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

Discovering Drug-Drug Interactions Using Association Rule Mining from Electronic Health Records

In this paper, we propose utilising Electronic Health Records (EHR) to discover previously unknown drug-drug interactions (DDI) that may result in high rates of hospital readmissions. We used association rule mining and categorised drug combinations as high or low risk based on the adverse events they caused. We demonstrate that the drug combinations in the high-risk group contain significantly more drug-drug interactions than those in the low-risk group. This approach is efficient for discovering potential drug interactions that lead to negative outcomes, thus should be given priority and evaluated in clinical trials. In fact, severe drug interactions can have life-threatening consequences and result in adverse clinical outcomes. Our findings were achieved using a new association rule metric, which better accounts for the adverse drug events caused by DDI

Exploiting formyl peptide receptor 2 to promote microglial resolution: a new approach to Alzheimer's disease treatment.

Alzheimer’s disease and dementia are among the most significant current healthcare challenges given the rapidly growing elderly population, and the almost total lack of effective therapeutic interventions. Alzheimer’s disease pathology has long been considered in terms of accumulation of amyloid beta and hyperphosphorylated tau, but the importance of neuroinflammation in driving disease has taken greater precedence over the last 15–20 years. Inflammatory activation of the primary brain immune cells, the microglia, has been implicated in Alzheimer’s pathogenesis through genetic, preclinical, imaging and postmortem human studies, and strategies to regulate microglial activity may hold great promise for disease modification. Neuroinflammation is necessary for defence of the brain against pathogen invasion or damage but is normally self-limiting due to the engagement of endogenous pro-resolving circuitry that terminates inflammatory activity, a process that appears to fail in Alzheimer’s disease. Here, we discuss the potential for a major regulator and promoter of resolution, the receptor FPR2, to restrain pro-inflammatory microglial activity, and propose that it may serve as a valuable target for therapeutic investigation in Alzheimer’s disease

Stimulation of the Pro-Resolving Receptor Fpr2 Reverses Inflammatory Microglial Activity by Suppressing NFκB Activity

Neuroinflammation driven primarily by microglia directly contributes to neuronal death in many neurodegenerative diseases. Classical anti-inflammatory approaches aim to suppress pro-inflammatory mediator production, but exploitation of inflammatory resolution may also be of benefit. A key driver of peripheral inflammatory resolution, formyl peptide receptor 2 (Fpr2), is expressed by microglia, but its therapeutic potential in neurodegeneration remains unclear. Here, we studied whether targeting of Fpr2 could reverse inflammatory microglial activation induced by the potent bacterial inflammogen lipopolysaccharide (LPS). Exposure of murine primary or immortalised BV2 microglia to LPS triggered pro-inflammatory phenotypic change and activation of ROS production, effects significantly attenuated by subsequent treatment with the Fpr2 agonist C43. Mechanistic studies showed C43 to act through p38 MAPK phosphorylation and reduction of LPS-induced NFκB nuclear translocation via prevention of IκBα degradation. Here, we provide proof-of-concept data highlighting Fpr2 as a potential target for control of microglial pro-inflammatory activity, suggesting that it may be a promising therapeutic target for the treatment of neuroinflammatory disease

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

Stimulation of the Pro-Resolving Receptor Fpr2 Reverses Inflammatory Microglial Activity by Suppressing NFκB Activity

Neuroinflammation driven primarily by microglia directly contributes to neuronal death in many neurodegenerative diseases. Classical anti-inflammatory approaches aim to suppress pro-inflammatory mediator production, but exploitation of inflammatory resolution may also be of benefit. A key driver of peripheral inflammatory resolution, formyl peptide receptor 2 (Fpr2), is expressed by microglia, but its therapeutic potential in neurodegeneration remains unclear. Here, we studied whether targeting of Fpr2 could reverse inflammatory microglial activation induced by the potent bacterial inflammogen lipopolysaccharide (LPS). Exposure of murine primary or immortalised BV2 microglia to LPS triggered pro-inflammatory phenotypic change and activation of ROS production, effects significantly attenuated by subsequent treatment with the Fpr2 agonist C43. Mechanistic studies showed C43 to act through p38 MAPK phosphorylation and reduction of LPS-induced NFκB nuclear translocation via prevention of IκBα degradation. Here, we provide proof-of-concept data highlighting Fpr2 as a potential target for control of microglial pro-inflammatory activity, suggesting that it may be a promising therapeutic target for the treatment of neuroinflammatory disease

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 (α-MSH, BMS-470539 dihydrochloride) and MC3 ([DTrp8]-γ-MSH, PG-990) receptors and C-terminal peptide α-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 μg/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]-γ-MSH. Prophylactic and therapeutic regimes of [DTrp8]-γ-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

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

Calcineurin and Protein kinase G regulate C. elegans behavioral quiescence during locomotion in liquid

<p>Abstract</p> <p>Background</p> <p>Most rhythmic motor behaviors in nature are episodic i.e. they alternate between different behavioral states, including quiescence. Electrophysiological studies in invertebrate behavioral switching, maintenance and quiescence have elucidated several neuronal mechanisms that generate a temporal pattern in behavior. However, the genetic bases of these processes are less well studied. We have previously uncovered a novel episodic behavior exhibited by <it>C. elegans </it>in liquid media where they alternate between distinct phases of rhythmic swimming and quiescence. Here, we have investigated the effect of several genes and their site of action on the behavioral quiescence exhibited in liquid by the nematode <it>C. elegans</it>.</p> <p>Results</p> <p>We have previously reported that high cholinergic signaling promotes quiescence and command interneurons are critical for timing the quiescence bout durations. We have found that in addition to command interneurons, sensory neurons are also critical for quiescence. We show that the protein phosphatase calcineurin homolog <it>tax-6 </it>promotes swimming whereas the protein kinase G homolog <it>egl-4 </it>promotes quiescence. <it>tax-6 </it>expression in the sensory neurons is sufficient to account for its effect. <it>egl-4 </it>also acts in multiple sensory neurons to mediate its effect on quiescence. In addition our data is consistent with regulation of quiescence by <it>egl-4 </it>acting functionally downstream of release of acetylcholine (ACh) by motor neurons.</p> <p>Conclusions</p> <p>Our study provides genetic evidence for mechanisms underlying the maintenance of a behavioral state operating at multiple neuronal levels through the activities of a kinase and a phosphatase. These results in a genetically tractable organism establish a framework for further dissection of the mechanism of quiescence during episodic behaviors.</p