Regulation of interferon lambda-1 (IFNL1/IFN-λ1/IL-29) expression in human colon epithelial cells.

The efficient regulation of intestinal immune responses is critical to colon health. Viruses, for example noraviruses, are key pathogens of the intestine. The lambda interferons (comprising three ligands: IFNL1, L2 and L3 - the so-called "Type III" interferons) constitute the most recently discovered IFN family and are known to be important in intestinal anti-viral defense. A fourth family member, IFNL4, was recently described. Expression of the IFN-lambda receptor is restricted to epithelial and immune cells; together, these ligands and their receptor represent an important anti-viral and immunoregulatory component of the immune/epithelial inteface. We investigated control of IFNL1 expression in human colon epithelial cells. We used the TLR3 agonist poly I:C to drive expression of IFNL1 in SW480 cells, and small interfering RNA (siRNA) to knockdown target transcription factors. We identified ZEB1 and BLIMP-1 as transcription factors that strongly inhibited IFNL1 expression in SW480 cells. Interestingly, while BLIMP-1 inhibited both type-III and type-I interferons (IFN-ß), the inhibitory action of ZEB1 was specific for IFNL1. We also defined the NF-κB family member, p65 as a key activator of IFNL1 and NF-κB p50 as a key inhibitor. Finally, we demonstrated that siRNA targeting of ZEB1 or NF-κB p50 resulted in a significant elevation of secreted IFN-λ1 protein and expression of the anti-viral gene OAS1, while knockdown of p65 inhibited these events. Our data provide insight to the regulation of IFNL1 expression in the human colon and suggest novel therapeutic approaches to elevate IFNλ-1 protein where required.

Regulation of IFN-λ1 promoter activity (IFN-λ1/IL-29) in human airway epithelial cells.

The type III (λ) IFNs (IFN-λ1, IFN-λ2, and IFN-λ3) and their receptor are the most recently discovered IFN family. They are induced by viruses and mediate antiviral activity, but type III IFNs have an important, specific functional niche at the immune/epithelial interface, as well as in the regulation of Th2 cytokines. Their expression appears diminished in bronchial epithelial cells of rhinovirus-infected asthmatic individuals. We investigated the regulation of IFN-λ1 expression in human airway epithelial cells using reporter genes analysis, chromatin immunoprecipitation, small interfering RNA knockdown, and DNase footprinting. In this article, we define the c-REL/p65 NF-κB heterodimer and IRF-1 as key transcriptional activators and ZEB1, B lymphocyte-induced maturation protein 1, and the p50 NF-κB homodimer as key repressors of the IFN-λ1 gene. We further show that ZEB1 selectively regulates type III IFNs. To our knowledge, this study presents the first characterization of any type III IFN promoter in its native context and conformation in epithelial cells and can now be applied to understanding pathogenic dysregulation of IFN-λ1 in human disease.

The lambda interferons: guardians of the immune-epithelial interface and the T-helper 2 response.

The type-III interferons (IFNs) are the most recently discovered IFNs in the human immune system and have important, but as yet poorly characterized, functions in innate and adaptive immunity that complement their antiviral functions. It is now becoming clear that these type-III IFNs have a functional niche where epithelial surfaces interact with the adaptive immune system, that their antiviral capability is not as highly developed as that of the type-I IFNs, and that they have their own profile of immunomodulatory functions; specifically, they are key modulators of the T-helper (Th)2 response.

Interferon-lambda1 (interleukin-29) preferentially down-regulates interleukin-13 over other T helper type 2 cytokine responses in vitro.

Interferon (IFN)-lambda1 [interleukin (IL)-29] is a member of the interferon lambda family (also known as type III interferons), whose members are distantly related to both the type I interferons and members of the IL-10 family. While IFN-lambda1 has significant antiviral activity, it is also becoming apparent that it has important immunoregulatory properties, especially with regard to the T helper type 2 (Th2) response. Previously, we have shown that IFN-lambda1 is capable of down-regulating IL-13 production in an IFN-gamma-independent manner and that this is mediated in part via monocyte-derived dendritic cells. Here, we have extended our knowledge of IFN-lambda1 regulation of the human in vitro Th2 response by examining the regulation of three major Th2 cytokines, IL-4, IL-5 and IL-13, by IFN-lambda1. Our results reveal that IFN-lambda1 preferentially inhibits IL-13 production, compared with IL-4 or IL-5. Levels of IL-13 mRNA, the amount of secreted IL-13 protein and numbers of IL-13-positive CD3(+) CD4(+) cells were all significantly diminished by IFN-lambda1. IFN-lambda1 significantly decreased some aspects of IL-4 and IL-5 production, but its effects were not as consistent as those seen on IL-13. IFN-lambda1 was also effective at decreasing IL-13 secretion under conditions designed to support the generation of Th2 cells. Irrespective of whether Concanavalin-A or T-cell-stimulatory microbeads were used, IFN-lambda1 markedly diminished IL-13 secretion in cultures where IL-4 had been added. Thus, IFN-lambda1 appears to be an inhibitor of human Th2 responses whose action is primarily directed towards IL-13 but which may also affect Th2 responses generally and does not invoke a complementary elevation of IFN-gamma secretion.

Human beta-defensin 2 induces a vigorous cytokine response in peripheral blood mononuclear cells.

beta-Defensins are a family of small cationic peptides involved in the innate response to microbial infection. Although their role in microbial killing is well established, the mechanisms through which this occurs remain largely undefined. Here, using protein array technology, we describe a role for human beta-defensins in the induction of an inflammatory cytokine response by human peripheral blood mononuclear cells (PBMCs). Human beta-defensins 1, 2, and 3 were examined for induction of an array of cytokines and chemokines. Some cytokines, such as interleukin 8 (IL-8) and monocyte chemoattractant protein 1, were up-regulated by all three defensins, while others, such as IL-6 and IL-10, were induced more selectively. It was notable that each defensin induced a unique pattern of cytokines. This report documents, for the first time, an analysis of the composite cytokine response of human PBMCs to beta-defensins. The induction or up-regulation of a number of cytokines involved in the adaptive immune response suggests a possible role for these defensins in linking innate and acquired immunity.

Human IL-19 regulates immunity through auto-induction of IL-19 and production of IL-10.

IL-19 is a novel, recently identified member of the IL-10 family of cytokines. We identified IL-10 as a cytokine that was strongly induced in IL-19-stimulated PBMC. IL-19-induced IL-10 secretion was dose-dependent and could be detected in culture supernatants after 3 h of stimulation. Furthermore, quantitative RT-PCR analysis demonstrated that IL-19 stimulation increased the level of IL-10 mRNA present within cells, suggesting that IL-19 is a transcriptional activator of IL-10. IL-19 was also able to induce its own expression, with IL-10 potently down-regulating this IL-19 'auto-induction'. LPS induction of IL-19 expression was also regulated by IL-10, demonstrating that IL-10 is likely an important regulator of human IL-19 induction. Maturation of dendritic cells from human PBMC in the presence of IL-19 resulted in an increase in IL-10 levels within these cells, whereas IL-12 was not affected. These results advance our understanding of the function of this novel cytokine and its regulation within the human immune system, in addition to providing a new insight into the control of the important immunoregulatory cytokine, IL-10.

Novel hairpin-shaped primer assay to study the association of the -44 single-nucleotide polymorphism of the DEFB1 gene with early-onset periodontal disease.

A powerful, cost-effective new method for studying single-nucleotide polymorphisms (SNPs) is described. This method is based on the use of hairpin-shaped primers (HP), which give a sensitive and specific PCR amplification of each specific allele, without the use of costly fluorophore-labeled probes and any post-PCR manipulation. The amplification is monitored in real-time using SYBR Green I dye and takes only 2 h to yield results. The HP assay has a simple design and utilizes a conventional real-time PCR apparatus. The -44 C-->G transversion in the DEFB1 gene (which encodes human beta-defensin 1) has been previously associated with Candida carriage in oral epithelia. In this study, we analyzed the association between early-onset periodontal disease (EOP) and the -44 SNP. We used an HP assay to study the distribution of the -44 SNP in 264 human DNAs obtained from two cohorts of EOP patients and healthy controls from different ethnic backgrounds. The results indicate that the -44 SNP has a similar distribution between EOP and healthy patients, suggesting that it is not associated with the disease.

Human interleukin-19 and its receptor: a potential role in the induction of Th2 responses.

Interleukin-19 (IL-19) is a newly discovered member of the IL-10 family of ligands whose function is presently undefined. We recently described its cloning and initial characterization and in so doing, noted that the induction of IL-19 by LPS in human monocytes was down-regulated by interferon-gamma (IFN-gamma) and up-regulated by IL-4. This preliminary observation led us to speculate that IL-19 may play a role in the Th1/Th2 system and we examined this hypothesis further. Our results suggested that IL-19 is able to influence the maturation of human T-cells. CD4+ T-cells resulting from SEB stimulation in the presence of IL-19 contained a higher proportion of IL-4 producing cells than those developing in the absence of IL-19. This observation was complimented by the observation that fewer IFN-gamma cells accrued in the presence of IL-19, thereby suggesting that IL-19 altered the balance of Th1/Th2 cells in favour of Th2. Furthermore, in whole PBMC cultures, IL-19 up-regulated IL-4 and down-regulated IFNgamma in a dose-dependent manner. These results are presented here in review format, in the context of an overall discussion of IL-19 and its receptor.

Evidence for duplication of the human defensin gene DEFB4 in chromosomal region 8p22-23 and implications for the analysis of SNP allele distribution.

Defensins constitute a primary mechanism in the innate immune system of humans and all mammals. Defensins are short, processed peptide molecules that are classified by structure into three groups: alpha-defensins, beta-defensins and theta-defensins. In humans, four beta-defensins have been described so far, corresponding to the products of the genes DEFB1 (hBD1, NM_005218), DEFB4 (hBD2, NM_004942.2), DEFB103 (hBD3, NM_018661), and DEFB104 (hBD4, NM_080389), respectively. All these genes have been mapped to chromosome 8p22-23. Much interest has been shown in genetic variation in the population at defensin loci to understand individual differences in disease susceptibility and severity. In this study, we have used an electronic search and then fluorescence in situ hybridization (FISH) on elongated chromosomes to demonstrate that the region containing the DEFB4 gene is duplicated on human chromosome 8p, making difficult the discovery of new SNPs in this gene and compromising the assessment of their allelic distribution in various ethnic populations for disease association studies.

Interleukin 10 (IL-10) influences autoimmune response in primary Sjögren's syndrome and is linked to IL-10 gene polymorphism.

OBJECTIVE: To investigate the association between serum levels of interleukin 10 (IL-10), the synthesis of autoantibodies, salivary gland disease activity, clinical manifestations, and IL-10 microsatellite polymorphism in patients with primary Sjögren's syndrome (pSS). METHODS: Serum IL-10 and autoantibody levels [IgG anti-Ro and anti-La, total and IgA rheumatoid factor (RF)] were measured by ELISA. A minor salivary gland (MSG) biopsy was performed in all patients and the focus score was determined as a measure of salivary gland disease activity. In addition, IL-10 microsatellite typing was performed by polymerase chain reaction technique. RESULTS: IL-10 concentration was higher in patients (n = 39) than in controls (n = 15) (21.4 +/- 6.7 vs 2.5 +/- 3.5 pg/ml; p = 0.001). We found a significant positive correlation between IL-10 levels and titers of IgA RF, anti-Ro, and anti-La antibodies, as well as focus score. In comparison with patients with low IL-10 production (< 9.5 pg/ml), patients producing high IL-10 had significantly more episodes of cutaneous vasculitis and a higher proportion of them carried the IL-10.G9 allele. CONCLUSION: Autoimmune response in pSS patients as well as salivary gland disease activity and cutaneous involvement appears to be mediated by IL-10 levels; in turn, there is a linkage with IL-10 gene polymorphism.

Frequency of functional interleukin-10 promoter polymorphism is different between relapse-onset and primary progressive multiple sclerosis.

Interleukin-10 (IL-10) secretion affects the inducibility of experimental autoimmune encephalomyelitis and the outcome of multiple sclerosis (MS). Here, we report that a G to A polymorphism in the IL-10 promoter at position -2849 is significantly associated with low IL-10 production. The frequency of this polymorphism is lower among patients with primary progressive compared with patients with relapse-onset MS and control persons.