Leukotriene B4 Receptor Research Articles

What happens to basophils and tryptase, LXA4 and CysLTs during aspirin desensitization?

Introduction Aspirin desensitization (AD) is an effective treatment in patients with non-steroidal anti-inflammatory drugs (NSAID)-exacerbated respiratory disease (NERD) by providing inhibitory effect on symptoms and polyp recurrence. However, limited data is available on how AD works. We aimed to study comprehensively the mechanisms underlying AD by examining basophil activation (CD203c upregulation), mediator-releases of tryptase, CysLT, and LXA4, and LTB4 receptor expression for the first 3 months of AD. Methods The study was conducted in patients with NERD who underwent AD (group 1: n = 23), patients with NERD who received no desensitization (group 2: n = 22), and healthy volunteers (group 3, n = 13). All participants provided blood samples for flow cytometry studies (CD203c and LTB4 receptor), and mediator releases (CysLT, LXA4, and tryptase) for the relevant time points determined. Results All baseline parameters of CD203c and LTB4 receptor expressions, tryptase, CysLT, and LXA4 releases were similar in each group (p > 0.05). In group 1, CD203c started to be upregulated at the time of reactions during AD, and continued to be high for 3 months when compared to controls. All other study parameters were comparable with baseline and at the other time points in each group (p > 0.05). Conclusion Although basophils are active during the first 3 months of AD, no releases of CysLT, tryptase or LXA4 exist. Therefore, our results suggest that despite active basophils, inhibition of mediators can at least partly explain underlying the mechanism in the first three months of AD.

Intestinal epithelial BLT1 promotes mucosal repair.

Acute and chronic intestinal inflammation is associated with epithelial damage, resulting in mucosal wounds in the forms of erosions and ulcers in the intestinal tract. Intestinal epithelial cells (IECs) and immune cells in the wound milieu secrete cytokines and lipid mediators to influence repair. Leukotriene B4 (LTB4), a lipid chemokine, binds to its receptor BLT1 and promotes migration of immune cells to sites of active inflammation; however, a role for intestinal epithelial BLT1 during mucosal wound repair is not known. Here we report that BLT1 was expressed in IECs both in vitro and in vivo, where it functioned as a receptor not only for LTB4 but also for another ligand, resolvin E1. Intestinal epithelial BLT1 expression was increased when epithelial cells were exposed to an inflammatory microenvironment. Using human and murine primary colonic epithelial cells, we reveal that the LTB4/BLT1 pathway promoted epithelial migration and proliferation leading to accelerated epithelial wound repair. Furthermore, in vivo intestinal wound repair experiments in BLT1-deficient mice and bone marrow chimeras demonstrated an important contribution of epithelial BLT1 during colonic mucosal wound repair. Taken together, our findings show a potentially novel prorepair in IEC mechanism mediated by BLT1 signaling.

Leukotriene B4 Receptor 2 Mediates the Production of G-CSF That Plays a Critical Role in Steroid-Resistant Neutrophilic Airway Inflammation.

Granulocyte colony-stimulating factor (G-CSF) has been suggested to be closely associated with neutrophilic asthma pathogenesis. However, little is known about the factors regulating the production of G-CSF in neutrophilic asthma. We previously reported that a leukotriene B4 receptor 2, BLT2, played an important role in neutrophilic airway inflammation. Therefore, in the current study, we investigated whether BLT2 plays a role in the production of G-CSF in lipopolysaccharide/ovalbumin (LPS/OVA)-induced steroid-resistant neutrophilic asthma. The data showed that BLT2 critically mediated G-CSF production, contributing to the progression of neutrophilic airway inflammation. We also observed that 12-lipoxygenase (12-LO), which catalyzes the synthesis of the BLT2 ligand 12(S)-HETE, was also necessary for G-CSF production. Together, these results suggest that the 12-LO-BLT2-linked signaling network is critical for the production of G-CSF, contributing to the development of neutrophilic airway inflammation. Our findings can provide a potential new target for the therapy of severe neutrophilic asthma.

Specialized pro-resolvin mediators induce cell growth and improve wound repair in intestinal epithelial Caco-2 cell cultures

Specialized pro-resolvin mediators (SPMs) are a superfamily of bioactive molecules synthesized from polyunsaturated fatty acids (arachidonic, eicosapentaenoic and docosahexaenoic acids) that include resolvins, protectins and maresins. These metabolites are important to control the resolution phase of inflammation and the epithelial repair, which is essential in restoring the mucosal barriers. Unfortunately, the effects of SPMs on intestinal epithelial cell growth remain poorly understood. Caco-2 cell were used as intestinal epithelial cell model. Cell growth/DNA synthesis, cell signalling pathways, western blot and wound repair assay were performed. Our data demonstrated that SPMs such as lipoxin LxA4, resolvin (Rv) E1, RvD1, protectin D 1 and maresin 1 were able to enhance intestinal epithelial Caco-2 cell growth and DNA synthesis. Furthermore, our results provide evidence that these effects of RvE1 and RvD1 were associated with a pertussis toxin-sensitive G protein-coupled receptor, and that leukotriene B4 receptor 2 could be involved, at least in part, in these effects of RvE1/RvD1. Moreover, these mitogenic effects induced by SPMs were dependent on the ERK 1/2 and p38 MAPK pathways as well as phospholipase C and protein kinase C activation. Thus, these mitogenic effects of RvE1/RvD1 on intestinal epithelial cells could be involved in this signalling circuit involved in wounded epithelium and the catabasis process.

LTB4R Promotes the Occurrence and Progression of Clear Cell Renal Cell Carcinoma (ccRCC) by Regulating the AKT/mTOR Signaling Pathway.

ccRCC is highly immunogenic, yet its underlying immune-related molecular mechanisms are unknown. Leukotriene B4 Receptor 1 (LTB4R), a novel immune-related gene associated in our previous research with the prognosis of ccRCC patients, has been found in many cancers; however, its potential mechanism in renal clear carcinoma is unclear. This study was conducted to investigate LTB4R's action mechanism in renal clear cell carcinoma. First, a CCK8 assay was performed to verify LTB4R's effect on the proliferation viability of renal clear cell carcinoma cells. Scratch and transwell assays verified LTB4R's effect on the migration and invasion ability of renal clear cell carcinoma cells. Flow cytometry validated LTB4R's effect on renal clear cell carcinoma cells' apoptosis and cell cycle. A Western blot assay was conducted to further investigate LTB4R's effect on apoptosis, cell cycle, EMT process, and AKT/mTOR signaling pathway in renal clear cell carcinoma at the protein level. In vitro experiments showed that LTB4R knockdown inhibited the proliferation, migration, and invasion of renal clear cell carcinoma cells and promoted their apoptosis, whereas LTB4R overexpression promoted the proliferation, migration, and invasion of renal clear cell carcinoma cells and inhibited their apoptosis. In addition, we found that LTB4R regulated the proliferation and apoptosis of renal clear cell carcinoma cells by regulating the AKT/mTOR signaling pathway's phosphorylation process. Furthermore, we verified some of these results using bioinformatic analysis. LTB4R plays an oncogenic role in renal clear cell carcinoma; it is expected to be a molecular target for renal clear cell carcinoma treatment and a predictive biomarker for prognosis.

Abstract 12174: Inflammation-Dependent Hydroxyeicosatetraenoic Acids Dysregulate Macrophage Resolution Function

Introduction and Hypothesis: Macrophage efferocytosis is a critical step in the resolution of inflammation. Dysregulated macrophage efferocytosis has been implicated in the pathogenesis of chronic inflammatory disorders including atherosclerosis and IBD. Nonetheless, the nature of possible dysregulating mechanisms remains incompletely understood. We reported that non-enzymatic lipid peroxidation and the mucosal levels of monohydroxylated PUFAs including 12- and 15-hydroxyeicosatetraenoic acid (HETE) correlated with disease in murine models of IBD. HETEs can form enzymatically and non-enzymatically and can signal through the leukotriene (LT) receptor BLT2. LTB4-BLT1/2 signaling can suppress macrophage efferocytosis capacity. We hypothesized that non-enzymatically derived mucosal HETEs correlate with disease in a murine Crohn’s disease (CD) model and in IBD patient biopsies; and that HETEs suppress murine and human macrophage efferocytosis capacity. Methods: We exploited the stereospecificity of enzymatic HETE formation to determine by chiral-LC-MS/MS the levels of non-enzymatically derived HETEs in intestine from a murine myeloid Cox2 knock-out model of Crohn’s disease (CD) and in human IBD patient biopsies. We assessed the effect of racemic HETEs on the efferocytosis capacity of murine BMDMs and human THP1 macrophages for apoptotic MPRO neutrophils. We performed proteomics using accurate mass LC-MS/MS followed by GO enrichment analysis. Results: Non-enzymatically derived mucosal 5-, 12-, and 15HETE were elevated with disease: a) by 3-4 fold in the murine CD model (p<0.01 to p<0.0001); and b) by 2-3 fold in ileal and colonic biopsies from areas of active inflammation in CD and ulcerative colitis patients compared to remission controls (p<0.05). Racemic HETEs suppressed efferocytosis of apoptotic neutrophils by murine (12- and 15-HETE: p<0.0001) and human (12-HETE: p<0.0001) macrophages. Racemic 12-HETE altered the proteome of efferocytic BMDM by inducing 200 proteins while silencing 200 more (<1% FDR), and by suppressing pathways associated with efferocytosis. Conclusion: Inflammation-dependent HETEs may suppress macrophage efferocytosis in chronic inflammatory disorders including IBD and atherosclerosis.

Role of leukotriene B4 (LTB4)-LTB4 receptor 1 signaling in post-incisional nociceptive sensitization and local inflammation in mice.

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.

Hepatocyte leukotriene B4 receptor 1 promotes NAFLD development in obesity.

NAFLD is the most prevalent chronic liver disease worldwide and has emerged as a serious public health issue with no approved treatment. The development of NAFLD is strongly associated with hepatic lipid content, and patients with NAFLD have significantly higher rates of hepatic de novo lipogenesis (DNL) than lean individuals. Leukotriene B4 (LTB4), a metabolite of arachidonic acid, is dramatically increased in obesity and plays important role in proinflammatory cytokine production and insulin resistance. But the role of liver LTB4/LTB4 receptor 1 (Ltb4r1) in lipid metabolism is unclear. Hepatocyte-specific knockout (HKO) of Ltb4r1 improved hepatic steatosis and systemic insulin resistance in both diet-induced and genetically induced obese mice. The mRNA level of key enzymes involved in DNL and fatty acid esterification decreased in Ltb4r1 HKO obese mice. LTB4/Ltb4r1 directly promoted lipogenesis in HepG2 cells and primary hepatocytes. Mechanically, LTB4/Ltb4r1 promoted lipogenesis by activating the cAMP-protein kinase A (PKA)-inositol-requiring enzyme 1α (IRE1α)-spliced X-box-binding protein 1 (XBP1s) axis in hepatocytes, which in turn promoted the expression of lipogenesis genes regulated by XBP1s. In addition, Ltb4r1 suppression through the Ltb4r1 inhibitor or lentivirus-short hairpin RNA delivery alleviated the fatty liver phenotype in obese mice. LTB4/Ltb4r1 promotes hepatocyte lipogenesis directly by activating PKA-IRE1α-XBP1s to promote lipogenic gene expression. Inhibition of hepatocyte Ltb4r1 improved hepatic steatosis and insulin resistance. Ltb4r1 is a potential therapeutic target for NAFLD.

The In Vitro and In Vivo Effect of Lipoxygenase Pathway Inhibitors Nordihydroguaiaretic Acid and Its Derivative Tetra-O-methyl Nordihydroguaiaretic Acid against Brucella abortus 544.

This study investigated the contribution of lipoxygenase (LOX) inhibitors, nordihydroguaiaretic acid (NDGA), tetra-O-methyl nordihydroguaiaretic acid (M4N) and zileuton (ZIL), and thromboxane A2 (TXA2) inhibitor 4,5-diphenylimidazole (DPI) in the proliferation of Brucella abortus infection. None of the compounds affected the uptake of Brucella into the macrophages. We determined the effect of neutralizing leukotriene B4 (LTB4) receptor and showed that the uptake of the bacteria was inhibited at 30 min post-infection. M4N treatment attenuated intracellular survival of Brucella at 2 h post-incubation but it was not observed in the succeeding time points. DPI treatment showed reduced survival of Brucella at 24 h post-incubation while blocking LTB4 receptor was observed to have a lower intracellular growth at 48 h post-incubation suggesting different action of the inhibitors in the course of the survival of Brucella within the cells. Reduced proliferation of the bacteria in the spleens of mice was observed in animals treated with ZIL or DPI. Increased serum cytokine level of TNF-α and MCP-1 was observed in mice treated with M4N or ZIL while a lower IFN-γ level in ZIL-treated mice and a higher IL-12 serum level in DPI-treated mice were observed at 7 d post-infection. At 14 d post-infection, ZIL-treated mice displayed reduced serum level of IL-12 and IL-10. Overall, inhibition of 5-LOX or TXA2 or a combination therapy promises a potential alternative therapy against B. abortus infection. Furthermore, strong ligands for LTB4 receptor could also be a good candidate for the control of Brucella infection.

Compilation and evaluation of a fatty acid mimetics screening library

Focused compound libraries are well-established tools for hit identification in drug discovery and chemical probe development. We present the compilation and application of a focused screening library of fatty acid mimetics (FAMs), which are compounds designed to bind the orthosteric site of proteins that endogenously accommodate natural fatty acids and lipid metabolites. This set complies with chemical properties of FAM and was found suitable for use also in cellular setting. Several hits were retrieved in screening the focused library against diverse fatty acid binding targets including the enzymes soluble epoxide hydrolase (sEH) and leukotriene A4 hydrolase (LTA4H), the nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RXRα), the carrier proteins fatty acid binding protein 4 and 5 (FABP4 and FABP5), as well as the G-protein coupled receptors leukotriene B4 receptor 1 (BLT1) and free-fatty acid receptor 1 (FFAR1). Thus, the focused FAM library is suitable to obtain chemical starting matter for fatty acid binding proteins and provides a valuable extension to available screening collections.

Isoflurane targets BLT1 to attenuate lung injury

Abstract Sepsis-associated lung injury is one of major comorbidities in sepsis, largely responsible for its mortality. Lack of specific therapeutic for lung injury is an urgent issue to be addressed. We found that volatile anesthetic isoflurane attenuated lung injury associated with experimental polymicrobial abdominal sepsis. This was associated with a reduction in the number of recruited neutrophils in the lung and bronchoalveolar space. Furthermore, isoflurane attenuated neutrophil chemotaxis induced by major chemoattractant leukotriene B4 (LTB4), not by C5a. Although LTB4 level did not differ by isoflurane exposure, isoflurane was shown to interact with LTB4’s neutrophil receptor BLT1 in silico. The interaction site was predicted to overlap with BLT1 antagonist BIIL260 binding site, suggesting isoflurane as a BLT1 antagonist. Five biotinylated compounds with a range of spacer lengths were developed and subjected to calcium mobilization test using CHO-BLT1 cells to determine their property as BLT1 antagonist. Two compounds showed adequate inhibition and we performed the competition assay for BLT1 between the compound and isoflurane. Isoflurane significantly competed with the BIIL260 biotinylated compound, indicating that isofluane bound to the BIIL260 binding site on BLT1. Isoflurane is considered as an alternative sedative in Europe and Canada, and this result could support the potential use of isoflurane as an ICU sedative in lung injury setting. Supported by GM118277

Cysteinyl–leukotriene and prostaglandin pathways in bronchial versus alveolar lavage in allergic asthmatics

Cysteinyl–leukotriene and prostaglandin pathways in bronchial versus alveolar lavage in allergic asthmatics

Prognostic implications and functional enrichment analysis of LTB4R in patients with acute myeloid leukemia

To explore the expression patterns, prognostic implications, and biological role of leukotriene B4 receptor (LTB4R) in patients with acute myeloid leukemia (AML). We collected the data of mRNA expression levels and clinical information of patients with AML from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database for mRNA expression analyses, survival analyses, Cox regression analyses and correlation analyses using R studio to assess the expression patterns and prognostic value of LTB4R. The correlation of LTB4R expression levels with clinical characteristics of the patients were analyzed using UALCAN. The co-expressed genes LTB4R were screened from Linkedomics and subjected to functional enrichment analysis. A protein-protein interaction network was constructed using STRING. GSEA analyses of the differentially expressed genes (DEGs) were performed based on datasets from TCGA-LAML stratified by LTB4R expression level. We also collected peripheral blood mononuclear cells (PBMCs) from AML patients and healthy donors for examination of the mRNA expression levels of LTB4R and immune checkpoint genes using qRT-PCR. We also examined serum LTB4R protein levels in the patients using ELISA. The mRNA expression level of LTB4R was significantly increased in AML patients (4.898±1.220 vs 2.252±0.215, P < 0.001), and an elevated LTB4R expression level was correlated with a poor overall survival (OS) of the patients (P=0.004, HR=1.74). LTB4R was identified as an independent prognostic factor for OS (P=0.019, HR=1.66) and was associated with FAB subtypes, cytogenetic risk, karyotype abnormalities and NPM1 mutations. The co- expressed genes of LTB4R were enriched in the functional pathways closely associated with AML leukemogenesis, including neutrophil inflammation, lymphocyte activation, signal transduction, and metabolism. The DEGs were enriched in differentiation, activation of immune cells, and cytokine signaling. Examination of the clinical serum samples also demonstrated significantly increased expressions of LTB4R mRNA (P=0.044) and protein (P=0.008) in AML patients, and LTB4R mRNA expression was positively correlated with the expression of the immune checkpoint HAVCR2 (r= 0.466, P=0.040). LTB4R can serve as a novel biomarker and independent prognostic indicator of AML and its expression patterns provide insights into the crosstalk of leukemogenesis signaling pathways involving tumor immunity and metabolism.

Structural basis of leukotriene B4 receptor 1 activation

Leukotriene B4 receptor 1 (BLT1) plays crucial roles in the acute inflammatory responses and is a valuable target for anti-inflammation treatment, however, the mechanism by which leukotriene B4 (LTB4) activates receptor remains unclear. Here, we report the cryo-electron microscopy (cryo-EM) structure of the LTB4 -bound human BLT1 in complex with a Gi protein in an active conformation at resolution of 2.91 Å. In combination of molecule dynamics (MD) simulation, docking and site-directed mutagenesis, our structure reveals that a hydrogen-bond network of water molecules and key polar residues is the key molecular determinant for LTB4 binding. We also find that the displacement of residues M1013.36 and I2717.39 to the center of receptor, which unlock the ion lock of the lower part of pocket, is the key mechanism of receptor activation. In addition, we reveal a binding site of phosphatidylinositol (PI) and discover that the widely open ligand binding pocket may contribute the lack of specificity and efficacy for current BLT1-targeting drug design. Taken together, our structural analysis provides a scaffold for understanding BLT1 activation and a rational basis for designing anti-leukotriene drugs.

Isoflurane attenuates sepsis-associated lung injury

Acute lung injury is one of major complications associated with sepsis, responsible for morbidity and mortality. Patients who suffer from acute lung injury often require respiratory support under sedations, and it would be important to know the role of sedatives in lung injury. We examined volatile anesthetic isoflurane, which is commonly used in surgical setting, but also used as an alternative sedative in intensive care settings in European countries and Canada. We found that isoflurane exposure attenuated neutrophil recruitment to the lungs in mice suffering from experimental polymicrobial abdominal sepsis. We found that isoflurane attenuated one of major neutrophil chemoattractants LTB4 mediated response via its receptor BLT1 in neutrophils. Furthermore, we have shown that isoflurane directly bound to BLT1 by a competition assay using newly developed labeled BLT1 antagonist, suggesting that isoflurane would be a BLT1 antagonist.

Multipathway In Vitro Pharmacological Characterization of Specialized Proresolving G Protein-Coupled Receptors.

Specialized proresolving mediators (SPMs) and their cognate G protein-coupled receptors are implicated in autoimmune disorders, including chronic inflammation, rheumatoid arthritis, systemic scleroderma, and lupus erythematosus. To date, six G protein-coupled receptors (GPCRs) have been paired with numerous endogenous and synthetic ligands. However, the function and downstream signaling of these receptors remains unclear. To address this knowledge gap, we systematically expressed each receptor in a human embryonic kindney 293 (HEK293)-Flp-In-CD8a-FLAG cell system. Each receptor was pharmacologically characterized with both synthetic and putative endogenous ligands across different signaling assays, covering both G protein-dependent (Gs, Gi, and Gq) and independent mechanisms (β-arrestin2 recruitment). Three orphan GPCRs previously identified as SPM receptors (GPR 18, GPR32 and GPR37) failed to express in HEK 293 cells. Although we were unsuccessful in identifying an endogenous ligand for formyl peptide receptor 2 (FPR2)/lipoxin A4 receptor (ALX), with only a modest response to N-formylmethionine-leucyl-phenylalanine (fMLP), we did reveal clear signaling bias away from extracelluar signal-related kinase (ERK) 1/2 phosphorylation for the clinically tested agonist N-(2-{[4-(1,1-difluoroethyl)-1,3-oxazol-2-yl]methyl}-2H-1,2,3-triazol-4-yl)-2-methyl-5-(3-methylphenyl)-1,3-oxazole-4-carboxamide (ACT-389949), adding further evidence for its poor efficacy in two phase I studies. We also identified neuroprotectin D1 as a new leukotriene B4 receptor 1 (BLT1) agonist, implying an alternative target for the neuroprotective effects of the ligand. We confirmed activity for resolvin E1 (RvE1) at BLT1 but failed to observe any response at the chemerin1 receptor. This study provides some much-needed clarity around published receptor-ligand pairings but indicates that the expression and function of these SPM GPCRs remains very much context-dependent. In addition, the identification of signaling bias at FPR2/ALX may assist in guiding design of new FPR2/ALX agonists for the treatment of autoimmune disorders. SIGNIFICANCE STATEMENT: To our knowledge, this is the first study to comprehensibly show how several natural mediators and synthetic ligands signal through three specialized proresolving mediator GPCRs using multiple ligands from different classes across four-six endpoint signaling assays. This study discovers new ligand pairings, refutes others, reveals poly-pharmacology, and identifies biased agonism in formyl peptide receptor 2/lipoxin A4 receptor pharmacology. This study highlights the potential of these receptors in treating specific autoimmune diseases, including rheumatoid arthritis, systemic scleroderma, and systemic lupus erythematosus.

POS-099 Identification of the pathological mechanism of glomerulonephritis by the LTB4-BLT1 axis and its application to therapy

ANCA-associated nephritis is a rapidly progressive glomerulonephritis in which neutrophils are strongly involved in the pathogenesis. Although immunosuppressive drugs are the mainstay of treatment, there is an urgent need to establish a treatment based on the pathogenesis. Leukotriene B4 (LTB4) is one of the eicosanoids generated through the metabolism of arachidonic acid by 5-lipoxygenase and leukotriene A4hydrolase (LTA4H). Although it is generally known that LTB4functions as a potent chemotactic factor for neutrophils via its receptor BLT1, the role of LTB4-BLT1 axis on glomerulonephritis has not been clarified.

Development and Characterization of a Fluorescent Ligand for Leukotriene B4 Receptor 2 in Cells and Tissues.

The leukotriene B4 receptor 2 (BLT2) is a G-protein coupled receptor activated by 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), which has been proposed as a promising therapeutic target for diabetic wound healing and gastrointestinal lesions. In this study, the rational design of a fluorescent probe based on the synthetic BLT2 agonist CAY10583 is described. The synthesis of several derivatives of CAY10583 coupled to fluorescein resulted in a traceable ligand suitable for different fluorescence-based techniques. An HTRF-based displacement assay (Tag-lite) on stably transfected CHO-K1 cells was developed to characterize binding properties of diverse BLT2 ligands. Highly specific binding to the BLT2 receptor was demonstrated in staining experiments on mouse skin tissue, and specific modulation of BLT2-induced cAMP signaling provided further evidence for receptor binding and ligand functionality. In conclusion, the fluorescent ligands developed in this study are suitable to investigate the pharmacology of BLT2 receptor ligands in a variety of assay systems.

BLT1 and BLT2: Leukotriene B4 Receptors, Their Characteristics and Role in Various Diseases

LTB4는 호중구를 포함한 림프구의 모집 및 활성화에 중요한 역할을 하는 지질 매개체이다. LTB4는 두 가지 수용체를 통해 면역세포를 모집하거나 사이토카인을 분비함으로써 면역 반응을 조절한다. LTB4는 염증성 질환과 밀접한 관련이 있다. BLT1 저해제 개발에 대한 연구가 활발히 진행 중이지만 성공한 사례가 거의 없다. BLT2의 역할은 많이 연구되지 않았지만 최근 연구에 따르면 기도 염증과 암에서 염증을 유도하는 데 중요한 역할을 하는 것으로 나타났다. 본 논문에서는 다양한 질병에서의 LTB4와 LTB4 수용체인 BLT1과 BLT2의 특성과 역할에 대한 연구결과들을 살펴보고자 한다.

Leukotriene B4 Is a Major Determinant of Leukocyte Recruitment During Otitis Media.

BackgroundPathogens of otitis media (OM) induce inflammatory responses in the middle ear (ME), characterized by mucosal hyperplasia, leukocyte infiltration, and inflammatory mediators, including arachidonic acid metabolites. We studied the role of the eicosanoid leukotriene B4 (LTB4) in OM.MethodsExpression of LTB4-related genes was evaluated by gene array and single-cell RNA-Seq in MEs infected with nontypeable Haemophilus influenzae (NTHi). An inhibitor of LTB4 receptor 1 (i.e. U75302) was also used to block LTB4 responses.ResultsME expression of LTB4-related genes was observed by gene arrays and scRNA-Seq. However, not all genes involved in LTB4 generation occurred in any one specific cell type. Moreover, LTB4 receptor inhibition significantly reduced mucosal hyperplasia and virtually eliminated leukocyte infiltration.ConclusionsME expression of LTB4-related genes suggest a functional role in OM disease. The fact that LTB4-generation is spread across different cell types is consistent with a transcellular pathway of eicosanoid biosynthesis involving cell-to-cell signaling as well as transfer of biosynthetic intermediates between cells. The dramatic reduction in ME leukocyte infiltration caused by U75302 indicates that LTB4 plays a major role in ME inflammatory cell recruitment, acting via the LTB4R1 receptor. Given that there are many other chemotactic factors that occur in the ME during OM, the ability of LTB4 to activate leukocytes and stimulate their extravasation may explain the effects of inhibition. Reduction in mucosal hyperplasia due to U75302 administration may be secondary to the reduction in leukocytes since LTB4R1 is not expressed by mucosal epithelial or stromal cells. The results suggest that LTB4 receptor antagonists could be useful in treating OM.