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Abstract While cardiovascular disease (CVD) is one of the major co-morbidities in patients with rheumatoid arthritis (RA), the mechanism(s) that contribute to CVD in patients with RA remain to be fully elucidated. Herein, we observe that plasma concentrations of 13-series resolvin (RvT)4 negatively correlate with vascular lipid load in mouse inflammatory arthritis. Administration of RvT4 to male arthritic mice fed an atherogenic diet significantly reduces atherosclerosis. Assessment of the mechanisms elicited by this mediator demonstrates that RvT4 activates cholesterol efflux in lipid laden macrophages via a Scavenger Receptor class B type 1 (SR-BI)-Neutral Cholesterol Ester Hydrolase-dependent pathway. This leads to the reprogramming of lipid laden macrophages yielding tissue protection. Pharmacological inhibition or knockdown of macrophage SR-BI reverses the vasculo-protective activities of RvT4 in vitro and in male mice in vivo. Together these findings elucidate a RvT4-SR-BI centered mechanism that orchestrates macrophage responses to limit atherosclerosis during inflammatory arthritis.

Aims: The cardio-protective and immuno-regulatory properties of RTP-026, a synthetic peptide that spans the Annexin-A1 (AnxA1) N-terminal region, were tested in rat acute myocardial infarction. Methods and results: In vitro, selective activation of formyl-peptide receptor type 2 (FPR2) by RTP-026 occurred with apparent EC50 in the 10–30 nM range. With human primary cells, RTP-026 counteracted extension of neutrophil life-span and augmented phagocytosis of fluorescent E.coli by blood myeloid cells. An in vivo model of rat acute infarction was used to quantify tissue injury and phenotype immune cells in myocardium and blood. The rat left anterior descending coronary artery was occluded and then reopened for 2-hour or 24-hour reperfusion. For the 2-hour reperfusion protocol, RTP-026 (25–500 µg/kg; given i.v. at the start of reperfusion) significantly reduced infarct size by ∼50 %, with maximal efficacy at 50 µg/kg. Analyses of cardiac immune cells showed that RTP-026 reduced neutrophil and classical monocyte recruitment to the damaged heart. In the blood, RTP-026 (50 µg/kg) attenuated activation of neutrophils and monocytes monitored through CD62L and CD54 expression. Modulation of vascular inflammation by RTP-026 was demonstrated by reduction in plasma levels of mediators like TNF-α, IL-1β, KC, PGE2 and PGF2α⊡ For the 24-hour reperfusion protocol, RTP-026 (30 µg/kg given i.v. at 0, 3 and 6 h reperfusion) reduced necrotic myocardium by ∼40 %. Conclusions: RTP-026 modulate immune cell responses and decreases infarct size of the heart in preclinical settings. Tempering over-exuberant immune cell activation by RTP-026 is a suitable approach to translate the biology of AnxA1 for therapeutic purposes.

Background: Many genes associated with asthma explain only a fraction of its heritability. Most genome-wide association studies (GWASs) used a broad definition of ‘doctor-diagnosed asthma’, thereby diluting genetic signals by not considering asthma heterogeneity. The objective of our study was to identify genetic associates of childhood wheezing phenotypes. Methods: We conducted a novel multivariate GWAS meta-analysis of wheezing phenotypes jointly derived using unbiased analysis of data collected from birth to 18 years in 9568 individuals from five UK birth cohorts. Results: Forty-four independent SNPs were associated with early-onset persistent, 25 with pre-school remitting, 33 with mid-childhood remitting, and 32 with late-onset wheeze. We identified a novel locus on chr9q21.13 (close to annexin 1 [ANXA1], p

Using a global formyl-peptide receptor (Fpr)2 knockout mouse colony, we have reported the modulatory properties of this pro-resolving receptor in polymicrobial sepsis. Herein, we have used a humanized FPR2 (hFPR2) mouse colony bearing an intact or a selective receptor deficiency in myeloid cells to dwell on the cellular mechanisms. hFPR2 mice and myeloid cell-specific hFPR2 KO (abbreviated to KO) mice were subjected to cecal ligation and puncture (CLP)-induced polymicrobial sepsis. Compared with hFPR2 mice, CLP caused exacerbated cardiac dysfunction (assessed by echocardiography), worsened clinical outcome and impaired bacteria clearance in KO mice. This pathological scenario was paralleled by increased recruitment of pro-inflammatory monocytes and reduced M2-like macrophages within the KO hearts. In peritoneal exudates of KO mice, we quantified increased neutrophil and MHC II+ macrophage numbers, but decreased monocyte/macrophage and MHC II- macrophage recruitment. hFPR2 up-regulation was absent in myeloid cells and local production of lipoxin A4 was reduced in septic KO mice. Administration of the FPR2 agonist Annexin A1 (AnxA1) improved cardiac function in hFPR2 septic mice, but had limited beneficial effects in KO mice, in which the FPR2 ligand failed to polarize macrophages towards an MHC II- phenotype. In conclusion, FPR2 deficiency in myeloid cells exacerbates cardiac dysfunction and worsens clinical outcome in polymicrobial sepsis. The improvement of cardiac function and the host immune response by AnxA1 is more effective in hFPR2 competent septic mice.

BackgroundMelanocortins are peptides endowed with anti-inflammatory and pro-resolving activities. Many of these effects are mediated by the Melanocortin receptor 1 (MC1) as reported in several experimental settings. As such, MC1 can be a viable target for the development of new therapies that mimic endogenous pro-resolving mediators. The aim of this study was to assess the immunopharmacology of a selective MC1 agonist (PL8177) in vitro and in a mouse model of inflammatory arthritis.MethodsPL8177 and the natural agonist αMSH were tested for activation of mouse and human Melanocortin receptors (MC1,3,4,5), monitoring cAMP accumulation and ERK1/2 phosphorylation, using transiently transfected HEK293A cells. The anti-inflammatory and pro-resolving effects of PL8177 and αMSH were evaluated using mouse peritoneal Macrophages. Finally, a model of K/BxN serum transfer induced arthritis was used to determine the in vivo potential of PL8177.ResultsPL8177 activates mouse and human MC1 with apparent EC50 values of 0.01 and 1.49 nM, respectively, using the cAMP accumulation assay. Similar profiles were observed for the induction of ERK phosphorylation (EC50: 0.05 and 1.39 nM). PL8177 displays pro-resolving activity (enhanced Macrophage efferocytosis) and counteracts the inflammatory profile of zymosan-stimulated macrophages, reducing the release of IL-1β, IL-6, TNF-α and CCL-2. In the context of joint inflammation, PL8177 (3mg/kg i.p.) reduces clinical score, paw swelling and incidence of severe disease as well as the recruitment of immune cells into the arthritic joint.ConclusionThese results demonstrate that the MC1 agonism with PL8177 affords therapeutic effects in inflammatory conditions including arthritis.SignificanceDrugs targeting the Melanocortin system have emerged as promising therapeutics for several conditions including inflammation or obesity. Multiple candidates are under clinical development, and some have already reached approval. Here we present the characterization of a novel drug candidate, PL8177, selective for the Melanocortin 1 receptor (MC1), demonstrating its selectivity profile on cAMP and ERK1/2 phosphorylation signaling pathways, of relevance as selective drugs will translate into lesser off-target effect. PL8177 also demonstrated, not only anti-inflammatory activity, but pro-resolving actions due to its ability to enhance efferocytosis (i.e. the phagocytosis of apoptotic cells), endowing this molecule with therapeutic advantages compared to classical anti-inflammatory drugs. Using a mouse model of inflammatory arthritis, the compound demonstrated in vivo efficacy by reducing clinical score, paw swelling and overall disease severity. Taken together, these results present Melanocortin-based therapies, and specifically targeting MC1 receptor, as a promising strategy to manage chronic inflammatory diseases.

Peritoneal adhesions are a major cause of complications after abdominal surgery. Here the authors use a post-operative abdominal adhesion model in mice to show that resident F4/80HighCD206− macrophages form a protective barrier that can be enhanced by IL-4 administration or adoptive transfer of these cells.

Fibroblast hyper-activation and proliferation is a major feature in arthritis, yet scarcely addressed for anti-arthritic therapies. Here, the authors show that activation of the MC1 receptor induces fibroblast senescence associated with a reparative phenotype, ultimately regulating experimental inflammatory arthritis.

Host immune responses contribute to dengue’s pathogenesis and severity, yet the possibility that failure in endogenous inflammation resolution pathways could characterise the disease has not been contemplated. The pro-resolving protein Annexin A1 (AnxA1) is known to counterbalance overexuberant inflammation and mast cell (MC) activation. We hypothesised that inadequate AnxA1 engagement underlies the cytokine storm and vascular pathologies associated with dengue disease. Levels of AnxA1 were examined in the plasma of dengue patients and infected mice. Immunocompetent, interferon (alpha and beta) receptor one knockout (KO), AnxA1 KO, and formyl peptide receptor 2 (FPR2) KO mice were infected with dengue virus (DENV) and treated with the AnxA1 mimetic peptide Ac2-26 for analysis. In addition, the effect of Ac2-26 on DENV-induced MC degranulation was assessed in vitro and in vivo. We observed that circulating levels of AnxA1 were reduced in dengue patients and DENV-infected mice. Whilst the absence of AnxA1 or its receptor FPR2 aggravated illness in infected mice, treatment with AnxA1 agonistic peptide attenuated disease manifestationsatteanuated the symptoms of the disease. Both clinical outcomes were attributed to modulation of DENV-mediated viral load-independent MC degranulation. We have thereby identified that altered levels of the pro-resolving mediator AnxA1 are of pathological relevance in DENV infection, suggesting FPR2/ALX agonists as a therapeutic target for dengue disease.

Non-responsiveness to anti-TNF-α therapies presents relevant rates in inflammatory bowel disease patients, presenting the need to find biomarkers involved in therapeutic efficacy. Herein, we demonstrate that higher levels of colonic formyl peptide receptor 1 and annexin A1 correlate with histological recovery in Crohn’s disease patients under remission. Using the dextran sulfate sodium colitis model in mice, we suggest that infliximab induces annexin A1 expression and secretion in activated intestinal leukocytes. Conversely, this mechanism might stimulate epithelial formyl peptide receptors, inducing wound healing and consequent histological remission. Our data indicate that assessing intestinal expressions of formyl peptide receptors and annexin A1 might provide precious information on the disease activity and responsiveness to infliximab in inflammatory bowel disease patients.

Chikungunya (CHIKV) is an arthritogenic alphavirus that causes a self-limiting disease usually accompanied by joint pain and/or polyarthralgia with disabling characteristics. Immune responses developed during the acute phase of CHIKV infection determine the rate of disease progression and resolution. Annexin A1 (AnxA1) is involved in both initiating inflammation and preventing over-response, being essential for a balanced end of inflammation. In this study, we investigated the role of the AnxA1-FPR2/ALX pathway during CHIKV infection. Genetic deletion of AnxA1 or its receptor enhanced inflammatory responses driven by CHIKV. These knockout mice showed increased neutrophil accumulation and augmented tissue damage at the site of infection compared with control mice. Conversely, treatment of wild-type animals with the AnxA1 mimetic peptide (Ac2–26) reduced neutrophil accumulation, decreased local concentration of inflammatory mediators and diminished mechanical hypernociception and paw edema induced by CHIKV-infection. Alterations in viral load were mild both in genetic deletion or with treatment. Combined, our data suggest that the AnxA1-FPR2/ALX pathway is a potential therapeutic strategy to control CHIKV-induced acute inflammation and polyarthralgia.

Abstract Cartilage defects repair poorly. Recent genetic studies suggest that WNT3a may contribute to cartilage regeneration, however the dense, avascular cartilage extracellular matrix limits its penetration and signalling to chondrocytes. Extracellular vesicles actively penetrate intact cartilage. This study investigates the effect of delivering WNT3a into large cartilage defects in vivo using exosomes as a delivery vehicle. Exosomes were purified by ultracentrifugation from conditioned medium of either L‐cells overexpressing WNT3a or control un‐transduced L‐cells, and characterized by electron microscopy, nanoparticle tracking analysis and marker profiling. WNT3a loaded on exosomes was quantified by western blotting and functionally characterized in vitro using the SUPER8TOPFlash reporter assay and other established readouts including proliferation and proteoglycan content. In vivo pathway activation was assessed using TCF/Lef:H2B‐GFP reporter mice. Wnt3a loaded exosomes were injected into the knees of mice, in which large osteochondral defects were surgically generated. The degree of repair was histologically scored after 8 weeks. WNT3a was successfully loaded on exosomes and resulted in activation of WNT signalling in vitro. In vivo, recombinant WNT3a failed to activate WNT signalling in cartilage, whereas a single administration of WNT3a loaded exosomes activated canonical WNT signalling for at least one week, and eight weeks later, improved the repair of osteochondral defects. WNT3a assembled on exosomes, is efficiently delivered into cartilage and contributes to the healing of osteochondral defects.

Abstract Extracellular vesicles (EVs) are emerging as key players in different stages of atherosclerosis. Here we provide evidence that EVs released by mixed aggregates of monocytes and platelets in response to TNF‐α display pro‐inflammatory actions on endothelial cells and atherosclerotic plaques. Tempering platelet activation with Iloprost, Aspirin or a P2Y12 inhibitor impacted quantity and phenotype of EV produced. Proteomics of EVs from cells activated with TNF‐α alone or in the presence of Iloprost revealed a distinct composition, with interesting hits like annexin‐A1 and gelsolin. When added to human atherosclerotic plaque explants, EVs from TNF‐α stimulated monocytes augmented release of cytokines. In contrast, EVs generated by TNF‐α together with Iloprost produced minimal plaque activation. Notably, patients with coronary artery disease that required percutaneous coronary intervention had elevated plasma numbers of monocyte, platelet as well as double positive EV subsets. In conclusion, EVs released following monocyte/platelet activation may play a potential role in the development and progression of atherosclerosis. Whereas attenuating platelet activation modifies EV composition released from monocyte/platelet aggregates, curbing their pro‐inflammatory actions may offer therapeutic avenues for the treatment of atherosclerosis.

Inflammation is a life-saving immune reaction occurring in response to invading pathogens. Nonetheless, inflammation can also occur in an uncontrolled, unrestricted manner, leading to chronic disease and organ damage. Mechanisms triggering an inflammatory response, hindering such a response, or leading to its resolution are well-studied but so far insufficiently elucidated with regard to precise therapeutic interventions. Notably, as an immune reaction evolves, requirements and environments for immune cells change, and thus cellular phenotypes adapt and shift, leading to the appearance of distinct cellular subpopulations with new functional features. In this article, we aim to highlight properties of, and overarching regulatory factors involved in, the occurrence of immune cell phenotypes with a special focus on neutrophils, macrophages and platelets. Additionally, we point out implications for both diagnostics and therapeutics in inflammation research.

BackgroundAtherosclerosis is a chronic inflammatory disease driven by macrophage accumulation in medium and large sized arteries. Macrophage polarization and inflammation are governed by microRNAs (miR) that regulate the expression of inflammatory proteins and cholesterol trafficking. Previous transcriptomic analysis led us to hypothesize that miR-155-5p (miR-155) is regulated by conjugated linoleic acid (CLA), a pro-resolving mediator which induces regression of atherosclerosis in vivo. In parallel, as extracellular vesicles (EVs) and their miR content have potential as biomarkers, we investigated alterations in urinary-derived EVs (uEVs) during the progression of human coronary artery disease (CAD).MethodsmiR-155 expression was quantified in aortae from ApoE−/− mice fed a 1% cholesterol diet supplemented with CLA blend (80:20, cis-9,trans-11:trans-10,cis-12 respectively) which had been previously been shown to induce atherosclerosis regression. In parallel, human polarized THP-1 macrophages were used to investigate the effects of CLA blend on miR-155 expression. A miR-155 mimic was used to investigate its inflammatory effects on macrophages and on ex vivo human carotid endarterectomy (CEA) plaque specimens (n = 5). Surface marker expression and miR content were analyzed in urinary extracellular vesicles (uEVs) obtained from patients diagnosed with unstable (n = 12) and stable (n = 12) CAD.ResultsHere, we report that the 1% cholesterol diet increased miR-155 expression while CLA blend supplementation decreased miR-155 expression in the aorta during atherosclerosis regression in vivo. CLA blend also decreased miR-155 expression in vitro in human THP-1 polarized macrophages. Furthermore, in THP-1 macrophages, miR-155 mimic decreased the anti-inflammatory signaling proteins, BCL-6 and phosphorylated-STAT-3. In addition, miR-155 mimic downregulated BCL-6 in CEA plaque specimens. uEVs from patients with unstable CAD had increased expression of miR-155 in comparison to patients with stable CAD. While the overall concentration of uEVs was decreased in patients with unstable CAD, levels of CD45+ uEVs were increased. Additionally, patients with unstable CAD had increased CD11b+ uEVs and decreased CD16+ uEVs.ConclusionmiR-155 suppresses anti-inflammatory signaling in macrophages, is decreased during regression of atherosclerosis in vivo and is increased in uEVs from patients with unstable CAD suggesting miR-155 has potential as a prognostic indicator and a therapeutic target.

G-protein-coupled receptor 40 (GPR40) is known to play a role in the regulation of fatty acids, insulin secretion, and inflammation. However, the function of this receptor in human neutrophils, one of the first leukocytes to arrive at the site of infection, remains to be fully elucidated. In the present study, we demonstrate that GPR40 is upregulated on activated human neutrophils and investigated the functional effects upon treatment with a selective agonist; GW9508. Interestingly, GPR40 expression was up-regulated after neutrophil stimulation with platelet-activating factor (10 nM) or leukotriene B4 (LTB4, 10 nM) suggesting potential regulatory roles for this receptor during inflammation. Indeed, GW9508 (1 and 10 μM) increased neutrophil chemotaxis in response to the chemokine IL-8 (30 ng/ml) and enhanced phagocytosis of Escherichia coli by approximately 50% when tested at 0.1 and 1 μM. These results were translated in vivo whereby administration of GW9508 (10 mg/kg, i.p.) during E. coli infections resulted in elevated peritoneal leukocyte infiltration with a higher phagocytic capacity. Importantly, GW9508 administration also modulated the lipid mediator profile, with increased levels of the pro-resolving mediators resolvin D3 and lipoxins. In conclusion, GPR40 is expressed by activated neutrophils and plays an important host protective role to aid clearance of bacterial infections.

The targeted delivery of therapies to diseased tissues offers a safe opportunity to achieve optimal efficacy while limiting systemic exposure. These considerations apply to many disease indications but are especially relevant for rheumatoid arthritis (RA), as RA is a systemic autoimmune disease which affects multiple joints. We have identified an antibody that is specific to damaged arthritic cartilage (anti-ROS-CII) that can be used to deliver treatments specifically to arthritic joints, yielding augmented efficacy in experimental arthritis. In the current study, we demonstrate that scaffolds enriched with bioactive payloads can be delivered precisely to an inflamed joint and achieve superior efficacy outcomes consistent with the pharmacological properties of these payloads. As a scaffold, we have used extracellular vesicles (EVs) prepared from human neutrophils (PMNs), which possess intrinsic anti-inflammatory properties and the ability to penetrate inflamed arthritic cartilage. EV fortified with anti-ROS-CII (EV/anti-ROS-CII) retained anti-ROS-CII specificity and bound exclusively to the damaged cartilage. Following systemic administration, EV/anti-ROS-CII (a) exhibited the ability to localize specifically in the arthritic joint in vivo and (b) was able to specifically target single (viral IL-10 or anti-TNF) or combined (viral IL-10 and anti-TNF) anti-inflammatory treatments to the arthritic joint, which accelerated attenuation of clinical and synovial inflammation. Overall, this study demonstrates the attainability of targeting a pro-resolving biological scaffold to the arthritic joint. The potential of targeting scaffolds such as EV, nanoparticles, or a combination thereof alongside combined therapeutics is paramount for designing systemically administered broad-spectrum of anti-inflammatory treatments.

Annexin-A1 (AnxA1) and its N-terminal derived peptide Ac2-26 regulate the inflammatory response in several experimental models of disorders. This study evaluated the effect of endogenous AnxA1 and its N-terminal peptide Acetyl 2-26 (Ac2-26) on allergic asthma triggered by house dust mite (HDM) extract in mice. ANXA1−/− and wildtype (WT) mice were exposed to intranasal instillation of HDM every other day for 3 weeks, with analyses performed 24 h following the last exposure. Intranasal administration of peptide Ac2-26 was performed 1 h before HDM, beginning 1 week after the initial antigen application. ANXA1−/− mice stimulated with HDM showed marked exacerbations of airway hyperreactivity (AHR), eosinophil accumulation, subepithelial fibrosis, and mucus hypersecretion, all parameters correlating with overexpression of cytokines (IL-4, IL-13, TNF-α, and TGF-β) and chemokines (CCL11/eotaxin-1 and CCL2/MCP-1). Intranasal treatment with peptide Ac2-26 decreased eosinophil infiltration, peribronchiolar fibrosis, and mucus exacerbation caused by the allergen challenge. Ac2-26 also inhibited AHR and mediator production. Collectively, our findings show that the AnxA1-derived peptide Ac2-26 protects against several pathological changes associated with HDM allergic reaction, suggesting that this peptide or related AnxA1-mimetic Ac2-26 may represent promising therapeutic candidates for the treatment of allergic asthma.

Summary: An abdominal aortic aneurysm (AAA) is a progressive aortic dilation that may lead to rupture, which is usually lethal. This study identifies the state of failure in the resolution of inflammation by means of decreased expression of the pro-resolving receptor A lipoxin/formyl peptide receptor 2 (ALX/FPR2) in the adventitia of human AAA lesions. Mimicking this condition by genetic deletion of the murine ALX/FPR2 ortholog in hyperlipidemic mice exacerbated the aortic dilation induced by angiotensin II infusion, associated with decreased vascular collagen and increased inflammation. The authors also identified key roles of lipoxin formation through 12/15-lipoxygenase and neutrophil p38 mitogen-activated protein kinase. In conclusion, this study established pro-resolving signaling by means of the ALX/FPR2 receptor in aneurysms and vascular inflammation. Key Words: abdominal aortic aneurysms, cardiovascular disease, eicosanoids, inflammation, lipoxygenase

Microvesicles (MVs) are emerging as a novel means to enact cell-to-cell communication in inflammation. Here, we aimed to ascertain the ability of neutrophil-derived MVs to modulate target cell behaviour, the focus being the macrophage.MVs were generated in response to tumour necrosis factor-α, from healthy control neutrophils or those from rheumatoid arthritis patients. MVs were used to stimulate human monocyte-derived macrophages in vitro, or administered intra-articularly in the K/BxN mouse model of arthritis. A macrophage/fibroblast-like synoviocyte co-culture system was used to study the effects of vesicles on the crosstalk between these cells.We demonstrate a direct role for phosphatidylserine and annexin-A1 exposed by the MVs to counteract classical activation of the macrophages, and promote the release of transforming growth factor-β, respectively. Classically-activated macrophages exposed to neutrophil MVs no longer activated fibroblast-like synoviocytes in subsequent co-culture settings. Finally, intra-articular administration of neutrophil MVs from rheumatoid arthritis patients in arthritic mice affected the phenotype of joint macrophages.Altogether these data, with the identification of specific MV determinants, open new opportunities to modulate on-going inflammation in the synovia – mainly by affecting macrophage polarization and potentially also fibroblast-like synoviocytes - through the delivery of autologous or heterologous MVs produced from neutrophils. Keywords: Neutrophils, Macrophages, Vesicles, Rheumatoid arthritis

Objective: platelets possess not only haemostatic but also inflammatory properties, which combined are thought to play a detrimental role in thromboinflammatory diseases such as acute coronary syndromes and stroke. Phosphodiesterase (PDE) 3 and -5 inhibitors have demonstrated efficacy in secondary prevention of arterial thrombosis, partially mediated by their antiplatelet action. Yet it is unclear whether such inhibitors also affect platelets’ inflammatory functions. Here, we aimed to examine the effect of the PDE3A inhibitor cilostazol and the PDE5 inhibitor tadalafil on platelet function in various aspects of thromboinflammation. Approach and results: cilostazol, but not tadalafil, delayed ex vivo platelet-dependent fibrin formation under whole blood flow over type I collagen at 1000 s−1. Similar results were obtained with blood from Pde3a deficient mice, indicating that cilostazol effects are mediated via PDE3A. Interestingly, cilostazol specifically reduced the release of phosphatidylserine-positive extracellular vesicles (EVs) from human platelets while not affecting total EV release. Both cilostazol and tadalafil reduced the interaction of human platelets with inflamed endothelium under arterial flow and the release of the chemokines CCL5 and CXCL4 from platelets. Moreover, cilostazol, but not tadalafil, reduced monocyte recruitment and platelet-monocyte interaction in vitro. Conclusions: this study demonstrated yet unrecognised roles for platelet PDE3A and platelet PDE5 in platelet procoagulant and proinflammatory responses.

Exosomes are known to contain microRNAs (miRs). Here the authors show that dorsal root ganglion neurons release exosomes containing miR-21-5p, which contributes to inflammatory cell recruitment following peripheral nerve injury.

Mast cell stabilizers like cromoglycate and nedocromil are mainstream treatments for ocular allergy. Biochemical studies in vitro suggest that these drugs prevent mast cell degranulation through the release of Annexin-A1 (Anx-A1) protein. However, the direct effect of Anx-A1 gene deletion on mast cell function in vitro and in vivo is yet to be fully investigated. Hence, we aim to elucidate the role of Anx-A1 in mast cell function, both in vivo and in vitro, using a transgenic mouse model where the Anx-A1 gene has been deleted. Bone marrow-derived mast cells (BMDMCs) were cultured from wild-type animals and compared throughout their development to BMDMCs obtained from mice lacking the Anx-A1 gene. The mast cell differentiation, maturity, mediator, and cytokine release were explored using multiple biochemical techniques, such as Western blots, ELISA, and flow cytometry analysis. Electron microscopy was used to identify metachromatic granules content of cells. For in vivo studies, Balb/C wild-type and Anx-A1-deficient mice were divided into the following groups: group 1, a control receiving only saline, and group 2, which had been sensitized by prior exposure to short ragweed (SRW) pollen by topical contact with the conjunctival mucosae. Allergic conjunctivitis was evaluated blind after 24 h by trained observers scoring clinical signs. Electron micrographs of BMDMCs from Anx-A1-null mice revealed more vacuoles overall and more fused vacuoles than wild-type cells, suggesting enhanced secretory activity. Congruent with these observations, BMDMCs lacking the Anx-A1 gene released significantly increased amounts of histamine both spontaneously as well as in response to Ig-E-FcεRI cross-linking compared to those from wild-type mice. Interestingly, the spontaneous release of IL-5, IL-6, IL-9, and monocyte chemoattractant protein-1 (MCP-1) were also markedly increased with a greater production observed upon IgE cross-linking. This latter finding is congruent with augmented calcium mobilization in BMDMCs lacking the Anx-A1 gene. In vivo, when compared to wild-type animals, Anx-A1-deficient mice exposed to SRW pollen displayed exacerbated signs and symptoms of allergic conjunctivitis. Taken together, these results suggest Anx-A1 is an important non-redundant regulator of mast cell reactivity and particularly in allergen mediated allergic reactions.

Inflammation resolution is an active process that functions to restore tissue homeostasis. Clearance of apoptotic leukocytes by efferocytosis at inflammatory sites plays an important role in inflammation resolution and induces remarkable macrophage phenotypic and functional changes. Here, we investigated the effects of deletion of either plasminogen (Plg) or the Plg receptor, Plg-RKT, on the resolution of inflammation. In a murine model of pleurisy, the numbers of total mononuclear cells recruited to the pleural cavity were significantly decreased in both Plg−/− and Plg-RKT−/− mice, a response associated with decreased levels of the chemokine CCL2 in pleural exudates. Increased percentages of M1-like macrophages were determined in pleural lavages of Plg−/− and Plg-RKT−/− mice without significant changes in M2-like macrophage percentages. In vitro, Plg and plasmin (Pla) increased CD206/Arginase-1 expression and the levels of IL-10/TGF-β (M2 markers) while decreasing IFN/LPS-induced M1 markers in murine bone-marrow-derived macrophages (BMDMs) and human macrophages. Furthermore, IL4-induced M2-like polarization was defective in BMDMs from both Plg−/− and Plg-RKT−/− mice. Mechanistically, Plg and Pla induced transient STAT3 phosphorylation, which was decreased in Plg−/− and Plg-RKT−/− BMDMs after IL-4 or IL-10 stimulation. The extents of expression of CD206 and Annexin A1 (important for clearance of apoptotic cells) were reduced in Plg−/− and Plg-RKT−/− macrophage populations, which exhibited decreased phagocytosis of apoptotic neutrophils (efferocytosis) in vivo and in vitro. Taken together, these results suggest that Plg and its receptor, Plg-RKT, regulate macrophage polarization and efferocytosis, as key contributors to the resolution of inflammation.

Pancreatic cancer (PC) is one of the most aggressive cancers in the world. Several extracellular factors are involved in its development and metastasis to distant organs. In PC, the protein Annexin A1 (ANXA1) appears to be overexpressed and may be identified as an oncogenic factor, also because it is a component in tumor-deriving extracellular vesicles (EVs). Indeed, these microvesicles are known to nourish the tumor microenvironment. Once we evaluated the autocrine role of ANXA1-containing EVs on PC MIA PaCa-2 cells and their pro-angiogenic action, we investigated the ANXA1 paracrine effect on stromal cells like fibroblasts and endothelial ones. Concerning the analysis of fibroblasts, cell migration/invasion, cytoskeleton remodeling, and the different expression of specific protein markers, all features of the cell switching into myofibroblasts, were assessed after administration of wild type more than ANXA1 Knock-Out EVs. Interestingly, we demonstrated a mechanism by which the ANXA1-EVs complex can stimulate the activation of formyl peptide receptors (FPRs), triggering mesenchymal switches and cell motility on both fibroblasts and endothelial cells. Therefore, we highlighted the importance of ANXA1/EVs-FPR axes in PC progression as a vehicle of intercommunication tumor cells-stroma, suggesting a specific potential prognostic/diagnostic role of ANXA1, whether in soluble form or even if EVs are captured in PC.

Embryo implantation into the uterine wall is a highly modulated, complex process. We previously demonstrated that Annexin A1 (AnxA1), which is a protein secreted by epithelial and inflammatory cells in the uterine microenvironment, controls embryo implantation in vivo. Here, we decipher the effects of recombinant AnxA1 in this phenomenon by using human trophoblast cell (BeWo) spheroids and uterine epithelial cells (Ishikawa; IK). AnxA1-treated IK cells demonstrated greater levels of spheroid adherence and upregulation of the tight junction molecules claudin-1 and zona occludens-1, as well as the glycoprotein mucin-1 (Muc-1). The latter effect of AnxA1 was not mediated through IL-6 secreted from IK cells, a known inducer of Muc-1 expression. Rather, these effects of AnxA1 involved activation of the formyl peptide receptors FPR1 and FPR2, as pharmacological blockade of FPR1 or FPR1/FPR2 abrogated such responses. The downstream actions of AnxA1 were mediated through the ERK1/2 phosphorylation pathway and F-actin polymerization in IK cells, as blockade of ERK1/2 phosphorylation reversed AnxA1-induced Muc-1 and claudin-1 expression. Moreover, FPR2 activation by AnxA1 induced vascular endothelial growth factor (VEGF) secretion by IK cells, and the supernatant of AnxA1-treated IK cells evoked angiogenesis in vitro. In conclusion, these data highlight the role of the AnxA1/FPR1/FPR2 pathway in uterine epithelial control of blastocyst implantation.

Resolution Pharmacology identifies drugs developed on the biology of the resolution phase of inflammation, the complex molecular and cellular network of events that ensure the tight temporal and spatial control on the inflammatory response. As such, new anti-inflammatory and pro-resolving drugs could derive from pro-resolving mediators and receptors. To implement faithful screening programs, however, it is important to rely on predictive signaling pathway relevant for the ultimate bio-action of interest. Herein we performed an analysis with four prototypical melanocortin receptor (MC1,3,4,5) agonists. The choice fell on the natural agonist αMSH, the small molecule BMS-470539, and the synthetic derivatives [D-Trp8]-γMSH and [Nle4,D-Phe7]-αMSH. We used human macrophages and quantified the effect of the four agonists on inhibition of cytokine release and promotion of efferocytosis. All agonists (1–10 μM) significantly inhibited cytokine release by LPS-stimulated cells whereas [D-Trp8]-γMSH was the most effective in inducing efferocytosis (∼60% increase). To study the signaling profile, we monitored cAMP accumulation and ERK1/2 phosphorylation, and constructed biased plots that revealed a marked biased profile of [D-Trp8]-γMSH toward phospho-ERK1/2. Correlation matrix analysis of all data pointed at phospho-ERK1/2 at any receptor as the most prominent pathway to attain pro-phagocytic actions, and MC1 receptor as the most relevant to drive anti-cytokine effects. In conclusion, the present study highlights the need to associate single-target signaling data with relevant functional outcomes. In this manner, we would increase our chances to optimize drug discovery programs during the early target validation and hit-to-lead phases.

Inflammation in now appreciated to be at the centre of may diseases that affect Western civilization. Current therapeutics for managing these conditions may interfere with the host response leading to immune suppression. We recently developed an annexin (Anx) A1-derived peptide, coined CR-AnxA12-50, which displays potent pro-resolving and tissue protective actions. Herein, we designed a novel peptide using CR-AnxA12-50 as a template that was significantly more resistant to neutrophil-mediated degradation. This peptide, termed CR-AnxA12-48, retained high affinity and specificity to the pro-resolving Lipoxin A4 receptor (ALX) with an IC50 of ~20nM. CR-AnxA12-48 dose dependently (100fM-10nM) promoted the efferocytosis of apoptotic neutrophils, an action that was mediated by the murine orthologue of human ALX. The neutrophil-directed actions were also retained with human primary cells were CR-AnxA12-48 reduced human neutrophil recruitment to activated endothelial cells at concentrations as low as 100 pM. This protective action was mediated by human ALX, since incubation of neutrophils with an anti-ALX antibody reversed this anti-inflammatory actions of CR-AnxA12-48. Administration of this peptide to mice during dermal inflammation led to a significant and dose dependent decrease in neutrophil recruitment. This reduction in neutrophil numbers was more pronounced than that displayed by the parent peptide CR-AnxA12-50. CR-AnxA12-48 was also cardioprotecitve reducing infarct size and systemic chemokine (C-C motif) ligand 5 concentration following ischemia reperfusion injury. These findings identify CR-AnxA12-48 as a new ALX agonist that regulates phagocyte responses and displays tissue-protective actions.

We have recently demonstrated that mesoglycan, a fibrinolytic compound, may be a promising pro-healing drug for skin wound repair. We showed that mesoglycan induces migration, invasion, early differentiation, and translocation to the membrane of keratinocytes, as well as the secretion of annexin A1 (ANXA1), further involved in keratinocytes activation. These events are triggered by the syndecan-4 (SDC4)/PKCα pathway. SDC4 also participates to the formation and secretion of microvesicles (EVs) which may contribute to wound healing. EVs were isolated from HaCaT cells, as human immortalized keratinocytes, and then characterised by Western blotting, Field Emission-Scanning Electron Microscopy, and Dynamic Light Scattering. Their autocrine effects were investigated by Wound-Healing/invasion assays and confocal microscopy to analyse cell motility and differentiation, respectively. Here, we found that the mesoglycan increased the release of EVs which amplify its same effects. ANXA1 contained in the microvesicles is able to promote keratinocytes motility and differentiation by acting on Formyl Peptide Receptors (FPRs). Thus, the extracellular form of ANXA1 may be considered as a link to intensify the effects of mesoglycan. In this study, for the first time, we have identified an interesting autocrine loop ANXA1/EVs/FPRs in human keratinocytes, induced by mesoglycan.

Abstract 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.

Abstract Resolution of inflammation is a coordinated and active process aimed at restoration of tissue integrity and function. This review integrates the key molecular and cellular mechanisms of resolution. We describe how abrogation of chemokine signalling blocks continued neutrophil tissue infiltration and how apoptotic neutrophils attract monocytes and macrophages to induce their clearance. Uptake of apoptotic neutrophils by macrophages reprograms macrophages towards a resolving phenotype, a key event to restore tissue homeostasis. Finally, we highlight the therapeutic potential that derives from understanding the mechanisms of resolution.

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The mechanism by which the platelet-endothelial cell adhesion molecule PECAM-1 regulates leukodiapedesis, vascular endothelial integrity, and proinflammatory cytokine expression in vivo is not known. We recently identified PECAM-1 as a negative regulator of Kv11.1, a specific voltage-gated potassium channel that functioned in human macrophages to reset a resting membrane potential following depolarization. We demonstrate here that dofetilide (DOF), a selective inhibitor of the Kv11.1 current, had a profound inhibitory effect on neutrophil recruitment in mice following TLR/IL-1R–elicited peritonitis or intrascrotal injection of IL-1β, but had no effect on responses seen with TNFα. Furthermore, inhibitors of Kv11.1 (DOF, E4031, and astemizole), but not Kv1.3 (margatoxin), suppressed the expression of IL-6 and MCP-1 cytokines by murine resident peritoneal macrophages, while again having no effect on TNFα. In contrast, IL-6 expression by peritoneal mesothelial cells was unaffected. Using murine P388 cells, which lack endogenous C/EBPβexpression and are unresponsive to LPS for the expression of both IL-6 and MCP-1, we observed that DOF inhibited LPS-induced expression of IL-6 mRNA following ectopic expression of wild-type C/EBPβ, but not a serine-64 point mutant. Finally, DOF inhibited the constitutive activation of cdk2 in murine peritoneal macrophages; cdk2 is known to phosphorylate C/EBPβ at serine-64. Taken together, our results implicate a potential role for Kv11.1 in regulating cdk2 and C/EBPβ activity, where robust transactivation of both IL-6 and MCP-1 transcription is known to be dependent on serine-64 of C/EBPβ. Our data might also explain the altered phenotypes displayed by PECAM-1 knockout mice in several disease models.

Lactate has long been considered a "waste" by-product of cell metabolism, and it accumulates at sites of inflammation. Recent findings have identified lactate as an active metabolite in cell signalling, although its effects on immune cells during inflammation are largely unexplored. Here we ask whether lactate is responsible for T cells remaining entrapped in inflammatory sites, where they perpetuate the chronic inflammatory process. We show that lactate accumulates in the synovia of rheumatoid arthritis patients. Extracellular sodium lactate and lactic acid inhibit the motility of CD4+ and CD8+ T cells, respectively. This selective control of T cell motility is mediated via subtype-specific transporters (Slc5a12 and Slc16a1) that we find selectively expressed by CD4+ and CD8+ subsets, respectively. We further show both in vitro and in vivo that the sodium lactate-mediated inhibition of CD4+ T cell motility is due to an interference with glycolysis activated upon engagement of the chemokine receptor CXCR3 with the chemokine CXCL10. In contrast, we find the lactic acid effect on CD8+ T cell motility to be independent of glycolysis control. In CD4+ T helper cells, sodium lactate also induces a switch towards the Th17 subset that produces large amounts of the proinflammatory cytokine IL-17, whereas in CD8+ T cells, lactic acid causes the loss of their cytolytic function. We further show that the expression of lactate transporters correlates with the clinical T cell score in the synovia of rheumatoid arthritis patients. Finally, pharmacological or antibody-mediated blockade of subtype-specific lactate transporters on T cells results in their release from the inflammatory site in an in vivo model of peritonitis. By establishing a novel role of lactate in control of proinflammatory T cell motility and effector functions, our findings provide a potential molecular mechanism for T cell entrapment and functional changes in inflammatory sites that drive chronic inflammation and offer targeted therapeutic interventions for the treatment of chronic inflammatory disorders.

To investigate therapeutic effects of annexin A1 (anxA1) on atherogenesis in LDLR-/- mice.Human recombinant annexin A1 (hr-anxA1) was produced by a prokaryotic expression system, purified and analysed on phosphatidylserine (PS) binding and formyl peptide receptor (FPR) activation. Biodistribution of 99mTechnetium-hr-anxA1 was determined in C57Bl/6J mice. 12 Weeks old LDLR-/- mice were fed a Western Type Diet (WTD) during 6 weeks (Group I) or 12 weeks (Group P). Mice received hr-anxA1 (1 mg/kg) or vehicle by intraperitoneal injection 3 times per week for a period of 6 weeks starting at start of WTD (Group I) or 6 weeks after start of WTD (Group P). Total aortic plaque burden and phenotype were analyzed using immunohistochemistry.Hr-anxA1 bound PS in Ca2+-dependent manner and activated FPR2/ALX. It inhibited rolling and adherence of neutrophils but not monocytes on activated endothelial cells. Half lives of circulating 99mTc-hr-anxA1 were

The anti-inflammatory actions of the nonapeptide antiflammin-2, identified by homology with uteroglobin and annexin-A1 sequences, have been described in some detail, yet its mechanisms of action remain elusive. Since recent data indicate an involvement of the formyl peptide receptor (FPR)-like 1 (or FPRL-1) in the effects of annexin-A1, we have tested here the effect of antiflammin-2 with respect to this receptor family. Using HEK-293 cells expressing either human FPR and FPRL-1, and an annexin-A1 peptide as tracer ([125I-Tyr]-Ac2-26), we found that antiflammin-2 competed for binding only at FPRL-1, and not FPR, with an approximate EC50 of 1 μM. In line with data produced for the full-length protein, genuine receptor activation by antiflammin-2 was confirmed by rapid phosphorylation of extracellular-regulated kinase 1 and 2. Finally, study of the neutrophil interaction with activated endothelium under flow demonstrated an inhibitory effect of antiflammin-2, thus providing functional support to a role for the antiflammin-2/FPRL-1 anti-inflammatory axis.

The concept of anti-inflammation is currently evolving with the definition of several endogenous inhibitory circuits that are important in the control of the host inflammatory response. Here we focus on one of these pathways, the annexin 1 (ANXA1) system. Originally identified as a 37 kDa glucocorticoid-inducible protein, ANXA1 has emerged over the last decade as an important endogenous modulator of inflammation. We review the pharmacological effects of ANXA1 on cell types involved in inflammation, from blood-borne leukocytes to resident cells. This review reveals that there is scope for more research, since most of the studies have so far focused on the effects of the protein and its peptido-mimetics on neutrophil recruitment and activation. However, many other cells central to inflammation, e.g. endothelial cells or mast cells, also express ANXA1: it is foreseen that a better definition of the role(s) of the endogenous protein in these cells will open the way to further pharmacological studies. We propose that a more systematic analysis of ANXA1 physio-pharmacology in cells involved in the host inflammatory reaction could aid in the design of novel anti-inflammatory therapeutics based on this endogenous mediator.

Background: Human and rodent leukocytes express high levels of the glucocorticoid-inducible protein annexin 1 (ANXA1) (previously referred to as lipocortin 1). Neutrophils and monocytes have abundant ANXA1 levels.

Formyl peptide receptors (FPR) belong to a family of sensors of the immune system that detect microbe-associated molecules and inform various cellular and sensorial mechanisms to the presence of pathogens in the host. Here we demonstrate that Fpr2/3-deficient mice show a distinct profile of behaviour characterised by reduced anxiety in the marble burying and light-dark box paradigms, increased exploratory behaviour in an open-field, together with superior performance on a novel object recognition test. Pharmacological blockade with a formyl peptide receptor antagonist, Boc2, in wild type mice reproduced most of the behavioural changes observed in the Fpr2/3(-/-) mice, including a significant improvement in novel object discrimination and reduced anxiety in a light/dark shuttle test. These effects were associated with reduced FPR signalling in the gut as shown by the significant reduction in the levels of p-p38. Collectively, these findings suggest that homeostatic FPR signalling exerts a modulatory effect on anxiety-like behaviours. These findings thus suggest that therapies targeting FPRs may be a novel approach to ameliorate behavioural abnormalities present in neuropsychiatric disorders at the cognitive-emotional interface.

This study compares the signal transduction pathway which leads to the upregulation of intercellular adhesion molecule-1 (ICAM-1) expression with that of the increase in the expression of inducible nitric oxide synthase (iNOS) protein and activity caused by endotoxin in cultured J774.2 macrophages. Treatment of J774.2 cells with lipopolysaccharide E. coli (LPS) induced a concentration-dependent increase in the expression of ICAM-1 on the cell surface within 4 h and an increase in iNOS protein and activity at 24 h. The upregulation of ICAM-1 expression on J774.2 macrophages caused by LPS was significantly inhibited by pretreatment of the cells with inhibitors of the activation of the nuclear transcription factor NF-κB, such as L-1-tosylamido-2-phenylethylchloromethyl ketone (TPCK), pyrrolidine dithiocarbamate (PDTC), rotenone or calpain inhibitor I, but not by the tyrosine kinase inhibitors, tyrphostin AG126 or genistein. In contrast, genistein or tyrphostin AG126 also prevented the induction of iNOS protein and activity in J774.2 macrophages elicited by LPS. Thus, the increase in the expression of ICAM-1 on J774.2 macrophages by endotoxin involves the activation of NFκB, but not of protein tyrosine kinase.

Background. NOS/•NO inhibitors are potential therapeutics for sepsis, yet they increase clinical mortality. However, there has been no in vivo investigation of the (in vitro) •NO scavenger, cobalamin’s (Cbl) endogenous effects on NOS/•NO/inflammatory mediators during the immune response to sepsis. Methods. We used quantitative polymerase chain reaction (qPCR), ELISA, Western blot, and NOS Griess assays, in a C57BL/6 mouse, acute endotoxaemia model. Results. During the immune response, pro-inflammatory phase, parenteral hydroxocobalamin (HOCbl) treatment partially inhibits hepatic, but not lung, iNOS mRNA and promotes lung eNOS mRNA, but attenuates the LPS hepatic rise in eNOS mRNA, whilst paradoxically promoting high iNOS/eNOS protein translation, but relatively moderate •NO production. HOCbl/NOS/•NO regulation is reciprocally associated with lower 4 h expression of TNF-α, IL-1β, COX-2, and lower circulating TNF-α, but not IL-6. In resolution, 24 h after LPS, HOCbl completely abrogates a major late mediator of sepsis mortality, high mobility group box 1 (HMGB1) mRNA, inhibits iNOS mRNA, and attenuates LPS-induced hepatic inhibition of eNOS mRNA, whilst showing increased, but still moderate, NOS activity, relative to LPS only. experiments (LPS+D-Galactosamine) HOCbl afforded significant, dose-dependent protection in mice Conclusions. HOCbl produces a complex, time- and organ-dependent, selective regulation of NOS/•NO during endotoxaemia, corollary regulation of downstream inflammatory mediators, and increased survival. This merits clinical evaluation.

The resolution of inflammation is now known to be an active process, armed with a multitude of mediators both lipid and protein in nature. Melanocortins are peptides endowed with considerable promise with their proresolution and anti-inflammatory effects in preclinical models of inflammatory disease, with tissue protective effects. These peptides and their targets are appealing because they can be seen as a natural way of inducing these effects as they harness endogenous pathways of control. Whereas most of the information generated about these mediators derives from several acute models of inflammation (such as zymosan induced peritonitis), there is some indication that these mediators may inhibit chronic inflammation by modulating cytokines, chemokines, and leukocyte apoptosis. In addition, proresolving mediators and their mimics have often been tested alongside therapeutic protocols, hence have been tested in settings more relevant to real life clinical scenarios. We provide here an overview on some of these mediators with a focus on melanocortin peptides and receptors, proposing that they may unveil new opportunities for innovative treatments of inflammatory arthritis.

BACKGROUND: Improvement of patient quality of life is the ultimate goal of biomedical research, particularly when dealing with complex, chronic and debilitating conditions such as inflammatory bowel disease (IBD). This is largely dependent on receiving an accurate and rapid diagnose, an effective treatment and in the prediction and prevention of side effects and complications. The low sensitivity and specificity of current markers burden their general use in the clinical practice. New biomarkers with accurate predictive ability are needed to achieve a personalized approach that take the inter-individual differences into consideration. METHODS: We performed a high throughput approach using microarray gene expression profiling of colon pinch biopsies from IBD patients to identify predictive transcriptional signatures associated with intestinal inflammation, differential diagnosis (Crohn's disease or ulcerative colitis), response to glucocorticoids (resistance and dependence) or prognosis (need for surgery). Class prediction was performed with self-validating Prophet software package. RESULTS: Transcriptional profiling divided patients in two subgroups that associated with degree of inflammation. Class predictors were identified with predictive accuracy ranging from 67 to 100%. The expression accuracy was confirmed by real time-PCR quantification. Functional analysis of the predictor genes showed that they play a role in immune responses to bacteria (PTN, OLFM4 and LILRA2), autophagy and endocytocis processes (ATG16L1, DNAJC6, VPS26B, RABGEF1, ITSN1 and TMEM127) and glucocorticoid receptor degradation (STS and MMD2). CONCLUSIONS: We conclude that using analytical algorithms for class prediction discovery can be useful to uncover gene expression profiles and identify classifier genes with potential stratification utility of IBD patients, a major step towards personalized therapy.

Obesity and type 2 diabetes are associated with increased production of Galectin-3 (Gal-3), a protein that modulates inflammation and clearance of glucose adducts. We used Lean and Diet-induced Obese (DIO) WT and Gal-3 KO mice to investigate the role of Gal-3 in modulation of adiposity, glucose metabolism and inflammation. Deficiency of Gal-3 lead to age-dependent development of excess adiposity and systemic inflammation, as indicated by elevated production of acute-phase proteins, number of circulating pro-inflammatory Ly6C(high) monocytes and development of neutrophilia, microcytic anemia and thrombocytosis in 20-week-old Lean and DIO male Gal-3 KO mice. This was associated with impaired fasting glucose, heightened response to a glucose tolerance test and reduced adipose tissue expression of adiponectin, Gal-12, ATGL and PPARγ, in the presence of maintained insulin sensitivity and hepatic expression of gluconeogenic enzymes in 20-week-old Gal-3 KO mice compared to their diet-matched WT controls. Expression of PGC-1α and FGF-21 in the liver of Lean Gal-3 KO mice was comparable to that observed in DIO animals. Impaired fasting glucose and altered responsiveness to a glucose load preceded development of excess adiposity and systemic inflammation, as demonstrated in 12-week-old Gal-3 KO mice. Finally, a role for the microflora in mediating the fasting hyperglycemia, but not the excessive response to a glucose load, of 12-week-old Gal-3 KO mice was demonstrated by administration of antibiotics. In conclusion, Gal-3 is an important modulator of glucose metabolism, adiposity and inflammation.

INTRODUCTION: Previous work reported the anti-arthritic synergy afforded by combining calcitonin (CT) and glucocorticoids (GC). Here we focus on the pairing of elcatonin (eCT) and dexamethasone (Dex), querying whether: i) this was a class-effect action; ii) mechanistic insights could be unveiled; iii) the synergy affected canonical GC adverse effects. METHODS: Using the rat collagen-induced arthritis model, different combinations of eCT and Dex, were administered from disease onset to peak (day 11 to 18). Macroscopic disease score was monitored throughout, with biochemical and histological analyses conducted on plasma and tissues at day 18. The effect on acute hyperglycaemia and liver enzyme message were also assessed. RESULTS: Whilst eCT alone was inactive, it synergised at 1 µg/kg with low doses of Dex (7.5 or 15 µg/kg) to yield an anti-arthritic efficacy equivalent to a 4- to 7-fold higher Dex dose. Mechanistically, the anti-arthritic synergy corresponded to a marked attenuation in RA-relevant analytes. CXCL5 expression, in both plasma and joint, was markedly inhibited by the co-therapy. Finally, co-administration of eCT did not exacerbate metrics of GC adverse effects, and rescued some of them. CONCLUSIONS: We present evidence of a class-effect action for the anti-arthritic synergy of CT/GC combination, underpinned by the powerful inhibition of joint destruction markers. Furthermore, we identify CXCL5 as a marker for the combination therapy with potential diagnostic and prognostic utility. Substantial GC dose reduction, together with the absence of exacerbated adverse effects, indicated a significant clinical potential for this co-therapy in RA and beyond.

BackgroundOne of the characteristics of an active episode of ulcerative colitis (UC) is the intense mucosal infiltration of leukocytes. The pro-resolution mediators Annexin-A1 (AnxA1) and lipoxin A(4) (LXA(4)) exert counter-regulatory effects on leukocyte recruitment, however to date, the dual/cumulative effects of these formyl peptide receptor-2 (FPR2/ALX) agonists in the context of human intestinal diseases are unclear. To define the contribution of these mediators, we measured their expression in biopsies from individuals with UC.MethodsColonic mucosal biopsies were collected from two broad patient groups: healthy volunteers without ('Ctrl' n = 20) or with a prior history of UC ('hx of UC' n = 5); individuals with UC experiencing active disease ('active' n = 8), or in medically-induced remission ('remission' n = 16). We assessed the mucosal expression of LXA(4), AnxA1, and the FPR2/ALX receptor in each patient group using a combination of fluorescence microscopy, biochemical and molecular analyses.ResultsMucosal expression of LXA(4) was elevated exclusively in biopsies from individuals in remission (3-fold, PConclusionsOur results demonstrate a specific up-regulation of this pro-resolution circuit in individuals in remission from UC, and suggest a significant role for LXA(4) and AnxA1 in promoting mucosal homeostasis.

Our understanding of pro-resolution factors in determining the outcome of inflammation has recently gained ground, yet not many studies have investigated whether specific genes or patterns of genes, are modified by these mediators. Here, we have focussed on the glucocorticoid modulated pro-resolution factor annexin A1 (AnxA1), studying if its interaction with the ALX receptor would affect downstream genomic targets.Using microarray technology in ALX transfected HEK293 cells, we discovered an over-lapping, yet distinct gene activation profile for AnxA1 compared to its N-terminal mimetic peptide Ac2-26, which may be suggestive of unique downstream inflammatory outcomes for each substance. When the up-regulated genes were explored, consistently induced was the sphingosine phosphate phosphatase-2 gene (SGPP2), involved in regulation of the sphingosine 1 phosphate chemotactic system. Up-regulation of this gene, as well as JAG1 (and down-regulation of JAM3), was confirmed using real time PCR both with transfected HEK293 cells and human peripheral blood leukocytes. Furthermore, lymph nodes taken from AnxA1(null) mice displayed lower SGPP2 gene activity. Finally, connectivity map analysis for AnxA1 and peptide Ac2-26 indicated striking similarities with known anti-inflammatory therapeutics, glucocorticoids and aspirin-like compounds, as well as with histone deacetylase inhibitors.We believe these new data raise the profile of AnxA1 from being solely a short-term anti-inflammatory factor, to being a 'trigger' of the endogenous pro-resolution arsenal.