Alpha-MSH SF Feb 18.ppt

Modulatory Functions of  -MSH Neuropeptide in the Immune System Saray Felix Department of Biological Sciences- California State University Los Angeles Winter 2009

Evolution
Prokaryotes
Eubacteria (true bacteria)
Archaeabacteria
Protists
Eukaryotes
Fungi
Plants
Invertebrates and Arthropods
Vertebrates
(Murphy, K. Janeway’s Immunobiology 2008) www.bordalierinstitute.com/.../evolutionTree.gif

Evolution of the Immune System
Ubiquitous Immune System
Every organism is attacked by pathogens
Response Mechanism
Provides protection against invading pathogens
Similar strategies across phyla with diversity in response
biology.creighton.edu/courses/BIO432/ Evolution %2005.htm (Murphy, K. Janeway’s Immunobiology 2008)

Evolution of the Innate Immune System www.nature.com/.../n6/images/ni0607-547-F1.jpg (Litman and Cooper, Nature Immunology 2007) Fly Snail Mollusk Amphioxus Lamprey Teleost Chicken Mouse/Human

Evolution of Innate Immunity
Phagocytosis
Not observed in plants
Many invertebrates and all vertebrates
Antimicrobial Peptides
Defensins- in many multicellular organisms including plants
Differ in structural detail
Pattern Recognition Receptors
Toll homologs found in many species- from plants to mammals
Complement System
Not observed in plants
Many invertebrates and vertebrates
biology.creighton.edu/courses/BIO432/ Evolution %2005.htm (Murphy, K. Janeway’s Immunobiology 2008)

First Line of Defense: Skin
Skin acts as a barrier for pathogens
Epidermal cells contain necessary immune cells to activate a response against pathogens
α -MSH and skin
Express and secrete α -MSH
(Maaser et al., N.Y. Acad. Sci. 2006)

POMC
P ro- o pio m elano c ortin
Precursor Polypeptide
Contains 241 aa residues
Undergoes post-translational cleavage
Proteases
Subtilisin enzymes- a pro-hormone convertase (PC) that belongs to the Serine protease group
Tissue-specific
(Bohm et al J. Invest. Dermatology 2006)

POMC Production
It can be synthesized by:
Corticotroph cells in the anterior pituitary gland
Melanotrope cells in the intermediate lobe of the pituitary gland
Most Neurons in the arcuate nucleus or the hypothalamus
Few neurons in the dorsomedial hypothalamus and in the brainstem
Melanocytes in the skin

 -MSH
Alpha- M elanocyte S timulating H ormone
Neuropeptide- 13 amino acid peptide with the tripeptide 11-13 (KPV) aa containing the “active” sequence
A hormone secreted by the anterior lobe of the pituitary gland that regulates skin color in humans and other vertebrates by stimulating melanin synthesis in melanocytes and melanin granule dispersal in melanophores
Anti-inflammatory properties
Down-regulates pro-inflammatory cytokines (IL-1, IL-6, IL-8, TNF-α, etc.)
Up-regulates anti-inflammatory cytokines (IL-10, etc.
Anti-microbial properties
Stimulates melanin production to generate oxygen radicals
Indirectly stimulates stimulates immune function
Directly inhibits pathogen proliferation
(Maaser et al., N.Y. Acad. Sci. 2006)

 -MSH Receptors
M elano c ortin R eceptors (MCR): G-protein-linked receptors whose transduction
occurs via induction of adenylyl cyclase and an increase in cAMP
MC1R- highest α -MSH affinity
Key player in regulation of pigmentation in mammals and inflammation
Detected in melanocytes, neutrophils, monocytes, dendritic cells, endothelial cells, B-lymphocytes, glioma cells, and astrocytes
MC2R
Stimulates secretion of adrenal steroids
Detected in adrenal cortex, skin, murine adipocytes
MC3R
Activation effects are related to feeding and energy homeostasis
Expressed in the CNS, gastro-intestinal system (stomack, duodenum, pancreas), and kidneys
MC4R
Activation effects are related to feeding behavior, energy homeostasis, erectile function, inflammation, and neuroprotection
Expressed primarily in CNS (hypothalamus, vagus nerve, basal ganglia, hippocampus, and cerebral cortex
MC5R
Might mediate stimulation of the production of pheromones, influencing sexual behavior
Expressed in exocrine glands (sebaceous, Harderian, lacrimal, and preputial glands)
(Lasaga et al., Peptides 2008)

MelanoCortin Receptors
Receptors for  -MSH in the skin can be found on:
Epidermal melanocytes and keratinocytes
Endothelial cells
Mast cells
Adipocytes
(Bohm et al. J. Invest. Dermatology 2006)
Fibroblasts
Dermal Papilla cells
Sebocytes

 -MSH and Melanin
Stimulates Melanocytes via MC1R
 -MSH increases cAMP and PKA in melanocytes
In turn increases PKC and Tyrosinase mRNA synthesis
PKC activates Tyrosinase in the melanosome (lysosome related organelles in melanocytes enriched with tyrosinase proteins)
Tyrosine is synthesized into Melanin
bric.postech.ac.kr/…/images/kcib-5-3.jpg

Melanogenesis
Tyrosine, DOPA or L-DOPA are Melanin precursors
End products are Eumelanins (brown and black pigments), Pheomelanins (red and light colored pigments) and O2 radicals
Melanins
Bind metal ions and organic cations to efficiently scavenge for free reactive radicals
Synthesized by organisms representative of all biological kingdoms
(MacKintosh, J. Theoretical Biology 2001)

Melanin Storage
Keratinocytes store the melanin granules
Melanocytes transfer melanin granules via dendrites

Invertebrate Melanin
Chromatophores
Pigment containing and light-reflecting cells
Melanin: negatively charged hydrophobic pigments
Synthesized by the pro-Phenoloxidase (pro-PO) pathway activation system activated upon microbe infection
Dopa decarboxylase (Ddc) coverts dopa to dopamine
Involved in wound healing, parasite defense, cuticle hardening, and melanization
Phenoloxidase (PO) catalyses several steps of the eumelanin pathway
last component of the pro-PO activation system that ultimately results in melanin production
May be involved in blood clotting (activity may strengthen protein-protein cross-links)
Acts as anti-microbial via direct killing
(Cerenius et al., Trends in Immunology 2008) (Kan et al, J. Biological Chemistry 2008)

Hypothesis
 -MSH is a melanin activator with innate immune functions in vertebrates and invertebrates

Experimental Findings

POMC, the ACTH/melanocortin precursor, is secreted by human epidermal keratinocytes and melanocytes and stimulates melanogenesis Rousseau et al, FASEB Journal 2007
(A) POMC and derivative peptides induce melanogenesis
 -MSH is most potent stimulator molecule
(B & C) Higher levels of POMC are needed to stimulate melanogenesis
(D) POMC stimulated pigment cell dendricity in a dose-dependent manner
85% 63% 23% Increasing [POMC] POMC stimulated cell pellets

Human melanocytes express functional Toll-Like Receptor 4 Ahn et al., Experimental Dermatology 2008
RT-PCR analysis demonstrate that human melanocytes express TLR4, CD14, and MyD88 mRNA
LPS treatment induced upregulation of TLR4 and MyD88 mRNA in human melanocytes
LPS stimulated melanin granule production resulting in increased melanin pigmentation
Microbial induced melanogenesis may be activated via TLR4
Lane 1: G3PDH Lane 2: TLR4 Lane 3: CD14 Lane 4: MyD88

Activation of MC4R reduces the inflammatory response and prevents apoptosis induced by LPS and IFN- γ Caruso et al., Endocrinology 2007
RT-PCR determined that MC4R RNA but not MC3R RNA is detected in astrocytes
Western blotting detected MC4R protein in astrocytes
 -MSH attenuated the stimulatory effects of LPS/IFN- γ on iNOS mRNA and protein expression
MC4R is involved in the anti-inflammatory effect of  -MSH in astroglial cells
Lane 1: blank Lane 2: HP Lane 3: Astrocytes Lane 4: Liver Lane 1: HP Lane 2: Astrocytes Lane 3: Liver Lane 4: (-) control

Antimicrobial effects of  -MSH peptides Cutuli et al., J. Leukocyte Biology 2000
(1)  -MSH peptides, at various concentrations, inhibited S. aureus colony formation
(2)  -MSH peptides had inhibitory effects on C. albicans viability at somewhat higher concentrations
 -MSH peptides have anti-microbial effects on gram-positive (S.a.) bacteria and fungus (C.a.)
Fig. 1 Fig. 2

Anti-microbial action of melanocortin peptides and identification of a novel X-Pro-D/L-Val sequence in Gram-positive and Gram-negative bacteria Charnley et al., Peptide 2008
 -MSH peptide 11-13 containing the Lysine-Proline-Valine sequence as well as the Alanine-Proline-Valine sequence decreased microbe viability in a dose-dependent manner
 -MSH peptide sequence 1-13 and 11-13 possess anti-microbial effects
There was no significant differences between the peptides
Control peptide: AAA KPV peptide APV peptide

 -MSH reduces the internalization of S. aureus and down-regulates HSP 70, integrins and cytokine expression in human keratinocyte cell lines Donnarumma et al., Experimental Dermatology 2004
S. aureus , protein A (PA), and lipoteichoic acid (LTA) induced the release of TNF- α , IL-8, and ICAM-1 from human keratinocytes
 -MSH pre-incubated human keratinocytes strongly down-regulated TNF- α , IL-8, and ICAM-1 release
 -MSH lowers pro-inflammatory molecules in LTA and PA stimulated keratinocytes
 -MSH has protective roles at the cutaneous level by reducing infection and inflammatory process
Ctrl S.a. (S.a + α -MSH) TNF- α IL-8 ICAM-1 2hr Ctrl PA PA + α -MSH LTA LTA + α -MSH 24hr

The immunomodulating neuropeptide  -MSH suppresses LPS-stimulated TLR4 with IRAK-M in macrophages Taylor, J. Neuroimmunology 2005
APC spleen cells incubated with LPS-contaminated ovalbumin enhanced IFN- γ production
APC cells incubated with S.a. ovalbumin were limited in stimulating a strong IFN- γ production
LPS-stimulated APC cells pre-incubated with  -MSH could not enhance IFN- γ production
S.a-stimulated APC cells pre-incubated with  -MSH did not change nor enhaced the IFN- γ production
 -MSH is limited to antagonizing a TLR4-associated response in APC

Innate immunity in insects: surface-associated dopa decarboxylase-dependent pathways regulate phagocytosis, nodulation and melanization in medfly haemocytes Sideri et al., Immunology 2007
(b and c) Nodulation (aggregates) and melanization is evident after 10+ mins of bacterial infection in medfly haemocytes
(d- g) No aggregates and reduced melanization resulted in haemocytes pre-incubated with Ddc inhibitors (benserazide, carbidopa, anti-Ddc, or anti-proPO)
(h and i) haemocytes pre-incubated with anti-proPO and then with either dopa or dopamine failed to induce melanization but formed small nodules
PO and Ddc activities are key regulatory molecules of haemocyte nodulation and melanization in presence of bacteria
0 min E.coli infection 10 min E.coli 60 min E.coli benserazide carbidopa Anti-Ddc Anti-proPO Anti- pro-PO + dopa Anti- pro-PO + dopamine

Conclusions

 -MSH is a melanin activator with innate immune functions in vertebrates and invertebrates Melanin as antimicrobial was not tested thoroughly No No PO and Ddc are key for melanin production during microbe invasion Sideri et al. None N/A Yes MSH inhibited TLR4 activity on pro-inflmmatory cytokines Taylor et al. None N/A Yes MSH down-regulated pro-inflammatory cytokines in human keratinocytes Donnarumma et al. No cell lineages used (only bacterial cells) N/A Yes  -MSH peptides 11-13 possess anti-microbial properties Charnley et al. One cell lineage used at end of experiments N/A Yes α -MSH directly inhibited S.a. colony formation and C.a. tube formation Cutuli et al. No future experiments stated N/A Yes α -MSH inhibits pro-inflammatory effects (LPS induced) in astrocytes Caruso et al. Melanin production was not well discussed Yes Yes Melanin granule production resulted in LPS-stimulted melanocytes Ahn et al. Various cell lines were used under differing treatments Yes N/A POMC stimulates melanogenesis; α -MSH is a potent melanin stimulator molecule Rousseau et al. Critique Hypothesis Supported Key Findings Author

Follow-Up Experiments
Melanocytes and/or keratinocytes lacking all melanin pre-cursors
Infect  -MSH pretreated cells with S. aureus
Test for microbe inhibition/killing
Examines role of melanin granule/production in microbe host defense
BALB/c (immuno-deficient) mice
treated and untreated with varying concentrations of  -MSH
Injected with S. aureus or PBS (control)
Tested for immune response and/or microbe inhibition
Investigates whether  -MSH anti-microbial properties enhance immune response
BALB/c mice with POMC-KO genes
Treated and untreated with  -MSH at various concentrations
Injected with S. aureus or control
Testes for immune response and/or microbe killing
Analyses whether  -MSH treatment is effective drug therapy

Take Home Message
 -MSH can be processed (from POMC) in many tissues and various cells
 -MSH neuropeptide is an immunomodulator in the innate immune response
Down-regulates pro-inflammatory cytokines via up-regulation of anti-inflammatory cytokines
Anti-microbial properties can be direct (kills microbe) or indirect (enhances immune response)
 -MSH activates melanogenesis in skin cells
Melanin granules may have anti-microbial properties in vertebrates
Melanin synthesis is key molecule in invertebrate host defense against invading pathogens

References
Ahn, J.H., Park, T.J., Jin, S.H., Kang, H.J. (2008) Human melanocytes express functional Toll-like receptor 4. Experimental Dermatology 17, 412-417
Bohm, M., Luger, T.A., Tobin, D.J., Garcia-Borron, J.C. (2006) Melanocortin Receptor Ligands: New Horizons for Skin Biology and Clinical Dermatology. Journal of Investigative Dermatology 126, 1966-1975
Caruso, C., Durand, D., Schioth, H.B., Rey, R., Seilicovich, A., Lasaga, M. (2007) Melanocortin 4 Receptors Reduces the Inflammatory Response and Prevents Apoptosis Induced by Lipopolysaccharide and Interferon-  in Astrocytes. Endocrinology 148, 4918-4926
Cerenius, L., Lee, B.L., Soderhall, K. (2008) The proPO system: pros and cons for its role in invertebrate immunity. Trends in Immunology 29, 263-271
Charnley, M., Moir, A.J.G., Douglas, C.W.I., Haycock, J.W., (2008) Anti-microbial action of melanocortin peptides and identification of a novel X-Pro-D/L-Val sequence in Gram-positive and Gram-negative bacteria. Peptides 29, 1004-1009
Cutuli, M., Cristiani, S., Lipton, J.M., Catania, A., (2000) Antimicrobial effects of  -MSH peptides. Journal of Leukocyte Biology 67, 233-239
Donnarumma, G., Paoletti, I., Buommino, E., Tufano, M.A., Baroni, A. (2004)  -MSH reduces the internalization of Staphylococcus aureus and down-regulates HSP 70, integrins and cytokine expression in human keratinocyte cell lines. Experimental Dermatology 13, 748-754
Hillyer, J.F., Christensen, B.M. (2005) Mosquito Phenoloxidase and Defensin Colocalize in Melanization Innate Immune Responses. Journal of Histochemistry and Cytochemistry 53, 689-698
Kan, H., Kim, C., Kwon, H., Park, J., Roh, K., Lee, H., Park, B., Zhang, R., Zhang, J., Soderhall, K., Ha, N., Lee, B.L. (2008) Molecular Control of Phenoloxidase-induced Melanin Synthesis in an Insect. Journal of Biological Chemistry 283, 25316-25323
Lasaga, M., Debeljuk, L., Durand, D., Scimonelli, T.N., Caruso, C. (2008) Role of  -melanocyte stimulating hormone and melanocortin 4 receptor in brain inflammation. Peptides 29, 1825-1835
Litman, G.W., Cooper, M.D. (2007) Why study the evolution of immunity? Nature Immunology 8, 547-548
Maaser, C., Kannengiesser, K., Kucharzik, T. (2006) Role of the Melanocortin System in Inflammation. Annals New York Academy of Science 1072, 123-134

References Cont’d
Mackintosh, J.A. (2001) The Antimicrobial Properties of Melanocytes, Melanosomes, and Melanin and the Evolution of Black Skin. Journal of Theoretical Biology 211, 101-113
Nosanchuk, J.D., Casadevall, A. (2003) The contribution of melanin to microbial pathogenesis. Cellular Microbiology 5, 203-223
Popovich, P.G., Longbrake, E.E. (2008) Can the immune system be harnessed to repair the CNS? Nature Reviews Neuroscience 9. 481-493
Rosas, A.L., MacGill, R.S., Nosanchuk, J.D., Kozel, T.R., Casadevall, A. (2002) Activation of the Alternative Complement Pathway by Fungal Melanins. Clinical and Diagnostic Laboratory Immunology 9, 144-148
Rousseau, K., Kauser, S., Pritchard, L.E., Warhurst, A., Oliver, R.L., Slominski, A., Wei, E.T., Thody, A.J., Tobin, D.J., White, A. (2007) Proopiomelanocortin (POMC), the ACTH/melanocortin precursor, is secreted by human epidermal keratinocytes and melanocytes and stimulates melanogenesis. FASEB Journal 21, 1844-1856
Sideri, M., Tsakas, S., Markoutsa, E., Lampropoulou, M., Marmaras, V.J. (2008) Innate immunity in insects: surface-associated dopa decarboxylase-dependent pathways regulate phagocytosis, nodulation and melanization in medfly haemocytes. Immunology 123, 528- 537
Scholzen, T.E., Sunderkotter, C., Kalden, D.H., Brzoska, T., Fastrich, M., Fisbeck, T., Armstrong, C.A., Ansel, J.C., Luger, T.A. (2003) Alpha-Melanocyte Stimulating Hormone Prevents Lipopolysaccharide-Induced Vasculitis by Down-Regulating Endothelial Cell Adhesion Molecule Expression. Endocrinology 144, 360-370
Tang, H., Kambris, Z., Lemaitre, B., Hashimoto, C. (2006) Two Proteases Defining a Melanization Cascade in the Immune System of Drosophila. Journal of Biological Chemistry 281, 28097-28104
Tang, H., Kambris, Z., Lemaitre, B., Hashimoto, C. (2008) A Serpin that Regulates Immune Melanization in the Respiratory System of Drosophila. Developmental Cell 15, 617-626
Taylor, A.W. (2005) The immunomodulating neuropeptide alpha-melanocyte-stimlating hormone (alpha-MSH) suppresses LPS-stimulated TLR4 with IRAK-M in macrophages. Journal of Neuroimmunology 162, 43-50
www.copewithcytokines.org
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Alpha-MSH SF Feb 18.ppt

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