Obesity is characterized by chronic low-grade inflammation which promotes numerous complications such as type 2 diabetes (T2D) and metabolic dysfunction associated fatty liver disease (MAFLD). A class of lipid mediators known as Specialized Pro-resolving Mediators (SPMs) has garnered interest in this field due to their capacity to promote inflammation resolution pathways. Some SPMs also appear to act directly on inflammation disrupted biological functions to aid in the return to homeostasis. One such SPM is Protectin DX (PDX), the stereoisomer of Protectin D1 (PD1). Our group previously demonstrated that PDX ameliorates metabolic inflammation and glycemic control and attenuates end-stage renal failure in two different T2D murine models. Our group recently developed cost-efficient synthetic routes for PDX and structural analogues to accelerate research on PDX functions and to scale-up production of these molecules for preclinical therapeutic studies. Thus, over 30 PDX analogues were synthesized and screened to evaluate their bioactivity on relevant cellular models. Fifteen of these small PDX structural mimics reduced inducible nitric oxide synthase (iNOS) activity in LPS treated J774 macrophages. Six analogues, but not PD1, increased glucose uptake in L6 and C2C12 myocytes. None of them, however, replicated PDX and PD1 capacity to activate PPARγ transcriptional activity in a cell-based luciferase reporter assay. A few bioactive analogues were next tested in vivo for their ability to suppress LPSinduced inflammation in golden Syrian hamsters. One analogue, RM-598-48 was found to significantly decrease plasma TNF-α, specifically in female hamsters. Lastly, a low dose of RM-598-48 (50 ng/g) orally administered daily was found to reduce hepatic steatosis and to prevent fasting hyperinsulinemia in hamsters fed an obesogenic high fat high fructose mixed protein diet. These results suggest that cost-efficient analogues of PDX display a high potential as new therapeutic drugs against obesity-linked inflammation, T2D, and MAFLD.

Most patients with advanced cancer suffer from cachexia, which results in the death of 20% of patients. There are no approved therapies for cancer cachexia as the underlying mechanisms remain poorly characterized. The hallmark of cachexia is unresolved hyperinflammation resulting in a devastating muscle wasting syndrome. Cachexia-induced apoptotic cell death occurs in many tissues by pro-inflammatory cytokines. A paradigm shift is emerging in understanding the resolution of inflammation as an active biochemical process with the discovery of novel specialized pro-resolving mediators (SPMs). SPMs stimulate clearance of debris, promote muscle regeneration, and counter-regulate cytokines. We hypothesized that cachexia results from disrupted resolution of inflammation. We profiled lipid mediators in cachexia models via state-of-the-art targeted metabololipidomics. Here, we’ve identified dysregulated SPMs in six cancer cachexia models. SPMs (e.g., RvD2 and MaR1) were markedly reduced in colon cancer (CT26)-induced cachectic mice on day 35 post-tumor cell injection vs. non-tumor bearing mice (NTB). SPMs (RvD1, RvD2, LXA4, and MaR1) were also dramatically dysregulated in Lewis lung carcinoma (LLC)-induced cachectic mice, including the gastrocnemius and tibialis anterior muscles, heart, liver, and spleen, on day 20 post-tumor cell injection. Cachexia induced a pro-inflammatory eicosanoid storm in plasma from CT26-induced cachectic mice. Chemotherapy also induced cachexia via loss of SPMs in a lymphoma (EL4) and ovarian cancer (ID8) mouse model. At 10 days post-LLC tumor resection, the RvD1 receptor (ALX/FPR2) KO and RvE1 receptor (ChemR23/ERV) KO mice exhibited 20-23% loss in body weight compared to WT mice. Thus, cancer cachexia is resolvin receptor-dependent. RvD2 and PCTR2 prevented LLC- and B16F10 melanoma-induced cachexia at 15 nanograms/day compared to control without immunosuppression. RvD2 prevented pancreatic cancer (KPC)-induced loss of grip strength and prolonged survival compared to control. In contrast to celecoxib, SPMs prevented the cachexia-induced cytokine storm, including inhibition of TNF-α, CCL2, CCL3, CCL4, CXCL2, G-CSF, and PAI-1. SPMs were sharply reduced by up to 85% in the plasma of pancreatitis patients at risk for cachexia compared to healthy individuals. Thus, our studies shall provide the basis for the clinical translation of SPM-directed treatments in humans as a new direction to potentially prevent and/or reverse cancer cachexia.

Platelets, traditionally known for maintaining blood balance, are now recognized as crucial cells in antimicrobial defense. Upon SARS-CoV-2 infection, platelets become hyperactivated, releasing various molecules such as cytokines, granule contents, and bioactive lipids. The key biolipids produced in platelets are regulated by enzymes: group IV cytosolic phospholipase A2 (cPLA2a) responsible for the hydrolysis of precursor lipids from membrane phospholipids; 12-lipoxygenase (12-LOX), producing 12-hydroxyeicosatetraenoic acid (12-HETE); and cyclooxygenase-1 (COX-1), implicated in prostaglandins (PG) and thromboxane (TX) production. While PGE2 and TXB2 were previously associated with lung inflammation in severe COVID-19, the role of platelet 12-LOX in SARS-CoV-2 infection remains unclear. Using Pla2g4a and Alox12 knockout mice, we found increased lung inflammation in Pla2g4a knockout mice post-infection. Conversely, Alox12-deficient mice exhibited not only higher lung inflammation characterized by increased leukocyte infiltrates and cytokine productions but also premature mortality and distinct lung transcriptomic changes, including alterations in NLRP1 inflammasome-related gene expression. Additionally, lipidomic analysis in Alox12 knockout mice revealed significant changes, including reduced levels of the 12-LOX product 12-HETrE and Maresin-2, known for their anti-inflammatory and pro-resolving effects, inversely correlating with disease severity. This study highlights the complex interplay between 12-LOX-related lipid metabolism, and inflammatory responses during SARS-CoV-2 infection. The findings provide valuable insights into potential therapeutic targets aimed at mitigating severe outcomes, emphasizing the pivotal role of platelet enzymes in the host response to viral infections.

Dysregulated neutrophil function underlies acute respiratory distress syndrome (ARDS) that is associated with high mortality. We reported that bacterial DNA (CpG DNA) through TLR9 activation impairs bacterial clearance by neutrophils and identified IFN-β as a macrophage-derived cytokine that facilitate bacterial clearance and promotes the resolution of ARDS. However, little is known about the underlying mechanisms; albeit these would be essential for implementing precision treatment with IFN-β. Since the resolution of inflammation is skewed toward a pro-resolving lipid profile, we investigated whether IFN-β can exert its beneficial actions through modulating pro-resolving lipid mediator-based resolution mechanisms. Culture of human neutrophils with IFN-β, 15-epi-LXA4 or RvD1 countered the survival cues from CpG DNA through reducing Mcl-1 expression. CpG DNA stimulated production of leukotriene B4 (LTB4), but not 15-epi-lipoxin A4 (15-epi-LXA4) and resolvin D1 (RvD1). IFN-β reduced LTB4 release without evoking detectable increases in 15-epi-LXA4 and RvD1 production. Unlike 15-epi-LXA4 and RvD1, IFN-β did not restore CpG DNA-impaired phagocytosis and bacterial clearance. In a mouse model of ARDS evoked by CpG DNA and intratracheal instillation of E. coli, treatment with IFN-β at the peak of inflammation, restored impaired neutrophil phagocytosis, accelerated bacterial clearance, enhanced neutrophil apoptosis and efferocytosis, resulting in accelerated resolution of airway inflammation. CpG DNA or neutralizing endogenous IFN-β markedly enhanced bronchoalveolar lavage fluid levels of LTB4, while reduced 15-epi-LXA4 and RvD1 levels. Conversely, treatment with IFN-β markedly enhanced lavage fluid levels of 15-epi-LXA4 and RvD1 and reduced LTB4. Furthermore, selective blockade of the receptor ALX/FPR2 (which binds both 15-epi-LXA4 and RvD1) with WRW4 partially blocked IFN-β-mediated resolution, leading to impaired bacterial clearance and persisting lung injury. Our results identify an intricate interplay between IFN-β and pro-resolving lipid mediators that signal through ALX/FPR2 to restore impaired neutrophil function and to facilitate the timely resolution of neutrophil driven ARDS. Grant support: Canadian Institutes of Health Research MOP-97742 and MOP-102619.

A growing body of evidence indicates that specialized pro-resolving mediators (SPMs) play a pivotal role in the resolution of inflammation (Nature, 2014, 510:92-101). Recently, we achieved the first total synthesis of 4S,5S-epoxy-17S-hydroxy-6E,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (RSC Adv., 2022, 12:11613-18), the biosynthetic precursor to potent resolvin D3, resolvin D4 and 4S,5R-RCTR1. We also accomplished the total synthesis of resolvin 7,8(S,S)-epoxytetraene and are currently investigating its critical role in natural formation of pro-resolving mediators. Stereospecific and chiral pool-based synthetic approaches were employed, with key synthetic features including Sonogashira coupling, Sharpless asymmetric epoxidation, and a late-stage Wittig olefination to forge the core carbon skeleton. Next, we demonstrated experimental evidence supporting the role of this highly labile epoxide (T1/2 ≈ 5 sec; aq. solutions) in the biosynthesis of resolvin D3 (4S,11R,17S-trihydroxy-5Z,7E,9E,13Z,15E,19Z-docosahexaenoic acid) and resolvin D4 (4S,5R,17S-trihydroxy-6E,8E,10Z,13Z,15E,19Z-docosahexaenoic acid) by human neutrophils and M2 macrophages (PNAS, 2021, e2116559118). Of interest, this transient epoxide was also converted to a novel cysteinyl-resolvin, 4S,5R-RCTR1 (5R-glutathionyl-4S,17S-dihydroxy-6E,8E,10Z,13Z,15E,19Z-docosahexaenoic acid), by M2 macrophages. Both resolvins D3 and D4 are potent signaling molecules with protective actions, i.e., limit neutrophilic transmigration and enhance macrophage phagocytosis and efferocytosis, as well as modulate the severity of deep vein thrombosis (Nat Rev Cardiol., 2024, PMID: 38216693). Total synthesis of the newly uncovered cys-resolvin enabled its structural elucidation and biological activity studies including clearance of live bacteria and erythrophagocytosis of senescent human red cells (Am. J. Hematol., 2023, 98:1000-16). Together, these results confirm the role of 4S,5S-epoxy-resolvin intermediate in the biosynthesis of bioactive resolvins D3, D4 and 4S,5R-RCTR1 in a cell-type specific manner. Currently, biosynthetic studies involving these allylic epoxide intermediates are underway and results will be presented. The authors gratefully acknowledge support by US NIH grants P01GM095467 and R35GM139430.