Lipidomic Analysis of Dynamic Eicosanoid Responses during the Induction and Resolution of Lyme Arthritis
2009; Elsevier BV; Volume: 284; Issue: 32 Linguagem: Inglês
10.1074/jbc.m109.003822
ISSN1083-351X
AutoresVictoria A. Blaho, Matthew W. Buczynski, Charles R. Brown, Edward A. Dennis,
Tópico(s)Alcohol Consumption and Health Effects
ResumoEicosanoids and other bioactive lipid mediators are indispensable regulators of biological processes, as demonstrated by the numerous inflammatory diseases resulting from their dysregulation, including cancer, hyperalgesia, atherosclerosis, and arthritis. Despite their importance, a robust strategy comparable with gene or protein array technology for comprehensively analyzing the eicosanoid metabolome has not been forthcoming. We have developed liquid chromatography-tandem mass spectrometry methodology that quantitatively and comprehensively analyzes the eicosanoid metabolome and utilized this approach to characterize eicosanoid production during experimental Lyme arthritis in mice infected with the bacterium Borrelia burgdorferi. Eicosanoids were extracted throughout infection from the joints of Lyme arthritis-resistant and -susceptible mice and subjected to lipidomic profiling. We identified temporal and quantitative differences between these mouse strains in the production of eicosanoids, which correlated with differences in arthritis development. The eicosanoid biosynthetic enzyme cyclooxygenase (COX)-2 has been implicated in the regulation of Lyme arthritis pathology, and subsequent lipidomic profiling of B. burgdorferi-infected COX-2−/− mice identified reductions not only in COX-2 products but, surprisingly, also significant off-target reductions in 5-lipoxygenase metabolites. Our results demonstrate the utility of a comprehensive lipidomic approach for identifying potential contributors to disease pathology and may facilitate the development of more precisely targeted treatment strategies. Eicosanoids and other bioactive lipid mediators are indispensable regulators of biological processes, as demonstrated by the numerous inflammatory diseases resulting from their dysregulation, including cancer, hyperalgesia, atherosclerosis, and arthritis. Despite their importance, a robust strategy comparable with gene or protein array technology for comprehensively analyzing the eicosanoid metabolome has not been forthcoming. We have developed liquid chromatography-tandem mass spectrometry methodology that quantitatively and comprehensively analyzes the eicosanoid metabolome and utilized this approach to characterize eicosanoid production during experimental Lyme arthritis in mice infected with the bacterium Borrelia burgdorferi. Eicosanoids were extracted throughout infection from the joints of Lyme arthritis-resistant and -susceptible mice and subjected to lipidomic profiling. We identified temporal and quantitative differences between these mouse strains in the production of eicosanoids, which correlated with differences in arthritis development. The eicosanoid biosynthetic enzyme cyclooxygenase (COX)-2 has been implicated in the regulation of Lyme arthritis pathology, and subsequent lipidomic profiling of B. burgdorferi-infected COX-2−/− mice identified reductions not only in COX-2 products but, surprisingly, also significant off-target reductions in 5-lipoxygenase metabolites. Our results demonstrate the utility of a comprehensive lipidomic approach for identifying potential contributors to disease pathology and may facilitate the development of more precisely targeted treatment strategies. Systems biology has advanced exponentially during the past decade, producing a wealth of data relating to disease susceptibility and pathology. Because of its immense scope and complex chemical composition, metabolomics has lagged behind its genomics and proteomics counterparts, hampering the advancement of sequential systems biology as a cohesive approach to disease investigation and drug discovery (1Dennis E.A. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 2089-2090Crossref PubMed Scopus (115) Google Scholar). The unique challenges faced by metabolomics researchers are evident in the emerging field of lipidomics, the analysis and characterization of the enormous and diverse set of lipid molecules. For this reason, the Lipid Metabolites and Pathway Strategy (LIPID MAPS) consortium was created to provide an intellectual infrastructure for the advancement of lipidomics (2Schmelzer K. Fahy E. Subramaniam S. Dennis E.A. Methods Enzymol. 2007; 432: 171-183Crossref PubMed Scopus (115) Google Scholar). To expedite the elevation of lipidomics to a level comparable with that of genomics and proteomics, efforts have included the establishment of a comprehensive lipid classification system (3Fahy E. Subramaniam S. Brown H.A. Glass C.K. Merrill Jr., A.H. Murphy R.C. Raetz C.R. Russell D.W. Seyama Y. Shaw W. Shimizu T. Spener F. van Meer G. VanNieuwenhze M.S. White S.H. Witztum J.L. Dennis E.A. J. Lipid Res. 2005; 46: 839-861Abstract Full Text Full Text PDF PubMed Scopus (1140) Google Scholar), the adoption of liquid chromatography-tandem mass spectrometry (LC-MS/MS) 5The abbreviations used are: LCliquid chromatographyMS/MStandem mass spectrometryC3Harthritis-susceptible C3H/HeJ miceCOXcyclooxygenaseCYPcytochrome P450ddayDBAarthritis-resistant DBA/2J miceDHAdocosahexaenoic acidDHETdihydroxyeicosatrienoic aciddiHDoHEdihydroxydocosahexaenoic aciddiHOMEdihydroxyoctadecamonoenoic acidEETepoxyeicosatrienoic acidHDoHEhydroxydocosahexaenoic acidHETEhydroxyeicosatetranoic acidHODEhydroxyoctadecadienoic acidHXhepoxilinLOXlipoxygenaseLTleukotrieneLXlipoxinMRMmultiple reaction monitoringoxoETEoxoeicosatetraenoic acidPDprotectinPGprostaglandinRvresolvinWTwild-type. as the preferred methodology for lipid analysis (2Schmelzer K. Fahy E. Subramaniam S. Dennis E.A. Methods Enzymol. 2007; 432: 171-183Crossref PubMed Scopus (115) Google Scholar), and the development of a publicly accessible data base of lipid mass spectra and standards (see Lipidomics Gateway web site). These integrated components provide a springboard for the investigation of potential interactions between large classes of lipid signaling molecules and their impact on the induction and modulation of disease pathology. liquid chromatography tandem mass spectrometry arthritis-susceptible C3H/HeJ mice cyclooxygenase cytochrome P450 day arthritis-resistant DBA/2J mice docosahexaenoic acid dihydroxyeicosatrienoic acid dihydroxydocosahexaenoic acid dihydroxyoctadecamonoenoic acid epoxyeicosatrienoic acid hydroxydocosahexaenoic acid hydroxyeicosatetranoic acid hydroxyoctadecadienoic acid hepoxilin lipoxygenase leukotriene lipoxin multiple reaction monitoring oxoeicosatetraenoic acid protectin prostaglandin resolvin wild-type. Eicosanoids comprise a diverse class of over one hundred bioactive lipid mediators derived from the metabolism of polyunsaturated fatty acids by cyclooxygenase (COX) (4Buczynski M.W. Dumlao D.S. Dennis E.A. J. Lipid Res. 2009; 50: 1015-1038Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 5Funk C.D. Science. 2001; 294: 1871-1875Crossref PubMed Scopus (3049) Google Scholar), lipoxygenase (LOX) (4Buczynski M.W. Dumlao D.S. Dennis E.A. J. Lipid Res. 2009; 50: 1015-1038Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 5Funk C.D. Science. 2001; 294: 1871-1875Crossref PubMed Scopus (3049) Google Scholar, 6Serhan C.N. Chiang N. Van Dyke T.E. Nat. Rev. Immunol. 2008; 8: 349-361Crossref PubMed Scopus (2215) Google Scholar), and cytochrome P450 (CYP) (4Buczynski M.W. Dumlao D.S. Dennis E.A. J. Lipid Res. 2009; 50: 1015-1038Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 7Roman R.J. Physiol. Rev. 2002; 82: 131-185Crossref PubMed Scopus (1170) Google Scholar) as well as other non-enzymatic pathways (4Buczynski M.W. Dumlao D.S. Dennis E.A. J. Lipid Res. 2009; 50: 1015-1038Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). As a class, these molecules act as spatially localized hormone-like signaling molecules that have a wide range of effects, and under non-disease conditions contribute to the maintenance of homeostasis in almost every organ system (5Funk C.D. Science. 2001; 294: 1871-1875Crossref PubMed Scopus (3049) Google Scholar). However, it is because of their participation in inflammatory diseases that eicosanoids have been the target of intense study. For example, cyclooxygenases can generate pro-inflammatory prostaglandins (PG), which are responsible for many of the hallmark signs of inflammation such as heat, redness, swelling and pain, whereas cytochrome P450s create epoxyeicosatrienoic acids (EET), which may affect local inflammation by promoting vasodilatation, and the lipoxygenases, which generate molecules with diverse signaling effects such as pro-inflammatory leukotrienes (LT) or anti-inflammatory/pro-resolution lipoxins (LX), resolvins (Rv), and protectins (PD). Metabolic and non-enzymatic breakdown products of these lipids can have significantly altered bioactivity, further complicating our understanding of these intricate pathways. The currently accepted model of inflammation proposes that both the induction of inflammation and its eventual resolution and return to homeostasis are not passive events but instead are coordinated, orderly processes actively signaled by specific protein and lipid molecules (6Serhan C.N. Chiang N. Van Dyke T.E. Nat. Rev. Immunol. 2008; 8: 349-361Crossref PubMed Scopus (2215) Google Scholar). The inflammatory response is the first step in the immune response and is characterized by an influx of innate immune cells, the production of soluble pro-inflammatory mediators, and tissue destruction. Conversely, resolution sees an influx of anti-inflammatory macrophages, pro-resolution lipid production, and tissue remodeling and repair. Although the roles of some individual eicosanoids in inflammation have been well studied, the overall biological significance of how these signals act in vivo to properly initiate and resolve inflammation has yet to be fully elucidated. Studies specifically addressing pathway interactions have investigated the connections between PGE2 and LX signaling in several animal models of autoimmunity (8Bannenberg G.L. Chiang N. Ariel A. Arita M. Tjonahen E. Gotlinger K.H. Hong S. Serhan C.N. J. Immunol. 2005; 174: 4345-4355Crossref PubMed Scopus (0) Google Scholar, 9Levy B.D. De Sanctis G.T. Devchand P.R. Kim E. Ackerman K. Schmidt B.A. Szczeklik W. Drazen J.M. Serhan C.N. Nat. Med. 2002; 8: 1018-1023Crossref PubMed Scopus (335) Google Scholar), and Schmelzer et al. (10Schmelzer K.R. Inceoglu B. Kubala L. Kim I.H. Jinks S.L. Eiserich J.P. Hammock B.D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 13646-13651Crossref PubMed Scopus (161) Google Scholar) have identified in vivo changes in prostaglandin signaling following modulation of endogenous EETs. Although these studies have examined isolated sectors of the eicosanoid metabolome, no reported study has comprehensively examined the temporal production of these metabolites. Our understanding of eicosanoid involvement in regulating the response to infection is even more fragmented and has been focused primarily on a few select metabolites (11Blaho V.A. Mitchell W.J. Brown C.R. Arthritis Rheum. 2008; 58: 1485-1495Crossref PubMed Scopus (27) Google Scholar, 12Bafica A. Scanga C.A. Serhan C. Machado F. White S. Sher A. Aliberti J. J. Clin. Invest. 2005; 115: 1601-1606Crossref PubMed Scopus (192) Google Scholar). Thus, a complete metabolomics approach on a scale comparable with current gene microarray technology is essential to achieve a thorough understanding of the inflammatory and immune responses and to facilitate the design of more effective disease therapies. To date, lipidomics studies have been capable of measuring, at most, only ∼40 eicosanoids, fewer than half of the known eicosanoid species, with a per sample run time of over 50 min (13Masoodi M. Mir A.A. Petasis N.A. Serhan C.N. Nicolaou A. Rapid Commun. Mass Spectrom. 2008; 22: 75-83Crossref PubMed Scopus (118) Google Scholar, 14Kiss L. Röder Y. Bier J. Weissmann N. Seeger W. Grimminger F. Anal. Bioanal. Chem. 2008; 390: 697-714Crossref PubMed Scopus (19) Google Scholar). Additionally, extraction methods have been limited to primarily soft tissue samples such as lung or brain, excluding the possibility of lipidomic analysis of more complex tissues such as arthritic joints. These constraints create significant impediments toward the general applicability of previous methodology and require considerable technical development when applying them to other biological systems. For this reason, we have developed a protocol for complete eicosanoid extraction from complex tissue combined with a comprehensive LC-MS/MS methodology with a run time of 25 min/sample to identify and quantify 104 unique lipid species, which encompasses about 90% of the known eicosanoid metabolome (4Buczynski M.W. Dumlao D.S. Dennis E.A. J. Lipid Res. 2009; 50: 1015-1038Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). These advances allow this technique to be robustly applied to diverse biological samples at a systems biology level. To demonstrate the applicability of such an approach, we utilized this strategy to investigate the role of eicosanoids in regulating the development and resolution of arthritis pathology in mice infected with Borrelia burgdorferi, the causative agent of Lyme disease. Lyme disease is the most common vector-borne disease in the Northern hemisphere and presents diverse pathologies, the most common of which is arthritis (15Steere A.C. Glickstein L. Nat. Rev. Immunol. 2004; 4: 143-152Crossref PubMed Scopus (185) Google Scholar). Lyme arthritis manifests in about 60–80% of individuals not treated with antibiotics and is characterized by cellular infiltrate consisting of primarily neutrophils and macrophages, a pathology that is recapitulated in the murine model (16Barthold S.W. Beck D.S. Hansen G.M. Terwilliger G.A. Moody K.D. J. Infect. Dis. 1990; 162: 133-138Crossref PubMed Scopus (330) Google Scholar). The resolution of arthritis is thought to be dependent upon the clearance of B. burgdorferi from the joints by specific antibodies. Genomic profiling of mouse strains that are resistant (C57BL/6, DBA/2) or susceptible (C3H/He) to the development of Lyme arthritis has yielded intriguing data (17Crandall H. Dunn D.M. Ma Y. Wooten R.M. Zachary J.F. Weis J.H. Weiss R.B. Weis J.J. J. Immunol. 2006; 177: 7930-7942Crossref PubMed Scopus (61) Google Scholar) but has generated more questions than answers as to the determining factor(s) for arthritis development. Although previous studies indicate that eicosanoids may play a role in regulating Lyme arthritis pathology, it is unknown which eicosanoids are involved or what their specific roles might be in this pathology (11Blaho V.A. Mitchell W.J. Brown C.R. Arthritis Rheum. 2008; 58: 1485-1495Crossref PubMed Scopus (27) Google Scholar). Here we present the first comprehensive lipidomic analysis of an in vivo inflammatory response, from initiation to resolution. Analysis of joints from Lyme arthritis-resistant and -susceptible mouse strains revealed significant differences in basal and infection-induced eicosanoid production between the two strains. We further demonstrate the utility of this approach by performing lipidomic analysis on joints from COX-2-deficient (COX-2−/−) animals during infection with B. burgdorferi at the peak of the inflammatory response. Lipidomics of COX-2−/− joints exhibited the expected decrease in COX-2 products but also revealed surprising decreases in multiple products from the 5-LOX pathway. These data establish the robustness of this protocol, as well as its potential to uncover unexpected regulatory pathways. LC grade solvents were purchased from EMD Biosciences (San Diego, CA). Synergy C18 reverse phase high pressure liquid chromatography (HPLC) column and Strata-X solid phase extraction columns were purchased from Phenomenex (Torrance, CA). Eicosanoids were purchased from Cayman Chemicals (Ann Arbor, MI) and Biomol (Plymouth Meeting, PA). With the exception of dihomo-PGF2α, primary standards for dihomo (adrenic acid-derived)-prostaglandins were not commercially available; these metabolites were analyzed qualitatively using previously characterized extracts from RAW264.7 murine macrophage cells prepared as described (18Harkewicz R. Fahy E. Andreyev A. Dennis E.A. J. Biol. Chem. 2007; 282: 2899-2910Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Female C3H/HeJ (C3H) and DBA/2J (DBA) mice, 4–6 weeks of age, were purchased from The Jackson Laboratory (Bar Harbor, ME). COX-2 heterozygous mice (B6;129S7-Ptgs2tm1Jed) were purchased as breeders from The Jackson Laboratory and backcrossed onto the C3H genetic background for 10 generations. Heterozygous mice were then intercrossed to produce knock-out and wild-type littermates. Animals were housed in a specific pathogen-free facility and given sterile food and water ad libitum. All studies were conducted in accordance with the Animal Care and Use Committee of the University of Missouri. A virulent, low passage, clonal isolate of the B. burgdorferi N40 strain was used for all infections. Frozen stocks were placed in 7.5 ml of Barbour, Stoenner, Kelly (BSK) II medium (Sigma-Aldrich) with 6% rabbit serum (Sigma-Aldrich) and grown to log phase by incubation for 5–6 days at 32 °C. Spirochetes were enumerated using dark field microscopy and a Petroff-Hausser counting chamber (Hausser Scientific, Horsham, PA). Spirochete dilutions were made in sterile BSK II medium, and mice were inoculated in both hind footpads with 5 × 104 B. burgdorferi organisms in 50 μl of medium for a final inoculum of 1 × 105, a concentration that reliably produces arthritis in susceptible animals (19Brown C.R. Blaho V.A. Loiacono C.M. J. Immunol. 2003; 171: 893-901Crossref PubMed Scopus (107) Google Scholar). Sham-infected control animals were injected with 50 μl of BSK II medium/hind footpad. To determine arthritis severity scores, a histologic analysis of the left tibiotarsal joint from each mouse was performed following sacrifice. The tibiotarsal joint was excised, fixed in 10% buffered zinc-formalin, and embedded in paraffin, and 5-μm sections were stained with hematoxylin and eosin. The sections were evaluated in a blind manner by two independent observers and were assessed for arthritis severity on a scale of 0 to 4. A score of 0 indicated normal tissue, 1, 2, and 3 indicated minimal, mild, and moderate inflammation, respectively, and 4 indicated severe arthritis. The pathology present in histologic sections was characterized by neutrophil and monocyte infiltration into the joints, tendons, and ligament sheaths; hypertrophy and hyperplasia of the synovium; and fibrin exudates. The extent of the observed inflammatory changes formed the basis for the arthritis severity scores. B. burgdorferi-specific IgM and IgG levels in the sera of infected animals were determined by enzyme-linked immunosorbent assay as described previously (11Blaho V.A. Mitchell W.J. Brown C.R. Arthritis Rheum. 2008; 58: 1485-1495Crossref PubMed Scopus (27) Google Scholar). All tissues were snap-frozen in liquid nitrogen immediately after collection and then stored at −80 °C until extraction. Tissues were homogenized in TRIzol (Invitrogen), and DNA was extracted according to the manufacturer's instructions. Multiplex quantitative PCR for determination of B. burgdorferi loads was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) as described previously (19Brown C.R. Blaho V.A. Loiacono C.M. J. Immunol. 2003; 171: 893-901Crossref PubMed Scopus (107) Google Scholar). Bacterial loads are expressed as copies of B. burgdorferi flagellin per 1000 copies of mouse nidogen. Tissues were snap-frozen in liquid nitrogen and stored at −80 °C until extraction. The extraction protocol is a modification of Chen et al. (20Chen M. Lam B.K. Kanaoka Y. Nigrovic P.A. Audoly L.P. Austen K.F. Lee D.M. J. Exp. Med. 2006; 203: 837-842Crossref PubMed Scopus (230) Google Scholar). For extraction, each frozen tibiotarsal joint was wrapped in foil and placed in liquid nitrogen. The joint was then pulverized and the resulting frozen powder placed immediately in 3 ml of ice-cold 50% ethanol and then weighed. This protocol resulted in a nominal loss of tissue; the weight was later used for the normalization of eicosanoid levels in each sample. 10 μl of antioxidant mixture (0.2 mg/ml butylated hydroxytoluene, 0.2 mg/ml EDTA, 2 mg/ml triphenylphosphine, 2 mg/ml indomethacin in a solution of 2:1:1 methanol:ethanol:H2O) was added to each sample, and all tubes were kept on ice until incubation at −20 °C for 72 h. Samples were then centrifuged at 3500 × g for 30 min, and the clear ethanolic supernatant was removed to a new tube and dried under nitrogen gas. Samples were reconstituted with 2 ml of 10% MeOH and supplemented with 50 μl of internal standard containing the following deuterated eicosanoids (50 pg/μl, 2.5 ng total): (d4) 6k PGF1α, (d4) TXB2, (d4) PGF2α, (d4) PGE2, (d4) PGD2, (d4) 15d PGJ2, (d11) 5-iso PGF2α VI, (d4) dhk PGF2α, (d4) dhk PGD2, (d4) LTB4, (d8) 5-hydroxyeicosatetranoic acid (HETE), (d8) 15-HETE, (d6) 20-HETE, (d4) 9-HODE, (d4) 13-HODE, (d7) 5-oxoETE, (d8) 8,9-EET, (d8) 11,12-EET, (d8) 14,15-EET, (d4) 9,10-diHOME, and (d4) 12,13-diHOME. Samples were sonicated for 30 s with a probe sonicator and purified by solid phase extraction as described previously (21Buczynski M.W. Stephens D.L. Bowers-Gentry R.C. Grkovich A. Deems R.A. Dennis E.A. J. Biol. Chem. 2007; 282: 22834-22847Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 22Deems R. Buczynski M.W. Bowers-Gentry R. Harkewicz R. Dennis E.A. Methods Enzymol. 2007; 432: 59-82Crossref PubMed Scopus (139) Google Scholar). Prior to LC-MS/MS analysis, samples were evaporated using a SpeedVac and reconstituted in 50 μl of LC solvent A (water-acetonitrile-acetic acid (70:30:0.02, v/v/v)). Eicosanoids were separated by reverse-phase LC on a Synergy C18 column (2.1 × 250 mm) at a flow rate of 300 μl/min at 50 °C. The column was equilibrated in solvent A (water-acetonitrile-acetic acid (70:30:0.02; v/v/v)), and 40 μl of sample (80%) was injected using a 50-μl injection loop and eluted with 0% solvent B (acetonitrile-isopropyl alcohol (50:50, v/v)) between 0 and 1 min. Solvent B was increased in a linear gradient to 25% solvent B until 3 min, to 45% until 11 min, to 60% until 13 min, to 75% until 18 min, and to 90% until 18.5 min. Solvent B was held at 90% until 20 min, dropped to 0% by 21 min, and held until 25 min. Eicosanoids were analyzed using a tandem quadrupole mass spectrometer (ABI 4000 Q-Trap®, Applied Biosystems) via multiple reaction monitoring (MRM) in negative ion mode. The electrospray voltage was −4.5 kV, and the turbo ion spray source temperature was 525 °C. Collisional activation of eicosanoid precursor ions used nitrogen as a collision gas. Eicosanoids were measured using precursor→product (MRM) pairs, and the MS analysis was divided into six periods (Fig. 1, supplemental Table 1). The duty cycle for each period ranged between 390 and 840 ms, with an average time of 565 ms. The declustering potential and collision energy for each eicosanoid was optimized for maximal signal using flow injection mass spectrometry. Eicosanoids were identified in samples by matching their MRM signal and LC retention time with those of a pure standard. Extraction and ionization efficiencies were determined for the internal standards by comparing the ion intensity of the samples with those of the extraction controls. The range of efficiency for a typical experiment was between 20 and 75%, with an average of around 50%. Quantitative eicosanoid determination was performed by the stable isotope dilution method (21Buczynski M.W. Stephens D.L. Bowers-Gentry R.C. Grkovich A. Deems R.A. Dennis E.A. J. Biol. Chem. 2007; 282: 22834-22847Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 22Deems R. Buczynski M.W. Bowers-Gentry R. Harkewicz R. Dennis E.A. Methods Enzymol. 2007; 432: 59-82Crossref PubMed Scopus (139) Google Scholar). Deuterated internal standards (2.5 ng) were added to each sample to account for extraction efficiency and mass spectrometry ion suppression. To calculate the amount of eicosanoid in a sample, the ratio of the natural eicosanoid peak area to the deuterated eicosanoid peak area (termed "eicosanoid ratio") was determined for each sample. The eicosanoid ratio, proportional to ng of eicosanoid, was converted using the standard curve, 2.5 ng of each internal (deuterated) eicosanoid standard mixed with the following amounts of natural eicosanoid (nondeuterated) primary standard: 0.1, 0.3, 1, 3, 10, 30, and 100 ng. The hepoxilin standards that are commercially available from Biomol each contain a mixture of two diasteriomers, which are not distinguishable by this methodology and are identified only as hepoxilin (HX) A3 and HXB3, respectively. For PD1 and Δ15t-PD1, the isomer 10S,17S-diHDoHE was used as the quantitative primary standard; for 11Β PGE2, the isomer PGE2 was used. Performing linear regression using Y = mX, where (Y = eicosanoid ratio, X = ng of eicosanoid, m = slope), the eicosanoid ratio was multiplied by (1/m) to determine the ng eicosanoid in a sample. The deuterated internal standards contained a small but detectable level of natural, undeuterated eicosanoid; to account for this, extraction controls were performed in quadruplicate and the ng of eicosanoid subtracted from each sample. The final values were normalized to the original weight of the tissue and expressed as ng of eicosanoid/mg of tibiotarsal tissue. In addition to quantitative analysis, data were expressed as a heat map to identify relative increases and decreases in metabolites over time. Similar to the quantitative analysis, the extraction eicosanoid ratio was subtracted from the sample ratio and normalized to the original weight of the tissue; this value was then normalized to the corresponding value at day 0. The (1/m) value determined from the quantitative curve cancels out of relative fold calculations. One advantage to eliminating (1/m) is that, for eicosanoids with a limited availability, analysis can be performed in the absence of full quantitation. Results are expressed as the means ± S.E. Comparisons were made using one-way analysis of variance followed by a multiple comparison procedure, or t test, and p values ≤ 0.05 were considered significant. Upon infection with B. burgdorferi, arthritis-susceptible C3H/HeJ (C3H) mice developed a severe inflammatory response as measured by arthritis severity scores, which peaked at 2 weeks post-infection and then spontaneously resolved. Resistant DBA/2J (DBA) mice developed only a very mild inflammation (Fig. 2A). Bacterial loads in the joint tended to mirror the course of pathology (Fig. 2B), peaking just before the arthritic response. However, inoculum size does not affect susceptibility or resistance to arthritis development in most mouse strains (23Ma Y. Seiler K.P. Eichwald E.J. Weis J.H. Teuscher C. Weis J.J. Infect. Immun. 1998; 66: 161-168Crossref PubMed Google Scholar). The difference in histopathology was most evident at the peak of arthritis, the development of which is dependent upon neutrophil recruitment into the infected joint (Fig. 2C, day 10) (19Brown C.R. Blaho V.A. Loiacono C.M. J. Immunol. 2003; 171: 893-901Crossref PubMed Scopus (107) Google Scholar), whereas macrophages predominated during the resolution phase (Fig. 2C, day 35) (16Barthold S.W. Beck D.S. Hansen G.M. Terwilliger G.A. Moody K.D. J. Infect. Dis. 1990; 162: 133-138Crossref PubMed Scopus (330) Google Scholar). Resolution of arthritis is attributed to B. burgdorferi-specific IgM- and IgG-mediated (Fig. 2D) clearance of bacteria from infected joints (24Barthold S.W. Feng S. Bockenstedt L.K. Fikrig E. Feen K. Clin. Infect. Dis. 1997; 25 (Suppl. 1): S9-S17Crossref PubMed Scopus (81) Google Scholar). Overall, DBA mice demonstrated less cellular infiltration into the joints with almost no differences in the bacterial loads and antibody responses, demonstrating that Lyme arthritis susceptibility is genetically regulated and independent of the numbers of bacteria in the joints (25Brown C.R. Reiner S.L. J. Immunol. 2000; 165: 1446-1452Crossref PubMed Scopus (8) Google Scholar). Tibiotarsal (ankle) joints from B. burgdorferi-infected C3H and DBA mice were collected at various time points post-infection and analyzed for eicosanoid levels during the progression of arthritis (Fig. 3). The majority of joint mass is bone, making it difficult to extract the low levels of eicosanoids from the surrounding tissue comprised of infiltrating cells, muscle, synovium, intrarticular fluid, and cellular exudate. Joints were kept frozen with liquid nitrogen and then pulverized. The resultant powder was placed in cold 50% ethanol containing an antioxidant mixture to prevent lipid degradation. After 72 h of incubation at −20 °C, samples were clarified by centrifugation and supernatants dried completely under nitrogen gas. Repeated analysis found dramatically decreased eicosanoid levels with −20 °C incubation times less than 72 h and little to no increase in lipid concentrations with incubation times greater than 72 h. Samples were reconstituted with 10% methanol containing 21 deuterated internal standards representative of products from all major eicosanoid biosynthetic pathways. Subsequently, the samples were purified by solid phase extraction and stored in methanol for analysis (21Buczynski M.W. Stephens D.L. Bowers-Gentry R.C. Grkovich A. Deems R.A. Dennis E.A. J. Biol. Chem. 2007; 282: 22834-22847Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 22Deems R. Buczynski M.W. Bowers-Gentry R. Harkewicz R. Dennis E.A. Methods Enzymol. 2007; 432: 59-82Crossref PubMed Scopus (139) Google Scholar). Eicosanoids were analyzed using liquid chromatography coupled with tandem mass spectrometry. Upon injection, eicosanoids were eluted using a 25-min reverse phase liquid chromatography grad
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