Increased Saturated Fatty Acids in Obesity Alter Resolution of in Part by Stimulating Production

This information is current as Jason Hellmann, Michael J. Zhang, Yunan Tang, Madhavi of October 2, 2021. Rane, Aruni Bhatnagar and Matthew Spite J Immunol published online 19 June 2013 http://www.jimmunol.org/content/early/2013/06/17/jimmun ol.1203369 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 19, 2013, doi:10.4049/jimmunol.1203369 The Journal of Immunology

Increased Saturated Fatty Acids in Obesity Alter Resolution of Inflammation in Part by Stimulating Prostaglandin Production

Jason Hellmann,* Michael J. Zhang,* Yunan Tang,* Madhavi Rane,† Aruni Bhatnagar,*,‡ and Matthew Spite*,‡,x

Extensive evidence indicates that nutrient excess associated with obesity and type 2 diabetes activates innate immune responses that lead to chronic, sterile low-grade inflammation, and obese and diabetic humans also have deficits in wound healing and increased susceptibility to infections. Nevertheless, the mechanisms that sustain unresolved inflammation during obesity remain unclear. In this study, we report that saturated free fatty acids that are elevated in obesity alter resolution of acute sterile inflammation by promoting survival and decreasing macrophage . Using a targeted mass spectrometry–based lipidomics Downloaded from approach, we found that in db/db mice, PGE2/D2 levels were elevated in inflammatory exudates during the development of acute peritonitis. Moreover, in isolated macrophages, palmitic acid stimulated cyclooxygenase-2 induction and prostanoid production.

Defects in macrophage phagocytosis induced by palmitic acid were mimicked by PGE2 and PGD2 and were reversed by cyclo- oxygenase inhibition or prostanoid receptor antagonism. Macrophages isolated from obese-diabetic mice expressed prostanoid receptors, EP2 and DP1, and contained significantly higher levels of downstream effector, cAMP, compared with wild-type mice. Therapeutic administration of EP2/DP1 dual receptor antagonist, AH6809, decreased neutrophil accumulation in the peritoneum of db/db mice, as well as the accumulation of apoptotic cells in the thymus. Taken together, these studies provide new insights into http://www.jimmunol.org/ the mechanisms underlying altered innate immune responses in obesity and suggest that targeting specific prostanoid receptors may represent a novel strategy for resolving inflammation and restoring phagocyte defects in obese and diabetic individuals. The Journal of Immunology, 2013, 191: 000–000.

besity has emerged as one of the most important public metabolic dysregulation in obesity is intimately linked with the health concerns of our time. It is a leading risk factor for development of chronic inflammation, which in turn contributes to O the development of type 2 diabetes (T2D), and nearly alterations in metabolism in a vicious positive feed-forward cycle (8). Thus, understanding how nutrient excess impacts immune re-

90% of adults with T2D are obese (1). Obesity is also a risk factor by guest on October 2, 2021 for cardiovascular disease and liver disease, due largely to meta- sponses is required to identify novel strategies for treatment and bolic disturbances, such as insulin resistance and dyslipidemia (2, management of obesity and its secondary complications. 3). Obesity is associated with chronic elevation of free fatty acids Effector cells of the innate immune system, including polymor- (FFAs), which promote insulin resistance and contribute to the phonuclear (PMNs) and macrophages, play key roles in development of systemic hyperglycemia (4, 5). High plasma levels mediating immune responses. These leukocytes also sense nutrient of FFAs are also independently associated with hypertension and imbalances. Saturated fatty acids, which are elevated in obesity and sudden cardiac death (6, 7). Accumulating evidence indicates that diabetes (5), initiate inflammatory responses in part via signaling through pattern recognition receptors, that is, TLR2, TLR4, and nucleotide-binding oligomerization domain–like receptors (9–12). *Diabetes and Obesity Center, University of Louisville School of Medicine, Louis- Other endogenous mediators of metabolism, such as insulin, leptin, ville, KY 40202; †Division of Nephrology, Department of Medicine, University of Louisville School of Medicine, Louisville, KY 40202; ‡Division of Cardiovascular and adiponectin, also regulate leukocyte effector functions, sug- Medicine, Department of Medicine, Institute of Molecular Cardiology, University of x gesting that the immune system is exquisitely sensitive to the nu- Louisville School of Medicine, Louisville, KY 40202; and Department of Microbi- trient tone (13, 14). Several studies have shown that low-grade ology and Immunology, University of Louisville School of Medicine, Louisville, KY 40202 inflammation associated with obesity promotes the development of Received for publication December 10, 2012. Accepted for publication May 24, chronic diseases such as atherosclerosis, cancer, and nonalcoholic 2013. fatty liver disease and impairs host defense and wound healing (15– This work was supported by National Institutes of Health Grants HL106173 (to M.S.) 17). Nevertheless, the mechanisms by which metabolic imbalance and RR024489 (to A.B. and M.S.). J.H. is the recipient of a National Research affects inflammatory processes remain unclear. The initiation and Service Award from the National Institutes of Health (Grant HL116186). the resolution of inflammatory responses involve different cellular Address correspondence and reprint requests to Dr. Matthew Spite, University of Louisville School of Medicine, 580 South Preston Street, Delia Baxter Building, responses that are temporally controlled, in part, by specific lipid Room 404F, Louisville, KY 40202. E-mail address: [email protected] mediators (18–21). Upon trafficking to sites of tissue infection and Abbreviations used in this article: ALOX5, arachidonate 5-lipoxygenase; ALOX15, injury, PMNs encounter and attempt to eradicate invading patho- arachidonate 15-lipoxygenase; ASA, acetylsalicylic acid; COX, cyclooxygenase; gens. Subsequently, they undergo rapid and must be ef- DPBS, Dulbecco’s PBS; FFA, free fatty acid; HETE, hydroxyeicosatetraenoic acid; HF, high fat; hPGDS, hematopoietic PGD synthase; LC-MS/MS, liquid chromatog- fectively cleared by macrophages to prevent inadvertent tissue raphy–tandem mass spectrometry; LTB4, ; mPGES, microsomal PGE injury (21, 22). As such, defects in macrophage phagocytosis can synthase; PA, palmitic acid; PKA, protein kinase A; PMN, polymorphonuclear neu- give rise to altered clearance of apoptotic cells (23, 24). Our recent trophil; PTGS, PG endoperoxide synthase; T2D, type 2 diabetes; WT, wild-type. studies have shown that in db/db mice, which are a model of human Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 obesity and T2D, defective macrophage-dependent clearance of

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203369 2 PGs AND ALTERED INFLAMMATION IN OBESITY apoptotic cells delays the resolution of acute peritonitis (15). allowed to adhere for 1 h. Macrophages were then washed to remove any Moreover, we found that restoring defective macrophage phago- nonadherent cells and were incubated with IgG-opsonized FITC-labeled cytosis with proresolving lipid mediators, such as D1, en- zymosan A (Invitrogen) for 60 min at 37˚C in fresh DMEM containing 10% FBS. Trypan blue was used to quench extracellular fluorescence for 1 hances diabetic wound healing, suggesting that defects in min, followed by a DPBS+/+ (pH 7.45) wash. Phagocytosis was quantified phagocytosis are causally related to clinically relevant complica- using a spectrophotometer (PerkinElmer). To determine how saturated FFAs tions of diabetes and obesity (15). Nonetheless, the mechanisms impact macrophage phagocytosis, BSA conjugates of PA were prepared to underlying altered resolution of inflammation and phagocyte func- increase the solubility of the FFAs. For this, BSA and sodium palmitate were first dissolved in 150 mM NaCl. Sodium palmitate (1 mM) was then tion in conditions of nutrient excess remain incompletely under- slowly introduced into the BSA (0.17 mM) solution at 37˚C and allowed to stood. In this study, we show that resolution of acute peritonitis in incubate for 1 h, and the pH was adjusted to 7.4. To examine the effects of obesity is delayed in part because elevated FFAs prolong PMN FFAs on macrophages, RAW 264.7 macrophages (American Type Culture survival and inhibit macrophage phagocytosis. Our results suggest Collection) were pretreated with either BSA alone or palmitate-BSA con- m that defects in macrophage phagocytosis induced by palmitic acid jugates (500 M) for 6 h, and phagocytosis of opsonized zymosan was assessed. The effects of PGD /E on macrophage phagocytosis were de- (PA) are due to autocrine actions of PGs E and D and downstream 2 2 2 2 termined by preincubating RAW macrophages with PGE2 or PGD2 (100 modulation of the cAMP pathway. Overall, this study provides nM) for 30 min prior to the addition of opsonized zymosan, whereas the novel insights into the mechanisms by which nutrient excess involvement of cAMP-dependent signaling was assessed by preincubating impacts acute inflammation and its resolution. These findings sug- the cells with cAMP analogs specific for PKA (6-Bnz-cAMP, 2 mM) or Epac-1 (8-pCPT-29-O-Me-cAMP, 2mM). To assess the role of endogenous gest that targeting prostanoid receptors or downstream signaling prostanoid generation in deficits in macrophage phagocytosis induced by mediators may be an effective new means of controlling excessive PA, cells were pretreated with ASA (200 mM) or prostanoid receptor an- inflammatory responses and promoting the resolution of inflam- tagonist, AH6809 (100 mM), initially and every hour thereafter and mation in obese and/or diabetic individuals. phagocytosis of opsonized zymosan was assessed as above. In some Downloaded from experiments, RAW macrophages were incubated with plasma (10% in DMEM, 6 h) collected from 12-wk-old WT and db/db mice and phago- Materials and Methods cytosis was assessed. In all studies, uptake of FITC-zymosan was con- Mice and reagents firmed by fluorescence microscopy prior to quantification using an EVOS fluorescence microscope (Advanced Microscopy Group, Bothell, WA). Zymosan A and acetylsalicylic acid (ASA) were purchased from Sigma- Aldrich (St. Louis, MO). Male leptin receptor–deficient mice (B6.BKS Assessment of apoptotic thymocyte clearance (D)-Leprdb/J; db/db) and their appropriate wild-type (WT) controls (C57BL/ http://www.jimmunol.org/ 6J) were purchased from The Jackson Laboratory (Bar Harbor, ME) at 8 wk WT and db/db mice were treated i.p. with dexamethasone (15 mg/kg body weight in sterile saline). Mice were administered EP/DP prostanoid receptor of age and maintained on normal chow. PGE2 and PGD2 were purchased from Cayman Chemical (Ann Arbor, MI). Specific cAMP analogs for protein antagonist AH6809 (10 ng/g body weight) or vehicle (0.1% ethanol in sterile kinase A (PKA) and Epac-1 activation, 6-Bnz-cAMP and 8-pCPT-29-O-Me- saline) by i.v. injection initially and 4 h later. The mice were euthanized 6 h cAMP, respectively, were obtained from BioLog Life Science Institute after dexamethasone administration and the thymus was collected and (Bremen, Germany). formalin-fixed. Sections of the tissue were stained using the DeadEnd col- orimetric TUNEL system kit (Promega, Madison, WI). The TUNEL+ area Measurement of plasma nonesterified fatty acids was quantified using MetaMorph software (7–11 random fields per animal). Mice maintained on a 60% high-fat diet for 12 wk or 12-wk-old db/db mice Western blotting and appropriate WT controls were anesthetized according to a University of by guest on October 2, 2021 Louisville Institutional Animal Care and Use Committee approved protocol. Cell lysates were normalized for protein concentration and boiled in Blood was collected by cardiac puncture using EDTA as the anticoagulant. Laemmli buffer, followed by standard SDS-PAGE separation. Gels were then Whole blood was then centrifuged at 4˚C for 20 min. Plasma was separated transblotted onto Protran BA 83 nitrocellulose membranes (MIDSCI, St. from RBCs and stored at 280˚C until use. Nonesterified FFAs were mea- Louis, MO) and probed for caspase-3 (Cell Signaling Technology, Danvers, b sured using a colorimetric assay (Wako Diagnostics) and a spectrophotom- MA), pERK1/2, or -actin (Santa Cruz Biotechnology, Santa Cruz, CA). eter according to the manufacturer’s instructions. Alternatively, gels were transblotted onto polyvinylidene difluoride mem- branes and probed for cyclooxygenase (COX)-2 (Cayman Chemical), DP1, Acute peritonitis and flow cytometry EP2, EP4 (Cayman Chemical), or GAPDH (Cell Signaling Technology). Blots were then developed using ECL Plus reagent followed by lumines- Acute peritonitis was initiated in WT and db/db mice by i.p. administration cence detection using a Typhoon 9400 variable mode imager (GE Health- of zymosan A (0.04 mg/g body weight) in 1 ml sterile saline (15). After 6, care, Chalfont St. Giles, U.K.). Quantification of band intensities was 24, or 48 h, mice were sacrificed and the peritoneum was lavaged with performed using ImageQuant TL software (GE Healthcare). Dulbecco’s PBS (DPBS)2/2 (5 ml; without calcium and magnesium). Leukocytes in the total exudate were enumerated by light microscopy. Real-time PCR Specific leukocyte populations and nonapoptotic cells were identified by flow cytometry. For this, peritoneal exudates were suspended in FACS For real-time PCR, mRNA was isolated from cells using the RNeasy tissue buffer (1% FBS in PBS) and incubated with Fc Block (BD Biosciences, San kit (Qiagen, Germantown, MD), followed by cDNA synthesis. Real-time Jose, CA) for 10 min at 4˚C. Cells were then stained with fluorescence- PCR amplification was performed with SYBR Green qPCR Master Mix tagged primary Abs (FITC-conjugated anti-F4/80 and PE-conjugated anti- (SABiosciences, Frederick, MD) using a 7900HT Fast real-time PCR system Ly6G; BioLegend, San Diego, CA) or appropriate isotype controls for 30 (Applied Biosystems, Foster City, CA) and commercially available primers min at 4˚C. To quantify nonapoptotic neutrophils, Ly6G+ cells that were for PG endoperoxide synthase (Ptgs)1, Ptgs2, microsomal PGE synthase negative for annexin V were assessed using FITC-conjugated annexin V (mPges)1, hematopoietic PGD synthase (hPgds), arachidonate 5-lip- oxygenase (Alox5), and arachidonate 15-lipoxygenase (Alox15) (SABio- (BioLegend). Flow cytometry was performed using a BD LSRII cytometer 2DDCt equipped with FACSDiva version 6.0 (BD Biosciences). FlowJo version 7.6 sciences). Relative expression was determined by the 2 method after software (Tree Star, Ashland, OR) was used for gating analysis and quan- internal normalization to hprt. tification. In selected experiments, db/db mice undergoing peritonitis were Measurement of cAMP treated with PGE2/D2 dual receptor antagonist, AH6809 (Cayman Chem- ical; 10 ng/g body weight; i.p.) in 1 ml sterile saline 32 h after zymosan Resident peritoneal macrophages were isolated from WT or db/db mice and challenge. Peritoneal exudates were then obtained after 16 h and leukocyte were plated onto 24-well plates at a density of 7.5 3 105 cells per well for populations were assessed by flow cytometry as described above. 1 h in DPBS+/+. Adherent cells were then lysed with 0.1 M HCl for 15 min at room temperature. Levels of cAMP were measured using an ELISA Assessment of macrophage phagocytosis (Cayman Chemical) as per the manufacturer’s instructions. To measure macrophage phagocytosis, resident peritoneal macrophages Mass spectrometry–based targeted lipidomics analysis were isolated from WT, obese-diabetic (db/db), and WT mice fed a low-fat (10%) or high-fat (60%) diet for 12 wk. Macrophages were seeded in 96- Exudates or macrophage supernatants were collected and added to two 5 +/+ well plates at a density of 4 3 10 cells per well in 100 ml DPBS and volumes of cold containing deuterium-labeled PGE2 (1 ng) as The Journal of Immunology 3 internal standard (25). After protein precipitation, samples were subjected to macrophage infiltration were observed throughout the time course solid-phase extraction (C18) and methyl formate fractions were collected. of peritonitis. Lipid mediators were profiled by liquid chromatography–tandem mass We previously demonstrated that altered resolution of acute in- spectrometry (LC-MS/MS) using an HPLC system (Shimadzu Prominence) equipped with an Eclipse Plus reverse-phase C18 column (4.6 mm 3 50 mm flammation in db/db mice was associated with the accumulation of 3 1.8 mm; Agilent Technologies) coupled to a triple quadrupole mass apoptotic PMNs, as well as FITC-zymosan, and that these events spectrometer (API 2000; Applied Biosystems/SCIEX). The instrument was were associated with defective macrophage-mediated clearance operated in negative ionization mode, and the mobile phase consisted of (15). In this study, we found similar changes in high fat (HF)–fed methanol/water/acetic acid (60:40:0.01 [v/v/v]), which was ramped to 80:20:0.01 during 7.5 min and to 95:5:0.01 in the next 4.5 min at a flow rate WT mice. Similar to macrophages from db/db mice, resident peri- of 400 ml/min. The mobile phase was ramped up during the next 6 min to toneal macrophages from WT mice fed a HF diet for 12 wk showed 100:0:0.01 before returning to 60:40:0.01. Lipid mediators of interest were significant defects in their ability to phagocytose IgG-osponized identified by multiple reaction monitoring using established transitions for zymosan compared with mice fed a low-fat (LF) diet (Fig. 1D). . . . PGE2/D2 (351 271), PGF2a (353 193), leukotriene B4 (LTB4; 335 To determine whether this defect was due to changes in plasmatic 195), 12-hydroxyeicosatetraenoic acid (HETE; (319 . 179), 15-HETE (319 factors, macrophages isolated from WT mice were incubated with . 175), 5-HETE (319 . 115), and thromboxane B2 (369 . 169) (25). Given that PGE2 and PGD2 are not readily separated using these HPLC conditions plasma obtained from WT and db/db mice and macrophage phago- and that they have nearly identical mass spectral fragmentation patterns, we cytosis was measured. As shown in Fig. 1E, macrophages incubated present data pertaining to PGE2 and PGD2 as PGE2/D2. For quantification, with plasma from db/db mice showed diminished phagocytosis rel- external calibration curves were constructed for each compound using au- thentic standards (Cayman Chemical) and recoveries were calculated using ative to macrophages incubated with WT plasma. The extent of this internal deuterium-labeled PGE2. defect was similar to that in macrophages isolated from HF-fed mice (Fig. 1E). These observations indicate that the defective macrophage Human neutrophil isolation and survival analysis Downloaded from phagocytosis in diabetes and obesity may be related to the presence Blood was obtained from healthy, medication-free human donors on a vol- of plasmatic factors that suppress phagocytosis. untary basis using standard venipuncture. The study was approved by the Because obesity is associated with an increase in circulating FFAs, University of Louisville Institutional Review Board, and all donors received we measured FFA levels in WT, db/db, and HF-fed mice. As ex- material regarding the research study and signed consent forms prior to blood donation. Following blood collection, PMNs were isolated using plasma- pected, in comparison with WT mice fed a normal chow diet, Percoll gradients (26). After isolation, PMNs were washed and resus- plasma FFA levels were significantly elevated in both db/db and HF- pended with LPS-free (sterile) Krebs-Ringer phosphate buffer (pH 7.2) fed mice (Fig. 1F). To determine whether FFAs can directly sup- http://www.jimmunol.org/ containing 0.2% dextrose (Krebs). Microscopic evaluation of isolated cells press macrophage phagocytosis, we incubated WT macrophages by trypan blue exclusion indicated that .95% of cells were PMNs, and at least 95% were viable. PMNs were gently centrifuged for 5 min at 2000 rpm with PA, which is one of the major FFAs in the plasma in obese mice at room temperature and resuspended in Krebs-Ringer buffer (with calcium (27). For these and following experiments we used PA within the and magnesium) at 5 3 106 cells/ml. Neutrophils (0.9 ml/condition) were dose range found in the plasma of obese mice (Fig. 1F). As shown in aliquoted into sterile 1.5 ml Eppendorf tubes and prewarmed in a 37˚C Fig. 1G, we found that PA suppressed macrophage phagocytosis to water bath for 5 min. Neutrophils were left untreated (positive control for the extent similar to that observed with plasma isolated from db/db constitutive apoptosis) or treated with LPS (a positive control for neutrophil survival), PA-BSA (100 mM), or BSA-control for 10 min followed by ad- mice, as well as the level of suppression observed in macrophages dition of 100 ml FBS. Neutrophils were incubated overnight (24 h) at 37˚C isolated from HF-fed mice (vide supra). Taken together, these ob- by guest on October 2, 2021 in a 5% CO2 incubator. The following day, PMNs were spun down in servations, in agreement with previous reports (27), suggest that a microfuge and total cell lysates were prepared by resuspending cell pellets saturated fatty acids suppress macrophage phagocytosis. in a lysis buffer containing 137 mM NaCl, 20 mM Tris-HCl (pH 7.5), 10% glycerol, 1% Nonidet P-40, 1% Triton X-100, 20 mM sodium fluoride, and In addition to defective macrophage phagocytosis, altered reso- 2 mM sodium orthovanadate. PMSF (4 mM) and protease and phosphatase lution of inflammation could also be due to prolongation of PMN inhibitor cocktails (Sigma-Aldrich) were added (1:100) to lysis buffer prior survival (22, 28, 29). Therefore, we quantified the number of non- to use. Lysed cells were left on ice for 15 min, followed by centrifugation at apoptotic PMNs (Ly6G+annexin V2) remaining in the peritoneum 3 13,000 g for 10 min at 4˚C. Supernatants were saved as total lysate and of WT and db/db mice 48 h after zymosan challenge. As expected, stored at 270˚C until analysis. For survival analysis, 50 mg protein in each condition was loaded onto 4–12% gradient gels and proteins were separated levels of nonapoptotic PMNs were significantly elevated in the by SDS-PAGE, transblotted, and probed with cleaved caspase-3 Ab (a peritoneum of db/db mice compared with WT mice (Fig. 1H), in- marker of cellular apoptosis), pERK (marker of cell survival), and b-actin dicating that PMN survival is prolonged in db/db mice. To test this (as a loading control). directly, we assessed spontaneous apoptosis of human PMNs Statistical analysis in vitro. As shown in Fig. 1I, human PMNs readily underwent ap- optosis when incubated at 37˚C for 24 h, as assessed by caspase-3 Results are presented as mean 6 SEM. Statistical significance was assessed using a two-tailed Student t test or one-way ANOVA followed by either cleavage. The extent of apoptosis was markedly diminished when Bonferroni or Dunnett posttests, as appropriate. In all cases, p , 0.05 was the PMNs were incubated with PA-BSA, but not BSA alone, as considered significant. assessed by reduced caspase-3 cleavage and enhanced signaling through the prosurvival ERK1/2 pathway. As a positive control, we Results examined the effects of LPS, which markedly delays apoptosis. We To evaluate how nutrient excess associated with obesity and T2D found that incubation with LPS led to a significant decrease in alters innate immune responses, we used an acute model of sterile caspase-3 cleavage and increased pERK1/2 levels (Fig. 1I) (30). peritonitis. Consistent with our recent studies (15), zymosan chal- The extent of increase in PMN survival by LPS was similar to that lenge provoked a large influx of PMNs into the peritoneum 6 h after observed with PA-BSA. Collectively, these results demonstrate that the injection (Fig. 1A). Maximal infiltration was observed 24 h elevated levels of saturated FFAs could potentially alter acute in- postinjection (Fig. 1B). As reported before (15), the extent and the flammation and its resolution by increasing PMN survival and de- time course of PMN infiltration was similar in both WT and db/db creasing macrophage phagocytosis. mice at 6 and 24 h. However, whereas levels of PMNs declined Among the mediators of acute inflammation and its resolution, during the resolution phase (i.e., 48 h) in WT mice, PMNs accu- lipid mediators play key roles in regulating leukocyte trafficking mulated to significantly higher levels in db/db mice (Fig. 1C), and cytokine production, as well as leukocyte effector functions, suggesting that resolution of inflammation was delayed in db/db such as phagocytosis (18–20, 31). To understand how nutrient mice, rather than the initiation of inflammation. No differences in excess in obesity alters the process of inflammation and its reso- 4 PGs AND ALTERED INFLAMMATION IN OBESITY Downloaded from http://www.jimmunol.org/

FIGURE 1. Nutrient excess alters cellular events in the resolution of inflammation. (A–C) Time course of neutrophil (PMNs; Ly6G+F4/802) and macrophage (Mf; Ly6G2F4/80+) accumulation during acute sterile peritonitis stimulated by zymosan in WT and db/db mice, as determined by flow cytometry (n = 4/group). (D) Phagocytosis of IgG-opsonized zymosan by peritoneal macrophages isolated from WT mice fed a LF (10%) or HF (60%) diet by guest on October 2, 2021 for 12 wk (n = 4/group). (E) Phagocytosis of IgG-opsonized zymosan by RAW macrophages pretreated with plasma (10%) isolated from either WT or db/db mice (n = 3–4/group). (F) Fasting plasma levels of nonesterified FFAs in WT, db/db, and WT mice fed a HF diet (60%) for 12 wk (n = 3/group). (G) Phagocytosis of IgG-opsonized zymosan in peritoneal macrophages isolated from WT mice and incubated with BSA alone or PA-BSA (500 mM; n =3/ group). (H) Levels of nonapoptotic PMNs (annexin V2Ly6G+) in inflammatory exudates obtained from WT and db/db mice 48 h after zymosan challenge (n = 4/group). (I) Representative Western blots of cleaved caspase-3 and phosphorylated ERK1/2 in human PMNs undergoing spontaneous apoptosis and incubated without or with LPS (100 ng/ml), BSA, or PA-BSA conjugates (100 mM) for 24 h at 37˚C (representative of three independent donors). Data are means 6 SEM. *p , 0.05 by one-way ANOVA (A–C, F) or Student t test (D, E, G, H).

lution, we profiled the production of lipid mediators during the PGs such as PGE2 and PGD2 have well-defined roles in acute development of acute peritonitis in WT and db/db mice using a inflammation, such as the regulation of blood flow, cytokine gen- targeted LC-MS/MS–based approach. This analysis revealed that eration, and leukocyte trafficking (20, 32). Additionally, both PGE2 6 h after zymosan challenge, the arachidonic acid–derived proin- and PGD2 can counterregulate leukocyte phagocytosis (33, 34). flammatory lipid mediators LTB4, 12-HETE, and PGE2/D2 were Therefore, to determine whether the production of these prostanoids significantly elevated in the peritoneal exudates of db/db mice is causally related to phagocyte defects induced by nutrient excess, compared with WT mice, whereas the levels of 15-HETE were we examined how the production of these mediators was affected by significantly decreased (Fig. 2A). By 24 h, the production of LTB4 PA in macrophages. Inhibition of phagocytosis by PA was concen- and 12-HETE largely abated, whereas PGE2/D2 levels remained tration dependent and we found that PA at concentrations $300 mM significantly elevated (Fig. 2B). Interestingly, this pattern persisted (0.3 mEq/l) induced significant defects in the uptake of IgG- for 48 h (Fig. 2C), at a time when more leukocyte accumulation opsonized zymosan by RAW macrophages (Fig. 3A, 3B). These was observed in db/db mice than in WT mice (see Fig. 1). The observations demonstrate that at concentrations measured in the increase in PGE2/D2 appears to be selective, because other pros- plasma of obese mice (see Fig. 1F), PA prevents macrophage tanoids, such as PGF2a and thromboxane (measured as throm- phagocytosis. To examine how FFAs affect lipid mediator produc- boxane B2), were not significantly elevated throughout the time tion, we assessed mRNA expression of genes encoding the bio- course of peritonitis. Overall, this unbiased approach revealed synthetic enzymes of lipid mediators profiled in inflammatory that, although the levels of several lipid mediators were affected exudates, including Ptgs1, Ptgs2, mPges1, hPgds, Alox5,and initially, the production of COX-2–derived prostanoids, PGE2/D2, Alox15. This analysis revealed that Ptgs2, encoding murine COX-2, was increased throughout the entire time course and this increase and downstream prostanoid synthase, hPgds, were selectively and coincided with PMN accumulation. This suggests that the increase significantly upregulated in macrophages treated with PA-BSA, but in these prostanoids may be related to delayed resolution of in- not BSA alone (Fig. 3C). Consistent with this observation, levels of flammation in obese mice. COX-2 protein were also significantly increased by PA-BSA (Fig. The Journal of Immunology 5

and DP1/2, respectively, and that activation of these receptors mediates their biological effects (20, 32). In alveolar macrophages, inhibition of phagocytosis by PGE2 has been shown to be mediated primarily by EP2, whereas less is known about the role of the PGD2 receptor, DP1, in macrophage phagocytosis (34). Both EP2 and DP1 couple to Gas and activate the cAMP pathway (32). Accumulation of cAMP in turn activates downstream kinases, including PKA and the exchange protein activated by cAMP-1 (Epac-1). To test the involvement of this pathway, we incubated macrophages with cAMP analogs that specifically activate either the PKA-dependent pathway (6-bnz-cAMP) or the Epac-1 pathway (8-pCPT-29-O-Me- cAMP). We found that the selective PKA activator 6-bnz-cAMP significantly inhibited phagocytosis in macrophages, whereas the Epac-1 selective activator did not (Fig. 4A). To determine whether the cAMP pathway is affected by obesity, we isolated resident peritoneal macrophages from WT and db/db mice and found that cAMP levels were significantly elevated in db/db macrophages compared with WT cells (Fig. 4B). Moreover, both EP2 and DP1 receptors were expressed in macrophages isolated from both WT and db/db mice (Fig. 4C), whereas EP4 was not readily detected Downloaded from (data not shown). Quantification of band intensities revealed that whereas DP1 levels are maintained in WT and db/db macrophages, the EP2 levels were significantly decreased (Fig. 4C, lower panel). Because our LC-MS/MS analysis revealed that PGE2/D2 are significantly elevated in inflammatory exudates isolated from obese- http://www.jimmunol.org/ diabetic mice, we next sought to determine whether blocking PGE2 and PGD2 receptors would alter the accumulation of leukocytes in the inflamed peritoneum. To this end, peritonitis was initiated in FIGURE 2. Altered resolution of inflammation in obesity is associated obese-diabetic mice using zymosan, and leukocytes were allowed to with increased prostanoid generation. (A–C) Peritoneal exudates were accumulate (see scheme in Fig. 4D and results in Fig. 1B). After collected from WT or db/db mice during the time course of acute sterile 32 h, the EP2/DP1 dual receptor antagonist AH6809 was adminis- peritonitis. Targeted lipidomics analysis was carried out on cell-free exu- tered by i.p. delivery and leukocyte differentials in inflammatory dates using LC-MS/MS following solid phase extraction (n = 3-4/group/ exudates were determined 48 h after the zymosan challenge, the time point). Data are means 6 SEM and normalized to exudate volume. time at which PMN accumulation was maximal in db/db mice (see by guest on October 2, 2021 Recovery was calculated based on internal standard, deuterium-labeled Fig. 1C). We found that mice treated with AH6809 had significantly , PGE2.*p 0.05 by Student t test. TXB2, Thromboxane B2. less PMNs remaining in the peritoneum as compared with mice given the vehicle alone, although the number of macrophages was 3D). To determine whether this increase in COX-2 expression re- unaffected (Fig. 4E). We also performed LC-MS/MS analysis on sults in an increase in prostanoid production, we measured these cell-free exudates to determine whether levels of prostanoids were lipid mediators in supernatants of cells stimulated with BSA alone affected by the treatment. As shown in Fig. 4F, levels of PGE2/D2 or PA-BSA. The results of this analysis showed that incubation with were not significantly different in vehicle- and AH6809-treated PA led to a significant increase in PGE2/D2 levels (Fig. 3E), indi- mice, suggesting that blocking downstream receptors for these cating that induction of COX-2 is associated with increased prota- mediators is sufficient to blunt their effects despite their continued sonoid production. Previous studies in alveolar macrophages show production. that prostanoids, including PGE2, decrease phagocytosis (34, 35). Because the results of these studies suggested that elevated pro- Therefore, to test this in our experimental conditions, we pre- duction of prostanoids alters macrophage phagocytosis in obesity incubated macrophages with vehicle alone, PGE2, or PGD2 and and that this in turn could alter the clearance of PMNs, we tested assessed the phagocytosis of IgG-opsonized zymosan. Consistent whether increased prostanoid generation in obesity alters macro- with previous reports, we found that both PGE2 and PGD2 (100 nM) phage-dependent clearance of apoptotic cells in vivo. We assessed significantly decreased phagocytosis at concentrations similar to the clearance of apoptotic cells in the thymus, because this process that generated by the cells in the presence of PA (∼85 nM; Fig. 3F). is dependent on resident macrophages and is not complicated by To examine whether PGE2/D2 production is causally related to PA- changes in infiltrating cells (23). To measure macrophage-mediated induced deficits in macrophage phagocytosis, we tested whether clearance, we used dexamethasone to induce thymocyte apoptosis inhibition of COX1/2 or antagonism of prostanoid receptors specific in WT and db/db mice. At the same time, we also treated these mice for PGE2 and PGD2 (EP2 and DP1, respectively) would abolish this with vehicle or prostanoid receptor antagonist AH6809 and mea- defect. Indeed, pretreatment with ASA (aspirin) or prostanoid re- sured the accumulation of apoptotic cells after 6 h. Consistent with ceptor antagonist AH6809 significantly reversed PA-induced defi- our previous report (25), we found higher levels of apoptotic cell cits in macrophage phagocytosis (Fig. 3G, 3H), suggesting that accumulation in db/db mice compared with WT mice (Fig. 4G); autocrine actions of prostanoids play a causal role in this process. however, administration of AH6809 significantly reduced the ac- Given our results showing that PA stimulates macrophage pros- cumulation of apoptotic cells in the thymus of db/db but not WT tanoid production and that this is related to defects in phagocytosis, mice. Importantly, the levels of apoptotic cells in AH6809-treated we examined the downstream signaling pathways that could be db/db mice were similar to those observed in WT mice, suggesting involved in this process. It has been shown before that both PGE2 and that deficits in macrophage clearance of apoptotic cells in obesity PGD2 specifically activate the G protein–coupled receptors EP1–4 could be attributed to prostanoids. Taken together, these results 6 PGs AND ALTERED INFLAMMATION IN OBESITY

FIGURE 3. PA impairs macrophage phagocytosis by increasing PG generation. (A) Representative images of RAW macrophages undergoing phagocytosis of FITC- labeled IgG-opsonized zymosan and counterstained with PE-conjugated anti-F4/80 Abs following incuba- tion with BSA alone or PA (500 mM)-BSA conjugates. Original magnification, 320 (upper panels) and 340 (lower panels). (B) Concentration-dependent effects of PA-BSA on phagocytosis in RAW macrophages com- pared with cells treated with BSA alone (n = 3–6/ group). (C) Analysis of mRNA expression of Ptgs1 (COX1), Ptgs2 (COX2), mPges1, hPgds, Alox5 (5- LOX), and Alox15 (15-LOX) in RAW macrophages incubated with BSA alone or PA-BSA (500 mM; n =6/ group). (D) Representative Western blot of COX-2 expression in RAW macrophages incubated with BSA alone or PA-BSA. Right panel, Quantification of total

COX-2 protein normalized to b-actin (n = 3/group). (E) Downloaded from

Quantification of PGE2/D2 in supernatants collected from RAW macrophages incubated with BSA alone or PA-BSA (500 mM) by LC-MS/MS analysis (n = 3). (F) Phagocytosis of IgG-opsonized zymosan in RAW macrophages stimulated with vehicle, PGE2 (100 nM), or PGD2 (100 nM; n = 6–7). (G) Phagocytosis of IgG- opsonized zymosan in RAW macrophages incubated http://www.jimmunol.org/ with BSA or PA-BSA (300 mM) in the presence or absence of COX inhibitor ASA (200 mM) (n = 5–8/ group). (H) Phagocytosis as in (G) in the presence or absence of prostanoid receptor antagonist AH6809 (100 mM) (n = 4/group). Data are means 6 SEM. *p , 0.05 by one-way ANOVA (B, G, H) or Student t test (C–F). by guest on October 2, 2021 suggest that excessive prostanoid production may be an important 5). Whereas shifts in glucose metabolism can arise from increased underlying reason for the delayed resolution of inflammation under fatty acid flux (i.e., the Randle cycle), it is also becoming apparent conditions of nutrient excess such as obesity and diabetes. that FFAs can initiate inflammatory signaling and that inflam- mation links obesity with the development of insulin resistance Discussion (8). Indeed, insulin resistance induced by direct FFA infusion is The major findings of this study are that elevated levels of free fatty largely prevented in mice deficient in Tlr4, a pattern recognition acids that accompany metabolic diseases such as obesity and di- receptor involved in sensing Gram-negative bacteria (9). More- abetes alter resolution of acute inflammation by promoting PMN over, FFAs activate inflammatory signaling, including the NF-kB survival and blocking macrophage phagocytosis. We found that pathway, and increase the production of inflammatory cytokines in in macrophages, the saturated fatty acid PA induced COX-2 ex- the vasculature and in leukocytes, and these effects are regulated pression and promoted the generation of PGE2/D2, which in turn in part by TLR receptor (i.e., TLR2 and TLR4) activation (10, 11). play a causal role in defective phagocytosis induced by PA. Our In addition to activating TLRs, FFAs can also induce inflammation results identify cAMP as a critical downstream effector of pros- by activating the inflammasome, as mice lacking components of tanoids in macrophages that could potentially contribute to defects the inflammasome, such as Nlrp3 and Pycard, have been shown to in phagocytosis. We found that increased generation of prosta- be resistant to the inflammatory effects of FFAs (12). Furthermore, noids during acute inflammation in obese mice coincided with the other studies have demonstrated that FFAs induce defects in accumulation of PMNs in the inflamed peritoneum, which was phagocyte function independent of TLR signaling (27). Thus, even decreased by the PGE2/D2 receptor antagonist AH6809. Overall, though several mechanisms by which FFAs can induce inflam- these studies suggest that nutrient excess alters innate immune cell matory signaling in phagocytes have been identified, the down- survival and effector functions, in part by stimulating prostanoid stream mediators of their effects on phagocyte function are not generation, and that targeting specific prostanoid receptors or their entirely known. The results of our study show that inflammatory downstream mediators may be a novel strategy for controlling signaling induced by FFAs leads to the generation of lipid medi- inflammation in obese or diabetic individuals. ator autacoids that regulate phagocytosis and that these mediators It is well documented that obesity and diabetes are associated might be involved in defective resolution of acute inflammatory with an increase in circulating levels of FFAs, due in part to in- events in the context of nutrient excess. creased lipolysis in adipocytes. This increase in plasma FFA levels Macrophage phagocytosis is required for the immunologically contributes to insulin resistance in several tissues, including the muted process of apoptotic cell uptake and it is a critical event in skeletal muscle, which is the major site of glucose consumption (4, the active resolution of inflammation, as failed clearance of apo- The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021 FIGURE 4. Role of prostanoid receptors in macrophage phagocytosis, acute inflammation, and apoptotic cell clearance in obese-diabetic mice. (A) Phagocytosis of IgG-opsonized zymosan by RAW macrophages incubated with vehicle, selective exchange protein activated by cAMP 1 (Epac-1) cAMP analog (8-pCPT-29-O-Me-cAMP), or selective PKA cAMP analog (6-BNZ-cAMP) (n = 7–9). (B) Intracellular cAMP levels measured in resident peritoneal macrophages isolated from WT and db/db mice (n = 3–4). (C) Representative Western blots of PGD2 receptor, DP1, and PGE2 receptor, EP2, in peritoneal macrophages isolated from WT and db/db mice. Quantification of DP1 and EP2 levels in isolated WT and db/db peritoneal macrophages normalized to GAPDH is shown in the lower panel.(D) Scheme illustrating treatment protocol for in vivo administration of AH6809 during the time course of acute sterile peritonitis in db/db mice. (E) Flow cytometry analysis of PMNs (Ly6G+F4/802) and macrophages (Mf; Ly6G2F4/80+) from peritoneal exudates in vehicle and AH6809-treated db/db mice 48 h following zymosan challenge (n = 8–9/group). (F) Quantification of PGE2/D2 in peritoneal exudates of db/db mice undergoing peritonitis and treated without or with AH6809 by LC-MS/MS (n = 3–5). (G) Representative images of TUNEL+ cells (counterstained with methyl green) in the thymus of WT or db/db mice treated without or with AH6809. Scale bars, 200 mm (top panels) and 50 mm (bottom panels)(n =5/ group). Quantification of TUNEL+ cells as a percentage of the total imaged area is shown in the right panel. Data are means 6 SEM. *p , 0.05 by one-way ANOVA (A, G) or Student t test (B, C, E). ptotic cells can give rise to necrosis and resultant tissue damage (21, have well-described roles in regulating macrophage phagocytosis. 22). Recent results from our group and others show that macro- We found that PA suppresses macrophage phagocytosis in part by phage-dependent clearance of apoptotic cells is perturbed in inducing Ptgs2 (COX-2) and the downstream production of pros- obesity and diabetes and that this delays the resolution of an in- tanoids. These findings are consistent with other studies demon- flammatory insult (15, 36–39). Defects in apoptotic cell clearance strating that FFAs induce COX-2 in skeletal muscle and that are an important feature of delayed wound healing in obesity and macrophages isolated from obese/diabetic mice, as well as those diabetes, and such defects are also observed in advanced athero- treated with FFA mixtures, have elevated COX-2 levels (10, 37, sclerosis (15, 27, 39, 40). Thus, elucidation of the mechanisms by 42). Additionally, we found that phagocytosis was compromised in which nutrient excess alters this critical function of macrophages macrophages isolated from mice fed a HF diet, as well as normal is important for understanding the development of several clini- macrophages incubated with plasma collected from db/db mice. cally relevant disease features in obese and diabetic humans. These observations are consistent with a direct, model-independent In addition to phagocytosis of apoptotic cells, macrophages also effect of FFAs on macrophage function. Given that both db/db mice serve an important role in the clearance of microbial pathogens, and HF-fed mice have elevated FFA levels, these results underscore a process that is also defective in metabolic disease (41). Our the importance of nutrient excess in driving acute defects in leu- results are concordant with these findings and demonstrate further kocyte function. Although we cannot rule out effects of other cir- that nutrient excess induced defects in FcR-mediated macrophage culating mediators, such as glucose, leptin, and insulin, it is unlikely phagocytosis are due in part to enhanced production of PGs that that these mediators play a major role in the defects in macrophages 8 PGs AND ALTERED INFLAMMATION IN OBESITY observed because in our study glucose was not significantly elevated mation also delays resolution, our results suggest that the nutrient in mice fed a HF diet (data not shown). Moreover, the plasma used excess alters resolution of acute inflammation by deranging both in phagocytosis assays was collected from db/db mice at a time PMN apoptosis and their clearance by macrophages. This is im- when their fasting glucose values were within the normal range. portant because prolonged accumulation of activated leukocytes in Additionally, previous studies have shown that defects in macro- tissues can cause further tissue damage by releasing pro-oxidant phage phagocytosis induced by FFAs persist in macrophages iso- species and proteolytic enzymes (21). Indeed, prolonged and height- lated from insulin receptor–deficient mice and were not rescued by ened accumulation of leukocytes is a critical feature of delayed leptin treatment (27), indicating that the defect in macrophage wound healing in obesity and diabetes (49). Activation of MAPK phagocytosis may be leptin-independent. signaling in leukocytes, such as the ERK1/2 pathway, leads to As noted, one of the main findings of our study is that PA sup- prolonged PMN survival, and our results demonstrate that this presses phagocytosis in macrophages by inducing COX-2 and in- pathway is robustly activated by FFAs (30). The extent of activation creasing the downstream productionof PGs. These PGs play multiple of this pathway by FFAs was similar to that elicited by LPS and was roles in the acute inflammatory response, including the regulation of also associated with decreased activation of caspase-3. Of interest, blood flow, pain sensitization, thrombosis, cytokine production, and PGE2 is generated by PMNs and it promotes survival signaling in phagocytosis (20, 32, 33). At sites of inflammation, inducible COX- PMNs, as do other agonists that elevate cAMP (28, 50, 51). We did 2 is commonly upregulated and generates high local levels of spe- not observe any increase in PGE2 by FFAs in PMNs at the end of the cific prostanoids dependent on the expression of downstream syn- incubation protocol (data not shown), although we cannot rule out thases, such as mPGES-1 and hPGDS (20). We found that in that there was a transient increase in PGE2 during the 24-h incu- addition to COX-2, PA stimulated an increase in hPgds mRNA, but bation. Moreover, because PGE2 was elevated in vivo during the Downloaded from not mPges1. Additional studies using improved HPLC conditions to time at which PMNs accumulate, it is likely that PGE2 plays a dual separate PGE2 and PGD2 demonstrated that whereas RAW mac- role in promoting PMN survival, as well as blocking macrophage rophages produce more PGD2 overall, both PGE2 and PGD2 were phagocytosis in vivo. That blockade of EP2/DP1 receptors de- significantly increased with PA (data not shown). Moreover, both creased accumulation of PMNs in the inflamed peritoneum when PGE2 and PGD2 induced defects in phagocytosis, and the receptor the receptor antagonist was delivered after maximal PMN infil- antagonist used in these studies blocks both EP2 and DP1 (43). tration adds further support of this dual role for PGE2. Finally, Thus, further studies using genetic manipulation of these prostanoid because previous studies have shown that db/db mice have elevated http://www.jimmunol.org/ receptors will be required to fully elucidate the role of each mediator peripheral blood PMNs, it is plausible that sustained infiltration of in the context of obesity. Importantly, note that in addition to in- PMNs gives rise to PMN accumulation in the peritoneum (52). duction, COX-2 activity may be further stimulated by the increase in However, as pointed out above, there was no difference in maximal FFAs, as it has been shown that saturated fatty acids such as PA, PMN infiltration between WT and db/db mice, although the de- which are not COX-2 substrates, can directly bind one monomer of crease in PMN levels was much slower in db/db mice, indicating COX-2 and increase substrate turnover in the other monomer (44, that diet-induced obesity selectively affects PMN clearance. 45). Hence, it is possible that in addition to inducing COX-2 ex- In this study, we used an unbiased LC-MS/MS approach to profile pression, saturated FFAs could also potentiate prostanoid generation lipid mediators generated during the initiation and resolution of acute by guest on October 2, 2021 by directly activating COX-2. inflammation in vivo because of the well-documented temporal Prostanoids, such as PGE2 and PGD2, elicit their biological relationships between different classes of lipid mediators in regu- actions by binding to and activating specific G protein–coupled lating leukocyte trafficking (18, 53). This approach revealed that receptors. In leukocytes, PGE2 and PGD2 activate G protein–cou- specific prostanoids, PGE2/D2, are elevated upon initiation of in- pled receptors, such as EP2 and DP1, that couple to G proteins (GaS) flammation in obesity and that they persist at high levels throughout and lead to the activation of adenylate cyclase, resulting in an ele- the time when inflammation resolves in WT mice. This suggests that vation of cAMP and its downstream effectors, including PKA and normal temporal orchestration of inflammation is perturbed in Epac-1 (32, 34, 46). Our results show that cAMP analogs specific obesity and that these changes are associated with altered cellular for PKA activation induce a phagocytosis defect similar to that dynamics. Interestingly, previous studies have shown that prosta- observed with FFAs and PGE2/D2. Other studies have demonstrated noids play important roles in both the initiation and resolution of that prostanoids block phagocytosis in a cAMP-dependent manner, inflammation. Indeed, Gilroy et al. (31) reported that there are two although PKA and Epac-1 play differential roles depending on the distinct waves of COX-2–dependent production of prostanoids mode of phagocytosis and the macrophage subset. For instance, during inflammation and resolution, and additional studies have Serezani et al. (46) demonstrated that phagocytosis of Candida shown that inhibiting COX-2 delays resolution (54). Moreover, albicans was blocked by a PKA-dependent pathway, whereas other results from Levy et al. (53) show that lipid mediator class switching studies using alveolar macrophages demonstrate that blockade of during inflammation/resolution occurs as PGE2 drives biosynthesis FcR-mediated macrophage phagocytosis was dependent on Epac-1 of proresolving lipid mediators in PMNs by stimulating the up- (47). There also seems to be some differential involvement of PKA regulation of 15-lipoxygenase. Thus, the temporal relationships and Epac-1 effectors in cytokine generation and macrophage mat- between lipid mediator classes are important determinants of uration, highlighting the complexity of cAMP-dependent signaling whether inflammation will resolve or persist. Our results suggest in macrophages (48). Thus, it is tempting to speculate that cAMP that these temporal relationships are disrupted in metabolic disease elevation in obesity and diabetes could play a key role in delayed and that nutrient excess alters the innate immune response from the wound healing and inappropriate inflammatory responses, despite very initiation of inflammation. This is demonstrated by our results leukocyte accumulation in tissues. Further studies are required to showing that macrophages isolated from obese mice display defects understand fully how aberrant cAMP signaling alters innate im- in phagocytosis and have elevated levels of intracellular cAMP even munity in the context of obesity and other related chronic inflam- in the absence of an inflammatory challenge. This observation is matory diseases. consistent with other reports showing that proresolving lipid me- In addition to inducing defects in macrophage phagocytosis, we diators, such as the lipoxins and , counterregulate the found that FFAs directly prolong PMN survival. Because delayed cAMP pathway in macrophages and reduce the induction of COX-2, apoptosis, or prolonged survival, of PMNs during acute inflam- indicating that counterregulatory lipid mediators may offer an at- The Journal of Immunology 9 tractive treatment strategy in the context of obesity and T2D (15, 18, 8. Hotamisligil, G. S., and E. Erbay. 2008. 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