Identification and Actions of a Novel Third Maresin Conjugate in Tissue Regeneration: MCTR3

Maresin conjugates in tissue regeneration (MCTR) are a new family of evolutionarily conserved chemical signals that orchestrate host responses to promote tissue regeneration and resolution of infections. Herein, we identified the novel MCTR3 and established rank order potencies and matched the stereochemistries of MCTR1, MCTR2 and MCTR3 using material prepared by total organic synthesis and mediators isolated from both mouse and human systems. MCTR3 was produced from endogenous substrate by E. coli activated human macrophages and identified in sepsis patients. Each of the three synthetic MCTR dose-dependently (1–100nM) accelerated tissue regeneration in planaria by 0.6–0.9 days. When administered at the onset or peak of inflammation, each of the MCTR promoted resolution of E. coli infections in mice. They increased bacterial phagocytosis by exudate leukocytes (~15–50%), limited neutrophil infiltration (~20–50%), promoted efferocytosis (~30%) and reduced eicosanoids. MCTR1 and MCTR2 upregulated human neutrophil and macrophage phagocytic responses where MCTR3 also proved to possess potent actions. These results establish the complete stereochemistry and rank order potencies for MCTR1, MCTR2 and MCTR3 that provide novel resolution moduli in regulating host responses to clear infections and promote tissue regeneration.


Introduction
We recently identified, in self-resolving exudates, novel mediators that resolve infections [1][2][3] as well as regulate tissue regeneration [2].These novel chemical signals link the resolution of infectious inflammatory exudates to the process of tissue regeneration.The acute inflammatory response, when self-contained, plays a critical role in limiting bacterial invasion to promote wound repair and tissue regeneration (for recent reviews, see [3,4]).This protective response is orchestrated by autacoids at the site of inflammation [3,5,6].Arachidonic acid-derived eicosanoids, namely prostaglandins and leukotrienes, are produced during the initiation stage of the inflammatory response leading to vascular leak and leukocyte recruitment [5,6].During resolution of acute inflammation, a process termed lipid mediator class switching occurs that leads to the formation of novel potent pro-resolving mediators [3].This new genus of specialized proresolving mediators (SPM) include the n-3 essential fatty acid derived resolvins, protectins and maresins that possess leukocyte directed actions promoting the resolution of acute inflammation [1,3,7].These mediators also stimulate the clearance of apoptotic cells and debris, playing fundamental role(s) in inflammation-resolution [3,4,8].Resolution mediators such as Annexin A1 and Lipoxin A 4 are critical in human control of tissue repair [9], and maresins in regeneration [2].
The biological actions of these pro-resolving mediators are stereochemically selective reflecting their routes of biosynthesis [3].Therefore, establishing the complete stereochemical assignment and rank order potencies of lipid mediators are of considerable interest.Indeed establishing the complete stereochemistry of resolvins [3] enabled the confirmation and extension of the potent and diverse biological actions of these new mediators, for example, identification of Resolvin D1's actions in treating postoperative cognitive decline [10] and protective actions of Maresin 1 (MaR1) in murine colitis [11].
Maresins (macrophage mediators in resolving inflammation) by definition are formed via 14-lipoxygenation of docosahexaenoic acid and promote resolution of acute inflammation and tissue regeneration [2,3,12].Recently, we uncovered a new series of bioactive peptide-lipid conjugated mediators that are produced during the later stages of self-resolving infections [2] that are also present in human sepsis patients [13].These were termed maresin conjugates in tissue regeneration (MCTR) because they regulate mechanisms in inflammation-resolution as well as tissue regeneration establishing links between local inflammatory exudates and tissue regeneration via novel chemical signals [2,3].The structure for MCTR1 is 13-glutathionyl, 14-hydroxy-docosahexaenoic acid, and that of MCTR2, 13-cysteinylglycinyl, 14-hydroxydocosahexaenoic acid.In this report, we identified MCTR3 as 13-cysteinyl, 14-hydroxy-docosahexaenoic acid and established the potencies of MCTRs in the clearance of infections and tissue regeneration as well as their complete stereochemistry.
To monitor and quantify the levels of targeted LMs, we used multiple reaction monitoring (MRM) with signature ion fragments for each molecule (six diagnostic ions and calibration curves).The following transitions were employed for MCTRs: MCTR1 m/z 650>191, MCTR2 521>191 and MCTR3 464>191 [13].

Planaria tissue regeneration
Planaria (Dugesia japonica) were kept in water (Poland Spring; Nestle Waters North America, Stamford, CT, USA) at 18°C.All animals were starved for at least 7 days before the experiments.Tissue regeneration was assessed as described [2].In brief, planaria were subjected to head resection postocularly (surgical injury).The posterior portions of the planaria were then placed in spring water containing 0.01% EtOH, or MCTR at indicated concentrations.The extent of tissue regeneration during a 6-day period was determined using captured images of regenerating blastemas at regular intervals (24 h).These images were analyzed using ImageJ software (NIH, Bethesda, MD, USA) to give a tissue regeneration index (TRI) [2].

E. coli peritonitis
Peritonitis experiments were conducted as in [2].Briefly, FVB mice (6-8-weeks old), purchased from Charles River Laboratories (Wilmington, MA, USA) were fed ad libitum Laboratory RodentDiet 20-5058 (LabDiet; Purina Mills, St. Louis, MO, USA) and housed at a maximum of four animals per cage and maintained in 12:12 hour light-dark cycles.Mouse experimental procedures were approved by the Standing Committee on Animals of Harvard Medical School (Protocol 02570) and complied with institutional and U.S. National Institutes of Health (NIH) guidelines.No surgical procedures were conducted and no analgesics were administered.Mice were euthanized by overdose of isoflurane via inhalation in saturated atmosphere, followed by cervical dislocation.If recovery from procedure is compromised as assessed by increase in self grooming, aggressive behavior or rapid movements, self-mutilation, absent activity, lack of eating or drinking, abdominal distention or redness and heat over abdominal area, humane endpoint in Protocol 02570 requires that mice must be euthanized immediately by isoflurane overdose, followed by cervical dislocation.During this procedure, animals did not experience more than slight or momentary pain or distress and no animals died prior to experimental endpoint.E. coli (serotypeO6:K2:H1) was cultured in Luria-Bertani broth and harvested at midlog phase (OD600 nm, ~0.5 absorbance units).In determined experiments mice were administered MCTR1, MCTR2, MCTR3 (50ng/mouse) or vehicle via intraperitoneal (i.p) injection and after 5 minutes inoculated with E. coli (10 5 CFU/mouse).Exudates were collected at the indicated intervals and leukocyte populations as well as bacterial phagocytosis and macrophage efferocytosis determined using flow cytometry and fluorescent-labeled antibodies [2].In select experiments, mice were inoculated with E. coli (10 5 CFU/mouse), after 12h administered MCTR1, MCTR2, MCTR3 (100ng/mouse) or vehicle i.p. and exudates collected after 24h with leukocyte populations and bacterial phagocytosis determined as above.
Additionally, PMN survival was assessed.PMN were plated in 12-well plates at 5x10 6 cells/ mL in PBS with calcium and magnesium.At time zero, either PBS alone or 10nM MCTR1, MCTR2, or MCTR3 was added to PMN.Cells were incubated at 37°C with 5% CO 2 for 24 or 48 hours, collected and analyzed by flow cytometry.Cells were surface-stained against Annexin V and Propidium Iodide (Molecular Probes, FITC AnnexinV/Dead Cell Apoptosis, Eugene, OR).Cell staining was evaluated using FACSCanto II flow cytometer (BD Biosciences, San Jose, CA) and results were analyzed using FlowJo software (Tree Star, Ashland, OR).
For real-time imaging, macrophages were plated onto 8-well chamber slides (0.1 x10 6 cells/ well in PBS +/+ ).The Nunc™ Lab-Tek™ II chamber system (Thermo Scientific, cat.# 15434) consists of a removable polystyrene media chamber attached to a glass microscope slide.The chamber slides were kept in a Stage Top Incubation system for microscopes equipped with a built-in digital gas mixer and temperature regulator (TOKAI HIT model INUF-K14).MCTR1, MCTR2 MCTR3 (1nM) or vehicle (PBS containing 0.01% EtOH) was added to macrophages for 15 min, followed by BacLight Green-labeled E. coli (2.5 x 10 6 CFU).Images were then acquired every 6 min for 2h (37°C) with a Keyence BZ-9000 (BIOREVO) inverted fluorescence phase-contrast microscope (20X objective) equipped with a monochrome/color switching camera using BZ-II Viewer software (Keyence, Itasca, IL, USA).Green fluorescence intensity was quantified using BZ-II Analyzer.

Statistical analysis
All results are expressed as the mean ± SEM.Differences between groups were compared using Student's T-test (for two groups), 1-way ANOVA (multiple groups) followed by post hoc Bonferroni test, or 2-way ANOVA (multiple groups, multiple time points) followed by post hoc Bonferroni or Tukey tests as appropriate.The criterion for statistical significance was p < 0.05.

Results
We recently determined the complete stereochemistry of a key intermediate in maresin biosynthetic pathway 13S,14S-epoxy maresin [12], the proposed intermediate and immediate precursor in MCTR production (Fig 1A and [2]).Given that MCTR are produced in several biological systems, we obtained MCTR isolated from human macrophages, murine infectious exudates, and sepsis patients (Fig 1B -1D) to assess if they coeluted in LC-MS-MS and give MS-MS spectra identical to compounds independently prepared by total organic synthesis (Fig 1E).To determine the rank order potencies and stereochemistry for each MCTR, it was essential to use stereochemically pure precursors [14] because the endogenous mediators MCTR are produced in pico-to nanogram quantities that are not suitable for NMR analysis to obtain stereochemical assignments.In liquid chromatography tandem mass spectrometry, MCTR1 gave a distinct sharp peak with retention time (T R ) = 9.8 min, MCTR2 gave a sharp peak with T R = 9.1 min and the new MCTR3 gave a sharp peak with T R = 10.3 min.
Co-injection of the synthetic with authentic MCTR1 demonstrated co-elution at T R = 9.8 min (n = 3 macrophage preparations).Similarly, co-injection of synthetic MCTR2 and MCTR3 with MCTR isolated from human macrophages demonstrate co-elution with the respective authentic products (n = 3 macrophage preparations).Assessment of the MS-MS spectra for the material obtained by total organic synthesis gave essentially identical fragmentation spectra to those obtained with authentic material (Fig 1F and cf.[2,13]).These mass Thus, the stereochemically defined MCTR1, 2 and 3 each matched the endogenous molecules, permitting the identification of MCTR3.Having matched the physical properties of synthetic, stereochemically defined materials with those of endogenously produced MCTRs from both human and murine systems, we next required assessment of their potent biological actions.Given that MCTRs display tissue regenerative actions, we assessed the ability of each synthetic MCTR to promote tissue regeneration.Planaria were surgically injured by rapidly removing the head portion of the animals and tissue regeneration was followed over time [2].Planaria undergo both physiological and restorative regeneration via evolutionarily conserved pathways, making this an ideal system to characterize novel chemical signals involved in tissue regeneration [15].When planaria were kept with vehicle alone, maximum tissue regeneration (TRI max ) occurred within ~6 days with time to 50% regeneration (T 50 ) occurring by ~2.7 days.Incubation of planaria with MCTR1 led to acceleration in tissue regeneration with a reduction in T 50 from ~2.7 days to ~2 days when planaria were incubated with 100nM of MCTR1 and to ~1.9 days when incubated with 1nM (Fig 2A).MCTR2 also accelerated tissue regeneration with planaria, where incubation of surgically injured planaria with MCTR2 reduced T 50 to ~1.8 days (Fig 2B ) and at 1nM reduced T 50 to ~1.9 days.The new MCTR3 also demonstrated potent tissue regenerative actions that were dose dependent (Fig 2C).Assessment of their relative potencies at stimulating tissue regeneration demonstrated that MCTR3 and MCTR2 were the most potent of the three, followed by MCTR1.Hence, MCTR3 % MCTR2>MCTR1 (Fig 2D ).
Given the increased incidence of bacterial infections [1,16] and that MCTR1 and MCTR2 each displays potent actions promoting resolution of infections, we could now test whether each of the stereochemically pure MCTR1, 2 and 3 also carried these actions in murine infections.E. coli (1x10 5 CFU/mouse) inoculation gave a self-resolving inflammatory response with maximal neutrophil infiltration at ~12 h that subsequently declined, which gave a calculated resolution interval (R i ) of ~12h (Fig 3A).MCTR1 (50ng/mouse) injection immediately prior to E. coli inoculation gave a significant reduction in neutrophil infiltration at the 12 h interval, and shortened the resolution interval from ~12h to ~4h (Fig 3A).Administration of MCTR2 also gave significant reductions in exudate leukocyte counts (~50%), a shortening in the R i to ~2h and an increase in phagocytosis of E. coli (~50%) by exudate leukocytes.These results with synthetic MCTRs are in accordance with published findings with biologically produced MCTRs [2] and confirm the potent actions of these novel mediators in infections.Of note, MCTR3 given immediately prior to E. coli inoculation reduced exudate neutrophil levels by ~54%, shortening the R i from 12 h to 3 h (Fig 3C).In addition to reducing neutrophil infiltration, each of three MCTR significantly increased leukocyte phagocytosis of E. coli, with MCTR2 and MCTR3 displaying the highest potencies (Fig 3D).MCTR1 was the most potent in stimulating macrophage efferocytosis (Fig 3E ), a key defining pro-resolving action.
We also tested whether MCTR carry pro-resolving actions when administered at the peak of inflammation during E. coli infections.Administration of MCTR1 at the peak of inflammation (12h interval) gave reductions in exudate neutrophil counts at 24h and shortened R i to ~10 h (Fig 3F).In mice given MCTR2 we found reductions in exudate neutrophil counts (~50%) giving an R i of ~7 h (Fig 3G).At equal doses, MCTR3 also promoted the resolution of E. coli infections, reducing exudate neutrophil counts by ~50% and shortening the R i to ~9 h (Fig 3G and 3H).Together these results demonstrate the potent biological actions of MCTR1 and MCTR2, confirming their original structural assignments (Fig 3I and 3J).Moreover, we describe here the unexpected actions of MCTR3 in accelerating the resolution of E. coli infections and establish the rank order potencies in promoting the resolution of infectiousinflammation.
Toward human translation, we validated the bioactions of these novel molecules with human primary leukocytes.Incubation of MCTR1 (1pM-10nM) with human macrophages gave dose-dependent increases (15-50%) in macrophage phagocytosis of E. coli with a maximum at ~1pM (Fig 4A).In addition, MCTR1 stimulated the efferocytosis of apoptotic neutrophils by macrophages (10-20% increase above vehicle; n = 8 macrophage preparations p<0.05), a key macrophage response in resolution cellular mechanisms [3,4].MCTR1, at a MCTRs also regulated human neutrophil responses.Each of these molecules dose-dependently regulated human neutrophil phagocytosis of E. coli with MCTR3 displaying higher potency at promoting neutrophil phagocytosis at doses as low as 1pM-100pM, with ~60% increase in phagocytosis when compared to both MCTR2 and MCTR3 (S1 Fig) .MCTR1 and MCTR3 each gave a bell-shaped curve that is characteristic of G protein-coupled receptor activation by SPM [17] that may reflect cellular shape changes at higher concentrations of the mediators.MCTR2 gave two distinct peaks, suggesting this mediator may activate two distinct receptors, a high affinity and low affinity receptor, on macrophages that mediate its actions in regulating phagocytosis of E. coli.
Highly effective bioactive metabolic structure-function units are retained and copied throughout evolution.This is exemplified by the diverse roles of the isoprenoid unit throughout the biosphere with many biosynthetic pathways and wide ranging functions.Indeed this structural unit is the building block of many biologically significant molecules including steroids, retinoids and terpenoids [18].By comparison, the enzymatically-produced allylic epoxides (Fig 1) central to maresin and SPM biosynthesis also appear to be highly conserved in chemical evolution as a central building block in the biosynthesis of diverse bioactive molecules with wide ranging functions such the MCTR from 13S,14S-eMAR [2] and, for example, CysLT from the epoxide intermediate leukotriene A 4 [5,19].Of relevance to our present studies is the conjugation of allylic epoxides with sulfur-containing peptides that give rise to molecules that carry potent infection-resolution properties (shown in Fig 1).The biological actions of these molecules are also separate, whereby the cysteinyl leukotrienes carry potent bronchoconstrictive and inflammation-initiating actions [5,20], whereas MCTR are potently proresolving and function in tissue regeneration [2,3] ).Of interest, MCTR3 proved to be a potent bioactive molecule with planaria, mouse, and human cells.Thus the present findings provide new, highly conserved targets and pathways linking resolution of infections with tissue regeneration.
spectra gave fragmentations and ions that were consistent with the deduced structures, where MCTR1 gave a parent ion with m/z of 650, MCTR2 an m/z of 521 and MCTR3 that of m/z 464.MCTR3 gave diagnostic ions at m/z 191, 205, 235 and 343 (see Fig 1F inset) as well as 464 (M+H), 325 (343-H 2 O), 227 (245-H 2 O), 213 (231-H 2 O), The ultraviolet chromophores for each of the three molecules were characteristic of conjugated triene double bond systems with an allylic auxochrome where the λ max MeOH for MCTR1, MCTR2 and MCTR3 was 281nm.