LTB4 Production Research Articles

ALOX5 drives the pyroptosis of CD4+ T cells and tissue inflammation in rheumatoid arthritis.

Pyroptosis, an inflammatory form of programmed cell death, is linked to the pathology of rheumatoid arthritis (RA). Here, we investigated the molecular mechanism underlying pyroptosis in T cells isolated from patients with RA. Compared with healthy individuals, patients with RA had more pyroptotic CD4+ T cells in blood and synovia, which correlated with clinical measures of disease activity. Moreover, the mRNA expression and protein abundance of arachidonate 5-lipoxygenase (ALOX5), which converts arachidonic acid to leukotriene A4 (LTA4), were increased in CD4+ T cells from patients with RA and, among patients with RA, were lowest in those in clinical remission. Knockdown or pharmacological inhibition of ALOX5 suppressed CD4+ T cell pyroptosis and improved symptoms in two rodent models of RA. Mechanistically, the increase in ALOX5 activity in RA CD4+ T cells enhanced the production of the LTA4 derivative LTB4, which stimulated Ca2+ influx through ORAI3 channels, leading to the activation of NLRP3 inflammasomes and pyroptosis. Our findings reveal a role for ALOX5 in RA and provide a molecular basis for further exploring the clinical utility of ALOX5 inhibition in RA and for using ALOX5 as a biomarker to distinguish active disease and remission in RA.

Insight Into Invitro Immune Stimulatory Effect Of Phyllathus Amarus Extracts

The medicinal property of Phyllanthus amarus (P. amarus) has been well appreciated and well documented in Indian system of traditional medicines namely Ayurvedic, Siddha and Unani. P. amarus has been long used to treat various ailments, however its mechanism of action is still obscure because of the lack of scientific proofs to support its medical benefits. The current study was performed to elucidate the immune modulation properties of P. amarus by measuring free radical scavenging ability, nitric oxide stimulation potential and LTB4 production. Aqueous extracts of P. amarus (100 µg/ml) had quenched the free radicals by 81.4 ± 1.1 % of activity followed by 76.1 ± 2.4 % quenching by methanolic extract. Aqueous extract of P. amarus (100 µg/ml) produced a profound NO to a tune of 580 ± 10 µMol. Methanolic extract produced 530 ± 17.3 µMol of NO. There was an increase of NO with aqueous extract in comparison to methanolic extract however these data are not statistically significant (p> 0.05). LTB4 production with 100 µg/ml concentration of aqueous extract was 103 ± 6.2 pg/ml and it was 113.2 ± 7.8 with methanolic extract.

Lipoxin receptor agonist and inhibition of LTA4 hydrolase prevent tight junction disruption caused by P. aeruginosa filtrate in airway epithelial cells.

Airway diseases can disrupt tight junction proteins, compromising the epithelial barrier and making it more permeable to pathogens. In people with pulmonary disease who are prone to infection with Pseudomonas aeruginosa, pro-inflammatory leukotrienes are increased and anti-inflammatory lipoxins are decreased. Upregulation of lipoxins is effective in counteracting inflammation and infection. However, whether combining a lipoxin receptor agonist with a specific leukotriene A4 hydrolase (LTA4H) inhibitor could enhance these protective effects has not to our knowledge been investigated. Therefore, we explored the effect of lipoxin receptor agonist BML-111 and JNJ26993135 a specific LTA4H inhibitor that prevents the production of pro-inflammatory LTB4 on tight junction proteins disrupted by P. aeruginosa filtrate (PAF) in human airway epithelial cell lines H441 and 16HBE-14o. Pre-treatment with BML-111 prevented an increase in epithelial permeability induced by PAF and conserved ZO-1 and claudin-1 at the cell junctions. JNJ26993135 similarly prevented the increased permeability induced by PAF, restored ZO-1 and E-cadherin and reduced IL-8 but not IL-6. Cells pre-treated with BML-111 plus JNJ26993135 restored TEER and permeability, ZO-1 and claudin-1 to the cell junctions. Taken together, these data indicate that the combination of a lipoxin receptor agonist with a LTA4H inhibitor could provide a more potent therapy.

Lipid mediators in the regulation of innate and adaptive immunity.

Lipid mediators in the regulation of innate and adaptive immunity.

ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production.

Chemotaxis is an essential physiological process, often harnessed by tumors for metastasis. CXCR4, its ligand CXCL12 and the atypical receptor ACKR3 are overexpressed in many human cancers. Interfering with this axis by ACKR3 deletion impairs lymphoma cell migration towards CXCL12. Here, we propose a model of how ACKR3 controls the migration of the diffused large B-cell lymphoma VAL cells in vitro and in vivo in response to CXCL12. VAL cells expressing full-length ACKR3, but not a truncated version missing the C-terminus, can support the migration of VAL cells lacking ACKR3 (VAL-ko) when allowed to migrate together. This migration of VAL-ko cells is pertussis toxin-sensitive suggesting the involvement of a Gi-protein coupled receptor. RNAseq analysis indicate the expression of chemotaxis-mediating LTB4 receptors in VAL cells. We found that LTB4 acts synergistically with CXCL12 in stimulating the migration of VAL cells. Pharmacologic or genetic inhibition of BLT1R markedly reduces chemotaxis towards CXCL12 suggesting that LTB4 enhances in a contact-independent manner the migration of lymphoma cells. The results unveil a novel mechanism of cell-to-cell-induced migration of lymphoma.

Spatiotemporal dynamics of calcium signals during neutrophil cluster formation

Neutrophils form cellular clusters or swarms in response to injury or pathogen intrusion. Yet, intracellular signaling events favoring this coordinated response remain to be fully characterized. Here, we show that calcium signals play a critical role during mouse neutrophil clustering around particles of zymosan, a structural fungal component. Pioneer neutrophils recognizing zymosan or live Candida albicans displayed elevated calcium levels. Subsequently, a transient wave of calcium signals in neighboring cells was observed followed by the attraction of neutrophils that exhibited more persistent calcium signals as they reached zymosan particles. Calcium signals promoted LTB4 production while the blocking of extracellular calcium entry or LTB4 signaling abrogated cluster formation. Finally, using optogenetics to manipulate calcium influx in primary neutrophils, we show that calcium signals could initiate recruitment of neighboring neutrophils in an LTB4-dependent manner. Thus, sustained calcium responses at the center of the cluster are necessary and sufficient for the generation of chemoattractive gradients that attract neutrophils in a self-reinforcing process.

Topical Aspirin Administration Improves Cutaneous Wound Healing in Diabetic Mice Through a Phenotypic Switch of Wound Macrophages Toward an Anti-inflammatory and Proresolutive Profile Characterized by LXA4 Release.

Patients with diabetes present a persistent inflammatory process, leading to impaired wound healing. Since nonhealing diabetic wound management shows limited results, the introduction of advanced therapies targeting and correcting the inflammatory status of macrophages in chronic wounds could be an effective therapeutic strategy to stop the sustained inflammation and to return to a healing state. In an excisional skin injury in a diet-induced diabetic murine model, we demonstrate that topical administration of low-dose aspirin (36 μg/wound/day) improves cutaneous wound healing by increasing wound closure through the promotion of the inflammation resolution program of macrophages. This treatment increased efferocytosis of wound macrophages from aspirin-treated diabetic mice compared with untreated diabetic mice. We also show that aspirin treatment of high-fat-fed mice oriented the phenotype of wound macrophages toward an anti-inflammatory and proresolutive profile characterized by a decrease of LTB4 production. The use of diabetic mice deficient for 5-LOX or 12/15-LOX demonstrated that these two enzymes of acid arachidonic metabolism are essential for the beneficial effect of aspirin on wound healing. Thus, aspirin treatment modified the balance between pro- and anti-inflammatory eicosanoids by promoting the synthesis of proresolving LXA4 through 5-LOX, LTA4, 12/15-LOX signaling. In conclusion, the restoration of an anti-inflammatory and proresolutive phenotype of wound macrophages by the topical administration of low-dose aspirin represents a promising therapeutic approach in chronic wounds.

Bitis arietans Snake Venom and Kn-Ba, a Snake Venom Serine Protease, Induce the Production of Inflammatory Mediators in THP-1 Macrophages.

Bitis arietans is a snake of medical importance found throughout sub-Saharan Africa and in savannas and pastures of Morocco and western Arabia. The effects of its venom are characterized by local and systemic alterations, such as inflammation and cardiovascular and hemostatic disturbances, which can lead to victims’ death or permanent disability. To better characterize the inflammatory process induced by this snake’s venom, the participation of eicosanoids and PAF (platelet- activating factor) in this response were demonstrated in a previous study. In addition, edema and early increased vascular permeability followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity were accompanied by the production of the eicosanoids LTB4, LTC4, TXB2, and PGE2, and local and systemic production of IL-6 and MCP-1. In this context, the present study focused on the identification of inflammatory mediators produced by human macrophages derived from THP-1 cells in response to Bitis arietans venom (BaV), and Kn-Ba, a serine protease purified from this venom. Here, we show that Kn-Ba, and even the less intensive BaV, induced the production of the cytokine TNF and the chemokines RANTES and IL-8. Only Kn-Ba was able to induce the production of IL-6, MCP-1, and IP-10, whereas PGE2 was produced only in response to BaV. Finally, the release of IL-1β in culture supernatants suggests the activation of the inflammasomes by the venom of Bitis arietans and by Kn-Ba, which will be investigated in more detail in future studies.

Complex and Controversial Roles of Eicosanoids in Fungal Pathogenesis.

The prevalence of fungal infections has increased in immunocompromised patients, leading to millions of deaths annually. Arachidonic acid (AA) metabolites, such as eicosanoids, play important roles in regulating innate and adaptative immune function, particularly since they can function as virulence factors enhancing fungal colonization and are produced by mammalian and lower eukaryotes, such as yeasts and other fungi (Candida albicans, Histoplasma capsulatum and Cryptococcus neoformans). C. albicans produces prostaglandins (PG), Leukotrienes (LT) and Resolvins (Rvs), whereas the first two have been well documented in Cryptococcus sp. and H. capsulatum. In this review, we cover the eicosanoids produced by the host and fungi during fungal infections. These fungal-derived PGs have immunomodulatory functions analogous to their mammalian counterparts. Prostaglandin E2 (PGE2) protects C. albicans and C. parapsilosis cells from the phagocytic and killing activity of macrophages. H. capsulatum PGs augment the fungal burden and host mortality rates in histoplasmosis. However, PGD2 potentiates the effects and production of LTB4, which is a very potent neutrophil chemoattractant that enhances host responses. Altogether, these data suggest that eicosanoids, mainly PGE2, may serve as a new potential target to combat diverse fungal infections.

An IgG/IgY sandwich-ELISA for the detection of heat-labile enterotoxin B subunit of enterotoxigenic Escherichia coli

ObjectiveEnterotoxinogenic Escherichia coli is a health-threatening pathogen, especially in children. LT toxin this bacterium causes diarrhea and loss of body fluids. The development of rapid and cost-effective methods for diagnosis will help the patient survive and prevent the spread of this bacterium. The aim of this study was to design and set up the double sandwich ELISA diagnostic method for the detection of LT toxin using egg yolk (IgY) and rabbit antibody (IgG). MethodsRecombinant LTB protein was expressed in E coli BL21 (DE3) and purified using a Ni-NTA column. Chickens and rabbits were immunized subcutaneously and the generated antibodies were purified from egg yolks by polyethylene glycol (PEG) precipitation and from the rabbits' sera by protein G column. These antibodies were used to set up the ELISA method. The sensitivity of the designed ELISA method was evaluated using serial dilutions of the protein. ResultsExpression of recombinant protein led to the production of LTB with molecular weight of 15 kDa. Yield of protein was 6 mg/L. ELISA assay results showed, increased serum antibody levels against the protein in chickens and rabbits sera after each injection. The yield of purified IgG was 2/5 mg/mL. The yield of purified IgY was 27 mg per egg. The sensitivity of the ELISA method was about 39 ng for recombinant LTB. The results showed the high specificity of this technique. ConclusionResults suggest that IgY/IgG-based sandwich ELISA in this study provides a convenient preparation for the development of immune-based method for the detection of LTB.

Echinacea purpurea water extracts suppress the release of chemical mediators from mast cells

Histamine and leukotrienes (LTs), the chemical mediators released from mast cells, play an important role in type-I allergies such as hay fever. Echinacea purpurea (EP) has traditionally been used for herbal tea and has been reported to show biological functions. We evaluated the inhibitory activity of water extracts of EP petals, leaves, and stems against the chemical mediators released from mast cell lines. Petal and leaf extracts exhibited a significant inhibitory effect on histamine release from the stimulated cells, while the stem extract did not exert any effect. Activity of the petal extract was much stronger than that of the leaf extract. All the extracts significantly suppressed LTB4 production in the stimulated cells and displayed similar activities. The petal extract decreased Syk phosphorylation and Ca2+ influx associated with signal transduction in the stimulated cells. These results suggest that EP petal extract may have a relieving effect on allergic symptoms.

Protective effects of Dioscin on TNF-α-induced collagen-induced arthritis rat fibroblast-like synoviocytes involves in regulating the LTB4/BLT pathway

Background and Objective: LTB4 has been shown to be involved in rheumatoid arthritis (RA) pathogenesis. The effect of Dioscin(Dio) on the LTB4 pathway of RA have not been reported yet. This study aimed at further exploring whether Dioscin’s effects on TNF-α induced collagen-induced arthritis (CIA) rat fibroblast-like synoviocytes (FLS) connected with the LTB4 and its receptor pathway. <b>Materials & Methods:</b> In this experiment, control group, TNF-α group, and different concentrations of Dioscin groups were established. Cell viability was evaluated using MTT assay. The levels of LTB4 in the samples of above groups were measured using ELISA. The mRNA expression levels of LTA4H, BLT1, and BLT2 were detected by quantitative real time PCR, while the expression level of LTA4H proteins were detected using western blot. The distribution of LTA4H was assessed by immunofluorescence assay. Results: the LTB4 level of TNF-α group in sample supernatant was higher than both control group and Dioscin groups with decreased LTB4 levels (<i>p</i>< 0.05). Compared with the control group, the expression of LTA4H was significantly increased in TNF-α group (<i>p</i> < 0.05), whereas LTA4H expressions were significantly decreased in all Dioscin groups when compared to TNF-α group (<i>p</i> < 0.05). The mRNA expressions of BLT1 and BLT2 were markedly higher in TNF-α group than those in control group while Dioscin treatment significantly inhibited the increased expressions of BLT1 and BLT2 induced by TNF-α (<i>p</i> < 0.05). <b>Conclusions:</b> These results firstly demonstrate that the protective effect of Dioscin on TNF-α induced FLS may involve in its reducing LTB4 production by down-regulating LTA4H expression, and may inhibit its downstream pathway by decreasing LTB4 receptors levels. This findings suggest that dioscin produces a potential therapeutic effects for RA via its influencing LTA4H/LTB4/BLT pathway.

P.441 Impact of CYP2C19 and CYP2D6 polymorphism on antidepressants and antipsychotics exposure; a meta-analysis

Crotalus Neutralizing Factor (CNF) is an inhibitor of phospholipase A2 (PLA2), present in the blood plasma of Crotalus durissus terrificus snake. This inhibitor neutralizes the lethal and enzymatic activity of crotoxin, the main neurotoxin from this venom. In this study, we investigated the effects of CNF on the functionality of human peripheral blood mononuclear cells (PBMCs) and human neutrophils. The following parameters were evaluated: viability and proliferation, chemotaxis, cytokines and LTB4 production, cytosolic PLA2s activity, myeloperoxidase (MPO) and superoxide anion (O2−) production. CNF showed no toxicity on PBMCs or neutrophils, and acts by stimulating the release of TNF-α and LTB4, but neither stimulates IL-10 and IL-2 nor affects PBMCs proliferation and O2− release. In neutrophils, CNF induces chemotaxis but does not induce the release of both MPO and O2−. However, it induces LTB4 and IL-8 production. These data show the influence of CNF on PBMCs’ function by inducing TNF-α and LTB4 production, and on neutrophils, by stimulating chemotaxis and LTB4 production, via cytosolic PLA2 activity, and IL-8 release. The inflammatory profile produced by CNF is shown for the first time. Our present results suggest that CNF has a role in activation of leukocytes and exert proinflammatory effects on these cell.

Bitis arietans Snake Venom Induces an Inflammatory Response Which Is Partially Dependent on Lipid Mediators.

Bitis arietans is a snake of medical importance, as it is responsible for more accidents in humans and domestic animals than all other African snakes put together. The accidents are characterized by local and systemic alterations, such as inflammation, cardiovascular and hemostatic disturbances, which can lead victims to death or permanent disability. However, little is known about the envenomation mechanism, especially regarding the inflammatory response, which is related to severe clinical conditions triggered by the venom. Therefore, the aim of the present study was to evaluate the inflammatory response related to the B. arietans envenomation using a peritonitis mice model. By pharmacological interventions and use of mice genetically deficient of the 5-lipoxygenase enzyme (5-LO−/−) or platelet-activating factor (PAF) receptor (PAFR−/− the participation of eicosanoids and PAF in this response was also investigated. The obtained results demonstrated that the venom induces an in vivo inflammatory response, characterized by an early increased vascular permeability, followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity, accompanied by the production of the eicosanoids LTB4, LTC4, TXB2 and PGE2, as well as the local and systemic production of IL-6 and MCP-1. These inflammatory events were attenuated by the pre-treatment with anti-inflammatory drugs that interfere in lipid mediators’ functions. However, 5-LO−/− mice did not show a reduction of inflammatory response induced by the venom, while PAFR−/− mice showed a reduction in both the PMN leukocytes number and the local and systemic production of IL-6 and MCP-1. This study demonstrated that the Bitis arietans venom contains toxins that trigger an inflammatory process, which is partially dependent on lipid mediators, and may contribute to the envenomation pathology.

New isoflavone glucosides in yabumame (Amphicarpaea bracteata (L.) Fernald subsp. edgeworthii (Benth.) H.Ohashi var. japonica (Oliv.) H.Ohashi) and their effect on leukotriene B4 production in mast cells.

Yabumame (Amphicarpaea bracteata (L.) Fernald subsp. edgeworthii (Benth.) H.Ohashi var. japonica (Oliv.) H.Ohashi) is a legume plant that the Ainu people eat as a traditional food, although the bioactive ingredients other than vitamins have not been studied. In this study, the structures of yabumame isoflavone glucosides were determined and their effect on leukotriene (LT) B4, a chemical mediator of type I allergy, produced in mast cells, was investigated in vitro. Seven compounds were isolated from yabumame. Their structures were determined by spectroscopic and spectrometric analyses, which were genistein-7-O-β-D-glucoside (1), formononetin-7-O-(2″-O-β-D-glucosyl)-β-D-glucoside (2), formononetin-7-O-β-D-glucoside (3), biochanin A-7-O-(2″-O-β-D-glucosyl)-β-D-glucoside (4), formononetin-7-O-(6″-O-malonyl)-β-D-glucoside (5), biochanin A-7-O-(2″-O-β-D-glucosyl-6″-O-β-D-glucosyl)-β-D-glucoside (6), and biochanin A-7-O-(6″-O-malonyl)-β-D-glucoside (7). Compounds 2, 4, and 6 were determined as new compounds. Compound 3 showed statistically significant suppressive effect on LTB4 production in mast cells, although the activity was not strong. On the other hand, biochanin A, an aglycone common to compounds 4, 6, and 7, strongly inhibited the LTB4 production. The results suggest that some of yabumame isoflavone glucosides might contribute to mitigate type I allergy. Seven isoflavone glucosides including 3 new compounds were found in yabumame and their anti-allergic effect was evaluated.

Abstract 6364: LFA-9, a dual mPGES-1 and 5-LOX inhibitor, suppresses colon cancer stemness and inflammogen-induced inflammatory response

Abstract Chronic inflammation triggers tumorigenesis of the colorectum; hence several non-steroidal anti-inflammatory drugs (NSAIDs) have been explored for prevention and treatment of inflammatory diseases, including colorectal cancer (CRC). Although traditional NSAIDs and COX-2 inhibitors are effective chemopreventive agents for CRC, unwanted side effects (gastrointestinal and renal) hinder their use. Mechanistic studies suggest that the side effects are in part due to a COX-2 inhibition-associated increase in 5-lipoxygenase (5-LOX) metabolites and reduced PGI2 levels. Hence, agents targeting microsomal prostaglandin synthase-1 (mPGES-1) and 5-LOX inhibitors may prove to be more effective and safer agents for chemoprevention. LFA-9 was designed based on the pharmacophore of licofelone by adding 2-aminoethanoic acid (Glycine) at its carboxylic end to enhance renal clearance and selectivity towards dual mPGES-1 and 5-LOX inhibition. Here we characterized LFA-9 in detail for its mPGES1/5-LOX selectivity over COX-1/2 using in silico molecular modelling, in vitro enzyme activity assays, and ex vivo inhibition studies with human/mouse/rat mPGES-1 and 5-LOX enzymes to facilitate pre-clinical experimentation and clinical translation. In silico, LFA-9 showed strong binding affinity against 5-LOX (binding energies -199.85 and -215.3 Kcal/mol) and mPGES-1 (binding energies -238.27 and -257.76 Kcal/mol), but not against COX-1/2. LFA-9 substantially inhibited mPGES-1 (IC50 0.52 - 1.40 µM) and 5-LOX (IC50 0.89 - 2.75 µM) but not COX-1/2 (IC50&amp;gt;1mM) in cell-free assays. Ex-vivo, LFA-9 inhibited PGE2 (IC50 0.47 - 0.78 µM) and LTB4 (IC50 0.66 - 1.24 µM) production and spared PGI2 (IC50&amp;gt;1 mM) and TXB2 (IC50&amp;gt;1 mM) production in LPS-stimulated human/mouse/rat macrophages and whole blood (cell-based system). Circular dichroism and isothermal calorimetric-based studies demonstrated that LFA-9 strongly binds and induces changes in the secondary structure of human mPGES-1 and 5-LOX enzymes, thereby inhibiting their activities. Since PGE2 from mPGES1 activity promotes colon tumor stemness, LFA-9 was evaluated on colonic cancer organoids (spheroids) derived from PIRC (Polyposis in Rat Colon) rats that replicate human familial adenomatous polyposis. LFA-9 significantly inhibited the growth of colonic spheroids at 25 µM as compared to that of untreated as well as licofelone-treated spheroids. Anti-inflammatory properties of LFA-9 were evaluated in vivo using an inflammogen (carrageenan)-induced rat paw edema model. Dietary LFA-9 inhibited edema formation after carrageenan injection. In summary, LFA-9 showed anti-inflammatory and anti-tumor properties through mPGES1/5-LOX inhibition. Our data supports LFA-9, a novel mPGES-1 and 5-LOX inhibitor, as a safer drug candidate for prevention and therapy of colorectal cancer. (Supported by R01CA 213987, NIH/NCI HHSN261201500024I and VA merit). Citation Format: Nagendra Sastri Yarla, Gopal Pathuri, Hariprasad Gali, Venkateshwar Madka, Janani Panneerselvam, Parthasarathy Chandrakesan, Courtney W. Houchen, Elizabeth R. Glaze, Jennifer Fox, David McCormick, Chinthalapally V. Rao. LFA-9, a dual mPGES-1 and 5-LOX inhibitor, suppresses colon cancer stemness and inflammogen-induced inflammatory response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6364.

Berberine Inhibits the Apoptosis-Induced Metastasis by Suppressing the iPLA2/LOX-5/LTB4 Pathway in Hepatocellular Carcinoma.

PurposeHepatocellular carcinoma (HCC) is one of the most malignant cancers around the world. HCC is less sensitive to conventional cytotoxic agents and easily develops into systemic metastases. However, the molecular mechanisms of the metastasis of HCC are poorly understood and need elucidation.Materials and MethodsTranswell system of the chemotherapy-challenged and unchallenged HepG2 cells was established. Adhesion assay and scratch-wound assay were utilized to analyze the adhesion and migration of HepG2 cells. iPLA2 and LOX-5 expression were analyzed by Western blot. LTB4 level was analyzed by ELISA.ResultsChemotherapeutics are traditionally regarded as a way of killing tumor cells; on the other hand, we proved that the chemotherapeutics-induced tumor cell apoptosis can also change the tumor microenvironment by activating the LOX pathway and subsequently release inflammatory factors such as LTB4 which can stimulate the adhesion and migration of the small number of surviving cells. Berberine can reverse the adhesion and migration of HepG2 cells by inhibiting the expression of LOX-5 and reducing the LTB4 production in the tumor microenvironment.ConclusionOur study sheds light on a novel anti-metastasis strategy that the combination of Berberine and chemotherapy may prevent the chemotherapy-induced metastasis in HCC.

Toll-like receptor signaling induces a temporal switch towards a resolving lipid profile in monocyte-derived macrophages

Inflammation is a tightly regulated process. During the past decade it has become clear that the resolution of inflammation is an active process and its dysregulation can contribute to chronic inflammation. Several cells and soluble mediators, including lipid mediators, regulate the course of inflammation and its resolution. It is, however, unclear which signals and cells are involved in initiating the resolution process. Macrophages are tissue resident cells and key players in regulating tissue inflammation through secretion of soluble mediators, including lipids. We hypothesize that persistent inflammatory stimuli can initiate resolution pathways in macrophages.In this study, we detected 21 lipids in LPS-stimulated human monocyte-derived macrophages by liquid chromatography coupled to tandem mass spectrometry. Cyclooxygenase-derived Prostaglandins were observed in the first six hours of stimulation. Interestingly, a switch towards 15-lipoxygenase products, such as the pro-resolving lipid precursors 15-HEPE and 17-HDHA was observed after 24 h. The RNA and protein expression of cyclooxygenase and 15-lipoxygenase were in line with this trend. Treatment with 17-HDHA increased IL-10 production of monocyte-derived macrophages and decreased LTB4 production by neutrophils, indicating the anti-inflammatory property of this lipid.These data reveal that monocyte-derived macrophages contribute to the resolution of inflammation in time by the production of pro-resolving lipids after an initial inflammatory stimulus.

LFA‐9, a Selective Inhibitor of Microsomal Prostaglandin Synthase‐1 and 5‐Lipoxygenase: Prevention of Inflammatory and Oncologic Diseases

Cyclooxygenase‐1 (COX‐1), COX‐2 and 5‐lipoxygenase (5‐LOX) as targets for inflammatory diseases and many agents were developed and extensively used in the clinic. Chronic use of drugs that target COX‐1/COX‐2 are associated with unwanted side effects including GI toxicity and cardiovascular risk. Hence, the main aim of this work is to develop mechanistic based safer agents that would target microsomal prostaglandin synthase‐1 (mPGES‐1) (a downstream enzyme of COX‐2) and 5‐LOX, while sparing COX‐1&amp;‐2 activity. LFA‐9 was designed using structure‐based drug discovery and synthesized from licofelone by adding 2‐aminoethanoic acid (Glycine) at its carboxylic end that led to targeting mPGES‐1 and 5‐LOX but not COX‐1&amp;2. In silico, in vitro and ex vivo studies on LFA‐9 were performed using human/mouse/rat mPGES‐1 and 5‐LOX to facilitate pre‐clinical experimentation and clinical translation. In the molecular docking studies, LFA‐9 showed strong binding affinity against 5‐LOX (binding energies range between −199.85 &amp; −215.3 Kcal/mol) and mPGES‐1 (binding energies range between −238.27 &amp; −257.76 Kcal/mol), but not with COX‐1&amp;2. In cell free system studies, LFA‐9 substantially inhibited mPGES‐1 (range of IC50 between 0.52 – 1.40 μM) and 5‐LOX (range of IC50 between 0.89 – 2.75 μM) but not COX‐1&amp;2 (IC50&gt;1mM) when assayed using fluorometric and colorimetric methods. Moreover, LFA‐9 substantially inhibited PGE2 (IC50 range between 0.47 – 0.78 μM) and LTB4 (IC50 range between 0.56 – 1.24 μM) production and spared PGI2 (IC50&gt;1 mM) and TXB2 (IC50&gt;1 mM) production in LPS‐stimulated human/mouse/rat macrophages and whole blood as the cell‐based system using ELISA technique. Further, circular dichroism and isothermal calorimetric based studies demonstrated that LFA‐9 strongly binds and induces changes in secondary structure of human mPGES‐1 and 5‐LOX enzymes, thereby inhibits their activities. In addition, LFA‐9 (cLogP = 4.34) obeys Lipinsky’s rule of 5 for druglikeness and shows better water solubility unlike licofelone (cLogP = 6.15). Furthermore, LFA9 prevented tumor stemness of APC mutant colonic epithelial cells from PIRC rats in vitro and inhibited exogenous PGE2 in tumor like microenvironment. Therefore, LFA9 is a novel mPGES‐1 and 5‐LOX inhibitor and safer drug candidate for inflammatory and oncologic diseases.Support or Funding InformationAuthors acknowledged NIH/NCI R01CA 213987 and NIH/NCI HHSN261201500024I for funding support.Chemical structures of Licofelone and LFA9Figure 1

Two- and three-color STORM analysis reveals higher-order assembly of leukotriene synthetic complexes on the nuclear envelope of murine neutrophils

Over the last several years it has become clear that higher order assemblies on membranes, exemplified by signalosomes, are a paradigm for the regulation of many membrane signaling processes. We have recently combined two-color direct stochastic optical reconstruction microscopy (dSTORM) with the (Clus-DoC) algorithm that combines cluster detection and colocalization analysis to observe the organization of 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) into higher order assemblies on the nuclear envelope of mast cells; these assemblies were linked to leukotriene (LT) C4 production. In this study we investigated whether higher order assemblies of 5-LO and FLAP included cytosolic phospholipase A2 (cPLA2) and were linked to LTB4 production in murine neutrophils. Using two- and three-color dSTORM supported by fluorescence lifetime imaging microscopy we identified higher order assemblies containing 40 molecules (median) (IQR: 23, 87) of 5-LO, and 53 molecules (62, 156) of FLAP monomer. 98 (18, 154) molecules of cPLA2 were clustered with 5-LO, and 77 (33, 114) molecules of cPLA2 were associated with FLAP. These assemblies were tightly linked to LTB4 formation. The activation-dependent close associations of cPLA2, FLAP, and 5-LO in higher order assemblies on the nuclear envelope support a model in which arachidonic acid is generated by cPLA2 in apposition to FLAP, facilitating its transfer to 5-LO to initiate LT synthesis.