Caterina Lonati

The Melanocortin System in Control of Inflammation

Melanocortin peptides, the collective term for α-, β-, and γ-melanocyte-stimulating hormone (α-, β-, γ-MSH) and adrenocorticotropic hormone (ACTH), are elements of an ancient modulatory system. Natural melanocortins derive from the common precursor pro-opiomelanocortin (POMC). Five receptor subtypes for melanocortins (MC1-MC5) are widely distributed in brain regions and in peripheral cells. Melanocortin receptor activation by natural or synthetic ligands exerts marked anti-inflammatory and immunomodulatory effects. The anticytokine action and the inhibitory influences on inflammatory cell migration make melanocortins potential new drugs for treatment of inflammatory disorders. Effectiveness in treatment of acute, chronic, and systemic inflammatory disorders is well documented in preclinical studies. Further, melanocortins are promising compounds in neuroprotection. This review examines the main signaling circuits in anti-inflammatory and immunomodulatory actions of melanocortins, and the potential therapeutic use of these molecules.

Detrimental consequences of brain injury on peripheral cells

Acute brain injury and brain death exert detrimental effects on peripheral host cells. Brain-induced impairment of immune function makes patients more vulnerable to infections that are a major cause of morbidity and mortality after stroke, trauma, or subarachnoid hemorrhage (SAH). Systemic inflammation and organ dysfunction are other harmful consequences of CNS injury. Brain death, the most severe consequence of brain injury, causes inflammatory changes in peripheral organs that can contribute to the inferior outcome of organs transplanted from brain-dead donors. Understanding of the mechanisms underlying the detrimental effects of brain injury on peripheral organs remains incomplete. However, it appears that sympathetic nervous system (SNS)-activation contributes to elicit both inflammation and immunodepression. Indeed, norepinephrine (NE)-induced production of chemokines in liver and other organs likely participates in local and systemic inflammatory changes. Conversely, catecholamine-stimulated interleukin-10 (IL-10) production by blood monocytes exerts immunosuppressive effects. Activation of the hypothalamic-pituitary-adrenal axis (HPA) by increased inflammatory cytokines within the brain is a significant component in the CNS-induced immune function inhibition. Non-neurologic consequences of brain injury show impressive similarities regardless of the brain insult and appear to depend on altered neuroimmune circuits. Modulation of these circuits could reduce extra-brain damage and improve patient outcome in both vascular and traumatic brain injury.

Antimicrobial Properties of α-MSH and Related Synthetic Melanocortins

The natural antimicrobial peptides are ancient host defense effector molecules, present in organisms across the evolutionary spectrum. Several properties of α-melanocyte stimulating hormone (α-MSH) suggested that it could be a natural antimicrobial peptide. α-MSH is a primordial peptide that appeared during the Paleozoic era, long before adaptive immunity developed and, like natural antimicrobial molecules, is produced by barrier epithelia, immunocytes, and within the central nervous system. α-MSH was discovered to have antimicrobial activity against two representative pathogens, Staphylococcus aureus and Candida albicans. The candidacidal influences of α-MSH appeared to be mediated by increases in cell cyclic adenosine monophosphate (cAMP). The cAMP-inducing capacity of α-MSH likely interferes with the yeasts own regulatory mechanisms of this essential signaling pathway. It is remarkable that this mechanism of action in yeast mimics the influences of α-MSH in mammalian cells in which the peptide binds to G-protein-linked melanocortin receptors, activates adenylyl cyclase, and increases cAMP. When considering that most of the natural antimicrobial peptides enhance the local inflammatory reaction, the anti-inflammatory and antipyretic effects of α-MSH confer unique properties to this molecule relative to other natural antimicrobial molecules. Synthetic derivatives, chemically stable and resistant to enzymatic degradation, could form the basis for novel therapies that combine anti-inflammatory and antimicrobial properties.

Protective action of NDP-MSH in experimental subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is still a major cause of morbidity and mortality. α-Melanocyte stimulating hormone (α-MSH) and other melanocortin peptides exert potent neuroprotective action and they might modulate key molecules involved in SAH-induced vasospasm. The aim of this research was to determine whether treatment with the α-MSH analog Nle4,DPhe7-α-MSH (NDP-MSH) exerts protective effects in experimental SAH in the rat. Initial experiments examined effects of NDP-MSH on the basilar artery phenotype in the absence of injury. In these tests intrathecal injection of small concentrations (10ng) of the peptide induced a tolerant phenotype similar to that observed after ischemic preconditioning. Then the effect of systemic treatment with NDP-MSH (100μg i.v.) on experimental SAH was evaluated. SAH was induced by a single-blood injection into the cisterna magna. The basilar artery phenotype was examined at 4h and the artery caliber at 5days following SAH. Expression of 96 genes was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) using Custom Taqman Low-Density Arrays. Four hours after SAH, the transcriptional profile of the basilar artery was deeply disrupted. Transcript alteration included genes involved in inflammation, stress response, apoptosis, and vascular remodeling. Treatment with NDP-MSH prevented most of these transcription changes and decreased phosphorylation of extracellular-signal-regulated kinases (ERK1/2) and inhibitor protein IκBα. Vasospasm on day 5 was significantly reduced by NDP-MSH administration. These results combine with others on CNS inflammation to suggest that the melanocortins could be safe and effective therapeutic candidates to treat SAH-related complications.

Alteration in the transcriptional profile of livers from brain-dead organ donors

Background. There is evidence that brain death causes changes in peripheral organs. Marked inflammation is found in organs collected during experimental brain death and clinical studies indicate that, despite genetic mismatch, organs obtained from living donors show improved survival over those from brain-dead donors. The aim of the present clinical research was to explore changes in the transcriptional profile of livers from brain-dead organ donors. Methods. Using the cDNA macroarray technique, we compared gene expression in liver biopsies from 21 brain-dead organ donors and in normal liver tissue obtained during resection of benign focal lesions. Results. Analysis of gene expression showed significant differences in the mRNA levels of 117 genes. There was reduced expression of 93 genes whereas expression of 24 genes was enhanced. Downregulated pathways included transcripts related to morphogenesis, blood coagulation, complement cascade, amine metabolism, lipid metabolism, nucleic acid metabolism, biodegradation of xenobiotics, signal transduction, and transcription. Conversely, there was induction of genes related to acute phase response, damage-related response, electron transport, and energy metabolism. Conclusions. The present research demonstrates major changes in the transcriptional profile of livers from brain-dead organ donors. The presence of both down- and upregulated gene families suggests that the alteration in transcriptional profile is not a consequence of death-associated organ failure, but rather, an active change in regulatory mechanisms.

The peptide NDP-MSH induces phenotype changes in the heart that resemble ischemic preconditioning.

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a pro-opiomelanocortin (POMC)-derived peptide that exerts multiple protective effects on host cells. Previous investigations showed that treatment with alpha-MSH or synthetic melanocortin agonists reduces heart damage in reperfusion injury and transplantation. The aim of this preclinical research was to determine whether melanocortin treatment induces preconditioning-like cardioprotection. In particular, the plan was to assess whether melanocortin administration causes phenotype changes similar to those induced by repetitive ischemic events. The idea was conceived because both ischemic preconditioning and melanocortin signaling largely depend on cAMP response element binding protein (CREB) phosphorylation. Rats received single i.v. injections of 750microg/kg of the alpha-MSH analogue Nle(4),DPhe(7)-alpha-MSH (NDP-MSH) or saline and were sacrificed at 0.5, 1, 3, or 5h. Western blot analysis showed that rat hearts expressed melanocortin 1 receptor (MC1R) protein. Treatment with NDP-MSH was associated with early and marked increase in interleukin 6 (IL-6) mRNA. This was followed by signal transducer and activator of transcription 3 (STAT3) phosphorylation and induction of suppressor of cytokine signaling 3 (SOCS3). There were no changes in expression of other cytokines of the IL-6 family. Expression of IL-10, IL-1beta, and TNF-alpha was likewise unaltered. In hearts of rats treated with NDP-MSH there was increased expression of the orphan nuclear receptor Nur77. The data indicate that NDP-MSH induces phenotype changes that closely resemble ischemic preconditioning and likely contribute to its established protection against reperfusion injury. In addition, the increased expression of Nur77 and SOCS3 could be part of a broader anti-inflammatory effect.

Reduced expression of the melanocortin-1 receptor in human liver during brain death.

Objective: There is evidence that brain death has detrimental effects on peripheral organs. Clinical and experimental studies on organ donors showed marked inflammation in tissue samples of livers and kidneys collected during brain death. The inflammatory reaction is characterized by release of cytokines and inflammatory cell infiltration. Because melanocortins and their receptors are significant modulators of inflammation, we hypothesized that downregulation of melanocortin receptors during brain death could contribute to enhance inflammation. Methods: Using real-time polymerase chain reaction (PCR) analysis, we determined expression of melanocortin receptors in liver biopsies obtained from brain-dead organ donors before cold ischemia and in normal liver tissue during resection of benign focal lesions of the liver. Tissue biopsies were also analyzed for expression of intercellular adhesion molecule-1 (ICAM-1), which has a central function in inflammatory cell migration. Results: Expression of melanocortin-1 receptor (MC1R) mRNA was markedly reduced in liver samples obtained from brain-dead organ donors compared to hepatic tissue collected during resection of benign focal lesions of the liver. Conversely, expression of the adhesion molecule ICAM-1 was significantly increased in livers of brain-dead organ donors. Conclusions: Disruption of the endogenous anti-inflammatory circuit based on MC1R could contribute to tissue damage during brain death.

Molecular Changes Induced in Rat Liver by Hemorrhage and Effects of Melanocortin Treatment

Background: Melanocortin peptides improve hemodynamic parameters and prevent death during severe hemorrhagic shock. In the present research we determined influences of a synthetic melanocortin 1/4 receptor agonist on the molecular changes that occur in rat liver during hemorrhage. Methods: Controlled-volume hemorrhage was performed in adult rats under general anesthesia by a stepwise blood withdrawal until mean arterial pressure fell to 40 mmHg. Then rats received either saline or the synthetic melanocortin 1/4 receptor agonist Butir-His-D-Phe-Arg-Trp-Sar-NH2 (Ro27-3225; n = 6–8 per group). Hemogasanalysis was performed throughout a 60-min period. Gene expression in liver samples was determined at 1 or 3 h using quantitative real-time polymerase chain reaction. Results: At 1 h, in saline-treated shocked rats, there were significant increases in activating transcription factor 3 (Atf3), early growth response 1 (Egr1), heme oxygenase (decycling) 1 (Hmox1), FBJ murine osteosarcoma viral oncogene homolog (Fos), and jun oncogene (Jun). These changes were prevented by Ro27-3225 (mean ± SEM: Atf3 152.83 ± 58.62 vs. 579.00 ± 124.13, P = 0.002; Egr1 13.21 ± 1.28 vs. 26.63 ± 1.02, P = 0.001; Hmox1 3.28 ± 0.31 vs. 166.54 ± 35.03, P = 0.002; Fos 4.36 ± 1.03 vs. 14.90 ± 3.44, P < 0.001; Jun 6.62 ± 1.93 vs. 15.07 ± 2.09, P = 0.005; respectively). Increases in alpha-2-macroglobulin (A2m), heat shock 70kD protein 1A (Hspa1a), erythropoietin (Epo), and interleukin-6 (Il6) occurred at 3 h in shocked rats and were prevented by Ro27-3225 treatment (A2m 6.90 ± 0.82 vs. 36.73 ± 4.00, P < 0.001; Hspa1a 10.34 ± 3.28 vs. 25.72 ± 3.64, P = 0.001; Epo 0.49 ± 0.13 vs. 2.37 ± 0.73, P = 0.002; Il6 1.05 ± 0.15 vs. 1.88 ± 0.23, P < 0.001; respectively). Further, at 3 h in shocked rats treated with Ro27-3225 there were significant increases in tight junction protein 1 (Tjp1; 27.30 ± 2.43 vs. 5.03 ± 1.68, P < 0.001) and nuclear receptor subfamily 4, group A, member 1 (Nr4a1; 91.03 ± 16.20 vs. 30.43 ± 11.0, P = 0.01) relative to sham animals. Treatment with Ro27-3225 rapidly restored blood pressure, hemogasanalysis parameters, and lactate blood levels. Conclusions: Melanocortin treatment significantly prevents most of the systemic and hepatic detrimental changes induced by hemorrhage.

Production and effects of alpha-melanocyte-stimulating hormone during acute lung injury.

&agr;-Melanocyte-stimulating hormone (&agr;-MSH) is a peptide with broad anti-inflammatory effects. The present research was designed to determine production and effects of &agr;-MSH in acute bleomycin-induced lung injury in rats. Intratracheal bleomycin instillation induced &agr;-MSH expression in lung infiltrating cells and a marked peptide increase in the circulation. In experiments on the therapeutic potential of &agr;-MSH on lung injury, we determined influences of the synthetic &agr;-MSH analogue [Nle4-dPhe7]-&agr;-MSH (NDP-&agr;-MSH) on pulmonary edema, circulating nitric oxide, and gene expression profile in lungs 8 and 24 h after bleomycin instillation. Three main gene categories, known to be involved in the development of acute lung injury, were explored: stress response, inflammation, and fluid homeostasis. Peptide treatment was associated with a significant reduction in interstitial edema, with a virtually normal wet/dry weight ratio. Several stress-related genes, which were either upregulated or reduced by bleomycin, were only marginally altered during NDP-&agr;-MSH treatment. NDP-&agr;-MSH prevented bleomycin-related transcriptional alterations in genes involved in lung fluid homeostasis, including upregulation of Na+/K+-transporting ATPase and epithelial sodium channels and downregulation of cystic fibrosis transmembrane conductance regulator. Bleomycin-induced expression of proinflammatory and profibrotic factors (interleukin 6, tumor necrosis factor-&agr;, transforming growth factor-&bgr;1, and inducible nitric oxide synthase) and chemokines (chemokine [C-C motif] ligand 2 and chemokine [C-C motif] ligand 5) was likewise significantly reduced by NDP-&agr;-MSH. In conclusion, treatment with the &agr;-MSH analogue NDP-&agr;-MSH greatly improved the clinical and molecular picture of bleomycin-induced lung injury. Treatment with &agr;-MSH-related agents can exert beneficial effects in acute lung injury.

Protective Effects of Melanocortins in Systemic Host Reactions

Systemic inflammatory reactions are pivotal in many disorders and have important secondary influences in many more. Although inflammation is initially useful to limit infection, it can also be detrimental and cause organ failure. Modulation of systemic reactions is important to restrict mediator release and limit cell activation that could cause harmful consequences. Experiments in which different models and treatments were used show that melanocortins reduce host responses such as fever, shock, reperfusion injury and allograft rejection. Melanocortin-derived peptides could be an effective treatment to prevent organ failure caused by excessive production of pro-inflammatory mediators. The degree of the modulatory effect exerted by melanocortins should be sufficient to reduce severity of systemic inflammation without impairing the host defense mechanisms.