Doctoral Seminar

1. Doctoral Seminar Title: Neuropeptide Y (NPY) and The Immune System Presented by: M. Sh. Mojadadi Advisor: Dr. A. Zavaran

2. Outline • Overview of the NPY family of peptides (NPY, PYY and PP) and their receptors • Biological functions of the NPY family of peptides • Distribution of NPY family receptors • NPY and the immune system (the innate and adaptive immunity) • NPY in immune disorders (Autoimmunity and Allergy)

3. The NPY family of peptides (NPY, PYY and PP) Overview of the NPY family of peptides (NPY, PYY and PP) and their receptors • NPY and its related peptides, peptide YY (PYY) and pancreatic polypeptide (PP), are a highly conserved family of genes and proteins. • Chromosomal location: NPY: 7, PYY and PP:17 • The NPY family of peptides consist in vertebrates of 36 amino acids. • All peptides have a carboxyterminal amide group. • These peptides regulate fundamental cellular processes of growth, metabolism and cell-cell communication.

4. The NPY family of peptides (NPY, PYY and PP) Overview of the NPY family of peptides (NPY, PYY and PP) and their receptors • Pancreatic polypeptide (PP) was initially discovered in the chicken in 1975. • NPY and peptide YY (PYY) were initially discovered in the pig in 1982. • NPY occurs predominantly in the central nervous system and in peripheral nerves, primarily in blood vessels. • The most prominent effects of NPY in mammals are vasoconstriction, stimulation of appetite, and influence on circadian rhythm, pain, and the release of pituitary hormones. • PYY is primarily produced in gut endocrine cells and is released after meals contributing to inhibition of gut motility and pancreatic secretion. • PP is released from pancreatic F cells after meals and shares the effects of PYY by reducing pancreatic secretion and gut motility.

5. The NPY family of peptide receptors Overview of the NPY family of peptides (NPY, PYY and PP) and their receptors • Five Y receptor subtypes exist in most mammals: Y1, Y2, Y4, Y5 and Y6. • They belong to G-protein-coupled receptors family and seem to function primarily by inhibiting cAMP accumulation. • Chromosomal location: Y1, Y2 and Y5: 4, Y4: 10 and Y6: 5 • In the periphery, Y1 mediates vasoconstriction, and in the hypothalamus, along with Y5, it stimulates food intake. • In contrast to Y1 and Y5, the subtypes Y2 and Y4 inhibit food intake. • Subtype Y2 also stimulates angiogenesis.

6. Introduction Biological functions of the NPY family of peptides • Pancreatic polypeptide (PP) was the first member of the NPY peptide family to be discovered. • NPY was first isolated by Tatemoto and Mutt from pig brain. • The final member of these peptide family was isolated from preparations of pig intestine and called peptide YY (PYY). • While NPY is primarily localized to neurons and PP is found mainly in endocrine pancreas, PYY is found in both endocrine cells and neurons. • The hairpin-like three-dimensional structure has been called the PP-fold is present in all three peptides.

7. Introduction Biological functions of the NPY family of peptides • When comparing the amino acid sequences from a variety of mammals, only two of the 36 amino acids of NPY are variable. • On the other hand, the PYY sequence exhibits eight variable amino acids between different orders of mammals. • PP is the least conserved peptide of NPY family. • Despite the limited conservation observed in the PP and PYY amino acid sequences, the general three-dimensional structure is maintained across a wide variety of species indicating its potential importance in function.

8. NPY functions Biological functions of the NPY family of peptides • The NPY gene is located on human chromosome 7 at the locus 7p15.1. • It is composed of four exons and results in the synthesis of a 97 amino acid pre-pro NPY. • Prepro NPY is proteolytically processed into the C-terminal peptide of NPY (CPON) and NPY. • The C-terminal amidation of NPY is essential for its biological activity. • NPY is further processed to NPY3-36 and NPY2-36 by two enzymes, dipeptidyl peptidase IV (DPPIV) and aminopeptidase P, respectively.

9. NPY functions Biological functions of the NPY family of peptides • In the rat brain, NPY is found in numerous brain regions including the hypothalamus, amygdala, hippocampus, cerebral cortex. • The adrenal medulla is the primary source of circulating NPY. • NPY is abundant in peripheral neurons including sympathetic neurons where it is co-stored and co-released with norepinephrine (NE).

10. NPY functions Biological functions of the NPY family of peptides • The biological functions of NPY include: 1:The most potent orexigenic peptides known. when injected ICV it induces food intake with a preference for carbohydrate-rich food. 2: Other centrally mediated effects of NPY include decreased thermogenesis, anti-convulsant activity, anxiolysis and modulation of cognition. 3: NPY deficient mice were found to have an impaired development of neurons in the olfactory epithelium suggesting a role in neuronal development. 4: NPY is frequently co-localized with noradrenaline (NA) in sympathetic nerves and enhances NA-mediated vasoconstriction, especially upon strong stimulation. 5: NPY can act as an antinociceptive peptide, probably through inhibition of substance P release in the dorsal horn of the spinal cord.

11. PYY and PYY3-36 functions Biological functions of the NPY family of peptides • The human PYY gene are found 10 kb apart from PP gene on chromosome 17q21.1. • Like NPY, the PYY gene is organized with four exons and three introns. • The rat preproPYY sequence is 98 amino acids. • Like NPY, PYY is cleaved by dipeptidyl peptidase to the 3–36 fragment that is thought to be the principal active circulating peptide. • PYY is found primarily in the L cells of the gastrointestinal tract with the highest concentrations found in the rectum followed by the ileum and the colon.

12. PYY and PYY3-36 functions Biological functions of the NPY family of peptides • PYY is released in the gastrointestinal (GI) tract in response to meals. Its biological effects include: 1: Inhibition of gut motility 2: Inhibition of fluid and electrolyte secretion in the intestinal tract. 3: Inhibition of pancreatic secretion 4: vasoconstriction 5: Peripherally administered PYY3-36 decreases food intake in both human and rodents.

13. PP functions Biological functions of the NPY family of peptides • The human PP gene are found 10 kb apart from PYY gene on chromosome 17q21.1. • PP is almost exclusively expressed in endocrine pancreas and is released in response to meals. produces its biological effects mainly in the gastrointestinal tract, including: 1: Inhibition of pancreatic secretion 2: Inhibition of intestinal motility 3: Inhibition of ileum contractions 4: Inhibition of gastric emptying 5: Peripherally administered PP inhibits food intake.

14. Introduction Distribution of NPY family receptors • The human NPY-family peptides bind to four receptors, namely Y1, Y2, Y4 and Y5. • They all belong to the G-protein coupled receptor (GPCR) superfamily. • Some species including mouse have an additional receptor (y6) that is non-functional in humans and absent in rats. • NPY and PYY bind Y1, Y2 and Y5 with similar high affinities. • On the other hand, Y2 binds truncated versions of NPY and PYY such as PYY3-36 and NPY13-36 with similar affinity as the native peptides • PP binds to Y4 with higher affinity than NPY and PYY and has moderate affinity for Y5.

15. Y1 receptor Distribution of NPY family receptors • The Y1 receptor was the first NPY receptor to be cloned. • The gene for Y1 is located in a cluster together with Y2 and Y5 on human chromosome 4q31. • Full length NPY and PYY are potent agonists at Y1 whereas PP and C-terminally truncated NPY/PYY fragments display lower affinity. • The Y1 receptor is involved in the feeding response of NPY. • Most of the vascular effects of NPY and PYY are transduced via the Y1 receptor. • Antinociception of NPY through inhibition of substance P release are mediated by Y1.

16. Y1 receptor Distribution of NPY family receptors • This first indication for the expression of the Y1 receptor on immune cells was further substantiated by De la Fuente et al (1993) who demonstrated binding sites for radiolabeled NPY on peritoneal murine macrophages. • A more detailed investigation on this matter was conducted by Petitto et al. • They successfully cloned a Y1 receptor from human splenic lymphocytes, which displayed a 100% sequence homology upon comparison to the Y1 receptor cloned from the frontal cortex. • The Y1 receptor for NPY is widely expressed on leukocytes, including T, B cells and APCs such as DCs and macrophages.

17. Y2 receptor Distribution of NPY family receptors • Originally, the Y2 receptor was identified using vascular preparations. • The Y2 receptor gene codes for a 381 amino acid protein that is located close to the Y1 gene on chromosome 4. • The Y2 receptor is mainly located presynaptically where it acts as an autoreceptor inhibiting further release of neurotransmitter. • Y2 specific agonists increase blood pressure. • In pig spleen, a Y2 specific agonist evoked potent vasoconstriction. • The Y2 receptor is important in the feeding response to NPY.

18. Y2 receptor Distribution of NPY family receptors • The Y2 receptor is expressed on the surface membrane of neutrophils. • Nave et al. have found that NPY enhances the adherence of rat peritoneal macrophages activated with LPS in vitro and in vivo, with NPY Y2 receptor mediating this effect. • Parallel activation of both the Y1 and the Y2 receptor modulates phagocytosis of gram-negative bacteria.

19. Y4 receptor Distribution of NPY family receptors • The third Y receptor to be identified by cloning had high affinity for PP. • The most interesting feature of the Y4 receptor may be the low degree of sequence identity between species (only 75% homology when comparing the rat and human sequences). • Many of the GI effects produced by PP, like rabbit ileum contractions are mediated by Y4 receptor. • Y4 agonism (PP) may serve as a potential inhibitory signal on food intake.

20. Y5 receptor Distribution of NPY family receptors • the Y5 receptor is highly conserved (88–90%) between orders of mammals. • The Y5 receptor mediates the NPY feeding effects. • Other effect of NPY that is mediated by the Y5 receptor are reproduction through inhibition of luteinizing hormone release. • The Y5 receptor is expressed on the surface membrane of neutrophils. • The production and secretion of highly reactive oxygen species from neutrophil can be inhibited by activating the Y5 receptor.

21. Introduction NPY and the immune system • It is now well established that the immune system and the nervous system communicate functionally with each other. • A major pathway for this bidirectional interaction between the nervous and the immune system is provided by the sympathetic nervous system (SNS). • Although earlier studies focused mostly on the catecholamines, more recent work has established that other sympathetic transmitters, such as neuropeptide Y (NPY), also play a significant role within the neuroimmune crosstalk.

22. NPY containing nerves in lymphoid organs NPY and the immune system • For role of NPY in neuroimmune crosstalk it is necessary the presence of NPY containing nerve fibers in primary and secondary lymphoid organs from which NPY can be released within the local immunological microenvironment. • The presence of sympathetic fibers innervating lymphoid organs is well documented. • Several of these not only contain catecholamines, but also store NPY. • NPY+ sympathetic nerves supply lymph nodes in a variety of mammalian species (guinea pig, rat, cat, pig, mouse and human). • NPY+ nerves also innervate other important lymphoid organs, such as the thymus and the bone marrow.

23. NPY containing nerves in lymphoid organs NPY and the immune system • Electron microscopy studies revealed that NPY+ nerve terminals form close contacts with lymphocytes and macrophages in the marginal zone of the rat spleen. • Thus, the presence of NPY containing fibers in lymphoid organs and the release of NPY within the local microenvironment allows for a direct interaction between NPY and immune cells.

24. NPY receptors in lymphoid organs NPY and the immune system • If NPY truly functions as a transmitter in the neuroimmune crosstalk, leukocytes should be equipped with functional NPY receptors. • Initial investigations have revealed a high density of the Y1 receptor for NPY on immune cells of different species. • Petitto et al. have cloned a Y1 receptor for NPY from human splenic lymphocytes. • Furthermore the Y1 receptor for NPY is widely expressed on leukocytes, including T, B cells and APCs such as DCs and macrophages. • Thus, the presence of NPY containing fibers in lymphoid organs and its receptors on various immune cells provides a direct communication between NPY and the immune system.

25. NPY and the innate immunity NPY and the innate immunity • Immunity—the state of protection from infectious disease —has both a less specific and more specific component. • The less specific component, innate immunity, provides the first line of defense against infection. • Phagocytic cells, such as macrophages and neutrophils, NK cells and a variety of antimicrobial compounds synthesized by the host all play important roles in innate immunity. • It has been shown that NPY could affect some functions of innate immunity such as phagocytosis, NK cell activity and cytokine release.

26. NPY and the innate immunity NPY and the innate immunity • Immunity—the state of protection from infectious disease —has both a less specific and more specific component. • The less specific component, innate immunity, provides the first line of defense against infection. • Phagocytic cells, such as macrophages and neutrophils, NK cells and a variety of antimicrobial compounds synthesized by the host all play important roles in innate immunity. • It has been shown that NPY could affect some functions of innate immunity such as phagocytosis, NK cell activity and cytokine release.

27. Phagocytosis NPY and the innate immunity • Phagocytosis of extracellular particles and microorganisms represents a major function of monocytes and neutrophils. • For this function, phagocytic cells carry out the phagocytic process. This process involves several functions such as adherence to tissues, mobility to the infectious focus (chemotaxis), ingestion of the foreign material (phagocytosis) and digestion and destruction of this ingested material by respiratory burst. • There is convincing evidence that NPY profoundly interferes with phagocytosis of both monocytes and neutrophils.

28. Adherence NPY and the innate immunity • The first step of the phagocytic process involves the adherence of phagocytic cells to tissue substrate before migration to the site of inflammation. • Nave et al. have found that NPY enhances the adherence of rat peritoneal macrophages activated with LPS in vitro and in vivo. • Furthermore NPY Y2 receptor mediates this effect.

29. Chemotaxis NPY and the innate immunity • Phagocytes show great mobility, both spontaneous and directed towards a chemical gradient (chemotaxis) produced from an infectious focus. • NPY profoundly alters chemotaxis, a critical step of phagocytic cells, directly, and additionally modulates the outcome of other chemotactic stimuli. • It has been shown that NPY increases spontaneous mobility and chemotaxis of peritoneal murine macrophages. • On the other hand, NPY inhibits the fMLP-induced chemotaxis of human monocytes.

30. Ingestion NPY and the innate immunity • The phagocytic capacity of macrophages and neutrophils are the most representative of these cells. • There is convincing evidence that NPY profoundly interferes with phagocytosis of both monocytes and neutrophils. • Notably, the effect of NPY on phagocytosis by monocytes depends on the type of microorganism studied. • For example NPY increases phagocytosis of Candida albicans, whereas inhibits engulfment of Leishmania major.

31. Ingestion NPY and the innate immunity • NPY also potently modulates the ability of neutrophils to phagocytoze gram-negative bacteria. • Studies using fluorescence-labeled Escherichia coli on human neutrophil phagocytosis showed that NPY itself modulated phagocytosis in a bimodal manner. • When stimulated with low concentrations, NPY significantly inhibited phagocytosis of E. coli. However, with increasing concentrations of NPY this effect changed, eventually resulting in a significant enhancement of phagocytosis. • Furthermore The results demonstrated that NPY-mediated modulation of phagocytosis is mediated via the Y5 receptor subtype.

32. Respiratory burst NPY and the innate immunity • A remarkable feature of the host defense apparatus is its ability to produce highly reactive oxidizing reagents, including oxidizing radicals and singlet oxygen. • The importance of this so called “respiratory burst (RB)” became obvious when the syndrome chronic granulomatous disease (CGD), characterized by predisposition to bacterial and fungal infections, was shown to be associated with decreased oxygen consumption. • It has been shown that mediators of the SNS are the potent modulators of the RB. • Studies show whereas NPY has no influence on the respiratory burst of resting murine peritoneal monocytes, the stimulation of the respiratory burst resulting from activation of phagocytosis is directly increased by NPY.

33. Respiratory burst NPY and the innate immunity • Moreover studies have been shown that the RB of rat peritoneal macrophages induced PMA stimulation is potentiated not only by NPY, but also by NPY13-36, a selective Y2 receptor agonist. • This supports the role of Y2 receptors in PMA-induced RB.

34. Summary NPY and the innate immunity

35. NK cell activity NPY and the innate immunity • NK cells control host response to viruses and tumors. • NK cell activity is modulated, among several other mechanisms, by physical and psychological stress, such as exercise or mood disorders. • Most likely these alterations are mediated via the SNS. • Interestingly, a growing number of studies indicate that sympathetic control on NK cell activity also involves NPY.

36. NK cell activity NPY and the innate immunity • The first detailed study on the association of NPY levels and NK activity was performed in patients undergoing bereavement or other severely threatening life events. • It was found that the decrease in NK activity resulting from such events is inversely correlated with NPY plasma levels. • Increased NPY levels result in decreased NK cell activity, and vice versa. • Moreover the peripheral distribution of NK cells is also modulated by NPY. • It has been shown that intravenous application of NPY dose-dependently alters NK cell distribution in the blood.

37. Mast cells NPY and the innate immunity • Another innate branch of the immune system is formed by mast cells. • It has been shown that NPY can induce releasing of histamine from murine connective tissue mast cells in receptor-independent manner. • In contrast NPY has no effect on human mucosa mast cells.

38. NPY and the adaptive immunity NPY and the adaptive immunity • The induction of an adaptive immune response is a complex process involving the coordination of a number cell types and molecules. • Macrophages and dendritic cells (DCs) act as APCs, taking up antigen and presenting it to naive CD8+ and CD4+ T cells respectively. which then differentiate into antigen-specific effector cells. • Newly activated T cells can differentiate into T helper (Th) 1 or 2 cells, depending on the cytokine environment. • The interaction between T cells and APCs is a fundamental step in the induction of an adaptive immune response. • It has been shown that NPY can modulate components of the adaptive immune system.

39. Th1/Th2 balance NPY and the adaptive immunity • The differentiation into Th1 or Th2 cells is a crucial step in the development of immune responses and diseases, such as autoimmunity or allergy. • Using lymhocytes isolated from BALB/c spleens, it was demonstrated that NPY elevates IL-4 production and decreases IFN-γ production upon stimulation with a plate-bound anti-CD 3 antibody. • To confirm these findings, several different Th1 and Th2 clones were stimulated with their specific antigens in the presence or absence of NPY. • These results showed that NPY suppresses differentiated Th1 cells in their production of IFN-γ and stimulates the production of IL-4 by Th2 cells. • Thus, a typical Th2 shift is induced by NPY in vitro.

40. Th1/Th2 balance NPY and the adaptive immunity • On the other hand, intraperitoneal application of NPY significantly inhibits the exvivo production of IFN-γ in antigen-specific murine lymphocytes. • The induction of a Th2 shift in vivo is further substantiated by the observation that NPY also selectively inhibits the production of antigen-specific IgG2a.

41. A bimodal role for neuropeptide Y1 receptor NPY and the adaptive immunity • Two studies have described a novel role for NPY via its Y1 receptor in regulating the induction Th1 responses. • In the first study, treatment of mice with NPY or an agonist to the Y1 receptor suppressed experimental autoimmune encephalomyelitis (EAE), a Th1 T cell-driven autoimmunity model. Suggesting suppressive role for NPY, via signaling through its Y1 receptor on T cells. • The second study using another Th1-mediated model of inflammatory colitis, the dextran sodium salt (DSS)-induced colitis. In this model, Y1-deficient mice or those treated with a Y1 receptor antagonist were protected against weight loss and disease activity induced by DSS. Suggesting this time that an absence of Y1 receptor signaling has a protective effect in this model.

42. A bimodal role for neuropeptide Y1 receptor NPY and the adaptive immunity • Why Y1 signaling protected in some Th1-mediated settings and yet participated in inflammation in some others? • Julie Wheway et al. using Y1 deficient mice (Y1-/-) answered to this question. • They found that Y1-/- mice were resistant against DSS-induced colitis. • In this case, the data suggests that Y1 is an important inflammatory mediator. • On the other hand, Both male and female C57/B6 WT mice injected daily with 50 mg/kg NPY were protected against DTH compared to the PBS-treated control mice. • In this case the result suggests that NPY signaling on T cells inhibits the Th1 T cell response that drives DTH.

43. A bimodal role for neuropeptide Y1 receptor NPY and the adaptive immunity • To examine which defective immune cell type contributed to the reduction in Th1-mediated inflammation in Y1-/- mice, they decided to examine Y1-/- T cells and APCs separately. • The results showed: 1: Y1-/- T cells were hyper-responsive to activation and normally differentiated into Th1 cells in vitro and in vivo. 2: Y1-/- APCs were functionally impaired.

44. A bimodal role for neuropeptide Y1 receptor NPY and the adaptive immunity

45. B cells NPY and the adaptive immunity • Intravenous infusion of a low-dose of NPY results in a significant decrease in the number of circulating IgM+ B cells. • Moreover this work revealed the dose-dependent mobilization of a previously ndetected • B cell subpopulation in the rat, referred to as B-1-like B cells. • Intraperitoneal application of NPY in rats results in a dose-dependent inhibition of IgM and IgG antibody responses against keyhole limpet hemocyanine (KLH).

46. Direct antimicrobial activity of NPY Direct antimicrobi alactivity of NPY • In vitro, NPY has potent antifungal activity against C. albicans, C. neoformans and Arthroderma simii and probably has activity against Gram-negative and Gram-positive microbes. • The expression of NPY by non-neuronal cells, sustentacular cells and olfactory ensheathing cells may create a local antimicrobial barrier protecting the projecting axons, discouraging microbial invasion along the axonal tract and suppressing a need for the assistance of tissue destructive inflammatory cells in the clearance of microbes.

47. Direct antimicrobial activity of NPY Direct antimicrobi alactivity of NPY

48. Autoimmunity NPY in immune disorders: Autoimmunity • An important feature of T cells is their ability to develop into distinct subtypes that are characterized by specific cytokine patterns. • The Th1 phenotype predominantly secretes cytokines, such as IL-2 and IFN-γ, whereas the production of IL-4 is an important feature of T cells biased towards a Th2 subtype. • This Th1/Th2 balance is of critical significance for autoimmune disorders, such as multiple sclerosis (MS), and systemic lupus erythematosus (SLE). • In MS Th1 cells predominate and in SLE a Th2 bias is present. • It has been shown that NPY can modulate Th1/Th2 balance in some autoimmune disorders.

49. Multiple Sclerosis NPY in immune disorders: Autoimmunity • In MS, a disease characterized by the destruction of myelin sheaths, impaired sympathetic functions are indicated both experimentally and clinically. • The course of EAE, an animal model for MS, is much more severe when sympathetic nerves are chemically disrupted by the application of 6-hydroxydopamine (6-OHDA). • Conversely, clinical signs and the pathology of EAE can be significantly reduced by treating the animals with β-adrenergic agonists. • In support of the concept that the sympathetic influence on the immune system is alleviated, decreased concentrations of NPY in the cerebrospinal fluid and plasma of MS patients have been documented.

50. Multiple Sclerosis NPY in immune disorders: Autoimmunity • Moreover it has been shown that administration of exogenous NPY can suppress actively and also passively-induced EAE in a dose-dependent manner. • Pharmacological assessment of the involved receptor revealed that the suppression is due to a Y1 receptor-mediated induction of a Th2 shift of autoreactive lymphocytes in vivo.