IL‐10 polymorphisms and T‐cell subsets could affect the clinical presentation and outcome of childhood immune thrombocytopenia in Egyptian population

The aim is to study IL‐10 polymorphisms and IL‐10 level and assess their relation to T‐cell subsets in childhood immune thrombocytopenia (ITP). In all, 40 (25 acute, 15 chronic) ITP child patients were investigated at time of presentation, compared to 15 healthy, age‐ and gender‐matched controls and followed up for 1 year to determine chronic cases. Studying the effect of IL‐10 promoter polymorphism was done by PCR‐RFLP, IL‐10 level was determined by ELISA, natural killer cells and T‐cell subsets were evaluated by flow cytometry. Subjects with IL‐10 promoter (1082 AA and 592 AA) genotypes had lower IL‐10 levels and had lower CD4%, higher CD8%, lower CD4/CD8 ratio and lower T‐reg%. IL‐10 polymorphisms had no effect on NK%. IL‐10 serum levels and IL‐10 promoter polymorphic genotype frequencies are not different between ITP cases and controls; however, in ITP patients, IL‐10 promoter (1082 AA and 592 AA) genotypes and associated lower CD4, higher CD8, lower CD4/CD8 ratio is associated with more severe thrombocytopenia at presentation and had a poorer response to first‐line treatment. Patients with lower T‐reg cells had a higher tendency to develop chronic ITP. IL‐10 level and polymorphisms as well as disturbed T‐cell subsets percentages are demonstrable effectors of immune dysfunction in ITP and can affect the presentation and outcome of childhood ITP.     

Abnormalities in iNKT cells are associated with impaired ability of monocytes to produce IL‐10 and suppress T‐cell proliferation in sarcoidosis

Sarcoidosis is a multisystem granulomatous disorder characterized by marked T‐cell expansion of T helper 1 (Th1) cells. The cause of T‐cell overactivity is unknown. We hypothesized that interleukin‐10 (IL‐10) production by a yet undefined cell type might be defective, resulting in loss of regulation of T‐cell activity. Focusing on IL‐10‐producing monocytes, we first showed that monocytes isolated from the peripheral blood of corticosteroid‐naïve sarcoidosis patients (n = 51) produced less IL‐10 compared to controls, and were less able to suppress T‐cell proliferation. In addition, monocytic IL‐10 production correlated negatively with disease activity score. As invariant natural killer T (iNKT) cells are known to both interact with monocytes and be reduced in sarcoidosis patients, we then asked whether iNKT‐specific defects might be responsible for this reduced IL‐10 production. We found that greater numbers of circulating iNKT cells was associated with higher IL‐10 production. Moreover, iNKT cells enhanced monocytic IL‐10 production in vitro. Defective IL‐10 production and T‐cell suppression by sarcoidosis monocytes could be restored following their coculture with iNKT cells, in a CD1d‐ and cell contact‐dependent process. We suggest that reduced iNKT‐cell numbers in sarcoidosis may lead to impaired monocytic IL‐10 production and unchecked T‐cell expansion in sarcoidosis. These findings provide fresh insight into the mechanism of sarcoidosis disease, and interaction between iNKT cells and monocytes.     

Allele frequencies of polymorphisms of TNFA,IL‐6, IL‐10 and IFNG in an Italian Caucasian population

Polymorphisms in the regulatory and intronic regions of several cytokines have been associated with differential cytokine production. In this paper we genotyped, using the polymerase chain reaction–sequence‐specific primers (PCR‐SSP) method, a series of 363 healthy Italian Caucasians with the aim of obtaining a reference population for further studies on the role of cytokines in the inflammatory and immune responses. We also compared the results to those for other populations. The polymorphisms analysed were those of tumour necrosis factor alpha (TNFA), interleukin 6 (IL‐6), interleukin 10 (IL‐10) and interferon gamma (IFNG). We found that the frequency of allele TNFA*1 at position ?380 was 87.7% and that of TNFA*2 was 12.4%, significantly different from those of the UK and Japanese populations but not different from that of a population in Gambia. For IL‐10 the frequencies of alleles ?1082A and ?1082G were 63.0% and 37.0% and those of alleles ?819C, ? 819T, ?592C and ?592A were 70.8, 29.2, 70.8 and 29.2%, respectively, significantly different from those observed in south‐east England, in Manchester and in an Oriental population from southern China. The frequencies of IL‐6 alleles ? 174C and ?174G were 29.0 and 71.0%, respectively; for IFNG polymorphisms at position ?874, in the population under evaluation, the alleles ?874T and ?874A were present in 44.7 and 55.3% of the subjects, respectively. Genotype frequencies of IL‐6 were significantly different from those observed in populations from Germany and from the UK. The analysis carried out by our group indicates that there is heterogeneity in the frequencies of the cytokine polymorphisms among the different Caucasian populations, and this underlines the importance of a ‘local’ reference population when evaluating the clinical relevance of cytokine gene polymorphisms.     

Monocytes/macrophages and/or neutrophils are the target of IL‐10 in the LPS endotoxemia model

IL‐10 is a potent regulator of the innate and adaptive immune responses. Several cell types produce IL‐10 and its receptor chains and these may regulate different immune responses. Here we report that inactivation of the IL‐10 receptor (IL‐10R1) gene in mice leads to an increased susceptibility to chemically induced colitis as in the classical IL‐10‐deficient mutant. To identify the cells regulated by IL‐10 in immune responses, we generated several cell type specific IL‐10R1‐deficient mutants. We show that, in an IL‐10‐dependent LPS model of endotoxemia, dampening of the immune response requires expression of IL‐10R1 in monocytes/macrophages and/or neutrophils but not in T cells nor B cells. As the macrophage and/or neutrophil‐specific IL‐10‐deficient mutants also display the same phenotype, our results suggest that an autocrine loop in monocytes/macrophages is the most probable mechanism for the regulation of an LPS‐induced septic shock. In contrast, in an IL‐10‐regulated T‐cell response to Trichuris muris infection, IL‐10 acting on T cells or monocytes/macrophages/neutrophils is not critical for the control of the infection.     

Interleukin 10 gene promoter polymorphisms (rs1800896, rs1800871 and rs1800872) and haplotypes are associated with the activity of systemic lupus erythematosus and IL10 levels in an Iranian population

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with unknown aetiology. According to the role of interleukin 10 (IL10) in SLE pathogenesis, the genetic alterations in its promoter region could be associated with elevated IL10 levels and exacerbated disease. Here, we investigated the association of genotype and haplotype frequencies of three IL10 gene promoter polymorphisms with susceptibility to SLE, IL10 plasma levels and disease activity of patients in an Iranian population. A total of 116 SLE patients and 131 healthy subjects were enrolled. The PCR‐RFLP technique was used to detect IL10 promoter genotypes at the positions of ?1082 (G/A), ?819 (C/T) and ?592 (C/A) in association with IL10 plasma levels and SLEDAI scores. The GG genotype of ?1082 polymorphism was associated with the increased risk of SLE [OR = 2.65, 95% CI (1.21–5.82), p‐value = 0.046]. The CC genotype in ?819 region was associated with SLE susceptibility [OR = 3.38, 95% CI (1.26–9.07), p‐value = 0.034] and C allele was introduced as risk allele [OR = 1.86, 95% CI (1.15–3.01), p‐value = 0.009] in this region. IL10 plasma levels were overexpressed in CC genotype carriers of ?592 SNP and decreased in AA genotype carriers of ?1082. IL10 was also increased in SLE patients with CGT (?592/?1082/?819) haplotype. The SLEDAI score was higher among CC genotype carriers at the position of ?592 and TT genotype carriers at the region of ?819. SLEDAI was also elevated among patients with CGC (?592/?1082/?819) and CAC (p = 0.011) haplotypes. The present study suggests that the IL10 –819(C/T), ?1082(G/A) and ?592(C/A) polymorphisms and the haplotypes are associated with SLE susceptibility, increased disease activity and elevated IL10 levels. While this is the first time to report such an association in an Iranian population, further studies are needed to confirm these findings.     

B‐cell‐derived IL‐10 does not vitally contribute to the clinical course of glomerulonephritis

IL‐10‐secreting regulatory B cells have been postulated as negative mediators of inflammation. However, their impact on immune‐mediated diseases requires further investigation. We recently found that IL‐10‐secreting B cells infiltrate the kidney during crescentic glomerulonephritis (GN). We therefore studied the function of B‐cell‐derived IL‐10 in light of the potential risks associated with increasingly used B‐cell depleting therapies. Lack of IL‐10 production by B cells, however, did not influence acute or adaptively mediated progressive renal injury in terms of renal function and histological damage in the nephrotoxic nephritis model of GN. Renal leukocyte infiltration and cytokine expression were similar apart from increased macrophages in mice lacking B‐cell‐derived IL‐10. Systemic immune responses as assessed by cytokine production, leukocyte composition, proliferation, and activation were indistinguishable, while production and renal deposition of Ag‐specific IgG were mildly impaired in the absence of B‐cell‐produced IL‐10. Importantly, detailed analysis of systemic and renal regulatory T cells did not show any differences between nephritic mice bearing IL‐10‐deficient B cells and WT controls. Finally, studies in reporter mice revealed that B cells are only a minor source of systemic IL‐10. In summary, our data reveal that endogenous B‐cell‐derived IL‐10 does not play a major role in the nephrotoxic nephritis model of crescentic GN.     

IL‐12B and IL‐10 gene polymorphisms in the development of Hashimoto's thyroiditis

Functional genetic polymorphisms that altered gene expression of cytokines are candidate genetic factors that could modulate the development and progression of Hashimoto's thyroiditis (HT). IL‐12B gene encoded the IL‐12p40 subunit, which is included in the pro‐inflammatory heterodimeric cytokines IL‐12p70 and IL‐23. IL‐10 is an important Treg cytokine suppressing inflammatory cytokine production and autoimmunity. This study was designed to compare ?1082A/GIL‐10 and +1188A/C3′UTRIL‐12B genotype distribution in 130 patients with HT to a group of 157 healthy controls in attempts to determine an association with HT development. Genotyping for the 3′UTRA/C IL‐12B polymorphism was performed using RFLP‐PCR and genotyping for ?1082A/G IL‐10 by ARMS‐PCR assay. Patients with HT were divided into euthyroid and hypothyroid stages. There were no significant differences in the genotype and allele frequencies of the IL‐12B polymorphism between patients with HT and controls. We observed higher euthyroid HT risk for individuals with CC genotype, unlike to develop hypothyroidism with OR = 1.68. Regarding the polymorphism rs1800896, it was shown the significantly higher frequency of homozygous genotype GG in cases vs controls (OR = 2.19; P = 0.024). Moreover, the combination of genotype AA of 3′UTRIL‐12B with GG of ?1082IL‐10 was associated with a threefold increasing risk (OR = 3.188; P = 0.022) of developing HT compared to individuals with the presence of 3′UTR allele C (AC+CC) simultaneously with AA genotype of ?1082IL‐10. Our data raise the possibility that the combined effect of polymorphisms from proinflammatory and anti‐inflammatory cytokines may be more decisive to HT development.     

Chronic non-bacterial osteomyelitis is associated with impaired Sp1 signaling, reduced IL10 promoter phosphorylation, and reduced myeloid IL-10 expression

Chronic non-bacterial osteomyelitis (CNO) is an auto-inflammatory disorder that affects the skeletal system. Interleukin (IL-)10 is an immune-modulatory cytokine that controls inflammation, and limits inflammatory cytokine responses. Dysregulation of IL-10 expression has been shown to result in autoimmune and infectious diseases.We investigated IL-10 expression by monocytic cells from CNO patients and controls. In response to stimulation with LPS, IL-10 expression from CNO monocytes was reduced (p < 0.001). This was independent of IL10 promoter polymorphisms. Thus, we investigated Sp1 recruitment to the IL10 promoter and saw markedly reduced binding in CNO monocytes. This was accompanied with reduced phosphorylation of histone H3 serine 10 (H3S10), an activating epigenetic mark.Impaired recruitment of Sp1 to the IL10 promoter, and reduced H3S10 phosphorylation, may be a reflection of deficient MAPK signaling in CNO monocytes in response to LPS stimulation. Thus, we have discovered a mechanism that may be central in the pathophysiology of CNO.     

Differential IL‐10 production by DCs determines the distinct adjuvant effects of LPS and PTX in EAE induction

Pertussis toxin (PTX) is used as an adjuvant to induce EAE in mice as a model of MS. Although LPS and PTX are similar in their ability to mature dendritic cells (DCs) and in their adjuvant properties, here we demonstrate that LPS does not induce EAE development. We also demonstrate that DCs treated with PTX (PTX‐DCs) are able to induce EAE, whereas DCs treated with LPS (LPS‐DCs) fail to induce EAE. We determine that a key difference between LPS‐DCs and PTX‐DCs is that LPS‐DCs produce larger amounts of IL‐10. IL‐10^?/?‐DCs treated with LPS promote stronger IFN‐γ and IL‐17 production and T‐cell proliferation than WT DCs treated with LPS in a coculture system. Finally, we demonstrate that EAE can be successfully induced when IL‐10^?/?‐DCs treated with LPS are used as an adjuvant, whereas the use of PTX‐DCs overexpressing IL‐10 as an adjuvant markedly controls EAE development. These results indicate that the inability of LPS to induce EAE is based on the induction of high levels of IL‐10 in the targeted DCs, providing insight into the mechanisms responsible for the induction of EAE.     

Association of interleukin‐10 promoter region polymorphisms with risk factors of Atherosclerosis

Atherosclerosis is considered as an inflammatory disease, and carotid artery intima‐media thickness (IMT) and carotid plaque are generally used as intermediated phenotype of atherosclerosis. The aim of this study was to investigate whether carotid IMT and plaque are associated with promoter region polymorphisms of interleukin 10 (IL‐10) gene. We recruited 135 subjects from a rural area of south‐eastern part of South Korea. Three polymorphisms in the promoter region of IL‐10 (?1082 A/G, ?819 T/C and ?592 A/C) were genotyped by pyrosequencing. Carotid IMT was measured at common carotid arteries, and carotid bulbs and cardiovascular risk factors such as cholesterol, blood pressure, uric acid and homocysteine were measured using blood samples. Subjects with the minor allele (C) of ?819 T/C or the minor allele (C) of ?592A/C showed lower values in carotid IMT than those with major allele homozygote of each polymorphism (P = 0.018 and P = 0.031, respectively). Subjects with carotid plaque were significantly older and showed higher values in carotid IMT, uric acid and homocysteine than those without plaque (P < 0.01, respectively). In conclusion, the promoter region polymorphisms of IL‐10 gene associate with carotid IMT and plaque. Further studies with larger samples are needed to provide stronger evidence to justify anti‐atheromatous properties of IL‐10.     

Jagged2‐signaling promotes IL‐6‐dependent transplant rejection

The Notch pathway is an important intercellular signaling pathway that plays a major role in controlling cell fate. Accumulating evidence indicates that Notch and its ligands present on antigen‐presenting cells might be important mediators of T helper cell differentiation. In this study, we investigated the role of Jagged2 in murine cardiac transplantation by using a signaling Jagged2 mAb (Jag2) that activates recombinant signal‐binding protein‐Jκ. While administration of Jag2 mAb had little effect on graft survival in the fully allogeneic mismatched model BALB/c→B6, it hastened rejection in CD28‐deficient recipients. Similarly, Jag2 precipitated rejection in the bm12→B6 model. In this MHC class II‐mismatched model, allografts spontaneously survive for >56 days due to the emergence of Treg cells that inhibit the expansion of alloreactive T cells. The accelerated rejection was associated with upregulation of Th2 cytokines and proinflammatory cytokine IL‐6, despite expansion of Treg cells. Incubation of Treg cells with recombinant IL‐6 abrogated their inhibitory effects in vitro. Furthermore, neutralization of IL‐6 in vivo protected Jag2‐treated recipients from rejection and Jagged2 signaling was unable to further accelerate rejection in the absence of Treg cells. Our findings therefore suggest that Jagged2 signaling can affect graft acceptance by upregulation of IL‐6 and consequent resistance to Treg‐cell suppression.