IL-12 and IL-10 polymorphisms and their effects on cytokine production

Interleukin (IL)-12 is an inducer of differentiation of T helper (Th) cells towards Th1, whereas IL-10 is mainly an anti-inflammatory cytokine inhibiting Th1 functions. Single nucleotide polymorphisms (SNPs) in the regulatory sequences of genes are presumed to be associated with the differential production of cytokines. One IL12B 3'untranslated region (UTR) and five of IL10 gene promoter region SNPs were screened in 152 individuals by genotyping. IL-10 and IL-12 secretion of lipopolysaccharide (LPS), purified protein derivative (PPD) and Staphylococcus aureus Cowan strain I (SAC) stimulated peripheral blood mononuclear cells (PBMCs) was determined. The frequencies of the less frequent IL12B +16974 C, IL10 -2763 A -3575 A, -1082 G, -819 T and -592 A alleles were 27.4, 32.2, 25.9, 14.8, 9.3 and 8.6%, respectively. Individuals CC homozygous at IL12B 3'UTR had significantly higher IL-12 secretion levels from LPS and PPD stimulated PBMCs than AC heterozygotes (P = 0.03 and P = 0.02) or AA homozygotes (P = 0.02 and P = 0.05, respectively). IL10 -2763 and -3575 SNPs did not show any effect on in vitro secretion levels, whereas the association of proximal promoter -1082 SNP with IL-10 production is confirmed. IL10 proximal and distal promoter SNP distribution with estimated haplotype variations has implicated considerable similarities with the Caucasian populations in Turkey.     

Racial differences in selected cytokine allelic and genotypic frequencies among healthy,pregnant women in North Carolina

BACKGROUND: Genetic susceptibility to diseases is likely influenced by common DNA variants in the form of single nucleotide polymorphisms (SNPs). The value of SNPs for linkage and association mapping studies may depend on the distribution of SNP allele frequencies across populations. OBJECTIVES: To establish the SNP allelic frequencies among Caucasian and African American women for tumor necrosis factor (TNF)alpha, transforming growth factor (TGF)beta1, interleukin-10 (IL10), interleukin-6 (IL6), and interferon (IFN)gamma. MATERIALS AND METHODS: DNA was extracted from whole blood from 123 healthy, pregnant women. PCR-based genotyping was performed for the genes encoding TNFalpha (-308G/A), TGFbeta1 (codon 10C/T, codon 25C/G), IL10 (-1082A/G, -819T/C, -592A/C), IL6 (-174C/G) and IFNgamma (874T/A). Allele frequencies were determined by Hardy-Weinberg Equilibrium and Linkage Disequilibrium tests. Differences in the SNP allelic frequencies between Caucasians and African Americans were assessed by the chi(2) of Amitage trend test. RESULTS: SNP allelic and genotypic frequencies for IL6 and IFNgamma, but not for TNFalpha, TGFbeta1, and IL10, differed significantly between the Caucasian and African American women. CONCLUSIONS: Recognition of racial differences in SNP allelic and genotypic frequencies for selected cytokines is important for designing and powering future linkage and association mapping studies investigating the role of cytokines in human disease.     

Analysis of the association of allelic variants of apolypoprotein E and interleukin 1 beta genes with multiple sclerosis in ethnic Tatars

Multiple sclerosis (MS) is a multifactorial disease of the central nervous system. The apolipoprotein E (APOE) and interleukin 1 beta (IL1B) genes are considered to be candidate genes of MS. The aim of the study was to examine the hypothesis of the importance of APOE and IL1B gene polymorphisms in MS development in ethnic Tatars. DNA samples isolated by phenol-chloroform extraction from peripheral blood of 383 ethnic Tatars (120 MS patients and 263 healthy donors) were studied. 112C/R and 158R/C APOE gene polymorphisms as well as -511T/C IL1B gene polymorphism were analyzed by polymerase chain reaction (PCR) followed by PCR product digestion by endonuclease. Odds ratio (OR) values were used for evaluation of the relative risk of alleles and(or) genotype combinations. It has been shown that APOE*2/*3 genotype is associated with low risk of the disease development (OR = 0.20) in women. A combined effect of APOE and IL1B allelic variants has been discovered indicating the increased risk of the disease development in the carriers of APOE*4 and IL1B*T/*T alleles (OR = 4.76).     

Combined promoter haplotypes of the IL10R genes are associated with protection against severe malaria in Gabonese children

The critical barrier in control of infections remains the failure of the immune system to clear parasites despite antigen recognition. We examined and validated possible association of regulatory immune gene polymorphisms in a cohort of children with mild and severe malaria. We focussed on two precursors of the Interleukin 10 Receptor (IL10R) gene namely the IL10R alpha and IL10R beta that play a fundamental role in initiation of signal transduction. Initial screening across 40 Gabonese adult individuals revealed two promoter variants for the IL10R alpha and three for the IL10R beta precursor, respectively. Validation of these variants for their allelic gene expression by transient transfection assays exhibited an altered expression in rs56356146 and rs7925112 of the IL10R alpha (P < 0.5); rs8178435 and rs999788 in the IL10R beta constructs (P < 0.0001), respectively. We further investigated the functional role of those SNP variants exhibiting altered expression in a cohort of children with mild and severe malaria. We genotyped 145 children with mild and 185 children with severe malaria for IL10R alpha; for IL10R beta, 102 children with mild and 101 children with severe malaria. We found that none of the SNP variants had any significant association neither in children with mild or severe malaria. The haplotype −185/−116 of IL10R alpha (TT) in combination with the haplotype −754/−750 of IL10R beta (AC) contributed towards mild malaria in comparison to severe malaria [TT + AC odds ratio of 0.73 (95% CI 0.56–0.94) P = 0.01]. This study may provide a better understanding on the role of IL10R promoter allelic variants contribution to a protective effect on the development of severe malaria.     

<Emphasis Type="Italic">IL4</Emphasis> in the 5q31 context: association studies of type 1 diabetes and rheumatoid arthritis in the Spanish population

IL4, the gene coding the prototypic Th2 cytokine, has been frequently studied in the context of several inflammatory conditions, but conclusive results have not been obtained. This gene is located in the 5q31-33 complex genetic region, which shows some susceptibility factors to type 1 diabetes (T1D) and rheumatoid arthritis (RA) among other inflammatory conditions. Our aim was to assess the involvement on T1D and RA of IL4 polymorphisms considered individually and in combination with other polymorphisms in 5q31-33, specifically in the OCTN locus, where the L503F polymorphism has been associated with Crohn's disease and other Th1 diseases. We performed a case-control study including 316 T1D patients, 599 RA patients and 540 healthy controls, all of them corresponding to white Spanish individuals. The IL4 single-nucleotide polymorphisms (SNPs) -590C/T (rs2243250) and the OCTN1 exonic SNP L503F (rs1050152) were analysed in all samples. Frequency comparisons of -590C/T and stratified analysis including both cited SNPs were performed using chi-square tests. The -590C/T IL4 SNP was not found associated with T1D or RA when individual analyses were performed. However, a significant association with T1D emerged after stratification by L503F [p=0.02, odds ratio=1.95, 95% CI=1.07-3.55]. The location of the IL4 gene in the complex 5q31-33 genetic region, which contains many genes involved in immunological responses and presents linkage disequilibrium extended along many kilobases, makes necessary to interpret cautiously the previous IL4-association studies.     

Polymorphisms of TNFalpha, IL1beta, and IL1RN genes in chronic obstructive pulmonary disease

It is recognized that genetic factors play a role in the susceptibility to COPD. COPD is characterized by airflow limitation. Chronic inflammation causes small airway disease and parenchymal destruction, leading to the airflow limitation. Polymorphisms in pro-inflammatory cytokine genes may confer a risk for the development of COPD. A case-control association study was performed in Japanese population (88 COPD patients and 61 controls) and Egyptian population (106 patients and 72 controls). Genotype and allele frequencies of the TNFalpha -308 G/A and +489 G/A polymorphisms, the IL1beta -511 C/T, -31 T/C, and +3954 C/T polymorphisms, and a VNTR polymorphism in intron 2 of the IL1RN gene were investigated. In addition, pairwise haplotype frequencies were analyzed. When studied independently, none of the polymorphisms were associated with the development of COPD in both populations. However, in the Egyptian population, the distributions of the haplotype (IL1beta -31 T/C : IL1beta +3954 C/T) were significantly different between the COPD patients and the controls (p(corr)=0.0037). Our findings suggest that this haplotype within the IL1beta gene may be involved in the pathogenesis of COPD and that the genetic factors of COPD susceptibility might be different between different populations.     

A novel polymorphic CAAT/enhancer-binding protein beta element in the FasL gene promoter alters Fas ligand expression: a candidate background gene in African American systemic lupus erythematosus patients

A single-nucleotide polymorphism (SNP), identified at nucleotide position -844 in the 5' promoter of the FasL gene, lies within a putative binding motif for CAAT/enhancer-binding protein beta (C/EBPbeta). Electrophoretic mobility shift and supershift assays confirmed that this element binds specifically to C/EBPbeta and demonstrated that the two alleles of this element have different affinities for C/EBPbeta. In luciferase reporter assays, the -844C genotype had twice the basal activity of the -844T construct, and basal expression of Fas ligand (FasL) on peripheral blood fibrocytes was also significantly higher in -844C than in -844T homozygous donors. FasL is located on human chromosome 1q23, a region that shows linkage to the systemic lupus autoimmune phenotype. Analysis of 211 African American systemic lupus erythematosus patients revealed enrichment of the -844C homozygous genotype in these systemic lupus erythematosus patients compared with 150 ethnically matched normal controls (p = 0.024). The -844C homozygous genotype may lead to the increased expression of FasL, to altered FasL-mediated signaling in lymphocytes, and to enhanced risk for autoimmunity. This functionally significant SNP demonstrates the potential importance of SNPs in regulatory regions and suggests that differences in the regulation of FasL expression may contribute to the development of the autoimmune phenotype.     

A new inducible adenoviral expression system that responds to inflammatory stimuli in vivo

BACKGROUND: Gene transfer using inducible promoters, which control expression of transgenic proteins in response to physiological conditions, may have significant advantages. In this study, we tried to achieve an inducible adenoviral expression system for physiologically responsive gene therapy of autoimmune or inflammatory diseases. METHODS: A luciferase reporter vector with a hybrid promoter containing the human IL-1beta enhancer region (-3690 to - 2720) and the human CIITA promoter IV (-399 to + 2) was constructed. A replication-deficient adenovirus was engineered with luciferase controlled by the IL1beta/CIITApIV promoter (Ad-IL1beta/CIITApIV-Luc). The reporter vector or adenovirus was transfected to C57Bl/6 myeloid dendritic cells (DCs), RAW264.7, and Hep G2 to study the in vitro characteristics of this hybrid promoter. An inflammation model was prepared by injecting lipopolysaccharide (LPS) into Balb/c mice intraperitoneally (i.p.), and infected with Ad-IL1beta/CIITApIV-Luc or Ad-CMV-Luc to study the in vivo characteristics of the IL1beta/CIITApIV promoter. RESULTS: The IL1beta/CIITApIV hybrid promoter has pronounced promoter activity, broad-range responsiveness to cytokines or LPS, and can be rechallenged after first induction. In the inflammation model, IL1beta/CIITApIV could drive hepatic luciferase expression increasedly rapidly after LPS challenge and in a LPS dose-dependent manner. CONCLUSIONS: Using the IL1beta/CIITApIV hybrid promoter in gene transfer vectors may make it possible to produce transgenic proteins in vivo in direct relationship with the intensity and duration of an individual's status. By providing endogenously controlled production of transgenic proteins, this approach might limit the severity of autoimmune or inflammatory response without interfering with the beneficial components of host defense and immunity.