A Multiomics Approach to Identify Genes Associated with Childhood Asthma Risk and Morbidity.

Childhood asthma is a complex disease. In this study, we aim to identify genes associated with childhood asthma through a multiomics "vertical" approach that integrates multiple analytical steps using linear and logistic regression models. In a case-control study of childhood asthma in Puerto Ricans (n = 1,127), we used adjusted linear or logistic regression models to evaluate associations between several analytical steps of omics data, including genome-wide (GW) genotype data, GW methylation, GW expression profiling, cytokine levels, asthma-intermediate phenotypes, and asthma status. At each point, only the top genes/single-nucleotide polymorphisms/probes/cytokines were carried forward for subsequent analysis. In step 1, asthma modified the gene expression-protein level association for 1,645 genes; pathway analysis showed an enrichment of these genes in the cytokine signaling system (n = 269 genes). In steps 2-3, expression levels of 40 genes were associated with intermediate phenotypes (asthma onset age, forced expiratory volume in 1 second, exacerbations, eosinophil counts, and skin test reactivity); of those, methylation of seven genes was also associated with asthma. Of these seven candidate genes, IL5RA was also significant in analytical steps 4-8. We then measured plasma IL-5 receptor α levels, which were associated with asthma age of onset and moderate-severe exacerbations. In addition, in silico database analysis showed that several of our identified IL5RA single-nucleotide polymorphisms are associated with transcription factors related to asthma and atopy. This approach integrates several analytical steps and is able to identify biologically relevant asthma-related genes, such as IL5RA. It differs from other methods that rely on complex statistical models with various assumptions.

IL-5 receptor α levels in patients with marked eosinophilia or mastocytosis.

BACKGROUND: IL-5 plays a central role in the development and maintenance of eosinophilia (EO) and eosinophil activation in a wide variety of eosinophilic disorders. Although IL-5, IL-3, and GM-CSF can modulate the expression of IL-5 receptor α (IL-5Rα) on eosinophils in vitro, little is known about soluble and surface IL-5Rα levels in vivo. OBJECTIVE: To assess soluble and surface IL-5Rα levels in patients with EO and/or mastocytosis. METHODS: Surface IL-5Rα expression was assessed by flow cytometry in blood and/or bone marrow from subjects with EO (n = 39) and systemic mastocytosis (n = 8) and from normal volunteers (n = 28). Soluble IL-5Rα (sIL-5Rα) level was measured in a cohort of 177 untreated subjects and correlated with EO, eosinophil activation, and serum tryptase and cytokine levels. RESULTS: IL-5Rα expression on eosinophils inversely correlated with EO (r = -0.48; P < .0001), whereas serum levels of sIL-5Rα increased with the eosinophil count (r = 0.56; P < .0001) and serum IL-5 (r = 0.40; P < .0001) and IL-13 (r = 0.29; P = .004) levels. Of interest, sIL-5Rα level was significantly elevated in patients with systemic mastocytosis without EO. Although sIL-5Rα levels correlated with serum tryptase levels in these patients, eosinophil activation, assessed by CD69 expression on eosinophils and serum eosinophil-derived neurotoxin levels, was increased compared with that in normal subjects. CONCLUSIONS: These data are consistent with an in vivo IL-5Rα regulatory pathway in human eosinophils similar to that described in vitro and involving a balance between soluble and surface receptor levels. This may have implications with respect to the use of novel therapeutic agents targeting IL-5 and its receptor in patients with EO and/or mastocytosis.

Leukoaraiosis is associated with genes regulating blood-brain barrier homeostasis in ischaemic stroke patients.

BACKGROUND: The biologic agents causing leukoaraiosis are unknown. Our aim was to study the genetic basis of leukoaraiosis. METHODS: We analyzed 212 single nucleotide polymorphisms (SNPs) in 142 patients with ischaemic stroke, generating a total of 30,104 genotypes. Seventy-nine subjects (55.6%) presented leukoaraiosis measured by the Fazekas scale and 69 (48.6%) by ARWMC scale. We analyzed the presence of synergic associations between SNPs using the hfcc software. Finally, functional studies were performed in 56 subjects. The Ingenuity Pathways software (ipa) was used to examine the role of the identified genes. RESULTS: Six SNPs were associated with leukoaraiosis using both measuring scales. After logistic regression adjusted for leukoaraiosis risk factors, the rs2252070 of MMP13 (OR = 4.9, 95%CI: 1.34-17.9, P = 0.016), rs662 of PON1 (OR = 0.37, 95%CI: 0.15-0.87, P = 0.024) and rs1800779 of NOS3 (OR = 3.9, 95%CI: 1.38-11.38, P = 0.01) were independently associated with leukoaraiosis under a dominant/recessive model and the rs2290608 of IL5RA (OR = 0.46, 95%CI: 0.25-0.85, P = 0.013) and rs669 of A2M (OR = 2.5, 95%CI: 1.36-4.83, P = 0.004) under an additive model. Computational analysis showed a synergic association of rs10497212-AA of ITGB6 and rs2290608-GG of IL5RA with leukoaraiosis using both scales. (i) ARWMC (P = 1.3 × 10(-4) ) and (ii) Fazekas (P = 4.5 × 10(-5) ). Functional studies showed that the rs669 SNP was associated with plasma levels of A2M (P = 0.012) and A2M levels with leukoaraiosis in Fazekas scale (P = 0.02). ipa analysis revealed that the genes associated with leukoaraiosis were involved in blood-brain barrier (BBB) homeostasis. CONCLUSIONS: Amongst patients with ischaemic stroke, several genes associated with BBB homeostasis could be involved with a higher risk of leukoaraiosis.

MiRNA-495-3p Attenuates TNF-α Induced Apoptosis and Inflammation in Human Nucleus Pulposus Cells by Targeting IL5RA.

Intervertebral disc degeneration (IVDD) is considered to be the fundamental cause of the occurrence and development of lumbar disc herniation (LDH). The degeneration of IVDD is mainly caused by the participation of inflammatory factors. Thus, it is of great significance to analyze the pathogenesis of IVDD, which may guide clinical prevention and treatment of LDH. Our current study aims to identify the role of miR-495-3p in LDH and to further unravel the underlying mechanisms. Results in the current study showed that TNF-α treatment markedly inhibited cell viability of HNPC, increased the IL-1ß level, and decreased the mRNA level of miR-495-3p in HNPC in a time-dependent manner. Up-regulation of miR-495-3p promoted cell proliferation and inhibited inflammation and apoptosis in TNF-α-induced HNPCs. To investigate the underlying molecular mechanism through which miR-495-3p regulates TNF-α-induced inflammation and apoptosis in HNPCs, we explored the possible target gene of miR-495-3p. Bioinformatics analysis indicated that IL5RA, which is an important gene for TNF-α-induced HNPC injury, was also a target gene of miR-495-3p. A luciferase reporter assay was applied to test and verify the direct target association between miR-495-3p and IL5RA. The results discovered that down-regulation of miR-495-3p markedly reversed the anti-apoptosis and anti-inflammation of sh-IL5RA. In short, the present study evaluated the roles of miR-495-3p and IL5RA in IVDD development and progression. All the data indicated that miRNA-495-3p may play a protective role via inhibiting inflammation and apoptosis in human nucleus pulposus cells by targeting IL5RA pathway. Therefore, miRNA-495-3p may be a potential agent for LDH, and our study may provide a novel strategy in LDH treatment.

IL-5 and IL-5 receptor alpha polymorphisms are associated with atopic dermatitis in Koreans.

BACKGROUND: Eosinophils are recruited into the affected tissue of asthma and atopic dermatitis (AD) patients. IL-5 and IL-5R are highly expressed in the AD skin lesions, yet the reported levels of IL-8 are controversial. METHOD: We genotyped 17 singlenucleotide polymorphisms (SNPs) from five genes of the 1120 case-control samples (646 AD and 474 controls). We measured the serum IL-5 concentrations in 87 individuals [36 ADe (AD extrinsic), 18 ADi (AD intrinsic) and 33 controls] by ELISA, and compared the results among these groups. RESULT: The rs2522411SNP and haplotype T-A in the IL-5 gene were significantly associated with the ADe. The serum IL-5 concentration was higher in the ADe than that in the ADi patients without any correlation with the rs2522411SNP. In the IL-5RA gene, the rs334809SNP showed a weak association with AD, and the rs6771148SNP and the haplotype T-C-T of the three adjacent tagged SNPs had an effect on the blood eosinophil counts and the serum ECP levels in the AD patients. However, we could not detect any relationship between AD and the SNPs in the IL-8 and IL-8R genes. CONCLUSION: We found that the rs2522411SNP and the haplotype T-A in the IL-5 gene and the serum IL-5 levels were strongly associated with the allergic type of AD, but not with the nonallergic type of AD. The association of the rs6771148SNP and the haplotype T-C-T in the IL5RA gene with the blood eosinophil counts and the serum ECP levels indicates that the IL5RA gene has a role for controlling eosinophils in the peripheral blood.

Nasal cholera toxin elicits IL-5 and IL-5 receptor alpha-chain expressing B-1a B cells for innate mucosal IgA antibody responses.

In this study, we examine whether native cholera toxin (nCT) as a mucosal adjuvant can support trinitrophenyl (TNP)-LPS-specific mucosal immune responses. C57BL/6 mice were given nasal TNP-LPS in the presence or absence of nCT. Five days later, significantly higher levels of TNP-specific mucosal IgA Ab responses were induced in the nasal washes, saliva, and plasma of mice given nCT plus TNP-LPS than in those given TNP-LPS alone. High numbers of TNP-specific IgA Ab-forming cells were also detected in mucosal tissues such as the nasal passages (NPs), the submandibular glands (SMGs), and nasopharyngeal-associated lymphoreticular tissue of mice given nCT. Flow cytometric analysis showed that higher numbers of surface IgA+, CD5+ B cells (B-1a B cells) in SMGs and NPs of mice given nasal TNP-LPS plus nCT than in those given TNP-LPS alone. Furthermore, increased levels of IL-5R alpha-chain were expressed by B-1a B cells in SMGs and NPs of mice given nasal TNP-LPS plus nCT. Thus, CD4+ T cells from these mucosal effector lymphoid tissues produce high levels of IL-5 at both protein and mRNA levels. When mice were treated with anti-IL-5 mAb, significant reductions in TNP-specific mucosal IgA Ab responses were noted in external secretions. These findings show that nasal nCT as an adjuvant enhances mucosal immune responses to a T cell-independent Ag due to the cross-talk between IL-5Ralpha+ B-1a B cells and IL-5-producing CD4+ T cells in the mucosal effector lymphoid tissues.

The severity of FIP1L1-PDGFRA-positive chronic eosinophilic leukaemia is associated with polymorphic variation at the IL5RA locus.

We have investigated the hypothesis that constitutional genetic variation in IL-5 signalling may be associated with the development or severity of FIP1L1-PDGFRA-positive chronic eosinophilic leukaemia (CEL) in humans. We genotyped six single-nucleotide polymorphisms (SNP) within or close to the IL5RA or IL5 genes in 82 patients with FIP1L1-PDGFRA-positive CEL plus, as controls, healthy individuals (n=100), patients with FIP1L1-PDGFRA-negative eosinophilia (n=100) or patients with chronic myeloid leukaemia (CML) (n=100). We found no association between SNP allele frequency between FIP1L1-PDGFRA-positive and control cases. However, for FIP1L1-PDGFRA cases, we found an association between the genotype at rs4054760, an SNP in the 5'-UTR of IL5RA and peripheral blood eosinophil count (P=0.026) as well as the presence or absence of tissue infiltration (P=0.032). Although these associations fell below the level of significance once corrected for multiple testing, no such association was seen in FIP1L1-PDGFRA-negative cases and no difference in allele frequencies for rs4054760 was seen in control populations across Europe. Furthermore, in an analysis of 112 patients with CML, IL5RA expression was strongly related to rs4054760 genotype (P<0.001). These data suggest that the variations in IL5RA expression are linked to constitutional IL5RA genotype and severity of FIP1L1-PDGFRA disease.

Recombinant-protein production in insect cells utilizing a hollow-fibre bioreactor.

Here, we demonstrate for the first time that the hollow-fibre bioreactor is an excellent tool for the production of Drosophila-expressed recombinant proteins. Using the example of the soluble extracellular portion of the human IL-5 (interleukin 5) receptor alpha expression in S2 (Schneider's Drosophila melanogaster cell line 2) cells, we found that it is possible to produce multi-milligram amounts of functional recombinant protein continuously for several months on a laboratory scale with minimal maintenance requirements. The insect cells grow to high density and express concentrated functional recombinant protein in a small volume, simplifying and economizing downstream purification.