An optimised protocol for the detection of lipofuscin, a versatile and quantifiable marker of cellular senescence.

Lipofuscin is a yellow-brown pigment typically found in the lysosomes that contains a mixture of molecules including lipids, metals and misfolded proteins. The use of Sudan black B to detect lipofuscin accumulation, a well described marker of cellular senescence and ageing, was first described in 2013 by Georgakopoulou, et al. Here, we provide an optimisation of the original protocol. Firstly, we adjusted the staining methodology for increased ease of use on cultured cells. Secondly, we show that Sudan black B-stained lipofuscin emits strong fluorescence in the far-red channel making it suitable for fluorescence microscopy detection and quantification. Moreover, we also demonstrate that this optimised protocol can be utilised in conjunction with standard immunofluorescence staining techniques, making possible the simultaneous detection of lipofuscin and other cellular proteins of interest, like additional markers of senescence. This is a significant advantage over the most commonly used method for senescence detection, based on beta galactosidase enzymatic activity. We therefore believe that these findings and the provided optimised protocol will represent a useful tool for the scientific community in the field of cellular senescence

Annexin A1 N-Terminal Derived Peptide Ac2-26 Exerts Chemokinetic Effects on Human Neutrophils

It is postulated that peptides derived from the N-terminal region of Annexin A1, a glucocorticoid-regulated 37-kDa protein, could act as biomimetics of the parent protein. However, recent evidence, amongst which the ability to interact with distinct receptors other then that described for Annexin A1, suggest that these peptides might fulfill other functions at variance to those reported for the parent protein. Here we tested the ability of peptide Ac2-26 to induce chemotaxis of human neutrophils, showing that this peptide can elicit responses comparable to those produced by the canonical activator formyl-Met-Leu-Phe (or FMLP). However, whilst disruption of the chemical gradient abolished the FMLP response, addition of peptide Ac2-26 in the top well of the chemotaxis chamber did not affect (10 μM) or augmented (at 30 μM) the neutrophil locomotion to the bottom well, as elicited by 10 μM peptide Ac2-26. Intriguingly, the sole addition of peptide Ac2-26 in the top wells produced a marked migration of neutrophils. A similar behavior was observed when human primary monocytes were used. Thus, peptide Ac2-26 is a genuine chemokinetic agent toward human blood leukocytes. Neutralization strategies indicated that engagement of either the GPCR termed FPR1 or its cognate receptor FPR2/ALX was sufficient to sustain peptide Ac2-26 induced neutrophil migration. Similarly, application of pharmacological inhibitors showed that cell locomotion to peptide Ac2-26 was mediated primarily by the ERK, but not the JNK and p38 pathways. In conclusion, we report here novel in vitro properties for peptide Ac2-26, promoting neutrophil and monocyte chemokinesis; a process that may contribute to accelerate the resolution phase of inflammation. We postulate that the generation of Annexin A1 N-terminal peptides at the site of inflammation may expedite the egress of migrated leukocytes thus promoting the return to homeostasis

Translational advances of melanocortin drugs: Integrating biology, chemistry and genetics.

Melanocortin receptors have emerged as important targets with a very unusual versatility, as their widespread distribution on multiple tissues (e.g. skin, adrenal glands, brain, immune cells, exocrine glands) together with the variety of physiological processes they control (pigmentation, cortisol release, satiety mechanism, inflammation, secretions), place this family of receptors as genuine therapeutic targets for many disorders. This review focuses in the journey of the development of melanocortin receptors as therapeutic targets from the discovery of their existence in the early 1990 s to the approval of the first few drugs of this class. Two major areas of development characterise the current state of melanocortin drug development: their role in obesity, recently culminated with the approval of setmelanotide, and their potential for the treatment of chronic inflammatory and autoimmune diseases like rheumatoid arthritis, multiple sclerosis or fibrosis. The pro-resolving nature of these drugs offers the advantage of acting by mimicking the way our body naturally resolves inflammation, expecting fewer side effects and a more balanced (i.e. non-immunosuppressive) response from them. Here we also review the approaches followed for the design and development of novel compounds, the importance of the GPCR nature of these receptors in the process of drug development, therapeutic value, current challenges and successes, and the potential for the implementation of precision medicine approaches through the incorporation of genetics advances

Senescence under appraisal: hopes and challenges revisited.

In recent years, cellular senescence has become the focus of attention in multiple areas of biomedical research. Typically defined as an irreversible cell cycle arrest accompanied by increased cellular growth, metabolic activity and by a characteristic messaging secretome, cellular senescence can impact on multiple physiological and pathological processes such as wound healing, fibrosis, cancer and ageing. These unjustly called 'zombie cells' are indeed a rich source of opportunities for innovative therapeutic development. In this review, we collate the current understanding of the process of cellular senescence and its two-faced nature, i.e. beneficial/detrimental, and reason this duality is linked to contextual aspects. We propose the senescence programme as an endogenous pro-resolving mechanism that may lead to sustained inflammation and damage when dysregulated or when senescent cells are not cleared efficiently. This pro-resolving model reconciles the paradoxical two faces of senescence by emphasising that it is the unsuccessful completion of the programme, and not senescence itself, what leads to pathology. Thus, pro-senescence therapies under the right context, may favour inflammation resolution. We also review the evidence for the multiple therapeutic approaches under development based on senescence, including its induction, prevention, clearance and the use of senolytic and senomorphic drugs. In particular, we highlight the importance of the immune system in the favourable outcome of senescence and the implications of an inefficient immune surveillance in completion of the senescent cycle. Finally, we identify and discuss a number of challenges and existing gaps to encourage and stimulate further research in this exciting and unravelled field, with the hope of promoting and accelerating the clinical success of senescence-based therapies

Melanocortin Receptors as Novel Effectors of Macrophage Responses in Inflammation

Macrophages have crucial functions in initiating the inflammatory reaction in a strict temporal and spatial manner to provide a “clear-up” response required for resolution. Hormonal peptides such as melanocortins modulate macrophage reactivity and attenuate inflammation ranging from skin inflammation to joint disease and reperfusion injury. The melanocortins (e.g., adrenocorticotrophin, ACTH and αMSH) elicit regulatory properties through activation of a family of GPCRs, the melanocortin (MC) receptors; MC1–MC5. Several studies have focused on MC1 and MC3 as anti-inflammatory receptors expressed on cells of the macrophage lineage. We review here elements of the melanocortin pathway with particular attention to macrophage function in anti-inflammatory and pro-resolving inflammatory settings. Evidence shows that ACTH, αMSH, and other MC agonists can activate MC1 and MC3 on macrophage through cAMP and/or NFκB-dependent mechanisms to abrogate pro-inflammatory cytokines, chemokines, and NO and enhance anti-inflammatory mediators such as IL-10 and HO-1. Melanocortins and their receptors regulate inflammation by inhibiting leukocyte recruitment to and interaction with inflamed tissue. An intensely exciting addition to this field of research has been the ability of an αMSH analog; AP214 to activate MC3 expressed on macrophage to enhance their clearance of both zymosan particles and apoptotic neutrophils thus putting melanocortins in line with other pro-resolving mediators. The use of mouse colonies mutated or nullified for MC1 or MC3, respectively as well as availability of selective MC receptor agonist/antagonists have been key to deciphering mechanisms by which elements of the melanocortin system play a role in these phenomena. We review here melanocortin pathway components with attention to the macrophage, reiterating receptor targets required for pro-resolving properties. The overall outcome will be identification of selective MC agonists as a strategy for innovative anti-inflammatory therapeutics

Old drugs with new skills: fenoprofen as an allosteric enhancer at melanocortin receptor 3

This project was supported by Queen Mary Innovation Ltd Proof of Concept Fund (2012/13) and The William Harvey Research Foundation

Pro-resolving and anti-arthritic properties of the MC1 selective agonist PL8177

BACKGROUND: Melanocortins are peptides endowed with anti-inflammatory and pro-resolving activities. Many of these effects are mediated by the Melanocortin receptor 1 (MC(1)) as reported in several experimental settings. As such, MC(1) 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 MC(1) agonist (PL8177) in vitro and in a mouse model of inflammatory arthritis. METHODS: PL8177 and the natural agonist αMSH were tested for activation of mouse and human Melanocortin receptors (MC(1,3,4,5)), 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. RESULTS: PL8177 activates mouse and human MC(1) with apparent EC(50) values of 0.01 and 1.49 nM, respectively, using the cAMP accumulation assay. Similar profiles were observed for the induction of ERK phosphorylation (EC(50): 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. CONCLUSION: These results demonstrate that the MC(1) agonism with PL8177 affords therapeutic effects in inflammatory conditions including arthritis. SIGNIFICANCE: Drugs 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 (MC(1)), 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 MC(1) receptor, as a promising strategy to manage chronic inflammatory diseases