P1049
Investigation of inflammasome components in the process of cell migration in FMF patients
Tayfun H. Akbaba1, Z. Yeliz Akkaya-Ulum1, Selcan Demir2, Zeynep Tavukcuoglu1, Seza Ozen2, Banu Balci-Peynircioglu1
1
Medical Biology;2Pediatric Nephrology and Rheumatology, Hacettepe University, Ankara, Turkey
Correspondence:Tayfun H. Akbaba
Pediatric Rheumatology2019,17(Suppl 1):P1049
Introduction:Autoinflammatory diseases, periodic fever syndromes, are a group of diseases that develop recurrent inflammatory re- sponse in the absence of infection. Familial Mediterranean fever, which is the most common disease among autoinflammatory dis- eases, is caused by mutant pyrin production due to mutations in
MEFVgene. Pyrin protein is thought to be involved in inflammatory pathways by means of protein-protein interactions. There are studies supporting the role of pyrin as a proinflammatory regulator in the lit- erature and involved in pathways associated with cell migration. Objectives:The aim of this study was to investigate the effect of pyrin inflammasome on cell migration in FMF patients, in CAPS pa- tient as disease control and healthy controls.
Methods:Within the scope of the thesis; pyrin inflammasome was examined in mononuclear cells isolated from the blood samples of 11 FMF patients homozygous for M694V mutation, 1 CAPS patient with somatic mosaicism and 7 healthy individuals in the process of inflammatory cell migration. Lipopolysaccharide was used to induce the activation of inflammasome in the cells, while arachidonic acid was used for the inhibition of inflammasome. IL-1 beta secretion was analyzed by western blot as an indicator of inflammasome activation, and transwell filter assay was performed in order to examine the dif- ferences in cell migration.
Results: We demonstrated that the inhibition of inflammasome in the cells of FMF patients compared to the controls was less under the same application conditions. Following inflammasome activation and inhibition, filter experiments were performed and fetal bovine serum was used to induce inflammatory cell migration. In both inflammasome activation and suppression conditions; we observed a statistically significant increase in the ratio of cell migration of the mononuclear cells of the FMF patients compared to CAPS patient and control indivuals.
Conclusion:These findings support the idea of increased cell migration ratio in patients with FMF due to the more active pyrin inflammasome seen in patients. Although the involvement of other inflammasome components remains to be defined, this study has made significant contributions to illuminate the role of pyrin protein in inflammatory cell migration through the structure of inflammasome.
Keywords:FMF, pyrin inflammasome, cell migration.
This thesis was supported by Hacettepe University Scientific Research CoordinationUnit (Project Number: TYL-2018-17354)
Disclosure of Interest
None Declared
P1050
Cell migration defect in hyperimmunoglobulin D syndrome
Tayfun H. Akbaba1, Selcan Demir2, Z. Yeliz Akkaya-Ulum1, Seza Ozen2,
Banu Balci-Peynircioglu1
1Medical Biology;2Pediatric Nephrology and Rheumatology, Hacettepe
University, Ankara, Turkey
Correspondence:Tayfun H. Akbaba
Pediatric Rheumatology2019,17(Suppl 1):P1050
Introduction:Hyper-IgD syndrome or Mevalonate Kinase Deficiency(- HIDS/MKD, MIM #260920) is a ultra rare, autosomal recessive heredi- tary autoinflammatory syndrome caused by compound heterozygous or homozygous mutations in the mevalonate kinase(MVK) gene. It is
characterized by recurrent febrile episodes with abdominal pain, lymphadenopathy and an increased serum immunoglobulin D (IgD) level.Anti-IL-1 and anti-TNF treatment are applied to alleviate the symptoms of the disease and to reduce the number of attacks. Objectives:In this study, we aimed to analyze cell migration, an im- portant process of inflammation,in HIDS patients and compare with FMF patients and controls in inflamasome activated and inhibited conditions.
Methods:Peripheral blood mononuclear cell isolation from HIDS pa- tients, FMF patients and controls was performed. LPS was used to in- duce inflammasome in isolated cells while arachidonic acid was used for inhibition of inflammasome.After activation and inhibition of inflammasome, transwell filter experiments were performed with iso- lated cells from patients. As a result of the experiment, the migrating cells were stained with calcein-AM and analyzed by Image J programme.
Results:We did not observe cell migration in HIDS patients’mono- nucleer cells under normal conditions, after LPS stimulation. We ob- served increased cell migration in FMF patients and normal rate in controls as a positive control of these experiments.Considering that 2 HIDS and 1 FMF patients involved in these experiments were receiv- ing anti-IL-1 treatment, it was thought that the drug used by the pa- tients had no effect in cell migration pattern that we observed. Conclusion:Our preliminary data is the first study identified cell mi- gration defect seen in HIDS. Due to the mutant protein produced as a result of MVK gene mutations, the deficiency in the function of the mevolanate kinase enzyme observed in HIDS leads to the depletion of geranylgeranyl pyrophosphate, a key substrate for protein prenyla- tion.Considering the close relationship between protein prenylation and cytoskeleton elements related with cell migration, a possible de- crease in prenylation may be a potential explanation of cell migra- tion defect seen in HIDS patients.
Keywords:HIDS, cell migration defect, prenylation
This project was supported by Hacettepe University Scientific Re- search Coordination Unit (Project Number: TYL-2018-17354) Disclosure of Interest
None Declared
P1051
Possible regulatory effects of miRNAs in the pathogenesis of systemic auto inflammatory diseases, from the perspective of familial Mediterranean fever
Z. Yeliz Akkaya-Ulum1, Zeynep Tavukcuoglu1, Ezgi Deniz Batu-Akal2, Tayfun Hilmi Akbaba1, Hafize Emine Sonmez2, Banu Balci-Peynircioglu1,
Seza Ozen2
1Medical Biology;2Pediatric Nephrology and Rheumatology, Hacettepe
University, Ankara, Turkey
Correspondence:Tayfun Hilmi Akbaba
Pediatric Rheumatology2019,17(Suppl 1):P1051
Introduction:Systemic Auto Inflammatory Diseases (SAID) is a group of rare and hereditary periodic fever syndromes with recurrent in- flammatory involvement developing in the absence of infection. Among same SAID patients, phenotypic heterogeneity is common, and modifier mechanisms such as epigenetic factors may be consid- ered as one of the reason of these variations. MicroRNAs (miRNAs), a type of epigenetic mechanisms, are regulated in most of the bio- logical processes like inflammation.
Objectives:This study aimed to explore the potential effect of miR- NAs in the auto inflammation mechanism seen in SAID from the per- spective of Familial Mediterranean fever (FMF) which is the most common auto inflammatory disorder.
Methods:The expression levels of miRNAs were analyzed by miRNA array, performed on whole blood RNA samples from healthy controls, homozygous FMF patients with severe phenotype, homozygous FMF patients with mild phenotype, carriers who displayed the disease phenotype, healthy carriers and other rare SAIDs, in pediatric term. The raw data was analyzed by Multi Experiment Viewer (MeV) and TAC programs. Then we performed pathway analyses using DAVID
v6.8. and Panther analysistools. Candidate miRNAs shown to be re- lated with inflammatory pathways by bioinformatics analysis, are fur- ther studied functionally for their possible effect on expression levels of inflammatory genes, caspase I activation and cell migration (trans- well and wound healing experiments) in SW982 (synovial fibroblast) cell lines.
Results: The four patient groups were compared in between and miR-30e-3p, miR-374b-5p, miR-329-3p, miR-29c-3p, miR-25-5p were found to be significantly down regulated in the patient groups. The expression levels of these miRNAs were validated with qRT-PCR. All these miRNAs were found to be known regulators in TGF-beta and Toll-like receptor signaling pathway, apoptosis and actin cytoskeleton regulation by bioinformatics. After pre-miR transfection of miR-30e- 3p, miR-374b-5p, miR-29c-3p; expression levels of inflammatory genes (IL-1β, IL-18, TNF-α, TGF-β) were decreased, caspase I activa- tion and cell migration ratio were significantly decreased (p<0.05). Conclusion:Functional results of this study showed that these miR- NAs may possibly have an anti-inflammatory effect and can regulate the expression of genes found in inflammatory pathways. The inves- tigation of potential target genes of these miRNAs by bioinformatics tools and 3’ UTR luciferase assay is underway. The results of this study will be informative for understanding and investigating the possible effect of miRNAs in other auto inflammatory diseases. This project has been funded by E-RARE-3 project (INSAID, grant 003037603) and The Technical and Scientific Research Council of Turkey (TUBITAK), Grant number: TUBITAK 1001-SBAG 315S096. Keywords: SAID, FMF, epigenetics, miRNA, inflammation. Disclosure of Interest
None Declared
P1052
MIR-197 regulates inflammation in monocytes and synovial fibroblasts by targeting IL1R1
Yeliz Z. Akkaya Ulum1, Zeynep Tavukcuoglu1, Tayfun Hilmi Akbaba1, Engin Yilmaz2, Banu Balci-Peynircioglu1
1
Medical Biology, Hacettepe University, Ankara;2Medical Biology, Acibadem Mehmet Ali Aydinlar University, Istanbul, Turkey Correspondence:Yeliz Z. Akkaya Ulum
Pediatric Rheumatology2019,17(Suppl 1):P1052
Introduction:Familial Mediterranean Fever (FMF); is an autosomal reces- sively inherited autoinflammatory disease. FMF is caused by the muta- tions in the Mediterranean Fever (MEFV) gene which encodes the pyrin protein. In many studies, pyrin has been implicated in important cellular processes like apoptosis, cytoskeleton dynamics, signal transduction, and inflammation. Recent studies have shown that epigenetic control mecha- nisms, particularly non-coding RNAs, may play a role in the pathogenesis of autoinflammation. microRNAs (miRNAs) are small non-coding RNAs that play critical roles in regulating host genome expression at the post- transcriptional level. Dysregulated miRNA expression patterns have been documented in a broad range of diseases including cancer, inflammatory, and autoimmune diseases.
Objectives:Phenotypic heterogeneity seen in FMF disease indicated that FMF is not a simple monogenic disease. Therefore it has been suggested that epigenetic factors can be one of the reason for the variations.
Methods:Previously we identified miR-20a-5p and miR-197-3p as signifi- cantly differentially expressed among healthy controls (-/-) and patients (M694V/M694V). The validation of differentially expressed miRNAs was done by quantitative reverse transcription polymerase chain reaction (qRT-PCR). miRNA target genes were determined in miRWalk, the data- base on predicted and validated miRNA targets. Then pathway analysis was performed in DAVID. For functional assays, SW982 (synovial fibro- blast) and THP-1 (monocyte) cell lines were cultured and transfected with miR-197 mimic and scramble control. After transfection, cytokines expres- sion levels (MEFV, IL-1b, IL-18, TNF-α, TGF-β), caspase I activity, apoptosis and cell migration rate were determined. Migration was analyzed in two ways by Transwell migration chamber and wound healing assays. For tar- get gene studies, 3’UTR lusiferase activity assay was done.
Results:qRT-PCR analysis confirmed the results of the miRNA profil- ing for 2 miRNAs. From two of them; miR-20a showed induction and the other one; miR-197 showed reduction in the homozygote group compared to controls (Akkaya-Ulum et al., 2017).
For functional studies, after pre-miR-197 transfection, the expression levels of cytokines were decreased, cells showed less migration and caspase 1 activity. There was no effect in apoptosis. These results showed that miR-197 could have an anti-inflammatory effect by causing less migration and cytokine secretion. miR-197 was found to bind to the interleukin-1beta (IL-1β) receptor, type I (IL1R1), which is one of the key molecules of the inflammatory pathways.
Conclusion:These findings provide evidence that miR-197 may play role in FMF pathogenesis. Therefore, this study may contribute to understand the inflammatory process seen in FMF disease, as well as to development of the new drug targets and biomarkers in addition to the existing colchicine and similar treatments.
As miRNA-based therapeutics are promising approaches for treating autoinflammatory diseases, we are planning to continue to study on potential therapeutic usage of miR-197 in mouse model of FMF disease.
This study was supported by The Scientific and Technological Re- search Council of Turkey TUBITAK 1001-SBAG Project Number: 214S106 and Hacettepe University Scientific Research Projects Coord- ination Unit, Thesis Support, PhD Project Number: TDK-2017-16253 and BAP Comprehensive Project Number: 013D05101005 and Turkey Rheumatology Association.
Keywords: Familial Mediterranean Fever, inflammation, microRNA, miR-197, IL1R1.
Reference
Akkaya-Ulum YZ, Balci-Peynircioglu B, Karadag O, Eroglu FK, Kalyoncu U, Kiraz S, et al. Alteration of the microRNA expression profile in familial Mediterranean fever patients. Clin Exp Rheumatol. 2017 Nov-Dec;35 Suppl 108(6):90-94.
Disclosure of Interest
None Declared
P1053
Comparison of FMF patients with age of onset before 20 versus 40 years and over
Okan Aydin, Serdal Ugurlu, Huri Ozdogan
Division of Rheumatology, Department of Internal Medicine, Cerrahpasa Medical Faculty, University of Istanbul - Cerrahpasa, Istanbul, Turkey
Correspondence:Okan Aydin
Pediatric Rheumatology2019,17(Suppl 1):P1053
Introduction:Familial Mediterranean fever (FMF) isa disease withan onset before 20 years of age in 90% of the patients.However late on- set FMF defined as age of onset over 40 years is being recognised more frequently.
Objectives:To better define patients with FMF who had their first at- tack before age 40 and compare them with early onset patient group in Turkish population
Methods:The files of 2180 FMF patients followed in a single cen- ter between 2008-2017 who have fulfilled Tel-Hashomer criteria, were reviewed with regard to age of onset 40 years and over (index patients, Group 1).For control purposesfiles before and after the index patients were browsed and
first patients with an onset before age 20 years (Group 2) were in- cluded. The demographic, clinical and geneticcharacteristics are com- pared between these 2 subgroups.
Results: Patients with an onset after 40 years consisted 2.7% of our FMF population. 50 of the 59 patients with an onset 40 year- sor over were re-evaluated and compared with early onset group consisting of 100 patients (Table 1).The delay in diagnosis, and disease durationwere significantly longer and number of patients with M694V homozygosity and M694V allele frequencywere sig- nificantly more frequent among group 2. In general, phenotypes of both onset groups were similar, the only significant differences
being the frequency of fever and myositis which were less com- mon amonggroup 1. Also response to colchicine was more pro- nouncedingroup 1. One other interesting observation was the low incidence ofamyloidosis in a group with such a significant delay in diagnosis and thus treatment.
Conclusion:FMF should be included among the differential diagno- sis of patients over 40 years of age with recurrent autoinflammatory manifestations. Less than 3% of FMF patients experience their first at- tacks after 40 years of age. The frequency of M694V is significantly less in the late onset group, pointing out a milder disease.
Disclosure of Interest
None Declared
P1054
What history of appendectomy will tell us about the course of familial Mediterranean fever in adulthood?
Erdal Bodakçi1, Nazife S. A. Bilge1, Nuh Ataş2, Berkan Armağan3, Hasan Satış2, Alper Sarı3, Hakan Babaoğlu2, Gözde K. Yardımcı3, Reyhan B.
Salman2, Levent Kılıç3, Mehmet A. Öztürk2, Berna Göker2, Seminur Haznedaroğlu2, Umut Kalyoncu3, Abdurrahman Tufan2, Timuçin
Kaşifoğlu1
1Deparment of Internal Medicine, Division of Rheumatology, Eskişehir
Osmangazi University Faculty of Medicine, Eskişehir;2Deparment of Internal Medicine, Division of Rheumatology, Gazi University Faculty of Medicine;3Deparment of Internal Medicine, Division of Rheumatology, Hacettepe University Faculty of Medicine, Ankara, Turkey
Correspondence:Erdal Bodakçi
Pediatric Rheumatology2019,17(Suppl 1):P1054
Introduction:Peritonitis attacks of FMF usually requires emergency medical admissions and it’s hard to distinguish a typical attack from surgical causes of acute abdomen. Therefore, unrevealing abdominal surgery history, particularly appendectomy, is very common in pa- tients with FMF. However, history of appendectomy might also give some clues about the disease course of FMF.
Objectives:to determine whether history of appendectomy is associ- ated with disease course of FMF in adulthood.
Methods:All patients recruited from FMF in Central Anatolia (FiCA) cohort, comprising 970 (mean age 35.3±12 years, 61.5%female) adult subjects. All patients fulfilled Tel Hashomer criteria. Demographic data, FMF disease characteristics, co-morbid conditions, past medical history including surgeries, disease complications were meticulously questioned and laboratory features and genotype data (if available) were recruited from patient files. Disease severity and FMF associated damage were assessed with International Severity Scoring System (ISSF) for FMF and Autoinflammatory Disease Damage Index (ADDI), respectively.
Results:Appendectomy history was evident in 240 (24.7%) subject- s.Peritonitis (4.4±6.7 vs 2.9±4.3 attacks/per year, p<0.001) and pleuri- tis (3.9±5.2 vs 2.8±4.4 attacks/per year, p=0.03) attacks were more frequent in appendectomy performed (AP) group than appendix in- tact (AI) group. However, there were no difference between AP and AI groups for the attack frequencies of musculoskeletal and skin components. Considering all types of attacks, AP group had more at- tacks (6.4±8.2 vs 4.3±6.6 attacks, p<0.001), despite they had used more higher doses of colchicine (1.43±0.6 mg/day vs 1.27±0.5 mg/ day, p=0.002). ISSF scores were also higher in AP group 3.13±1.68 vs 2.73±1.48, p=0.001). Colchicine nonresponse were more prevalent in AP group as well (15.1% vs 6.7%, pearson X2=20.2, p<0.001). How- ever, ADDI damage scores were similar between AP and AI groups. Conclusion:Appendectomy history is common in FMF patients and associated with frequent serositis attacks in adulthood. These pa- tients require more colchicine doses with a lower response rate. Hence, history of appendectomy would be a worthy clue for the management of FMF patients.
Disclosure of Interest
None Declared
P1055
Complement endorse the pathogenesis in autoinflammation
Juergen Brunner1, Wilfried Posch2, Doris Wilflingseder2 1
Pediatrics;2Division of Hygiene and Medical Microbiology, Medical University Innsbruck, Innsbruck, Austria
Correspondence:Juergen Brunner
Pediatric Rheumatology2019,17(Suppl 1):P1055
Introduction:The complement system represents a major part of the innate immune system, consisting of more than 30 different proteins in plasma and on cell surfaces and can be activated through three differ- ent pathways.Inflammasomes are also part of the innate immune sys- tem.A group of disorders in inflammasomes have been associated with autoinflammatory diseases (AIDs). Familial cold autoinflammatory syn- drome (FCAS), Muckle-Wells syndrome (MWS) and chronic infantile neurological, cutaneous and articular syndrome/neonatal onset multi- system inflammatory disease (CINCA/NOMID) were originally described as three distinct diseases. After the identification of their common gen- etic origin, i.e. mutations in the NLRP3 gene on chromosome 1q44, they are perceived as a continuum of one disease entity and labelled cryopyrin-associated periodic syndromes (CAPS).
Objectives:Aim of this preliminary study in a patient with MWS was to find a correlation between the complement system and a disorder of autoinflammation.
Methods:PBMCs (peripheral blood mononuclear cells) were isolated from blood of a healthy donor and of an individual suffering from MWS by density gradient centrifugation using a Ficoll Paque Pre- mium (GE Healthcare). After washing, PBMCs were incubated with anti-human CD14 Magnetic Beads (BD) to obtain CD14+ monocytes. These were stimulated by addition of cytokines (IL-4 and GM-CSF) for five days to generate immature moDCs (iDCs), which were used for cytokine ELISAs and flow cytometric analyses. IL-6 and IL-1βcytokine ELISAs were performed according to the manufacturer (Biolegend) following stimulation of cells using either LPS or differentially com- plement opsonized HIV-1. Phenotypical characterization of pathogen- exposed DCs was performed by analyzing characteristic surface markers (CD11c, DC-SIGN, CD86) by multi-color flow cytometry. Table 1 (abstract P1053).Demographic, clinical and genetic features of
the study groups
≥40 years n=50
≤20 years n= 100
p Sex (F:M); present age (mean±SD)
(yr) 32:18;57.2 ±7.9 62:38;31.8 ±9.1 NS; <0.001 Mean age at onset,(mean±sd) (yr) 45.6±5.2 8.7±4.8 <0.001 Mean age at diagnosis (mean ±sd)
(yr)
50.4±7.3 19.1±11.2 <0.001 Delay in diagnosis (mean ±sd) (yr) 4.8±5.5 10.4±11.8 <0.001 Mean disease duration (mean ±sd)
(yr) 11.5±6.4 23.1±10.8 <0.001 Abdominal pain, n (%) 44(88) 89(89.0) NS Chest pain, n (%) 7(14.0) 27 (27.0) NS Fever, n (%) 30(60.0) 81(81.0) 0.005 Arthritis, n (%) 12(24.0) 33(33.0) 0.25 Myalgia, n (%) 1(2.0) 12(12.0) 0.04 Amyloidosis, n (%) 1(2.0) 3(3.0) NS
Positive family history, n (%) 33(68.7) 62 (65.2) NS Response to colchicine, n (%) 37(82.2) 93 (94.8) 0.014 M694VHomozygous, n (%) 2(4.5) 23(25.8) 0.003 N of M694Vallelles 24( 48 ) 82 (82) 0.014