Leukotriene B4 Signaling Research Articles

Blocking LTB4 signaling-mediated TAMs recruitment by Rhizoma Coptidis sensitizes lung cancer to immunotherapy

BackgroundImmune checkpoint blockade (ICB) induces durable immune responses across a spectrum of advanced cancers and revolutionizes the oncology field. However, only a subset of patients achieves long-lasting clinical benefits. Tumor-associated macrophages (TAMs) usually secrete immunosuppressive cytokines and contribute to the failure of ICB therapy. Therefore, it is crucial to mechanically manipulate the abundance and function of TAMs in the tumor microenvironment (TME), which can offer a promising molecular basis to improve the clinical response efficacy of ICB in cancer patients. PurposeThis study aims to investigate TAMs in the immunosuppressive microenvironment to identify new therapeutic targets, improve the ability to predict and guide responses to clinical immunotherapy, and develop new strategies for immunotherapy of lung tumors. MethodsLewis lung carcinoma (LLC) xenograft-bearing mouse models were established to analyze the antitumor activity of Rhizoma Coptidis (RC) in vivo. A systems pharmacology strategy was used to predict the correlation between RC and M2 macrophages. The effect of RC on the abundance of M2 macrophages was analyzed by flow cytometry of murine samples. Western blot was performed to analyze the expression of Leukotriene A4 hydrolase (LTA4H) and LTB4 receptor 1 (BLT1) in harvested lung cancer tissues. The impact of blocking leukotriene B4 (LTB4) signaling by RC on the recruitment of M2 macrophages was assessed in vitro and in vivo. Transwell migration assays were conducted to clarify the inhibition of macrophage migration by blocking LTB4. Lta4h−/− mice were used to investigate the sensitivity of immunotherapy to lung cancer by blocking the LTB4 signaling. ResultsHere, we report that RC, an herbal medicine from the family Ranunculaceae, suppresses the recruitment and immunosuppressive function of TAMs, which in turn sensitizes lung cancer to ICB therapy. Firstly, a systems pharmacology strategy was proposed to identify combinatorial drugs for ICB therapy with a systems biology perspective of drug-target-pathway-TME phenotype. We predicted and verified that RC significantly inhibits tumor growth and the infiltration of M2-TAMs into TME of LLC tumor-bearing mice. Then, RC inhibits the recruitment of macrophages to the tumor TME via blocking LTB4 signaling, and suppresses the expression of immunosuppressive factors (IL-10, TGF-β and VEGF). As a result, RC enables CD8+ T cells to retain their proliferative and infiltrative abilities within the TME. Ultimately, these events promote cytotoxic T-cell-mediated clearance of tumor cells, which is further enhanced by the addition of anti-PD-L1 therapy. Furthermore, we employed LTA4H deficient mice (Lta4h−/− mice) to evaluate the antitumor efficiency, the results showed that the efficacy of immunotherapy was enhanced due to the synergistic effect of LTB4 signaling blockage and ICB inhibition, leading to remarkable inhibition of tumor growth in a mouse model of lung adenocarcinoma. ConclusionsTaken together, these findings suggest that RC enhances antitumor immunity, providing a rationale for combining RC with immunotherapies as a potential anti-cancer treatment strategy.

Lipoxin A4 Receptor Stimulation Attenuates Neuroinflammation in a Mouse Model of Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is caused by the rupture of blood vessels in the brain. The excessive activation of glial cells and the infiltration of numerous inflammatory cells are observed during bleeding. Thrombin is a key molecule that triggers neuroinflammation in the ICH brain. In this study, we focused on lipoxin A4 (LXA4), an arachidonic acid metabolite that has been reported to suppress inflammation and cell migration. LXA4 and BML-111, an agonist of the LXA4 receptor/formyl peptide receptor 2 (ALX/FPR2), suppressed microglial activation; LXA4 strongly inhibited the migration of neutrophil-like cells in vitro. ALX/FPR2 was expressed on neutrophils in the ICH mouse brain and the daily administration of BML-111 attenuated the motor coordination dysfunction and suppressed the production of proinflammatory cytokines in the ICH mouse brain. On the other hand, BML-111 did not show a significant reduction in the number of microglia and neutrophils. These results suggest that systemic administration of ALX/FPR2 agonists may suppress the neuroinflammatory response of microglia and neutrophils without a change in cell numbers. Additionally, their combination with molecules that reduce cell numbers, such as modulators of leukotriene B4 signaling, may be required in future studies.

The LTB4-BLT1 axis regulates actomyosin and β2-integrin dynamics during neutrophil extravasation.

The eicosanoid leukotriene B4 (LTB4) relays chemotactic signals to direct neutrophil migration to inflamed sites through its receptor BLT1. However, the mechanisms by which the LTB4-BLT1 axis relays chemotactic signals during intravascular neutrophil response to inflammation remain unclear. Here, we report that LTB4 produced by neutrophils acts as an autocrine/paracrine signal to direct the vascular recruitment, arrest, and extravasation of neutrophils in a sterile inflammation model in the mouse footpad. Using intravital subcellular microscopy, we reveal that LTB4 elicits sustained cell polarization and adhesion responses during neutrophil arrest in vivo. Specifically, LTB4 signaling coordinates the dynamic redistribution of non-muscle myosin IIA and β2-integrin, which facilitate neutrophil arrest and extravasation. Notably, we also found that neutrophils shed extracellular vesicles in the vascular lumen and that inhibition of extracellular vesicle release blocks LTB4-mediated autocrine/paracrine signaling required for neutrophil arrest and extravasation. Overall, we uncover a novel complementary mechanism by which LTB4 relays extravasation signals in neutrophils during early inflammation response.

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.

Leukotriene-B4 modulates macrophage metabolism and fat loss in type 1 diabetic mice.

Serum levels of leukotriene-B4 (LTB4) are increased in type 1 diabetes (T1D) and it mediates systemic inflammation and macrophage reprogramming associated with this condition. Herein, we investigated the involvement of LTB4 in adiposity loss, hyperlipidemia, and changes in macrophage metabolism in a mouse model of streptozotocin-induced T1D. LTB4 receptor (BLT1) antagonist u75302 was employed to block LTB4 effects. As expected, hypoinsulinemia in T1D was associated with hyperglycemia, low levels of glucagon, hyperlipidemia, significant body fat loss, and increased white adipose tissue expression of Fgf21, a marker for lipolysis. With the exception of hyperglycemia and hypoglucagonemia, blockade of LTB4 signaling reverted these parameters in T1D mice. Along with hyperlipidemia, macrophages from T1D mice exhibited higher lipid uptake and accumulation. These cells also had enhanced glycolysis and oxidative metabolism and these parameters were dependent on the mitochondrial uncoupling respiration, as evidenced by elevated expression of oxidation markers carnitine palmitoyltransferase and uncoupling protein 1. Interestingly, all these parameters were at least partially reverted in T1D mice treated with u75302. Altogether, these findings suggest that in T1D mice LTB4/BLT1 is involved in the fat loss, hyperlipidemia, and increased macrophage lipid uptake and metabolism with an important involvement of mitochondrial uncoupling activity. These previously unrecognized LTB4/BLT1 functions may be explored in future to therapeutically alleviate severity of hyperlipidemia and systemic inflammation in T1D.

Lymphatic dysfunction in critical illness.

The essential role of the lymphatic system in fluid homeostasis, nutrient transport, and immune trafficking is well recognized; however, there is limited understanding of the mechanisms that regulate lymphatic function, particularly in the setting of critical illness. The lymphatics likely affect disease severity and progression in every condition, from severe systemic inflammatory states to respiratory failure. Here, we review structural and functional disorders of the lymphatic system, both congenital and acquired, as they relate to care of the pediatric patient in the intensive care setting, including novel areas of research into medical and procedural therapeutic interventions. The mainstay of current therapies for congenital and acquired lymphatic abnormalities has involved nonspecific medical management or surgical procedures to obstruct or divert lymphatic flow. With the development of dynamic contrast-enhanced magnetic resonance lymphangiography, image-directed percutaneous intervention may largely replace surgery. Because of new insights into the mechanisms that regulate lymphatic biology, pharmacologic inhibitors of mTOR and leukotriene B4 signaling are each in Phase II clinical trials to treat abnormal lymphatic structure and function, respectively. As our understanding of normal lymphatic biology continues to advance, we will be able to develop novel strategies to support and augment lymphatic function during critical illness and through convalescence.

Let-7 microRNA-dependent control of leukotriene signaling regulates the transition of hematopoietic niche in mice

Hematopoietic stem and progenitor cells arise from the vascular endothelium of the dorsal aorta and subsequently switch niche to the fetal liver through unknown mechanisms. Here we report that vascular endothelium-specific deletion of mouse Drosha (DroshacKO), an enzyme essential for microRNA biogenesis, leads to anemia and death. A similar number of hematopoietic stem and progenitor cells emerge from Drosha-deficient and control vascular endothelium, but DroshacKO-derived hematopoietic stem and progenitor cells accumulate in the dorsal aorta and fail to colonize the fetal liver. Depletion of the let-7 family of microRNAs is a primary cause of this defect, as it leads to activation of leukotriene B4 signaling and induction of the α4β1 integrin cell adhesion complex in hematopoietic stem and progenitor cells. Inhibition of leukotriene B4 or integrin rescues maturation and migration of DroshacKO hematopoietic stem and progenitor cells to the fetal liver, while it hampers hematopoiesis in wild-type animals. Our study uncovers a previously undefined role of innate leukotriene B4 signaling as a gatekeeper of the hematopoietic niche transition.

Natural products isolated from Tetragonula carbonaria cerumen modulate free radical-scavenging and 5-lipoxygenase activities in vitro

BackgroundPropolis and cerumen are plant-derived products found in honeybees and stingless bees, respectively. Although propolis is an ancient folk medicine, the bioactivities of cerumen obtained from Australian native stingless bees (Tetragonula carbonaria) have not been widely studied. Therefore, we investigated selected anti-oxidant and anti-inflammatory properties of T. carbonaria cerumen.MethodsA methanolic extract was prepared from the combined cerumen of 40 T. carbonaria hives, and HPLC was used to screen for chemical constituents that scavenged 2,2-azobis(2-methylpropionamidine) dihydrochloride (AAPH). The ability of cerumen extracts to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) and to interfere with leukotriene B4 (LTB4) production in ionomycin-stimulated human neutrophils was also examined.ResultsThe extract dose-dependently scavenged DPPH (EC50 = 27.0 ± 2.3 μg/mL); and inhibited the 5-lipoxygenase (5-LOX)-mediated oxidation of linoleic acid (IC50 = 67.1 ± 9.6 μg/mL). Pre-treatment of isolated human neutrophils with the methanolic cerumen extract additionally inhibited the ionomycin-stimulated production of LTB4 from these cells (IC50 = 13.3 ± 5.3 μg/mL). Following multi-solvent extraction, the free radical-scavenging and 5-LOX-inhibiting activities of the initial cerumen extract were retained in a polar, methanol-water extract, which contained gallic acid and a range of flavonone and phenolic natural products.ConclusionsThe findings identify free radical scavenging activity, and interference by extracts of T. carbonaria cerumen in 5-LOX–LTB4 signaling. Further investigation is needed to determine whether the extracts will provide therapeutic benefits for medical conditions in which oxidative stress and inflammation are implicated, including cardiovascular disease and impaired wound healing.

Expression and cell distribution of leukotriene B4 receptor 1 in the rat brain cortex after experimental subarachnoid hemorrhage

Convincing evidence supports that nuclear factor kappa B (NF-κB)-meditated inflammation contributes to the adverse prognosis of aneurysmal subarachnoid hemorrhage (SAH), and pathologic neutrophil accumulation after SAH in the brain parenchyma enhances the inflammatory process. Leukotriene B4 (LTB4) is a highly potent lipid chemoattractant of neutrophils, and its biological effects are mediated primarily through the high-affinity LTB4 receptor 1 (BLT1). It is verified that NF-κB-dependent BLT1 mediates LTB4 signaling and LTB4 stimulates NF-κB-dependent inflammation via BLT1. This study aimed to determine the expression and cell distribution of BLT1 in the brain cortex after SAH and investigate the potential relationship between protein expressions of BLT1 and NF-κB. Male Sprague-Dawley rats were randomly assigned into sham group and SAH groups at 6h, 12h and on day 1, day 2 and day 3 (n=6 for each subgroup). SAH groups suffered experimental SAH by injecting 0.3ml autologous blood into the prechiasmatic cistern. BLT1 expression was measured by real-time PCR, western blot, immunohistochemistry and immunofluorescence. Nuclear expression of p65 protein, the major subunit of NF-κB, was also detected by western blot. Our data showed that the expression levels of BLT1 and nuclear p65 protein were both markedly increased after SAH. Moreover, there was a significant positive correlation between BLT1 and nuclear p65 protein expressions in the same specific time course. Double immunofluorescence staining showed that BLT1 were mainly expressed in neurons, microglia and endothelial cells rather than astrocytes after SAH. These results suggest that BLT1 may participate in the NF-κB-mediated inflammatory response after SAH, and there might be important implications for further studies using specific BLT1 antagonists to attenuate the NF-κB-mediated inflammation after SAH.

To Attract Others, Immune Cells Release a Packet Which Releases a Signal.

When you cut your finger, molecules released by damaged cells (and later, by growing bacteria) are sensed by roving immune cells called neutrophils, which respond to the signal by moving directly and rapidly toward the site of damage. They also release their own set of chemicals, especially leukotriene B4 (LTB4), essentially signaling to other neutrophils, “Hey, follow me!” The release of LTB4 both amplifies the primary damage signal and casts it more widely, bringing in far more reinforcements than the damaged cells themselves could muster on their own. The neutrophil knows which direction it should go by following the signal up its concentration gradient. However, it has been unclear how a diffusible secondary attractant such as LTB4 can be disseminated far from where it is released, and thereby amplify the diffusible primary attractants and recruit more neutrophils to the site of injury and inflammation. In a new study in PLOS Biology, Ritankar Majumdar, Aidin Tavakoli Tameh, and Carole Parent show that neutrophils release not the LTB4 itself but vesicles that contain it along with the enzymatic machinery to make it, allowing LTB4 to be dispersed over larger areas and for longer time periods than through direct release (Fig 1). Fig 1 Cartoon depicting the events involved in the relay of primary chemotactic signals to the secondary chemoattractant LTB4 during neutrophil chemotaxis. Neutrophils release large amounts of LTB4 only upon activation by a primary chemoattractant, such as N-formylmethionyl-leucyl-phenylalanine (fMLP), released by both damaged cells and bacteria. The authors began by activating neutrophils and then separating their subcellular components by density. They found that LTB4 was not increased in the density fractions associated with the most common secretory pathways, but its distribution pattern did match that for a protein known to be packaged in multivesicular bodies (MVBs). As their name implies, MVBs are membrane-bound structures that contain numerous smaller vesicles (called intraluminal vesicles). When an MVB fuses with the plasma membrane, its intraluminar vesicles are released into the extracellular space (at which point they are called exosomes). Using electron microscopy, the authors found that one of the enzymes that makes LTB4, called 5-lipoxygenase (5-LO), could be detected on MVBs, and activated neutrophils released exosomes containing 5-LO from their trailing edges. Exosomes purified from activated neutrophils contained high levels of 5-LO and other LTB4-synthesizing enzymes, and activation of resting neutrophils with fMLP led to an increase in the LTB4 concentration in exosomes. The authors also showed that the purified exosomes could mobilize resting neutrophils, inducing their polarization and adhesion, two key properties of activated, chemotaxing cells. The neutrophils migrated toward the exosomes, an effect that could be reduced significantly by treating the neutrophils with an antagonist for the LTB4 receptor. These results clearly showed that exposure to a primary chemoattractant induced release of exosomes that contained and released LTB4, and that release of LTB4 by one neutrophil could promote chemotaxis in another neutrophil, a “paracrine” effect. But the authors found that LTB4 also had an autocrine effect, inducing stronger chemotaxis in the releasing cell as well. Knocking down two proteins critical for exosome docking and secretion, thereby reducing the amount of LTB4 released, led to a reduction in the directional specificity of and distance covered by the neutrophils containing the knockdowns, without affecting their speed of movement. More work remains to be done to understand how LTB4 is released from the exosomes, whether through diffusion or transport or vesicle lysis. But this study makes it clear that the rapid and dramatic swarming of neutrophils to the site of an injury is mediated by release of exosomes carrying both LTB4 and the enzymes needed to synthesize it. These findings are likely to be directly relevant to developing treatments for several chronic inflammatory diseases, including asthma, in which LTB4 signaling plays an important role. The authors also suggest that exosome-mediated signaling is likely to be at work in other cell types and other situations requiring extracellular gradients that might otherwise be difficult to establish or maintain.

The proinflammatory LTB4/BLT1 signal axis confers resistance to TGF-β1-induced growth inhibition by targeting Smad3 linker region.

Leukotriene B4 (LTB4) is a potent pro-inflammatory eicosanoid that is derived from arachidonic acid, and its signaling is known to have a tumor-promoting role in several cancer types. In this study, we investigated whether enhanced LTB4 signaling confers resistance to the cytostatic transforming growth factor-β1 (TGF-β1) response. We found that LTB4 pretreatment or ectopic expression of BLT1, a high affinity LTB4 receptor, fully abrogated TGF-β1-induced cell cycle arrest and expression of p15INK4B and p27KIP1. Mechanism study revealed that LTB4-mediated suppression of TGF-β1-induced Smad3 activation and growth inhibition was due to enhanced phosphorylation of Smad3 linker region (pSmad3L) through activation of BLT1-NAD(P)H oxidase (NOX)-reactive oxygen species (ROS)-epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3-K)-extracellular signal-activated kinase1/2 (ERK1/2)-linked signaling cascade. Furthermore, the LTB4/BLT1 signaling pathway leading to pSmad3L was constitutively activated in breast cancer cells and was correlated with TGF-β1-resistant growth of the cells in vitro and in vivo. In human breast cancer tissues, the expression level of pSmad3L (Thr179) had a positive correlation with BLT1 expression. Collectively, our data demonstrate for the first time that the induction of pSmad3L through BLT1-NOX-ROS-EGFR-PI3K-ERK1/2 signaling pathway is a key mechanism by which LTB4 blocks the anti-proliferative responses of TGF-β1, providing a novel mechanistic insight into the connection between enhanced inflammatory signal and cancer cell growth.

The BLT1 Inhibitory Function of α-1 Antitrypsin Augmentation Therapy Disrupts Leukotriene B4 Neutrophil Signaling.

Leukotriene B4 (LTB4) contributes to many inflammatory diseases, including genetic and nongenetic forms of chronic obstructive pulmonary disease. α-1 Antitrypsin (AAT) deficiency (AATD) is characterized by destruction of lung parenchyma and development of emphysema, caused by low AAT levels and a high neutrophil burden in the airways of affected individuals. In this study we assessed whether AATD is an LTB4-related disease and investigated the ability of serum AAT to control LTB4 signaling in neutrophils. In vitro studies demonstrate that neutrophil elastase is a key player in the LTB4 inflammatory cycle in AATD, causing increased LTB4 production, and associated BLT1 membrane receptor expression. AATD patients homozygous for the Z allele were characterized by increased neutrophil adhesion and degranulation responses to LTB4. We demonstrate that AAT can bind LTB4 and that AAT/LTB4 complex formation modulates BLT1 engagement and downstream signaling events, including 1,4,5-triphosphate production and Ca(2+) flux. Additionally, treatment of ZZ-AATD individuals with AAT augmentation therapy decreased plasma LTB4 concentrations and reduced levels of membrane-bound neutrophil elastase. Collectively, these results provide a mechanism by which AAT augmentation therapy impacts on LTB4 signaling in vivo, and not only reinforces the utility of this therapy for resolving inflammation in AATD, but supports useful future clinical applications in treatment of other LTB4-related diseases.

Distinct cellular sources of hepoxilin A3 and leukotriene B4 are used to coordinate bacterial-induced neutrophil transepithelial migration.

Neutrophilic infiltration is a leading contributor to pathology in a number of pulmonary disease states, including cystic fibrosis. Hepoxilin A3 (HXA3) is a chemotactic eicosanoid shown to mediate the transepithelial passage of neutrophils in response to infection in several model systems and at multiple mucosal surfaces. Another well-known eicosanoid mediating general neutrophil chemotaxis is leukotriene B4 (LTB4). We sought to distinguish the roles of each eicosanoid in the context of infection of lung epithelial monolayers by Pseudomonas aeruginosa. Using human and mouse in vitro transwell model systems, we used a combination of biosynthetic inhibitors, receptor antagonists, as well as mutant sources of neutrophils to assess the contribution of each chemoattractant in driving neutrophil transepithelial migration. We found that following chemotaxis to epithelial-derived HXA3 signals, neutrophil-derived LTB4 is required to amplify the magnitude of neutrophil migration. LTB4 signaling is not required for migration to HXA3 signals, but LTB4 generation by migrated neutrophils plays a significant role in augmenting the initial HXA3-mediated migration. We conclude that HXA3 and LTB4 serve independent roles to collectively coordinate an effective neutrophilic transepithelial migratory response.

Leukotriene B4 activates intracellular calcium and augments human osteoclastogenesis.

IntroductionBone erosion in inflammatory arthritis depends on the recruitment and activation of bone resorbing cells, the osteoclasts. Interleukin-23 (IL-23) has been primarily implicated in mediating inflammatory bone loss via the differentiation of Th17 receptor activator of nuclear factor κB ligand (RANKL)–producing cells. In this article, we describe a new role of IL-23 in activating the synthesis and production of leukotriene B4 (LTB4) in innate immune cells.MethodsWe utilized whole blood–derived human peripheral blood mononuclear cells (PBMCs), differentiated them towards an osteoclast lineage and then performed immunofluorescence and cytochemical staining to detect the expression of LTB4-associated receptors and enzymes such as phospholipase A2, 5-lipoxygenase and leukotriene A4 hydrolase, as well as the presence of tartrate-resistant acid phosphatase (TRAP) and F-actin rings on fully mature osteoclasts. We used enzyme immunoassays to measure LTB4 levels in culture media derived from IL-23-treated human PBMCs. We used real-time calcium imaging to study the effect of leukotrienes and requirements of different calcium sources and signaling proteins in activating intracellular calcium flux using pharmacological inhibitors to phospholipase C (U73122), membrane calcium channels (2-APB) and phosphatidylinositol 3-kinase (Wortmannin) and utilized qPCR for gene expression analysis in macrophages and osteoclasts.ResultsOur data show that LTB4 engagement of BLT1 and BLT2 receptors on osteoclast precursors leads to activation of phospholipase C and calcium release–activated channel–mediated intracellular calcium flux, which can activate further LTB4 autocrine production. IL-23-induced synthesis and secretion of LTB4 resulted in the upregulation of osteoclast-related genes NFATC1, MMP9, ACP5, CTSK and ITGB3 and the formation of giant, multinucleated TRAP+ cells capable of F-actin ring formation. These effects were dependent on Ca2+ signaling and were completely inhibited by BLT1/BLT2 and/or PLC and CRAC inhibitors.ConclusionsIn conclusion, IL-23 can initiate osteoclast differentiation independently from the RANK-RANKL pathway by utilizing Ca2+ signaling and the LTB4 signaling cascade.

Leukotriene B4 amplifies eosinophil accumulation in response to nematodes.

Eosinophil accumulation is a defining feature of the immune response to parasitic worm infection. Tissue-resident cells, such as epithelial cells, are thought to initiate eosinophil recruitment. However, direct recognition of worms by eosinophils has not been explored as a mechanism for amplifying eosinophil accumulation. Here, we report that eosinophils rapidly migrate toward diverse nematode species in three-dimensional culture. These include the mammalian parasite Nippostrongylus brasiliensis and the free-living nematode Caenorhabditis elegans. Surprisingly, collective migration toward worms requires paracrine leukotriene B4 signaling between eosinophils. In contrast, neutrophils show a minimal response to nematodes, yet are able to undergo robust leukotriene-dependent migration toward IgG-coated beads. We further demonstrate that eosinophils accumulate around C. elegans in the lungs of mice. This response is not dependent on bacterial products, CCR3, or complement activation. However, mice deficient in leukotriene signaling show markedly attenuated eosinophil accumulation after injection of C. elegans or N. brasiliensis. Our findings establish that nematode-derived signals can directly induce leukotriene production by eosinophils and that leukotriene signaling is a major contributor to nematode-induced eosinophil accumulation in the lung. The similarity of the eosinophil responses to diverse nematode species suggests that conserved features of nematodes are recognized during parasite infection.

Critical role of leukotriene B4 receptor signaling in mouse 3T3-L1 preadipocyte differentiation

BackgroundVarious inflammatory mediators related to obesity might be closely related to insulin resistance. Leukotrienes (LTs) are involved in inflammatory reactions. However, there are few reports regarding the role of LTs in adipocyte differentiation. Therefore, we investigated the role of leukotriene B4 (LTB4)-leukotriene receptor (BLT) signaling in mouse 3T3-L1 fibroblastic preadipocyte differentiation to mature adipocytes.MethodsMouse 3T3-L1 preadipocytes were treated with lipoxygenase (LOX) inhibitors, BLT antagonist, and small interfering RNA (siRNA) for BLT1 and BLT2 to block the LTB4-BLT signaling pathway, then the adipocyte differentiation such as lipid accumulation and the increase in triglyceride was evaluated.ResultsBlockade of BLT signaling by treatment with a LOX inhibitor or a BLT antagonist suppressed preadipocyte differentiation into mature adipocytes. In addition, knockdown of BLT1 and BLT2 by siRNAs dramatically inhibited differentiation. These results indicate the LTB4-BLT signaling pathway may positively regulate preadipocyte differentiation and be a rate-limiting system to control adipocyte differentiation.ConclusionsThe LTB4-BLT signaling pathway provides a potent regulatory signal that accelerates the differentiation of mouse 3T3-L1 preadipocytes. Further investigations are necessary to confirm the exact role of LTB4 and BLTs signaling pathways in preadipocyte differentiation.

Cancer-Produced Metabolites of 5-Lipoxygenase Induce Tumor-Evoked Regulatory B Cells via Peroxisome Proliferator–Activated Receptor α

Breast cancer cells facilitate distant metastasis through the induction of immunosuppressive regulatory B cells, designated tBregs. We report in this study that, to do this, breast cancer cells produce metabolites of the 5-lipoxygenase pathway such as leukotriene B4 to activate the peroxisome proliferator-activated receptor α (PPARα) in B cells. Inactivation of leukotriene B4 signaling or genetic deficiency of PPARα in B cells blocks the generation of tBregs and thereby abrogates lung metastasis in mice with established breast cancer. Thus, in addition to eliciting fatty acid oxidation and metabolic signals, PPARα initiates programs required for differentiation of tBregs. We propose that PPARα in B cells and/or tumor 5-lipoxygenase pathways represents new targets for pharmacological control of tBreg-mediated cancer escape.

Macrophage Dectin-1 Expression Is Controlled by Leukotriene B4 via a GM-CSF/PU.1 Axis

Pattern recognition receptors for fungi include dectin-1 and mannose receptor, and these mediate phagocytosis, as well as production of cytokines, reactive oxygen species, and the lipid mediator leukotriene B(4) (LTB(4)). The influence of G protein-coupled receptor ligands such as LTB(4) on fungal pattern recognition receptor expression is unknown. In this study, we investigated the role of LTB(4) signaling in dectin-1 expression and responsiveness in macrophages. Genetic and pharmacologic approaches showed that LTB(4) production and signaling through its high-affinity G protein-coupled receptor leukotriene B(4) receptor 1 (BLT1) direct dectin-1-dependent binding, ingestion, and cytokine production both in vitro and in vivo. Impaired responses to fungal glucans correlated with lower dectin-1 expression in macrophages from leukotriene (LT)- and BLT1-deficent mice than their wild-type counterparts. LTB(4) increased the expression of the transcription factor responsible for dectin-1 expression, PU.1, and PU.1 small interfering RNA abolished LTB(4)-enhanced dectin-1 expression. GM-CSF controls PU.1 expression, and this cytokine was decreased in LT-deficient macrophages. Addition of GM-CSF to LT-deficient cells restored expression of dectin-1 and PU.1, as well as dectin-1 responsiveness. In addition, LTB(4) effects on dectin-1, PU.1, and cytokine production were blunted in GM-CSF(-/-) macrophages. Our results identify LTB(4)-BLT1 signaling as an unrecognized controller of dectin-1 transcription via GM-CSF and PU.1 that is required for fungi-protective host responses.

WS17.8 Inhibition of leukotriene B4 signaling by alpha-1 antitrypsin: support for the use of aerosolized alpha-1 antitrypsin therapy in cystic fibrosis

WS17.8 Inhibition of leukotriene B4 signaling by alpha-1 antitrypsin: support for the use of aerosolized alpha-1 antitrypsin therapy in cystic fibrosis

Cysteinyl-Leukotrienes in Cerebrovascular Disease

Although the initial interest in the field of leukotriene (LT) research was focused on their potent bronchoconstrictive effects, subsequent studies have suggested LTs as key mediators in several inflammatory diseases. Genetic variations within the enzymes transforming arachidonic acid into LTs (Figure) have for example been associated with atherosclerosis and an increased risk of cardiovascular events.1 Figure Metabolism of arachidonic acid into leukotrienes (LTs) by 5-lipoxygenase (5-LO), 5-LO activating protein (FLAP), LTA4 hydrolase (LTA4H), and LTC4 synthase (LTC4S). Cysteinyl-LTs (LTC4, LTD4, LTE4) activate CysLT receptors, which can be targeted by antiasthmatic CysLT1 receptor antagonists. Inset shows the 5 exons (I-V) of the LTC4S gene and its 5′-flanking untranscribed region (UTR). The 2 reported single nucleotide polymorphisms (SNP) are located at 444 and 1072 bp upstream, respectively. The −444 A to C substitution has been associated with increased LTC4 synthesis in eosinophils,6 aspirin intolerant asthma,5 increased risk for coronary calcium,10 increased carotid artery intima media thickness (IMT),10 and a decreased risk of ischemic cerebrovascular events.4 See accompanying article on page 990 Leukotriene C4 synthase (LTC4S; Figure) is a microsomal glutathione-S-transferase (mGST), which conjugates LTA4 with glutathione.2 The product, LTC4, shares its following enzymatic steps with glutathione to …