The translational relevance of the Annexin A1 pathway in inflammatory pathologies: opportunities for novel therapeutic development

PhDEndogenous anti-inflammatory mediators form a complex network triggered in the host to dampen cell activation and promote resolution of inflammation. The glucocorticoidregulated 37-kDa protein Annexin A1 is one such endogenous checkpoint effector, acting on human neutrophils via a specific GPCR termed FPR like-1 (FPRL1). FPRL1 belongs to the family of formyl-peptide receptors, of which FPR (the receptor for formylated peptides) is the prototype. However, very little information is available on the mechanisms governing the export of this protein from activated cells, hence the machinery required to activate the counter-regulatory circuit centred on Annexin A1. Furthermore, little data is available describing the status of Annexin A1 and the FPR receptor family in human disease conditions. These questions have been addressed in my thesis. The analysis of PMNs and monocytes from both Wegener granulomatosis and Giant cell arthritis (GCA) patients provided strong evidence for a deregulation in the Annexin A1 pathway as I observed an increased expression of both FPRL-1 and FPR, along with an elevated cell surface Annexin A1 expression. Moreover, Western blotting against Annexin A1 membrane expression also evidenced elevated protein cleavage suggesting that this is potentially the mechanism responsible for the reported hyper-activated status of the PMNs in these pathologies, a feature I could confirm also in the flow chamber assay, where marked adhesion to endothelial cell monlayers was measured. Significantly, pharmacological manipulation of the Annexin A1 axis could correct cell behaviour, further supporting the notion of a deregulated Annexin A1 system in these conditions. Interestingly, very little difference was observed at the mRNA levels for both the FPRL-1 and Annexin A1 genes in samples analysed from patients suffering from Wegener granulomatosis whilst there was a significant increase in expression of both these genes in the GCA samples when compared to aged matched healthy volunteers. An observation, which suggests that the underlying mechanisms governing the regulation in these two conditions, and their potential impact on the Annexin A1 pathway, might be different. Annexin A1 lacks a signal peptide but nonetheless is abundantly released from activated PMNs; our understanding of the route that is employed for its release is still modest. The analysis of PMN derived microparticles confirmed the presence of the Annexin A1 protein in these microstructures; I was then able to demonstrate that this protein was responsible for microparticle-induced inhibition of PMN recruitment to an 4 activated endothelium in vitro and into the airpouch in vivo. Furthermore, when monitoring levels of Annexin A1 positive microparticles in plasma samples from a number of human inflammatory disease, I could observe that both these microparticle subsets were altered when compared to those found in healthy age matched controls, with higher extent of PMN-derived microparticles (CD62L positive) and Annexin A1 positive microparticles. I had the opportunity to monitor these microparticles longitudinally in RA patients treated with prednisolone over a 2-week period: both CD62L and Annexin A1 positive microparticles were restored back to the values (as percentage and median fluorescence intensity) of healthy volunteers. This result occurred in parallel to an amelioration of the clinical symptoms. The final part of my project involved assessing the anti-inflammatory properties of 5 novel Annexin A1 N-terminal derived peptides developed in collaboration with Unigene (Fairfield, NJ), that are modification of peptide Ac2-26 (which conserves the natural amino acid sequence). In vitro analyses of these peptides identified two peptides as the ones with the highest anti-inflammatory capabilities. In radioligand binding assays, I observed these peptides possessed similar binding affinities to the FPRL-1 as the natural peptide. More in detail, peptide 57 did not bind to FPR in a dose dependent faishon as opposed to peptide 84, even though when assessing p-ERK activity it was noted that both peptides equally activated ERK. When tested in vivo it was observed that both peptides were able to inhibit PMN recruitment into an inflamed mouse airpouch, with peptide 84 showing the highest potency. The findings of this thesis provided evidence for a deregulated Annexin A1 system in the human inflammatory pathologies under observation, hence vasculitis and RA. Treatment of patients with an acute glucocorticoid regimen modulated the Annexin A1 pathway suggesting that the re-establishment of this effector of anti-inflammation, likely to occur also at the functional level, could contribute – at least partly – to the positive clinical effect of glucocorticoids in RA patients. Captivatingly, this acute glucocorticoid treatment was also observed to restore the plasma Annexin A1 positive microparticle levels to those observed in healthy age-matched volunteers, suggesting that these microstructures can potentially be used both as biomarkers of disease and also a measure of treatment effectiveness. Finally I have provided evidence for the antiinflammatory properties of two novel Annexin A1 N-terminal derived peptides that may serve as guidance for the development of novel treatments for inflammatory disorders, depicted on the biology of this intriguing protein that is Annexin A1

Novel n-3 Immunoresolvents: Structures and Actions

Resolution of inflammation is now held to be an active process where autacoids promote homeostasis. Using functional-metabololipidomics and in vivo systems, herein we report that endogenous n-3 docosapentaenoic (DPA) acid is converted during inflammation-resolution in mice and by human leukocytes to novel n-3 products congenerous to D-series resolvins (Rv), protectins (PD) and maresins (MaR), termed specialized pro-resolving mediators (SPM). The new n-3 DPA structures include 7,8,17-trihydroxy-9,11,13,15E,19Z-docosapentaenoic acid (RvD1n-3 DPA), 7,14-dihydroxy-8,10,12,16Z,19Z-docosapentaenoic acid (MaR1n-3 DPA) and related bioactive products. Each n-3 DPA-SPM displayed protective actions from second organ injury and reduced systemic inflammation in ischemia-reperfusion. The n-3 DPA-SPM, including RvD1n-3 DPA and MaR1n-3 DPA, each exerted potent leukocyte directed actions in vivo. With human leukocytes each n-3 DPA-SPM reduced neutrophil chemotaxis, adhesion and enhanced macrophage phagocytosis. Together, these findings demonstrate that n-3 DPA is converted to novel immunoresolvents with actions comparable to resolvins and are likely produced in humans when n-3 DPA is elevated

Elucidation of novel 13-series resolvins that increase with atorvastatin and clear infections

Endogenous mechanisms leading to host protection and resolution of infections without immunosuppression are of wide interest1,2. Here we elucidated the structures of four new host-protective molecules produced in neutrophil-endothelial co-cultures, and present in human and mouse tissues after sterile inflammation or infection. These bioactive molecules contained conjugated triene and diene double bonds with each carrying a 13-carbon position alcohol and were derived from n-3 docosapentaenoic acid (DPA, C22:5). These compounds, termed 13-series resolvins (RvT), demonstrated potent protective actions increasing mice survival during Escherichia coli infections. RvT also regulated human and mouse phagocyte responses stimulating bacterial phagocytosis and regulating inflammasome components. Their biosynthesis during neutrophil-endothelial cell interactions was initiated by endothelial cyclooxygenase-2 (COX-2) and increased by atorvastatin via S-nitrosylation of COX-2. The actions of atorvastatin and RvT were additive in E. coli infections in mice where they accelerated resolution of inflammation and increased survival >60%. These results document novel host protective molecules in bacterial infections, namely 13-series resolvins, derived from n-3 DPA via transcellular biosynthesis and increased by atorvastatin. These novel molecules regulate key innate protective responses in the resolution of infectious-inflammation

Vagus nerve controls resolution and pro-resolving mediators of inflammation

Resolution of inflammation is now recognized as a biosynthetically active process involving pro-resolving mediators. Here, we show in zymosan-initiated peritoneal inflammation that the vagus nerve regulates local expression of netrin-1, an axonal guidance molecule that activates resolution, and that vagotomy reduced local pro-resolving mediators, thereby delaying resolution. In netrin-1+/− mice, resolvin D1 (RvD1) was less effective in reducing neutrophil influx promoting resolution of peritonitis compared with Ntn1+/+. Netrin-1 shortened the resolution interval, decreasing exudate neutrophils, reducing proinflammatory mediators, and stimulating the production of resolvins, protectins, and lipoxins. Human monocytes incubated with netrin-1 produced proresolving mediators, including resolvins and lipoxins. Netrin-1 and RvD1 displayed bidirectional activation in that they stimulated each other’s expression and enhanced efferocytosis. These results indicate that the vagus nerve regulates both netrin-1 and pro-resolving lipid mediators, which act in a bidirectional fashion to stimulate resolution, and provide evidence for a novel mechanism for local neuronal control of resolution

Self-Limited versus Delayed Resolution of Acute Inflammation: Temporal Regulation of Pro-Resolving Mediators and MicroRNA

Mechanisms underlying delays in resolution programs of inflammation are of interest for many diseases. Here, we addressed delayed resolution of inflammation and identified specific microRNA (miR)-metabolipidomic signatures. Delayed resolution initiated by high-dose challenges decreased miR-219-5p expression along with increased leukotriene B4 (5-fold) and decreased (~3-fold) specialized pro-resolving mediators, e.g. protectin D1. Resolvin (Rv)E1 and RvD1 (1 nM) reduced miR-219-5p in human macrophages, not shared by RvD2 or PD1. Since mature miR-219-5p is produced from pre-miRs miR-219-1 and miR-219-2, we co-expressed in human macrophages a 5-lipoxygenase (LOX) 3′UTR-luciferase reporter vector together with either miR-219-1 or miR-219-2. Only miR-219-2 reduced luciferase activity. Apoptotic neutrophils administered into inflamed exudates in vivo increased miR-219-2-3p expression and PD1/NPD1 levels as well as decreased leukotriene B4. These results demonstrate that delayed resolution undermines endogenous resolution programs, altering miR-219-2 expression, increasing pro-inflammatory mediators and compromising SPM production that contribute to failed catabasis and homeostasis

Maresin Biosynthesis and Identification of Maresin 2, a New Anti-Inflammatory and Pro-Resolving Mediator from Human Macrophages

Maresins are a new family of anti-inflammatory and pro-resolving lipid mediators biosynthesized from docosahexaenoic acid (DHA) by macrophages. Here we identified a novel pro-resolving product, 13R,14S-dihydroxy-docosahexaenoic acid (13R,14S-diHDHA), produced by human macrophages. PCR mapping of 12-lipoxygenase (12-LOX) mRNA sequence in human macrophages and platelet showed that they are identical. This human 12-LOX mRNA and enzyme are expressed in monocyte-derived cell lineage, and enzyme expression levels increase with maturation to macrophages or dendritic cells. Recombinant human 12-LOX gave essentially equivalent catalytic efficiency (kcat/KM) with arachidonic acid (AA) and DHA as substrates. Lipid mediator metabololipidomics demonstrated that human macrophages produce a novel bioactive product 13,14-dihydroxy-docosahexaenoic acid in addition to maresin-1, 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (MaR1). Co-incubations with human recombinant 12-LOX and soluble epoxide hydrolase (sEH) demonstrated that biosynthesis of 13,14-dihydroxy-docosahexaenoic acid (13,14-diHDHA) involves the 13S,14S-epoxy-maresin intermediate produced from DHA by 12-LOX, followed by conversion via soluble epoxide hydrolase (sEH). This new 13,14-diHDHA displayed potent anti-inflammatory and pro-resolving actions, and at 1 ng reduced neutrophil infiltration in mouse peritonitis by ∼40% and at 10 pM enhanced human macrophage phagocytosis of zymosan by ∼90%. However, MaR1 proved more potent than the 13R,14S-diHDHA at enhancing efferocytosis with human macrophages. Taken together, the present findings demonstrate that macrophages produced a novel bioactive product identified in the maresin metabolome as 13R,14S-dihydroxy-docosahexaenoic acid, from DHA via conversion by human 12-LOX followed by sEH. Given its potent bioactions, we coined 13R,14S-diHDHA maresin 2 (MaR2)

Lipid mediators in platelet concentrate and extracellular vesicles: Molecular mechanisms from membrane glycerophospholipids to bioactive molecules

Platelets are collected for transfusion to patients with different hematological disorders, and for logistical reasons, platelets are stored as concentrates. Despite the carefully controlled conditions, platelets become activated during storage, and platelet concentrates (PLCs) may cause adverse inflammatory reactions in the recipients. We studied by mass spectrometry the lipidomic changes during storage of the clinical PLCs, the platelets isolated from PLCs, and the extracellular vesicles (EVs) thereof. The release of EVs from platelets increased with the prolonged storage time. The molar percentages of arachidonic acid -containing species were increased during storage especially in the phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine classes of glycerophopholipids. The increase of these species in the membrane glycerophopholipid composition paralleled the production of both proinflammatory and proresolving lipid mediators (LMs) as the amount of the arachidonic acid-derived LMs such as thromboxane B2 and prostaglandin E2 also increased in time. Moreover, several monohydroxy pathway markers and functionally relevant proinflammatory and proresolving LMs were detected in the PLC and the EVs, and some of these clearly accumulated during storage. By Western blot, the key enzymes of these pathways were shown to be present in the platelets and in many cases also in the EVs. Since the EVs were enriched in the fatty acid precursors of LMs, harbored LM-producing enzymes, contained the related monohydroxy pathway markers, and also secreted the final LM products, the PLC-derived EVs appear to have the potential to regulate inflammation and healing, and may thereby aid the platelets in exerting their essential physiological functions.Peer reviewe

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

Polyunsaturated fatty acids modify the extracellular vesicle membranes and increase the production of proresolving lipid mediators of human mesenchymal stromal cells

Human mesenchymal stromal/stem cells (hMSCs) are used in experimental cell therapy to treat various immunological disorders, and the extracellular vesicles (hMSC-EVs) they produce have emerged as an option for cell-free therapeutics. The immunomodulatory function of hMSCs resembles the resolution of inflammation, in which proresolving lipid mediators (LMs) play key roles. Multiple mechanisms underlying the hMSC immunosuppressive effect has been elucidated; however, the impact of LMs and EVs in the resolution is poorly understood. In this study, we supplemented hMSCs with polyunsaturated fatty acids (PUFAs); arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, which serve as precursors for multiple LMs. We then determined the consequent compositional modifications in the fatty acid, phospholipid, and LM profiles. Mass spectrometric analyses revealed that the supplemented PUFAs were incorporated into the main membrane phospholipid classes with different dynamics, with phosphatidylcholine serving as the first acceptor. Most importantly, the PUFA modifications were transferred into hMSC-EVs, which are known to mediate hMSC immunomodulation. Furthermore, the membrane-incorporated PUFAs influenced the LM profile by increasing the production of downstream prostaglandin E-2 and proresolving LMs, including Resolvin E2 and Resolvin D6. The production of LMs was further enhanced by a highly proinflammatory stimulus, which resulted in an increase in a number of mediators, most notably prostaglandins, while other stimulatory conditions had less a pronounced impact after a 48-h incubation. The current findings suggest that PUFA manipulations of hMSCs exert significant immunomodulatory effects via EVs and proresolving LMs, the composition of which can be modified to potentiate the therapeutic impact of hMSCs.Peer reviewe

Microparticle alpha-2-macroglobulin enhances pro-resolving responses and promotes survival in sepsis

Incorporation of locally produced signaling molecules into cell-derived vesicles may serve as an endogenous mediator delivery system. We recently reported that levels alpha-2-macroglobulin (A2MG)-containing microparticles are elevated in plasma from patients with sepsis. Herein, we investigated the immunomodulatory actions of A2MG containing microparticles during sepsis. Administration of A2MG-enriched (A2MG-E)-microparticles to mice with microbial sepsis protected against hypothermia, reduced bacterial titers, elevated immunoresolvent lipid mediator levels in inflammatory exudates and reduced systemic inflammation. A2MG-E microparticles also enhanced survival in murine sepsis, an action lost in mice transfected with siRNA for LRP1, a putative A2MG receptor. In vitro, A2MG was functionally transferred onto endothelial cell plasma membranes from microparticles, augmenting neutrophil–endothelial adhesion. A2MG also modulated human leukocyte responses: enhanced bacterial phagocytosis, reactive oxygen species production, cathelicidin release, prevented endotoxin induced CXCR2 downregulation and preserved neutrophil chemotaxis in the presence of LPS. A significant association was also found between elevated plasma levels of A2MG-containing microparticles and survival in human sepsis patients. Taken together, these results identify A2MG enrichment in microparticles as an important host protective mechanism in sepsis