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Leukotriene B4 (LTB4) is a potent lipid mediator involved in the recruitment and activation of neutrophils, which is an important feature of tissue injury and inflammation. The biological effects of LTB4 are primarily mediated through the high-affinity LTB4 receptor, BLT1. Postoperative incisional pain is characterized by persistent acute pain at the site of tissue injury and is associated with local inflammation. Here, we compared the role of LTB4-BLT1 signaling in postoperative incisional pain between BLT1-knockout (BLT1KO) and wild-type (BLT1WT) mice. A planter incision model was developed, and mechanical pain hypersensitivity was determined using the von Frey test before and after incision. Local infiltration of neutrophils and inflammatory monocytes was quantified by flow cytometry. Inflammatory cytokine levels in the incised tissue were also determined. Mechanical pain hypersensitivity was significantly reduced in BLT1KO mice compared to BLT1WT mice at 2, 3, and 4 days after incision. LTB4 levels in the tissue at the incision site peaked 3 hours after the incision. Infiltrated neutrophils peaked 1 day after the incision in both BLT1KO and BLT1WT mice. The accumulation of inflammatory monocytes increased 1-3 days after the incision and was significantly more reduced in BLT1KO mice than in BLT1WT mice. In BLT1KO mice, Interleukin-1β and Tumor Necrosis Factor-α levels 1 day after the incision were significantly lower than those of BLT1WT mice. Our data suggest that LTB4 is produced and activates its receptor BLT1 in the very early phase of tissue injury, and that LTB4-BLT1 signaling exacerbates pain responses by promoting local infiltration of inflammatory monocytes and cytokine production. Thus, LTB4-BLT1 signaling is a potential target for therapeutic intervention of acute and persistent pain induced by tissue injury., Competing Interests: The authors have declared that no competing interests exist.

Leukotriene B4 (LTB4) is a lipid mediator that acts as a potent chemoattractant for inflammatory leukocytes. Kidney fibrosis is caused by migrating inflammatory cells and kidney-resident cells. Here, we examined the role of the high-affinity LTB4 receptor BLT1 during development of kidney fibrosis induced by unilateral ureteral obstruction (UUO) in wild-type (WT) mice and BLT1 knockout (BLT1-/-) mice. We found elevated expression of 5-lipoxygenase (5-LOX), which generates LTB4, in the renal tubules of UUO kidneys from WT mice and BLT1-/- mice. Accumulation of immunoreactive type I collagen in WT UUO kidneys increased over time; however, the increase was less prominent in BLT1-/- UUO kidneys. Accumulation of S100A4-positive fibroblasts increased temporally in WT UUO kidneys, but was again less pronounced in-BLT1-/- UUO kidneys. The same was true of mRNA encoding transforming growth factor-β (TGF)-β and fibroblast growth factor (FGF)-2. Finally, accumulation of F4/80-positive macrophages, which secrete TGF-β, increased temporally in WT UUO and BLT1-/- UUO kidneys, but to a lesser extent in the latter. Following LTB4 stimulation in vitro, macrophages showed increased expression of mRNA encoding TGF-β/FGF-2 and Col1a1, whereas L929 fibroblasts showed increased expression of mRNA encoding α smooth muscle actin (SMA). Bone marrow (BM) transplantation studies revealed that the area positive for type I collagen was significantly smaller in BLT1-/-BM→WT than in WT-BM→WT. Thus, LTB4-BLT1 signaling plays a critical role in fibrosis in UUO kidneys by increasing accumulation of macrophages and fibroblasts. Therefore, blocking BLT1 may prevent renal fibrosis., Competing Interests: The authors have declared that no competing interests exist.

Background: Transient receptor potential cation channel subfamily V member 1 (TRPV1) are sensitive to heat, capsaicin, pungent chemicals and other noxious stimuli. They play important roles in the pain pathway where in concert with proinflammatory factors such as leukotrienes they mediate sensitization and hyperalgesia. TRPV1 is the target of several novel analgesics drugs under development and therefore, TRPV1 genetic variants might represent promising candidates for pharmacogenetic modulators of drug effects., Methods: A next-generation sequencing (NGS) panel was created for the human TRPV1 gene and in addition, for the leukotriene receptors BLT1 and BLT2 recently described to modulate TRPV1 mediated sensitisation processes rendering the coding genes LTB4R and LTB4R2 important co-players in pharmacogenetic approaches involving TRPV1. The NGS workflow was based on a custom AmpliSeq™ panel and designed for sequencing of human genes on an Ion PGM™ Sequencer. A cohort of 80 healthy subjects of Western European descent was screened to evaluate and validate the detection of exomic sequences of the coding genes with 25 base pair exon padding., Results: The amplicons covered approximately 97% of the target sequence. A median of 2.81 x 106 reads per run was obtained. This identified approximately 140 chromosome loci where nucleotides deviated from the reference sequence GRCh37 hg19 comprising the three genes TRPV1, LTB4R and LTB4R2. Correspondence between NGS and Sanger derived nucleotide sequences was 100%., Conclusions: Results suggested that the NGS approach based on AmpliSeq™ libraries and Ion Personal Genome Machine (PGM) sequencing is a highly efficient mutation detection method. It is suitable for large-scale sequencing of TRPV1 and functionally related genes. The method adds a large amount of genetic information as a basis for complete analysis of TRPV1 ion channel genetics and its functional consequences.

Cytochrome P-450 epoxygenase (EPOX)-derived epoxyeicosatrienoic acids (EETs), 5-lipoxygenase (5-LO), and leukotriene B4 (LTB4), the product of 5-LO, all play a pivotal role in the vascular inflammatory process. We have previously shown that EETs can alleviate oxidized low-density lipoprotein (ox-LDL)-induced endothelial inflammation in primary rat pulmonary artery endothelial cells (RPAECs). Here, we investigated whether ox-LDL can promote LTB4 production through the 5-LO pathway. We further explored how exogenous EETs influence ox-LDL-induced LTB4 production and activity. We found that treatment with ox-LDL increased the production of LTB4 and further led to the expression and release of both monocyte chemoattractant protein-1 (MCP-1/CCL2) and intercellular adhesion molecule-1 (ICAM-1). All of the above ox-LDL-induced changes were attenuated by the presence of 11,12-EET and 14,15-EET, as these molecules inhibited the 5-LO pathway. Furthermore, the LTB4 receptor 1 (BLT1 receptor) antagonist U75302 attenuated ox-LDL-induced ICAM-1 and MCP-1/CCL2 expression and production, whereas LY255283, a LTB4 receptor 2 (BLT2 receptor) antagonist, produced no such effects. Moreover, in RPAECs, we demonstrated that the increased expression of 5-LO and BLT1 following ox-LDL treatment resulted from the activation of nuclear factor-κB (NF-κB) via the p38 mitogen-activated protein kinase (MAPK) pathway. Our results indicated that EETs suppress ox-LDL-induced LTB4 production and subsequent inflammatory responses by downregulating the 5-LO/BLT1 receptor pathway, in which p38 MAPK phosphorylation activates NF-κB. These results suggest that the metabolism of arachidonic acid via the 5-LO and EPOX pathways may present a mutual constraint on the physiological regulation of vascular endothelial cells.

The leukotriene B4 (LTB4) receptor 1 (BLT1) is a high affinity receptor for LTB4, a chemotactic and inflammatory eicosanoid. The LTB4 receptor 2 (BLT2) was originally identified as a low affinity receptor for LTB4, and, more recently, as a high affinity receptor for 12-hydroxyheptadecatrienoic acid (12-HHT). The zebrafish BLT receptors have not been previously identified and the in vivo functions of these receptors have been unknown. In this paper, we describe one zebrafish BLT1-like receptor, Blt1, and two zebrafish BLT2-like receptors, Blt2a and Blt2b. Cells expressing Blt1 exhibited LTB4-induced intracellular [Ca2+] increases, inhibition of cAMP production, ligand-dependent [35S]GTPγS binding, and transforming growth factor-α (TGFα) shedding activity in a dose-dependent manner, similar to human BLT1. Cells expressing Blt2a and Blt2b exhibited 12-HHT- and LTB4-induced intracellular [Ca2+] increases, inhibition of cAMP production, [35S]GTPγS binding, and TGFα shedding activity, with a dose-dependency similar to human BLT2. Reverse transcription (RT)-PCR analysis and whole-mount in situ hybridization revealed that blt1, blt2a, blt2b, zebrafish LTA4 hydrolase (lta4h), and zebrafish 5-lipoxiganase (5lo) are expressed in zebrafish embryos. Knockdown of blt1 by morpholino antisense oligonucleotides resulted in delayed epiboly at gastrulation. Consistently, knockdown of lta4h, an enzyme mediating LTB4 production, induced a phenotype similar to knockdown of blt1. These results suggest that the LTB4-BLT1 axis is involved in epiboly in zebrafish development.

The bioactive lipid mediator leukotriene B4 (LTB4) greatly enhances phagocyte antimicrobial functions against a myriad of pathogens. In murine histoplasmosis, inhibition of the LT-generating enzyme 5-lypoxigenase (5-LO) increases the susceptibility of the host to infection. In this study, we investigated whether murine resistance or susceptibility to Histoplasma capsulatum infection is associated with leukotriene production and an enhancement of in vivo and/or in vitro antimicrobial effector function. We show that susceptible C57BL/6 mice exhibit a higher fungal burden in the lung and spleen, increased mortality, lower expression levels of 5-LO and leukotriene B4 receptor 1 (BLT1) and decreased LTB4 production compared to the resistant 129/Sv mice. Moreover, we demonstrate that endogenous and exogenous LTs are required for the optimal phagocytosis of H. capsulatum by macrophages from both murine strains, although C57BL/6 macrophages are more sensitive to the effects of LTB4 than 129/Sv macrophages. Therefore, our results provide novel evidence that LTB4 production and BLT1 signaling are required for a histoplasmosis-resistant phenotype.

Background: The elevated production of interleukin (IL)-8 is critically associated with invasiveness and metastatic potential in breast cancer cells. However, the intracellular signaling pathway responsible for up-regulation of IL-8 production in breast cancer cells has remained unclear., Methodology/principal Findings: In this study, we report that the expression of BLT2 is markedly up-regulated in the highly aggressive human breast cancer cell lines MDA-MB-231 and MDA-MB-435 compared with MCF-10A immortalized human mammary epithelial cells, as determined by RT-PCR, real-time PCR and FACS analysis. Blockade of BLT2 with BLT2 siRNA knockdown or BLT2 inhibitor treatment downregulated IL-8 production and thereby diminished the invasiveness of aggressive breast cancer cells, analyzed by Matrigel invasion chamber assays. We further characterized the downstream signaling mechanism by which BLT2 stimulates IL-8 production and identified critical mediatory roles for the generation of reactive oxygen species (ROS) and the consequent activation of the transcription factor NF-κB. Moreover, blockade of BLT2 suppressed the formation of metastatic lung nodules by MDA-MB-231 cells in both experimental and orthotopic metastasis models., Conclusions/significance: Taken together, our study demonstrates that a BLT2-ROS-NF-κB pathway up-regulates IL-8 production in MDA-MB-231 and MDA-MB-435 cells, thereby contributing to the invasiveness of these aggressive breast cancer cells. Our findings provide insight into the molecular mechanism of invasiveness in breast cancer.

Background: During their blood meal, ticks secrete a wide variety of proteins that can interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response., Methodology/principal Findings: We previously identified 14 new lipocalin genes in the tick Ixodes ricinus. One of them codes for a protein that specifically binds leukotriene B4 with a very high affinity (Kd: +/-1 nM), similar to that of the neutrophil transmembrane receptor BLT1. By in silico approaches, we modeled the 3D structure of the protein and the binding of LTB4 into the ligand pocket. This protein, called Ir-LBP, inhibits neutrophil chemotaxis in vitro and delays LTB4-induced apoptosis. Ir-LBP also inhibits the host inflammatory response in vivo by decreasing the number and activation of neutrophils located at the tick bite site. Thus, Ir-LBP participates in the tick's ability to interfere with proper neutrophil function in inflammation., Conclusions/significance: These elements suggest that Ir-LBP is a "scavenger" of LTB4, which, in combination with other factors, such as histamine-binding proteins or proteins inhibiting the classical or alternative complement pathways, permits the tick to properly manage its blood meal. Moreover, with regard to its properties, Ir-LBP could possibly be used as a therapeutic tool for illnesses associated with an increased LTB4 production.