Resolvin T-series reduce neutrophil extracellular traps.

The newly identified 13-series (T-series) resolvins (RvTs) regulate phagocyte functions and accelerate resolution of infectious inflammation. Because severe acute respiratory syndrome coronavirus 2 elicits uncontrolled inflammation involving neutrophil extracellular traps (NETs), we tested whether stereochemically defined RvTs regulate NET formation. Using microfluidic devices capturing NETs in phorbol 12-myristate 13-acetate-stimulated human whole blood, the RvTs (RvT1-RvT4; 2.5 nM each) potently reduced NETs. With interleukin-1ß-stimulated human neutrophils, each RvT dose and time dependently decreased NETosis, conveying ∼50% potencies at 10 nM, compared with a known NETosis inhibitor (10 µM). In a murine Staphylococcus aureus infection, RvTs (50 ng each) limited neutrophil infiltration, bacterial titers, and NETs. In addition, each RvT enhanced NET uptake by human macrophages; RvT2 was the most potent of the four RvTs, giving a >50% increase in NET-phagocytosis. As part of the intracellular signaling mechanism, RvT2 increased cyclic adenosine monophosphate and phospho-AMP-activated protein kinase (AMPK) within human macrophages, and RvT2-stimulated NET uptake was abolished by protein kinase A and AMPK inhibition. RvT2 also stimulated NET clearance by mouse macrophages in vivo. Together, these results provide evidence for novel pro-resolving functions of RvTs, namely reducing NETosis and enhancing macrophage NET clearance via a cyclic adenosine monophosphate-protein kinase A-AMPK axis. Thus, RvTs open opportunities for regulating NET-mediated collateral tissue damage during infection as well as monitoring NETs.

Cysteinyl-specialized proresolving mediators link resolution of infectious inflammation and tissue regeneration via TRAF3 activation.

The recently elucidated proresolving conjugates in tissue regeneration (CTR) maresin-CTR (MCTR), protectin-CTR (PCTR), and resolvin-CTR (RCTR), termed cysteinyl-specialized proresolving mediators (cys-SPMs) each promotes regeneration, controls infection, and accelerates resolution of inflammation. Here, we sought evidence for cys-SPM activation of primordial pathways in planaria (Dugesia japonica) regeneration that might link resolution of inflammation and regeneration. On surgical resection, planaria regeneration was enhanced with MCTR3, PCTR3, or RCTR3 (10 nM), each used for RNA sequencing. The three cys-SPMs shared up-regulation of 175 known transcripts with fold-change > 1.25 and combined false discovery rate (FDR) < 0.002, and 199 canonical pathways (FDR < 0.25), including NF-κB pathways and an ortholog of human TRAF3 (TNFR-associated factor 3). Three separate pathway analyses converged on TRAF3 up-regulation by cys-SPMs. With human macrophages, three cys-SPMs each dose-dependently increased TRAF3 expression in a cAMP-PKA-dependent manner. TRAF3 overexpression in macrophages enhanced Interleukin-10 (IL-10) and phagocytosis of Escherichia coli IL-10 also increased phagocytosis in a dose-dependent manner. Silencing of mouse TRAF3 in vivo significantly reduced IL-10 and macrophage phagocytosis. TRAF3 silencing in vivo also relieved cys-SPMs' actions in limiting polymorphonuclear neutrophil in E. coli exudates. These results identify cys-SPM-regulated pathways in planaria regeneration, uncovering a role for TRAF3/IL-10 in regulating mammalian phagocyte functions in resolution. Cys-SPM activation of TRAF3 signaling is a molecular component of both regeneration and resolution of infectious inflammation.

Stereochemistry and functions of the new cysteinyl-resolvin, 4S,5R-RCTR1, in efferocytosis and erythrophagocytosis of human senescent erythrocytes.

Specialized pro-resolving lipid mediators play key functions in the resolution of the acute inflammatory response. Herein, we elucidate the stereochemical structure of the new 4S,5R-RCTR1, a cysteinyl-resolvin, recently uncovered in human leukocytes incubated with a 4S,5S-epoxy-resolvin intermediate, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and ultra-violet (UV) spectrophotometry. With this approach, the physical properties of the new mediator prepared by total organic synthesis were matched to enzymatically produced biogenic material. In addition, we confirmed the potent biological actions of 4S,5R-RCTR1 with human M2-like macrophage phagocytosis of live bacteria, efferocytosis of apoptotic neutrophils, and erythrophagocytosis of senescent human red blood cells in a concentration-dependent manner from 0.1 to 10 nM. Taken together, these results establish the complete stereochemistry of 4S,5R-RCTR1 as 5R-glutathionyl-4S,17S-dihydroxy-6E,8E,10Z,13Z,15E,19Z-docosahexaenoic acid and give evidence of its novel bioactivities in human phagocyte responses. Moreover, they confirm and extend the stereoselective functions of the 4S,5R-RCTR1 with isolated human phagocytes of interest in the resolution of inflammation.

Resolvins and cysteinyl-containing pro-resolving mediators activate resolution of infectious inflammation and tissue regeneration.

This review is a synopsis of the main points from the opening presentation by the authors in the Resolution of Inflammation session at the 8th European Workshop on Lipid Mediators held at the Karolinska Institute, Stockholm, Sweden, June 29th, 2022. Specialized pro-resolving mediators (SPM) promote tissue regeneration, control infections and resolution of inflammation. These include resolvins, protectins, maresins and the newly identified conjugates in tissue regeneration (CTRs). We reported mechanisms of CTRs in activating primordial regeneration pathways in planaria using RNA-sequencing. Also, the 4S,5S-epoxy-resolvin intermediate in the biosynthesis of resolvin D3 and resolvin D4 was prepared by total organic synthesis. Human neutrophils convert this to resolvin D3 and resolvin D4, while human M2 macrophages transformed this labile epoxide intermediate to resolvin D4 and a novel cysteinyl-resolvin that is a potent isomer of RCTR1. The novel cysteinyl-resolvin significantly accelerates tissue regeneration with planaria and inhibits human granuloma formation.

Plasticity of leukocytic exudates in resolving acute inflammation is regulated by MicroRNA and proresolving mediators.

The magnitude and duration of acute inflammation are controlled by active resolution programs involving specialized proresolving mediators (SPMs; resolvins and maresins) and microRNAs (miRNAs). Here, we report that miR-466l was temporally regulated in murine exudate-infiltrating leukocytes. Neutrophil miR-466l overexpression in vivo promoted initiation of inflammation that anteceded macrophage expression of this miRNA, which accelerated resolution when overexpressed. In macrophages, miR-466l overexpression increased prostanoids and SPMs (e.g., resolvin D1 [RvD1] and RvD5), which enhanced resolution. RvD1, RvD2, maresin 1 (MaR1), and apoptotic neutrophils reduced miR-466l expression within human macrophages, a feedback regulation that most likely prepares for homeostasis. miR-466l was upregulated in peripheral blood of sepsis patients, and its increase correlated with nonsurvival from sepsis. SPMs and miR-466l regulated transcription factors activator protein 1 and nuclear factor κB1 in miRNA biogenesis. These results demonstrate pivotal roles for SPMs and miR-466l in dynamic leukocyte plasticity during resolution of acute inflammatory responses.

Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132.

Epoxyeicosatrienoic acids (EETs) are lipid-derived signaling molecules with cardioprotective and vasodilatory actions. We recently showed that 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via G protein-coupled receptors, with evidence supporting the existence of a specific high-affinity receptor. Identification of a hematopoietic-specific EET receptor would enable genetic interrogation of EET signaling pathways, and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify an EET receptor based on the expression of G protein-coupled receptors in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are expressed in three EET-responsive cell lines, but not expressed in an EET-unresponsive line. Of these, only recombinant GPR132 showed EET-responsiveness in vitro, using a luminescence-based ß-arrestin recruitment assay. Knockdown of zebrafish gpr132b prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to high-affinity EET receptors, GPR132 is reported to respond to additional hydroxy-fatty acids in vitro, and we found that these same hydroxy-fatty acids enhance hematopoiesis in the zebrafish. We conducted structure-activity relationship analyses using both cell culture and zebrafish assays on diverse medium-chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, whereas unoxygenated or saturated fatty acids had lower activity. Absence of the carbon-1 position carboxylic acid prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of oxygenated polyunsaturated fatty acids to enhance both embryonic and adult hematopoiesis.

Identification and Complete Stereochemical Assignments of the New Resolvin Conjugates in Tissue Regeneration in Human Tissues that Stimulate Proresolving Phagocyte Functions and Tissue Regeneration.

Resolvin conjugates in tissue regeneration (RCTRs) are new chemical signals that accelerate resolution of inflammation, infection, and tissue regeneration. Herein, using liquid chromatography-tandem mass spectrometry-based metabololipidomics, we identified RCTRs in human spleen, lymph node, bone marrow, and brain. In human spleen incubated with Staphylococcus aureus, endogenous RCTRs were increased along with conversion of deuterium-labeled docosahexaenoic acid, conferring pathway activation. Physical and biological properties of endogenous RCTRs were matched with those prepared by total organic synthesis. The complete stereochemical assignment of bioactive RCTR1 is 8R-glutathionyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, RCTR2 is 8R-cysteinylglycinyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, and RCTR3 is 8R-cysteinyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid. These stereochemically defined RCTRs stimulated human macrophage phagocytosis, efferocytosis, and planaria tissue generation. Proteome profiling demonstrated that RCTRs regulated both proinflammatory and anti-inflammatory cytokines with human macrophages. In microfluidic chambers, the three RCTRs limited human polymorphonuclear cell migration. In hind-limb ischemia-reperfusion-initiated organ injury, both RCTR2 and RCTR3 reduced polymorphonuclear cell infiltration into lungs. In infectious peritonitis, RCTR1 shortened the resolution intervals. Each RCTR (1 nmol/L) accelerated planaria tissue regeneration by approximately 0.5 days, with direct comparison to both maresin and protectin CTRs. Together, these results identify a new bioactive RCTR (ie, RCTR3) in human tissues and establish the complete stereochemistry and rank-order potencies of three RCTRs in vivo. Moreover, RCTR1, RCTR2, and RCTR3 each exert potent anti-inflammatory and proresolving actions with human leukocytes.

Resolving Inflammation: Synthesis, Configurational Assignment, and Biological Evaluations of RvD1n-3 DPA.

New drugs that can resolve inflammation without immunosuppressive effects are at the medicinal chemistry frontier. Pro-resolving endogenously formed small molecules, that is, the resolvins, are excellent candidates displaying such bioactions. The first total synthesis of the specialized pro-resolving mediator RvD1n-3 DPA has been achieved using the underutilized sp3 -sp3 Negishi cross coupling reaction and an alkyne hydrosilylation-protodesilylation protocol. Biological evaluations revealed that this novel mediator displays low nanomolar pro-resolving properties and potently activates the human DRV1/GPR32 receptor. As such, this endogenous natural product is a lead compound for the development of novel immunoresolvents.

New maresin conjugates in tissue regeneration pathway counters leukotriene D4-stimulated vascular responses.

Resolution of acute inflammation is governed, in part, by lipid mediator class switching from proinflammatory eicosanoids to specialized proresolving mediators, including a recently identified new pathway of mediators, termed maresin conjugates in tissue regeneration (MCTR), which includes MCTR1, MCTR2, and MCTR3. Here, we addressed whether each MCTR can impact the known vascular actions of cysteinyl leukotrienes. Leukotriene D4 (LTD4; 1.5 nmol/mouse) initiated vascular leakage in mouse cremaster vessels, which was reduced (>75%) by MCTR1 and MCTR2 (0.15 nmol each). With isolated Ciona intestinalis (sea squirt) primordial hearts, LTD4 (1-100 nM) induced negative inotropic action and lowered heartbeats 20-30%. Each MCTR (1-100 nM) prevented LTD4-reduced heart rates. With human cysteinyl leukotriene receptor-1 (CysLT1) expressed in CHO cells, each MCTR (10-100 nM) significantly reduced LTD4-initiated signaling. To assess the contribution of CysLT1 in the proresolving actions of MCTR, we carried out human macrophage (MΦ) phagocytosis. Each MCTR (0.1-10 nM) stimulated human MΦ phagocytosis of live Escherichia coli, whereas LTD4 did not stimulate phagocytosis. MCTR-activated phagocytosis was significantly blocked by a pharmacologic receptor antagonist (MK571). With both CHO-CysLT1 and human MΦs, each MCTR competed for specific [3H]-LTD4 binding with apparent lower affinity than LTD4. Thus, each MCTR functionally interacts with human CysLT1 to pharmacologically counter-regulate vascular responses and stimulate physiologic phagocytosis with MΦs.-Chiang, N., Riley, I. R., Dalli, J., Rodriguez, A. R., Spur, B. W., Serhan, C. N. New maresin conjugates in tissue regeneration pathway counters leukotriene D4-stimulated vascular responses.

Novel Resolvin D2 Receptor Axis in Infectious Inflammation.

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 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 resolution code of acute inflammation: Novel pro-resolving lipid mediators in resolution.

Studies into the mechanisms in resolution of self-limited inflammation and acute reperfusion injury have uncovered a new genus of pro-resolving lipid mediators coined specialized pro-resolving mediators (SPM) including lipoxins, resolvins, protectins and maresins that are each temporally produced by resolving-exudates with distinct actions for return to homeostasis. SPM evoke potent anti-inflammatory and novel pro-resolving mechanisms as well as enhance microbial clearance. While born in inflammation-resolution, SPM are conserved structures with functions discovered in microbial defense, pain, organ protection and tissue regeneration, wound healing, cancer, reproduction, and neurobiology-cognition. This review covers these SPM mechanisms and other new omega-3 PUFA pathways that open their path for functions in resolution physiology.

Maresin conjugates in tissue regeneration biosynthesis enzymes in human macrophages.

Macrophages are central in coordinating immune responses, tissue repair, and regeneration, with different subtypes being associated with inflammation-initiating and proresolving actions. We recently identified a family of macrophage-derived proresolving and tissue regenerative molecules coined maresin conjugates in tissue regeneration (MCTR). Herein, using lipid mediator profiling we identified MCTR in human serum, lymph nodes, and plasma and investigated MCTR biosynthetic pathways in human macrophages. With human recombinant enzymes, primary cells, and enantiomerically pure compounds we found that the synthetic maresin epoxide intermediate 13S,14S-eMaR (13S,14S-epoxy- 4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid) was converted to MCTR1 (13R-glutathionyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) by LTC4S and GSTM4. Incubation of human macrophages with LTC4S inhibitors blocked LTC4 and increased resolvins and lipoxins. The conversion of MCTR1 to MCTR2 (13R-cysteinylglycinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) was catalyzed by γ-glutamyl transferase (GGT) in human macrophages. Biosynthesis of MCTR3 was mediated by dipeptidases that cleaved the cysteinyl-glycinyl bond of MCTR2 to give 13R-cysteinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid. Of note, both GSTM4 and GGT enzymes displayed higher affinity to 13S,14S-eMaR and MCTR1 compared with their classic substrates in the cysteinyl leukotriene metabolome. Together these results establish the MCTR biosynthetic pathway and provide mechanisms in tissue repair and regeneration.

Identification and Actions of the Maresin 1 Metabolome in Infectious Inflammation.

Maresin 1 (MaR1) is an immunoresolvent that governs resolution of acute inflammation, and its local metabolism in the context of infectious inflammation is of interest. In this study, we investigated the MaR1 metabolome in infectious exudates and its bioactions in regulating leukocyte responses in the context of bacterial infection. In Escherichia coli infectious exudates, MaR1 was temporally regulated with maximal levels at 4 h (2.2 ± 0.4 pg/lavage). In these exudates we also identified two novel products, and their structure elucidation gave 22-hydroxy-MaR1 and 14-oxo-MaR1. Using human primary leukocytes, we found that neutrophils primarily produced 22-OH-MaR1, whereas the main macrophage product was 14-oxo-MaR1. Both 22-OH-MaR1 and 14-oxo-MaR1 incubated with human primary macrophages gave dose-dependent increases in macrophage phagocytosis of ∼75% at 1 pM 22-OH-MaR1 and ∼25% at 1 pM 14-oxo-MaR1, whereas 14-oxo-MaR1 was less active than MaR1 at higher concentrations. Together these findings establish the temporal regulation of MaR1 during infectious inflammation, and elucidate the structures and actions of two novel MaR1 further metabolites that carry bioactivities.

Infection regulates pro-resolving mediators that lower antibiotic requirements.

Underlying mechanisms for how bacterial infections contribute to active resolution of acute inflammation are unknown. Here, we performed exudate leukocyte trafficking and mediator-metabololipidomics of murine peritoneal Escherichia coli infections with temporal identification of pro-inflammatory (prostaglandins and leukotrienes) and specialized pro-resolving mediators (SPMs). In self-resolving E. coli exudates (10(5) colony forming units, c.f.u.), the dominant SPMs identified were resolvin (Rv) D5 and protectin D1 (PD1), which at 12 h were at significantly greater levels than in exudates from higher titre E. coli (10(7) c.f.u.)-challenged mice. Germ-free mice had endogenous RvD1 and PD1 levels higher than in conventional mice. RvD1 and RvD5 (nanograms per mouse) each reduced bacterial titres in blood and exudates, E. coli-induced hypothermia and increased survival, demonstrating the first actions of RvD5. With human polymorphonuclear neutrophils and macrophages, RvD1, RvD5 and PD1 each directly enhanced phagocytosis of E. coli, and RvD5 counter-regulated a panel of pro-inflammatory genes, including NF-κB and TNF-α. RvD5 activated the RvD1 receptor, GPR32, to enhance phagocytosis. With self-limited E. coli infections, RvD1 and the antibiotic ciprofloxacin accelerated resolution, each shortening resolution intervals (R(i)). Host-directed RvD1 actions enhanced ciprofloxacin's therapeutic actions. In 10(7) c.f.u. E. coli infections, SPMs (RvD1, RvD5, PD1) together with ciprofloxacin also heightened host antimicrobial responses. In skin infections, SPMs enhanced vancomycin clearance of Staphylococcus aureus. These results demonstrate that specific SPMs are temporally and differentially regulated during infections and that they are anti-phlogistic, enhance containment and lower antibiotic requirements for bacterial clearance.

Specialized proresolving lipid mediators in patients with coronary artery disease and their potential for clot remodeling.

Inflammation in arterial walls leads to coronary artery disease (CAD). Because specialized proresolving lipid mediators (SPMs; lipoxins, resolvins, and protectins) stimulate resolution of inflammation in animal models, we tested whether n-3 fatty acids impact SPM profiles in patients with CAD and promote clot remodeling. Six patients with stable CAD were randomly assigned to either treatment with daily 3.36 g Lovaza for 1 yr or without. Targeted lipid mediator-metabololipidomics showed that both groups had absence of resolvin D1 (RvD1), RvD2, RvD3, RvD5 and resolvin E1-all of which are present in healthy patients. Those not taking Lovaza had an absence of aspirin-triggered resolvin D3 (AT-RvD3) and aspirin-triggered lipoxin B4 (AT-LXB4). Lovaza treatment restored AT-RvD3 and AT-LXB4 and gave levels of RvD6 and aspirin-triggered protectin D1 (AT-PD1) twice as high (resolvin E2 ∼5 fold) as well as lower prostaglandins. Principal component analysis indicated positive relationships for patients with CAD who were receiving Lovaza with increased AT-RvD3, RvD6, AT-PD1, and AT-LXB4 SPMs identified in Lovaza-treated patients with CAD enhanced ∼50% at 1 nM macrophage uptake of blood clots. These results indicate that patients with CAD have lower levels and/or absence of specific SPMs that were restored with Lovaza; these SPMs promote macrophage phagocytosis of blood clots. Together, they suggest that low vascular SPMs may enable progression of chronic vascular inflammation predisposing to coronary atherosclerosis and to thrombosis.-Elajami, T. K., Colas, R. A., Dalli, J., Chiang, N., Serhan, C. N., Welty, F. K. Specialized proresolving lipid mediators in patients with coronary artery disease and their potential for clot remodeling.

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

Upon infection and inflammation, tissue repair and regeneration are essential in reestablishing function. Here we identified potent molecules present in self-limited infectious murine exudates, regenerating planaria, and human milk as well as macrophages that stimulate tissue regeneration in planaria and are proresolving. Characterization of their physical properties and isotope tracking indicated that the bioactive structures contained docosahexaenoic acid and sulfido-conjugate (SC) of triene double bonds that proved to be 13-glutathionyl, 14-hydroxy-docosahexaenoic acid (SCI) and 13-cysteinylglycinyl, 14-hydroxy-docosahexaenoic acid (SCII). These molecules rescued Escherichia coli infection-mediated delay in tissue regeneration in planaria, improving regeneration intervals from ∼ 4.2 to ∼ 3.7 d. Administration of SCs protected mice from second-organ reflow injury, promoting repair via limiting neutrophil infiltration, up-regulating Ki67, and Roof plate-specific spondin 3. At nanomolar potencies these conjugates also resolved E. coli infections by limiting neutrophil infiltration and stimulating bacterial phagocytosis and clearance as well as efferocytosis of apoptotic cells. Together, these findings identify previously undescribed conserved chemical signals and pathways in planaria, mouse, and human tissues that enhance host responses to contain infections, stimulate resolution of inflammation, and promote the restoration of function.

Protectins and maresins: New pro-resolving families of mediators in acute inflammation and resolution bioactive metabolome.

Acute inflammatory responses are protective, yet without timely resolution can lead to chronic inflammation and organ fibrosis. A systems approach to investigate self-limited (self-resolving) inflammatory exudates in mice and structural elucidation uncovered novel resolution phase mediators in vivo that stimulate endogenous resolution mechanisms in inflammation. Resolving inflammatory exudates and human leukocytes utilize DHA and other n-3 EFA to produce three structurally distinct families of potent di- and trihydroxy-containing products, with several stereospecific potent mediators in each family. Given their potent and stereoselective picogram actions, specific members of these new families of mediators from the DHA metabolome were named D-series resolvins (Resolvin D1 to Resolvin D6), protectins (including protectin D1-neuroprotectin D1), and maresins (MaR1 and MaR2). In this review, we focus on a) biosynthesis of protectins and maresins as anti-inflammatory-pro-resolving mediators; b) their complete stereochemical assignments and actions in vivo in disease models. Each pathway involves the biosynthesis of epoxide-containing intermediates produced from hydroperoxy-containing precursors from human leukocytes and within exudates. Also, aspirin triggers an endogenous DHA metabolome that biosynthesizes potent products in inflammatory exudates and human leukocytes, namely aspirin-triggered Neuroprotectin D1/Protectin D1 [AT-(NPD1/PD1)]. Identification and structural elucidation of these new families of bioactive mediators in resolution has opened the possibility of diverse patho-physiologic actions in several processes including infection, inflammatory pain, tissue regeneration, neuroprotection-neurodegenerative disorders, wound healing, and others. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

Cutting edge: Parathyroid hormone facilitates macrophage efferocytosis in bone marrow via proresolving mediators resolvin D1 and resolvin D2.

Bone marrow macrophages stimulate skeletal wound repair and osteoblastic bone formation by poorly defined mechanisms. Specialized proresolving mediators of inflammation drive macrophage efferocytosis (phagocytosis of apoptotic cells) and resolution, but little is known regarding this process in the bone marrow. In this study, metabololipidomic profiling via liquid chromatography mass spectrometry revealed higher levels of specialized proresolving mediators in the bone marrow relative to the spleen. The endocrine and bone anabolic agent parathyroid hormone increased specialized proresolving mediator levels, including resolvins (Rvs), in bone marrow. Human and murine primary macrophages efferocytosed apoptotic osteoblasts in vitro, and RvD1 and RvD2 (10 pM-10 nM) enhanced this process. These findings support a unique profile of specialized lipid mediators in bone marrow that contribute to a feedback system for resolution of inflammation and maintenance of skeletal homeostasis.

The Regulation of Proresolving Lipid Mediator Profiles in Baboon Pneumonia by Inhaled Carbon Monoxide.

Strategies for the treatment of bacterial pneumonia beyond traditional antimicrobial therapy have been limited. The recently discovered novel genus of lipid mediators, coined "specialized proresolving mediators" (SPMs), which orchestrate clearance of recruited leukocytes and restore epithelial barrier integrity, have offered new insight into the resolution of inflammation. We performed lipid mediator (LM) metabololipidomic profiling and identification of LMs on peripheral blood leukocytes and plasma from a baboon model of Streptococcus pneumoniae pneumonia. Leukocytes and plasma were isolated from whole blood of S. pneumoniae-infected (n = 5-6 per time point) and control, uninfected baboons (n = 4 per time point) at 0, 24, 48, and 168 hours. In a subset of baboons with pneumonia (n = 3), we administered inhaled carbon monoxide (CO) at 48 hours (200-300 ppm for 60-90 min). Unstimulated leukocytes from control animals produced a proresolving LM signature with elevated resolvins and lipoxins. In contrast, serum-treated, zymosan-stimulated leukocytes and leukocytes from baboons with S. pneumoniae pneumonia produced a proinflammatory LM signature profile with elevated leukotriene B4 and prostaglandins. Plasma from baboons with S. pneumoniae pneumonia also displayed significantly reduced LM-SPM levels, including eicosapentaenoic acid-derived E-series resolvins (RvE) and lipoxins. CO inhalation increased levels of plasma RvE and lipoxins relative to preexposure levels. These results establish the leukocyte and plasma LM profiles biosynthesized during S. pneumoniae pneumonia in baboons and provide evidence for pneumonia-induced dysregulation of these proresolution programs. Moreover, these SPM profiles are partially restored with inhaled low-dose CO and SPM, which may shorten the time to pneumonia resolution.

Maresin 1 Biosynthesis and Proresolving Anti-infective Functions with Human-Localized Aggressive Periodontitis Leukocytes.

Localized aggressive periodontitis (LAP) is a distinct form of early-onset periodontitis linked to periodontal infection with uncontrolled inflammation and leukocyte-mediated tissue destruction. The resolution of inflammation is an active process orchestrated by specialized proresolving lipid mediators (SPMs). Since the level of the Maresin pathway marker 14-hydroxy-docosahexaenoic acid (14-HDHA) was lower in activated peripheral blood from LAP patients, we investigated the Maresin 1 (MaR1) biosynthetic pathway in these subjects and its role in regulating phagocyte functions. Macrophages from LAP patients had a lower level of expression of 12-lipoxygenase (∼30%) and reduced MaR1 (LAP versus healthy controls [HC], 87.8 ± 50 pg/10(6) cells versus 239.1 ± 32 pg/10(6) cells). Phagocytosis by LAP macrophages was reduced ∼40% compared to that of HC, and killing of periodontal pathogens, including Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, were similarly reduced. LAP neutrophils also displayed slower kinetics (∼30%) and decreased maximal phagocytosis (∼20% lower) with these pathogens than those of HC. The administration of MaR1 at 1 nM enhanced phagocytosis (31 to 65% increase), intracellular antimicrobial reactive oxygen species production (26 to 71% increase), bacterial killing of these periodontal pathogens (22 to 38% reduction of bacterial titers), and restored impairment of LAP phagocytes. Together, these results suggest that therapeutics targeting the Maresin pathway have clinical utility in treating LAP and other oral diseases associated with infection, inflammation, and altered phagocyte functions.