- Open Access
Differential gene expression mediated by 15-hydroxyeicosatetraenoic acid in LPS-stimulated RAW 264.7 cells
© Schrimpe and Wright; licensee BioMed Central Ltd. 2009
Received: 26 March 2009
Accepted: 11 August 2009
Published: 11 August 2009
Given the immuno-modulatory activity of native haemozoin (Hz), the effects of constitutive Hz components on immune response are of interest. Recently, gene expression changes mediated by HNE and the synthetic analogue of Hz, beta-haematin (BH), were identified and implicated a significant role for lipid peroxidation products in Hz's activity. The study presented herein examines gene expression changes in response to 15(S)-hydroxyeicosatetraenoic acid (HETE) in a model macrophage cell line.
LPS-stimulated RAW 264.7 macrophage-like cells were treated with 40 μM 15(S)-HETE for 24 h, and microarray analysis was used to identify global gene expression alterations. Fold changes were calculated relative to LPS-stimulated cells and those genes altered at least 1.8-fold (p value ≤ 0.025) were considered to be differentially expressed. Expression levels of a subset of genes were assessed by qRT-PCR and used to confirm the microarray results.
Network analysis revealed that altered genes were primarily associated with "lipid metabolism" and "small molecule biochemistry". While several genes associated with PPAR-gamma receptor-mediated signaling were differentially expressed, a number of genes indicated the activation of secondary signaling cascades. Genes related to cytoadherence (cell-cell and cell-matrix), leukocyte extravasation, and inflammatory response were also differentially regulated by treatment, supporting a potential role for 15(S)-HETE in malaria pathogenesis.
These results add insight and detail to 15-HETE's effects on gene expression in macrophage-like cells. Data indicate that while 15-HETE exerts biological activity and may participate in Hz-mediated immuno-modulation, the gene expression changes are modest relative to those altered by the lipid peroxidation product HNE.
Although haem is a vital cofactor for a diverse set of proteins involved in respiration, oxygen transport, and drug detoxification, the accumulation of free haem has deleterious effects. Haem is capable of binding to lipid bilayers, catalyzing lipid peroxidation, inhibiting enzymatic activity, and lysing cells and parasites [1, 2]. Many organisms utilize the haem oxygenase pathway to degrade free haem. Blood-feeding Plasmodium parasites, the source of malaria infection, lack such a pathway. Consequently, haem released during haemoglobin catabolism is sequestered as the insoluble crystalline "malaria pigment" (i.e., haemozoin [Hz]). As most of the haem is occluded within the crystal, the parasite is protected.
Hz is composed of five-coordinate Fe (III) protoporphyrin IX dimers covalently bound by reciprocal iron-carboxylate bonds . The remaining propionate side chains of adjacent dimers form hydrogen bonds, resulting in an extended dimeric network producing the Hz crystal. In its native state, Hz is coated by an array of host- and parasite-derived lipids, proteins, and nucleic acids . Analysis of the lipid component identified peroxidation products including a racemic mixture of 5-, 8-, 9-, 11-, 12-, and 15-hydroxyeicosatetraenoic acids (HETEs) and 9- and 13- hydroxyoctadecadienoic acids (HODEs) . Elevated levels of 4-hydroxynonenal (HNE) were also detected in haemozoin-laden monocytes  at the highest reported concentration of any biological system to date .
Rupture of parasitized red blood cells (RBCs) releases cellular debris, including residual bodies containing Hz, into the host's vasculature and triggers an innate immune response. The typical response of phagocytic cells to such foreign material includes oxidative burst and rephagocytosis, however, phagocytosis of Hz impairs these innate functions [8–10]. It has been suggested that Hz's immunological activity may not stem from the haem moiety but from nonspecific toxins , such as lipid peroxidation products, present on its surface and introduced into the cell during phagocytosis
The cellular response to several lipid peroxidation species associated with Hz is well documented and indicates an involvement in malaria pathophysiology. Recently, two components of native Hz were targeted as potential players involved in macrophage dysfunction . Microarray analysis of the response to HNE and Hz's biologically naïve synthetic analogue, β-haematin (BH), indicated a potential role for HNE in malaria pathogenesis. It seemed probable, given HNE-mediated gene expression changes, that other biologically active lipid peroxidation products generated by Hz, including 15-HETE, may be active in the disease's pathogenesis.
Macrophage-like cells treated with 15-HETE exhibited impaired PMA-activated NADPH oxidase and LPS-stimulated inducible nitric oxide synthase (iNOS) activities, mimicking Hz-mediated monocyte immunomodulation . 15-HETE was also reported to enhance vascular permeability/oedema  and RBC adherence to endothelia , two hallmarks of malarial infection. The present study examined steady-state gene expression changes induced by 15-HETE in activated RAW 264.7 model macrophage cells in the context of a nonspecific malaria toxin that may be involved in disease pathophysiology.
Murine macrophage-like RAW 264.7 cells (American Type Culture Collection TIB-71, Monassas, VA) were cultured under standard incubation conditions (37°C, 5% CO2) and grown in RPMI supplemented with 10% FBS (Atlanta Biologicals, Atlanta, GA) and 1 μg/mL P/S (Cellgro MediaTech, Herndon, VA). Cells were plated at a density of 4 × 106 cells/well in six well plates and incubated for 24 h prior to treatment.
Cell treatment and RNA isolation
Cells were washed once with Dulbecco's PBS (DPBS) and treated with 40 μM 15(S)-HETE. Immediately following treatment, LPS was added to all wells at a final concentration of 1 μg/mL. After 24 h, cells were washed three times with DPBS and scraped from the wells. Three biological replicates (composed of six pooled wells each) per sample were prepared. Total RNA was isolated using the Versagene RNA purification and DNase treatment kits, following the manufacturer's recommendations.
Microarray analysis was performed by the Vanderbilt Microarray Shared Resource. Three biological replicates of each treatment were analyzed for quality (Agilent 2100 Bioanalyzer, Agilent Technologies, Palo Alto, CA). One microgram of total RNA (30 ng mRNA) was used to generate first strand cDNA using the NanoAmp RT-IVT labeling kit according to the manufacturer's protocol. Following first strand synthesis, second strand synthesis was completed. The resulting cDNA was then purified using an ABI kit-provided column and the entire reaction was used in an IVT reaction to generate DIG-labeled cRNA. The cRNA was then purified using a kit-provided column and assessed for quality on an Agilent Bioanalyzer. All reactions meeting ABI criteria in terms of quantity and size of target produced were fragmented and then hybridized to an ABI mouse genome survey microarray for 16 h with agitation at 55°C per the manufacturer's protocol. Following the addition of the chemiluminescence reaction substrate, each array was immediately imaged on the 1700 Chemiluminescent Analyzer, and a primary analysis was completed by the AB1700 Expression Array System Software (v 1.1.1). Expression values were quantile normalized and filtered based on S/N (> 3) and flag value (< 5000). GeneSpring GX 7.3.1 software (Agilent Silicon Genetics, Redwood City, CA) was used to determine statistically significant differentially expressed genes. T-tests were performed on probes altered by ≥ 1.8-fold in 2 of 3 samples (0.025 p value cut-off, Benjamini-Hochberg multiple testing correction, parametric test, variances not assumed equal) in treated stimulated cells (experimental) relative to stimulated cells (control). Genes were classified according to genes ontology (GO) terms using GeneSpring. In accordance with MIAME procedure, microarray data have been submitted to the NCBI Gene Expression Omnibus and can be found under series number GSE15070. Nomenclature for genes and proteins is as described by the Mouse Genome Informatics (MGI) database guidelines.
Ingenuity pathway analysis
Ingenuity Pathways Analysis (IPA) was used for gene expression analysis (Ingenuity Systems®). A data set containing gene identifiers and corresponding expression values was uploaded into the application, and each identifier was mapped to its corresponding gene object in the Ingenuity knowledge base (IKB). A functional analysis was performed to determine biological functions that were most significant to the genes in the data set. A network analysis was also performed whereby focus genes were overlaid onto a global molecular network developed from information contained in the IKB. Networks of focus genes were then algorithmically generated based on their connectivity. A functional analysis of each network identified the biological functions that were most significant to the genes in the network, and canonical pathway analysis identified the pathways from the IPA library of canonical pathways that were most significant to the data set. Fischer's exact test was used to calculate a p value determining the probability that that each biological function assigned to a network or data set, or the association between the genes in the data set and the canonical pathway, are explained by chance alone.
Real-time reverse transcription polymerase chain reaction
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to validate the expression levels of genes identified as differentially expressed by microarray analysis. Quadruplicate measurements for n = 3 independent biological replicates per sample were performed. cDNA was reverse-transcribed from 0.5 μg of total RNA using random hexamer primers and Superscript II Reverse Transcriptase (Invitrogen). Reactions were purified using Qiagen's PCR Purification Kit following the manufacturer's protocol. Following RT, all assays were performed with Applied Biosystems TaqMan FAM labeled 20× probes: Arf3 (Taqman assay Mm00500194_m1), Cldn11 (Mm00500915_m1), Cxcl11 (Mm00444662_m1), Mapk14 (Mm00442497_m1), Prdx1 (Mm01621996_s1), Sdc1 (Mm00448918_m1), and Egr1 (Mm00656724_m1). Ywhaz was chosen as the endogenous control based on results obtained from an Applied Biosystems mouse endogenous control array. cDNA amplification was performed using TaqMan 2× Universal PCR Master Mix (Applied Biosystems), and standard Taqman cycling conditions were used as specified by the manufacturer. Cycling and data collection were performed using the Applied Biosystems 7900 HT instrument, and analysis was performed using SDS software to calculate Ct values for each detector. Ct values were processed based on the comparative Ct method where the relative transcript level of each target gene was calculated according to the equation 2-ΔCt, where ΔCt is defined as Ct target gene – Ct Ywhaz.
Functional analysis of gene expression changes induced by 15(S)-HETE
Ingenuity Pathway Analysis (IPA) software was used to examine biological relationships associated with 15-HETE-mediated expression changes. Identifiers and relative levels of altered genes comprising the data set were imported and mapped by IPA for comparison to molecules within the Ingenuity knowledge base (IKB). Two types of IPA analyses were performed. First, a network analysis was employed to reveal direct and indirect relationships that exist between specific genes in the data set. This analysis resulted in the generation of a network map which illustrates direct and indirect connections between focus genes. Second, a functional analysis was performed to identify the biological processes that are most relevant to the entire set of differentially expressed genes. This analysis resulted in a list of significant biological functions associated with the data set as a whole. Functional analyses were also used to find biological processes associated with individual networks. Focus genes, imported genes that are eligible for generating interaction networks based on incorporation in IKB, were used to identify relationships based on known interactions in the literature. Each network is associated with a score indicating the likelihood that the focus genes occur in the network by random chance. Networks scoring 10 or higher (score is defined as -log (p value)) are considered significant.
Figure 2b shows the second most significant interaction network identified by IPA network analysis. Eighteen focus genes were incorporated into the network with a score of 29. Functional analysis of the network revealed that the genes were involved in "molecular transport" (p = 9.42 × 10-7) and "cellular movement" (p = 9.77 × 10-6). This network is enriched with focus genes encoding molecules associated with the plasma membrane such as Pkd2, Cd300a, Cldn11, Gypc, Klra4, peptidase Adam9 and transporters Atp1a2, Slc16a1, and Slc16a3. Consistent with these genes, the network predicted interactions with several other plasma membrane molecules (Tjp2, Bsg, Cdh1, Tspan3, Tspan4, Cd247, Itgb1, and Atp1b2) that were not present in the data file.
Molecular and cellular functions controlled by 15(S)-HETE
Functional Analysis of 15-S-HETE Data Set a
1.28 × 10-4
1.93 × 10-5
2.53 × 10-4
cellular assembly and organization
5.30 × 10-4
4.52 × 10-4
3.42 × 10-5
cellular growth and proliferation
3.81 × 10-4
3.81 × 10-4
2.22 × 10-4
1.25 × 10-3
3.81 × 10-4
RNA damage and repair
3.79 × 10-4
RNA post-transcriptional modification
3.79 × 10-4
small molecule biochemistry
3.81 × 10-4
Both IPA network and functional analyses identified a large group of "lipid metabolism" and "carbohydrate metabolism" expression changes. Given that Il1b acts upstream of Cyp3a4, Ugdh, Gnas, Gm2a, Psen1, and Il15, stimulated Il1b expression may be indirectly involved in the up-regulation of each of these genes in this study. Expression of several "small molecule biochemistry" transcriptional regulators (Bclaf1, Med1, Rnf4, Noc2l, and Zfp36l1) was also identified.
Differential gene expression in the context of malaria pathogenesis
Select Gene Expression Changes Mediated by 15-HETE a
Electron Transport 
Cytochrome P450, family 3, subfamily a, polypeptide 11
Regulation of Transcription, DNA-Dependent 
BCL2-associated transcription factor 1
Cyclin-dependent kinase 9 (CDC2-related kinase)
Cellular repressor of E1A-stimulated genes 1
Early growth response 1
F-box and leucine-rich repeat protein 11
Friend leukemia integration 1
Flightless I homologue (Drosophila)
H2.0-like homeo box 1 (Drosophila)
MAX dimerization protein 1
MYST histone acetyltransferase 2
POU domain, class 2, transcription factor 2
Peroxisome proliferator activated receptor binding protein
Paraspeckle protein 1
RAB11a, member RAS oncogene family
Ring finger protein 4
TSC22 domain family 3
Zinc finger protein 482
Protein Folding 
Caseinolytic peptidase X (E. coli)
Heat shock protein 4
Ubiquitin Cycle 
F-box only protein 3
Ubiquitin-conjugating enzyme E2L 6
Intracellular Protein Transport 
Adaptor-related protein complex 1, sigma 2 subunit
ADP-ribosylation factor 3
Response to Stress 
Mitogen activated protein kinase 14
Bone marrow stromal cell antigen 1
Killer cell lectin-like receptor, subfamily A, member 18
TAP binding protein
ATP binding cassette, sub-family B (MDR/TAP), member 1A
Carbonic anhydrase 2
Caspase recruitment domain family, member 12
Cytochrome P450, family 3, subfamily a, polypeptide 11
Complement component (3b/4b) receptor 1-like
Cytochrome P450, family 3, subfamily a, polypeptide 11
Histocompatibility 2, Q region locus 8
Heterogeneous nuclear ribonucleoprotein A3
Mannose receptor, C type 1
Polymerase (DNA-directed), ε 4 (p12 subunit)
Protein phospatase 3, regulatory subunit B, α isoform (calcineurin B, type I)
Protein C receptor, endothelial
Ribonucleotide reductase M1
Serine (or cysteine) proteinase inhibitor, clade B, member 2
Leukocyte Extravasation and Signalling
ρ GTPase activating protein 12
V-crk sarcoma virus CT10 oncogene homologue (avian)-like
PTK2 protein tyrosine kinase 2 β
Chemokine (C-X-C motif) ligand 11
DEAD (Asp-Glu-Ala-Asp) box polypeptide 58
Fc receptor, IgG, α chain transporter
Guanylate nucleotide binding protein 1
Guanylate nucleotide binding protein 3
Interferon-induced protein with tetratricopeptide repeats 3
Interleukin 1 α
Interleukin 1 β
Cell Cycle 
Cyclin-dependent kinase inhibitor 1B (P27)
Peripheral myelin protein
Ras homologue gene family, member B
Cell Adhesion 
Scavenger receptor class B, member 2
Tumor necrosis factor alpha induced protein 6
Signal Transduction 
Chemokine (C-C motif) receptor 4
GNAS (guanine nucleotide binding protein, alpha stimulating) complex locus
Membrane-spanning 4-domains, subfamily A, member 4C
Olfactory receptor 472
Protein-kinase, interferon-inducible double stranded RNA dependent inhibitor, repressor of (P58 repressor)
Prostaglandin E receptor 2 (subtype EP2)
Ras and Rab interactor 1
3-monooxygenase/tryptophan 5-monooxygenase activation protein, gamma polypeptide
G-Protein Coupled Receptor Protein Signaling Pathway 
Olfactory receptor 1303
Olfactory receptor 316
Olfactory receptor 435
Solute carrier family 19 (thiamine transporter), member 2
Cell-Cell Signaling 
Wingless-related MMTV integration site 6
EGF-like-domain, multiple 4
Low density lipoprotein receptor-related protein 6
Placental growth factor
ATPase, Na+/K+ transporting, α 2 polypeptide
ATPase, Ca2+ sequestering
Enoyl Coenzyme A hydratase domain containing 3
Hydroxysteroid (17-beta) dehydrogenase 4
Matrix metalloproteinase 9
2'-5' oligoadenylate synthetase 3
Carbohydrate Transport 
Solute carrier family 35, member A4
Protein Transport 
Exocyst complex component 2
Nucleoporin like 2
RAB20, member RAS oncogene family
RAP2B, member of RAS oncogene family
RAS-homologue enriched in brain
Zinc finger, FYVE domain containing 20
Protein Ubiquitination 
Tripartite motif protein 12
Tripartite motif protein 34
N-myc downstream regulated gene 2
Phosphoinositide-3-kinase adaptor protein 1
Validation of microarray results
Global responses to malaria infection have been examined at the molecular level in the blood of human victims [16, 21], malaria positive tissue , and both murine [17, 18, 20] and monkey  malaria models using microarray technology. Perturbations of gene expression associated with erythropoiesis, glycolysis, metabolism, B-cell activation, and inflammation were frequently identified in these analyses; however, the specific agents responsible for mediating expression changes remain unknown. Accumulating evidence supports the hypothesis that many of the adverse effects of malaria are not caused directly by the parasite, but by endogenous toxins generated during interactions with parasite-derived species such as Hz .
The immuno-modulatory response to native Hz has been recapitulated using individual components of Hz (i.e., membrane lipids from erythrocyte ghosts incubated with BH) in a model system . Macrophage-like cells treated with the reaction supernatant exhibited a dose-dependent impairment of PMA-activated NADPH oxidase and LPS-stimulated iNOS activities. Neither BH- nor ghost-supernatant alone altered NADPH or iNOS activity, indicating that lipid peroxidation products generated during reactions between BH and ghost membranes were responsible for the inhibitory effects. Several laboratories have shown that biologically relevant levels of the individual lipid peroxidation products HNE and 15-HETE were capable of mimicking the dysfunctional response to Hz phagocytosis, suggesting the basis of Hz activity [13, 25, 26].
Considering these results, global responses to individual Hz constituents are of particular interest. The ability of specific and nonspecific malaria toxins to stimulate changes in gene expression has recently been examined in macrophage-like cells . Microarray analyses of two individual Hz components (i.e., BH and HNE) indicated that while BH primarily elicited a phagocytic response, HNE significantly perturbed a myriad of biological processes. These results substantiated further exploration of a potential role for the Hz-associated lipid peroxidation product 15-HETE.
In the current study, activated RAW 264.7 macrophage-like cells were treated with 15-HETE, and mRNA levels were assessed at 24 h to mimic a steady-state response that would be relevant to an established malaria infection. IPA software was utilized to perform complementary network (Figure 2) and functional (Table 1) analyses for identification of biological relationships within the data. Figure 3 shows that the number of expression changes mediated by 15-HETE was modest relative to the previously reported global response to HNE . Unlike the mode of action behind HNE's biological activity (forming adducts to cellular nucleophiles and subsequently modulating intracellular signaling), 15-HETE serves as a ligand for the nuclear PPARγ receptor . As expected, downstream PPARγ signaling transcripts (e.g., Adfp, Ca2, Cyp3a4, M6pr, M6prbp1, Med1, Med7, and Sdc1) were elevated in response to 15-HETE.
A balance between removal of Plasmodium from circulation and sequestration inside host cells is crucial for parasite survival during infection. Sequestration is mediated by cytoadherence, specifically, the adherence of parasitized RBCs (PRBCs) and leukocytes to capillary and post-capillary venular endothelial cells (EC). This cytoadherence reduces blood flow and causes metabolic dysfunction  and is thought to be a major factor associated with cerebral malaria (CM). The mechanism(s) used for adhesion and migration involve the expression of constitutive ligands (i.e., adhesion molecules) and receptors on PRBCs or leukocytes and EC. Cell-cell and cell-matrix interactions are also mediated by the secretion of microbial products or cytokines, which enhance the expression of inducible adhesion molecules.
Investigation of potential arachidonic acid metabolite involvement in cytoadherence identified 15-HETE as an agent capable of stimulating basal adhesion of erythrocytes  and monocytes to EC [29, 30]. In this study, 15-HETE induced the expression of several transcripts involved in integrin signaling (e.g., Crkl, Rap2b, Arf3). The expression of genes encoding Pkd2 and Sdc1, which are involved in cell-cell and cell-matrix interactions, and Ptpn14, which has alleged involvement in cell adhesion, were also induced by 15-HETE.
Leukocyte extravasation and chemotaxis
The inflammatory response to malaria, both acute and chronic, follows a predictable sequence of events. Initial vascular changes precede increases in permeability, which ultimately causes oedema. Enhanced cytoadherence results in the accumulation, adherence, and migration of leukocytes through vascular endothelium. Molecular mediators are subsequently released and contribute to both the immune response and recruitment/activation of effector cells. Overwhelming evidence demonstrates that the pathophysiology of malaria involves both systemic and local cytokine release. The recruitment of phagocytes around cerebral capillaries has been observed in CM and likely explains increased chemotaxis and chemokinesis . CM is a severe complication of P. falciparum infection that is characterized by cytoadherence in cerebral microvasculature. Accumulation of Hz-loaded monocytes has been observed in brains of CM victims  and may contribute to the disruption of endothelial basement membrane and subsequent extravasation of blood cells . Importantly, blood brain barrier (BBB) destruction and enhanced vascular permeability/oedema are major factors associated with CM [33, 34].
A potential contribution of 15-HETE toward increased vascular permeability has been examined in the lung. Administration of this hydroxylated fatty acid was shown to increase respiratory oedema fluid production , suggesting a role as an inflammatory mediator. The current analysis identified the "Leukocyte Extravasation Signaling" pathway as being significantly (p = 0.015) affected by 15-HETE. Specifically, the steady-state expression of Arhgap12, Cldn11, Crkl, Mapk14, and Ptk2b was up-regulated. Although 15-HETE is generally considered to have anti-inflammatory properties, activation of a large group of genes encoding inflammatory response molecules was observed (Table 2).
15-HETE and MMP9 Regulation
15-HETE was recently shown to enhance IL1β expression and MMP9 activity in human monocytes . The current study identified a different response to 15-HETE. While Il1b mRNA was elevated in 15-HETE treated LPS-stimulated RAW 264.7 cells, Mmp9 mRNA was down-regulated (-4.8-fold by qRT-PCR). Mmp9 expression can be regulated through a variety of signaling cascades including NF-κB, p38 MAPK, and ERK1/2 pathways . It was proposed that enhanced regulation of IL1B and MMP9 by 15-HETE in human monocytes may be associated with NF-κB signaling  based on reports demonstrating NF-κB-mediated MMP9 expression in LPS-stimulated RAW 264.7 cells . This mechanism seems unlikely given that 15-HETE has been shown to impair NF-κB-mediated expression of iNOS in LPS-stimulated RAW 264.7 cells . Furthermore, PPARγ ligands have been shown to repress NF-κB signal transduction [37, 38] and inhibit MMP9 expression, secretion, and activity in macrophages and vascular smooth muscle cells [39–41], in accord with the results of this study.
The complex innate and adaptive host immune responses to malaria are triggered by the presence of P. falciparum parasites, composite native Hz, Hz-derived lipid peroxidation products, and other cellular debris. A reductionist examination of the cellular response to two individual constituents of native Hz, BH and HNE , implicated a significant role for lipid peroxidation products in macrophage immuno-modulation. Consequently, the global response of macrophage-like cells to 15-HETE was explored. 15-HETE has previously been implicated as having a functional role in a variety of cellular processes such as inflammation, asthma, carcinogenesis, and atherosclerosis.15-HETE can be incorporated into membrane lipids and alter both vascular tone and EC permeability , supporting a potential role in malaria pathogenesis as well. In the current study, the response to 15-HETE was significantly associated with altered expression of "lipid metabolism" and "small molecule biochemistry" genes. Several genes related to "cytoadherence", "leukocyte extravasation", and "inflammatory response" were also differentially regulated by 15-HETE treatment. While these change uphold a potential role for 15-HETE in malaria pathogenesis, the small number of expression changes indicates that 15-HETE does not elicit a major response from macrophage-like cells in this model. These results add insight and detail to 15-HETE's effect on gene expression in macrophage-like cells, but there are limitations to any model system. For example, 15-HETE represents but one structural HETE isomer that is associated with Hz; 5-, 8-, 9-, 11-, and 12-HETE have also been identified [13, 42]. 5- and 15-HETE are reported to be the predominant isomers formed during iron catalyzed or Hz-mediated oxidation of AA [42, 43], yet 12-HETE may exert greater biological activity . Current findings suggest that upon phagocytosis, the sum of all species adsorbed to the surface of Hz likely mediate a synergistic immuno-modulatory response. Future studies will address expression changes in primary human monocytes and macrophages in response to native Hz and Hz-associated lipid peroxidation products.
The Vanderbilt Microarray Shared Resource is supported by the Vanderbilt Ingram Cancer Center (P30 CA68485), the Vanderbilt Digestive Disease Center (P30 DK58404), and the Vanderbilt Vision Center (P30 EY08126). The VMC Flow Cytometry Shared Resource is supported by the Vanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Digestive Disease Research Center (DK058404). We thank M. F. Richards for editorial assistance.
- Schmitt TH, Wilson A, Frezzatti J, Schreier S: Hemin-induced lipid membrane disorder and increased permeability: a molecular model for the mechanism of cell lysis. Arch Biochem Biophys. 1993, 307: 96-103. 10.1006/abbi.1993.1566.View ArticlePubMedGoogle Scholar
- Gluzman IY, Francis SE, Oksman A, Smith CE, Duffin KL, Goldberg DE: Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway. J Clin Invest. 1994, 93: 1602-1608. 10.1172/JCI117140.PubMed CentralView ArticlePubMedGoogle Scholar
- Pagola S, Stephens PW, Bohle DS, Kosar AD, Madsen SK: The Structure of malaria pigment β-haematin. Nature. 2000, 404: 307-310. 10.1038/35005132.View ArticlePubMedGoogle Scholar
- Goldie P, Roth EF, Oppenheim J, Vanderberg JP: Biochemical characterization of Plasmodium falciparum hemozoin. Am J Trop Med Hyg. 1990, 43: 584-596.PubMedGoogle Scholar
- Schwarzer E, Kuhn H, Valente E, Arese P: Malaria-parasitized erythrocytes and hemozoin nonenzymatically generate large amounts of hydroxy fatty acids that inhibit monocyte functions. Blood. 2003, 101: 722-728. 10.1182/blood-2002-03-0979.View ArticlePubMedGoogle Scholar
- Schwarzer E, Muller O, Arese P, Siems WG, Grune T: Increased levels of 4-hydroxynonenal in human monocytes fed with malarial pigment hemozoin. FEBS Lett. 1996, 338: 119-122. 10.1016/0014-5793(96)00523-6.View ArticleGoogle Scholar
- Poli G, Schaur RJ, Siems WG, Leonarduzzi G: 4-Hydroxynonenal: a membrane lipid oxidation product of medicinal interest. Med Res Rev. 2008, 28: 569-631. 10.1002/med.20117.View ArticlePubMedGoogle Scholar
- Schwarzer E, Turrini F, Ulliers D, Giribaldi G, Ginsburg H, Arese P: Impairment of Macrophage Functions after Ingestion of Plasmodium falciparum-infected Erythrocytes or Isolated Malarial Pigment. J Exp Med. 1992, 176: 1033-1041. 10.1084/jem.176.4.1033.View ArticlePubMedGoogle Scholar
- Schwarzer E, Arese P: Phagocytosis of malarial pigment hemozoin inhibits NADPH-oxidase activity in human monocyte-derived macrophages. Biochim Biophys Acta. 1996, 1316: 169-175.View ArticlePubMedGoogle Scholar
- Fiori PL, Rappelli P, Mirkarimi SN, Ginsburg H, Cappuccinelli P, Turrini F: Reduced microbicidal and anti-tumour activities of human monocytes after ingestion of Plasmodium falciparum-infected red blood cells. Parasite Immunol. 1993, 15: 647-655. 10.1111/j.1365-3024.1993.tb00579.x.View ArticlePubMedGoogle Scholar
- Nguyen PH, Day N, Pram TD, Ferguson DJ, White NJ: Intraleucocytic malaria pigment and prognosis in severe malaria. Trans R Soc Trop Med Hyg. 1995, 89: 200-204. 10.1016/0035-9203(95)90496-4.View ArticlePubMedGoogle Scholar
- Schrimpe AC, Wright DW: Comparative analysis of gene expression changes mediated by individual constituents of hemozoin. Chem Res Toxicol. 2009, 22: 433-445. 10.1021/tx8002752.PubMed CentralView ArticlePubMedGoogle Scholar
- Carney CK, Schrimpe AC, Halfpenny K, Harry RS, Miller CM, Broncel M, Sewell SL, Schaff JE, Deol R, Carter MD, Wright DW: The basis of the immunomodulatory activity of malaria pigment (hemozoin). The Journal of Biological Inorganic Chemistry. 2006, 11: 917-929. 10.1007/s00775-006-0147-0.View ArticlePubMedGoogle Scholar
- Spector AA, Gordon JA, Moore SA: Hydroxyeicosatetraenoic acids (HETEs). Prog Lipid Res. 1988, 27: 271-323. 10.1016/0163-7827(88)90009-4.View ArticlePubMedGoogle Scholar
- Setty BNY, Dampier CD, Stuart MJ: Arachidonic acid metabolites are involved in mediating red blood cell adherence to endothelium. J Lab Clin Med. 1995, 125: 608-617.PubMedGoogle Scholar
- Griffiths MJ, Mohammed JS, Popper SJ, Hemingway CA, Kortok MM, Wathen A, Rockett KA, Mott R, Levin M, Newton CR, Marsh K, Relman DA, Kwiatkowski DP: Genomewide Analysis of the Host Response to Malaria in Kenyan Children. The Journal of Infectious Diseases. 2005, 191: 1599-1611. 10.1086/429297.View ArticlePubMedGoogle Scholar
- Sexton AC, Good RT, Hansen DS, D'Ombrain MC, Buckingham L, Simpson K, Schofield L: Transcriptional Profiling Reveals Suppressed Erythropoiesis, Up-Regulated Glycolysis, and Interferon-Associated Responses in Murine Malaria. The Journal of Infectious Diseases. 2004, 189: 1245-1256. 10.1086/382596.View ArticlePubMedGoogle Scholar
- Lovegrove FE, Pena-Castillo L, Mohammad N, Liles C, Hughes TR, Kain KC: Simultaneous host and parasite expression profiling identifies tissue-specific transcriptional programs associated with susceptibility or resistance to experimental cerebral malaria. BMC Genomics. 2006, 7: 295-311. 10.1186/1471-2164-7-295.PubMed CentralView ArticlePubMedGoogle Scholar
- Sherry BA, Alava G, Tracey KJ, Martiney J, Cerami A, Slater AFG: Malaria-specific metabolite hemozoin mediates the release of several potent endogenous pyrogens (TNF, MIP-1α, and MIP-1β) in vitro, and altered thermoregulation in vivo. Journal of Inflammation. 1995, 45: 85-96.PubMedGoogle Scholar
- Schaecher K, Kumar S, Yadava A, Vahey M, Ockenhouse CF: Genome-Wide Expression Profiling in Malaria Infection Revelas Transcriptional Changes Associated with Lethal and Nonlethal Outcomes. Infection and Immunity. 2005, 73: 6091-6100. 10.1128/IAI.73.9.6091-6100.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Ockenhouse CF, Hu W-c, Kester KE, Cummings JF, Stewart A, Heppner DG, Jedlicka AE, Scott AL, Wolfe ND, Vahey M, Burke DS: Common and Divergent Immune Response Signaling Pathways Discovered in Peripheral Blood Mononuclear Cell Gene Expression Patterns in Presymptomatic and Clinically Apparent Malaria. Infection and Immunity. 2006, 74: 5561-5573. 10.1128/IAI.00408-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Muehlenbachs A, Fried M, Lachowitzer J, Mutabingwa TK, Duffy PE: Genome-Wide Expression Analysis of Placental Malaria Reveals Features of Lymphoid Neogenesis during Chronic Infection. The Journal of Immunology. 2007, 179: 557-565.View ArticlePubMedGoogle Scholar
- Ylostalo J, Randall AC, Myers TA, Metzger M, Krogstad DJ, Cogswell FB: Transcriptome Profiles of Host Gene Expression in a Monkey Model of Human Malaria. The Journal of Infectious Diseases. 2005, 191: 400-409. 10.1086/426868.View ArticlePubMedGoogle Scholar
- Boutlis CS, Yeo TW, Anstey NM: Malaria tolerance – For whom the cell tolls?. Trends Parasitol. 2006, 22: 371-377. 10.1016/j.pt.2006.06.002.PubMed CentralView ArticlePubMedGoogle Scholar
- Schwarzer E, Skorokhod OA, Barrera V, Arese P: Hemozoin and the human monocyte – a brief review of their interactions. Parassitologia. 2008, 50: 143-145.PubMedGoogle Scholar
- Prato M, Giribaldi G, Polimeni M, Gallo V, Arese P: Phagocytosis of hemozoin enhances matrix metalloproteinase-9 activity and TNF-α production in human monocytes: role of matrix metalloproteinases in the pathogenesis of Falciparum malaria. The Journal of Immunology. 2005, 175: 6436-6442.View ArticlePubMedGoogle Scholar
- Skorokhod OA, Alessio M, Mordmuller B, Arese P, Schwarzer E: Hemozoin (malarial pigment) inhibits differentiation and maturation of human monocyte-derived dendritic cells: a peroxisome proliferator-activated receptor-γ-mediated effect. The Journal of Immunology. 2004, 173: 4066-4074.View ArticlePubMedGoogle Scholar
- Hage T, Sebald W, Reinemer P: Crystal structure of the interleukin-4/receptor α chain complex revelas a mosaic binding interface. Cell. 1999, 97: 271-281. 10.1016/S0092-8674(00)80736-9.View ArticlePubMedGoogle Scholar
- Bolick DT, Orr AW, Whetzel A, Srinivasan S, Hatley ME, Schwartz MA, Hedrick CC: 12/15-Lipoxygenase regulates intercellular adhesion molecule-1 expression and monocyte adhesion to endothelium through activation of RhoA and Nuclear Factor-kappaB. Arterioscler Thromb Vasc Biol. 2005, 25: 2301-2307. 10.1161/01.ATV.0000186181.19909.a6.View ArticlePubMedGoogle Scholar
- Patricia MK, Kim JA, Harper CM, Shih PT, Berliner JA, Natarajan R, Nadler JL, Hedrick CC: Lipoxygenase products increase monocyte adhesion to human aortic endothelial cells. Arterioscler Thromb Vasc Biol. 1999, 19: 2615-2622.View ArticlePubMedGoogle Scholar
- Howard RJ, Gilladoga AD: Molecular studies related to the pathogenesis of cerebral malaria. Blood. 1989, 74: 2603-2618.PubMedGoogle Scholar
- Silamut K, Phu NH, Whitty C, Turner GDH, Louwrier K, Mai NTH, Simpson JA, Hien TT, White NJ: A quantitative analysis of the microvascular sequestration of malaria parasites in the human brain. Am J Pathol. 1999, 155: 395-410.PubMed CentralView ArticlePubMedGoogle Scholar
- Patnaik JK, Das BS, Mishra SK, Mohanty S, Satpathy SK, Mohanty D: Vascular clogging, mononuclear cell margination, and enhanced vascular permeability in the pathogenesis of human cerebral malaria. Am J Trop Med Hyg. 1994, 51: 642-647.PubMedGoogle Scholar
- Adams S, Brown H, Turner G: Breaking down the blood-brain barrier: Signaling a path to cerebral malaria?. Trends Parasitol. 2002, 18: 360-366. 10.1016/S1471-4922(02)02353-X.View ArticlePubMedGoogle Scholar
- Johnson H, McNee M, Sun F: 15-Hydroxyeicosatetraenoic acid is a potent inflammatory mediator and agonist of canine tracheal mucus secretion. Am Rev Respir Dis. 1985, 131: 917-922.PubMedGoogle Scholar
- Rhee JW, Lee KW, Kim D, Lee Y, Jeon OH, Kwon HJ, Kim DS: NF-kappaB-dependent regulation of matrix metalloproteinase-9 gene expression by lipopolysaccharide in a macrophage cell line RAW 264.7. J Biochem Mol Biol. 2007, 40: 88-94.View ArticlePubMedGoogle Scholar
- Zambon A, Gervois P, Pauletto P, Fruchart J-C, Staels B: Modulation of hepatic inflammatory risk markers of cardiovascular diseases by PPAR-alpha activators: clinical and experimental evidence. Arterioscler Thromb Vasc Biol. 2006, 26: 977-986. 10.1161/01.ATV.0000204327.96431.9a.View ArticlePubMedGoogle Scholar
- Jiang C, Ting AT, Seed B: PPAR-γ agonists inhibit production of monocyte inflammatory cytokines. Nature. 1998, 391: 82-86. 10.1038/35154.View ArticlePubMedGoogle Scholar
- Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK: The peroxisome proliferator-activated receptor-γ is a negative regulator of macrophage activation. Nature. 1998, 391: 79-82. 10.1038/34178.View ArticlePubMedGoogle Scholar
- Marx N, Schonbeck U, Lazar MA, Libby P, Plutzky J: Peroxisome proliferator-activated receptor gamma activators inhibit gene expression and migration in human vascular smooth muscle cells. Circ Res. 1998, 83: 1097-1103.PubMed CentralView ArticlePubMedGoogle Scholar
- Marx N, Sukhova G, Murphy C, Libby P, Plutzky J: Macrophages in human atheroma contain PPARgamma: differentiation-dependent peroxisomal proliferator-activated receptor gamma (PPARgamma) expression and reduction of MMP-9 activity through PPARgamma activation in mononuclear phagocytes in vitro. Am J Pathol. 1998, 153: 17-23.PubMed CentralView ArticlePubMedGoogle Scholar
- Green MD, Xiao L, Lal AA: Formation of hydroxyeicosatetraenoic acids from hemozoin-catalyzed oxidation of arachidonic acid. Mol Biochem Parasitol. 1996, 83: 183-188. 10.1016/S0166-6851(96)02769-7.View ArticlePubMedGoogle Scholar
- Fridovich SE, Porter NA: Oxidation of arachidonic acid in micelles by superoxide and hydrogen peroxide. J Biol Chem. 1981, 256: 260-265.PubMedGoogle Scholar
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