PL8177 (provided by Palatin Technologies Inc) and αMSH (Tocris, Bristol, UK) solutions were prepared at 1mM stocks in DMSO (for in vitro studies) or PBS (in vivo studies) and single-use aliquots were frozen at -20°C. All other chemicals were obtained from Sigma-Aldrich, Poole, UK, unless otherwise indicated.

B16-F1 melanocyte cell line naturally expressing MC₁ were maintained in RPMI containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin and kept at 37°C with 5% CO₂. Confluent monolayers were washed with PBS, harvested by gentle scrapping and centrifuged at 600 x g for 10 min. Pellets were resuspended in harvesting buffer and incubated with radioligand [³H]-PL8177 at a range of 1x10^-6M to 1x10^-13M for 90 min at room temperature. The endogenous Melanocortin agonist peptide αMSH was used as control. Binding was detected by scintillation counting and results are expressed as a percent of control specific binding and as a percent inhibition of control specific binding obtained in the presence of the test compound, PL8177. The inhibition constants (K_i) were calculated using the Cheng Prusoff equation.

Vectors for human and mouse MC₁, MC₃, MC₄ and MC₅ and empty vector pCMV6 were originally purchased from Origene (Rockville, Maryland, USA) and in-house transformed into bacteria. Bacterial clones were grown overnight in 150ml LB medium supplemented with kanamycin (25µg/ml). Plasmid DNA isolation was performed using Zyppy™ Plasmid Maxiprep kit (Zymo Research; Irvine, California, USA).

HEK293A cells were maintained in DMEM containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin and kept at 37°C with 5% CO₂. Cells were seeded in 96-well plates at 2x10⁴ cells/well and transfected 24 hours later with 50ng of plasmid DNA (encoding for human and mouse MC₁, MC₃, MC₄ and MC₅, TrueORF cDNA clones (Origene) and Lipofectamine 2000 (Invitrogen; Waltham, Massachusetts, USA) according to manufacturer’s instructions. Cells were used 24 h later.

Twenty-four hours after transfection, cells were serum-starved for 3 hours to reduce and stabilize basal levels of cAMP. Melanocortin agonists were tested using 1/5 serial dilutions to generate concentration response curves starting at 10 or 0.4 µM. Vehicle was used as negative control, and forskolin (3 µM, Tocris Bioscience) as positive control. Compound solutions were prepared in serum-free DMEM containing 10 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma), to inhibit phosphodiesterase activity. Cells were stimulated for 15 min with the respective compounds and lysed immediately with 0.1M HCl followed by scrapping and freezing at -80°C until the assay was performed. cAMP was quantified using the Cyclic AMP Select ELISA kit (Cayman Chemicals, CAY501040, Ann Arbor, Michigan, USA) according to manufacturer instructions. Optical Density (OD) measurements were converted to cAMP concentration using a standard curve and results were normalized subtracting background signal and calculated as % of forskolin. The endogenous agonist αMSH (10 µM) was used to define the 100% agonistic effect for each receptor subtype.

Twenty-four hours after transfection, cells were serum-starved for 3 hours to reduce and stabilize basal levels of phospho-ERK1/2. Concentration response curves were generated by 1/5 serial dilutions as above. Drug solutions were prepared in serum-free DMEM. Cells were stimulated for 5 min with the different compounds and lysed immediately with ice-cold extraction buffer supplied in the ERK1/2 (pT202/Y204) SimpleStep ELISA Kit (Abcam ab176640; Cambridge, UK), according to manufacturer instructions. Total protein was also quantified by Bradford to normalize p-ERK1/2 response. Basal absorbance levels (vehicle treated cells) were subtracted form test samples and data expressed as percentage of 10 µM αMSH effect, normalized to 100%.

All animal studies were approved by and performed under the guidelines of the Ethical Committee for the Use of Animals, Barts and The London School of Medicine and Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act, 1986). C57BL/6J wild-type (WT) mice were purchased from Charles River Laboratories (UK). Male mice (7-8 weeks old; body weight ~30 g) were maintained on a standard chow pellet diet and had free access to water, with a 12-hour light-dark cycle.

C57BL/6J male mice were injected with 1 ml of 2% Biogel intraperitoneally (Bio-Rad; Hercules, California, USA). Four days later, peritoneal cells were collected by lavage using 4 ml of 3mM EDTA in PBS and plated in RPMI 1640 containing 10% FCS. After 1 h of incubation of peritoneal lavage, wells were washed to remove non-adherent cells and experiment initiated. For cytokine production, the different conditions were tested in duplicate using 24-well plates with 0.5x10⁶ cells/well. For mRNA analyses, 2.5x10⁶ cells/well in 6-well plates were used. Compounds or vehicle were added 30 min before stimulation with 25 µg/ml zymosan A (Sigma-Aldrich) for 6 h. Then, supernatants and cells were collected for cytokines measurements and gene expression analyses, respectively.

Total RNA was extracted using RNAeasy Plus Mini Kit (Qiagen; Hilden, Germany) following manufacturer’s instructions. cDNA was synthesized using the SuperScript III VILO Master Mix (Invitrogen). Real time-PCR was performed in duplicates, with 4µl cDNA (20 ng), 1µl of primers and 5µl iTaq™ Universal SYBR^® Green PCR Master Mix (Applied Biosystems), using the ABI Prism 7900HT Sequence Detection System. Dissociation step was always included to confirm the absence of unspecific products. For relative gene expression, fold increase was calculated with the formula 2^(-ΔΔCt), using Gapdh as reference gene. Quantitect primers (QIAGEN) used were the following: Alox15 (QT00111034), Alox5 (QT00258622), Ano6, (QT01038660), Anxa1 (QT00145915), Cd14 (QT00246190), Gapdh (QT01658692), Hmgb1 (QT00247786), Hmox1 (QT00159915), Il1b (QT01048355), Il6 (QT00098875), Lgals3 (QT00152558), Lgals9 (QT00173236), Lta4h (QT00160475), Nos2 (QT00100275), Pparg (QT00100296), Ptges (QT00118223), Tlr4 (QT00259042).

IL-6, TNF-α (both 1/10 dilution), IL-1β and CCL-2 (undiluted) were measured by ELISA (Invitrogen; Waltham, Massachusetts, USA). Concentration values were extrapolated from a standard curve and expressed as increase over basal cytokine levels for each individual donor mouse.

Experiments using healthy volunteers (written consent provided) were approved by Queen Mary Ethics of Research Committee QMREC2014.61. Blood was collected into 3.2% sodium citrate (1:10) and diluted 1:1 in RPMI 1640 before separation through a double-density gradient using Histopaque 10771 and 11191 (Sigma-Aldrich). Contaminating erythrocytes were removed by hypotonic lysis. Polymorphonuclear cells were incubated in 10% FCS overnight at 37°C, 5% CO₂ to let neutrophils undergo spontaneous apoptosis.

Mouse primary peritoneal Macrophages (0.5x10⁶ cells/well in 24-well plates, in duplicate) were stimulated with compounds/vehicle for 30 min before the addition of apoptotic neutrophils (1:5 ratio, Macrophage:neutrophil) for 1 h. Cells were washed 3 times and fixed for 30 min with 2.5% glutaraldehyde and neutrophils stained using the myeloperoxidase assay by adding 0.1 mg/ml dimethoxybenzidine (Sigma-Aldrich) and 0.03% (v/v) hydrogen peroxide for 1 h. Cells were analysed by light microscopy. Ten random fields were acquired, and more than 400 cells were blindly counted per well. Clearance index was calculated as (% phagocytic cells x % multiple ingestions cells) x100.

Arthritis was induced in C57BL/6J male mice (10 weeks old) by two i.p. injections of 100μl of K/BxN serum (diluted 1:1 in PBS, i.e. 200μl volume injected) on days 0 and 2. Arthritic mice (n=24) were randomized in 4 cages, each one including 2 mice from each different experimental group (n=6 mice per cage). Compound PL8177 was prepared as a 0.1mg/ml solution in PBS and stored frozen in single use aliquots. From day 2 to 7, vehicle PBS or PL8177 (3 or 0.3 mg/kg) daily treatment was administered by 0.8ml i.p. injection. The development of systemic arthritis was monitored daily measuring clinical score, paw volume (plethysmometer) and knee width (caliper). To obtain the clinical score, each of the four limbs was evaluated for signs of inflammation in wrist/ankle, pad and digits (one point each of these 3 parts), thus reaching a maximum of 12 (3 per limb). Mice were sacrificed at day 7, and different tissue samples were collected for evaluation. An additional group of non-arthritic mice (n=6) was also included for baseline measurements.

For the evaluation of joint immune infiltration, cells were isolated from the hind paws by digestion in serum-free media containing DNAse I (40µg/ml) and collagenase D (0.5µg/ml) for 1 h. Upon tissue digestion, 30,000 precision counting beads (BioLegend; San Diego, California, USA) were added for total cell quantification and the cell suspension was incubated with live/dead discrimination marker (Fixable Viability eF780, Thermo Fisher Scientific; Waltham, Massachusetts, USA), Fc Receptor blocking reagent (BioLegend) and subsequently stained with an antibody cocktail to characterize the different immune cell populations. The antibodies used were from BioLegend, unless otherwise specified: Ly6C-BV421 (clone HK1.4), CD45-VioGreen (clone 30F11; Miltenyi; Bergisch Gladbach, Germany), MHC-II-BV650 (clone M5/114.15.2), CD11b-BV785 (clone M1/70), B220-FITC (clone RA3-6B2), F4/80-PE (clone BM8), CD3e-PE-Cy5 (clone 145-2C11), SiglecF-PE-Vio770 (clone REA798; Miltenyi), CD11c-APC (clone N418) and Ly6G-AF700 (clone 1A8). A sample containing at least 20,000 beads was acquired in a Fortessa Cytometer (BD; Franklin Lakes, Nueva Jersey, USA) and flow cytometry data was analyzed with FlowJo software v10.4 (ThreeStars).

Non-linear regression models were used to generate dose–response curves. Statistical analysis was performed with GraphPad Prism v8 using either t-test (2 groups) or one-way ANOVA test (>2 groups), and paired tests for repeated measures when appropriate, followed by Tukey’s multiple comparison test. Values of p<0.05 were considered statistically significant.

PL8177 is a synthetic analog derived from the sequence of the natural αMSH peptide. It is cyclized by an amide bond between glutamic and diaminopropionic acid side chains and includes one D-amino acid (D-phenylalanine) ( Figure 1A ). In binding affinity studies with mouse MC₁ expressed in B16-F1 cell line, PL8177 displayed high affinity for this receptor (Ki =75 pM; Figure 1B ), while the affinity for MC₃ and MC₄ was substantially weaker using transiently transfected HEK293 cells (3500nM and 360nM respectively -data not shown).

To characterize the signaling profile and selectivity of PL8177 for the different MC receptors, concentration-response curves were generated in human embryonic kidney cells, HEK293A, transiently transfected with mouse or human MC₁, MC₃, MC₄ or MC₅ receptors. Both cAMP accumulation and ERK1/2 phosphorylation were used as readouts to evaluate their agonistic activity.

Quantification of cAMP accumulation ( Figure 2A ) showed that PL8177 peptide triggered MC₁-mediated cAMP production at picomolar-nanomolar range (EC₅₀ values of 0.01 and 1.49nM for mouse and human receptors respectively, see Table 1 ), while reduced or no significant effect was observed on cells transfected with MC_3,4,5 receptors subtypes at those concentrations. By contrast, the natural agonist αMSH activated all MC receptor subtypes, both mouse and human, showing no receptor selectivity as expected ( Figure 2A and Table 1 ). The potency at MC₁ was lower than that obtained for PL8177, with EC₅₀ values of 4.37 and 37.07nM for mouse and human MC₁ respectively. A similar pattern was observed when phosphorylation of ERK1/2 was measured ( Figure 2B ): whilst αMSH induced ERK1/2 phosphorylation following activation of all four receptor subtypes, the synthetic peptide PL8177 displayed a strong selectivity for MC₁ receptor, with EC₅₀ values of 0.05nM and 1.39nM for mouse and human receptors respectively. On the other MC receptors, PL8177 had only partial or null phospho-ERK1/2 activity ( Figure 2B and Table 1 ).

Altogether, the results obtained suggest that the pharmacological activity of PL8177 corresponds to a selective MC₁ agonist with a non-biased profile with respect to cAMP and ERK, and improved potency compared to the endogenous agonist αMSH.

Treatment of biogel-elicited peritoneal macrophages with Melanocortin peptides (2 µM) for 30 minutes was followed by a 6-hour incubation period with zymosan (25 μg/ml). This inflammogen produced a classical activation of inflammatory genes like interleukins 1 beta and 6 (Il1b, Il6), inducible nitric oxide synthase (Nos2), prostaglandin E synthase (Ptges), galectins 3 and 9 (Lgals3, Lgals9), heme-oxygenase 1 (Hmox1) and the LPS co-receptor Cd14 ( Figures 3A, B ). On the other hand, gene expression levels were reduced for annexin A1 (Anxa1), anoctamin 6 (Ano6), high motility group box 1 (Hmgb1), lipoxygenases 5 and 15 (Alox5, Alox15), leukotriene A4 hydrolase (Lta4h) and toll-like receptor 4 (Tlr4). Both PL8177 and αMSH modulated the response to zymosan in a selective manner, with reduction in the expression of Il1b, Il6, Nos2, Lgals9 and Ptges ( Figures 3A, B ), without reverting the effect on anti-inflammatory genes, at least at this 6-hour time-point. These effects were mirrored by a partial modulation of cytokine levels released in the supernatants ( Figure 3C ). Herein, PL8177 and αMSH were tested at 80 nM-10 µM range, with more prominent reductions (~60%) on IL-1β, IL-6 and CCL-2 release.

To complete the characterization of PL8177 as a genuine pro-resolving compound, we tested the ability of this peptide to enhance Macrophage efferocytosis (i.e. the phagocytosis of apoptotic cells), using apoptotic neutrophils to feed the macrophages and quantifying efferocytosis by performing the myeloperoxidase assay ( Figure 4A ). At 2 µM concentration, PL8177 augmented the already marked efferocytosis (~16% increase over basal levels, Figure 4B ) as well as the presence of multi-phagocytic macrophages (i.e. cells that have engulfed more than one apoptotic neutrophil, Figure 4C ). Such properties of PL8177 yielded a highly significant effect on the clearance index ( Figure 4D ).

Injection of K/BxN serum to mice (day 0 and 2) promoted a rapid onset of polyarthritis with parameters elevated from day 4 onwards, including clinical score, paw oedema and knee width ( Figures 5A–C ). Drug treatment started at day 2, with animals being randomized to the distinct treatment groups. PL8177 was tested at two doses (0.3 and 3mg/kg), with the higher one significantly attenuating clinical score (-35%; Figure 5A ), the development of oedema ( Figure 5B ), the incidence of severe disease ( Figure 5D ), and to a lesser extent, knee swelling ( Figure 5C ). Representative images of paws collected at the end of the study show evident signs of swelling and inflammation in vehicle-treated arthritic mice and improvement with the PL8177 treatment ( Figure 5E ). The lower dose of PL8177 (0.3mg/kg) offered a degree of improvement on the parameters under analyses in the STIA model, but none of them reached statistical significance ( Figure 5 ).

The effects of PL8177 on the macroscopic signs of arthritis were matched by a modulation of immune cell phenotype in the arthritic joints ( Figure 6 and Supplementary Figure 1 ). At day 7, the paw immune infiltrate was mostly myeloid, with ~65% of cells being neutrophils (CD11b⁺Ly6G⁺), ~2% monocytes (CD11b⁺Ly6C⁺) and ~20% macrophages (CD11b⁺F4/80⁺). At the dose of 3 mg/kg, PL8177 did not affect the small number of B cells (B220⁺) and T cells (CD3e⁺), while producing a marked reduction in myeloid cell infiltration ( Figure 6 ). The latter effect was due to significant differences in the number of neutrophils (-81%), eosinophils (CD11b⁺SingleF⁺) (-66%) and monocytes-macrophages (-70%). Further analyses of the different subsets of monocytes and macrophages using the combination of Ly6C, F4/80 and MHC-II markers, revealed a slightly higher inhibitory effect in the abundance of the immature monocyte populations Ly6C⁺/MCH-II^- and Ly6C⁺/MCH-II⁺ cells.