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Natural Product Anacardic Acid from Cashew Nut Shells Stimulates Neutrophil Extracellular Trap Production and Bactericidal Activity*

Open AccessPublished:May 13, 2016DOI:https://doi.org/10.1074/jbc.M115.695866
      Emerging antibiotic resistance among pathogenic bacteria is an issue of great clinical importance, and new approaches to therapy are urgently needed. Anacardic acid, the primary active component of cashew nut shell extract, is a natural product used in the treatment of a variety of medical conditions, including infectious abscesses. Here, we investigate the effects of this natural product on the function of human neutrophils. We find that anacardic acid stimulates the production of reactive oxygen species and neutrophil extracellular traps, two mechanisms utilized by neutrophils to kill invading bacteria. Molecular modeling and pharmacological inhibitor studies suggest anacardic acid stimulation of neutrophils occurs in a PI3K-dependent manner through activation of surface-expressed G protein-coupled sphingosine-1-phosphate receptors. Neutrophil extracellular traps produced in response to anacardic acid are bactericidal and complement select direct antimicrobial activities of the compound.

      Introduction

      The rapid increase in multidrug-resistant bacterial strains is a problem of great concern to the medical and public health communities. Currently, the emergence of antibiotic resistance outpaces the development of antibiotic compounds (
      World Health Organization
      Antimicrobial Resistance: Global Report on Surveillance.
      ), stimulating interest in novel approaches to treat difficult infections. Therapies that enhance the bactericidal activity of host immune cells (e.g. neutrophils) represent one such alternative.
      Natural products have garnered substantial interest as lead points for identification of new pharmaceutical agents (
      • Newman D.J.
      • Cragg G.M.
      • Snader K.M.
      Natural products as sources of new drugs over the period 1981–2002.
      ). Leaves or nut shell extracts from Anacardium occidentale, commonly known as the cashew tree, have long been used to treat inflammation and other conditions, including asthma, ulcers, and cancer (
      • Hemshekhar M.
      • Sebastin Santhosh M.
      • Kemparaju K.
      • Girish K.S.
      Emerging roles of anacardic acid and its derivatives: a pharmacological overview.
      ). Although the efficacy of these compounds for treating such disorders has not been established in controlled trials, the major component of cashew nut shell extract, anacardic acid, has been shown to exert a variety of effects on both prokaryotic and eukaryotic cells (
      • Mamidyala S.K.
      • Ramu S.
      • Huang J.X.
      • Robertson A.A.
      • Cooper M.A.
      Efficient synthesis of anacardic acid analogues and their antibacterial activities.
      ,
      • Seong Y.
      • Shin P.-G.
      • Yoon J.-S.
      • Yadunandam A.K.
      • Kim G.-D.
      Induction of the endoplasmic reticulum stress and autophagy in human lung carcinoma A549 cells by anacardic acid.
      ).
      Anacardic acid is a blanket term applied to a family of closely related compounds consisting of salicylic acid with a 15-carbon alkyl chain, which exist either in a fully saturated form or as a monoene, diene, or triene. Anacardic acid has been shown to exhibit direct antimicrobial activity against a number of bacterial species, including Propionibacterium acnes, Staphylococcus aureus, and Helicobacter pylori (
      • Mamidyala S.K.
      • Ramu S.
      • Huang J.X.
      • Robertson A.A.
      • Cooper M.A.
      Efficient synthesis of anacardic acid analogues and their antibacterial activities.
      ,
      • Kubo J.
      • Lee J.R.
      • Kubo I.
      Anti-Helicobacter pylori agents from the cashew apple.
      ,
      • Sharma R.
      • Kishore N.
      • Hussein A.
      • Lall N.
      Antibacterial and anti-inflammatory effects of Syzygium jambos L. (Alston) and isolated compounds on acne vulgaris.
      ). However, it is perhaps best known as an inhibitor of eukaryotic histone acetyltransferase (
      • Balasubramanyam K.
      • Swaminathan V.
      • Ranganathan A.
      • Kundu T.K.
      Small molecule modulators of histone acetyltransferase p300.
      ) that has been shown to inhibit NFκB activities (
      • Sung B.
      • Pandey M.K.
      • Ahn K.S.
      • Yi T.
      • Chaturvedi M.M.
      • Liu M.
      • Aggarwal B.B.
      Anacardic acid (6-nonadecyl salicylic acid), an inhibitor of histone acetyltransferase, suppresses expression of nuclear factor-κB–regulated gene products involved in cell survival, proliferation, invasion, and inflammation through inhibition of the inhibitory subunit of nuclear factor-κBα kinase, leading to potentiation of apoptosis.
      ) and, more recently, matrix metalloproteinase activity (
      • Omanakuttan A.
      • Nambiar J.
      • Harris R.M.
      • Bose C.
      • Pandurangan N.
      • Varghese R.K.
      • Kumar G.B.
      • Tainer J.A.
      • Banerji A.
      • Perry J.J.
      • Nair B.G.
      Anacardic acid inhibits the catalytic activity of matrix metalloproteinase-2 and matrix metalloproteinase-9.
      ). Through these mechanisms, anacardic acid may induce autophagy (
      • Pietrocola F.
      • Lachkar S.
      • Enot D.P.
      • Niso-Santano M.
      • Bravo-San Pedro J.M.
      • Sica V.
      • Izzo V.
      • Maiuri M.C.
      • Madeo F.
      • Mariño G.
      • Kroemer G.
      Spermidine induces autophagy by inhibiting the acetyltransferase EP300.
      ) and enhance apoptosis (
      • Harsha Raj M.
      • Yashaswini B.
      • Rössler J.
      • Salimath B.P.
      Combinatorial treatment with anacardic acid followed by TRAIL augments induction of apoptosis in TRAIL resistant cancer cells by the regulation of p53, MAPK and NFκβ pathways.
      ) of mammalian cells. It has been proposed that the potentiation of apoptosis by anacardic acid results from inhibition of genes involved in cell survival and proliferation (
      • Sung B.
      • Pandey M.K.
      • Ahn K.S.
      • Yi T.
      • Chaturvedi M.M.
      • Liu M.
      • Aggarwal B.B.
      Anacardic acid (6-nonadecyl salicylic acid), an inhibitor of histone acetyltransferase, suppresses expression of nuclear factor-κB–regulated gene products involved in cell survival, proliferation, invasion, and inflammation through inhibition of the inhibitory subunit of nuclear factor-κBα kinase, leading to potentiation of apoptosis.
      ). Although anacardic acid has antioxidant properties in vitro (
      • Trevisan M.T.
      • Pfundstein B.
      • Haubner R.
      • Würtele G.
      • Spiegelhalder B.
      • Bartsch H.
      • Owen R.W.
      Characterization of alkyl phenols in cashew (Anacardium occidentale) products and assay of their antioxidant capacity.
      ,
      • Melo Cavalcante A.A.
      • Rubensam G.
      • Picada J.N.
      • Gomes da Silva E.
      • Fonseca Moreira J.C.
      • Henriques J.A.
      Mutagenicity, antioxidant potential, and antimutagenic activity against hydrogen peroxide of cashew (Anacardium occidentale) apple juice and cajuina.
      ), it may also stimulate cellular superoxide production by inhibiting the SUMOylation of NADPH oxidase (
      • Pandey D.
      • Chen F.
      • Patel A.
      • Wang C.-Y.
      • Dimitropoulou C.
      • Patel V.S.
      • Rudic R.D.
      • Stepp D.W.
      • Fulton D.J.
      SUMO1 Negatively regulates reactive oxygen species production from NADPH oxidases.
      ). Given the importance of these pathways in the regulation of neutrophil function and innate immune activities, we investigated the actions of anacardic acid on human neutrophils, with an emphasis on the formation of neutrophil extracellular traps (NETs),
      The abbreviations used are: NET
      neutrophil extracellular trap
      ROS
      reactive oxygen species
      S1P
      sphingosine-1-phosphate
      S1PR
      S1P receptor
      MRSA
      methicillin-resistant S. aureus
      DPI
      diphenyleneiodonium
      PMA
      phorbol 12-myristate 13-acetate
      CNSE
      cashew nut shell-extracted
      HBSS
      Hanks' balanced salt solution.
      reactive oxygen species (ROS)-dependent, DNA-based structures coated with antimicrobial compounds that entrap and kill pathogens (
      • Brinkmann V.
      • Reichard U.
      • Goosmann C.
      • Fauler B.
      • Uhlemann Y.
      • Weiss D.S.
      • Weinrauch Y.
      • Zychlinsky A.
      Neutrophil extracellular traps kill bacteria.
      ).

      Discussion

      Anacardic acid has long been used as a therapeutic agent in herbal medicine; although the molecular mechanisms underlying its activity are still poorly understood, its ability to modulate superoxide formation led us to investigate its effects on innate immune function and bacterial clearance. In addition to exhibiting direct antimicrobial effects against a series of important human Gram-positive pathogens, we found that anacardic acid enhanced neutrophil antibacterial function by promoting extracellular trap production. NETs are DNA-based structures that have been shown to play key role in pathogen clearance by neutrophils (
      • von Köckritz-Blickwede M.
      • Nizet V.
      Innate immunity turned inside-out: antimicrobial defense by phagocyte extracellular traps.
      ,
      • Wartha F.
      • Henriques-Normark B.
      ETosis: A novel cell death pathway.
      ). We show that anacardic acid-induced NETs are capable of ensnaring and killing bacteria, which likely contributes to enhanced bactericidal activity of anacardic acid-treated neutrophils in vitro.
      Using computation approaches and known crystal structures, we identified the G protein-coupled sphingosine-1-phosphate receptors as likely targets of anacardic acid. This complements our previous finding that sphigosine-1-phosphate-related compounds, such as ceramide, can induce NET production in human neutrophils (
      • Corriden R.
      • Hollands A.
      • Olson J.
      • Derieux J.
      • Lopez J.
      • Chang J.T.
      • Gonzalez D.J.
      • Nizet V.
      Tamoxifen augments the innate immune function of neutrophils through modulation of intracellular ceramide.
      ). We found that the S1P4-selective antagonist CYM 50358 (
      • Guerrero M.
      • Urbano M.
      • Velaparthi S.
      • Zhao J.
      • Schaeffer M.T.
      • Brown S.
      • Rosen H.
      • Roberts E.
      Discovery, design and synthesis of the first reported potent and selective sphingosine-1-phosphate 4 (S1P4) receptor antagonists.
      ), but not an antagonist of the S1P1/S1P3 receptors, was a potent inhibitor of anacardic acid-induced ROS production. The S1P4 receptor is predominantly expressed by immune cells, including neutrophils (
      • Kihara Y.
      • Mizuno H.
      • Chun J.
      Lysophospholipid receptors in drug discovery.
      ); intriguingly, previous work has shown that mice deficient in S1P lyase, an enzyme that catalyzes degradation of S1P, exhibit features of an inflammatory response that is ameliorated by deletion of the S1P4 receptor (
      • Allende M.L.
      • Bektas M.
      • Lee B.G.
      • Bonifacino E.
      • Kang J.
      • Tuymetova G.
      • Chen W.
      • Saba J.D.
      • Proia R.L.
      Sphingosine-1-phosphate lyase deficiency produces a pro-inflammatory response while impairing neutrophil trafficking.
      ).
      Consistent with investigations using other pharmacological agents to stimulate NET production (
      • Wartha F.
      • Henriques-Normark B.
      ETosis: A novel cell death pathway.
      ,
      • Rada B.
      • Jendrysik M.A.
      • Pang L.
      • Hayes C.P.
      • Yoo D.-G.
      • Park J.J.
      • Moskowitz S.M.
      • Malech H.L.
      • Leto T.L.
      Pyocyanin-enhanced neutrophil extracellular trap formation requires the NADPH oxidase.
      ), we found anacardic acid-induced NET production to be both ROS- and PI3K-dependent; this is in keeping with reports describing coupling of the S1P4 receptor to Gα12/13 (
      • Gräler M.H.
      • Grosse R.
      • Kusch A.
      • Kremmer E.
      • Gudermann T.
      • Lipp M.
      The sphingosine 1-phosphate receptor S1P4 regulates cell shape and motility via coupling to Gi and G12/13.
      ), which has been shown to trigger cell death pathways via activation of PI3K (
      • Lappano R.
      • Maggiolini M.
      G protein-coupled receptors: novel targets for drug discovery in cancer.
      ). Given the diverse array of GPCRs acting through Gα12/13, our findings could help guide future efforts to identify novel and potentially more selective/potent, immune-boosting compounds.
      Natural products, such as anacardic acid, represent an important resource for drug discovery efforts and can yield not only potential therapeutic agents but also scaffolds on which to develop more potent and efficacious drugs. Our findings reveal a role of the S1P4 receptor as a mediator of NET production and identify anacardic acid as a potential lead compound to boost neutrophil function in innate immune defense.

      Author Contributions

      G. B. K., B. G. N., and J. J. P. P. provided critical reagents and inspired the initial analysis of immune boosting properties of anacardic acid. A. H., R. C., and V. N. designed the experiments. A. H., R. C., G. G., S. D., J. O., S. R. A., and A. L. performed the experiments. M. T. K. and A. C. N. designed and conducted the FRET-based PKC activity assay and associated signaling studies. S. F. and J. J. P. P. performed the in silico docking studies of receptor-ligand interactions. A. H., R. C., and V. N. wrote the paper, and all authors reviewed the manuscript and provided critical input.

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