Leukotriene B4 Biosynthesis Research Articles

Structure-Guided Elaboration of a Fragment-Like Hit into an Orally Efficacious Leukotriene A4 Hydrolase Inhibitor.

Leukotriene A4 hydrolase (LTA4H) is the final and rate-limiting enzyme in the biosynthesis of pro-inflammatory leukotriene B4 (LTB4). Preclinical studies have provided strong evidence that LTA4H is an attractive drug target for the treatment of chronic inflammatory diseases. Here, we describe the transformation of compound 2, a fragment-like hit, into the potent inhibitor of LTA4H 3. Our strategy involved two key steps. First, we aimed to increase the polarity of fragment 2 to improve its drug-likeness, particularly its solubility, while preserving both its promising potency and low molecular weight. Second, we utilized structural information and incorporated a basic amino function, which allowed for the formation of an essential hydrogen bond with Q136 of LTA4H and consequently enhanced the potency. Compound 3 exhibited exceptional selectivity and showed oral efficacy in a KRN passive serum-induced arthritis model in mice. The anticipated human dose to achieve 90% target engagement at the trough concentration was determined to be 40 mg administered once daily.

Endothelial Protein kinase D1 is a major regulator of post-traumatic hyperinflammation.

Trauma is a major cause of death worldwide. The post-traumatic immune response culminates in the release of pro-inflammatory mediators, translating in the infiltration of neutrophils (PMNs) at injury sites. The extent of this inflammation is determined by multiple factors, such as PMN adhesion to the endothelium, transendothelial migration, endothelial barrier integrity as well as PMN swarming, mass infiltration and activation. This process is initiated by secondary lipid mediators, such as leukotriene B4 (LTB4). We here provide evidence that Protein kinase D1 (PRKD1) in endothelial cells is implicated in all these processes. Endothelial PRKD1 is activated by pro-inflammatory stimuli and amplifies PMN-mediated inflammation by upregulation of cytokine and chemokines as well as adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin. This induces enhanced PMN adhesion and trans-migration. PRKD1 activation also destabilizes endothelial VE-cadherin adhesion complexes and thus the endothelial barrier, fostering PMN infiltration. We even describe a yet unrecognized PRKD1-dependant mechanism to induce biosynthesis of the PMN-swarming mediator LTB4 directed via intercellular communication through small extracellular vesicles (sEVs) and enhanced CXCL8 secretion from activated endothelial cells. These endothelial sEVs transfer the LTB4 biosynthesis enzyme LTA4 hydrolase (LTA4H) to prime PMNs, while initiating biosynthesis also requires additional signals, like CXCL8. We further demonstrate the respective LTA4H-positive sEVs in the serum of polytrauma patients, peaking 12h post injury. Therefore, PRKD1 is a key regulator in the coordinated communication of the endothelium with PMNs and a vital signaling node during post-traumatic inflammation.

The leukotriene B4 /BLT1-dependent neutrophil accumulation exacerbates immune complex-mediated glomerulonephritis.

Crescent formation is the most important pathological finding that defines the prognosis of nephritis. Although neutrophils are known to play an important role in the progression of crescentic glomerulonephritis, such as anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, the key chemoattractant for neutrophils in ANCA-associated glomerulonephritis has not been identified. Here, we demonstrate that a lipid chemoattractant, leukotriene B4 (LTB4 ), and its receptor BLT1 are primarily involved in disease pathogenesis in a mouse model of immune complex-mediated crescentic glomerulonephritis. Circulating neutrophils accumulated into glomeruli within 1 h after disease onset, which was accompanied by LTB4 accumulation in the kidney cortex, leading to kidney injury. LTB4 was produced by cross-linking of Fc gamma receptors on neutrophils. Mice deficient in BLT1 or LTB4 biosynthesis exhibited suppressed initial neutrophil infiltration and subsequent thrombotic glomerulonephritis and renal fibrosis. Depletion of neutrophils before, but not after, disease onset prevented proteinuria and kidney injury, indicating the essential role of neutrophils in the early phase of glomerulonephritis. Administration of a BLT1 antagonist before and after disease onset almost completely suppressed induction of glomerulonephritis. Finally, we found that the glomeruli from patients with ANCA-associated glomerulonephritis contained more BLT1-positive cells than glomeruli from patients with other etiologies. Taken together, the LTB4 -BLT1 axis is the key driver of neutrophilic glomerular inflammation, and will be a novel therapeutic target for the crescentic glomerulonephritis.

Blockade of the BLT1-LTB4 axis does not affect mast cell migration towards advanced atherosclerotic lesions in LDLr−/− mice

Mast cells have been associated with the progression and destabilization of advanced atherosclerotic plaques. Reducing intraplaque mast cell accumulation upon atherosclerosis progression could be a potent therapeutic strategy to limit plaque destabilization. Leukotriene B4 (LTB4) has been reported to induce mast cell chemotaxis in vitro. Here, we examined whether antagonism of the LTB4-receptor BLT1 could inhibit mast cell accumulation in advanced atherosclerosis. Expression of genes involved in LTB4 biosynthesis was determined by single-cell RNA sequencing of human atherosclerotic plaques. Subsequently, Western-type diet fed LDLr−/− mice with pre-existing atherosclerosis were treated with the BLT1-antagonist CP105,696 or vehicle control three times per week by oral gavage. In the spleen, a significant reduction in CD11b+ myeloid cells was observed, including Ly6Clo and Ly6Chi monocytes as well as dendritic cells. However, atherosclerotic plaque size, collagen and macrophage content in the aortic root remained unaltered upon treatment. Finally, BLT1 antagonism did not affect mast cell numbers in the aortic root. Here, we show that human intraplaque leukocytes may be a source of locally produced LTB4. However, BLT1-antagonism during atherosclerosis progression does not affect either local mast cell accumulation or plaque size, suggesting that other mechanisms participate in mast cell accumulation during atherosclerosis progression.

Transcellular biosynthesis of leukotriene B4 orchestrates neutrophil swarming to fungi

SummaryNeutrophil swarming is an emergent host defense mechanism triggered by targets larger than a single neutrophil’s capacity to phagocytose. Swarming synergizes neutrophil functions, including chemotaxis, phagocytosis, and reactive oxygen species (ROS) production, and coordinates their deployment by many interacting neutrophils. The potent inflammatory lipid mediator leukotriene B4 (LTB4) has been established as central to orchestrating neutrophil activities during swarming. However, the details regarding how this eicosanoid choreographs the neutrophils involved in swarming are not well explained. Here we leverage microfluidics, genetically deficient mouse cells, and targeted metabolipidomic analysis to demonstrate that transcellular biosynthesis occurs among neutrophils to generate LTB4. Furthermore, transcellular biosynthesis is an entirely sufficient means of generating LTB4 for the purposes of orchestrating neutrophil swarming. These results further our understanding of how neutrophils coordinate their activities during swarming, which will be critical in the design of eventual therapies that can harness the power of swarming behavior.

Drug discovery strategies for novel leukotriene A4 hydrolase inhibitors

ABSTRACT Introduction Leukotriene A4 hydrolase (LTA4H) is the final and rate limiting enzyme regulating the biosynthesis of leukotriene B4 (LTB4), a pro-inflammatory lipid mediator implicated in a large number of inflammatory pathologies. Inhibition of LTA4H not only prevents LTB4 biosynthesis but also induces a lipid mediator class-switch within the 5-lipoxygenase pathway, elevating biosynthesis of the anti-inflammatory lipid mediator Lipoxin A4. Ample preclinical evidence advocates LTA4H as attractive drug target for the treatment of chronic inflammatory diseases. Areas covered This review covers details about the biochemistry of LTA4H and describes its role in regulating pro- and anti-inflammatory mediator generation. It summarizes recent efforts in medicinal chemistry toward novel LTA4H inhibitors, recent clinical trials testing LTA4H inhibitors in pulmonary inflammatory diseases, and potential reasons for the discontinuation of former development programs. Expert opinion Given the prominent role of LTB4 in initiating and perpetuating inflammation, LTA4H remains an appealing drug target. The reason former attempts targeting this enzyme have not met with success in the clinic can be attributed to compound-specific liabilities of first-generation inhibitors and/or choice of target indications to test this mode of action. A new generation of highly potent and selective LTA4H inhibitors is currently undergoing clinical testing in indications with a strong link to LTB4 biology.

Dapsone Suppresses Disease in Preclinical Murine Models of Pemphigoid Diseases

Epidermolysis bullosa acquisita and mucous membrane pemphigoid are autoimmune blistering diseases characterized by mucocutaneous blisters elicited by an autoantibody-mediated immune response against specific proteins of the epidermal basement membrane. The antibiotic dapsone is frequently used to treat both diseases, but its therapeutic effectiveness is uncertain, and its mode of action in these diseases is largely unknown. We evaluated the effect of dapsone in antibody transfer mouse models of epidermolysis bullosa acquisita and mucous membrane pemphigoid, which do not allow the drawing of conclusions on clinical treatment regimens but can be instrumental to partially uncover the mode(s) of action of dapsone in these diseases. Dapsone significantly mitigated inflammation in both models, reducing the recruitment of neutrophils into the skin and disrupting their release of leukotriene B4 (LTB4) and ROS in response to immune complexes. LTB4 has been implicated in numerous diseases, but effective LTB4 inhibitors for clinical use are not available. Our findings indicate that the mode of action of dapsone in these models may be based on the inhibition of LTB4 and ROS release from neutrophils. Moreover, they encourage testing the use of dapsone as an effective, albeit nonspecific, inhibitor of LTB4 biosynthesis in other LTB4-driven diseases.

Discovery of LYS006, a Potent and Highly Selective Inhibitor of Leukotriene A4 Hydrolase.

The cytosolic metalloenzyme leukotriene A4 hydrolase (LTA4H) is the final and rate-limiting enzyme in the biosynthesis of pro-inflammatory leukotriene B4 (LTB4). Preclinical studies have validated this enzyme as an attractive drug target in chronic inflammatory diseases. Despite several attempts, no LTA4H inhibitor has reached the market, yet. Herein, we disclose the discovery and preclinical profile of LYS006, a highly potent and selective LTA4H inhibitor. A focused fragment screen identified hits that could be cocrystallized with LTA4H and inspired a fragment merging. Further optimization led to chiral amino acids and ultimately to LYS006, a picomolar LTA4H inhibitor with exquisite whole blood potency and long-lasting pharmacodynamic effects. Due to its high selectivity and its ability to fully suppress LTB4 generation at low exposures in vivo, LYS006 has the potential for a best-in-class LTA4H inhibitor and is currently investigated in phase II clinical trials in inflammatory acne, hidradenitis suppurativa, ulcerative colitis, and NASH.

Molecules and Prostaglandins Related to Embryo Tolerance.

It is generally understood that the entry of semen into the female reproductive tract provokes molecular and cellular changes facilitating conception and pregnancy. We show a broader picture of the participation of prostaglandins in the fertilization, implantation and maintenance of the embryo. A large number of cells and molecules are related to signaling networks, which regulate tolerance to implantation and maintenance of the embryo and fetus. In this work, many of those cells and molecules are analyzed. We focus on platelets, polymorphonuclear leukocytes, and group 2 innate lymphoid cells involved in embryo tolerance in order to have a wider view of how prostaglandins participate. The combination of platelets and neutrophil extracellular traps (Nets), uterine innate lymphoid cells (uILC), Treg cells, NK cells, and sex hormones have an important function in immunological tolerance. In both animals and humans, the functions of these cells can be regulated by prostaglandins and soluble factors in seminal plasma to achieve an immunological balance, which maintains fetal-maternal tolerance. Prostaglandins, such as PGI2 and PGE2, play an important role in the suppression of the previously mentioned cells. PGI2 inhibits platelet aggregation, in addition to IL-5 and IL-13 expression in ILC2, and PGE2 inhibits some neutrophil functions, such as chemotaxis and migration processes, leukotriene B4 (LTB4) biosynthesis, ROS production, and the formation of extracellular traps, which could help prevent trophoblast injury and fetal loss. The implications are related to fertility in female when seminal fluid is deposited in the vagina or uterus.

Design and synthesis of Leukotriene A4 hydrolase inhibitors to alleviate idiopathic pulmonary fibrosis and acute lung injury

Idiopathic pulmonary fibrosis (IPF) and acute lung injury (ALI) are considered two severe public health issues, attributed to malfunctions of neutrophils. They can cause chronic inflammation and have association with subsequent tissue damages. There have been rare drugs applying to the efficient treatment in clinical practice. Existing research revealed that Leukotriene B4 (LTB4) is the critical endogenous molecule to induce neutrophil inflammatory response. LTB4 blocking biosynthesis is the potential strategy treating IPF and ALI. In the present study, 45 hydroxamic acid derivatives were produced, and compound 26 was screened out as a highly selective Lead compound of Leukotriene A4 Hydrolase (LTA4H), i.e., an enzyme critical to the biosynthesis of LTB4. This compound is capable of relieving neutrophilic inflammation in an IPF mouse model at early stage, as well as mitigating LPS-induced acute lung injury via a mechanism of LTB4 blocking biosynthesis in vivo. Whether this compound acts as the potential lead compound for the treatment of IPF and ALI requires further verification.

The Orphan G-Protein Coupled Receptor 182 Is a Negative Regulator of Definitive Hematopoiesis through Leukotriene B4 Signaling.

The G protein-coupled receptor 182 (GPR182) is an orphan GPCR, the expression of which is enriched in embryonic endothelial cells (ECs). However, the physiological role and molecular mechanism of action of GPR182 are unknown. Here, we show that GPR182 negatively regulates definitive hematopoiesis in zebrafish and mice. In zebrafish, gpr182 expression is enriched in the hemogenic endothelium (HE), and gpr182–/– display an increased expression of HE and hematopoietic stem cell (HSC) marker genes. Notably, we find an increased number of myeloid cells in gpr182–/– compared to wild-type. Further, by time-lapse imaging of zebrafish embryos during the endothelial-to-hematopoietic transition, we find that HE/HSC cell numbers are increased in gpr182–/– compared to wild-type. GPR182–/– mice also exhibit an increased number of myeloid cells compared to wild-type, indicating a conserved role for GPR182 in myelopoiesis. Using cell-based small molecule screening and transcriptomic analyses, we further find that GPR182 regulates the leukotriene B4 (LTB4) biosynthesis pathway. Taken together, these data indicate that GPR182 is a negative regulator of definitive hematopoiesis in zebrafish and mice, and provide further evidence for LTB4 signaling in HSC biology.

Identification of Human Leukotriene A4 Hydrolase Inhibitors Using Structure-Based Pharmacophore Modeling and Molecular Docking.

Leukotriene B4 (LTB4) is a potent, proinflammatory lipid mediator implicated in the pathologies of an array of inflammatory diseases and cancer. The biosynthesis of LTB4 is regulated by the leukotriene A4 hydrolase (LTA4H). Compounds capable of limiting the formation of LTB4, through selective inhibition of LTA4H, are expected to provide potent anti-inflammatory and anti-cancer agents. The aim of the current study is to obtain potential LTA4H inhibitors using computer-aided drug design. A hybrid 3D structure-based pharmacophore model was generated based on the crystal structure of LTA4H in complex with bestatin. The generated pharmacophore was used in a virtual screen of the Maybridge database. The retrieved hits were extensively filtered, then docked into the active site of the enzyme. Finally, they were consensually scored to yield five hits as potential LTA4H inhibitors. Consequently, the selected hits were purchased and their biological activity assessed in vitro against the epoxide hydrolase activity of LTA4H. The results were very promising, with the most active compound showing 73.6% inhibition of the basal epoxide hydrolase activity of LTA4H. The results from this exploratory study provide valuable information for the design and development of more potent and selective inhibitors.

Phenotypically Silent Bone Morphogenetic Protein Receptor 2 Mutations Predispose Rats to Inflammation-Induced Pulmonary Arterial Hypertension by Enhancing the Risk for Neointimal Transformation.

Bmpr2 (bone morphogenetic protein receptor 2) mutations are critical risk factors for hereditary pulmonary arterial hypertension (PAH) with approximately 20% of carriers developing disease. There is an unmet medical need to understand how environmental factors, such as inflammation, render Bmpr2 mutants susceptible to PAH. Overexpressing 5-LO (5-lipoxygenase) provokes lung inflammation and transient PAH in Bmpr2+/- mice. Accordingly, 5-LO and its metabolite, leukotriene B4, are candidates for the second hit. The purpose of this study was to determine how 5-LO-mediated pulmonary inflammation synergized with phenotypically silent Bmpr2 defects to elicit significant pulmonary vascular disease in rats. Monoallelic Bmpr2 mutant rats were generated and found phenotypically normal for up to 1 year of observation. To evaluate whether a second hit would elicit disease, animals were exposed to 5-LO-expressing adenovirus, monocrotaline, SU5416, SU5416 with chronic hypoxia, or chronic hypoxia alone. Bmpr2-mutant hereditary PAH patient samples were assessed for neointimal 5-LO expression. Pulmonary artery endothelial cells with impaired BMPR2 signaling were exposed to increased 5-LO-mediated inflammation and were assessed for phenotypic and transcriptomic changes. Lung inflammation, induced by intratracheal delivery of 5-LO-expressing adenovirus, elicited severe PAH with intimal remodeling in Bmpr2+/- rats but not in their wild-type littermates. Neointimal lesions in the diseased Bmpr2+/- rats gained endogenous 5-LO expression associated with elevated leukotriene B4 biosynthesis. Bmpr2-mutant hereditary PAH patients similarly expressed 5-LO in the neointimal cells. In vitro, BMPR2 deficiency, compounded by 5-LO-mediated inflammation, generated apoptosis-resistant and proliferative pulmonary artery endothelial cells with mesenchymal characteristics. These transformed cells expressed nuclear envelope-localized 5-LO consistent with induced leukotriene B4 production, as well as a transcriptomic signature similar to clinical disease, including upregulated nuclear factor Kappa B subunit (NF-κB), interleukin-6, and transforming growth factor beta (TGF-β) signaling pathways. The reversal of PAH and vasculopathy in Bmpr2 mutants by TGF-β antagonism suggests that TGF-β is critical for neointimal transformation. In a new 2-hit model of disease, lung inflammation induced severe PAH pathology in Bmpr2+/- rats. Endothelial transformation required the activation of canonical and noncanonical TGF-β signaling pathways and was characterized by 5-LO nuclear envelope translocation with enhanced leukotriene B4 production. This study offers an explanation of how an environmental injury unleashes the destructive potential of an otherwise silent genetic mutation.

The standardized herbal combination BNO 2103 contained in Canephron® N alleviates inflammatory pain in experimental cystitis and prostatitis

BackgroundUrinary tract infections are among the most common types of infections and give rise to inflammation with pain as one of the main symptoms. The herbal medicinal product Canephron® N contains BNO 2103, a defined mixture of pulverized rosemary leaves, centaury herb, and lovage root, and has been used in the treatment of urinary tract infections for more than 25 years. PurposeTo test the hypothesis that BNO 2103 reduces pain in cystitis and prostatitis by virtue of anti-inflammatory properties, and to reveal potential mechanisms underlying the anti-inflammatory features. Study designBNO 2103 was studied for anti-inflammatory and analgesic properties in three animal models in vivo, and the mode of action underlying the anti-inflammatory features was investigated in human leukocytes and cell-free assays in vitro. MethodsTo assess the anti-inflammatory and analgesic efficacy of BNO 2103 we employed cyclophosphamide-induced cystitis and carrageenan-induced prostatitis in rats, and zymosan-induced peritonitis in mice. Human neutrophils and monocytes as well as isolated human 5-lipoxygenase and microsomal prostaglandin E2 synthase-1-containing microsomes were utilized to assess inhibition of leukotriene and/or prostaglandin E2 production by HPLC and/or ELISA. ResultsWhen given orally, BNO 2103 reduced inflammation and hyperalgesia in experimental cystitis in rats, while individual components of BNO 2103 also reduced hyperalgesia. Furthermore, BNO 2103 reduced hyperalgesia in rats with carrageenan-induced prostatitis. Cell-based and cell-free studies implicate inhibition of prostaglandin E2 and leukotriene B4 biosynthesis as potential mechanisms underlying the analgesic and anti-inflammatory effects. ConclusionOur data support the hypothesis that BNO 2103 reduces pain by virtue of its anti-inflammatory properties, possibly related to suppression of prostaglandin E2 and leukotriene B4 formation, and suggest that this combination has the potential to treat clinical symptoms such as inflammatory pain. Thus BNO 2103 may represent an alternative to reduce the use of antibiotics in urinary tract infections.

Metabolic profiling of human plasma reveals the activation of 5-lipoxygenase in the acute attack of gouty arthritis.

Monosodium urate-induced inflammation plays a vital role in acute gout (AG). Inflammation is a multi-stage process involved in the acute release of arachidonic acid and its metabolites. However, the function of the metabolism of arachidonic acid and other polyunsaturated fatty acids in AG is not well understood. This study aimed to investigate the modification of polyunsaturated fatty acid metabolism by AG. Plasma samples from patients with an AG attack (n = 26) and gender-matched healthy controls (n = 26) were analysed by metabolic profiling of polyunsaturated fatty acids. The findings were further validated with a second cohort (n = 20 each group). The associated mechanisms were investigated in whole blood cells from the second cohort and neutrophils in vitro. Plasma metabolic profiling revealed a significant increase in leukotriene B4 (LTB4) for AG patients in both cohorts. The increase in plasma LTB4 was accounted for by the dynamic balance between the activation of 5-lipoxygenase and CYP4F3, the former mediating the biosynthesis of LTB4 and the latter mediating its metabolism. This was supported by significantly increased transcriptional levels of 5-lipoxygenase and CYP4F3 in whole blood cells from AG patients compared with those of controls, and the uric acid-caused dose-relevant and time-dependent activation of 5-lipoxygenase and CYP4F3 at the transcriptional and molecular levels in vitro. Increased LTB4 in AG patients is mainly due to activation of 5-lipoxygenase. 5-Lipoxygenase inhibition may be of therapeutic value clinically.

Feasibility and physiological relevance of designing highly potent aminopeptidase-sparing leukotriene A4 hydrolase inhibitors

Leukotriene A4 Hydrolase (LTA4H) is a bifunctional zinc metalloenzyme that comprises both epoxide hydrolase and aminopeptidase activity, exerted by two overlapping catalytic sites. The epoxide hydrolase function of the enzyme catalyzes the biosynthesis of the pro-inflammatory lipid mediator leukotriene (LT) B4. Recent literature suggests that the aminopeptidase function of LTA4H is responsible for degradation of the tripeptide Pro-Gly-Pro (PGP) for which neutrophil chemotactic activity has been postulated. It has been speculated that the design of epoxide hydrolase selective LTA4H inhibitors that spare the aminopeptidase pocket may therefore lead to more efficacious anti-inflammatory drugs. In this study, we conducted a high throughput screen (HTS) for LTA4H inhibitors and attempted to rationally design compounds that would spare the PGP degrading function. While we were able to identify compounds with preference for the epoxide hydrolase function, absolute selectivity was not achievable for highly potent compounds. In order to assess the relevance of designing such aminopeptidase-sparing LTA4H inhibitors, we studied the role of PGP in inducing inflammation in different settings in wild type and LTA4H deficient (LTA4H KO) animals but could not confirm its chemotactic potential. Attempting to design highly potent epoxide hydrolase selective LTA4H inhibitors, therefore seems to be neither feasible nor relevant.

Dioxin-induced increase in leukotriene B4 biosynthesis through the aryl hydrocarbon receptor and its relevance to hepatotoxicity owing to neutrophil infiltration

Dioxin and related chemicals alter the expression of a number of genes by activating the aryl hydrocarbon receptors (AHR) to produce a variety of disorders including hepatotoxicity. However, it remains largely unknown how these changes in gene expression are linked to toxicity. To address this issue, we initially examined the effect of 2,3,7,8-tetrachrolodibenzo-p-dioxin (TCDD), a most toxic dioxin, on the hepatic and serum metabolome in male pubertal rats and found that TCDD causes many changes in the level of fatty acids, bile acids, amino acids, and their metabolites. Among these findings was the discovery that TCDD increases the content of leukotriene B4 (LTB4), an inducer of inflammation due to the activation of leukocytes, in the liver of rats and mice. Further analyses suggested that an increase in LTB4 comes from a dual mechanism consisting of an induction of arachidonate lipoxygenase-5, a rate-limiting enzyme in LTB4 synthesis, and the down-regulation of LTC4 synthase, an enzyme that converts LTA4 to LTC4. The above changes required AHR activation, because the same was not observed in AHR knock-out rats. In agreement with LTB4 accumulation, TCDD caused the marked infiltration of neutrophils into the liver. However, deleting LTB4 receptors (BLT1) blocked this effect. A TCDD-produced increase in the mRNA expression of inflammatory markers, including tumor-necrosis factor and hepatic damage, was also suppressed in BLT1-null mice. The above observations focusing on metabolomic changes provide novel evidence that TCDD accumulates LTB4 in the liver by an AHR-dependent induction of LTB4 biosynthesis to cause hepatotoxicity through neutrophil activation.

The Endocannabinoid Metabolite Prostaglandin E2 (PGE2)-Glycerol Inhibits Human Neutrophil Functions: Involvement of Its Hydrolysis into PGE2 and EP Receptors.

The endocannabinoids 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine mediate an array of pro- and anti-inflammatory effects. These effects are related, in part, to their metabolism by eicosanoid biosynthetic enzymes. For example, N-arachidonoyl-ethanolamine and 2-arachidonoyl-glycerol can be metabolized by cyclooxygenase-2 into PG-ethanolamide (PG-EA) and PG-glycerol (PG-G), respectively. Although PGE2 is a recognized suppressor of neutrophil functions, the impact of cyclooxygenase-derived endocannabinoids such as PGE2-EA or PGE2-G on neutrophils is unknown. This study's aim was to define the effects of these mediators on neutrophil functions and the underlying cellular mechanisms involved. We show that PGE2-G, but not PGE2-EA, inhibits leukotriene B4 biosynthesis, superoxide production, migration, and antimicrobial peptide release. The effects of PGE2-G were prevented by EP1/EP2 receptor antagonist AH-6809 but not the EP4 antagonist ONO-AE2-227. The effects of PGE2-G required its hydrolysis into PGE2, were not observed with the non-hydrolyzable PGE2-serinol amide, and were completely prevented by methyl-arachidonoyl-fluorophosphate and palmostatin B, and partially prevented by JZL184 and WWL113. Although we could detect six of the documented PG-G hydrolases in neutrophils by quantitative PCR, only ABHD12 and ABHD16A were detected by immunoblot. Our pharmacological data, combined with our protein expression data, did not allow us to pinpoint one PGE2-G lipase, and rather support the involvement of an uncharacterized lipase and/or of multiple hydrolases. In conclusion, we show that PGE2-G inhibits human neutrophil functions through its hydrolysis into PGE2, and by activating the EP2 receptor. This also indicates that neutrophils could regulate inflammation by altering the balance between PG-G and PG levels in vivo.

Drug Repurposing of Histone Deacetylase Inhibitors That Alleviate Neutrophilic Inflammation in Acute Lung Injury and Idiopathic Pulmonary Fibrosis via Inhibiting Leukotriene A4 Hydrolase and Blocking LTB4 Biosynthesis

Acute lung injury (ALI) and idiopathic pulmonary fibrosis (IPF) are both serious public health problems with high incidence and mortality rate in adults, and with few drugs available for the efficient treatment in clinic. In this study, we identified that two known histone deacetylase (HDAC) inhibitors, suberanilohydroxamic acid (SAHA, 1) and its analogue 4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]benzamide (2), are effective inhibitors of Leukotriene A4 hydrolase (LTA4H), a key enzyme in the biosynthesis of leukotriene B4 (LTB4), across a panel of 18 HDAC inhibitors, using enzymatic assay, thermofluor assay, and X-ray crystallographic investigation. Importantly, both 1 and 2 markedly diminish early neutrophilic inflammation in mouse models of ALI and IPF under a clinical safety dose. Detailed mechanisms of down-regulation of proinflammatory cytokines by 1 or 2 were determined in vivo. Collectively, 1 and 2 would provide promising agents with well-known clinical safety for potential treatment in patients with ALI and IPF via pharmacologically inhibiting LAT4H and blocking LTB4 biosynthesis.

The Leukotriene B4 and its Receptor BLT1 Act as Critical Drivers of Neutrophil Recruitment in Murine Bullous Pemphigoid-Like Epidermolysis Bullosa Acquisita

Recruitment of neutrophils and eosinophils into the skin is a hallmark of pemphigoid diseases. The molecular cues regulating granulocyte recruitment into the skin and the individual contributions of neutrophils and eosinophils to pemphigoid diseases are, however, poorly understood. The lipid mediator leukotriene B4 (LTB4) is a potent granulocyte chemoattractant and is abundant in the skin blister fluid of bullous pemphigoid (BP) patients, but its pathogenic significance is unknown. Using mouse models of BP-like epidermolysis bullosa acquisita and of BP, we show that LTB4 and its receptor BLT1 act as critical drivers of neutrophil entry into the skin upon antibody deposition at the dermal-epidermal junction. Mice deficient in 5-lipoxygenase, a key enzyme in LTB4 biosynthesis, or in BLT1 exhibited dramatic resistance to neutrophil recruitment and, consequently, skin inflammation. Accordingly, liquid chromatography-mass spectrometry, used to comprehensively profile lipid mediator generation in the first 48 hours after antibody deposition, showed a pronounced parallel increase in LTB4 and in neutrophils in the skin. Subsequent mechanistic studies in BP-like epidermolysis bullosa acquisita uncovered that neutrophils are necessary for skin inflammation, whereas eosinophils are dispensable, thus identifying neutrophils as major culprits of blister formation. Our results highlight LTB4/BLT1 as absolutely critical drivers of murine pemphigoid disease-like skin inflammation.