The Intriguing Role of Neuropeptides at the Ocular Surface

Accepted Manuscript The intriguing role of neuropeptides at the ocular surface Francesco Sabatino, MD, Antonio Di Zazzo, MD, Luca De Simone, MD, Stefano Bonini, MD PII:

S1542-0124(16)30215-4

DOI:

10.1016/j.jtos.2016.10.003

Reference:

JTOS 206

To appear in:

Ocular Surface

Received Date: 4 March 2016 Revised Date:

5 October 2016

Accepted Date: 26 October 2016

Please cite this article as: Sabatino F, Di Zazzo A, De Simone L, Bonini S, The intriguing role of neuropeptides at the ocular surface, Ocular Surface (2016), doi: 10.1016/j.jtos.2016.10.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT SECTION: Laboratory Science, James V. Jester, PhD, Editor The intriguing role of neuropeptides at the ocular surface

Francesco Sabatino, MD,1 Antonio Di Zazzo, MD,2 Luca De Simone, MD,1 and Stefano Bonini,

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SHORT TITLE: PEPTIDES AT THE OCULAR SURFACE/Sabatino et al FOOTNOTES

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Accepted for publication October 2016.

From 1University Campus Bio-Medico of Rome, and 2IRCSS – GB Bietti Foundation, Rome, Italy.

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Financial support: None.

The authors have no commercial or proprietary interest in any concept or product discussed in this article.

Corresponding author: Stefano Bonini, Department of Ophthalmology, Campus Bio-Medico

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University of Rome, Via Alvaro del Portillo 21, 00128 Rome, Italy. Phone number: +3906

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225419185; Fax number: +3906225411456. Email: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT In recent decades, the role of neuropeptides in physiology and pathology has been elucidated. Various neuropeptides are expressed at the ocular surface, where they facilitate the crosstalk between immune and nervous systems. They actively regulate trophic and immune functions and orchestrate neuroinflammation. The purpose of this manuscript is to review the

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expression of the neuropeptides SP, VIP, CGRP, and NPY at the ocular surface, focusing on their

infection, dry eye, and allergic eye disease.

Outline

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KEY WORDS allergic eye disease, dry eye, neuropeptides

I.

Introduction

II.

Neural Expression of Neuropeptides

III.

Biochemistry of Neuropeptides and their Receptors Substance P

B.

Calcitonin Gene-related Peptide

C.

Vasoactive Intestinal Polypeptide

D.

Neuropeptide Y

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A.

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role in tropism and immunity, and to summarize their functions in ocular immune privilege,

Neuropeptides and their Trophic Functions

V.

Ocular Immune Privilege

VI.

Neuropeptides and their Immune Functions

VII.

Neuropeptides and Infection

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IV.

A.

Pseudomonas aeruginosa-induced Keratitis

B.

Herpes Simplex Virus

VIII.

Neuropeptides and Conjunctival Allergic Diseases

IX.

Neuropeptides and Dry Eye

X.

Future Directions

XI.

Conclusions

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I.

Introduction The term “neuropeptide” was first coined in the late 1960s by De Wied to describe

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neuroactive peptides.1 However, the first report of neuropeptidergic functions was earlier, in 1931, when von Euler and Gaddum described hypotension and bowel-spasm induced by a molecule that subsequently was called substance P.2 Later, a neuropeptide was defined as “a small proteinaceous

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substance produced and released by neurons through the regulated secretory route and acting on neural substrates.”3

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Neuropeptides orchestrate neuroinflammation by inducing vascular changes through a direct action on vascular endothelial and smooth muscle cells but also by activating innate (mast cells, dendritic cells) and adaptive immune cells (T lymphocytes).4-6 Tangling up the network, this neuroimmune interplay is bidirectional, given that immune cells actually synthetize and release

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neuropeptides.7 The classical paradigm of autonomy and self-regulation of the immune system has been challenged8; increasing evidence proposes the crucial participation of neuropeptides in the complex neuroimmune networks.8,9 The crosstalk between the nervous and immune system is

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crucial in regulating systemic homeostasis. Intriguingly, neuropeptides have been implicated in regulating immune responses in the lacrimal functional unit (LFU), a morphofunctional complex

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composed of the ocular surface (tear film, conjunctiva, and cornea), the lacrimal glands, and the innervation to these structures (Figure 1).10,11 The purpose of this review is to summarize the current knowledge of the function of neuropeptides at the ocular surface, as well as their physiological and pathological roles. We focus on the expression, as well as the trophic and immune roles of substance P (SP), calcitonin gene– related peptide (CGRP), vasoactive intestinal peptide (VIP), and neuropeptide Y (NPY) at the ocular surface. We discuss their clinical implications in ocular immune privilege, infection, dry eye disease, and allergic eye disease. 3

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II.

Neural Expression of Neuropeptides The cornea provides the densest innervation within the human body.12 Most of its nerves,

deriving from the trigeminal ganglion, are sensory, although a scarce contribution of the

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sympathetic nervous system from the superior cervical ganglion and an unclear participation of the parasympathetic nervous system have been reported as well (Table 1).13 Substance P (SP) and calcitonin gene-related peptide (CGRP) are expressed by 3-31% and 15-41% of corneal nerve

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fibers, respectively.14 While CGRP and SP nerve fibers are largely distributed throughout the anterior corneal stroma and the epithelium, forming a highly-branched subepithelial plexus,

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neuropeptide Y (NPY)- and vasoactive intestinal polypeptide (VIP) fibers are far fewer in number and are mainly distributed in the anterior stroma, seldom branching and rarely seen below the basal epithelial cells. Interestingly, denervation studies showed that CGRP and SP nerve fibers originate from sensory nerves, whereas VIP fibers originate from parasympathetic nerves and NPY mainly

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from sympathetic nerves.15

The conjunctiva is innervated by unmyelinated sensory, sympathetic, and parasympathetic nerve fibers originating from the trigeminal, superior cervical, and pterygopalatine ganglion,

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respectively.16-18 CGRP-immunoreactive fibers innervate the epithelium, substantia propria, and the stroma of rat conjunctiva. Specifically, nearly 90% of these fibers innervate the palpebral

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conjunctiva with increasing numbers closer to the mucocutaneous junction. Nerve fibers in the bulbar conjunctiva are mainly innervating the vessels, whereas they are almost absent in the goblet cell-rich fornices.19 In the epithelium, SP-immunoreactive nerve fibers are reduced by half compared to CGRP-immunoreactive fibers, but the SP and CGRP-secreting fibers encircling the stromal blood vessels are similarly distributed.19, 20 VIP has shown an immunoreactive function along the epithelial-stromal junction and adjacent to goblet cells, where it stimulates mucin secretion by conjunctival goblet cells.21 Finally, NPY-immunoreactive fibers have been described in close proximity to conjunctival blood vessels and glands in rat.22 4

ACCEPTED MANUSCRIPT The lacrimal gland is highly innervated from the parasympathetic (pterygopalatine ganglion), sympathetic (superior cervical ganglion), and sensory (trigeminal ganglion) nervous system.23 While nerve fibers in the human lacrimal gland interstice mainly express VIP and very little CGRP and NPY, nerve fibers associated with interlobular blood vessels are mainly CGRP-

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and NPY-positive, and rarely VIP-positive. Moreover, CGRP is expressed in the human epithelium of the interlobular and excretory ducts.24 Conversely, SP fibers have been found mainly around the ducts in rats and guinea pigs.25

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The neurobiology of meibomian glands was thoroughly reviewed in 2014; nerve fibers from the parasympathetic (pterygopalatine ganglion), sympathetic (cervical ganglion), and sensory

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(trigeminal ganglion) nervous system innervate the meibomian glands.26 VIP and CGRP fibers have been described close to the acini of human meibomian glands, CGRP more densely at the lid margin conjunctiva. SP immunoreactivity is sparse in human meibomian glands and more associated to their vasculature, whereas NPY nerve fibers are found around the blood vessels in rat,

Biochemistry of Neuropeptides and their Receptors A.

Substance P

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III.

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guinea pig, and rhesus and cynomolgous monkeys.26

SP belongs to the tachykinin family, a group of widely distributed peptides biologically

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active at a low concentration.27 The preprotachykinin-A (TAC-1) gene encodes for the SP precursor, a larger protein that eventually undergoes splicing, resulting in the biologically active fragment.28 Although able to activate the neurokinin receptors-2 (NK-2) and-3 (NK-3), SP preferentially binds to the neurokinin receptor-1 (NK-1), a seven-transmembrane domain and G protein-coupled receptor.28 The activation of NK-1 triggers phospholipase C, adenylate cyclase and protein kinase A, promoting numerous physiological and pathological effector mechanisms such as chemotaxis, cell proliferation, and neuroimmune crosstalk.29

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ACCEPTED MANUSCRIPT B.

Calcitonin Gene-related Peptide Alternative splicing of the calcitonin gene on chromosome 11 encodes for the CGRP.30 This

37–aminoacid neuropeptide exists in two isoforms: α and β. Both isoforms are regulated through different mechanisms and control indistinguishable functions.31 CGRP binds to a G-protein coupled

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receptor complex composed by three subunits: calcitonin-like receptor (CLR), receptor activitymodifying protein 1 (RAMP1), and receptor component protein (RCP).32 Additionally, CGRP can bind also to the receptors of two CGRP-related peptides –adrenomedullin and amylin.32 CGRP

Vasoactive Intestinal Polypeptide

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C.

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elicits several mechanisms ranging from vasodilatation to immune regulation.33

Given its pleiotropic functions, VIP has been also entitled “very important peptide.”34 This 28-aminoacid peptide can bind to three types of G protein-coupled plasma membrane receptors: VPAC1, VPAC2 and PAC1 (PACAP-preferring receptor 1).35 While the latter binds VIP with low

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affinity VPAC 1 and VPAC2 bind VIP with equal affinity.35 Interestingly, VPAC1 is constitutively expressed on T cells whereas the expression of VPAC2 expression is induced upon inflammation.36

Neuropeptide Y

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Extracted by porcine brain in the early 1980s, neuropeptide Y gets its name due to its

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enriched tyrosine (Y) residues.37 This 36-aminoacid peptide is one of the most copious neuropeptides within the brain. Its precursor undergoes cleavage by peptidases releasing biologically active fragments, subsequently binding with different affinity to Y-receptors.38 These G-protein coupled receptors are peculiar since they share low sequence homology and represent one of the most heterogeneous receptor families.38 Intriguingly, NPY yields both pro- and antiinflammatory functions, depending on which Y-receptor and immune cell is bound.38 The wide distribution of Y-receptors in the central nervous system as well as in the periphery accounts for the crucial role of NPY in cell proliferation and neurogenesis.39 6

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IV.

Neuropeptides and their Trophic Functions Neuropeptides function in a trophic manner at the ocular surface (Table 2). The effect of SP

on corneal epithelial migration, proliferation, and attachment to extracellular matrix (ECM)

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proteins has been recently and thoroughly reviewed by Nishida et al.40 SP is known to participate in the early phase of wound healing as a systemic, injury-inducible messenger by stimulating

mobilization of the stromal-like cells expressing CD29 (a β1 integrin subunit).41 The migration of

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corneal epithelial cells is synergistically promoted by SP and epidermal growth factor (EGF) or insulin-like growth factor-1 (IGF-1) or -2 (IGF-2), but it cannot be stimulated by SP or IGF

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alone.40 Specifically, SP and IGF-1 upregulate the expression of integrin α5, promoting migration and adhesion of corneal epithelial cells to the extracellular matrix proteins (precisely, fibronectin, type I collagen and laminin), but not modifying corneal epithelial mitosis.40 Moreover, SP induces an overexpression of the tight junction protein zonula occludens-1 (ZO-1), a membrane-associated

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protein expressed by superficial corneal epithelial cells; this finding was associated with increased transepithelial electrical resistance, an index of barrier function.42 The lack of SP has been suggested to promote neurotrophic keratitis because it regulates E-

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cadherin expression, a cell-cell adhesion molecule frequently found at the end of trigeminal nerve

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fibers that maintains epithelial cell attachment.43 Experimental studies on animals affected by corneal epithelial defects, caused by n-heptanol, capsaicin, or through thermocoagulation of the ophthalmic branch of the trigeminal nerve, have demonstrated that treatment with SP and IGF-1 promotes wound closure, independent of the integrity of the trigeminal nerve.40 Combined topical administration of SP (as well as FGLM-amide, a SP derived peptide) and IGF-1 effectively improves corneal epithelial barrier function and wound healing, proving their clinical relevance in the treatment of neurotrophic keratopathy.44-46 The ability of SP to promote wound healing has also been investigated in diabetes, where reduced corneal sensitivity, nerve fiber density, and wound healing are known features.13,47-49 7

ACCEPTED MANUSCRIPT However, SP levels are significantly reduced in tears of diabetic humans but not in diabetic rat corneas.50 Recently, Yang et al have demonstrated that 1) the impairment of SP-NK-1 signaling in normal corneal epithelium resulted in pathological diabetic epithelial changes and reduced corneal sensitivity in mice, 2) SP promotes wound healing in diabetic cornea, and 3) SP induces nerve fiber

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regeneration to restore corneal sensitivity.47

CGRP increased epithelial wound healing by 25% in an ex vivo rabbit model.51 This “mitotic” action might explain decreased CGRP levels at 24 hours in vesicant sulphur mustard-

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induced limbal stem cell deficiency, and increased CGRP levels at 1 to 4 weeks, the time point of reinnervation.52 In contrast, treatment of rabbit corneal cells with CGRP inhibited mitosis.53 The

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vasodilator or VEGF-upregulating functions of CGRP might explain these controversial findings.33,54 CGRP together with SP promotes proliferation and has been implicated in upregulating N-cadherin on corneal epithelial cells.55 N-cadherin represents the neural isoform of cadherins, calcium-dependent cell adhesion molecules involved in cell-cell adhesion, cell

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differentiation, cell migration and invasion, and signal transduction.56,57 Through the modulation of N-cadherin, CGRP and SP, once released by trigeminal neurons, are crucial for the adhesion of corneal epithelial cells.55 Additionally, increased corneal CGRP expression in keratoconus is related

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to the changes of corneal innervation observed in this dystrophy.58 Being a parasympathetic neuropeptide, VIP controls exocrine secretions of water and

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electrolytes through its action on the lacrimal gland and conjunctiva. Binding to the VPAC2 receptor expressed on the surface of conjunctival goblet cells, VIP has been found to stimulate the secretion of high molecular weight glycoconjugates, including the mucin MUC5AC, through increased levels of cAMP and Ca2+, independently from EGFR transactivation.59 Similarly to CGRP, VIP corneal levels are increased in keratoconus compared to leukoma or control corneas, suggesting that VIP is involved in altering corneal nerve architecture in keratoconus and/or its counteracting action to the degenerative changes observed in this dystrophy.58, 60 Finally, NPY has been demonstrated to have a potent mitogenic activity but its physiological 8

ACCEPTED MANUSCRIPT roles in the cornea still remain unclear.61

V.

Ocular Immune Privilege Ocular immune privilege has been described as an evasion of an immune response by

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inducing low immunogenicity and alloantigen tolerance. SP has been recognized to play a role in anterior chamber associated immune deviation (ACAID), a mechanism contributing to immune privilege and encompassing suppressed delayed hypersensitivity, preserved humoral immunity, and

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primed cytotoxic T cell responses.62 In retinal laser burn, the receptor NK-1 is increased first in the treated and later in the fellow nontreated eye, and SP knockout mice retain their ability to develop

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ACAID after retinal laser burn.63,64 These findings, together with the demonstration of bilateral loss of ACAID after unilateral retinal laser burn, suggest that SP mediates the transmission of early inflammatory signals from the injured retina to the fellow eye.64,65 A recent report has confirmed that SP prevents the generation and activity of regulatory T cells (Tregs), and promotes graft

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rejection in the treated and untreated eye.63 Finally, immune privilege and heightened graft survival can be restored by spantide II, a NK-1 antagonist.63 Corneal lymphatic and hematic avascularity is probably the utmost mechanism providing

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corneal immune privilege and guaranteeing corneal homeostasis.66 However, a myriad of ocular surface diseases can result in the development of new corneal vessels.67 In 1990, Ziche et al

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demonstrated that SP stimulates corneal neovascularization through NK-1.68 More recently, a selective NK-1 antagonist has been used to successfully reduce both corneal hemangiogenesis and lymphangiogenesis in two mouse models of corneal neovascularization. 69

VI.

Neuropeptides and their Immune Functions The nervous system controls the immune response by regulating homeostasis and supporting

immune-driven host defense in a rapid, directional, and regional course of action.8 Neuropeptides are critical in the interplay between nervous and immune system; once released from nerve 9

ACCEPTED MANUSCRIPT terminals, they are involved in a dynamic process called “neurogenic inflammation” (Table 3).10 Although hypotension was the first function reported in the scientific literature regarding SP, the immunovascular effects of this neuropeptide actually go beyond that, given that it contributes to the accumulation of leukocytes at the site of inflammation by inducing vasodilation

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and the chemotaxis of immune cells.2,70 In addition to stimulating the recruitment of monocytes and neutrophils, SP also stimulates their innate immune functions by increasing the production of arachidonic metabolites (i.e., prostaglandin E2 and thromboxane B2) and their secretion of granule

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constituents.70 Indeed, diclofenac sodium was found to decrease the amount of prostaglandins and SP at the ocular surface as well as in other tissues.50 SP, expressed in close proximity of mast cells,

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regulates allergic responses by stimulating mast cell degranulation and increasing the release of histamine and thromboxane B2 by eosinophils.71,72 Interestingly, the interaction of SP/NK-1 affects the maturation of dendritic cells (DCs), i.e., the linking branch between innate and adaptive immunity.73 SP inhibits IL-10 secretion of dendritic cells74 and promotes the switch of

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noncommitted human memory CD4 T cells into T-helper 17 lymphocytes, a T cell subset regulating autoimmunity.75,76 Taking together these findings, SP clearly has a proinflammatory role in both innate and adaptive immunity (Figure 2).

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Similarly to SP, CGRP-expressing fibers have been described close to mast cells, and CGRP is known to induce vasodilatation.77,78 In contrast to SP, CGRP dampens the production of pro-

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inflammatory cytokines (TNF-α, IL-1, IL-12p40 and CCL4), and promotes IL-10 secretion from monocytes.77 The anti-inflammatory properties of CGRP could be an explanation for its increased tear level after excimer laser keratectomy. 79 Further, CGRP reduces the expression of the costimulatory molecule CD86 and the major histocompatibility complex-II (MHC-II) on DCs and macrophages, and thus diminishes their capacity to present antigens to T cells and T cell proliferation.77, 80 CGRP inhibits the release of IFN-γ, CXCL9, and CXCL10 from T-helper 1 cells and stimulates the secretion of IL-4, CCL17, and CCL22 from T-helper 2 cells.81 Furthermore, Levite et al have shown that CGRP has the ability to change T-helper subset commitment: after 10

ACCEPTED MANUSCRIPT exposure to CGRP, Th-1 cells secrete IL-4, Th-2 cells secrete IFN-γ, and Th-0 cells secrete both.82 Reduced allogeneic T-cell proliferation observed after CGRP treatment of monocytes and dendritic cells, and its effect on T-cell cytokine expression influencing Langerhans cells, suggests its therapeutic potential, also in corneal transplantation.77,80

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Interestingly, both CGRP and SP contribute to the recruitment of neutrophils to the inflamed cornea through IL-8.83,84 Although the major inducers of interleukin (IL)-8 in the inflamed cornea are IL-1α and tumor necrosis factor (TNF)-α, CGRP has been proposed to regulate the expression

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of IL-8 in response to minor insults not sufficient to elicit IL-1α or TNF-α release from corneal epithelial cells or macrophages and lymphocytes, respectively.83,84 In this context, SP amplifies IL-8

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at the inflamed corneal surface through the stabilization of IL-8 transcripts.83,84 The anti-inflammatory functions of VIP in immunity have been extensively reviewed by Delgado et al.35 VIP is highly expressed by immune cells and actively reduces the functions of macrophages and dendritic cells through the inhibition of chemotactic proinflammatory chemokines

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(CXCL1/KC, CXCL2, CCL2, CCL3, CCL4 and CCL5) and the downregulation of toll-like receptors.35,85,86 VIP induces tolerogenic DCs characterized by low expression of costimulatory molecules (CD40, CD80, CD86), low production of pro-inflammatory cytokines (IL-12, and TNF-

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α), and increased production of anti-inflammatory IL-10. While VIP promotes T-helper 2 cell differentiation, migration, and survival, it has been shown to dampen the differentiation of T-helper

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17 cells.35 Gonzalez-Rey et al have demonstrated that VIP promotes the generation of regulatory DCs that subsequently induce CD4 and CD8 T-regulatory cells, which, in turn, dampened the activation and proliferation of antigen-specific T-helper 1 lymphocytes. Generating tolerogenic dendritic cells with neuropeptides is a great therapeutic strategy that requires further research.87 The immunological interest in NPY was raised in 1991 when sympathetic neurons innervating the lymphoid organs were found to express NPY.38,88 The function of NPY on granulocytes is dependent on the receptor it binds: Y1 receptor promotes stimulatory whereas other Y receptors induce inhibitory effects.61 NPY induces chemoattraction, adhesion, phagocytosis, 11

ACCEPTED MANUSCRIPT release of transforming growth factor (TGF)-β1 from macrophages and the secretion of IL-1β from monocytes.61 NPY prevents autoantigen processing and presentation, and thus contributes to ocular immune privilege.89 Interestingly, although stimulating the chemotaxis and adhesion of lymphocytes, NPY has not been shown to promote their capacity to penetrate tissues. This

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neuropeptide is also known for inhibiting lymphocyte proliferation promoted by mitogens, likely through the reduced production of IL-2.90 NPY has been hypothesized to participate in the immune response by recruiting dendritic cells to the inflammation site and to promote T-helper 2 immunity.

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Similar to CGRP and VIP, NPY has been shown to be able to change T-cell commitment:

VII.

Neuropeptides and Infection

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specifically to induce the secretion of T-helper 2 cytokines from T-helper 1 cells and vice versa.91

Recognition of pathogen-associated molecular patterns (PAMPs) is crucial for initiating the innate immune response, which then activates the adaptive response.92 Given their amphipathic

established.9,93

Pseudomonas aeruginosa-induced Keratitis

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structure, neuropeptides have antimicrobial features and their role in infections has been

SP and VIP are actively involved in the response to Pseudomonas aeruginosa-induced

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keratitis in mouse models.94,95 The increased levels of these neuropeptides regulate both the sentinels (monocytes/macrophages) and the infantry (polymorphonuclear and natural killer cells) of the innate immune system.9 The release of IL-1, IL-6, and IL-12, and TNF-α from monocytes occurs upon SP stimulation.96 SP/NK-1 interaction stimulates natural killer cells to secrete IFN-γ and promotes IL-18 release (a proinflammatory cytokine, known to induce IFN-γ secretion) from macrophages.94,97 Antagonizing NK-1 with spantide I results in decreased corneal perforation, bacterial count, polymorphonuclear cell infiltration, reduced expression of type I cytokines (IL-18, IL-12, IFN-γ and IL-1β), and increased IL-10 secretion.96 In addition to influencing natural killer 12

ACCEPTED MANUSCRIPT cells, SP amplifies the release of IL-8 by corneal epithelial cells, and SP blockade results in earlier apoptosis of polymorphonuclear cells.84,98 Blocking VIP in Pseudomonas aeruginosa-induced keratitis results in exacerbated disease, increased levels of IL-18, IL-12, IFN-γ and decreased levels of IL-10.99 Further, VIP has been

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shown to downregulate pro-inflammatory TLRs (TLR1, TLR4, TLR6, TLR8, TLR9, IRAK1, TRAF6, and Chuk) and upregulate anti-inflammatory TLRs.100 VIP has also been implicated in controlling leukocyte migration through its action on adhesion molecules. Namely, treatment of

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Pseudomonas aeruginosa-induced keratitis with recombinant VIP leads to reduced corneal

expression of intercellular adhesion molecule-1/leukocyte function-associated antigen-1 (ICAM-

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1/LFA-1), and vascular cell adhesion molecule-1/very late antigen-4 (VCAM-1/VLA-4) compared to PBS-treated animals.101 Furthermore, VIP influences corneal healing by promoting extracellular matrix reconstitution by acting on corneal fibroblasts via VIP-receptor 2. VIP treatment of Pseudomonas aeruginosa-induced keratitis (i) enhances the expression of α1 and α2 integrins,

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CD44, laminin-1, extracellular matrix protein 1, chondroitin sulphate proteoglycan 2, and secreted phosphoprotein 1 and (ii) stimulates both epithelial and stromal cells to produce growth factors (EGF, FGF, HGF and VEGF).100-102 In contrast, SP induces increased levels of hepatocyte growth

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factor (HGF) and fibroblast growth factor 7 (FGF-7), promotes pro-inflammatory cytokine expression and anti-apoptotic genes, while it downregulates anti-inflammatory cytokines and pro-

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apoptotic genes.103

Herpes Smplex Virus 1 Neuropeptides actively participate in regulating the immune response against herpetic

keratitis. Herpes simplex virus 1 (HSV-1) keratitis leads to a dramatic depletion of corneal sensory nerve fibers, causing neurotrophic keratopathy.12 In fact, the tear concentration of SP is decreased in patients with reduced corneal sensitivity, probably due to the depletion of nerve fibers caused by herpes simplex virus. Moreover, HSV-1 acute keratitis is characterized by a profound regression of 13

ACCEPTED MANUSCRIPT SP and CGRP nociceptive fibers and a loss of corneal sensitivity in mice. Nonetheless, after healing, peculiar patterns of re-innervation in the middle and posterior corneal stroma can be seen at 1 month after the infection, but the fine subepithelial fibers remain lost. Moreover, CGRP immunoreactivity was increased within stromal nerves when compared to uninfected corneas,

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whereas SP immunoreactivity was not retrieved.104

In particular, severe forms of herpetic stromal keratitis (HSK) show higher expression of SP compared to mild forms of HSK.105 Such increased SP levels are associated with higher amounts of

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proinflammatory chemokines (CCL3 and CXCL2) and cytokines (IL-6, IFN-γ).105-107 In accord, subconjunctival injection of spantide I, a NK-1 antagonist, significantly reduced the frequencies of

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neutrophils, CD4 T cells as well as IL-6 and CCL3 expression, as well as corneal opacity and angiogenesis.105

VIII. Neuropeptides and Conjunctival Allergic Diseases

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Neurogenic inflammation is an acknowledged player in the pathogenesis of allergies such as asthma and smoke-induced airway disease. The release of neuropeptides by nerve endings triggers allergic reactions characterized by bronchoconstriction, mucus secretion, and mucosal

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hyperaemia.108 Neuropeptides can be secreted after exposure to several substances, including

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allergens, ozone, and bronco-active agonists such as histamine, prostaglandins, and leukotrienes.109-

After the conjunctival allergen provocation test, the tear level of SP is increased together with the induction of a conjunctival allergic reaction108 and, in patients with allergic conjunctivitis and vernal keratoconjunctivitis (VKC), a chronic bilateral inflammation of the conjunctiva belonging to a group of diseases classified as allergic conjunctivitis.10, 50, 112-115 SP was found to influence tear secretion and goblet cell function in rats by inducing histamine release, mucus secretion, and increased vascular permeability,116-119 suggesting that this neuropeptide may contribute to the pathogenesis and severity of allergic conjunctivitis. 14

ACCEPTED MANUSCRIPT VIP has been suggested to participate in the pathogenesis of ocular allergic diseases, as 1) increased expression of VIP in the nasal secretion after nasal allergen challenge in patients with allergic rhinitis, 2) increased tear levels after conjunctival allergen provocation test, and 3) VIPimmunoreactivity is higher in upper tarsal conjunctiva biopsies of patients with VKC.109,116,120

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CGRP ameliorates neuroinflammation in allergic diseases such as irritant dermatitis and ovalbumin-induced airway inflammation.33 It promotes the biasing of Langerhans cells toward Thelper 2 immunity.80 Intradermal injection of CGRP and histamine in patients resulted in a

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suppression of histamine-produced wheals.121,122 The anti-inflammatory properties of CGRP together with its vasodilator and edemigenous effects might explain the increased tear levels of this

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neuropeptide after conjunctival allergen challenge.119

Although NPY tear levels were not increased after conjunctival allergen provocation test, NPY is known to promote eosinophil migration and T helper-2 cell generation91 by upregulating IL6 and IL-10 in DCs, inhibiting IFN-γ, and upregulating IL-4 in T helper-1 and T helper-2

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lymphocytes, respectively.82, 91 Furthermore, NPY has been suggested to play a bimodal role in allergic asthma119, 123, 124 by activating antigen-presenting cells but also negatively regulating T cells.123,125-127 The levels of NPY were found to be higher at later stages of allergic asthma and,

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interestingly, inversely correlate with the levels of proinflammatory molecules in the airways.127 The anti-inflammatory and repairing roles of NPY seen in allergic asthma suggest further research

IX.

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on the role of NPY as a treatment in allergic conjunctivitis.

Neuropeptides and Dry Eye Dry eye disease is considered a multifactorial inflammatory syndrome in which nerves play

a critical role.128-130 The architectural changes of the subbasal corneal nerve plexus have been extensively demonstrated with in vivo confocal microscopy studies showing decreased subbasal corneal nerves and altered corneal nerve morphology.130-134

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ACCEPTED MANUSCRIPT Moreover, the variations of circulating neuropeptides and neurotrophins, together with the impairment of salivary gland innervation, have been demonstrated in patients with Sjögren syndrome.128, 135, 136 SP is known for selectively polarizing human memory CD4+ T cells into T-helper 17 cells,

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pivotal cells in dry eye disease.76, 137-138 Given that topical cyclosporine A is a SP receptor

antagonist and has been shown to be an effective treatment for dry eye disease, it is tempting to postulate a role of SP in dry eye disease.139, 140

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In contrast, tear levels of CGRP are decreased in dry eye patients, particularly in patients with non-Sjögren syndrome dry eye and mucous membrane pemphigoid.141 Reduced CGRP

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expression inversely correlates with disease severity and directly correlates with the Schirmer test values.141 It has been speculated that changes in the lacrimal gland function might contribute to the decreased tear levels of CGRP.141 Interestingly, tear levels of CGRP increase after topical instillation of cyclosporine in dry eye likely due to the immunomodulatory role of cyclosporine.142

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Stimulation of VIP receptors expressed on lacrimal gland cells results in the secretion of water and electrolytes through increased cAMP intracellular levels.143 Similarly, topical installation of VIP decreases tear osmolarity due to increased fluid and protein secretion.144 However, patients

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with dry eye show no statistically significant different tear levels of VIP compared to healthy individuals, thus making the exact role of VIP in dry eye disease still unclear.141

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Tear levels of NPY are reduced and inversely correlate with the severity of dry eye,141 probably due to the anti-inflammatory properties of NPY, but whether the reduced levels of NPY are cause or consequence of dry eye disease has not been investigated yet.

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Future Directions Understanding the biochemistry of neuropeptides and their effects on the immune system

have led to the development of novel treatment strategies for ocular surface diseases.

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ACCEPTED MANUSCRIPT Epithelial resurfacing can be promoted by treating patients after neurotrophic keratopathy with a combination of SP and IGF-1 (and their derivates).44,46,145 However, despite the positive effects on tropism reported after SP and IGF-1 treatment, researchers have mostly focused on VIP and the inhibition of SP/NK1 interaction.

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VIP has successfully been used in managing Pseudomonas-induced keratitis in mice by reducing TLRs, growth factors, and adhesion molecules.100 SP promotes inflammation in a mouse model of Pseudomonas aeruginosa keratitis through the upregulation of type I cytokines and

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downregulation of VIP and anti-inflammatory molecules.94 Treatment of Pseudomonas aeruginosa keratitis in mice with spantide I reduces inflammation, due to increased secretion of IL-10 and

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reduced pro-inflammatory molecules, resulting in a decreased number of polymorphonucleates, bacterial count, and perforated corneas.96 Subconjunctival injection of spantide I has been successfully used to treat severe HSK by significantly diminishing the number of immune cells, corneal opacity and angiogenesis.105

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The effects of NK-1 antagonism have also been investigated in the context of ocular immune privilege. Immune privilege can be restored by spantide II, and lanepitant, a NK-1 competitive antagonist; both reduced corneal hemangiogenesis and lymphangiogenesis in two

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mouse models of corneal neovascularization.69

The anti-inflammatory properties, together with the impairment of angiogenesis, configure

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the NK-1 antagonists as an important breakthrough in ocular pharmacology. In fact, they represent an attractive treatment strategy for a large group of ocular diseases, ranging from dry eye and conjunctival allergic disease to infectious diseases and graft rejection. Similarly, the creation of an anti-inflammatory environment in both infectious and noninfectious inflammatory conditions affecting the ocular surface confirms the deserved title of “very important peptide” for VIP.

17

ACCEPTED MANUSCRIPT Nonetheless, the expression of NK-1 receptor and VIP receptors is widespread within the human body, and their activation promotes pleiotropic functions. Therefore, the involvement of cardiovascular, endocrine, nervous, gastrointestinal, and immune systems would potentially lead to serious adverse effects (i.e., hypotension, tachycardia, and cutaneous flushing induced by VIP) and

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poor patient tolerance if a systemic route of drug administration is used.146 Moreover, poor

metabolic stability and reduced biological activity intervals of neuropeptides might represent major barriers to their therapeutic usage.146

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While systemic side effects of neuropeptides could be avoided by using topical preparations,

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chemical manipulation is required to prolong their half-life and biological activity. Interestingly, nanoparticles and ligands of different chemical nature have been developed to encapsulate neuropeptides in different formulations, including liposomes, biodegradable protamine oligonucleotide and polyethylene glycol–poly (lactic acid) nanoparticles.146 Stark et al showed that

proteolytic degradation.146

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VIP encapsulated into liposomes maintained its biological activity and was protected from

Conventional or new generation liposomes could represent a valuable delivery strategy to

XI.

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neuropeptides.147

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improve corneal corneal adhesion and permeation as well as the pharmacokinetics of

Conclusions

Neuroimmune crosstalk embodies an interesting physiological mechanism that is involved in the pathogenesis of various immune diseases. Although increasing scientific evidence has been gathered so far, few studies concerning the role of neuropeptides in the LFU exist and further experimental studies are necessary. Their intriguing pleiotrophic roles on the neuroimmune axis represent a promising therapeutic strategy in the management of ocular surface diseases.

18

ACCEPTED MANUSCRIPT Acknowledgments: We thank Dr. Susanne Eiglmeier for writing assistance and proofreading the manuscript.

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Figure 1. The crosstalk between immune and nervous systems is bidirectional and regulates the ocular surface homeostasis: the immune cells synthetize and release neuropeptides that in turn

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Figure 2. Proinflammatory activities promoted by Substance P. IL-10 = Interleukin 10; SP =

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Substance P; Th17 = T-helper 17 lymphocytes; Treg = T regulatory lymphocytes. TXB2 =

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ACCEPTED MANUSCRIPT Neuropeptides

Distribution of nerve fibers CONJUNCTIVA LACRIMAL MEIBOMIAN GLAND GLAND (LG)

SP

Epithelium and stroma in rats14

Epithelium (half of CGRP fibers) and stroma around blood vessels in rats19

Around the ducts in rats and guinea pigs25

Associated to blood vessels in humans26

CGRP

Epithelium and stroma in rats14

Mostly palpebral conjunctiva, especially around mucocutaneous junction in rat.

Associated with interlobular blood vessels and epithelium of ducts in humans24

Close to the acini

Epithelialstromal junction and adjacent to goblet cells in human and mouse21

Interstice of the LG in humans24

Close to the acini in humans 26

Close to conjunctiva blood vessels and glands in rats22

Associated with interlobular blood vessels in humans24

Around the blood vessels in rat, guinea pig, rhesus and cynomolgous monkeys 26

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Around blood vessels in the bulbar conjunctiva in rats19

Anterior stroma in rats15

NPY

Mainly in the anterior stroma in rats15

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and lid margins in humans26

Table 1. Neural expression of neuropeptides in the lacrimal functional unit. CGRP = Calcitonin gene related peptide; NPY = Neuropeptide Y; SP = Substance P; VIP = Vasoactive intestinal peptide.

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Trophic effects Promotes migration of corneal epithelial cells in synergy with EGF or IGF (1 or 2)

SP Induces over-expression of the tight junction protein ZO-1 Unclear effect: increases epithelial wound healing in ex-vivo rabbit model; inhibits mitosis in rabbit corneal cells

CGRP + SP

Promote proliferation and upregulate Ncadherin on corneal epithelial cells

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Stimulates the secretion of high molecular weight glycoconjugates (MUC5AC)

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Potent mitogenic activity but unclear role in the cornea

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CGRP

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Table 2. Trophic effects of neuropeptides. CGRP = Calcitonin gene related peptide; EGF = Epidermal growth factor; IGF = Insulin-like growth factor; MUC5AC = Mucin 5AC; NPY = Neuropeptide Y; SP = Substance P; VIP = Vasoactive intestinal peptide; ZO-1 = Zonula occludens-1

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Immune effects Stimulates the recruitment and function of monocytes and neutrophils Stimulates mast cell degranulation and increases the release of histamine and TXB2 by eosinophils

SP

Affects the maturation of DCs

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Inhibits IL-10 secretion of DCs

Promotes the switch of non-committed CD4 T cells into Th17

Dampens the production of pro-inflammatory cytokines and promotes IL-10 secretion from monocytes

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Inhibits the release of IFN-γ, CXCL9, and CXCL10 from Th1 cells and stimulates the secretion of IL-4, CCL17, and CCL22 from Th2 cells Changes T-helper subset commitment: Th1 cells secrete IL-4, Th2 cells secrete IFN-γ, and Th0 cells secrete both CGRP + SP

Contribute to the recruitment of neutrophils to the inflamed cornea through IL-8 Reduces the functions of macrophages and dendritic cells

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Promotes Th2 cell differentiation, migration and survival Dampens the differentiation of Th17 cells

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Prevents autoantigen processing and presentation Inhibits lymphocyte proliferation promoted by mitogens Recruits DCs to the inflammation site and promotes Th2 immunity Changes T-cell commitment: induces the secretion of Th2 cytokines from Th1 cells and vice versa

Table 3. Immune roles of neuropeptides. CD4 = Cluster of Differentiation 4; CD86 = Cluster of Differentiation 86 ; CGRP = Calcitonin gene related peptide; CXCL9 = Chemokine (C-X-C motif) ligand 9; CXCL10 = Chemokine (C-X-C motif) ligand 10; CCL17 = Chemokine (C-C motif) ligand 17; CCL22 = Chemokine (C-C motif) ligand 22; DCs = dendritic cells; IFN-γ = Interferon γ; IL-1β = Interleukin 1β; IL-2 = Interleukin 2; IL-4 = Interleukin

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4; IL-8 = Interleukin 8; IL-10 = Interleukin 10; MHC-II = major histocompatibility complex class II; NPY = Neuropeptide Y; SP = Substance P; TGF-β1 = Transforming growth factor beta 1; Th0 = Thelper 0; Th1 = T-helper 1; Th2 = T-helper 2; Th17 = T-helper 17; TXB2 = Thromboxane B2; VIP = Vasoactive intestinal peptide.

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