Variations in the interferon and TLR3 genes may be associated with susceptibility to systemic lupus erythematosus and its clinical presentation.

The study aimed to explore the pontential impact of 10 polymorphisms within IFN-α, IFN-ß1, IFN-γ and TLR3 genes on SLE phenotype and susceptibility and to study the relationship between specific genotypes and clinics. Whole blood samples from SLE patients and healthy controls was obtained. DNA was extracted from the peripheral blood by the QIAamp DNA Blood Mini Kit (Qiagen). The quality and quantity of isolated DNA was estimated by the Quawell Q5000 spectrophotometer. We genotyped SLE patients and healthy subjects using real-time PCR (QuantStudio 5 thermocycler). The study suggests that IFN-γ rs2069705, IFN-γ rs2069718 and IFN-α rs3758236 polymorphisms have a protective role in SLE. We observed relations between TLR3 rs3775292, IFN-ß1 rs7873167, IFN-γ rs2069705, TLR3 rs3775291 and TLR3 rs5743305 polymorphisms and clinical picture of SLE patients. We found associations between the IFN-α rs3758236, IFN-γ rs2069705, IFN-γ rs2069718, IFN-γ rs1861493 and IFN-ß1 rs10964831 polymorphisms and the clinical manifestation of the SLE and/or its comorbidities. We perceived links between IFN-γ rs2069705, IFN-γ rs2069718, IFN-γ rs1861493, TLR3 rs3775291, TLR3 rs3775292 and TLR3 rs5743305 polymorphisms and the occurrence of autoantibodies. Our study presented the relationship between IFN and TLR gene polymorphisms with SLE susceptibility, phenotype and autoantibodies profile. This study propose that polymorphisms within interferons and TLR3 genes can be engaged in the SLE pathogenesis and course.

Variety of endosomal TLRs and Interferons (IFN-α, IFN-ß, IFN-γ) expression profiles in patients with SLE, SSc and MCTD.

We investigated Toll-like receptor (TLR)-3/-7/-8/-9 and interferon (IFN)-α/ß/γ mRNA expression in whole blood and serum IFN-α/ß/γ levels in patients with mixed connective tissue disease (MCTD), systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) and in healthy subjects to assess the association between the TLR-IFN expression and severity of and susceptibility to diseases, and identify potential biomarkers. Expression of the IFN-γ, TLR-3 and TLR-8 was detected only in SLE patients. TLR-7, IFN-α and IFN-ß expression was highest in SLE, while TLR-9 expression was highest in SSc patients. In SLE and MCTD patients a strong correlation was observed between TLR-7 and IFN-α expression and IFN-ß and IFN-α expression. In MCTD patients, negative correlation between IFN-α and TLR-9 and TLR-7 and TLR-9 was revealed. TLR-9 expression in anti-U1-70k-negative, anti-C negative and anti-SmB-negative MCTD patients was higher than in MCTD-positive patients. We observed negative correlations between serum IFN-α levels and TLR-7 expression and C3 and C4 levels in SLE patients. In SLE patients we observed that with increased IFN-γ, TLR-3 and TLR-8 expression increased the value of C3 and C4. Our results confirmed that the endosomal TLR-IFN pathway seems to be more important in SLE than in MCTD or SSc, and that IFN-α and IFN-ß may be possible biomarkers for SLE.

Epigenetics: The Future Direction in Systemic Sclerosis.

Systemic sclerosis (SSc) is an immune-mediated connective tissue disease of which the aetiology is still unclear. Previous genetic studies including candidate-gene studies and genomewide association studies have identified a number of genetic variations that confer risk to SSc. However, these variants, such as single nucleotide polymorphisms, cannot completely explain the SSc susceptibility and the diversity in the clinical symptoms of SSc patients. The contribution of epigenetic mechanisms as a link between genetics and environmental triggers represents promising field in understanding the pathogenesis of SSc. The aim of this review was to present the current knowledge on epigenetic mechanisms and highlight novel directions in diagnostic and therapeutical approaches.

IL-12B Gene Polymorphisms and IL-12 p70 Serum Levels Among Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is one of the autoimmune diseases, where different polymorphisms in cytokine genes play a pathogenic role. IL-12 is now recognized as a critical cytokine in terms of regulating the balance between Th1 and Th2 cells. We investigated the role of single nucleotide polymorphisms (SNPs) (rs3212227 (A/C) and rs17860508 (CTCTAA/GC)) of the IL-12B gene in the genetic susceptibility to RA and in the severity of the disease. Six hundred and thirty-four Caucasian RA patients and 341 healthy matched controls were studied using PCR-RFLP method and high-resolution melting analysis. Concentration of IL-12 cytokine level in serum was evaluated using ELISA. The genotype frequency did not deviate from HWE in each examined group. Frequencies of the rs3212227 CC genotype were statistically higher in patients with RA compared with the healthy control group in both codominant and recessive models (P = 0.037; P = 0.04, respectively). The frequency of rs3212227 C allele also showed similar tendency (P = 0.07). IL-12 level in serum was significantly higher in RA group compared with control (P < 0.0001). We observed that increased IL-12 serum level was correlated with higher number of tender and swollen joints, ExRA presence and higher levels of haemoglobin, CRP and PLT. Also higher IL-12 level in serum was observed within RA patients with hypertension. Present findings indicated that IL-12p40 + 1188A/C polymorphism as well as IL-12p70 protein levels may be associated with RA in the Polish population.

Genetic Variants in IL-12B and IL-27 in the Polish Patients with Systemic Lupus Erythematosus.

To investigate the potential association between IL-12B and IL-27 gene polymorphisms and systemic lupus erythematosus (SLE), we performed a case-control study based on the Polish population. Patients with SLE and healthy individuals were examined for -6415 CTCTAA/GC (rs17860508) and +1188A/C (rs3212227) in IL-12B and -924A/G (rs153109) and 4730T/C (rs181206) in IL-27 gene polymorphisms using the high-resolution melting method, PCR-RFLP method and TaqMan SNP genotyping assay, respectively. An increased frequency of GC/GC genotype as well as GC allele of the IL-12B rs17860508 was found in patients with SLE, as compared with healthy subjects (P < 0.001). We did not find differences in genotype and allele frequencies of the IL-12B rs3212227 and IL-27 rs153109 and rs181206 variants between patients with SLE and controls. IL-27 haplotype rs181206C/rs153109G indicated higher risk for SLE (P = 0.002), whereas haplotype rs181206T/rs153109G indicated reduced risk for SLE (P = 0.005). The IL-12B rs3212227 A/C polymorphism was associated with the mean value of the platelets (PLT), urea and complement C3 level. Furthermore, IL-12B rs17860508 genetic variant showed correlation with PLT, prothrombin time, international normalized ratio and alkaline phosphatase. Our results revealed that IL-12B rs17860508 and IL-27 haplotype CG are genetic risk factors for SLE and that both IL-12B rs17860508 and rs3212227 predict disease phenotype.

Association of HLA-DRB1 alleles with susceptibility to mixed connective tissue disease in Polish patients.

Mixed connective tissue disease (MCTD) is a systemic autoimmune disease, originally defined as a connective tissue inflammatory syndrome with overlapping features of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), polymyositis/dermatomyositis (PM/DM) and systemic sclerosis (SSc), characterized by the presence of antibodies against components of the U1 small nuclear ribonucleoprotein (U1snRNP). The aim of the study was to assess the frequency of (high-resolution-typed) DRB1 alleles in a cohort of Polish patients with MCTD (n = 103). Identification of the variants potentially associated with risk and protection was carried out by comparison with the DKMS Polish Bone Marrow Donor Registry (41306 alleles). DRB1*15:01 (odds ratio (OR): 6.06; 95% confidence interval (CI) 4.55-8.06), DRB1*04 (OR: 3.69; 95% CI 2.69-5.01) and *09:01 (OR: 8.12; 95% CI 2.15-21.75) were identified as risk alleles for MCTD, while HLA-DRB1*07:01 allele was found to be protective (OR: 0.50; 95% CI 0.28-0.83). The carrier frequency of the DRB1*01 was higher in MCTD patients compared with controls, although the differences were not statistically significant. Our results confirm the modulating influence of HLA-DRB1 genotypes on development of connective tissue diseases such as MCTD.

Association of the Smad3 and NFATc2 gene polymorphisms and their serum levels with susceptibility to rheumatoid arthritis in Polish cohorts.

One among many factors involved in induction of rheumatoid arthritis (RA) are T cells, the differentiation of which depends upon a unique combination of stimulants and subsequent activation of diverse transcription factors. The aim of this study was to identify polymorphic variants in Smad3 and NFATc2 genes and their possible association with susceptibility to and severity of RA. A total of 272 RA patients, 321 for Smad3 and 304 for nuclear factor of activated T cells (NFAT)c2 healthy individuals, were examined for rs6494629 C/T and rs2289263 T/G Smad3 and rs880324 NFATc2 gene polymorphisms using the polymerase chain reaction-fragment length polymorphism (PCR-RFLP) method and TaqMan single nucleotide polymorphism (SNP) genotyping assay, respectively. Serum Smad3 and NFATc2 levels in RA patients and controls were measured by enzyme-linked immunosorbent assay (ELISA). The rs6494629 C/T Smad3 gene polymorphism under the recessive (TT versus CC+CT) and over-dominant (CC+TT versus CT) models were associated with RA (P=0.014 and P=0.008, respectively). Smad3 rs2289263 T/G revealed differences in the case-control distribution in co-dominant, recessive and over-dominant models (P=0.037, P=0.010, P=0.034). Overall, rs6494629 C/T and rs2289263 T/G Smad3 gene polymorphisms were in a weak linkage disequilibrium (LD) with D'=0.116 and r(2)=0.004. After Bonferroni correction, the genotype-phenotype analysis showed no significant correlation of the Smad3 rs6494629 C/T and rs2289263 T/G and NFATc2 rs2289263 TT polymorphisms with disease activity, joint damage and extra-articular manifestation in RA patients. Serum Smad3 and NFATc2 levels were significantly higher in RA patients than in control groups (both P=0 0000). The present findings indicated that Smad3 genetic polymorphisms may be associated with the susceptibility to RA in the Polish population.

Interleukin 1 beta (IL1beta) gene polymorphisms (SNP-511 and SNP+3953) in Hashimoto's thyroiditis among the Polish population.

AIM: The association between the interleukin IL1 beta gene polymorphisms SNP-511 and SNP+3953 and susceptibility to the development of Hashimoto's thyroiditis among adult Caucasian-Polish population were analyzed. PATIENTS AND METHODS: The group studied comprised of 115 unrelated patients with Hashimoto's thyroiditis (112 women and 3 men, mean age 53.3 years). All patients were euthyroid on thyroid replacement therapy, had extremely high serum anti-TPO levels and in 53 patients anti-TG levels were also increased. The control group consisted of 103 healthy blood donors without raised anti-TPO antibodies, in whom a personal and familial history of thyroid, autoimmune and inflammatory diseases was excluded. No goiter or thyroid dysfunction was found.2 polymorphisms of the IL1 beta were studied by PCR-RFLP analysis. To confirm the accuracy of the method used, randomly selected patients were analyzed by direct sequencing. RESULTS: In both groups allele frequencies were in Hardy-Weinberg equilibrium. The significant statistical differences between the frequency of C and T allele for both SNPs (C-511T and C+3953T) in the group studied and in the controls were found (p=0.0081; OR=1.846; 95% CI: 1.183-2.878 and p=0.0099; OR=1.953; 95% CI: 1.183-3.224).The frequencies of the genotype C-511C compared to C-511T and T-511T as well as C+3953C compared to C+3953T and T+3953T also differed significantly (p=0.0057; OR=2.248; 95% CI: 1.292-3.912 and p=0.0043; OR=2.338; 95% CI: 1.305-4.191) between patients and controls. CONCLUSIONS: An association between the SNPs of the IL1 beta and susceptibility to Hashimoto's thyroiditis among the group of Caucasian-Polish population studied was found.

Association of single nucleotide polymorphisms in the IL27 gene with rheumatoid arthritis.

Rheumatoid arthritis (RA) is one of the autoimmune diseases, where different polymorphisms in cytokine genes play a pathogenic role. Interleukin 27 (IL-27) is a novel pro-/anti-inflammatory cytokine, an excellent candidate for chronic inflammatory disease studies. The aim of the study was to identify polymorphisms in the IL-27 gene and their possible association with susceptibility to and severity of RA. Two hundred and seventy-four patients with RA and of 295 healthy individuals were examined for -924A/G and 4730T/C IL27 gene polymorphisms using PCR-RFLP method and TaqMan SNP genotyping assay, respectively. Haplotype frequencies of IL-27 polymorphisms were estimated using SHEsis platform. Frequencies of the -924GG genotype and the -924G allele were statistically higher in RA patients comparing with the healthy control group (P = 0.008 and P = 0.004, respectively). Overall, strong LD was observed between the IL27 gene -924A/G and 4730 T/C polymorphisms (D' = 0.613, r2 = 0.199). From four possible haplotypes, frequencies of two (CA and CG) showed significant differences between both examined groups (respectively: P < 0.001 and P = 0.001062). The genotype-phenotype analysis showed significant association between the IL-27 4730 T/C polymorphism and HAQ score and means value of the ESR, additionally they revealed that individuals with the polymorphic allele -924G had more advanced disease than wild-type allele carriers. Present findings indicated that IL27 -924A/G polymorphism may be involved in susceptibility to RA in the Polish population.

Association between IL-17F gene polymorphisms and susceptibility to and severity of rheumatoid arthritis (RA).

Interleukin-17F (IL-17F) is a novel proinflammatory cytokine. IL-17F gene is an excellent candidate for chronic inflammatory disease. We investigated the association between rheumatoid arthritis (RA) and His161Arg (7488A/G; rs763780) and Glu126Gly (7383A/G; rs2397084) polymorphism of IL-17F gene. The gene polymorphisms in 220 Polish patients with RA and 106 healthy subjects were amplified by polymerase chain reaction with restriction endonuclease mapping. Overall, the polymorphisms of the IL-17F gene were not correlated with susceptibility to RA in Polish population. However, the IL-17F His161Arg variant was associated with parameters of disease activity, such as number of tender joints, HAQ score or DAS-28-CRP. Moreover, our findings have shown that Glu126Gly IL-17F gene polymorphism may be correlated with longer disease duration in patients with RA. Our results for the first time showed the relationship between IL-17F gene polymorphisms and severity of RA.

Interleukin-10 gene promoter polymorphism in Polish rheumatoid arthritis patients.

Interleukin (IL)-10 is an important multifunctional cytokine with both anti-inflammatory and immunoregulatory effects in rheumatoid arthritis (RA). In the present study, we evaluated the frequency and potential impact of IL-10 promoter polymorphisms on susceptibility to and severity of RA in Polish in - patients with a high disease activity (mean DAS 28 C-reactive protein 5.25). DNA was obtained from 244 RA patients and 106 healthy controls. The -592C/A and -1082G/A IL-10 gene polymorphisms were amplified by polymerase chain reaction with restriction endonuclease mapping. The frequency of the IL-10-592CA, -592AA genotypes (respectively: 30% vs 5% and 7% vs 0%) and allele -592A (37% vs 5%) were significantly higher in RA patients as compared with a control group. We did not find any association of the IL-10-592C/A genotype distribution with disease parameters, except for an increased ESR (erythrocyte sedimentation rate) in patients with the -592CC genotype as compared with those with -592CA or -592AA genotypes (P = 0.01). The frequency of the IL-10-1082GG genotype was lower (P = 0.0001), and that of the IL-10-1082GA genotype was higher (P = 0.009) in RA patients comparing with the control group. In RA patients with -1082GA or -1082AA genotypes the time duration of the disease (P = 0.03), Health Assessment Questionnaire (HAQ) Score (P = 0.04) and PLT count (P = 0.001) were significantly increased as compared with subjects with -1082GG genotype. Presented findings indicate that IL-10-592C/A and IL-10-1082G/A polymorphisms may be considered genetic risk factors for RA susceptibility and severity.

IL-23 in the pathogenesis of rheumatoid arthritis.

Interleukin-23 (IL-23) is a heterodimeric cytokine belonging to the IL-6/IL-12 family that plays a key role in several of autoimmune and inflammatory disorders. This family contains the 34 type I cytokine receptor chains and 27 ligands, which share structural and functional similarities, but on the other hand they display distinct roles in shaping Th cells responses. IL-12 family cytokines have not only proinflammatory effects but they also promote inflammatory responses. IL-23 is composed of the p40 subunit in common with IL-12, and with a unique p19 subunit. IL-23 binding to an IL-23 receptor expressed on dendritic cells, macrophages and monocytes triggers the activation of Jak2 and Tyk2, which in turn phosphorylates STAT1, STAT3, STAT4 and STAT5 as well as induce formation of STAT3-STAT4 heterodimers. IL-23 is one of the essential factors required for the survival and/or expansion of Th17 cells, which produce IL-17, IL-17F, IL-6 and TNF-alpha. Th17 cells stimulated by the IL-23 promote osteoclastogenesis through production of IL-17, which induce receptor activator of NF-kappa B ligand on mesenchymal cells. The IL-23-IL-17 axis includes Th17 cells and plays a key role in the development of autoimmune arthritis.