Photoperiod Affects Leptin Action on the Choroid Plexus in Ewes Challenged with Lipopolysaccharide-Study on the mRNA Level.

The ovine choroid plexus (ChP) expresses the long isoform of the leptin receptor, which makes this structure a potential target for leptin action. In sheep, leptin concentration in plasma is higher during long days (LD) than short days (SD). This study evaluates the influence a of photoperiod on leptin impact on the gene expression of Toll-like receptor 4 (TLR4), proinflammatory cytokines (IL1B, IL6), their receptors (IL1R1, IL1R2, ILRN, IL6R, IL6ST) and inflammasome components necessary for pro-IL-1ß activation (NLRP3, PYCARD, CASP1), chemokine (CCL2), leptin receptor isoforms (LEPRa, LEPRb) and a suppressor of cytokine signalling (SOCS3) in the ChP of ewes treated or not with lipopolysaccharide (LPS). Studies were conducted on adult female sheep divided into four groups (n = 6 in each): control, leptin (20 µg/kg), LPS (400 ng/kg), and LPS and leptin injected under SD and LD photoperiods. The leptin alone did not affect the gene expression but in co-treatment with LPS increased (p < 0.05) IL1B but only during SD, and SOCS3, IL1R2, IL1RN, IL6ST and CCL2 only during LD, and decreased (p < 0.05) the IL1R1 expression only during SD photoperiod. This indicates that the immunomodulatory action of leptin on the ChP is manifested only under the LPS challenge and is photoperiodically dependent.

Fine mapping of the GWAS loci identifies SLC35D1 and IL23R as potential risk genes for leprosy.

BACKGROUND: Previous genome-wide association study (GWAS) identified two new leprosy associated loci (1p31.3 [rs3762318] and 6q24.3 [rs2275606]). However, there were insufficient validations in independent populations. OBJECTIVE: To validate the association and to map the potentially causal variants/genes underlying the association between the confirmed GWAS hit and leprosy. METHODS: We genotyped 10 variants in the regions encompassing the two loci in 1110 Han Chinese subjects with and without leprosy, followed by expression quantitative trait loci (eQTL), mRNA expression profiling, and network analysis. We further sequenced the exon region of four genes that were located in the confirmed GWAS hit region in 80 leprosy patients and 99 individuals without leprosy. RESULTS: We validated the positive association of rs3762318 with multibacillary leprosy (P=7.5×10-4), whereas the association of rs2275606 could not be validated. eQTL analysis showed that both the GWAS locus rs3762318 and one surrounding positively associated SNP rs2144658 (P=1.8×10-3) significantly affected the mRNA expression of a nearby gene SLC35D1, which might be involved in metabolism. Moreover, SLC35D1 was differentially expressed in skin tissues of leprosy patients, and the differential expression pattern was consistent among leprosy subtypes. Rare damaging missense variants in IL23R were significantly enriched in leprosy patients. CONCLUSION: Our results supported the positive association between the GWAS reported rs3762318 and leprosy, and SLC35D1 and IL23R might be the causal genes.

Variations in IL-23 and IL-25 receptor gene structure, sequence and expression associated with the two disease forms of sheep paratuberculosis.

The immunopathology of paucibacillary and multibacillary sheep paratuberculosis is characterized by inflammatory T cell and macrophage responses respectively. IL-23 and IL-25 are key to the development of these responses by interaction with their complex receptors, IL-23R/IL-12RB1 and IL-17RA/IL-17RB. In humans, variations in structure, sequence and/or expression of these genes have been implicated in the different pathological forms of tuberculosis and leprosy, and in gastrointestinal inflammatory disorders such as Crohn's disease. Sequencing has identified multiple transcript variants of sheep IL23R, IL12RB1 and IL17RB and a single IL17RA transcript. RT-qPCR assays were developed for all the identified variants and used to compare expression in the ileo-caecal lymph node of sheep with paucibacillary or multibacillary paratuberculosis and uninfected animals. With IL-23 receptor, only the IL12RB1v3 variant, which lacks the receptor activation motif was differentially expressed and was significantly increased in multibacillary disease; this may contribute to high Th2 responses. Of the IL17RB variants only full length IL17RB was differentially expressed and was significantly increased in multibacillary pathology; which may also contribute to Th2 polarization. IL17RA expression was significantly increased in paucibacillary disease. The contrast between the IL17RA and IL17RB results may indicate that, in addition to Th1 cells, Th17 T cells are also involved in paucibacillary pathology.

A general efficient and flexible approach for genome-wide association analyses of imputed genotypes in family-based designs.

Genotype imputation is a critical technique for following up genome-wide association studies. Efficient methods are available for dealing with the probabilistic nature of imputed single nucleotide polymorphisms (SNPs) in population-based designs, but not for family-based studies. We have developed a new analytical approach (FBATdosage), using imputed allele dosage in the general framework of family-based association tests to bridge this gap. Simulation studies showed that FBATdosage yielded highly consistent type I error rates, whatever the level of genotype uncertainty, and a much higher power than the best-guess genotype approach. FBATdosage allows fast linkage and association testing of several million of imputed variants with binary or quantitative phenotypes in nuclear families of arbitrary size with arbitrary missing data for the parents. The application of this approach to a family-based association study of leprosy susceptibility successfully refined the association signal at two candidate loci, C1orf141-IL23R on chromosome 1 and RAB32-C6orf103 on chromosome 6.

IL12B SNPs and copy number variation in IL23R gene associated with susceptibility to leprosy.

BACKGROUND: Genome-wide studies have identified both human leucocyte antigen (HLA) and non-HLA regions in association with leprosy. Involvement of novel functional loci within these regions has been proposed by us earlier. METHODS: We investigated the role of 23 single nucleotide polymorphisms (SNPs) in IL12B and IL12RB2 in a total of 2345 individuals from India, using MassArray platform, along with the copy number variations in IL23R, IL12RB2 and IL10 genes in a representative set of 257 individuals, using real-time PCR. RESULTS: SNP rs2853694 in IL12B gene (AA vs AC+CC, p=2.6E-04, OR=1.42 (1.17-1.70)) showed an association with leprosy. Pairwise interaction analysis followed by combined analysis of multiple SNPs identified that IL12B, TNF and BTNL2-DRA inter-genic SNPs provided a major risk towards leprosy (p=2.6E-08, OR=3.94 (2.43-6.38)), showing a further increase (p=3.6E-14) for combined risk genotype interactions. On the other hand, IL12B, BAT1, NFKBIL1 and LTA SNPs together showed a disposition towards protection (p=0.000011, OR=0.32 (0.19-0.53)) with a further increase (p=6.38E-10) for combined protective genotype-interactions. Copy number variation analysis showed an increased copy number of the IL23R gene (PB=36.4%, controls=20.2%; p=0.026) associated with the pauci-bacillary form of leprosy, which correlated with a trend towards enhanced expression in memory T cells in a preliminary observation. CONCLUSIONS: The observations made here highlight the importance of interaction between specific genetic backgrounds of immune response related genes in the outcome of Mycobacterium leprae infection.

Identification of two new loci at IL23R and RAB32 that influence susceptibility to leprosy.

We performed a genome-wide association study with 706 individuals with leprosy and 5,581 control individuals and replicated the top 24 SNPs in three independent replication samples, including a total of 3,301 individuals with leprosy and 5,299 control individuals from China. Two loci not previously associated with the disease were identified with genome-wide significance: rs2275606 (combined P = 3.94 × 10(-14), OR = 1.30) on 6q24.3 and rs3762318 (combined P = 3.27 × 10(-11), OR = 0.69) on 1p31.3. These associations implicate IL23R and RAB32 as new susceptibility genes for leprosy. Furthermore, we identified evidence of interaction between the NOD2 and RIPK2 loci, which is consistent with the biological association of the proteins encoded by these genes (NOD2-RIPK2 complex) in activating the NF-κB pathway as a part of the host defense response to infection. Our findings have expanded the biological functions of IL23R by uncovering its involvement in infectious disease susceptibility and suggest a potential involvement of autophagocytosis in leprosy pathogenesis. The IL23R association supports previous observations of the marked overlap of susceptibility genes for leprosy and Crohn's disease, implying common pathogenesis mechanisms.

Polymorphism of the 5' flanking region of the IL-12 receptor beta2 gene partially determines the clinical types of leprosy through impaired transcriptional activity.

BACKGROUND: Individual differences in T cell responsiveness to interleukin 12 (IL-12), resulting from inherited factors, may be responsible for differences in the intensity of cell mediated immune (CMI) responses in patients with leprosy, a disease with a wide clinical spectrum. AIM: Polymorphisms in the 5' flanking region of the IL12RB2 gene were analysed to determine potential immunogenetic factors affecting CMI responses, using leprosy as a model. METHODS: Polymorphisms in the 5' flanking region of IL12RB2 were examined using direct sequencing techniques, and allele frequencies between patients with lepromatous leprosy and patients with tuberculoid leprosy were compared. The effect of these single nucleotide polymorphisms (SNPs) on IL12RB2 expression was estimated using the dual luciferase reporter gene assay in Jurkat T cells. RESULTS: Several SNPs, including -1035A>G, -1023A>G, -650delG, and -465A>G, were detected within the 5' flanking region of IL12RB2. The frequency of haplotype 1 (-1035A, -1023A, -650G, -464A) was high in the general Japanese population, but was significantly lower in lepromatous patients compared with tuberculoid patients and healthy controls. Reporter gene assays using Jurkat T cells revealed that all haplotypes carrying one or more SNP exhibited a lower transcriptional activity compared with haplotype 1. CONCLUSION: SNPs within the 5' flanking region of IL12RB2 affect the degree of expression of this gene and may be implicated in individual differences in CMI responsiveness to mycobacterial antigens, leading to lepromatous or tuberculoid leprosy.

Control of human host immunity to mycobacteria.

Infection with Mycobacterium tuberculosis results in disease in 5-10% of exposed individuals, whereas the remainder controls infection effectively. Similar inter-individual differences in disease susceptibility are characteristic features of leprosy, typhoid fever, leishmaniasis and other chronic infectious diseases, including viral infections. Although the outcome of infection is influenced by many factors, it is clear that genetic host factors play an important role in controlling disease susceptibility to intracellular pathogens. Knowledge of the genes involved and their downstream cellular pathways will provide new insights for the design of improved and rationalized strategies to enhance host-resistance, e.g. by vaccination. In addition, this knowledge will aid in identifying better biomarkers of protection and disease, which are essential tools for the monitoring of vaccination and other intervention trials. The recent identification of patients with deleterious mutations in genes that encode major proteins in the type-1 cytokine (IL-12/IL23-IFN-gamma) axis, that suffered from severe infections due to otherwise poorly pathogenic mycobacteria (non-tuberculous mycobacteria (NTM) or M. bovis Bacille Calmette-Guerin (BCG)) or Salmonella species has revealed the major role of this system in innate and adaptive immunity to mycobacteria and salmonellae. Clinical tuberculosis has now been described in a number of patients with IL-12/IL23-IFN-gamma system defects. Moreover, unusual mycobacterial infections were reported in several patients with genetic defects in NEMO, a key regulatory molecule in the NFkappaB pathway. These new findings will be discussed since they provide further insights into the role of type-1 cytokines in immunity to mycobacteria, including M. tuberculosis.

Human genetics of intracellular infectious diseases: molecular and cellular immunity against mycobacteria and salmonellae.

The ability to develop adequate immunity to intracellular bacterial pathogens is unequally distributed among human beings. In the case of tuberculosis, for example, infection with Mycobacterium tuberculosis results in disease in 5-10% of exposed individuals, whereas the remainder control infection effectively. Similar interindividual differences in disease susceptibility are characteristic features of leprosy, typhoid fever, leishmaniasis, and other chronic infectious diseases, including viral infections. The outcome of infection is influenced by many factors, such as nutritional status, co-infections, exposure to environmental microbes, and previous vaccinations. It is clear, however, that genetic host factors also play an important part in controlling disease susceptibility to intracellular pathogens. Recently, patients with severe infections due to otherwise poorly pathogenic mycobacteria (non-tuberculous mycobacteria or Mycobacterium bovis BCG) or Salmonella spp have been identified. Many of these patients were unable to produce or respond to interferon gamma, due to deleterious mutations in genes that encode major proteins in the type 1 cytokine (interleukin 12/interleukin 23/interferon gamma) axis (interleukin 12p40/interleukin 23p40, IL12 receptor beta1/IL23 receptor beta1, interferon gamma receptors 1 and 2, or signal transducer and activator of transcription 1). This axis is a major immunoregulatory system that bridges innate and adaptive immunity. Unusual mycobacterial infections were also reported in several patients with genetic defects in inhibitor of NFkappaB kinase gamma, a key regulatory molecule in the nuclear factor kappaB pathway. New findings discussed in this review provide further and sometimes surprising insights into the role of type 1 cytokines, and into the unexpected heterogeneity seen in these syndromes.

Missense mutations of the interleukin-12 receptor beta 1(IL12RB1) and interferon-gamma receptor 1 (IFNGR1) genes are not associated with susceptibility to lepromatous leprosy in Korea.

Interleukin-12 receptor beta 1 ( IL12RB1), interleukin-12 receptor beta 2 ( IL12RB2), and interferon gamma receptor 1 ( IFNGR1) perform important roles in the host defense against intracellular pathogens such as Mycobacteria. Several mutations within their genes have been confirmed as associated with increased susceptibility to mycobacterial infection. However, the association between mutations of the IL12RB1, IL12RB2, and IFNGR1 encoding genes and lepromatous leprosy has not been studied. This study screened for polymorphisms within IL12RB1, IL12RB2, and IFNGR1 encoding genes in the Korean populations using polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP) DNA sequencing assay, and an association study was performed using the missense mutations of 705 A/G (Q214R), 1196 G/C (G378R), 1637 G/A (A525T), and 1664 C/T (P534S) of the IL12RB1, 83 G/A (V14M), and 1443 T/C (L467P) for the IFNGR1 encoding genes. There were no differences in the genotype and allele frequencies of IL12RB1 and IFNGR1 genes between 93 lepromatous leprosy patients and 94 control subjects. In conclusion, missense mutations of 705 A/G (Q214R), 1196 G/C (G378R), 1637 G/A (A525T), 1664 C/T (P534S) of the IL12RB1, 83 G/A (V14 M), and 1443 T/C (L467P) of the IFNGR1 encoding genes have no association with the susceptibility to lepromatous leprosy in the Korean population.

Missense mutations of the interleukin-12 receptor beta 1(IL12RB1) and interferon-gamma receptor 1 (IFNGR1) genes are not associated with susceptibility to lepromatous leprosy in Korea

Interleukin-12 receptor beta 1 ( IL12RB1), interleukin-12 receptor beta 2 ( IL12RB2), and interferon gamma receptor 1 ( IFNGR1) perform important roles in the host defense against intracellular pathogens such as Mycobacteria. Several mutations within their genes have been confirmed as associated with increased susceptibility to mycobacterial infection. However, the association between mutations of the IL12RB1, IL12RB2, and IFNGR1 encoding genes and lepromatous leprosy has not been studied. This study screened for polymorphisms within IL12RB1, IL12RB2, and IFNGR1 encoding genes in the Korean populations using polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP) DNA sequencing assay, and an association study was performed using the missense mutations of 705 A/G (Q214R), 1196 G/C (G378R), 1637 G/A (A525T), and 1664 C/T (P534S) of the IL12RB1, 83 G/A (V14M), and 1443 T/C (L467P) for the IFNGR1 encoding genes. There were no differences in the genotype and allele frequencies of IL12RB1 and IFNGR1 genes between 93 lepromatous leprosy patients and 94 control subjects. In conclusion, missense mutations of 705 A/G (Q214R), 1196 G/C (G378R), 1637 G/A (A525T), 1664 C/T (P534S) of the IL12RB1, 83 G/A (V14 M), and 1443 T/C (L467P) of the IFNGR1 encoding genes have no association with the susceptibility to lepromatous leprosy in the Korean population.

Genetic and physical mapping of 2q35 in the region of the NRAMP and IL8R genes: identification of a polymorphic repeat in exon 2 of NRAMP.

Recent interest has focused on the region of conserved synteny between mouse chromosome 1 and human 2q33-q37, particularly over the region encoding the murine macrophage resistance gene Ity/Lsh/Bcg (candidate Nramp) and members of the Il8r interleukin-8 (IL8) receptor gene cluster. In this paper, identification of a restriction fragment length polymorphism in the IL8RB gene in 35 pedigrees previously typed for markers in the 2q33-q37 interval provided evidence (lod scores > 3) for linkage between IL8RB and the 2q34-q35 markers FN1, TNP1, VIL1, and DES. Physical mapping, using yeast artificial chromosomes isolated with VIL1, confirmed that IL8RA, IL8RB, and the IL8RB pseudogene map within the NRAMP-VIL1 interval, with the physical distance (155 kb) from 5' LSH to 3' VIL1 representing approximately 3-fold that observed in the mouse. Partial sequencing of NRAMP confirmed the presence of the N-terminal proline/serine-rich putative SH3 binding domain in exon 2 of the human gene. Further analysis of Brazilian leprosy and visceral leishmaniasis pedigrees identified a rare second allele varying in a 9-nucleotide repeat motif of the exon 2 sequence but segregating independently of the disease phenotype.