Leukotriene B4 Synthesis 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.

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.

ATP and Formyl Peptides Facilitate Chemoattractant Leukotriene-B4 Synthesis and Drive Calcium Fluxes, Which May Contribute to Neutrophil Swarming at Sites of Cell Damage and Pathogens Invasion.

Here, we demonstrate that human neutrophil interaction with the bacterium Salmonella typhimurium fuels leukotriene B4 synthesis induced by the chemoattractant fMLP. In this work, we found that extracellular ATP (eATP), the amount of which increases sharply during tissue damage, can effectively regulate fMLP-induced leukotriene B4 synthesis. The vector of influence strongly depends on the particular stage of sequential stimulation of neutrophils by bacteria and on the stage at which fMLP purinergic signaling occurs. Activation of 5-lipoxygenase (5-LOX), key enzyme of leukotriene biosynthesis, depends on an increase in the cytosolic concentration of Ca2+. We demonstrate that eATP treatment prior to fMLP, by markedly reducing the amplitude of the fMLP-induced Ca2+ transient jump, inhibits leukotriene synthesis. At the same time, when added with or shortly after fMLP, eATP effectively potentiates arachidonic acid metabolism, including by Ca2+ fluxes stimulation. Flufenamic acid, glibenclamide, and calmodulin antagonist R24571, all of which block calcium signaling in different ways, all suppressed 5-LOX product synthesis in our experimental model, indicating the dominance of calcium-mediated mechanisms in eATP regulatory potential. Investigation into the adhesive properties of neutrophils revealed the formation of cell clusters when adding fMLP to neutrophils exposed to the bacterium Salmonella typhimurium. eATP added simultaneously with fMLP supported neutrophil polarization and clustering. A cell-derived chemoattractant such as leukotriene B4 plays a crucial role in the recruitment of additional neutrophils to the foci of tissue damage or pathogen invasion, and eATP, through the dynamics of changes in [Ca2+]i, plays an important decisive role in fMLP-induced leukotrienes synthesis during neutrophil interactions with the bacterium Salmonella typhimurium.

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.

Type 3 secretion system induced leukotriene B4 synthesis by leukocytes is actively inhibited by Yersinia pestis to evade early immune recognition.

Subverting the host immune response to inhibit inflammation is a key virulence strategy of Yersinia pestis. The inflammatory cascade is tightly controlled via the sequential action of lipid and protein mediators of inflammation. Because delayed inflammation is essential for Y. pestis to cause lethal infection, defining the Y. pestis mechanisms to manipulate the inflammatory cascade is necessary to understand this pathogen's virulence. While previous studies have established that Y. pestis actively inhibits the expression of host proteins that mediate inflammation, there is currently a gap in our understanding of the inflammatory lipid mediator response during plague. Here we used the murine model to define the kinetics of the synthesis of leukotriene B4 (LTB4), a pro-inflammatory lipid chemoattractant and immune cell activator, within the lungs during pneumonic plague. Furthermore, we demonstrated that exogenous administration of LTB4 prior to infection limited bacterial proliferation, suggesting that the absence of LTB4 synthesis during plague contributes to Y. pestis immune evasion. Using primary leukocytes from mice and humans further revealed that Y. pestis actively inhibits the synthesis of LTB4. Finally, using Y. pestis mutants in the Ysc type 3 secretion system (T3SS) and Yersinia outer protein (Yop) effectors, we demonstrate that leukocytes recognize the T3SS to initiate the rapid synthesis of LTB4. However, several Yop effectors secreted through the T3SS effectively inhibit this host response. Together, these data demonstrate that Y. pestis actively inhibits the synthesis of the inflammatory lipid LTB4 contributing to the delay in the inflammatory cascade required for rapid recruitment of leukocytes to sites of infection.

Modeling population pharmacokinetics of morniflumate in healthy Korean men: extending pharmacometrics analysis to niflumic acid, its major active metabolite.

This study aimed to quantify and explain inter-subject variability in morniflumate pharmacokinetics and identify effective covariates through population pharmacokinetics modeling. Models were constructed using bioequivalence pharmacokinetics results from healthy Korean males and individual physiological and biochemical parameters. Additionally, we incorporated previously reported pharmacokinetics results of niflumic acid, a major active metabolite of morniflumate, to extend the established population pharmacokinetics model and predict niflumic acid pharmacokinetics. Moreover, we used quantitative reports of leukotriene B4 (LTB4) synthesis inhibition in response to niflumic acid exposure to predict drug efficacy using Sigmoid Emax model. Population pharmacokinetics profiles of morniflumate were described using a multi-absorption (5-sequential) two-compartment model, and analysis of inter-individual variability suggested that volume of distribution in peripheral compartment was correlated with body mass index (BMI). Model simulation results showed that individuals with lower BMI had higher plasma concentrations of morniflumate and niflumic acid, resulting in increased and sustained inhibition of LTB4 synthesis. Under steady-state conditions, average plasma concentrations of morniflumate and niflumic acid were 2.66-2.68 times higher in group with a BMI of 17.36kg/m2 compared to the group with a BMI of 28.41kg/m2. Additionally, inhibition of LTB4 synthesis was 1.02 times higher in group with a BMI of 17.36kg/m2 compared to group with a BMI of 28.41kg/m2, and the fluctuation was significantly reduced from 6.06 to 0.01%. These findings suggest that the concentration of active metabolite in plasma following morniflumate exposure was lower in the obese group compared to the normal group, thus potentially reducing the drug's efficacy.

The Role of Leukotriene B4 in Cow Metritis.

Metritis is a common postpartum disease in dairy cows. As a mast cell (MC) mediator, leukotriene B4 (LTB4) is the strongest phagocyte chemokine. It is important in inflammation for the recruitment of immune cells to resist infection. This study investigated the effect of LTB4 in metritis. Twenty Holstein cows 3 to 6 years old and at 6 to 10 days postpartum were selected, ten of which with postpartum metritis were the experimental group, and the other ten of which as healthy cows were the control group. The levels of LTB4, substance P (SP) and vasoactive intestinal peptide (VIP) were measured by ELISA, the expression of LTB4 receptor 2 (BLT2), matrix metalloproteinase (MMP)-2 and MMP-9 mRNA was measured by qPCR, and collagens I and IV were detected by immunohistochemical staining. Concentrations of SP and LTB4 were significantly higher, but those of VIP were significantly lower in the experimental group than those in the control group. The expression of BLT2, MMP-2 and MMP-9 mRNA was significantly higher in the experimental group than that in the control group. The expression of collagen Ⅰ and collagen Ⅳ was significantly lower in the experimental group than that in the control group. In metritis, SP promotes the activation of MC and the synthesis and release of LTB4. Leukotriene B4 chemotactic immune cells promote the high expression of collagenase, which accelerated the hydrolysis of collagen, while the inhibitory effect of VIP on MC was weakened. This may further aggravate the damage to uterine tissue.

Pharmacological and Therapeutic Applications of Esculetin.

Esculetin is a coumarin compound, which belongs to the class of benzopyrone enriched in various plants such as Sonchus grandifolius, Aesculus turbinata, etc. Free radicals lead to the development of oxidative stress causing inflammation, arthritis, cancer, diabetes, fatty liver disease, etc. These further reduce the efficacy of anticancer drugs, activate inflammatory signaling pathways, degrade joints and cartilage, and disrupt the glycemic index and normal function of liver enzymes. For instance, the current treatment modalities used in arthritis such as non-steroidal anti-inflammatory drugs, disease-modifying anti-rheumatoid drugs, and lipoxygenase inhibitors present limited efficacy and adverse effects. Thus, there is a constant need to find newer and safer alternatives. Esculetin has an immense antioxidative potential thereby alleviating arthritis, diabetes, malignancies, and hepatic disorders. Structurally, esculetin contains two hydroxyl groups, which enhance its ability to function as an antioxidant by inhibiting oxidative stress in pathological conditions. Leukotriene B4 synthesis, NF-κB and MPAK pathway activation, and inflammatory cytokine production are the main causes of bone and joint deterioration in arthritis, whereas esculetin treatment reverses these factors and relieves the disease condition. In contrast, lipid peroxidation caused by upregulation of TGF-β-mediated expression and dysfunction of antioxidant enzymes is inhibited by esculetin therapy, thus reducing liver fibrosis by acting on the PI3K/FoxO1 pathway. Therefore, targeting NF-κB, pro-inflammatory cytokines, TGF-β and oxidative stress may be a therapeutic strategy to alleviate arthritis and liver fibrosis.

Dietary supplements in lymphedema.

Lymphedema is a chronic inflammatory disorder resulting from ineffective fluid uptake by the lymphatic system, and the effects are principally felt in the lower limbs. The condition is said to be primary when caused by genetic mutations and secondary when caused by injuries, infections, or surgery. Lymphedema, a worldwide pathology, does not have an effective therapy so far. Leukotriene B4 has recently been identified as a key molecule in lymphedema pathogenesis. Surgical, nonsurgical, and pharmacological treatments have been proposed; however, they do not cure the disease and only ameliorate the symptoms. Nutrition and nutritional status are extremely important in lymphedema physiopathology. Obesity is a comorbidity that exacerbates the risk for secondary lymphedema and constitutes a negative prognostic factor. Indeed, anti-inflammatory foods and their effects on the inflammatory state and on oxidative stress are now being investigated for their possible therapeutic role in lymphedema. Although no special diet has so far been proven to be very effective, specific dietary tips could help in alleviating the edematous state of patients with lymphedema. A few supplements have been tested for lymphedema treatment. Among them, GARLIVE® containing hydroxytyrosol, hesperidin, spermidine and vitamin A, exhibited promising effects in the animal model. Hydroxytyrosol, a polyphenol from olives, showed anti-inflammatory effects and reduced leukotriene B4 synthesis, thus holding promise as a potential natural candidate for lymphedema treatment.

Influence of N-acyl-ethanolamine of arachidonic acid on the synthesis of cytokines and oxylipins by the blood leukocytes of patients with asthma under <i>in vitro</i> conditions

Introduction. The search for hihly effective methods for the treatment and control of asthma remains an urgent task of modern pathophysiology and pharmacology. N-acyl-ethanolamines of arachidonic acid (NAE 20: 4) are promising biomolecules for the regulation of the immune processes. Aim. To investigate the in vitro influence of NAE 20: 4 on the immune cells' ability from patients with asthma to synthesize pro-inflammatory cytokines and oxylipins. Materials and methods. The objective of the study was the blood of 11 patients with mild controlled asthma and 10 healthy volunteers. The in vitro experiment was carried out in stimulated and not stimulated by lipopolysaccharide (LPS) blood for 6 hours. NAE 20: 4 was added at 1.0 µM, 3.0 µM or 10.0 µM doses. The levels of cytokines (TNFα, IL-8), thromboxaneB2 (TXB2) and leukotriene B4 (LTB4) were analyzed by the ELISA method. Results. We found an increase in TNFα, IL-8, TXB2, LTB4 in the blood of persons with asthma before in vitro NAE 20: 4 exposure, which indicates systemic chronic inflammation. Incubation of blood cells without LPS did not affect the change in the spectrum of mediators studied. Whereas the treatment of NAE 20: 4 at a 10 µM reduced the synthesis of TNFα, IL-8, LTB4, TXB2. In vitro blood cells stimulation by LPS led to hyperproduction of cytokines and oxylipins. NAE 20: 4 at a dose of 1 µM in LPS-stimulated blood had no effect on the production of signaling molecules, while NAE 20:4 at a concentration of 3 µM reduced the level of TNFα, IL-8. NAE 20:4 at a dose of 10.0 µM inhibited the cytokines and LTB4 synthesis. The level of TXB2 underthe influence of NAE 20: 4 at 10.0 µM did not change. Conclusion. The results of the study revealed a dose-dependent anti-inflammatory effect of NAE 20:4, characterized by its ability to influence the synthesis of pro-inflammatory cytokines and oxylipins by the immune cells of individuals with asthma in vitro. Further study of NAE 20: 4 opens up new prospects for the development of targeted methods for regulating immune processes in asthma.

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.

Antineoplastic drugs and drug-induced liver damage with cholestasis

The number of cases of drug-induced liver injury (DILI) has been increasing since the 1990s. DILIs cause up to 40,000 deaths each year. One of the leaders in the number of DILIs are antineoplastic drugs ms, such as alkylating agents, antimetabolites, targeted drugs, monoclonal antibodies, etc. One of the most effective and safe strategies for the treatment and prevention of DILI is to use hepatoprotective drugs. Currently, on the market of the Russian Federation, is available novel drug Heptrong® (does not have an International Non-proprietary Name), which has anti-inflammatory, antioxidant activity and the ability to stabilize and reduce the permeability of hepatocyte membranes, suppress the activity 5-lipoxygenase, a decrease in the synthesis of leukotriene B4, interleukin-1, interleukin-6, which are pro-inflammatory cytokines. The drug activates the antitoxic function of the liver, improves its protein- and lipid-synthesizing functions. Heptrong® neutralizes the processes of inflammation in the liver, thereby reducing the severity of the clinical manifestations of drug-induced lesions.

Leukotriene A4 hydrolase deficiency protects mice from diet-induced obesity by increasing energy expenditure through neuroendocrine axis.

Obesity is a health problem worldwide, and brown adipose tissue (BAT) is important for energy expenditure. Here, we explored the role of leukotriene A4 hydrolase (LTA4 H), a key enzyme in the synthesis of the lipid mediator leukotriene B4 (LTB4 ), in diet-induced obesity. LTA4 H-deficient (LTA4 H-KO) mice fed a high-fat diet (HFD) showed a lean phenotype, and bone-marrow transplantation studies revealed that LTA4 H-deficiency in non-hematopoietic cells was responsible for this lean phenotype. LTA4 H-KO mice exhibited greater energy expenditure, but similar food intake and fecal energy loss. LTA4 H-KO BAT showed higher expression of thermogenesis-related genes. In addition, the plasma thyroid-stimulating hormone and thyroid hormone concentrations, as well as HFD-induced catecholamine secretion, were higher in LTA4 H-KO mice. In contrast, LTB4 receptor (BLT1)-deficient mice did not show a lean phenotype, implying that the phenotype of LTA4 H-KO mice is independent of the LTB4 /BLT1 axis. These results indicate that LTA4 H mediates the diet-induced obesity by reducing catecholamine and thyroid hormone secretion.

Promotion of Myofibroblast Differentiation and Tissue Fibrosis by the Leukotriene B4 -Leukotriene B4 Receptor Axis in Systemic Sclerosis.

To investigate the role of the inflammatory lipid mediator leukotriene B4 (LTB4 ) and its receptor, BLT1, in the development and progression of systemic sclerosis (SSc). Serum levels of LTB4 were compared in 64 patients with SSc and 80 healthy controls. Skin and lung tissue sections from patients with SSc and healthy donors were immunostained for leukotriene A4 hydrolase (LTA4 H), the critical enzyme for LTB4 synthesis, and BLT1, in combination with different cell markers. In mouse models of SSc using bleomycin or angiotensin II challenge or immunization with the DNA topoisomerase I, genetic or pharmacologic interruption of the LTB4 -BLT1 axis in mice was carried out to assess its effects on systemic disease features and myofibroblast markers. Immunoblotting was performed to examine the signaling pathway in fibroblasts and endothelial cells following stimulation with LTB4 or with serum from SSc patients. Serum LTB4 levels were 44.93% higher in patients with SSc than in matched healthy controls (mean ± SD 220.3 ± 74.75 pg/ml versus 152.0 ± 68.05 pg/ml; P < 0.0001), and this was associated with the patient subsets of SSc-associated interstitial lung disease and diffuse cutaneous SSc. Levels of LTA4 H and BLT1 were increased in lesional areas of the skin and lungs of SSc patients, and both were abundant in myofibroblasts and endothelial cells. Interruption of the LTB4 -BLT1 axis in mouse models of SSc significantly mitigated dermal and pulmonary fibrosis, with 54.00% and 52.65% fewer α-smooth muscle actin-positive myofibroblasts accumulating in the skin and lungs of mice, respectively, after bleomycin challenge. Immunoblotting of cultures with recombinant LTB4 -stimulated fibroblasts and endothelial cells or with serum from SSc patients showed that fibroblast-myofibroblast and endothelial-mesenchymal transitions were promoted via BLT1, and that this was dependent on activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway but independent of the release of transforming growth factor β (TGFβ) by fibroblasts or endothelial cells. The LTB4 -BLT1 axis may contribute to fibrosis in SSc by directly promoting myofibroblast differentiation via the PI3K/Akt/mTOR pathway, and this appears to operate independently of autocrine secretion of TGFβ.

NADPH oxidase controls pulmonary neutrophil infiltration in the response to fungal cell walls by limiting LTB4

AbstractLeukocyte reduced NADP (NADPH) oxidase plays a key role in host defense and immune regulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD), characterized by recurrent bacterial and fungal infections and aberrant inflammation. Key drivers of hyperinflammation induced by fungal cell walls in CGD are still incompletely defined. In this study, we found that CGD (CYBB−) neutrophils produced higher amounts of leukotriene B4 (LTB4) in vitro after activation with zymosan or immune complexes, compared with wild-type (WT) neutrophils. This finding correlated with increased calcium influx in CGD neutrophils, which was restrained in WT neutrophils by the electrogenic activity of NADPH oxidase. Increased LTB4 generation by CGD neutrophils was also augmented by paracrine cross talk with the LTB4 receptor BLT1. CGD neutrophils formed more numerous and larger clusters in the presence of zymosan in vitro compared with WT cells, and the effect was also LTB4- and BLT1-dependent. In zymosan-induced lung inflammation, focal neutrophil infiltrates were increased in CGD compared with WT mice and associated with higher LTB4 levels. Inhibiting LTB4 synthesis or antagonizing the BLT1 receptor after zymosan challenge reduced lung neutrophil recruitment in CGD to WT levels. Thus, LTB4 was the major driver of excessive neutrophilic lung inflammation in CGD mice in the early response to fungal cell walls, likely by a dysregulated feed-forward loop involving amplified neutrophil production of LTB4. This study identifies neutrophil LTB4 generation as a target of NADPH oxidase regulation, which could potentially be exploited therapeutically to reduce excessive inflammation in CGD.

Redundant and Cooperative Roles for Yersinia pestis Yop Effectors in the Inhibition of Human Neutrophil Exocytic Responses Revealed by Gain-of-Function Approach.

Yersinia pestis causes a rapid, lethal disease referred to as plague. Y. pestis actively inhibits the innate immune system to generate a noninflammatory environment during early stages of infection to promote colonization. The ability of Y. pestis to create this early noninflammatory environment is in part due to the action of seven Yop effector proteins that are directly injected into host cells via a type 3 secretion system (T3SS). While each Yop effector interacts with specific host proteins to inhibit their function, several Yop effectors either target the same host protein or inhibit converging signaling pathways, leading to functional redundancy. Previous work established that Y. pestis uses the T3SS to inhibit neutrophil respiratory burst, phagocytosis, and release of inflammatory cytokines. Here, we show that Y. pestis also inhibits release of granules in a T3SS-dependent manner. Moreover, using a gain-of-function approach, we discovered previously hidden contributions of YpkA and YopJ to inhibition and that cooperative actions by multiple Yop effectors are required to effectively inhibit degranulation. Independent from degranulation, we also show that multiple Yop effectors can inhibit synthesis of leukotriene B4 (LTB4), a potent lipid mediator released by neutrophils early during infection to promote inflammation. Together, inhibition of these two arms of the neutrophil response likely contributes to the noninflammatory environment needed for Y. pestis colonization and proliferation.

NADPH Oxidase Controls Neutrophil Infiltration in the Lung in Early Response to Fungal Cell Walls Via LTB4

The leukocyte NADPH oxidase generates superoxide, the precursor to reactive oxygen species important for host defense and immunoregulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD), characterized by serious bacterial and fungal infections and aberrant inflammation. Aspergillus pneumonia is frequent, and associated with pyogranulomatous inflammation. Of note, even sterile fungal cell walls elicit increased neutrophilic inflammation in CGD mice, demonstrating that that NADPH oxidase can limit inflammation independent of its antimicrobial effects. Leukotriene B4 (LTB4) is a potent inflammatory mediator, acting as a chemoattractant and activating other polymorphonuclear leukocyte (PMN) functions. PMN themselves are prominent source of LTB4, and sense LTB4 through its receptor BLT1, which stimulates additional LTB4 synthesis by autocrine and paracrine routes. This leads to feed forward amplification of LTB4 production locally and formation of PMN clusters. Ca2+ acts as an important second messenger in PMN, including regulation of LTB4 synthesis. Activated human CGD PMN have increased intracellular Ca2+ levels compared to PMN from healthy donors due to electrogenic effects of the NADPH oxidase (Geiszt M. et al JBC 1997). Intracellular Ca2+ overload in PMN is speculated to contribute to increased PMN pro-inflammatory activity, but specific pathways and in vivo relevance are not well defined. Therefore, we therefore investigated the production of LTB4 by murine CGD PMN and the role of LTB4 in PMN recruitment and lung hyperinflammation following pulmonary challenge with zymosan, a sterile fungal cell wall preparation. Zymosan-stimulated PMN from Cybb-null mice, a model for X-linked CGD, produced higher LTB4 in vitro compared to wild type (WT), even in the presence of SOD and catalase to counteract oxidative degradation. Higher LTB4 production correlated with higher levels of intracellular Ca2+ in stimulated X-CGD PMN. The increased LTB4 produced by X-CGD PMN was dependent on cell density and the LTB4 receptor BLT1, consistent with a feed-forward loop that amplified LTB4 production. Zymosan-stimulated X-CGD PMN also formed larger and more numerous clusters in vitro compared to WT PMN. Cluster formation was abrogated by inhibiting LTB4 synthesis with the 5-lipoxygenase (5-LO) inhibitor zileuton or by the BLT1 receptor antagonist U75302, demonstrating that cluster formation was LTB4 and BLT1 dependent. We next examined whether LTB4 regulated the response to zymosan in the lung. Following intranasal administration of zymosan, X-CGD mice had higher PMN numbers in bronchoalveolar lavage (BAL), larger PMN foci by lung histology, and increased lung LTB4 as compared to WT mice. These differences were evident by 8 hr and progressed over the first 24 hr post-challenge, in contrast to WT mice where inflammation plateaued at 8 hr. Treatment with zileuton or U75302 just prior to zymosan significantly reduced lung PMN numbers for both WT and X-CGD mice at 8 hr post challenge, and lung PMN remained substantially reduced in X-CGD mice at 24 hrs. Moreover, delaying administration of zileuton to 8 hr after zymosan challenge also significantly decreased PMN inflammation in X-CGD but not WT mice at 24 hrs, indicating that ongoing synthesis of LTB4 otherwise continued to promote PMN recruitment in CGD mice. These results demonstrate that PMN production of LTB4 in response to fungal cell walls is limited by NADPH oxidase via its effects on intracellular Ca2+. In oxidase-deficient CGD mice, LTB4 plays a major role in driving excessive PMN recruitment to the lung in the early response to zymosan. This study is the first to implicate LTB4 in promoting neutrophilic lung inflammation in response to fungal cell walls in CGD, likely by an amplified feed-forward loop involving increased production of LTB4 by CGD PMN. LTB4 could be a potential therapeutic target to ameliorate CGD hyperinflammation. Disclosures No relevant conflicts of interest to declare.

Leukotriene B4 Contributes to Development of Cardiac Allograft Vasculopathy

Purpose Cardiac allograft vasculopathy (CAV) is a highly prevalent vaso-occlusive disease that is a leading cause of graft failure and mortality after heart transplantation. While the pathogenesis of CAV remains incompletely understood, histologic evidence suggests that macrophages, which constitute an important part of the innate immune response, may play an important role. Prior studies in pulmonary hypertension have shown that macrophage-derived leukotriene B4 (LTB4) induces proliferation and hypertrophy of human pulmonary artery smooth muscle cells. We hypothesized that LTB4 plays a similar role in the development of CAV. Methods Human studies: We performed immunofluorescence staining of sections from human CAV+ and CAV- coronary arteries with CD68 (macrophage marker) and 5-LO (5-lipoxygenase, LTB4-producing enzyme), and measured serial LTB4 plasma levels in 14 patients with angiographic CAV and 14 matched CAV- controls. Rat model: We performed aortic transplants in a rat CAV model (Fischer into Lewis orthotopic aortic transplants, n=11) with allogeneic transplants as controls (n=6), and performed CD68 and 5-LO staining of aortic cross-sections of mice with and without aortic intimal hyperplasia. We then treated rat allogeneic transplants with bestatin (protease inhibitor that blocks LTB4 production) to determine whether LTB4 inhibition can prevent the intimal proliferation seen in this rat CAV model. Results Immunofluorescence staining showed that 5-LO co-localized with CD68+ macrophages in the intimal layer of CAV+ human arterioles and in allogeneic rat aortic transplant models. Plasma LTB4 levels were higher in CAV+ cases compared to CAV- controls (n=14 per group, p Conclusion Macrophage-derived leukotriene B4 may contribute to development of CAV. Blockade of LTB4 synthesis has the potential to prevent CAV development after heart transplantation.

Arachidonic Acid Induces the Migration of MDA-MB-231 Cells by Activating Raft-associated Leukotriene B4 Receptors.

The migration of tumor cells is critical in spreading cancers through the lymphatic nodes and circulatory systems. Although arachidonic acid (AA) and its soluble metabolites have been shown to induce the migration of breast and colon cancer cells, the mechanism by which it induces such migration has not been fully understood. The effect of AA on migratory responses of the MDA-MB-231 cell line (a triple-negative breast cancer cell) was examined and compared with MCF-7 (estrogen-receptor positive) breast cancer cells to elucidate the mechanism of AA-induced migration. Migrations of breast cancer cells were examined with the help of wound-healing assays. AA-induced eicosanoid synthesis was monitored by RP-HPLC. Cellular localizations of lipoxygenase and lipid rafts were assessed by immunoblot and confocal microscopy. AA treatment stimulated the synthesis of leukotriene B4 (LTB4) and HETE-8, but lowered the levels of prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), and HETE-5 in MDA-MB-231 cells. Further analysis indicated that AA increased the expression of 5-lipoxygenase (5-LOX) in this cell line and inhibiting its expression by small molecule inhibitors lowered the production of LTB4 and reduced migration. In contrast, MCF-7 cells did not show any appreciable changes in eicosanoid synthesis, 5-LOX expression, or cellular migration. Our results suggest that AA treatment activates the BLT1 receptor (present in membrane microdomains) and stimulates the synthesis of LTB4 production, which is likely to be associated with the migration of MDA-MB-231 cells.