophagy through regulation of small RabGTPases. Bioinformatic analyses predicted high cooperativity within the significantly deregulated miRNAs, with the clustering of 8 (out of 11) members of the miR-378/422a superfamily being the most pertinent example. Conclusions These findings reiterate the involvement of pyrin in innate immune responses and suggest a potential role of this protein in autophagy, probably through regulation of membrane trafficking RabGTPases. Validation of this data in monocytes from FMF patients is in progress. References The International FMF Consortium (1997). Cell 90:797–807. Booty M.G., Chae J.J., et al (2009). Ann. Rheum. Dis. 60:1851-1861. Touitou I, Picot MC, Domingo C, et al. (2001). Arthritis Rheum. 44:163–169. Acknowledgements Mir-Farzin Mashreghi, Helen Latsoudis and Joachim Gruen contributed equally. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.2470