Identification of 14-series sulfido-conjugated PNAS PLUS mediators that promote resolution of infection and organ protection

Jesmond Dalli, Nan Chiang, and Charles N. Serhan1

Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Harvard Institutes of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115

Edited by Carl F. Nathan, Weill Cornell Medical College, New York, NY, and approved September 25, 2014 (received for review August 5, 2014) Upon infection and inflammation, tissue repair and regeneration up-regulation of select SPMs [including Resolvin (Rv) D1, RvD5, are essential in reestablishing function. Here we identified potent and Protectin D1]. These mediators enhance bacterial killing and molecules present in self-limited infectious murine exudates, clearance along with regulating phagocyte recruitment (11). regenerating planaria, and human milk as well as macrophages The levels of these potent leukocyte agonists decline during that stimulate tissue regeneration in planaria and are proresolv- the later phase of the self-limited inflammatory response (11), ing. Characterization of their physical properties and isotope allowing the possibility that other signals may be produced that tracking indicated that the bioactive structures contained docosa- regulate leukocyte responses to promote tissue repair and re- hexaenoic acid and sulfido-conjugate (SC) of triene double bonds generation. Given the pivotal roles of chemical signals in infec- that proved to be 13-glutathionyl, 14-hydroxy-docosahexaenoic tions, we investigated whether mediators within self-resolving acid (SCI) and 13-cysteinylglycinyl, 14-hydroxy-docosahexaenoic infections could regulate tissue repair and regeneration without acid (SCII). These molecules rescued Escherichia coli infection- immunosuppression. Because maresin 1 (7R,14S-dihydroxy- mediated delay in tissue regeneration in planaria, improving re- docosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid; MaR1) displays generation intervals from ∼4.2 to ∼3.7 d. Administration of SCs potent proresolving and tissue-regenerative actions (12, 13), we protected mice from second-organ reflow injury, promoting repair investigated whether previously undescribed signals are produced via limiting neutrophil infiltration, up-regulating Ki67, and Roof during self-limited infections that regulate tissue regeneration. In plate-specific spondin 3. At nanomolar potencies these conjugates this report, we identify a new pathway and mediators in planaria, also resolved E. coli infections by limiting neutrophil infiltration mouse, and human tissues that promote repair and regeneration and stimulating bacterial phagocytosis and clearance as well as during infection. Identification of two new sulfido-conjugate (SC) efferocytosis of apoptotic cells. Together, these findings identify mediators provides the first evidence, to our knowledge, for previously undescribed conserved chemical signals and pathways autacoids produced during the resolution of infections that signal in planaria, mouse, and human tissues that enhance host responses innate host responses and accelerate repair. to contain infections, stimulate resolution of inflammation, and pro- mote the restoration of function. Results and Discussion To obtain self-resolving infectious exudates and assess tissue regeneration | omega 3 | leukocytes | inflammation | eicosanoids regeneration, we used murine Escherichia coli peritonitis rele- vant to human infections and mapped leukocyte trafficking. iven the rise in antibiotic-resistant infections and the critical E. coli inoculation at 105 colony-forming units (CFUs) per mouse Grole that barrier breach plays in microbial invasion, identifi- i.p. gave a self-limited host response that reached maximal cation of new endogenous signals that promote pathogen clear- neutrophil infiltration at 12 h and subsequently declined (Fig. 1A). ance and tissue repair/regeneration is of wide interest (1). When self-limited, acute inflammation is a host-protective response Significance (2, 3) that is orchestrated by chemical mediators and is an active process generated by evolutionarily conserved biosynthetic path- Inability of the body to contain infections may lead to collat- ways (3, 4). During initiation of inflammation, potent mediators eral organ damage resulting from unchecked innate immune are locally produced that promote vascular leakage, leukocyte responses. Here we investigated the chemical signals produced recruitment, and pain (5, 6). In disease, these pathways can be by immune cells to expedite clearance of bacteria and promote dysregulated, leading to heightened inflammatory responses and organ repair and tissue regeneration. We identified molecules perpetuation of the disease state (6–9). One approach to regu- produced during self-limited infections and in human milk that lating exuberant inflammatory responses is inhibition of initiating promote clearance of bacteria as well as accelerate tissue re- mediators (e.g., eicosanoids, including prostaglandins) via bio- generation. In addition, these molecules also protected organs synthetic enzyme inhibitors (6, 10) and receptor antagonists (5). In from exuberant inflammatory responses by limiting select the context of infection, this approach may be of limited clinical white blood cell recruitment and up-regulating the expression utility and can have potential drawbacks, including immune sup- of proteins involved in tissue repair. Therefore, these results pression (7, 8). identify new resolution moduli that regulate phagocytes to In self-limited inflammation, endogenous programs are acti- clear bacteria and activate the regeneration milieu. INFLAMMATION vated at the onset that regulate the amplitude of the inflammatory IMMUNOLOGY AND responses and stimulate resolution (3, 9). Central to these host- Author contributions: C.N.S. conceived the overall research plan and experimental design; protective responses are novel families of endogenous chemical J.D., N.C., and C.N.S. designed research; J.D. and N.C. performed research; J.D. and N.C. mediators termed specialized proresolving mediators (SPMs) (9). analyzed data; and J.D., N.C., and C.N.S. wrote the paper. In sterile inflammation and injury, these mediators actively limit The authors declare no conflict of interest. further neutrophil recruitment and promote macrophage clear- This article is a PNAS Direct Submission. ance of apoptotic cells and tissue debris (7–9). In self-resolving 1To whom correspondence should be addressed. Email: [email protected]. infections, endogenous resolution programs are also activated This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. during the early stages of inflammatory responses, with the 1073/pnas.1415006111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1415006111 PNAS | Published online October 16, 2014 | E4753–E4761 Downloaded by guest on September 25, 2021 Monocyte/macrophage numbers increased between 4 and dose-dependently accelerated head regeneration (r2 = 0.91) as 24 h, demarking onset of the resolution phase. We next isolated early as 2 d after surgery, shortening T50 to ∼3.3 d. For direct products from resolving infectious exudates (i.e., 24 h) and comparison, maresin 1 (Fig. 1B and Fig. S1A), the known dihy- assessed their ability, with planaria, to stimulate tissue regen- droxy-containing potent macrophage-derived proresolving me- eration. Because MaR1 stimulates tissue regeneration (12) and diator (12), accelerated this process to essentially the same elutes within methyl formate fractions from C18 solid-phase extent. Toward human translation, and because milk carries extraction [referred to as solid-phase extraction chromatographic nutrients and is appreciated to have products relevant to infant (SPE-C) isolate fraction 1], we sought evidence for signals in development and immune status (15), we also assessed tissue- distinct chromatographic fractions. Here we assessed eluates in regenerative properties of human milk isolates using the same methanol fractions (SPE-C isolate fraction 2) for previously chromatographic fractions (Fig. 1B). Incubation of planaria with undescribed signals that displayed tissue-regenerative properties. human milk isolates dose-dependently accelerated regeneration ∼ ∼ Planaria undergo both restorative and physiological regeneration and reduced T50 from 4.3 to 3.5 d (Fig. 1B and Fig. S1B). via evolutionarily conserved pathways, making this an ideal sys- These results demonstrate that both mouse resolving exudates tem to identify chemical signals involved in tissue regeneration and human milk possess tissue-regenerative properties that elute (14). To this end, planaria (Dugesia japonica) were injured on within the methanol fractions from solid-phase C18 extractions. day 0 and time-dependent head regeneration was monitored. To Next, we investigated whether these bioactive molecules reg- quantitate regeneration, we calculated tissue-regeneration in- ulated signaling pathways that trigger head-to-tail differentiation dices (TRIs) (12). Following head resection, regeneration en- in D. japonica (16). Two days postinjury, in regenerating blaste- mas, isolates from both mouse resolving exudates and human sued, giving a TRImax (maximum tissue regeneration) at 6 d and milk significantly increased expression of the fibroblast growth T50 (the interval at which 50% regeneration, TRI50, occurred) at ∼4.3 d (Fig. 1B). Isolates from 24-h infection-resolving exudates factor receptor-like gene nou-darake (Djndk) and mitogen-acti- vated protein kinase phosphatase gene (Djmpka;Fig.1C)and down-regulated DjAdb-Ba (Abdominal-B–like gene), a target Hox gene of posterior Wnt/β-catenin signaling in neoblast progeny Initiation Resolution +E.coli RE (14, 16). Because extracellular signal-regulated kinase (ERK) +Human Milk regulates expression of these genes (16), we tested whether ERK A B 600 40.9 0.6 +MaR1 6 105 PMN Surgical Injury signaling was responsible for the actions of molecules from re- 104 500 solving exudates. ERK inhibition abrogated regenerative actions Ly6G 103 * TRI of these infectious resolving exudates (Fig. S1C). Together, these 102 max 0 * 4 47.7 10.9 400 results suggest that ERK signaling is involved in the regenerative 0102 103 104 105 CD11b actions of molecules from resolving infectious exudates. Monocytes/ 300 * Macrophages are key in regulating host responses during in- /mouse) Macrophages * 6 flammation and infections, coordinating both the onset and 2 200 * TRI50 (x10 resolution of inflammatory responses (8, 17, 18). During sterile * inflammation, the resolution phase is characterized by increases 100

Tissue Regeneration Index (mm) Tissue in resolution-phase macrophages (rMs) (18) and production of T50

Number of infiltrated Leukocytes maresins (13). Using lipid mediator metabololipidomics, we 0 12 24 36 48 0 1 2 3 4 5 6 identified MaR1 and related isomers within SPE-C isolate frac- Time (h) Days post surgery tion 1 as well as previously undescribed signals in SPE-C isolate fraction 2 in infectious resolving exudates (n = 3 mice exudates). E.coli RE Products D C HM Products 600 +HM 12-LOX shRNA Hence, SPE-C isolate fraction 2 obtained from both mouse re- +HM CS shRNA solving exudates and human milk carried regenerative properties 500 Surgical (Fig. 1 B and C) that were unique and distinct from MaR1 (12), Djsfpr-A Injury ** which elutes within SPE-C isolate fraction 1 (Table S1). Djndk # Djmpk-A 400 Based on these findings, we next assessed the role of human 30 DjAdb-Ba * macrophage 12-lipoxygenase (LOX) (19) in the biosynthesis of ** 300 these products. Isolates obtained from human macrophages ** +E.coli RE TRI50 ϕ 20 +Human Milk (HM s) carried tissue-regenerative actions with D. japonica that Surgical Injury 200 were lost in cells transfected with shRNA targeting 12-LOX (Fig. 1D). In HMϕs, docosahexaenoic acid (DHA) is a 12-LOX sub- 10 **** 100 strate in maresin biosynthesis (19, 20); therefore, we investigated T50

Tissue Regeneration Index (mm) Tissue whether the bioactive products in SPE-C isolate fraction 2 were also from DHA. Incubation of infectious exudates obtained from Relative mRNA expression Relative mRNA 0 * * 0123456 self-resolving peritonitis with radiolabeled DHA demonstrated Djsfpr-A Djndk DjmpkA DjAdb-Ba Days post surgery accumulation of labeled material in SPE-C isolate fraction 2 at Fig. 1. New pathway promotes tissue regeneration. (A) Leukocyte re- both 12 h (peak of inflammation) and 24 h (resolution) (Fig. cruitment following E. coli (105 CFUs per mouse, i.p.) inoculation (Materials S2A). In these fractions, significantly higher radioactivity from and Methods). Results are mean ± SEM for n = 4 mice per time point. (B) DHA was present in exudates from mice with self-resolving Surgically injured planaria were incubated with SPE-C isolate fraction 2 from peritonitis compared with those with delayed resolving perito- E. coli resolving infectious exudates (REs), human milk, maresin 1 (100 nM), or nitis. The presence of four DHA-derived products in these iso- vehicle (surgical injury; water containing 0.01% EtOH). Tissue regeneration lates was confirmed using liquid chromatography tandem mass indices were determined. T50, time interval corresponding to 50% of maximal spectrometry (LC-MS-MS) (Fig. S2B). tissue regeneration (TRI50). (C) Gene expression in regenerating blastemas Peaks I and II gave essentially identical MS-MS fragmentation (Bottom). Results are mean ± SEM (n = 3 per group pooled from blastemas of + 9 animals). (D) Injured planaria were incubated with SPE-C isolate fraction patterns, with the parent ion (M H) displaying m/z at 521. Spectra 2 from human macrophages transfected with shRNA for 12-lipoxygenase, from peaks III and IV also gave identical MS-MS fragmentation control scrambled sequence (CS shRNA), or vehicle. (B and D) Results are with m/z for the parent ion at 650, suggesting that I and II were mean ± SEM for n = 9 planaria per incubation. *P < 0.05, **P < 0.0001 vs. likely related to each other and III and IV were related to each surgical injury group. #P < 0.01 vs. 12-LOX shRNA at day 4. other (Fig. 2 A and B). Similar results were also obtained with

E4754 | www.pnas.org/cgi/doi/10.1073/pnas.1415006111 Dalli et al. Downloaded by guest on September 25, 2021 human milk, regenerating planaria, and human macrophages (Fig. PNAS PLUS 2 C–F and Fig. S2C). In macrophages transfected with 12-LOX A B shRNA compared with mock transfected cells, quantification of Infectious resolving exudates 100 100 products in SPE-C isolate fraction 2 using LC-MS-MS demon- 650 521 strated a significant reduction in the levels of products identified under peaks I–IV (Fig. 2G). These results are in line with the observed reduction in biological activity of these isolates (Fig. 1D), ϕ implicating HM 12-LOX in the initiation of these signals. x3 503 Because 14S-hydro(peroxy)-docosahexaenoic acid (14S- x8 ϕ 503 179 HpDHA) is the product of the HM 12-LOX biosynthetic pre- 308 173187 459

Relative Intensity (%) 179

Relative Intensity (%) 343 325 205 cursor to maresins, we tested whether 14S-HpDHA was a pre- 109 191 400 187 343 521 109 191 cursor of these regenerative molecules. Incubation of HMϕswith 162 233 418 618 217 325 632 14S-HpDHA also gave products III and IV (Fig. 3A)aswell 0 0 100 200 300 400 500 600 100 200 300 400 500 as I and II (Fig. 3B). Products beneath III and IV each gave m/z, Da UV chromophores with maximum absorbance at 280 nm and m/z, Da C Regenerating planaria D shoulders at 270 and 295 nm in the reverse-phase high-pressure 100 650 100 liquid chromatography mobile phase (Fig. S3A), characteristic of a conjugated triene double-bond system coupled to an auxo- 147 chrome allylic to the triene such as sulfur (6, 21). This was cor- 109 521 roborated in incubations with a desulfurization reagent, Raney 173 nickel (6, 21), that gave 14-hydroxy-docosahexaenoic acid (14- 179 187 HDHA) (Fig. S3 B and C). These results together with MS-MS 191 632 fragmentation indicated a 13-glutathionyl,14-hydroxy-, and 22- 162 205 217 109 191 343 205 carbon backbone that originated from DHA (Fig. 3A), and 179 343 233 325 400 618 Relative Intensity (%) 503 Relative Intensity (%) 308 418 503 235 therefore was coined sulfido-conjugated product I (SCI). To gain 521 459 further evidence for this deduced structure, we assessed deu- 0 0 ϕ 100 200 300 400 500 600 100 200 300 400 500 terium incorporation in SCI with HM sandd5-14S-HpDHA m/z, Da m/z, Da (Fig. S4A). d5-SCI gave the expected 5-Da shift in the parent ion E F mass, from m/z 650 to m/z 655, as well as in the m/z of fragments Human macrophages 100 650 100 containing carbons 21 and 22, including that resulting from a di- 521

agnostic 14- to 15-carbon break, which increased in mass from x5 m/z 109 to m/z 114 (Fig. S4C). The proposed glutathione conju- 521 gate structure was further corroborated by treating SCI (Fig. S4 B and C) with diazomethane. This approach was also used to elu- x5 325 cidate the structure of the products beneath I and II, namely 13- 343 503 179 187 cysteinylglycinyl, 14-hydroxy-docosahexaenoic acid (SCII; Fig. 162 191 173191 325 308 217 343 3B and Figs. S5 and S6). Relative Intensity (%) 109 109 233 400 503 618 179 We next confirmed that these molecules carried tissue- 418 632 459 0 0 regenerative properties. Planaria incubated with SCI and SCII 100 200 300 400 500 600 100 200 300 400 500 gave accelerated tissue regeneration (T50 from ∼4to∼3 d; Fig. m/z, Da m/z, Da 3C). To investigate the role of endogenous SCs in planaria tissue CS shRNA regeneration, D. japonica was incubated with a LOX inhibitor G 5 6 () that reduced levels of both SCI and SCII measured 12-LOX shRNA 3 d postsurgery (Fig. 3D) and significantly delayed head regrowth 4 (Fig. 3E). This was rescued when planaria were incubated with 4 SCI and SCII (Fig. 3E). We next investigated the ability of SCs 4 3 to promote tissue regeneration during infection. Incubation of * D. japonica with E. coli gave a delay in regeneration, T50 from * ∼ ∼ 2 3.5 to 4.2 d, that was rescued by SCI and SCII addition (Fig. 2

3E). Having established that together, SCI and SCII displayed Ion counts x10 tissue-regenerative actions, we next sought evidence for their 1 individual biological actions. Incubation of injured planaria with either SCI or SCII accelerated head regrowth. This proved to be 0 0 concentration-dependent, reducing T50 from ∼5to∼3.5 d (Fig. 4 2 Fig. 2. Identification of previously undescribed signals in SPE-C isolate A and B and Fig. S7), with an r value of 0.85 for SCI and 0.97 for fraction 2. (A and B) Infectious exudates were obtained at 24 h after in- SCII (Fig. S7). In addition, SCI and SCII each up-regulated oculation with E. coli (105 CFUs per mouse). Exudates were then in- genes involved in head regeneration (Fig. 4C). cubatedwithDHA(1μg/mL, 37 °C, 45 min), products were extracted and Bioactive lipid mediators that are peptide conjugates such as signals investigated by LC-MS-MS. (A) MS-MS spectrum for signals under peaks III and IV. (B) MS-MS spectrum for signals under peaks I and II.

cysteinyl involve GST enzymes in their biosynthesis INFLAMMATION

(10, 22). Therefore, we assessed the role of D. japonica GST Results are representative of n = 4mice.(C and D) Planaria were surgically IMMUNOLOGY AND in SC biosynthesis. Whole-mount in situ hybridization (WISH) injured; after 3 d, products were extracted and signals were investigated of uninjured planaria confirmed the expression of D. japonica by LC-MS-MS. Results are representative of n = 20 planaria. (E–G)Human GST (DjGst), which was temporally regulated following injury macrophages were transfected with shRNA for 12-LOX or CS sequence; products were then extracted and signals were investigated by LC-MS- in regenerating blastemas (Fig. 5A). Double-stranded RNA MS. (G) MRM quantification for products under peaks III and IV (Left)and knockdown of DjGst (Fig. 5B) delayed tissue regeneration (Fig. 5C) peaks I and II (Right). Results for E and F are representative of n = 3 and reduced SC levels (>80%; Fig. 5D) in regenerating planaria. macrophage preparations. (G) Mean ± SEM expressed as peak area ion Alignment of deduced D. japonica GST amino acid sequence counts. n = 3 macrophage preparations. *P < 0.05 vs. CS shRNA trans- with human and mouse GST-mu4 (Table S2)demonstrated>70% fected macrophages.

Dalli et al. PNAS | Published online October 16, 2014 | E4755 Downloaded by guest on September 25, 2021 HOOC A NH3 B C Fig. 3. New sulfido conjugates promote regen- 7 593 O 115 521 eration. Human macrophages (1 × 10 cells per mL) +2H +2H NH3 O +H H NH 131 191 + + N +H -H / 191 650 100 109 329 were incubated with 14-HpDHA (1 μM, PBS )and 100 HOOC 418 NH S -H 700 O S 145 8 +2H +H +SC × HOOC 179 +H E. coli (1 10 CFUs per mL, 30 min, 37 °C), and 308 +H 205 205 343 600 Surgical 343 521 OH OH +H +H 5503 products were isolated by RP-UV-HPLC and assessed 235 235 Injury COOH COOH 109 147 109 500 173 521 by LC-MS-MS. Representative MS-MS spectra used in 325 632 179 400 * the identification of (A) SCI from peaks III and IV and 308 618 343 TRI50 179 233 (B) SCII from peaks I and II. Results represent n = 10 162 205 300 * 187 191 macrophage preparations. (C) After surgical injury, pla- 205 575 200 418 109 217 naria were incubated with SCI plus SCII (10 nM) or ve- Relative Intensity (%) Relative Intensity (%) 504 * 191 400 217 325 459 100 614 235 343 503 T50 hicle (surgical injury; water containing 0.01% EtOH) and 281 477 235 593 329 0 0 0 Regeneration Index (mm) Tissue regeneration indices determined. (D) After surgical in- 100 200 300 400 500 600 100 150 200 250 300 350 400 450 500 550 123456 m/z, Da m/z, Da Days post surgery jury, planaria were incubated with lipoxygenase in- hibitor (L.I.; 100 μM) or vehicle (surgical injury; water D E F Surgery 700 containing 0.01% EtOH). SCI (Left)andSCII(Right)were 25 ## Infected 700 +SC +LI 600 +LI+SC Uninfected Infected quantified by LC-MS-MS. Results are mean ± SEM. n = 3 +LI ## 600 600 20 500 Surgical * representative of 40 planaria per group. (E) Surgically

) 500 500

5 Injury injured planaria, were incubated with lipoxygenase in- 400 μ 15 ## 400 400 hibitor (L.I.; 100 M), L.I. plus SCI plus SCII (SC; 100 nM) or * TRI TRI * * 300 300 50 50 vehicle. (F) After surgical injury, planaria were incubated # * 300 8 * 10 * with E. coli (10 CFUs), E. coli plus SCI and SCII (100 nM), 200 200 * ** 200 or vehicle (surgical injury; water containing 0.01% (ion counts x10 5 100 * 100 100 EtOH), and regeneration indices were determined. Area beneath the curve T T * ** 50 50 Results are mean ± SEM for n = 9 planaria per group.

0 Regeneration Index (mm) Tissue Tissue Regeneration Index (mm) Tissue 0 0 123456 0 123456 123456 *P < 0.05, **P < 0.01 vs. the respective control group. Days post surgery Days post surgery Days post surgery #P < 0.01, ##P < 0.001 vs. the respective L.I. group.

sequence homology between the planarian and mammalian en- We tested SC isolates from regenerating planaria to deter- zymes, which is also expressed in HMϕs(n = 4). These results mine whether they displayed actions in mammalian species. indicate that a GST enzyme(s) is involved in SC biosynthesis and Here they significantly reduced neutrophil recruitment in murine is involved in tissue regeneration in planaria. peritonitis (Fig. S9A) and exudate eicosanoid levels including With human macrophages, we investigated product–precursor LTB4 and thromboxane (Tx) B2, actions that by direct compar- relationships for DHA with SCI and SCII. DHA was rapidly ison were shared with MaR1 (Fig. S9 B and C). Planarian SC converted by activated human macrophages to SCI, with levels isolates also stimulated human macrophage efferocytosis of ap- reaching a maximum at 15 min. SCII was also rapidly produced optotic neutrophils (Fig. S9D). Hence, these results indicated in these incubations, with levels that remained elevated between that SCs are antiinflammatory and proresolving, and that 15 and 60 min (Fig. 6A). Essentially similar relationships were their structure functions are conserved from planaria, mice, obtained with activated human macrophages and 14S-HpDHA and humans. (n = 3 separate incubations). To assess whether SCI was a precursor Vessel occlusion is a common consequence of many inflam- of SCII, we next incubated human macrophages with a γ-glutamyl matory conditions, leading to local ischemia that, upon re- transferase inhibitor (Acivicin) known to inhibit (LT) flow, can result in second-organ injury by activated leukocytes and, in extreme cases, organ failure (24). SCI and SCII gave D4 formation (23). Incubation of activated human macrophages with this inhibitor gave a significant increase in SCI and a re- significant protection from leukocyte-mediated tissue damage duction in SCII levels (Fig. 6B). Similar results were obtained with (Fig. 8A), decreasing leukocyte infiltration into lungs and regenerating planaria, where addition of the inhibitor increased spleens, actions comparable to RvD1 (Fig. S10A). SCs also re- SCI levels and decreased SCII levels 2 d postinjury (Fig. 6C). duced plasma eicosanoid levels, including LTB4, LTC4, and TxB2 Because SCs were biosynthesized in both resolving infectious (Fig. S10 B and C). In addition, immunofluorescence analysis of lung sections from mice given SCI and SCII demonstrated an up- exudates (Fig. 2 and Fig. S2) and regenerating planaria (Figs. 2, regulation of antigen Ki67, which plays a role in cell proliferation 5, and 6), we determined their ability to stimulate resolution (25), and Roof plate-specific Spondin 3 (RSPO3; Fig. 8B), which during infection in mice. For quantitative assessment of resolu- displays tissue-regenerative actions (26). E. coli tion components with infection, we used resolution in- Several lines of evidence support the proposed biosynthetic dices (11). Inoculation of mice with 105 CFUs E. coli (self- ∼ ∼ scheme for the formation of SC signals in Fig. 9. They are as limited inoculum) gave Tmax 12 h, T50 32 h, and resolution ϕ ∼ follows: (i) DHA is converted by HM 12-LOX to 14S-HpDHA interval (Ri) 20 h (Fig. 7A). SCI and SCII (50 ng each per and 13,14-epoxide intermediate, demonstrated using acid meth- mouse) significantly reduced neutrophil numbers at 24 h, anol trapping with human cells and recombinant enzyme (13, 19). ∼ shortening Ri to 10 h (Fig. 7A), and promoted an exudate Knockdown of macrophage 12-LOX reduced both SCI and macrophage phenotype switch toward an rM phenotype, in- SCII(Fig.2)aswellaslossofthe tissue-regenerative actions creasing rM markers including T-cell immunoglobulin and of SPE-C isolate fraction 2 (Fig. 1). (ii) Radiolabel from pre- ∼ 14 mucin domain containing 4 (TIMD4) ( 34%), interleukin-10 cursor [ C]DHA was recovered in SPE-C isolate fraction 2 that (IL-10) (∼15%), and Arginase 1 (∼76%) (n = 4 mice per group). was distinct from MaR1-containing fractions (Fig. S2). (iii) Reci- SCs also gave significant increases in leukocyte phagocytosis of procity existed in product–precursor relationships between DHA, E. coli in vivo in mice (Fig. 7B). For human translation, we de- SCI, and SCII (Fig. 6) as well as between 14-HpDHA and SCI termined the actions of SCI and SCII on human phagocytes, and SCII. (iv) Inhibition of planaria LOX reduced both SCI and where each dose-dependently increased macrophage effer- SCII in regenerating planaria and delayed tissue regeneration ocytosis of apoptotic cells, a key proresolving action (3), to that was rescued by addition of SCI and SCII (Fig. 3). (v) a similar extent as MaR1 (Fig. 7C). Of note, SCI and SCII each Knockdown of planaria GST reduces both SCI and SCII levels in enhanced bacterial phagocytosis and intracellular reactive oxy- regenerating planaria and delays tissue regeneration (Fig. 5). (vi) gen species (Fig. S8) used in bacterial killing (11, 17). Incubation of human macrophages and regenerating planaria

E4756 | www.pnas.org/cgi/doi/10.1073/pnas.1415006111 Dalli et al. Downloaded by guest on September 25, 2021 A B tion of E. coli infection and stimulate phagocytic functions, tissue PNAS PLUS regeneration in planaria, and tissue repair in mice, thereby ful- 500 600 +SCI +SCII filling criteria as immunoresolvents, namely agents that stimulate Surgical Surgical resolution of inflammation (12). The proposed biosynthesis of 400 Injury 500 Injury these mediators occurs via lipoxygenation of DHA, producing 14-hydro(peroxy)-docosahexaenoic acid and an epoxide intermedi- 400 * 300 * ate (Fig. 9) that is converted to SC. This step in planaria relies on * TRI50 a GST enzyme(s), giving 13-glutathionyl, 14-hydroxy-docosahex- TRI50 300 aenoic acid and 13-cysteinylglycinyl, 14-hydroxy-docosahexaenoic 200 * acid (Fig. 5), and opens the characterization of other peptide–lipid * 200 * conjugates in the SPM genus. These specific peptide–lipid con- 100 – 100 jugates carry a carbon-14 position alcohol and are biosynthesized T via the maresin-epoxide intermediate; thus, they belong to the T50 50 Tissue Regeneration Index (mm) Tissue Tissue Regeneration Index (mm) Tissue maresin (macrophage mediators in resolving inflammation) family 0 123456 0123456 (13). Given that these previously undescribed signals regulate Days post surgery Days post surgery the cardinal signs of resolution, namely clearance of debris and C infections by phagocytes, tissue regeneration, and regulation of proinflammatory chemical mediators, we coin these SC molecules 20 +SCI maresin conjugates in tissue regeneration. Together, these find- +SCII ings provide new signals and pathways in host responses to injury, Surgical Injury *** 15 acute inflammation, and infectious exudates that are resolution moduli promoting homeostasis. *** 10 *** *** *** *** A B 5 DjGst 5 DjGst WT

Relative mRNA expression Relative mRNA ** 0 *** 4 Djsfpr-A Djndk DjmpkA DjAdb-Ba 3 Fig. 4. SCI and SCII promote tissue regeneration and regulate key signaling pathways in planaria. After surgical injury, planaria were incubated with (A) 2 SCI (100 nM), (B) SCII (100 nM), or vehicle (surgical injury; water containing DjGst dsRNA 0.01% EtOH), and regeneration indices were determined. Results represent two independent experiments and are mean ± SEM for n = 9 planaria per 1

group. (C) After surgical injury, planaria were incubated with SCI (100 nM), Relative upregulation SCII (100 nM), or vehicle, and gene expression was assessed 2 d postinjury in regenerating head blastemas. Results are mean ± SEM for n = 3 per in- 0 12345 cubation pooled from blastemas of 9 animals. *P < 0.05, **P < 0.01, ***P < Days post surgery 0.001 vs. surgical injury group. C D 600 γ-glutamyl transferase inhibitor led to increased SCI and de- DjGst dsRNA 500 creased SCII (Fig. 6). (vii) SCI and SCII carry distinct structures Surgical Injury from that of MaR1 (12), as demonstrated by LC-MS-MS results 400 of the free acids (Figs. 2 and 3 and Fig. S2) and trimethyl and 400 dimethyl ester derivatives, as well as deuterium incorporation TRI50 300 from DHA (Figs. S4 and S6). (viii) Identification of 14-HDHA from incubations of SCI and SCII with Raney nickel as well as the ** 200 SCI and SCII respective UV chromophores supports the pres- 200 MCTR levels ence of a sulfido group allylic to a triene double-bond system in

(pg/100mg protein) 100 each of these molecules (Figs. S3 and S5). (ix) SCI and SCII * T isolated using RP-UV-HPLC each separately regulated human 50 * Tissue Regeneration Index (mm) Tissue phagocyte responses and promoted tissue regeneration in pla- 0 0 123456 Surgical +DjGst naria in a dose-dependent manner (Figs. 4 and 7 and Figs. S7 and Days post surgery Injury dsRNA S8). (x) When administered together in vivo, SCI and SCII reg- INFLAMMATION

ulated host mouse responses to E. coli infections promoting Fig. 5. Regulation of tissue regeneration and SC production by planaria IMMUNOLOGY AND clearance of bacteria and resolution of infections. (xi) SCI and GST. (A) DjGst expression by WISH in uninjured animals (Left; represen- SCII together protected against second-organ reflow injury (col- tative of n = 9) and time course for DjGst expression in regenerating lateral tissue damage) and promoted tissue repair (Figs. 7 and 8, blastemas (Right; mean ± SEM for n = 4 per interval pooled from blastemas – Fig. S10,andTable S1). of 12 animals). (B D) Planaria were fed homogenized beef liver containing In summary, using a systematic approach, we identified con- DjGst dsRNA or beef liver (WT). (B and C) After 8 d, DjGst expression was assessed by WISH (B) and tissue regeneration kinetics were determined (C). served chemical signals from planaria, mouse, and human tissues (D) SC levels 3 d postsurgery. Results are mean ± SEM for n = 13 planaria that are antiinflammatory, proresolving, and tissue-regenerative. per group. *P < 0.05, **P < 0.01 vs. surgical injury group. MCTR, maresin These new peptide–lipid conjugate molecules accelerate resolu- conjugates in tissue regeneration.

Dalli et al. PNAS | Published online October 16, 2014 | E4757 Downloaded by guest on September 25, 2021 Fig. 6. Endogenous SCI is converted to SCII with A B C human macrophages and planaria. (A)Human Surgical injury E.coli +Acivicin +Acivicin macrophages (3 × 107 cells) were incubated with 5 150 8 DHA SC 80 150 60 30 DHA (37 °C, pH 7.45) and E. coli (1.5 × 10 CFUs), levels (pg/10 and SC SC SC ** SC SC SC and product levels were assessed using LC-MS-MS. 4 100 60 40 * = 7 100 20

Results are mean for n 3 separate incubations. (B) macrophages)

macrophages) 3

7 ** Human macrophages were incubated with or with- 40 20 *

out γ-glutamyl transferase inhibitor (GTI, Acivicin; g/10 2 50 macrophages 7 50 10 2.5 mM, 37 °C, pH 7.45, 30 min) and then DHA (37 °C, 20 10 8 1 ** ** pH 7.45) and E. coli (1.5 × 10 CFUs), and precursor SC pg/100mg protein pg/10 ** and product levels were assessed by LC-MS-MS. 0 0 0 0 0 0 ± = 510154560 510154560 510154560 Results are mean SEM for n 3 distinct incu- levels ( DHA Time (min) Time (min) Time (min) bations. (C) Planaria were surgically injured and then incubated with or without GTI (2.5 mM). SCI and SCII were assessed 3 d after injury using LC-MS-MS. Results are mean ± SEM for n = 20 planaria per group. *P < 0.05 vs. surgical injury group, **P < 0.01 vs. macrophages plus E. coli.

Materials and Methods cold methanol were added. Isolates were obtained as detailed in Lipid Murine Peritonitis. Mouse experiments were conducted in accordance with Mediator Metabololipidomics and Isolation of Bioactive Fractions. guidelines from the Harvard Medical Area Standing Committee on Animals In select experiments 12 h after E. coli inoculation, mice were administered (protocol 02570). Mice were inoculated with E. coli (serotype 06:K2:H1) as in either 100 ng of SCI and SCII (50 ng each per mouse) or vehicle [saline con- ref. 11 and peritoneal exudates were collected by lavaging the peritoneal taining 0.1% ethanol (EtOH)] via i.p. injection. Peritoneal exudates were then collected and leukocyte counts were determined as above. Assessment of cavity with 4 mL of PBS (without calcium and magnesium) at the indicated leukocyte phagocytosis of E. coli in inflammatory exudates was conducted as intervals. Leukocytes were enumerated by light microscopy using nuclear described (11). Briefly, cells were incubated with anti–CD11b-PerCP/Cy5.5– morphology staining with Turk’s solution and flow cytometry probing for conjugated antibody; subsequently, the cells were permeabilized using a BD CD11b-PerCP/Cy5.5 (eBioscience; clone M1/70), F4/80-PE (eBioscience; clone Cytofix/Cytoperm Fixation/Permeabilization Kit following the manufacturer’s BM8), and Ly6G-FITC (eBioscience; clone 1A8) as described (11). For product instructions (BD Biosciences); cells were then incubated with a FITC-conju- isolation, 24-h peritoneal exudates were collected and two volumes of ice- gated anti-E. coli antibody (GeneTex; GTX40856), and phagocytosis was assessed as mean fluorescence in the CD11b-positive cell population.

Resolution indices were calculated as described (11), where Ψmax is the SCI and SCII maximal neutrophil (PMN) number in the exudates; Tmax is the time point A when PMN numbers reach the maximum; R50 is 50% of the maximal PMN 5 number; T50 is the time point when PMN numbers reduce to 50% of the maximum; and Ri = T50 − Tmax, the time period when 50% of PMNs are lost max E.coli from the exudates. 4 +SCI and SCII Mice were administered either 105 (self-resolving) or 107 (delayed resolving) CFUs E. coli, and exudates were harvested after 12 or 24 h. These were then 3 incubated with either DHA (Cayman Chemical) or 14C-labeled DHA (American Radiolabeled Chemicals; 1 μM, 37 °C, pH 7.45). The incubations were then

/mouse) Ri 50 6 stopped with two volumes of ice-cold methanol and products were extracted R ~20h 2 i using solid phase extraction (SPE) columns as detailed below. Radioactivity Ri ~10h

(x10 was measured using a scintillation counter, and DHA-derived products were 1 ** assessed using LC-MS-MS and product ion scan targeting m/z 343 (Fig. S2B). Peritoneal PMN counts In determined experiments, mice were administered SC isolates from regen- T max T50 erating planaria i.p., obtained as described in Lipid mediator Metabololipidomics and Isolation of Bioactive Fractions, immediately before zymosan adminis- 0 12 24 36 48 tration (i.p., 1 mg per mouse). After 4 h, peritoneal exudates were obtained, Time post E.coli inoculation (h) leukocytes were enumerated as detailed above, and eicosanoid levels were B C assessed by lipid mediator metabololipidomics as described below. 50 SCI SCII 20 MaR1 50 MaR1 Ischemia Reperfusion. Mice were anesthetized by i.p. injection of xylazine (80 mg/kg) and ketamine (10 mg/kg). To initiate hind-limb ischemia, tourni- * 40 ** 40 ** ) quets consisting of a rubber band were placed on each hind limb. Ten minutes 2 15 * * 30 30 before the initiation of reperfusion, vehicle (saline containing 0.1% EtOH), * SCI (50 ng) plus SCII (50 ng), or Resolvin D1 (500 ng) were administered by i.v. 10 * * * 20 20 ** injection. At the end of reperfusion (3 h), mice were euthanized, blood was * collected via cardiac puncture, and plasma was isolated for lipid mediator 5 10 * * 10 * * metabololipidomics. Lungs were harvested; left lungs were frozen in liquid −

(MFI Units x10 nitrogen and stored at 80 °C, and right lungs were stored in 10% (vol/vol) 0 0 0 buffered formalin and processed for histology and hematoxylin and eosin E.coli +SCI -11-10-9 -8 -7 -11-10-9 -8 -7 (H&E) staining by the Children’s Hospital Boston, Department of Pathology,

Leukocyte E.coli phagocytosis +SCII log [M] log [M] imaged using a Keyence BZ-9000 microscope and BZ II imaging software (Keyence). Expression of Ki67 and RSPO3 was assessed by immunofluores- Fig. 7. SCs resolve infection and stimulate efferocytosis. (A and B) Mice were cence staining. Briefly, sections were deparaffinized in xylene and rehy- inoculated with E. coli (105 CFUs per mouse, i.p.) followed by either SCI plus drated and then blocked in 10% (vol/vol) horse serum and stained with SCII (50 ng per mouse each, i.p.) or vehicle (saline containing 0.1% EtOH) 12 h either rat anti-mouse RSPON3 antibody (R&D Systems) for 1 h at room later (A) Peritoneal leukocyte counts and resolution indices were determined temperature and then with sheep anti-rat Alexa 594-conjugated antibody (Materials and Methods). (B) In vivo E. coli phagocytosis. Results are mean ± (BioLegend) or with rabbit anti-mouse antibody (Abcam) and then with SEM for n = 4 mice per interval. *P < 0.05, **P < 0.01 vs. E. coli.(C) Human donkey anti-rabbit Alexa 488 antibody (BioLegend). Slides were mounted in macrophages (5 × 104 cells per well) were incubated with SCI (Left), SCII VECTASHIELD mounting solution with DAPI (Vector Laboratories) and im- (Right), or MaR1, and fluorescently labeled apoptotic PMN and efferocytosis munofluorescence was assessed using a Zeiss LSM 510 Meta confocal micro- were assessed. Results are mean ± SEM for n = 4 macrophage preparations. scope. Images were processed using ImageJ software (National Institutes of *P < 0.05, **P < 0.01 vs. vehicle group. MFI, mean fluorescence intensity. Health) and Adobe Photoshop CS6 (Adobe Systems) software. Frozen lungs

E4758 | www.pnas.org/cgi/doi/10.1073/pnas.1415006111 Dalli et al. Downloaded by guest on September 25, 2021 PNAS PLUS A Sham B DAPI Sham Ki67 RSPO3 Merge

R.I. R.I. Fig. 8. SCs are organ-protective. Mice were sub- jected to hind-limb ischemia (1 h) followed by reperfusion (3 h). Ten minutes before reperfusion, vehicle (saline containing 0.1% EtOH; R.I., reflow injury), SCI plus SCII (50 ng each) were administered intravenously. Lungs were then collected. (A) Tissue μ R.I.+SCI+SCII R.I.+SCI+SCII H&E staining. (Scale bars, 100 m.) Black arrows indicate leukocyte-mediated tissue damage; blue arrows indicate intact alveolar regions. (B) Immuno- fluorescence staining. Nuclear material (DAPI, blue), Ki67 (Alexa 488, green), RSPO3 (Alexa 594, red). (Scale bars, 100 μm.) Results are representa- tive of n = 4 mice per group.

were gently dispersed and centrifuged (2,000 × g), and tissue myeloperox- 15 min, 37 °C) and E. coli (1:50 leukocytes to E. coli, 45 min, 37 °C). Intracellular idase (MPO) levels were determined using mouse MPO ELISA (R&D Systems). reactive oxygen species were determined by measuring fluorescence using an M3 SpectraMax plate reader. To assess bacterial phagocytosis, macrophages (5 × Leukocyte Phagocytosis. Macrophages were prepared from peripheral blood 104 cells per well) or neutrophils (1 × 105 cells per well) were incubated with SCI mononuclear cells purchased from Children’s Hospital Blood Bank, and phago- (10 pM to 100 nM), SCII (10 pM to 100 nM), or vehicle (0.1% EtOH in DPBS, cytosis was assessed as described (11). Briefly, macrophages (5 × 104 cells per well) 15 min, 37 °C) and then incubated with BacLight Green (Molecular Probes)-labeled were incubated with SCI (10 pM to 100 nM), SCII (10 pM to 100 nM), MaR1 E. coli (1:50 leukocytes to E. coli, 45 min, 37 °C). Extracellular fluorescence was (10 pM to 100 nM), or vehicle [0.1% EtOH in Dulbecco’s phosphate buffered quenched using Trypan blue (1:15 dilution), and phagocytosis was assessed saline (DPBS)] for 15 min at 37 °C, and then fluorescently labeled apoptotic cells using an M3 SpectraMax plate reader. were added and cells were incubated for 45 min at 37 °C. Extracellular fluo- In determined experiments, human macrophages were incubated with SC rescence was quenched using Trypan blue (1:15 dilution) and phagocytosis was isolates from regenerating planaria for 15 min at 37 °C, and then fluo- assessed using an M3 SpectraMax plate reader (Molecular Devices). In select rescently labeled apoptotic cells were added and efferocytosis was assessed experiments, macrophages (5 × 104 cells per well) or neutrophils (1 × 105 cells per as detailed above. well) were obtained from human healthy volunteers’ peripheral blood after obtaining informed consent as described (11), in accordance with the Partners Whole-Mount in Situ Hybridization, Real-Time PCR, and dsRNA Synthesis. Human Research Committee Protocol (1999P001297), and incubated with 2′,7′- Regenerating blastemas were obtained from planaria after surgical injury dichlorodihydrofluorescein (H2DCFDA) (5 μM, 30 min, 37 °C) and then with SCI and placed in TRIzol (Ambion). RNA was isolated following the manufacturer’s (10 pM to 100 nM), SCII (10 pM to 100 nM), or vehicle (0.1% EtOH in DPBS, instructions and cDNA was synthesized essentially as described (16) using

DHA

LOX Human milk Human macrophages Mouse infectious resolving exudates OOH Regenerating planaria COOH LOX

O

COOH Reduces neutrophil recruitment OH 13,14-epoxy-Maresin OH Stimulates efferocytosis Promotes tissue regeneration HOOC NH2 MaR1 COOH

O H N NH HOOC O S INFLAMMATION

OH IMMUNOLOGY AND SCI COOH Promote resolution of infections Stimulate bacterial phagocytosis and killing

O NH2 Promote tissue regeneration Protect organs by limiting neutrophil Fig. 9. Proposed SC biosynthetic scheme. Struc- NH S recruitment and stimulating repair tures are depicted in likely conformations based on HOOC biosynthetic evidence (see main text and Table S1 OH for further details). The stereochemistry of Maresin COOH 1 and the maresin-epoxide intermediate are estab- SCII lished (21).

Dalli et al. PNAS | Published online October 16, 2014 | E4759 Downloaded by guest on September 25, 2021 random hexamers, Oligo(dT)20, and SuperScript III (Invitrogen). Relative quanti- mediator metabololipidomics or ethanol for biological evaluation. For lipid tative analysis for each gene product was carried out using an ABI 7900HT Fast mediator metabololipidomics of known mediators and pathway products, Real-Time PCR machine (Applied Biosystems). DjGapdh:forward,5′-ACCAC- the LC-MS-MS system was operated as described (29). For identification and CAACTGTTTAGCTCCCTTA-3′; reverse, 5′-GATGGTCCATCAACAGTCTTTTGC-3′. quantification of SCs, a Shimadzu LC-20AD HPLC and a Shimadzu SIL-20AC Djsfpr-A:forward,5′-TTGCTCTCTTTACGCTCCGGT-3′;reverse5′-CGCATAGTTCC- autoinjector paired with a QTrap 6500 (AB SCIEX) were used. An Eclipse CTGCATGGT-3′. Djndk:forward,5′-TCACAAACTCCACCGCAGTACTTT-3′; reverse, Plus C18 column (50 mm × 4.6 mm × 1.8 μm; Agilent) was kept in a column oven 5′-GGTATGGATTAGCATTATTGAATTGTG-3′. DjmpkA:forward,5′-CACTGATAT- maintained at 50 °C (ThermaSphere; TS-130), and lipid mediators were eluted CTACTTCACGAAAGCCAG-3′;reverse,5′-AAGGCATCCAGTTCATTTCCTAAAT-3′. with a mobile phase consisting of methanol/water/acetic acid at 55:45:0.01 (vol: DjAdb-Ba:forward,5′-CGTAGGCAATACTTACATCACTAGACAAA-3′;reverse,5′- vol:vol) that was ramped to 85:15:0.01 (vol:vol:vol) over 0.1 min, to 86:14:0.01 TGTCTCTCCGACAAATGCAATTT-3′. DjGst:forward,5′-TGTTGGCTGAAGA- (vol:vol:vol) for the next 3 min, to 90:10:0.01 (vol:vol:vol) for the next 1 min, AGTGCAAG-3′;reverse,5′-TTCACCCATAAGCCAATGCT-3′. The constitutively and to 99.9:0:0.01 (vol:vol:vol) for the next 6 min. This was subsequently transcribed housekeeping gene GAPDH was used to normalize target gene maintained at 99.9:0:0.01 (vol:vol:vol) for 2 min, and the flow rate was expression levels as described (16). maintained at 0.65 mL/min. The QTrap 6500 was operated in positive ioni- Two separate PCR products containing T7 promoter sequences on the 5′ zation mode using scheduled multiple reaction monitoring (MRM) coupled ends of either sense or antisense strand DNA were used as templates to with information-dependent acquisition and enhanced product ion scan. transcribe antisense and sense RNA using the T7 RiboMAX Express RNAi Human milk (Biological Specialty) was placed in 2.5 volumes of ice-cold meth- System (Promega) (DjGst with T7 promoter sequence: forward, 5′-GGATCC- anol; proteins were then allowed to precipitate for 30 min at 4 °C and supernatants TAATACGACTCACTATAGGAGTGGCAATCACCACCAAAT-3′; reverse, 5′-GGA- were collected. These were then acidified to pH ∼3.5 and extracted using diethyl TCCTAATACGACTCACTATAGGAGTGGCAATCACCACCAAAT-3′. DjGst primers: ether. Samples were then brought to dryness under vacuum on a rotary evapo- ′ ′ ′ forward, 5 -GGCTCCTTTGTTAGGTTACTGGA-3 ; reverse, 5 -AGTGGCAATCAC- rator (Buchi); products were suspended in 1 mL of methanol and extracted as ′ CACCAAAT-3 ). These two complementary single-stranded RNA (ssRNA) were detailed above using C18 SPE columns to obtain SPE-C isolate fractions. mixed and incubated at 70 °C (10 min) and then slowly cooled to room Macrophages (1 × 107 cells in 175-cm2 flasks) were transfected with ∼ temperature ( 20 min) to allow annealing of the dsRNA. The remaining ALOX12 human shRNA in pRS vector (20 μg; OriGene) or mock vector (pRS template DNA and ssRNA were removed by treatment with DNase and RNase alone) using jetPEI transfection reagent (40 μL; following the manufacturer’s A (37 °C, 30 min) essentially as described (27). The dsRNA was then pre- instructions; Polyplus Transfection). Seventy-two hours later, 12-LOX ex- cipitated (0.1 volume of 3 M sodium acetate, pH 5.2, and 1 volume of iso- pression was assessed using immunofluorescence staining with human propanol) and further purified using MicroSpin G-25 Columns (GE Healthcare) 12-LOX antibody (Novus Biologicals) or relevant isotype control, and expres- to remove residual nucleotides. sion was determined by flow cytometry. Transfected cells (1 × 107 per mL) ′ PCR products containing T7 promoter sequences on the 5 end of the sense were also incubated with E. coli (5 × 108 CFUs per mL, RPMI supplemented strand DNA were used to produce in situ hybridization probes to DjGst using with 0.1% human serum, 37 °C) for 1 h. These incubations were stopped ’ FISH Tag Kits (Invitrogen) following the manufacturer s instructions. Planaria with two volumes of ice-cold methanol, and isolates (SPE-C isolate fractions ’ were treated with 2% hydrochloric acid in 5/8 Holtfreter s solution for 5 min 1 and 2) were obtained as described above. at 4 °C and fixed in 5/8 Holtfreter’s solution containing 4% paraformaldehyde Planaria (∼200 animals) were surgically injured as described above. After and 5% methanol for less than 2 h at 4 °C. Planaria were dehydrated and 3 d, they were placed in two volumes of ice-cold methanol and tissues were rehydrated and then bleached, and probes were hybridized as described (28). gently dispersed using a glass dounce. Homogenates were placed at −20 °C to allow for protein precipitation, and SPE-C isolate fraction 2 was obtained Planaria Regeneration. Planaria (D. japonica; Dj) were kept in water (Poland as described above. Spring) at 18 °C. All animals were starved for at least 7 d before the experiments. Tissue regeneration was assessed as described (12). Briefly, SC Biosynthesis, Isolation, and Derivatives. Human macrophages (1 × 107 cells planaria were subjected to head resection postoccularly (surgical injury). The + + per mL) were suspended in PBS / incubated with 14-HpDHA (1 μM), and E. posterior portions of the planaria were then placed in spring water con- coli (1 × 108 CFUs per mL, 37 °C, pH 7.45, 30 min). The 14-HpDHA was pro- taining 0.01% EtOH, SPE-C isolate fraction 2 from resolving exudates or milk duced by incubation of DHA with human macrophage 12-lipoxygenase, and at the indicated dilutions, U0126 [ERK inhibitor; Cell Signaling Technology; isolated as described (13). Two volumes of methanol were then added and 25 μM (16)], U0126 plus resolving exudates SPE-C isolate fraction 2, SCI products were extracted using C18 columns as outlined above. (100 nM), SCII (100 nM), lipoxygenase inhibitor (baicalein; 10 μM), or lipox- × 7 ygenase inhibitor plus SCI and SCII (100 nM). The extent of tissue re- In select experiments, human macrophages (1 10 cells per mL) were +/+ μ × 8 generation during a 6-d period for D. japonica was determined using suspended in PBS incubated with d5-14-HpDHA (1 M) and E. coli (1 10 captured images of the regenerating blastemas at regular intervals (24 h). CFUs per mL, 37 °C, pH 7.45, 30 min). Two volumes of methanol were added These images were analyzed using ImageJ software. A tissue regeneration and products were extracted using SPE columns as outlined above. index (TRI) was used that took into consideration the size of the regenerated SCs obtained as detailed above were isolated using online UV-RP-HPLC tissue total area (A) and the postocular width (W) of the animal, where TRI = (1100 Series; Agilent Technologies) and an Agilent Poroshell 120 C18 column A/W. In determined experiments, planaria were injured and incubated (100 mm × 4.6 mm × 2.7 μm) with the mobile phase consisting of methanol/ in water containing 108 CFUs E. coli (serotype O6:K2:H1) or with E. coli plus water at 55:45 (vol:vol) that was ramped to 85:15 (vol:vol) over 0.1 min, to SCI and SCII (100 nM), and tissue regeneration indices were assessed as 86:14 (vol:vol) for the next 3 min, to 90:10 (vol:vol) for the next 1 min, and to described above. 100:0 (vol:vol) for the next 6 min. This was subsequently maintained at In select experiments, planaria were fed homogenized beef liver or beef 100:0 (vol:vol) for 2 min, and the flow rate was maintained at 0.65 mL/min. liver containing dsRNA for DjGst as described (27). After 8 d, planaria were In select experiments, isolated SCI and SCII were incubated with diazo- either taken for whole-mount in situ hybridization or subjected to head re- methane in diethyl ether for 30 min at room temperature. Samples were section and tissue regeneration assessed every day for 7 d. SCs were identified then brought to dryness and products were assessed by LC-MS-MS using and quantified 3 d postinjury using lipid mediator metabololipidomics. MRM monitoring of the following ion pairs: 549>193 and 692>336. SCI and Planaria were surgically injured and incubated with or without Acivicin SCII were incubated with activated Raney nickel catalyst for 20 min at room (2.5 mM). After 3 d, products were extracted by solid-phase extraction (see temperature. The resulting products were then assessed by LC-MS-MS using below), and SCI and SCII levels were investigated by LC-MS-MS. MRM monitoring: 343>205 (Figs. S3 and S5). Human macrophages (3 × 107 cells per mL) were incubated with DHA 8 Lipid Mediator Metabololipidomics and Isolation of Bioactive Fractions. Peri- (15 μg, 37 °C, PBS, pH 7.45) and E. coli (1.5 × 10 CFUs). Incubations were toneal exudates and exudate cell incubations were immediately placed stopped with two volumes of ice-cold methanol, products were extracted, in two volumes of methanol. For lipid mediator profiling, 500 pg each of and levels were assessed by LC-MS-MS. In select experiments, macrophages

deuterium-labeled internal standards d8-5S-HETE, d4-LTB4, d5-LXA4, d4-PGE2, were incubated with Acivicin (2.5 mM, 37 °C, PBS, pH 7.45) before addition of μ and d5-LTC4 was added to facilitate quantification in each respective chro- DHA (15 g) and Acivicin (2.5 mM) and products were assessed by LC-MS-MS. matographic region and sample recovery. Samples were then held at −20 °C for 45 min to allow for protein precipitation and centrifuged (1,200 × g, 4 °C, Statistics. All results are expressed as means ± SEM. Differences between 10 min). Products were then extracted using solid-phase extraction as de- groups were compared using Student t test (two groups), one-way ANOVA scribed (29) and eluted using methyl formate (SPE-C isolate fraction 1) and (multiple groups) followed by post hoc Bonferroni test, or two-way ANOVA methanol (SPE-C isolate fraction 2). Eluted isolates were then brought to (multiple groups, multiple time points) followed by post hoc Bonferroni or dryness under nitrogen and suspended in methanol/water (50:50) for lipid Sidak tests. The criterion for statistical significance was P < 0.05.

E4760 | www.pnas.org/cgi/doi/10.1073/pnas.1415006111 Dalli et al. Downloaded by guest on September 25, 2021 ACKNOWLEDGMENTS. The authors thank Mary Halm Small for expert helpful discussions, and Dr. Romain Colas for assistance with material prep- PNAS PLUS assistance in manuscript preparation, Dr. Michael Levin and Dr. Junji aration. This work was supported in part by the National Institutes of Health Mokmura (Tufts University) for providing D. japonica seed colonies and for (Grant P01GM095467).

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