Modulatory Functions of  -MSH Neuropeptide in the Immune System

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

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

3. 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)

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

5. 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)

6. 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)

7. POMC • Pro-opiomelanocortin • 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)

8. 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

9. -MSH • Alpha-Melanocyte Stimulating Hormone • 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)

10. -MSH Receptors • Melanocortin Receptors (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)

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

12. -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

13. 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)

14. Melanin Storage • Keratinocytes store the melanin granules • Melanocytes transfer melanin granules via dendrites

15. 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 (Kan et al, J. Biological Chemistry 2008) (Cerenius et al., Trends in Immunology 2008)

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

17. Experimental Findings

18. POMC, the ACTH/melanocortin precursor, is secreted by human epidermal keratinocytes and melanocytes and stimulates melanogenesisRousseau 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 Increasing [POMC] 85% 63% 23% POMC stimulated cell pellets

19. Human melanocytes express functional Toll-Like Receptor 4Ahn et al., Experimental Dermatology 2008 Lane 1: G3PDH Lane 2: TLR4 Lane 3: CD14 Lane 4: MyD88 • LPS stimulated melanin granule production resulting in increased melanin pigmentation • Microbial induced melanogenesis may be activated via TLR4 • 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

20. Lane 1: blank Lane 2: HP Lane 3: Astrocytes Lane 4: Liver Lane 1: HP Lane 2: Astrocytes Lane 3: Liver Lane 4: (-) control Activation of MC4R reduces the inflammatory response and prevents apoptosis induced by LPS and IFN-γCaruso 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

21. Antimicrobial effects of -MSH peptidesCutuli 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

22. Anti-microbial action of melanocortin peptides and identification of a novel X-Pro-D/L-Val sequence in Gram-positive and Gram-negative bacteriaCharnley 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

23. -MSH reduces the internalization of S. aureus and down-regulates HSP 70, integrins and cytokine expression in human keratinocyte cell linesDonnarumma 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 TNF-α 2hr 24hr IL-8 ICAM-1 Ctrl PA PA + α-MSH LTA LTA + α-MSH Ctrl S.a. (S.a + α-MSH)

24. The immunomodulating neuropeptide -MSH suppresses LPS-stimulated TLR4 with IRAK-M in macrophagesTaylor, 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

25. Innate immunity in insects: surface-associated dopa decarboxylase-dependent pathways regulate phagocytosis, nodulation and melanization in medfly haemocytesSideri 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 Anti-Ddc benserazide carbidopa Anti- pro-PO + dopamine Anti- pro-PO + dopa Anti-proPO

26. Conclusions

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

28. 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

29. 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

30. 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

31. 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 • www.wikipedia.com

32. The End…Questions???