Activation of the pro-resolving receptor Fpr2 attenuates inflammatory microglial activation

Poster number: P-T099 Theme: Neurodegenerative disorders & ageing Activation of the pro-resolving receptor Fpr2 reverses inflammatory microglial activation Authors: Edward S Wickstead - Life Science & Technology University of Westminster/Queen Mary University of London Inflammation is a major contributor to many neurodegenerative disease (Heneka et al. 2015). Microglia, as the resident immune cells of the brain and spinal cord, provide the first line of immunological defence, but can become deleterious when chronically activated, triggering extensive neuronal damage (Cunningham, 2013). Dampening or even reversing this activation may provide neuronal protection against chronic inflammatory damage. The aim of this study was to determine whether lipopolysaccharide (LPS)-induced inflammation could be abrogated through activation of the receptor Fpr2, known to play an important role in peripheral inflammatory resolution. Immortalised murine microglia (BV2 cell line) were stimulated with LPS (50ng/ml) for 1 hour prior to the treatment with one of two Fpr2 ligands, either Cpd43 or Quin-C1 (both 100nM), and production of nitric oxide (NO), tumour necrosis factor alpha (TNFα) and interleukin-10 (IL-10) were monitored after 24h and 48h. Treatment with either Fpr2 ligand significantly suppressed LPS-induced production of NO or TNFα after both 24h and 48h exposure, moreover Fpr2 ligand treatment significantly enhanced production of IL-10 48h post-LPS treatment. As we have previously shown Fpr2 to be coupled to a number of intracellular signaling pathways (Cooray et al. 2013), we investigated potential signaling responses. Western blot analysis revealed no activation of ERK1/2, but identified a rapid and potent activation of p38 MAP kinase in BV2 microglia following stimulation with Fpr2 ligands. Together, these data indicate the possibility of exploiting immunomodulatory strategies for the treatment of neurological diseases, and highlight in particular the important potential of resolution mechanisms as novel therapeutic targets in neuroinflammation. References Cooray SN et al. (2013). Proc Natl Acad Sci U S A 110: 18232-7. Cunningham C (2013). Glia 61: 71-90. Heneka MT et al. (2015). Lancet Neurol 14: 388-40

Reversal of beta-Amyloid-Induced Microglial Toxicity In Vitro by Activation of Fpr2/3

Microglial inflammatory activity is thought to be a major contributor to the pathology of neurodegenerative conditions such as Alzheimer’s disease (AD), and strategies to restrain their behaviour are under active investigation. Classically, anti-inflammatory approaches are aimed at suppressing proinflammatory mediator production, but exploitation of inflammatory resolution, the endogenous process whereby an inflammatory reaction is terminated, has not been fully investigated as a therapeutic approach in AD. In this study, we sought to provide proof-of-principle that the major proresolving actor, formyl peptide receptor 2, Fpr2, could be targeted to reverse microglial activation induced by the AD-associated proinflammatory stimulus, oligomeric β-amyloid (oAβ). The immortalised murine microglial cell line BV2 was employed as a model system to investigate the proresolving effects of the Fpr2 ligand QC1 upon oAβ-induced inflammatory, oxidative, and metabolic behaviour. Cytotoxic behaviour of BV2 cells was assessed through the use of cocultures with retinoic acid-differentiated human SH-SY5Y cells. Stimulation of BV2 cells with oAβ at 100 nM did not induce classical inflammatory marker production but did stimulate production of reactive oxygen species (ROS), an effect that could be reversed by subsequent treatment with the Fpr2 ligand QC1. Further investigation revealed that oAβ-induced ROS production was associated with NADPH oxidase activation and a shift in BV2 cell metabolic phenotype, activating the pentose phosphate pathway and NADPH production, changes that were again reversed by QC1 treatment. Microglial oAβ-stimulated ROS production was sufficient to induce apoptosis of bystander SH-SY5Y cells, an effect that could be prevented by QC1 treatment. In this study, we provide proof-of-concept data that indicate exploitation of the proresolving receptor Fpr2 can reverse damaging oAβ-induced microglial activation. Future strategies that are aimed at restraining neuroinflammation in conditions such as AD should examine proresolving actors as a mechanism to harness the brain’s endogenous healing pathways and limit neuroinflammatory damage

Acute hypoxia reduces plasma myostatin independent of hypoxic dose

Background: Muscle atrophy is seen ~ 25 % of patients with cardiopulmonary disorders, such as chronic obstructive pulmonary disorder and chronic heart failure. Multiple hypotheses exist for this loss, including inactivity, inflammation, malnutrition and hypoxia. Healthy individuals exposed to chronic hypobaric hypoxia also show wasting, suggesting hypoxia alone is sufficient to induce atrophy. Myostatin regulates muscle mass and may underlie hypoxic-induced atrophy. Our previous work suggests a decrease in plasma myostatin and increase in muscle myostatin following 10 hours of exposure to 12 % O2. Aims: To establish the effect of hypoxic dose on plasma myostatin concentration. Concentration of plasma myostatin following two doses of normobaric hypoxia (10.7 % and 12.3 % O2) in a randomised, single-blinded crossover design (n = 8 lowlanders, n = 1 Sherpa), with plasma collected pre (0 hours), post (2 hours) and 2 hours following (4 hours) exposure. Results: An effect of time was noted, plasma myostatin decreased at 4 hours but not 2 hours relative to 0 hours (p = 0.01; 0 hours = 3.26 [0.408] ng.mL-1, 2 hours = 3.33, [0.426] ng.mL-1, 4 hours = 2.92, [0.342] ng.mL-1). No difference in plasma myostatin response was seen between hypoxic conditions (10.7 % vs. 12.3 % O2). Myostatin reduction in the Sherpa case study was similar to the lowlander cohort. Conclusions: Decreased myostatin peptide expression suggests hypoxia in isolation is sufficient to challenge muscle homeostasis, independent of confounding factors seen in chronic cardiopulmonary disorders, in a manner consistent with our previous work. Decreased myostatin peptide may represent flux towards peripheral muscle, or a reduction to protect muscle mass. Chronic adaption to hypoxia does not appear to protect against this response, however larger cohorts are needed to confirm this. Future work will examine tissue changes in parallel with systemic effects

Development of chitosan, pullulan, and alginate based drug-loaded nano-emulsions as a potential malignant melanoma delivery platform

Melanoma is the most aggressive form of skin cancer and various treatments have been investigated to treat this disease, but drug resistance remains an important factor in the failure of conventional therapeutics. Here we describe the development, optimisation and characterisation of alginate, chitosan, pullulan, and their combined nano-emulsions as drug delivery platforms for potential application for melanoma. A novel nano-emulsion delivery system was designed and assessed by determining in vitro drug release, cell viability (MTT), cellular apoptosis (ELISA) and confocal microscopy. A comparative analysis of the effect of the nano-emulsions on BRAF-mutant melanoma (A375) and keratinocyte (HaCaT) cells was conducted, with the “pullulan-chitosan” nano-emulsion chosen as an approach for melanoma drug delivery. Increased apoptosis induction of melanoma cells was recorded as 90% after 72 h of treatment with doxorubicin-loaded optimal nano-emulsion. Similarly, in the same treatment, the viability of melanoma cells was decreased by 70%. More importantly, A375 cells treated with naïve doxorubicin were 100% viable compared to cells treated with doxorubicin-loaded nano-emulsion which were only 30%viable. Achieved results are indicating the importance of the drug carrier’s polymeric combination and the impact of the drug release pattern on the efficiency of the treatment. This offers potential for the abrogation of drug- efflux-related chemo-resistance

Novel anti-inflammatory and chondroprotective effects of the human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride and human melanocortin MC3 receptor agonist PG-990 on lipopolysaccharide activated chondrocytes

Human melanocortin MC1 and MC3 receptors expressed on C-20/A4 chondrocytes exhibit chondroprotective and anti-inflammatory effects when activated by melanocortin peptides. Nearly 9 million people in the UK suffer from osteoarthritis, and bacterial infections play a role in its development. Here, we evaluate the effect of a panel of melanocortin peptides with different selectivity for human melanocortin MC1 (alpha-MSH, BMS-470539 dihydrochloride) and MC3 receptors ([DTrp8]-g-MSH, PG-990) and C-terminal peptide alpha-MSH11-13(KPV), on inhibiting LPS-induced chondrocyte death, pro-inflammatory mediators and induction of anti-inflammatory proteins. C-20/A4 chondrocytes were treated with a panel of melanocortin peptides prophylactically and therapeutically in presence of LPS (0.1 ug/ml). The chondroprotective properties of these peptides determined by cell viability assay, RT-PCR, ELISA for detection of changes in inflammatory markers (IL-6, IL-8 and MMP-1, -3 and -13) and western blotting for expression of the anti-inflammatory protein heme-oxygenase-1. C-20/A4 expressed human melanocortin MC1 and MC3 receptors and melanocortin peptides elevated cAMP. LPS stimulation caused a reduction in C-20/A4 viability, attenuated by the human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride, and MC3 receptor agonists PG-990 and [DTrp8]-g-MSH. Prophylactic and therapeutic regimes of [DTrp8]-g-MSH significantly inhibited LPS-induced modulation of cartilage-damaging IL-6, IL-8, MMPs -1,-3 and -13 mediators both prophylactically and therapeutically, whilst human melanocortin MC1 and MC3 receptor agonists promoted an increase in HO-1 production. In the presence of LPS, activation of human melanocortin MC1 and MC3 receptors provided potent chondroprotection, upregulation of anti-inflammatory proteins and downregulation of inflammatory and proteolytic mediators involved in cartilage degradation, suggesting a new avenue for osteoarthritis treatment

Both MC1 and MC3 Receptors Provide Protection From Cerebral Ischemia-Reperfusion-Induced Neutrophil Recruitment.

Objective Neutrophil recruitment is a key process in the pathogenesis of stroke, and may provide a valuable therapeutic target. Targeting the melanocortin receptors (MC) has previously shown to inhibit leukocyte recruitment in peripheral inflammation, however it is not known whether treatments are effective in the unique cerebral microvascular environment. Here, we provide novel research highlighting the effects of the melanocortin peptides on cerebral neutrophil recruitment, demonstrating important yet discrete roles for both MC1 and MC3. Approach and Results Using intravital microscopy, in two distinct murine models of cerebral ischemia-reperfusion (I/R) injury we have investigated melanocortin control over neutrophil recruitment. Following global I/R, pharmacological treatments suppressed pathological neutrophil recruitment. MC1 selective treatment rapidly inhibited neutrophil recruitment while a non-selective MC agonist provided protection even when co-administered with an MC3/4 antagonist, suggesting the importance of early MC1 signaling. However by 2h reperfusion, MC1 mediated effects were reduced, and MC3 anti-inflammatory circuits predominated. Mice bearing a non-functional MC1 displayed a transient exacerbation of neutrophil recruitment following global I/R, which diminished by 2h. However importantly, enhanced inflammatory responses in both MC1 mutant and MC3 -/- mice resulted in increased infarct size and poor functional outcome following focal I/R. Furthermore we utilized an in vitro model of leukocyte recruitment to demonstrate these anti-inflammatory actions are also effective in human cells. Conclusions These studies reveal for the first time melanocortin control over neutrophil recruitment in the unique pathophysiological context of cerebral I/R, whilst also demonstrating the potential therapeutic value of targeting multiple MCs in developing effective therapeutics

Activation of melanocortin receptors MC1 and MC5 attenuates retinal damage in experimental diabetic retinopathy

We hypothesize that melanocortin receptors (MC) could activate tissue protective circuit in a model of streptozotocin- (STZ-) induced diabetic retinopathy (DR) in mice. At 12–16 weeks after diabetes induction, fluorescein angiography (FAG) revealed an approximate incidence of 80% microvascular changes, typical of DR, in the animals, without signs of vascular leakage. Occludin progressively decreased in the retina of mice developing retinopathy. qPCR of murine retina revealed expression of two MC receptors, Mc1r and Mc5r. The intravitreal injection (5 \u1d707L) of the selective MC1 small molecule agonist BMS-470539 (33 \u1d707mol) and the MC5 peptidomimetic agonist PG-901 (7.32 nM) elicited significant protection with regular course and caliber of retinal vessels, as quantified at weeks 12 and 16 after diabetes induction. Mouse retina homogenate settings indicated an augmented release of IL-1\u1d6fc, IL-1\u1d6fd, IL-6, MIP-1\u1d6fc, MIP-2\u1d6fc, MIP-3\u1d6fc, and VEGF from diabetic compared to nondiabetic mice. Application of PG20N or AGRP and MC5 and MC1 antagonist, respectively, augmented the release of cytokines, while the agonists BMS-470539 and PG-901 almost restored normal pattern of these mediators back to nondiabetic values. Similar changes were quantified with respect to Ki-67 staining. Finally, application of MC3-MC4 agonist/antagonists resulted to be inactive with respect to all parameters under assessment