Novel Resolvin D2 Receptor Axis in Infectious Nan Chiang, Xavier de la Rosa, Stephania Libreros and Charles N. Serhan This information is current as of September 25, 2021. J Immunol 2017; 198:842-851; Prepublished online 19 December 2016; doi: 10.4049/jimmunol.1601650 http://www.jimmunol.org/content/198/2/842 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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Novel Resolvin D2 Receptor Axis in Infectious Inflammation

Nan Chiang,1 Xavier de la Rosa,1 Stephania Libreros, and Charles N. Serhan

Resolution of acute inflammation is an active process governed by specialized proresolving mediators, including resolvin (Rv)D2, that activates a cell surface G protein–coupled receptor, GPR18/DRV2. In this study, we investigated RvD2-DRV2–dependent resolution mechanisms using DRV2-deficient mice (DRV2-knockout [KO]). In polymicrobial sepsis initiated by cecal ligation and puncture, RvD2 (∼2.7 nmol/mouse) significantly increased survival (>50%) of wild-type mice and reduced hypothermia and bacterial titers compared with vehicle-treated cecal ligation and puncture mice that succumbed at 48 h. Protection by RvD2 was abolished in DRV2-KO mice. Mass spectrometry–based lipid mediator metabololipidomics demonstrated that DRV2-KO infectious exudates gave higher proinflammatory leukotriene B4 and procoagulating thromboxane B2, as well as lower specialized proresolving mediators, including RvD1 and RvD3, compared with wild-type. RvD2-DRV2–initiated intracellular signals were investigated using mass cytometry (cytometry by time-of-flight), which demonstrated that RvD2 enhanced phosphorylation of

CREB, ERK1/2, and STAT3 in WT but not DRV2-KO macrophages. Monitored by real-time imaging, RvD2–DRV2 interaction Downloaded from significantly enhanced phagocytosis of live Escherichia coli, an action dependent on protein kinase A and STAT3 in macrophages. Taken together, we identified an RvD2/DRV2 axis that activates intracellular signaling pathways that increase phagocytosis- mediated bacterial clearance, survival, and organ protection. Moreover, these results provide evidence for RvD2-DRV2 and their downstream pathways in pathophysiology of infectious inflammation. The Journal of Immunology, 2017, 198: 842–851.

nflammation is a protective response to defend the host conserved chemical structures derived from polyunsaturated fatty http://www.jimmunol.org/ against infection and injury (1). Ungoverned and excessive acids, including –derived E-series resolvins I inflammation is an underlying pathology of many prevalent and (DHA)–derived D-series resolvins, diseases, including cardiovascular diseases, diabetes, arthritis, and protectins, and . Complete stereochemistries of these sepsis (2–4). Complete resolution of acute inflammatory responses SPM are established and total organic synthesis achieved that also was thought to be a passive process with dissipation or dilution of confirmed their potent proresolving actions. SPM are potent me- local chemoattractants and proinflammatory mediators, allowing diators governing not only innate immune responses in host de- tissues to return to (1). In recent years, we obtained fense, but also pain, organ protection, and tissue remodeling (recently the first evidence, to our knowledge, that resolution of self-limited reviewed in Ref. 8). inflammation is not merely a passive termination, but rather an SPM derived from DHA including resolvin (Rv)D2 (7S,16R,17S- by guest on September 25, 2021 actively orchestrated programmed response that is rapidly turned trihydroxy-docosa-4Z,8E,10Z,12E,14E,19Z-hexaenoic acid) were on during acute inflammatory challenges, permitting inflamed and first identified and isolated from murine self-resolving exudates injured tissues to return via catabasis to function (5, 6). This event during the resolution phase of self-limited acute inflammation is driven in part by temporal lipid mediator (LM) class switching in vivo (12). The biosynthesis of RvD2 involves 17-lipoxygenation from generation of proinflammatory mediators (e.g., leukotriene of DHA to 17S-hydroperoxy-DHA that is further transformed en- [LT]B4) to the biosynthesis of (LX) (7) and specialized zymatically to a 7(8)epoxide-containing intermediate in leukocytes proresolving mediators (SPM) (8–11). SPM are evolutionally via 5-, followed by enzymatic hydrolysis to form RvD2. Endogenous RvD2 production is documented in human serum, plasma (13), adipose tissue (14), placenta (15), lung (16), Center for Experimental Therapeutics and Reperfusion Injury, Department of Anes- breast milk (17), and sepsis patients (18). With isolated human thesiology, Perioperative and Pain Medicine, Harvard Institutes of Medicine, Brig- polymorphonuclear (PMN), RvD2 increases intracel- ham and Women’s Hospital and Harvard Medical School, Boston, MA 02115 lular phagosomal reactive oxygen species generation for microbial 1 N.C. and X.d.l.R. contributed equally to this work. killing (19). In whole blood at a single cell level using microfluidic ORCID: 0000-0003-4627-8545 (C.N.S.). chambers, RvD2 limits PMN chemotaxis and direct travel as well Received for publication September 22, 2016. Accepted for publication November as increases random movement toward an IL-8 chemotactic gradi- 15, 2016. ent (20). RvD2 also decreases monocyte adhesion to adipocytes as This work was supported in part by National Institutes of Health Grants R01 well as their transadipose migration (14). RvD2 is a potent immu- GM38765 (to C.N.S.) and R01 GM38765-29S1 (to S.L.). noresolvent that stereoselectively reduces excessive PMN trafficking Address correspondence and reprint requests to Prof. Charles N. Serhan, Brigham and Women’s Hospital, Building for Transformative Medicine, Suite 3-016, 60 Fen- in peritonitis and improves survival in sepsis (19). RvD2’s potent wood Road, Boston, MA 02115. E-mail address: [email protected] nanogram actions are also protective in disease models where RvD2 The online version of this article contains supplemental material. prevents inflammatory bowel disease such as colitis (21), alleviates Abbreviations used in this article: BMDM, bone marrow–derived macrophage; CLP, inflammatory and fibromyalgia-induced pain (22, 23), increases cecal ligation and puncture; CyTOF, cytometry by time-of-flight; DHA, docosahexaenoic survival following burn wound and reduces kidney and liver injuries acid; KO, knockout; LM, lipid mediator; LT, leukotriene; LX, ; MMP, matrix metalloproteinase; MRM, multiple reaction monitoring; PKA, protein kinase A; PMN, in mice (24, 25), and reduces periodontitis (26) as well as nerve polymorphonuclear ; Rv, resolvin; RvD2, resolvin D2 (7S,16R,17S-trihydroxy- injuries as seen in Parkinson disease (27). docosa-4Z,8E,10Z,12E,14E,19Z-hexaenoic acid); SPM, specialized proresolving media- Resolution at the cellular level consists of cessation of PMN tor; STZ, serum-treated zymosan particle; TX, thromboxane; WT, wild-type. entry into the tissue and elevated (i.e., macrophage Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 phagocytosis of apoptotic PMN) (1). We introduced a quantitative www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601650 The Journal of Immunology 843

definition of resolution of self-limited sterile acute inflammation d4-5S-HETE, d4-PGE2, and d5-RvD2 internal standards (500 pg each) denoted resolution indices that permits assessment of the resolu- were added to facilitate quantification. All samples were kept at 220˚C for tion properties of SPM and pinpoints their unique mechanisms of 45 min to allow protein precipitation and then subjected to solid-phase extraction as described (13). Extracted samples were analyzed by a liquid action (6). SPM each lower the magnitude of leukocyte infiltration chromatography–tandem mass spectrometry system (QTRAP 5500; AB and/or shorten the resolution interval, which is the interval from Sciex) equipped with an LC-20AD HPLC (Shimadzu, Tokyo, Japan). A the time point of maximum PMN infiltration to the time point of Poroshell 120 EC-18 column (100 mm 3 4.6 mm 3 2.7 mm; Agilent 50% PMN reduction in peritoneal exudates (17, 28). We also Technologies, Santa Clara, CA) was kept in a column oven maintained at 50˚C, and LM were eluted with a gradient of methanol/water/acetic acid assessed the actions of SPM in resolution of Escherichia coli in- from 55:45:0.01 (v/v/v) to 100:0:0.01 at 0.5 ml/min flow rate. To monitor fection using these resolution indices together with cellular and quantify the levels of targeted LM, a multiple reaction monitoring composition and functions (29). During E. coli infection, RvD2 (MRM) method was devised with signature ion fragments for each mole- given at the peak of PMN infiltration (12 h) significantly shortens cule. Identification was conducted using published criteria including reten- the resolution interval by half and enhances PMN apoptosis and tion times and at least six diagnostic ions. Calibration curves were obtained using synthetic and authentic LM mixtures, including d4-LTB ,d5-LXA, macrophage efferocytosis in self-limited E. coli infection (30). 4 4 d4-PGE2, d5-RvD2, RvD1, RvD2, RvD3, RvD4, RvD5, PD1, MaR1, RvE1, 2a Based on these potent proresolving actions of SPM, we proposed RvE2, LXA4,LXB4,PGE2,PGD2,PGF , thromboxane (TX)B2,andLTB4 the “cardinal signs of resolution” including clearance of debris at 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 pg. Linear calibration curves for 2 (expurgatio reliquiorum), clearance of infective agents (expurgatio each compound were obtained with r values of 0.98–0.99. Quantification was carried out based on peak areas of the MRM transitions. contagionem agentis), analgesia (doloris absentia), and gain of function (muneris lucrum) (31). With these cellular, molecular, Proteome profiler array and quantitative definitions, we now appreciate the mechanisms Peritoneal lavages were collected as described at 12 h after CLP. Cell-free Downloaded from controlling the active resolution programs of inflammation and supernatants were collected by centrifugation. A 1:10 dilution of the su- have pinpointed the proresolving actions of SPM. pernatant (150 ml) was incubated with the precoated Proteome Profiler array Polymicrobial sepsis is a complex scenario containing phases of membranes (ARY028; R&D Systems) and processed according to the manufacturer’s instructions. Densitometric analysis of dot blots was per- both excessive inflammatory responses temporally associated with formed using ImageJ software (National Institutes of Health, Bethesda, MD). immunosuppressive states (32). Sepsis remains an unmet clinical Real-time imaging of phagocytosis challenge with high mortality rates and increasing incidence (33, http://www.jimmunol.org/ 34). Recently, we identified a G protein–coupled receptor for RvD2 Mouse bone marrow cells were collected and differentiated to macrophages termed DRV2/GPR18 and demonstrated specific binding of RvD2 to with mouse GM-CSF (10 ng/ml) for 6 d. These macrophages were plated 5 human recombinant DRV2 (30). In human macrophages, RvD2 onto eight-well chamber slides (0.5 3 10 cells per well) overnight before stimulates phagocytosis and efferocytosis in a DRV2-dependent the experiments. Chamber slides were kept in a Stage Top incubation system for microscopes equipped with a built-in digital gas mixer and manner. RvD2–DRV2 interaction stimulates phagocyte functions temperature regulator (Tokai Hit model INUF-K14). Cells were treated to accelerate resolution of bacterial infections. In the present study, with RvD2 (10 nM), PKA inhibitor H89 (3 mM; Sigma-Aldrich), RvD2 we tested the hypothesis that the DRV2 receptor is pivotal in sys- plus H89, or vehicle control for 15 min at 37˚C, followed by addition of temic infection and report that RvD2–DRV2 interactions stimulated BacLight Green–labeled E. coli at a proportion of 50:1 (E. coli/macro-

phage) to initiate phagocytosis. Fluorescent images were then recorded by guest on September 25, 2021 bacterial clearance and reduced mortality in microbial sepsis. This every 10 min for 100 min (37˚C) with Keyence BZ-9000 (Biorevo) ligand–receptor activation initiates specific intracellular signal inverted fluorescence phase-contrast microscope (320 objective) equipped transduction pathways (e.g., protein kinase A [PKA], STAT3) that with a monochrome/color switching camera using BZ-II Viewer software contribute to RvD2-DRV2–stimulated phagocytosis of live E. coli. (Keyence). Three separate experiments were performed. In each experi- ment, three fields (320) per condition (per well) were recorded. Green fluorescence intensity was quantified using a BZ-II analyzer. Materials and Methods Cecal ligation and puncture cAMP levels For cAMP measurements, resident peritoneal macrophages from naive WT Targeted deletion of mouse gpr18 (NM_182806) was constructed by 2 2 or DRV2-KO mice were collected with 5 ml of PBS / and plated in a Lexicon Pharmaceuticals in a 129/SvEv-C57B/6 mixed background (30). 12-well plate (0.5 3 106 cells per well) with RPMI 1640 supplemented Male mice (10–12 wk old) were used for all experiments and fed ad with 10% FBS. The next day, media were replaced with PBS+/+ and cells libitum laboratory rodent diet 20-5058 (Lab Diet; Purina Mills). All ex- incubated with RvD2 (0.1–100 nM), forskolin (10 mM; Sigma-Aldrich), or perimental procedures used were approved by the Standing Committee on vehicle control for 15 min at 37˚C. Lysis buffer (100 ml) was added to stop Animals of Harvard Medical School (protocol no. 02570) and complied incubation and cells were homogenized. cAMP levels were measured by with institutional and US National Institutes of Health guidelines. Cecal ELISA following the manufacturer’s instruction (Elite cAMP ELISA assay ligation and puncture (CLP) was carried out essentially as in Spite et al. kit; eEnzyme, CA-C315). (19). Mice were anesthetized with a mixture of oxygen and isoflurane 5% for anesthesia induction and 1.5% isoflurane for maintenance during sur- Macrophage phagocytosis of E. coli gery. After mice were shaved, basal temperature was monitored. A cut (0.5 cm) midline incision to the mouse’s left side was performed; cecum Resident peritoneal macrophages were collected from naive WTand DRV2- was pulled out and ligated below the ileocecal valve with a 4-0 silk suture. KOmiceandplatedonto96-wellplates(0.53 105 cells per well). RvD2 (1 pM Two punctures were performed with a 20-gauge needle, followed by a soft to 10 nM) or vehicle controls were incubated with macrophages for 15 min squeeze until stool was extruded. Cecum was then put back to the peri- at 37˚C, followed by incubation with FITC-labeled serum-treated zymosan toneal cavity and animals were sutured with a 6-0 silk suture. Mice then particles (STZ) at 10:1 ratio (zymosan/macrophage) or BacLight Green– received 1 mg of RvD2 i.p. or 500 ml of saline with 0.1% ethanol (vehicle), labeled E. coli at a 50:1 ratio (E. coli/macrophage) for 60 min at 37˚C. and survival was monitored during 120 h. In a second group of animals, Plates were gently washed, extracellular fluorescence was quenched by after 12 h of CLP, peritoneal exudates were collected by lavaging with 5 ml trypan blue, and phagocytosis was determined by measuring total fluo- of PBS2/2. Peritoneal bacterial titers were determined by plating lavages rescence (excitation 493/emission 535 nm) using a fluorescent plate reader on Luria–Bertani agar plates at 1003 dilution, and the CFU were counted (Molecular Probes). The following inhibitors were added together with 24 h later. Exudate cytokine levels were determined by proteome profiler RvD2 or vehicle control: PKA inhibitor H89 (3 mM; Sigma-Aldrich), mouse cytokine array following the manufacturer’s instruction. pERK1/2 inhibitor (50 mM; Tocris Bioscience), and STAT3 inhibitor (100 mM; Tocris Bioscience) LM metabololipidomics Mass cytometry Liquid chromatography–tandem mass spectrometry-based metab- ololipidomics were performed with infectious exudates. Prior to sample Peritoneal macrophages from DRV2-KO mice and WT mice were col- extraction, ice-cold methanol containing deuterium-labeled d4-LTB4, lected as in Chiang et al. (30). Peritoneal cells were incubated with RvD2 844 INNATE ANTIMICROBIAL FUNCTION OF HOST RESOLVIN D2

(10 nM) for 0, 1, 5, 15, and 30 min at 37˚C, followed by 1.6% para- Statistical analysis formaldehyde for 10 min at room temperature. Cells were barcoded following the manufacture’s protocol with palladium isotopes (Pd 102, Statistical analysis was performed using a Student t test (two-group 104, 105, 108, and 110) (Fluidigm Sciences). Briefly, cells were washed comparisons) and one-way ANOVA (multiple-group comparisons). twice using barcoding permeabilization buffer. Diluted barcodes were Kaplan–Meier survival curves were analyzed using a one-tailed log- , transferred to cells and incubated for 30 min at room temperature. rank (Mantel–Cox) test. In all cases a p 0.05 was considered Barcoded cells were washed twice in cytometry by time-of-flight significant. (CyTOF) staining buffer (PBS with 0.5% BSA and 0.1% sodium azide) and then pooled for staining. Pooled barcoded cells were incu- bated for 10 min with Fc Block (BioLegend, San Diego, CA) for Fc Results receptor–mediated nonspecific Ab binding. Cells were then stained for RvD2 protects mice from sepsis in a GPR18/DRV2 30 min with metal-labeled surface Abs at room temperature, then receptor–dependent manner washed twice in CyTOF staining buffer. Cells were then permeabilized in 80% ice-cold methanol for 10 min at 220˚C. After washing twice to Because RvD2 displays potent actions and RvD2 receptor, namely remove the methanol, cells were stained with metal-conjugated Abs for GPR18/DRV2, was identified (30), we set out to investigate whether intracellular markers at room temperature for 30 min. The Abs used for the RvD2/DRV2 receptor axis is protective in polymicrobial sepsis. CyTOF are listed in Supplemental Table III. Cells were then washed Using mice deficient in DRV2, we carried out CLP, a polymicrobial twice and stained in 500 ml of 1:1000 iridium intercalator (DVS Sci- ences, Toronto, ON, Canada) diluted in PBS overnight at 4˚C. Cells were systemic sepsis model that closely resembles human pathology then washed twice in CyTOF staining buffer and twice in MilliQ-filtered (37). In WT animals, RvD2 significantly increased survival (.50%) deionized water (35). Cells were reconstituted at a concentration of 5 3 in CLP compared with vehicle-treated mice that all perished before 6 10 cells/ml containing EQ calibration beads (EQ four elements cali- 48 h (Fig. 1A, upper panel). In contrast, in DRV2-deficient mice bration beads; Fluidigm Sciences) according to the manufacturer’s (DRV2-KO), there were no significant differences in survival be- Downloaded from protocol. Barcoded cells were analyzed on a Helios CyTOF (Fluidigm Sciences) at an event rate of 400–500 cells/s. The data were normalized tween RvD2- and vehicle-treated mice (Fig. 1A, bottom panel). We using v6.3.119 Helios software (Fluidigm Sciences) at the LMA CyTOF noted that with vehicle-treated CLP mice, DRV2-KO mice suc- facility at Dana Farber Cancer Institute (Boston, MA). Files were cumbed earlier (12 h) than did WT mice (24 h). Both WT and KO debarcoded using a Fluidigm debarcoder application. Gating was per- mice reached ∼50% mortality between 24 and 30 h. At 48 h the formed in a Cytobank platform (Cytobank, Mountain View, CA). Se- quential gating strategy was used to analyze signaling events in resident remaining WT mice perished whereas KO mice had 20% survival. peritoneal macrophages (CD11b+F4/80+). Phosphorylation levels of A log-rank test comparing the survival distributions of these two http://www.jimmunol.org/ signaling molecules were determined at 0, 1, 5, 15, and 30 min after groups (WT CLP plus vehicle versus KO CLP plus vehicle) did not exposure of RvD2 (10 nM) in WT and DRV2-KO macrophages. Phos- reach statistical significance (p = 0.87). It is possible, however, that phorylation levels were calculated as the difference between the inverse there was genetic and/or functional compensation in DRV2-KO hyperbolic sine of the median signal intensity at indicated time points and the inverse hyperbolic sine of the median signal intensity in the mice, for example, expressions of other proresolving receptors unstimulated (0 min) signal (36). and/or proinflammatory receptors might be altered in DRV2-KO by guest on September 25, 2021

FIGURE 1. RvD2 protects poly- microbial sepsis in a DRV2-dependent manner. Mice were administered with RvD2 (1 mg) or vehicle i.p. following CLP. (A) Percentage survival of (up- per) WT or (bottom) DRV2-KO mice. Results are from 10 mice in each group. **p , 0.01 log-rank (Mantel– Cox) test. (B) Changes in body tem- peratures 24 h after CLP. Results are expressed as mean 6 SEM from five to eight mice per group. *p , 0.05, CLP plus vehicle versus CLP plus RvD2 in WT group. #p , 0.05, versus naive mice using an unpaired Student t test. (C) Twelve hours after CLP, exudates were collected and microbial counts were determined. Results are expressed as mean 6 SEM from four to six mice per group. *p , 0.05, CLP plus vehicle versus CLP plus RvD2 in WT group using an unpaired Student t test. The Journal of Immunology 845 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. LM metabololipidomics: DRV2-KO versus WT mice. (A) MRM chromatographs. (B) Representative MS-MS of RvD1. (C) Principal component analysis of exudates LM three-dimensional loading plot. Exudate levels of (D) prostanoids (PGs, TxB2), (E)LTB4 plus 5-HETE, and (F) D-series resolvins (RvDs) (pg/ml) in WT and DRV2-KO mice 12 h after CLP are shown. Results are expressed as mean 6 SEM from four to five mice per group. *p , 0.05, WT versus DRV2-KO using unpaired Student t test. animals. These could contribute to the apparent different outcome CLP mice, we identified resolvins and other SPM, specifically eico- than that with WT mice survival following CLP. sapentaenoic acid–derived E-series resolvins, DHA-derived D-series We monitored body temperature at 24 h after CLP, which induced resolvins, protectins, and , as well as arachidonic acid–derived hypothermia in mice compared with naive mice. RvD2 reduced hy- LT, prostanoids, and LX (Fig. 2A, Supplemental Table I). A repre- pothermia (35.5 6 0.8˚C) compared with vehicle controls (31.7 6 sentative tandem mass spectrometry spectrum of RvD1 used for 1.2˚C) in WT mice but not in DRV2-KO mice (Fig. 1B). Addition- identification is shown in Fig. 2B. Principal component anal- ally, in WT mice RvD2 enhanced bacterial killing, significantly ysis was used for exploring cross-covariance between WT and reducing bacterial titers (105.5 6 32.3 CFU/cm2) compared with the DRV2-KO. The three-dimensional loading plot showed two distinct vehicle-treated group (234.8 6 82.3 CFU/cm2), a response dimin- clusters, one with mostly proinflammatory and procoagulating LM ished in DRV2-KO mice (Fig. 1C). Thus, RvD2–DRV2 interactions (i.e., PG, LT, and TX) that was associated with DRV2-KO. in vivo increased survival in CLP mice, protected hypothermia, and The other cluster contained proresolving mediators, for example, enhanced bacterial clearance from infectious exudates. RvE1–3, RvD1–5, PD1, MaR1, and LXA4, that were associated with WT animals (Fig. 2C). We quantified each LM and found LM metabololipidomics and proteome profiling significant increases ∼85% in proinflammatory and procoagulating We next questioned whether DRV2-KO gives heightened inflammatory LM, for example, PG plus TX (Fig. 2D) and LTB4 plus 5-HETE status in sepsis, and carried out mass spectrometry–based metab- (∼95% increase; Fig. 2E) in DRV2-KO compared with WT mice. ololipidomics focusing on local acting LM. Each LM was profiled Additionally, DRV2-KO mice showed significant decreases of ∼60% using multiple reaction monitoring and identified by direct comparison in D-series resolvins, including RvD1, AT-RvD1, RvD3, and AT-RvD3, with synthetic and authentic standards using matching criteria, in- compared with WT littermates (Fig. 2F). cluding retention time, characteristic fragmentation patterns, and at Next, we carried out proteome profiling for CLP. Cell-free su- least six diagnostic ions (13). In infectious exudates obtained from pernatants from infectious exudates showed significant upregulation 846 INNATE ANTIMICROBIAL FUNCTION OF HOST RESOLVIN D2 Downloaded from

FIGURE 3. RvD2 regulates se- http://www.jimmunol.org/ lected proteins in a DRV2-dependent manner. Proteome Profiler cytokine array was carried out using 12 h CLP infectious exudates. (A) Results are expressed as relative abundance to reference spots in a heat map with 111 proteins. (B)LevelsofMMP2,MMP3, and myeloperoxidase (MPO); mean 6 SEM from three to six mice per group.

Insets show representative dot plot im- by guest on September 25, 2021 ages. *p , 0.05, CLP versus CLP plus RvD2 in WT group. ##p , 0.01, CLP plus RvD2 in WT versus KO groups using an unpaired Student t test. The Journal of Immunology 847

FIGURE 4. RvD2-DRV2–dependent macro- phage intracellular signaling. (A) Heat maps of phosphorylated signaling molecules at 0, 1, 5, 15, and 30 min after exposure of RvD2 (10 nM) in WT and DRV2-KO macrophages were ob- tained using CyTOF (see Materials and Meth-

ods). Phosphorylation levels were calculated as Downloaded from the difference between the inverse hyperbolic sine (arcsinh) of the median signal intensity in RvD2-treated peritoneal resident macrophages (at 0, 1, 5, 15, and 30 min) and the arcsinh of the median signal intensity in vehicle-treated mac- rophages at 0 min. (Bottom) Gating strategy for + + macrophages (CD11b F4/80 ) and representa- http://www.jimmunol.org/ tive histograms of pCREB. (B) Flow cytometry for pCREB: gating strategy for macrophages (CD11b+F4/80+) and representative histograms. (C) Relative intensity of pERK1/2, pSTAT3, and pCREB. Results are mean 6 SEM from three independent experiments, and samples were pooled from five mice in each group (WT ver- sus DRV2-KO) for each experiment. *p , 0.05, **p , 0.01, WT plus RvD2 versus DRV2-KO plus RvD2 using a two-tailed Student t test. by guest on September 25, 2021

of a panel of cytokines compared with naive mice at 12 h with obionts via regulation of the complement system, promoting resistance both WT and DRV2-KO mice (Fig. 3A). RvD2 treatment signif- after pathogen-induced intestinal damage (39). Reg3G restricts bacte- icantly upregulated a panel of proteins, including matrix metal- rial colonization of mucosal surfaces and reduces bacterial transloca- loproteinase (MMP)-2, MMP-3, and myeloperoxidase in WT tion (40). Pentraxin 3/TSG-14 improves survival in endotoxic shock (Fig. 3B, Supplemental Table II). However, these cytokines were and CLP (41). Therefore, reduction in these proteins in KO-RvD2 not significantly altered in DRV2-KO by RvD2 treatment. It was mice might contribute to their higher mortality rate and impaired reported that loss of MMP-2 leads to increases in MCP-3 levels bacterial clearance compared with the WT-RvD2 group (Fig. 1A, 1C). and exacerbates myocarditis in mice, pointing to a potential pro- Taken together, our results demonstrated that DRV2-KO mice tective role of MMP-2 via its function in chemokine cleavage (38). showed dysregulated LM profiles, giving heightened proinflammatory Additionally, we carried out statistical analysis between WT-RvD2 LM and reduced SPM compared with WT in sepsis. Also, RvD2 and KO-RvD2 groups and found statistically significant reduction in regulates a selected panel of inflammation-related proteins in a DRV2- several proteins in KO mice compared with WT mice. These include dependent manner. IL-22, LIX (CXCL5), MMP-3, MMP-9, Pentraxin 2/SAP, Pentraxin 3/TSG-14, Reg3G, and Serpin F1 (Supplemental Table II). These RvD2-DRV2–dependent intracellular signaling proteins have been reported to have protective roles in regulating im- Next, we investigated RvD2-DRV2–initiated intracellular signals mune responses during infections. For example, IL-22 controls path- using mass cytometry (CyTOF) with naive mouse peritoneal 848 INNATE ANTIMICROBIAL FUNCTION OF HOST RESOLVIN D2 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. PKA and STAT3 pathways mediates RvD2-stimulated phagocytosis. (A) Differentiated mouse bone marrow macrophages collected from WT and DRV2-KO mice were plated onto eight-well chamber slides (0.5 3 105 cells per well) and incubated with vehicle or RvD2 (10 nM) for 15 min at 37˚C, followed by addition of BacLight Green–labeled E. coli to initiate phagocytosis. Fluorescent images were then recorded every 10 min. Three separate experiments were performed. In each experiment, three fields (320) per condition (per well) were recorded. Results are mean fluorescent intensity (MFI); mean from three independent experiments with three mice per group in triplicates (three fields per well) is shown. *p , 0.05 using unpaired Student t test. Insets, Representative fluorescent images at 100 min; scale bar, 50 mm. (B) Mouse resident naive macrophages were collected from WT and DRV2-KO mice and incubated with 0.1–100 nM RvD2 for 15 min and cAMP levels were determined. Results are expressed as percentage increase of cAMP. cAMP levels in the presence of forskolin (10 mM) was taken as 100%; mean 6 SEM from n =3.*p , 0.05, **p , 0.01, WT versus (Figure legend continues) The Journal of Immunology 849

markedly increased as early as 1 min, and their levels remained el- evated until 15–30 min. In macrophages from DRV2-KO mice, up- regulation of these phospho-proteins by RvD2 was abolished. In separate sets of experiments, pCREB levels were validated using flow cytometry (Fig. 4B). Quantification of pERK1/2, pSTAT3, and pCREB using CyTOF and flow cytometry are shown in Fig. 4C. These results indicate that RvD2 regulates phosphorylation of se- lected kinases and transcription factors with different kinetics in macrophages in a DRV2-dependent manner.

STAT3 and PKA pathways mediates RvD2-DRV2–stimulated phagocytosis Next, we investigated the role of selected signaling components in RvD2-DRV2–stimulated phagocytosis identified using CyTOF, including PKA (that phosphorylates CREB), STAT3 (that is in- volved in phagosome maturation), and ERK. First, we monitored phagocytosis of live fluorescence-labeled E. coli by bone marrow– derived macrophages (BMDM) using real-time imaging. Fluo- rescence intensity that increased with time (0–120 min) represents Downloaded from increased macrophage ingestion of E. coli (Fig. 5A). RvD2 ad- dition prior to E. coli (10 nM, 15 min) significantly enhanced phagocytosis (∼95% at 100 min; Fig. 5A). This RvD2 action was not observed in DRV2-KO BMDM, indicating the role of the RvD2/DRV2 axis in stimulating bacterial clearance via phagocy-

tosis. Because cAMP activates PKA, which can phosphorylate http://www.jimmunol.org/ pCREB (42), we determined cAMP levels with naive peritoneal resident macrophages and found that RvD2 (10–100 nM) signif- icantly increased cAMP levels in WT but not DRV2-KO (Fig. 5B). A suboptimal concentration of forskolin (10 mM) was used as a control and taken as 100% (Fig. 5B). This is consistent with earlier findings that RvD2 (10–100 nM) increases cAMP levels that are dependent on DRV2 in human macrophages (30). We next questioned whether PKA, STAT3, and ERK1/2 play a role by guest on September 25, 2021 FIGURE 6. Schematic representation of RvD2-DRV2–dependent signaling in RvD2-stimulated phagocytosis. RvD2 incubation prior to E. coli pathways involved in macrophage phagocytosis. RvD2–DRV2 interactions addition (10 nM, 15 min) significantly enhanced phagocytosis. In- initiate 1) Gas protein coupling, leading to activation of the cAMP/PKA cubation of macrophages with a STAT3 inhibitor (NSC 74859; signaling pathway, and 2) phosphorylation of STAT3, which contributed to 100 mM) or PKA inhibitor (H89; 3 mM) together with RvD2 sig- macrophage phagocytosis. nificantly reduced RvD2-stimulated phagocytosis (Fig. 5C). Similar results were obtained with BMDM, where H89 also reduced RvD2- macrophages. Macrophages collected from WT and DRV2-KO mice enhanced phagocytosis of live E. coli. In comparison, an ERK m were incubated with RvD2 (10 nM) for 0–30 min, and cells were inhibitor (FR 180204; 10 M) did not significantly change RvD2- collected for staining with specific Abs for cell surface markers, stimulated phagocytosis (Fig. 5C). Macrophage phagocytosis of followed by permeabilization and staining of intracellular targets, STZ was also carried out. STAT3 and PKA inhibition, but not ERK including a panel of phospho-proteins (see Materials and Methods). inhibition, significantly blocked RvD2-stimulated phagocytosis Macrophages were identified as CD11b+F4/80+ populations (see the (Fig. 5D). Taken together, our results demonstrate that DRV2 gating strategy in Supplemental Fig. 1). RvD2 time-dependently contributes to RvD2’s proresolving actions in macrophage clear- increased phosphorylation of pAKT, p-p38 MAPK, pCREB, pS6, ance of live E. coli and this action is dependent on cAMP/PKA and pERK1/2, pSTAT1, pSTAT3, and pSTAT5, each with different ki- STAT3 signaling pathways (Fig. 6). netics in macrophages. pAKT, p-p38 MAPK, pCREB, and pS6 levels reached maximum at 1 min and then gradually declined. pERK1/2 Discussion was detected at 1 min and peaked at 30 min after RvD2 stimulation RvD2 is a potent immunoresolvent and controller of leuko- (Fig. 4A). In comparison, pSTAT1, pSTAT3, and pSTAT5 were also cyte traffic and is protective in a wide range of disease models,

DRV2-KO. (C) Mouse resident naive macrophages collected from WT were plated onto eight-well chamber slides (1 3 105 cells per well) and incubated with vehicle or RvD2 (10 nM) in the presence or absence of a STAT3 inhibitor (NSC 74859; 100 mM), PKA inhibitor (H89; 3 mM), or ERK inhibitor (FR 180204; 10 mM) for 15 min at 37˚C, followed by addition of BacLight Green–labeled E. coli to initiate phagocytosis. Fluorescent images were then recorded as in panel (A). Results are (left) mean fluorescent intensity (MFI) from a representative of n = 3–4; (right) fold changes versus E. coli alone, mean 6 SEM from three to four independent experiments in triplicates or quadruplicates (three to four fields per well). (D) Mouse resident naive macrophages collected from WT mice were plated onto 96-well plates (0.5 3 105 cells per well) and incubated with vehicle or RvD2 (10 nM) in the presence or absence of a STAT3 inhibitor (NSC 74859; 100 mM), PKA inhibitor (H89; 3 mM), or ERK inhibitor (FR 180204; 10 mM) for 15 min at 37˚C, followed by addition of FITC-labeled STZ to initiate phagocytosis. Fluorescent images were then monitored using a plate reader. Results are mean 6 SEM from three to four independent experiments in triplicates. (C and D)*p , 0.05, **p , 0.01, ***p , 0.001 using one-way ANOVA with post hoc multiple comparison test (Newman–Keuls). MFI, mean fluorescence intensity. 850 INNATE ANTIMICROBIAL FUNCTION OF HOST RESOLVIN D2 including airway and gastrointestinal inflammation (8, 19). In the human monocytes (50). Inhibition of STAT3 phosphorylation de- present study, we demonstrated that RvD2–DRV2 interaction creases efferocytosis and M2 macrophage polarization in vitro (51). protected mice from sepsis, preventing hypothermia, enhancing Additionally, RAW264.7 murine macrophage phagocytosis of phagocytosis-based bacterial clearance, and increasing survival. In Staphylococcus aureus leads to upregulation of genes involved in infectious exudates collected from sepsis, DRV2-KO gave in- the JAK/STAT pathway, including STAT3 and STAT5. Phosphory- creased levels of PG, LT, and TX, and reduced SPM (i.e., RvD1, lation of STAT3 and STAT5 proteins is required for phagosome RvD3, AT-RvD1, AT-RvD3) in infectious exudates (Fig. 2). These acidification and maturation (52). Thus, it is likely that STAT3 results are consistent with those found in E. coli peritoneal in- phosphorylation is also required for phagosome maturation during fections where selected SPM, including AT-RvD1, RvD2, RvD5, human macrophage phagocytosis. Disruption of this pathway with PD1, and AT-PD1, were significantly reduced in DRV2-KO mice STAT3 inhibition diminished RvD2-enhanced phagocytosis of compared with WT mice. DRV2-KO also gave increased amounts E. coli and STZ (Fig. 5C, 5D). Along these lines, RvD1 upregulates of TX (30), indicating that DRV2-KO is associated with height- STAT3 in human monocytes (53). Taken together, these results ened inflammatory status during bacterial infection. Of interest, indicate that each SPM stereoselectively activates its own specific mice deficient in an RvD1 receptor, namely ALX, also gave receptor (e.g., RvD2-DRV2, RvD1-DRV1, and RvE1-ERV1). JAK/ heightened disease severity, including hypothermia and cardiac STAT and ERK/Akt/S6 are likely to be the common downstream dysfunction during sepsis (43). Conversely, ALX transgenic mice pathways following receptor activation by SPM that are involved in (i.e., mice overexpressing human ALX) gave reduced inflamma- SPM-initiated phagocytosis. tory status with markedly decreased PMN infiltrates in peritonitis In summary, we provide evidence for an RvD2/DRV2 resolution

(44). Along these lines, mice overexpressing the human RvE1 axis that protects microbial sepsis, increasing bacterial clearance and Downloaded from receptor, namely ChemR23/ERV1, also showed lower PMN in- improving survival. Additionally, we identified DRV2 receptor– filtration in peritonitis and, additionally, reduced ligature-induced dependent signaling pathways in macrophages. Our present results alveolar bone loss (reviewed in Ref. 8). Taken together, these suggest that these signaling components and pathways could rep- results point to an endogenous role of SPM receptors (e.g., ALX, resent “resolution signaling cassette/complex” in phagocytosis. ERV1, and DRV2) as checkpoint controllers of resolution during Earlier reports established that other SPM activation of their re-

inflammation and bacterial infections. ceptors also mediate proresolving actions. For example, RvD1 http://www.jimmunol.org/ We monitored a panel of phospho-proteins using CyTOF. regulates a panel of select micro-RNAs and their target genes in- These include transcription factors CREB, STAT1, STAT3, and volved in resolution of inflammation, including IkB kinase, IL-10, STAT5, and kinases such as ERK1/2, p38 MAPK, and Akt and 5-lipoxygenase in an ALX- and DRV1/GPR32-dependent (Fig. 4A). We found that RvD2 significantly increased phos- manner (53). RvE1-ERV1 enhanced phagocytosis via the Akt/S6 phorylation of CREB in WT but not DRV2-KO macrophages pathway (49). Resolvins and the other proresolution mediators from (Fig. 4A), suggesting that the RvD2/DRV2 axis could regulate a n-3 fatty acids are agonists of the resolution response and are panel of CRE-containing genes in a PKA-dependent manner conserved structures across the animal kingdom (8). Taken to- (42). In mouse macrophages, RvD2 enhanced cAMP levels in a gether, these findings provide new proresolution mechanisms of DRV2-dependent manner (Fig. 5B). Additionally, cAMP-activated the host that may be of interest, providing potential therapeutic by guest on September 25, 2021 protein kinase (PKA)–mediated RvD2-stimulated phagocytosis opportunities for the control of unwanted inflammation and in- (Fig. 5C, 5D). Earlier we reported that RvD2 activates recombinant fection that accompanies sepsis and infectious inflammatory dis- human DRV2 that is sensitive to cholera toxin, suggesting receptor eases. Thus, SPM and their receptors and signaling pathways coupling to a Gas-like protein. Also, in human macrophages, RvD2 documented in this study provide examples of the potential for does-dependently increases cAMP, which was abolished when resolution pharmacology. DRV2 was knocked down using specific shRNA (30). Thus, the present results together with earlier findings indicate that RvD2– Acknowledgments DRV2 interactions initiate Gas protein coupling, leading to acti- We thank Mary Small for assistance with manuscript preparation. vation of the cAMP/PKA signaling pathway that enhanced macro- phage phagocytosis (Fig. 6). Activation of PKA by cAMP enhances Disclosures Rac1 activity, leading to increased efferocytosis with peritoneal The authors have no financial conflicts of interest. resident macrophages (45). Also, a cAMP analog activates EPAC (a GTP/GDP exchange factor), which in turn increases the levels of GTP-Rap1, leading to F-actin formation and enhanced phagocytosis References 1. Cotran, R. S. 1999. Inflammation: historical perspectives. In Inflammation: Basic of STZ in RAW264.7 murine macrophages (46). These components, Principles and Clinical Correlates, 3rd Ed. J. I. Gallin, R. Snyderman, namely Rac1, EPAC, Rap1, and F-actin, might also contribute to D. T. Fearon, B. F. 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LM levels in mouse exudate (pg/ml)

AA bioactive Q1 Q3 WT veh. WT RvD2 DRV2-KO veh. DRV2-KO RvD2 metabolome

LXA4 351 115 8.0 ± 4.5 16.1 ± 14.2 9.3 ± 6.3 6.9 ± 2.9

LXB4 351 221 82.9 ± 24.3 156.5 ± 40.7 162.4 ± 37.3 177.1 ± 30.4 5S,15S-diHETE 335 235 53.0 ± 14.1 52.1 ± 10.8 63.4 ± 30.9 77.4 ± 13.3

AT-LXA4 351 115 13.7 ± 5.5 17.1 ± 9.4 14.3 ± 3.6 21.0 ± 4.6

AT-LXB4 351 221 14.4 ± 9.9 45.6 ± 38.7 23.1 ± 15.0 28.7 ± 14.8

LTB4 335 195 857.3 ± 227.3 903.3 ± 343.8 1825.3 ± 296.2 2459.0 ± 629.4

PGD2 351 233 312.9 ± 107.4 329.1 ± 66.3 389.0 ± 116.0 558.7 ± 158.7

PGE2 351 189 1326.1 ± 595.5 2554.3 ± 615.3 2800.8 ± 1079.5 3490.1 ± 889.0

PGF2α 353 193 1358.2 ± 540.4 608.3 ± 115.5 1817.8 ± 996.1 2050.9 ± 1046.0

TXB2 369 169 589.4 ± 72.7 755.1 ± 162.5 1128.9 ± 237.8 1492.0 ± 206.1 DHA bioactive metabolome RvD1 375 121 15.0 ± 9.8 7.6 ± 2.3 5.7 ± 1.9 8.9 ± 3.4 RvD2 375 175 12.0 ± 5.6 196.3 ± 81.2 8.9 ± 2.9 121.0 ± 41.4 RvD3 375 147 0.4 ± 0.1 1.2 ± 0.5 0.5 ± 0.2 1.0 ± 0.3 RvD4 359 255 30.4 ± 5.4 43.8 ± 17.2 54.8 ± 28.0 43.0 ± 11.4 RvD5 359 199 65.1 ± 19.9 74.9 ± 22.9 68.9 ± 36.8 115.6 ± 43.4 AT-RvD1 375 121 30.3 ± 12.9 35.0 ± 15.2 17.3 ± 9.7 21.6 ± 7.1 AT-RVD3 375 147 0.7 ± 0.2 3.4 ± 1.5 1.4 ± 0.8 2.6 ± 1.3 PD1 359 153 2.8 ± 0.5 4.2 ± 1.1 4.1 ± 0.6 5.7 ± 1.2 10S,17S-diHDHA 359 153 3.9 ± 0.4 2.3 ± 0.4 3.9 ± 0.7 2.6 ± 0.5 AT-PD1 359 153 11.0 ± 5.8 8.3 ± 1.3 17.2 ± 13.2 15.0 ± 4.7 MaR1 359 221 12.4 ± 6.3 6.7 ± 2.2 26.8 ± 18.0 21.6 ± 7.2 7S,14S-diHDHA 359 221 33.0 ± 18.5 30.3 ± 6.9 125.6 ± 93.3 99.3 ± 33.5 4S,14S-diHDHA 359 101 527.9 ± 193.9 126.3 ± 38.9 278.1 ± 80.6 155.9 ± 54.9 EPA bioactive metabolome RvE1 349 195 15.1 ± 3.5 45.5 ± 20.9 47.3 ± 22.8 34.7 ± 12.0 RvE2 333 253 4.4 ± 1.1 4.9 ± 2.2 6.4 ± 1.5 5.7 ± 2.2 RvE3 333 201 4.7 ± 1.5 3.0 ± 0.5 3.3 ± 1.3 2.2 ± 0.4

Exudates from mouse after CLP were collected as described then were extracted and LM levels investigated using LM metabolopidomics (see materials and methods for details). Values are express as mean ± SEM.

-1- Supplemental Table 2. Proteome profiling of CLP infectious exudates from WT and DRV2-KO with RvD2 treatment

Cytokines, chemokines and growth factor P value P value P value Adiponectin/Acrp30 0.431 DPPIV/CD26 0.175 IL-27 0.193 Amphiregulin 0.055 EGF 0.159 IL-28 0.093 Angiopoietin-1 0.170 Endoglin/CD105 0.258 IL-33 0.109 Angiopoietin-2 0.206 Endostatin 0.477 LDL R 0.193 Angiopoietin-like 3 0.128 Fetuin A/AHSG 0.376 Leptin 0.251 BAFF/BLyS/TNFSF13B 0.200 FGF acidic 0.249 LIF 0.119 C1q R1/CD93 0.239 FGF-21 0.086 Lipocalin-2/NGAL, 0.176 CCL2/JE/MCP-1 0.293 Flt-3 Ligand 0.062 LIX 0.007 * CCL3/CCL4 MIP-1 alpha/beta 0.242 Gas6 0.305 M-CSF 0.124 CCL5/RANTES 0.242 G-CSF 0.447 MMP-2 0.147 CCL6/C10 0.241 GDF-15 0.158 MMP-3 0.002 * CCL11/Eotaxin 0.145 GM-CSF -- MMP-9 0.016 * CCL12/MCP-5 0.411 HGF 0.258 Myeloperoxidase 0.057 CCL17/TARC 0.079 ICAM-1/CD54 0.334 Osteopontin (OPN) 0.204 CCL19/MIP-3 beta 0.223 IFN-gamma -- Osteoprotegerin/TNFRSF11B 0.383 PD-ECGF/Thymidine CCL20/MIP-3 alpha 0.170 IGFBP-1 0.402 phosphorylase 0.189 CCL21/6Ckine 0.120 IGFBP-2 0.471 PDGF-BB 0.052 CCL22/MDC 0.105 IGFBP-3 0.251 Pentraxin 2/SAP 0.036 * CD14 0.071 IGFBP-5 0.084 Pentraxin 3/ TSG-14 0.038 * CD40/TNFRSF5 0.083 IGFBP-6 0.283 Periostin/OSF-2 0.101 CD160 0.192 IL-1 alpha/IL1F1 0.326 Pref-1/DLK-1/FA1 0.085 Chemerin 0.184 IL-1 beta/IL-1F2 0.089 Proliferin 0.086 Chitinase 3-like 1 0.460 IL-1ra/IL-1F3 0.351 Proprotein Convertase 9/PCSK9 -- Coagulation Factor III/Tissue Factor 0.111 IL-2 -- RAGE 0.438 Complement Component C5/C5a 0.067 IL-3 -- RBP4 -- Complement Factor D 0.230 IL-4 0.231 Reg3G 0.018 * C-Reactive Protein/CRP 0.308 IL-5 0.095 Resistin 0.309 CX3CL1/Fractalkine 0.085 IL-6 0.338 E-Selectin/CD62E 0.099 CXCL1/KC 0.405 IL-7 -- P-Selectin/CD62P 0.126 CXCL2/MIP-2 0.479 IL-10 0.238 Serpin E1/PAI-1 0.282 CXCL9/MIG 0.198 IL-11 0.225 Serpin F1/PEDF 0.019 * CXCL10/IP-10 0.238 IL-12p40 0.167 Thrombopoietin 0.158 CXCL11/I-TAC 0.096 IL-13 0.107 TIM-1/KIM-1/HAVCR 0.086 CXCL13/BLC/BCA-1 0.350 IL-15 0.091 TNF-alpha 0.158 CXCL16 0.109 IL-17A 0.323 VCAM-1/CD106 0.261 Cystatin C 0.282 IL-22 0.036 * VEGF 0.205 Dkk-1 0.199 IL-23 0.112 WISP-1/CCN4 0.182 *p<0.05; - not detected

Peritoneal lavages were collected at 12h post CLP. Cell-free supernatants were collected by centrifugation. A 1:10 dilution of the supernatant (150µl) were incubated with the pre-coated Proteome Profiler array membranes (ARY028, Mouse Cytokine Antibody array kit; R&D Systems) and processed according to the manufacturer’s instructions. Densitometric analysis of dot blots was performed using Image J software (National Institute of Health, Bethesda, MD, USA). P values were obtained by comparing WT+ RvD2 vs. DRV2-KO + RvD2 groups using 2-tailed Student’s t test. *p<0.05; -- not detected.

-2-

Supplemental Table 3. Antibodies used for mass cytometry

Target epitope Clone Isotopic label Supplier CD45 30-F11 Pr 141 LMA CyTOF pAKT (S473) D9E Sm 152 Fluidigm p-p38 (T180/Y182) D3F9 Gd 156 Fluidigm pStat3 (Y705) 4 Gd 158 Fluidigm pStat1 (Y701) 58D6 Gd 160 LMA CyTOF pNF-κb p65 (S529) K10-895.12.50 ER166 Fluidigm pEER1/2 (T202/Y204) D13.14.4E Er 167 Fluidigm CD11b M1/70 Tm 169 LMA CyTOF pS6 (S235/S236) N7-548 Yb 172 Fluidigm pStat5 (Y694) D47/E7 XP Yb 174 LMA CyTOF F4/80 BM8 Lu 175 LMA CyTOF pCREB (S133) 87G3 Yb 176 Fluidigm

LMA CyTOF (Longwood Medical Areas CyTOF Antibody Resources Core; Brigham and Women’s Hospital)

-3- DRV2-KO! WT! (CD11b used toidentifyresidentperitonealmacrophages Supplementary Figure1.Sequentialgatingstrategy + F4/80 + ).for CyTOF -4-