Leukotriene B4 Production Research Articles

Distinct mechanisms of type 3 secretion system recognition control LTB4 synthesis in neutrophils and macrophages.

Leukotriene B4 (LTB4) is an inflammatory lipid produced in response to pathogens that is critical for initiating the inflammatory cascade needed to control infection. However, during plague, Yersinia pestis inhibits the timely synthesis of LTB4 and subsequent inflammation. Using bacterial mutants, we previously determined that Y. pestis inhibits LTB4 synthesis via the action of the Yop effector proteins that are directly secreted into host cells through a type 3 secretion system (T3SS). Here, we show that the T3SS is the primary pathogen associated molecular pattern (PAMP) required for production of LTB4 in response to both Yersinia and Salmonella. However, we also unexpectantly discovered that T3SS-mediated LTB4 synthesis by neutrophils and macrophages require the activation of two distinctly different host signaling pathways. We identified that phagocytosis and the NLRP3/CASP1 inflammasome significantly impact LTB4 synthesis by macrophages but not neutrophils. Instead, the SKAP2/PLC signaling pathway is required for T3SS-mediated LTB4 production by neutrophils. Finally, while recognition of the T3SS is required for LTB4 production, we also discovered that a second unrelated PAMP-mediated signal activates the MAP kinase pathway needed for synthesis. Together, these data demonstrate significant differences in the host factors and signaling pathways required by macrophages and neutrophils to quickly produce LTB4 in response to bacteria. Moreover, while macrophages and neutrophils might rely on different signaling pathways for T3SS-dependent LTB4 synthesis, Y. pestis has evolved virulence mechanisms to counteract this response by either leukocyte to inhibit LTB4 synthesis and colonize the host.

Neutrophils on the mast cell menu.

A recent article in Cell reported a new peculiar interaction between mast cells and neutrophils. Upon IgE/Ag-mediated degranulation, mast cells (MCs) produce leukotriene B4 (LTB4), which attracts migrating neutrophils. Some neutrophils are able to establish close contact with MCs and end up trapped inside the MC, forming a cell-in-cell structure. While the neutrophil eventually dies inside the vacuole, the MC benefits from the remains of its prisoner, using it as a nutrient reserve and reusing its antimicrobial weapons.

Distinct Mechanisms of Type 3 Secretion System Recognition Control LTB4 Synthesis in Neutrophils versus Macrophages.

Leukotriene B4 (LTB4) is critical for initiating the inflammatory cascade in response to infection. However, Yersinia pestis colonizes the host by inhibiting the timely synthesis of LTB4 and inflammation. Here, we show that the bacterial type 3 secretion system (T3SS) is the primary pathogen associated molecular pattern (PAMP) responsible for LTB4 production by leukocytes in response to Yersinia and Salmonella, but synthesis is inhibited by the Yop effectors during Yersinia interactions. Moreover, we unexpectedly discovered that T3SS-mediated LTB4 synthesis by neutrophils and macrophages require two distinct host signaling pathways. We show that the SKAP2/PLC signaling pathway is essential for LTB4 production by neutrophils but not macrophages. Instead, phagocytosis and the NLRP3/CASP1 inflammasome are needed for LTB4 synthesis by macrophages. Finally, while recognition of the T3SS is required for LTB4 production, we also discovered a second unrelated PAMP-mediated signal independently activates the MAP kinase pathway needed for LTB4 synthesis. Together, these data demonstrate significant differences in the signaling pathways required by macrophages and neutrophils to quickly respond to bacterial infections.

An LGR6 frameshift variant abrogates receptor expression on select leukocyte subsets and is associated with viral infections

AbstractThe leucine-rich repeat-containing G-protein–coupled receptor 6 (LGR6) was recently identified as the cognate receptor for the proresolving mediator maresin 1 (MaR1). To address the biological role of LGR6 in humans, we investigated the functional impact of a genetic variant in the gene encoding for LGR6, which is predicted to lead to a frameshift mutation in one of the receptor isoforms, on both receptor expression and immune cell responses. In neutrophils, monocytes, and natural killer (NK) cells from volunteers homozygous for this variant, we found a significant downregulation in the expression of LGR6 when compared with controls without the variant; whereas the LGR6 expression was essentially similar in monocyte-derived macrophages and CD8+ T cells. Functionally, loss of LGR6 expression was linked with a decreased ability of neutrophils and monocytes to phagocytose bacteria. We observed an increase in neutrophil chemotaxis and leukotriene B4 production and increased expression of activation markers, including markers for platelet-leukocyte phagocyte heterotypic aggregates, such as CD41, in neutrophils and monocytes from the variant group. Using data from the UK Biobank, we found that at a population level the rs4266947 variant, which is in high linkage disequilibrium with rs74355478, was associated with a higher incidence of viral infections. Intriguingly, neutrophils, NK cells, and CD8+ T cells from volunteers with the LGR6 variant displayed altered viral responses when stimulated with Toll-like receptor 3 (TLR3), TLR7/TLR8, and TLR9 agonists. Together, these findings shed new light on the cell type–specific regulation of LGR6 expression and the role of this receptor in directing host immune responses.

Leukotriene B4: A potential mediator and biomarker for cardiac allograft vasculopathy

BackgroundCardiac allograft vasculopathy (CAV) remains the leading cause of long-term graft failure and mortality after heart transplantation. Effective preventive and treatment options are not available to-date, largely because underlying mechanisms remain poorly understood. We studied the potential role of leukotriene B4 (LTB4), an inflammatory lipid mediator, in development of CAV. MethodsWe used an established pre-clinical rat CAV model to study the role of LTB4 in CAV. We performed syngeneic and allogeneic orthotopic aortic transplantation, after which neointimal proliferation was quantified. Animals were then treated with Bestatin, an inhibitor of LTB4 synthesis, or vehicle control for 30 days post-transplant, and evidence of graft CAV was determined by histology. We also measured serial LTB4 levels in a cohort of 28 human heart transplant recipients with CAV, 17 matched transplant controls without CAV, and 20 healthy non-transplant controls. ResultsWe showed that infiltration of the arterial wall with macrophages leads to neointimal thickening and a rise in serum LTB4 levels in our rat model of CAV. Inhibition of LTB4 production with the drug Bestatin prevents development of neointimal hyperplasia, suggesting that Bestatin may be effective therapy for CAV prevention. In a parallel study of heart transplant recipients, we found non-significantly elevated plasma LTB4 levels in patients with CAV, compared to patients without CAV and healthy, non-transplant controls. ConclusionsThis study provides key evidence supporting the role of the inflammatory cytokine LTB4 as an important mediator of CAV development, and provides preliminary data suggesting the clinical benefit of Bestatin for CAV prevention.

Redox processes are major regulators of leukotriene synthesis in neutrophils exposed to bacteria Salmonella typhimurium; the way to manipulate neutrophil swarming.

Neutrophils play a primary role in protecting our body from pathogens. When confronted with invading bacteria, neutrophils begin to produce leukotriene B4, a potent chemoattractant that, in cooperation with the primary bacterial chemoattractant fMLP, stimulates the formation of swarms of neutrophils surrounding pathogens. Here we describe a complex redox regulation that either stimulates or inhibits fMLP-induced leukotriene synthesis in an experimental model of neutrophils interacting with Salmonella typhimurium. The scavenging of mitochondrial reactive oxygen species by mitochondria-targeted antioxidants MitoQ and SkQ1, as well as inhibition of their production by mitochondrial inhibitors, inhibit the synthesis of leukotrienes regardless of the cessation of oxidative phosphorylation. On the contrary, antioxidants N-acetylcysteine and sodium hydrosulfide promoting reductive shift in the reversible thiol-disulfide system stimulate the synthesis of leukotrienes. Diamide that oxidizes glutathione at high concentrations inhibits leukotriene synthesis, and the glutathione precursor S-adenosyl-L-methionine prevents this inhibition. Diamide-dependent inhibition is also prevented by diphenyleneiodonium, presumably through inhibition of NADPH oxidase and NADPH accumulation. Thus, during bacterial infection, maintaining the reduced state of glutathione in neutrophils plays a decisive role in the synthesis of leukotriene B4. Suppression of excess leukotriene synthesis is an effective strategy for treating various inflammatory pathologies. Our data suggest that the use of mitochondria-targeted antioxidants may be promising for this purpose, whereas known thiol-based antioxidants, such as N-acetylcysteine, may dangerously stimulate leukotriene synthesis by neutrophils during severe pathogenic infection.

Staphylococcus aureus α-toxin impairs early neutrophil localization via electrogenic disruption of store-operated calcium entry

The pore-forming S.aureus α-toxin (Hla) contributes to virulence and disease pathogenesis. While high concentrations of toxin induce cell death, neutrophils exhibit relative resistance to lysis, suggesting that the action of Hla may not be solely conferred by lytic susceptibility. Using intravital microscopy, we observed that Hla disrupts neutrophil localization and clustering early in infection. Hla forms a narrow, ion-selective pore, suggesting that Hla may dysregulate calcium or other ions to impair neutrophil function. We found that sub-lytic Hla did not permit calcium influx but caused rapid membrane depolarization. Depolarization decreases the electrogenic driving force for calcium, and concordantly, Hla suppressed calcium signaling invitro and invivo and calcium-dependent leukotriene B4 (LTB4) production, a key mediator of neutrophil clustering. Thus, Hla disrupts the early patterning of the neutrophil response to infection, in part through direct impairment of neutrophil calcium signaling. This early mis-localization of neutrophils may contribute to establishment of infection.

Discovery of a novel BLT2 antagonist for the treatment of inflammatory airway diseases

Leukotriene B4 (LTB4) is a potent chemoattractant that can recruit and activate immune cells such as neutrophils, eosinophils, and monocytes to sites of inflammation. Excessive production of LTB4 has been linked to acute and chronic inflammatory diseases, including asthma, rheumatoid arthritis, and psoriasis. Inhibiting the binding of LTB4 to its receptors, BLT1 and BLT2, is a potential strategy for treating these conditions. While several BLT1 antagonists have been developed for clinical trials, most have failed due to efficacy and safety issues. Therefore, discovering selective BLT2 antagonists could improve our understanding of the distinct functions of BLT1 and BLT2 receptors and their pharmacological implications. In this study, we aimed to discover novel BLT2 antagonists by synthesizing a series of biphenyl analogues based on a BLT2 selective agonist, CAY10583. Among the synthesized compounds, 15b was found to selectively inhibit the chemotaxis of CHO-BLT2 cells with an IC50 value of 224 nM without inhibiting the chemotaxis of CHO-BLT1 cells. 15b also inhibited the binding of LTB4 and BLT2 with a Ki value of 132 nM. Furthermore, 15b had good metabolic stability in liver microsomes and moderate bioavailability (F = 34%) in in vivo PK studies. 15b also showed in vivo efficacy in a mouse model of asthma, reducing airway hyperresponsiveness by 59% and decreasing Th2 cytokines by up to 46%. Our study provides a promising lead for the development of selective BLT2 antagonists as potential therapeutics for inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease.

A genetically encoded sensor for visualizing leukotriene B4 gradients in vivo

Leukotriene B4 (LTB4) is a potent lipid chemoattractant driving inflammatory responses during host defense, allergy, autoimmune and metabolic diseases. Gradients of LTB4 orchestrate leukocyte recruitment and swarming to sites of tissue damage and infection. How LTB4 gradients form and spread in live tissues to regulate these processes remains largely elusive due to the lack of suitable tools for monitoring LTB4 levels in vivo. Here, we develop GEM-LTB4, a genetically encoded green fluorescent LTB4 biosensor based on the human G-protein-coupled receptor BLT1. GEM-LTB4 shows high sensitivity, specificity and a robust fluorescence increase in response to LTB4 without affecting downstream signaling pathways. We use GEM-LTB4 to measure ex vivo LTB4 production of murine neutrophils. Transgenic expression of GEM-LTB4 in zebrafish allows the real-time visualization of both exogenously applied and endogenously produced LTB4 gradients. GEM-LTB4 thus serves as a broadly applicable tool for analyzing LTB4 dynamics in various experimental systems and model organisms.

Inhibition of Leukotriene A4 Hydrolase Suppressed Cartilage Degradation and Synovial Inflammation in a Mouse Model of Experimental Osteoarthritis.

Chronic inflammation plays an important role in the osteoarthritis (OA) pathology but how this influence OA disease progression is unclear. Leukotriene B4 (LTB4) is a potent proinflammatory lipid mediator generated from arachidonic acid through the sequential activities of 5-lipoxygenase, 5-lipoxygenase-activating protein, Leukotriene A4 hydrolase (LTA4H) and its downstream product LTB4. The aim of this study is to investigate the involvement and the potential therapeutic target of the LTB4 pathway in OA disease progression. Both clinical human cartilage samples (n = 7) and mice experimental OA models (n = 6) were used. The levels of LTA4H and leukotriene B4 receptor 1 were first examined using immunostaining in human OA/non-OA cartilage and mice experimental OA models. We also determined whether the LTA4H pathway was associated with cartilage degeneration and synovitis inflammation in OA mice models and human articular chondrocytes. We found that both LTA4H and LTB4 receptor (BLT1) were highly expressed in human and mice OA cartilage. Inhibition of LTA4H suppressed cartilage degeneration and synovitis in OA mice model. Furthermore, inhibition of LTA4H promoted cartilage regeneration by upregulating chondrogenic genes expression such as aggrecan (ACAN), collagen 2A1 (COL2A1), and SRY-Box transcription factor 9 (SOX9). Our results indicate that the LTA4H pathway is a crucial regulator of OA pathogenesis and suggest that LTA4H could be a therapeutic target in combat OA.

NADPH oxidase 2 limits amplification of IL-1β-G-CSF axis and animmature neutrophil subset in murine lung inflammation.

The leukocyte NADPH oxidase 2 (NOX2) regulates inflammation independent of its antimicrobial activity. Inherited defects in NOX2 lead to chronic granulomatous disease (CGD), associated with recurrent bacterial and fungal infections, often with excessive neutrophilic inflammation that results in significant inflammatory burden and tissue damage. We previously showed that excessive leukotriene B4 (LTB4) production by NOX2-deficient mouse neutrophils was a key driver of elevated lung neutrophil infiltration in the initial response to pulmonary challenge with the model fungal particle zymosan. We now identify interleukin-1β (IL-1β) and downstream granulocyte colony-stimulating factor (G-CSF) as critical amplifying signals that augment and sustain neutrophil accrual in CGD mice. Neutrophils, delivered into the lung via LTB4, were the primary source of IL-1β within the airways, and their increased numbers in CGD lungs led to significantly elevated local and plasma G-CSF. Elevated G-CSF simultaneously promoted increased granulopoiesis and mobilized the release of higher numbers of an immature CD101- neutrophil subset from the marrow, which trafficked to the lung and acquired a significantly more proinflammatory transcriptome in CGD mice compared with wild-type mice. Thus, neutrophil-produced IL-1β and downstream G-CSF act sequentially but nonredundantly with LTB4 to deploy neutrophils and amplify inflammation in CGD mice after inhalation of zymosan. NOX2 plays a critical role in dampening multiple components of a feed-forward pipeline for neutrophil recruitment, and these findings highlight NOX2 as a key regulator of neutrophil number, subsets, and function at inflamed sites.

Mead acid inhibits retinol-induced irritant contact dermatitis via peroxisome proliferator-activated receptor alpha.

Retinol is widely used in topical skincare products to ameliorate skin aging and treat acne and wrinkles; however, retinol and its derivatives occasionally have adverse side effects, including the induction of irritant contact dermatitis. Previously, we reported that mead acid (5,8,11-eicosatrienoic acid), an oleic acid metabolite, ameliorated skin inflammation in dinitrofluorobenzene-induced allergic contact hypersensitivity by inhibiting neutrophil infiltration and leukotriene B4 production by neutrophils. Here, we showed that mead acid also suppresses retinol-induced irritant contact dermatitis. In a murine model, we revealed that mead acid inhibited keratinocyte abnormalities such as keratinocyte hyperproliferation. Consistently, mead acid inhibited p38 MAPK (mitogen-activated protein kinase) phosphorylation, which is an essential signaling pathway in the keratinocyte hyperplasia induced by retinol. These inhibitory effects of mead acid were associated with the prevention of both keratinocyte hyperproliferation and the gene expression of neutrophil chemoattractants, including Cxcl1 and Cxcl2, and they were mediated by a PPAR (peroxisome proliferator-activated receptor)-α pathway. Our findings identified the anti-inflammatory effects of mead acid, the use of which can be expected to minimize the risk of adverse side effects associated with topical retinoid application.

Neutrophil-activating therapy for the treatment of cancer.

Despite their cytotoxic capacity, neutrophils are often co-opted by cancers to promote immunosuppression, tumor growth, and metastasis. Consequently, these cells have received little attention as potential cancer immunotherapeutic agents. Here, we demonstrate in mouse models that neutrophils can be harnessed to induce eradication of tumors and reduce metastatic seeding through the combined actions of tumor necrosis factor, CD40 agonist, and tumor-binding antibody. The same combination activates human neutrophils invitro, enabling their lysis of human tumor cells. Mechanistically, this therapy induces rapid mobilization and tumor infiltration of neutrophils along with complement activation in tumors. Complement component C5a activates neutrophils to produce leukotriene B4, which stimulates reactive oxygen species production via xanthine oxidase, resulting in oxidative damage and Tcell-independent clearance of multiple tumor types. These data establish neutrophils as potent anti-tumor immune mediators and define an inflammatory pathway that can be harnessed to drive neutrophil-mediated eradication of cancer.

LC-MS/MS-based arachidonic acid metabolomics in acute spinal cord injury reveals the upregulation of 5-LOX and COX-2 products.

Arachidonic acid (AA) plays a critical role in inflammatory regulation and secondary injury after spinal cord injury (SCI). However, the overall AA metabolism profile in the acute phase of SCI remains elusive. Here we quantified AA metabolomics by High Performance Liquid Chromatography-Tandem Mass Spectrometry-Based Method (LC-MS/MS) using spinal cord tissue collected at 4h, 24h and 48h after contusive SCI in rats. Remarkably, Prostaglandin E2 (PGE2) and Leukotriene B4 (LTB4) were significantly increased throughout the acute SCI. Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), the key enzymes involved in the production of PGE2 and LTB4, were elevated in the lesioned spinal cord tissue, validated by both western blot and immunofluorecnce. The spatial-temporal changes of COX-2 and 5-LOX mainly occurs in neurons both in epicenter and rostral and caudal spinal cord segments after SCI. Our study sheds light on the dynamic microenvironment changes in acute SCI by characterizing the profile of AA metabolism. The COX-2 and 5-LOX may be promising therapeutic target for SCI.

Extracellular pH Controls Chemotaxis of Neutrophil Granulocytes by Regulating Leukotriene B4 Production and Cdc42 Signaling.

Neutrophil granulocytes are the first and robust responders to the chemotactic molecules released from an inflamed acidic tissue. The aim of this study was to elucidate the role of microenvironmental pH in neutrophil chemotaxis. To this end, we used neutrophils from male C57BL/6J mice and combined live cell imaging chemotaxis assays with measurements of the intracellular pH (pHi) in varied extracellular pH (pHe). Observational studies were complemented by biochemical analyses of leukotriene B4 (LTB4) production and activation of the Cdc42 Rho GTPase. Our data show that pHi of neutrophils dose-dependently adapts to a given pH of the extracellular milieu. Neutrophil chemotaxis toward C5a has an optimum at pHi ∼7.1, and its pHi dependency is almost parallel to that of LTB4 production. Consequently, a shallow pHe gradient, resembling that encountered by neutrophils during extravasation from a blood vessel (pH ∼7.4) into the interstitium (pH ∼7.2), favors chemotaxis of stimulated neutrophils. Lowering pHe below pH 6.8, predominantly affects neutrophil chemotaxis, although the velocity is largely maintained. Inhibition of the Na+/H+ exchanger 1 (NHE1) with cariporide drastically attenuates neutrophil chemotaxis at the optimal pHi irrespective of the high LTB4 production. Neutrophil migration and chemotaxis are almost completely abrogated by inhibiting LTB4 production or blocking its receptor (BLT1). The abundance of the active GTP-bound form of Cdc42 is strongly reduced by NHE1 inhibition or pHe 6.5. In conclusion, we propose that the pH dependence of neutrophil chemotaxis toward C5a is caused by a pHi-dependent production of LTB4 and activation of Cdc42. Moreover, it requires the activity of NHE1.

Toxoplasma gondii Infection Decreases Intestinal 5-Lipoxygenase Expression, while Exogenous LTB4 Controls Parasite Growth.

5-Lipoxygenase (5-LO) is an enzyme required for the production of leukotrienes and lipoxins and interferes with parasitic infections. In vitro, Toxoplasma gondii inhibits leukotriene B4 (LTB4) production, and mice deficient in 5-LO are highly susceptible to infection. The aim of this study was to investigate the effects of the pharmacological inhibition of the 5-LO pathway and exogenous LTB4 supplementation during experimental toxoplasmosis. For this purpose, susceptible C57BL/6 mice were orally infected with T. gondii and treated with LTB4 or MK886 (a selective leukotriene inhibitor through inhibition of 5-LO-activating protein [FLAP]). The parasitism, histology, and immunological parameters were analyzed. The infection decreased 5-LO expression in the small intestine, and treatment with MK886 reinforced this reduction during infection; in addition, MK886-treated infected mice presented higher intestinal parasitism, which was associated with lower local interleukin-6 (IL-6), interferon gamma (IFN-γ), and tumor necrosis factor (TNF) production. In contrast, treatment with LTB4 controlled parasite replication in the small intestine, liver, and lung and decreased pulmonary pathology. Interestingly, treatment with LTB4 also preserved the number of Paneth cells and increased α-defensins expression and IgA levels in the small intestine of infected mice. Altogether, these data demonstrated that T. gondii infection is associated with a decrease in 5-LO expression, and on the other hand, treatment with the 5-LO pathway product LTB4 resulted in better control of parasite growth in the organs, adding to the knowledge about the pathogenesis of T. gondii infection.

12/15-lipoxygenase is required for inflammation resolution in a murine model of Lyme arthritis

Abstract Lyme arthritis is a major sequela of disseminated Lyme disease that can persist despite antibiotic clearance of the etiological agent Borrelia burgdorferi. In the murine model of Lyme arthritis (mLA), infected mice develop an inflammatory arthritis characterized by robust innate cell infiltrate and synovial hyperplasia in the tibiotarsal joint, followed by spontaneous resolution around 4–5 weeks post-infection. Eicosanoids are bioactive lipid mediators that play a critical role in both the development and resolution of inflammation. As such, temporal regulation of eicosanoid class-switching from proinflammatory to proresolving is vital. In other inflammation models, late-phase cyclooxygenase (COX)-2 metabolite prostaglandin (PG) E2 skews the 5-lipoxygenase (LO) eicosanoid biosynthesis pathway away from leukotriene B4 production and towards proresolving lipoxin (LX) A4 production via 12/15-LO activity. Indeed, we have previously described a defect in mLA resolution in COX-2−/− and 5-LO−/− mice. Therefore, we hypothesize that 12/15-LO production of LXA4 is required to facilitate mLA resolution. To investigate this, we characterized mLA in 12/15-LO−/− mice and observed a pronounced resolution defect, as evidenced by significant ankle swelling and persistent innate immune infiltrate compared to wild-type mice after 4–5 weeks, despite efficient control of Borrelia burdens. We are currently investigating the mLA eicosanoid profile in 12/15LO−/− mice as well as whether exogenous treatment with LXA4 promotes mLA resolution. These experiments will determine how eicosanoids regulate inflammation resolution and may identify therapeutic targets for inflammatory diseases, including human Lyme arthritis. Supported by a grant from the NIH (R21 AR175160) and the G. Ellsworth Huggins Graduate Fellowship.

A novel core skeleton design and synthesis of N‐alkyl‐1′‐(substituted sulfonyl)spiro[chromene‐2,4′‐piperidin]‐6‐amine derivatives as 5‐lipoxygenase inhibitors

Abstract5‐LO inhibitors can potentially be employed for the treatment of various inflammatory disorders. In this study, we have designed and synthesized new N‐alkyl‐1′‐(substituted sulfonyl)spiro[chromene‐2,4′‐piperidin]‐6‐amine‐based library as potential and novel 5‐LO inhibitors. In vitro results showed that several synthesized compounds exhibited high 5‐LO inhibitory activity, in parallel with the inhibition of leukotriene B4 (LTB4) production in the rat basophilic leukemia (RBL‐1) cells. Among the synthesized compounds, 8l was selected for in vivo study using a mouse ear edema model: oral administration of 8l (100 mg/kg) inhibited arachidonic acid‐induced ear edema, myeloperoxidase (MPO) activity, and LTB4 synthesis. SAR analysis and molecular docking studies demonstrated the allosteric binding mode between 5‐LO and the synthesized compounds including 8l.

The IRE1α Inhibitor KIRA6 Blocks Leukotriene Biosynthesis in Human Phagocytes

The ER stress and Unfolded Protein Response (UPR) component inositol-requiring enzyme 1α (IRE1α) has been linked to inflammation and lipid mediator production. Here we report that the potent IRE1α inhibitor, KIRA6, blocks leukotriene biosynthesis in human phagocytes activated with lipopolysaccharide (LPS) plus N-formyl-methionyl-leucyl-phenylalanine (fMLP) or thapsigargin (Tg). The inhibition affects both leukotriene B4 (LTB4) and cysteinyl leukotriene (cys-LTs) production at submicromolar concentration. Macrophages made deficient of IRE1α were still sensitive to KIRA6 thus demonstrating that the compound’s effect on leukotriene production is IRE1α-independent. KIRA6 did not exhibit any direct inhibitory effect on key enzymes in the leukotriene pathway, as assessed by phospholipase A2 (PLA2), 5-lipoxygenase (5-LOX), LTA4 hydrolase (LTA4H), and LTC4 synthase (LTC4S) enzyme activity measurements in cell lysates. However, we find that KIRA6 dose-dependently blocks phosphorylation of p38 and ERK, mitogen-activated protein kinases (MAPKs) that have established roles in activating cytosolic PLA2α (cPLA2α) and 5-LOX. The reduction of p38 and ERK phosphorylation is associated with a decrease in cPLA2α phosphorylation and attenuated leukotriene production. Furthermore, KIRA6 inhibits p38 activity, and molecular modelling indicates that it can directly interact with the ATP-binding pocket of p38. This potent and unexpected, non-canonical effect of KIRA6 on p38 and ERK MAPKs and leukotriene biosynthesis may account for some of the immune-modulating properties of this widely used IRE1α inhibitor.

Empire-CF study: A phase 2 clinical trial of leukotriene A4 hydrolase inhibitor acebilustat in adult subjects with cystic fibrosis

BackgroundCystic fibrosis (CF) is characterized by neutrophilic inflammation in the airways. Leukotriene B4 (LTB4) is a neutrophil chemoattractant and has been implicated in CF pathogenesis. Acebilustat, a novel, synthetic, small-molecule leukotriene A4 hydrolase inhibitor, reduces LTB4 production. We report findings from a randomized placebo-controlled trial of acebilustat in adult subjects with mild-to-moderate lung disease. MethodsSubjects were randomized (1:1:1) to once-daily acebilustat 50 mg, 100 mg or placebo for 48 weeks, concomitantly with their current therapeutic regimen. Subjects were stratified by use of concomitant CF transmembrane conductance regulator (CFTR) modulators, baseline percent predicted forced expiratory volume in 1 second (ppFEV1) 50-75 and >75, and number of pulmonary exacerbations in the past year (1 or >1). Primary endpoints were the change from baseline in ppFEV1 and safety. Secondary endpoints included the rate of pulmonary exacerbations. ResultsOverall, 199 subjects were randomized and dosed (acebilustat 50 mg, n=67; acebilustat 100 mg, n=66; placebo, n=66). Baseline demographics and disease profile were well balanced among treatment groups. Acebilustat had no statistically significant effect on the primary endpoint of change in ppFEV1 at week 48 or the secondary endpoint pulmonary exacerbations. There was a trend towards reduced pulmonary exacerbations in subjects receiving acebilustat in pre-specified populations with ppFEV1>75 (35% rate reduction) and those on concomitant CFTR modulator therapy (20% rate reduction). Acebilustat was well tolerated. ConclusionsAcebilustat did not improve lung function. A trend towards reduced pulmonary exacerbations in subjects with an earlier stage of lung disease suggests a potential effect in this population.