Lipopolysaccharide/TLR4 Stimulates IL-13 Production through a MyD88-BLT2-Linked Cascade in Mast Cells, Potentially Contributing to the Allergic Response.

In an experimental asthma model, the activation of TLR4 by bacterial LPS occasionally exacerbates allergic inflammation through the production of Th2 cytokines, and mast cells have been suggested to play a central role in this response. However, the detailed mechanism underlying how LPS/TLR4 stimulates the production of Th2 cytokines, especially IL-13, remains unclear in mast cells. In the current study, we observed that the expression levels of leukotriene B4 receptor-2 (BLT2) and the synthesis of its ligands were highly upregulated in LPS-stimulated bone marrow-derived mast cells and that BLT2 blockade with small interfering RNA or a pharmacological inhibitor completely abolished IL-13 production, suggesting a mediatory role of the BLT2 ligand-BLT2 axis in LPS/TLR4 signaling to IL-13 synthesis in mast cells. Moreover, we demonstrated that MyD88 lies upstream of the BLT2 ligand-BLT2 axis and that this MyD88-BLT2 cascade leads to the generation of reactive oxygen species through NADPH oxidase 1 and the subsequent activation of NF-κB, thereby mediating IL-13 synthesis. Interestingly, we observed that costimulation of LPS/TLR4 and IgE/FcεRI caused greatly enhanced IL-13 synthesis in mast cells, and blockading BLT2 abolished these effects. Similarly, in vivo, the IL-13 level was markedly enhanced by LPS administration in an OVA-induced asthma model, and injecting a BLT2 antagonist beforehand clearly attenuated this increase. Together, our findings suggest that a BLT2-linked cascade plays a pivotal role in LPS/TLR4 signaling for IL-13 synthesis in mast cells, thereby potentially exacerbating allergic response. Our findings may provide insight into the mechanisms underlying how bacterial infection worsens allergic inflammation under certain conditions.

5-/12-Lipoxygenase-linked cascade contributes to the IL-33-induced synthesis of IL-13 in mast cells, thus promoting asthma development.

BACKGROUND: As asthma progresses, the levels of IL-33 in serum are markedly increased and contribute to asthmatic development and exacerbation. Mast cells, one of the principal effector cells in the pathogenesis of asthma, express high levels of the IL-33 receptor ST2 and have been shown to be activated by IL-33. Thus, IL-33 stimulates mast cells to produce Th2-type cytokines such as IL-13, thus contributing to asthmatic development. However, the signaling mechanism for IL-33-induced synthesis of Th2 cytokines, particularly IL-13, has not been fully elucidated in mast cells. METHODS: The role of 5- or 12-LO in the IL-33-induced synthesis of IL-13 was investigated using knockdown or pharmacological inhibitors in bone marrow-derived mast cells (BMMCs) and animal model. RESULTS: Blockade of 5- or 12-LO significantly suppressed IL-33-induced synthesis of IL-13 in BMMCs. The subsequent action of 5- and 12-LO metabolites through their specific receptor, BLT2, was also critical for IL-33-induced synthesis of IL-13. We also demonstrated that the MyD88-p38 kinase cascade lies upstream of 5-/12-LO and that NF-κB lies downstream of 5-/12-LO to mediate the IL-33-induced synthesis of IL-13 in mast cells. Consistent with these findings, we observed that in an IL-33-administered asthmatic airway inflammation model, IL-13 levels were markedly increased in bronchoalveolar lavage fluid, but its levels were markedly suppressed by treatment with inhibitors of 5-LO, 12-LO or BLT2, further suggesting roles of 5-/12-LO in IL-33-induced IL-13 production. CONCLUSION: Our results suggest that "MyD88-5-/12-LO-BLT2-NF-κB" cascade significantly contributes to the IL-33-induced synthesis of IL-13 in mast cells, thus potentially contributing to asthmatic development and exacerbation.

Leukotriene B4 Receptor 2 Is Critical for the Synthesis of Vascular Endothelial Growth Factor in Allergen-Stimulated Mast Cells.

Mast cells are among the principal effector cells in the pathogenesis of allergic asthma. In allergic reactions, allergen (Ag)-induced cross-linking of IgE bound to FcεRI on mast cells results in the production of vascular endothelial growth factor (VEGF), which is essential for the initiation and development of the allergic response. Despite the central role of VEGF in allergic asthma, the signaling events responsible for the production of VEGF remain unclear, particularly in Ag-stimulated mast cells. In the present study, we observed that blocking leukotriene B4 receptor 2 (BLT2) completely abrogated the production of VEGF in Ag-stimulated bone marrow-derived mast cells (BMMCs). The synthesis of BLT2 ligands (leukotriene B4 and 12(S)-hydroxyeicosatetraenoic acid) was also required for VEGF production, suggesting a mediating role of an autocrine BLT2 ligands-BLT2 axis in the production of VEGF in mast cells. The NADPH oxidase 1-reactive oxygen species-NF-κB cascade is downstream of BLT2 during Ag signaling to VEGF synthesis in mast cells. Furthermore, the level of VEGF synthesis in genetically mast cell-deficient Kit(W/Wv) mice was significantly lower than that in wild-type mice in the OVA-induced asthma model, suggesting that mast cells play a critical role in the synthesis of VEGF in OVA-induced allergic asthma. Importantly, VEGF production was restored to the levels observed in wild-type mice after adoptive transfer of normal BMMCs into Kit(W/Wv) mice but was not restored in BLT2(-/-) BMMC-reconstituted Kit(W/Wv) mice in the OVA-induced asthma model. Taken together, our results suggest that BLT2 expression in mast cells is essential for the production of VEGF in OVA-induced allergic asthma.

Association of the FGA and SLC6A4 genes with autistic spectrum disorder in a Korean population.

BACKGROUND: Autism spectrum disorder (ASD) is a neurobiological disorder characterized by distinctive impairments in cognitive function, language, and behavior. Linkage and population studies suggest a genetic association between solute carrier family 6 member 4 (SLC6A4) variants and ASD. METHOD: Logistic regression was used to identify associations between single-nucleotide polymorphisms (SNPs) and ASD with 3 alternative models (additive, dominant, and recessive). Linear regression analysis was performed to determine the influence of SNPs on Childhood Autism Rating Scale (CARS) scores as a quantitative phenotype. RESULTS: In the present study, we examined the associations of SNPs in the SLC6A4 gene and the fibrinogen alpha chain (FGA) gene. Logistic regression analysis showed a significant association between the risk of ASD and rs2070025 and rs2070011 in the FGA gene. The gene-gene interaction between SLC6A4 and FGA was not significantly associated with ASD susceptibility. However, polymorphisms in both SLC6A4 and the FGA gene significantly affected the symptoms of ASD. CONCLUSION: Our findings indicate that FGA and SLC6A4 gene interactions may contribute to the phenotypes of ASD rather than the incidence of ASD.

Contributory Role of BLT2 in the Production of Proinflammatory Cytokines in Cecal Ligation and Puncture-Induced Sepsis.

BLT2 is a low-affinity receptor for leukotriene B4, a potent lipid mediator of inflammation generated from arachidonic acid via the 5-lipoxygenase pathway. The aim of this study was to investigate whether BLT2 plays any role in sepsis, a systemic inflammatory response syndrome caused by infection. A murine model of cecal ligation and puncture (CLP)-induced sepsis was used to evaluate the role of BLT2 in septic inflammation. In the present study, we observed that the levels of ligands for BLT2 (LTB4 [leukotriene B4] and 12(S)-HETE [12(S)-hydroxyeicosatetraenoic acid]) were significantly increased in the peritoneal lavage fluid and serum from mice with CLP-induced sepsis. We also observed that the levels of BLT2 as well as 5-LO and 12-LO, which are synthesizing enzymes for LTB4 and 12(S)-HETE, were significantly increased in lung and liver tissues in the CLP mouse model. Blockade of BLT2 markedly suppressed the production of sepsis-associated cytokines (IL-6 [interleukin-6], TNF-α [tumor necrosis factor alpha], and IL-1ß [interleukin-1ß] as well as IL-17 [interleukin-17]) and alleviated lung inflammation in the CLP group. Taken together, our results suggest that BLT2 cascade contributes to lung inflammation in CLP-induced sepsis by mediating the production of inflammatory cytokines. These findings suggest that BLT2 may be a potential therapeutic target for sepsis patients.

Mediatory roles of leukotriene B4 receptors in LPS-induced endotoxic shock.

Sepsis, a systemic inflammatory response syndrome caused by infection, is the most common disease in patients treated in intensive care units. Endotoxic shock, the most critical form of sepsis, is caused by gram-negative bacterial infection. However, the detailed mechanism of endotoxic shock remains unclear. In the present study, we observed that the production of leukotriene B4 (LTB4) and 12(S)-hydroxyeicosatetraenoic acid (HETE), inflammatory lipid mediators acting on LTB4 receptors (BLT1 and BLT2), was significantly upregulated in peritoneal lavage fluid (PF) and serum from an LPS-induced endotoxic shock mouse model. Furthermore, BLT1/2-dependent signaling pathways mediated the expression of IL-17, IL-6, and IL-1ß, key cytokines for the development of endotoxic shock, via NF-κB activation in the LPS-induced endotoxic shock mouse model. Additionally, inhibition of BLT1/2 significantly attenuated inflammation and tissue damage associated with endotoxic shock and enhanced the survival rate of mice with this inflammatory complication. Together, these results suggest that LTB4 receptors play critical mediatory roles in the development of endotoxic shock. Our findings point to LTB4 receptors as potential therapeutic targets for the treatment of endotoxic shock.

Yeast-based assays for characterization of the functional effects of single nucleotide polymorphisms in human DNA repair genes.

DNA repair mechanisms maintain genomic integrity upon exposure to various types of DNA damage, which cause either single- or double-strand breaks in the DNA. Here, we propose a strategy for the functional study of single nucleotide polymorphisms (SNPs) in the human DNA repair genes XPD/ERCC2, RAD18, and KU70/XRCC6 and the checkpoint activation gene ATR that are essentially involved in the cell cycle and DNA damage repair. We analyzed the mutational effects of the DNA repair genes under DNA-damaging conditions, including ultraviolet irradiation and treatment with genotoxic reagents, using a Saccharomyces cerevisiae system to overcome the limitations of the human cell-based assay. We identified causal variants from selected SNPs in the present analyses. (i) R594C SNP in RAD3 (human XPD/ERCC2) caused severe reductions in the growth rate of mutant cells upon short-wavelength UV irradiation or chemical reagent treatment. (ii) The growth rates of the selected variants in RAD18, YKU70, and MEC1 were similar to those of wild-type cells on methyl methanesulfonate and hydroxyurea treated media. (iii) We also assessed the structural impact of the SNPs by analyzing differences in the structural conformation and calculating the root mean square deviation, which is a measure of the discordance of the Cα atoms between protein structures. Based on the above results, we propose that these analytical approaches serve as efficient methods for the identification of causal variants of human disease-causing genes and elucidation of yeast-cell based molecular mechanisms.

Associations between single-nucleotide polymorphism in the FNDC3A and autism spectrum disorder in a Korean population.

Autism spectrum disorder (ASD) is a neurodevelopmental syndrome associated with impairments of reciprocal communication and cognitive function. Associations between single-nucleotide polymorphisms (SNPs) and ASD were analysed by logistic regression. Polymorphisms in fibronectin type III domain-containing 3A (FNDC3A) exhibited significant associations in genotype and diplotype analyses. We conclude that FNDC3A influences the prevalence of ASD.

Association between peroxisomal biogenesis factor 7 and autism spectrum disorders in a Korean population.

Autism spectrum disorder is a neurodevelopmental disorder characterized by deficits in social communication, impaired reciprocal social interaction, and repetitive patterns of behaviors or interests. Although the cause of autism spectrum disorder remains elusive, the present study identified peroxisomal biogenesis factor 7 (PEX7) as a gene associated with autism spectrum disorder, and this association was examined in a Korean population. PEX7 encodes a cytosolic receptor for peroxisome targeting signal 2 of peroxisomal matrix enzymes that are targeted to and translocated into the peroxisome. PEX7 defects are associated with rhizomelic chondrodysplasia punctata type 1 and Refsum disease. Mutations in PEX7 are related to a variety of mild to severe clinical symptoms, including mental retardation. The analysis of 9 intronic single nucleotide polymorphisms in 214 patients with autism spectrum disorder and 258 controls revealed the association of 2 single nucleotide polymorphisms and 1 haplotype with autism spectrum disorder (P < .05).