cholinergic regulation of keratinocyte innate immunity

Cholinergic Regulation of Keratinocyte Innate Immunityand Permeability Barrier Integrity: New Perspectives inEpidermal Immunity and DiseaseBrenda J. Curtis1 and Katherine A. Radek1

Several cutaneous inflammatory diseases and theirclinical phenotypes are recapitulated in animal mod-els of skin disease. However, the identification ofshared pathways for disease progression is limited bythe ability to delineate the complex biochemicalprocesses fundamental for development of the dis-ease. Identifying common signaling pathways thatcontribute to cutaneous inflammation and immunefunction will facilitate better scientific and therapeuticstrategies to span a variety of inflammatory skindiseases. Aberrant antimicrobial peptide (AMP) ex-pression and activity is one mechanism behind thedevelopment and severity of several inflammatoryskin diseases and directly influences the susceptibilityof skin to microbial infections. Our studies haverecently exposed a newly identified pathway fornegative regulation of AMPs in the skin by thecholinergic anti-inflammatory pathway via acetylcho-line (ACh). The role of ACh in AMP regulation ofimmune and permeability barrier function in kerati-nocytes is reviewed, and the importance for a bettercomprehension of cutaneous disease progression bycholinergic signaling is discussed.

Journal of Investigative Dermatology (2012) 132, 28–42;doi:10.1038/jid.2011.264; published online 15 September 2011

INTRODUCTION AND SCOPE OF THE PROBLEMInflammatory skin diseases are a major cause of morbidityand mortality for patients, where the atypical cytokine and

cellular milieu creates a reservoir for cutaneous infection anddiminished barrier function. Loss of skin permeability barrierfunction leads to excessive water loss and penetration ofirritants, allergens, and microbes. Moreover, barrier functionstudies demonstrate that physical cutaneous permeabilitybarrier (e.g., electrolyte and water movement) and thechemical antimicrobial barrier (e.g., antimicrobial peptide(AMP)) are codependent (Ahrens et al., 2011). Studies haveshown that a diminished permeability barrier function isassociated with reduced lipid synthesis and AMP production(Aberg et al., 2008). Injury and infection function as a triggerfor the development of numerous skin diseases by providing astimulus for activation of distinct inflammatory pathways.Infections caused by viral, bacterial, or fungal agents can notonly initiate disease directly, but also further exacerbateinflammatory skin diseases. Although staphylococcal andstreptococcal infections often precede the development ofpsoriasis, which is generally considered the most stress-responsive skin disease compared with atopic dermatitis (AD)and acne vulgaris (Griffiths and Richards, 2001), psoriaticplaques are generally resistant to infection (Raychaudhuri andRaychaudhuri, 1993). AD is also initiated and worsened byprolonged psychological stress and injury or infection;however, lesions from these patients tend to be moresusceptible to colonization and infection following develop-ment of the disease (Ong et al., 2002; Nomura et al., 2003).Thus, the inflammatory pathways activated in response toprimary injury or infection further exacerbate the barrierdefect to facilitate disease progression. The current paradigmtends to categorize inflammatory responses as either pro- oranti-inflammatory, but fails to recognize the unifyingconcepts that initiate these injury and infection responsepatterns.

Both anecdotal and clinical evidence for the role ofperceived stress in the pathophysiology of skin diseases hasexisted for decades (Kimyai-Asadi and Usman, 2001; Picardiand Abeni, 2001). This effect is 2-fold, where stress caninitiate the onset of symptoms and, at a later time, the diseasemanifestations can become a major source of stress for thepatients to further exacerbate the clinical manifestations oftheir disease. Although the influence of glucocorticoids (GCs)and catecholamines on skin barrier and innate immunefunction have been more thoroughly studied in the context ofskin infection and disease progression, less attention has beengiven to the role of the cholinergic system on epidermalpermeability barrier and innate immune function. Our recent

REVIEW

28 Journal of Investigative Dermatology (2012), Volume 132 & 2012 The Society for Investigative Dermatology

Received 27 January 2011; revised 10 June 2011; accepted 25 June 2011;published online 15 September 2011

1Department of Surgery, Loyola University Medical Center, Burn and ShockTrauma Institute, Maywood, Illinois, USA

Correspondence: Katherine A. Radek, Department of Surgery, LoyolaUniversity Medical Center, Burn and Shock Trauma Institute, 2160 South FirstAvenue, Maywood, Illinois 60153, USA. E-mail: [email protected]

Abbreviations: ACh, acetylcholine; AD, atopic dermatitis; AMP, antimicrobialpeptide; CAMP, cathelicidin antimicrobial peptide (human);CRH, corticotropin-releasing hormone; Cst, catestatin; FFA, free fatty acid;GC, glucocorticoid; hbD-2, human beta-defensin; mAChR, muscarinic AChreceptor; MALP-2, macrophage-activating lipoprotein-2; nAChR, nicotinicACh receptor; NHEK, normal human epidermal keratinocyte; TNF-a, tumornecrosis factor-a; VD3, 1,25-dihydroxy vitamin D3

http://dx.doi.org/10.1038/jid.2011.264

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investigations into how the neuroendocrine system influencesepidermal AMP function has uncovered a newly recognizedpathway for the negative regulation of AMPs in the skinthrough the activation of nicotinic acetylcholine (ACh)receptors (nAChRs) by ACh. Here, we will review the currentmechanisms related to cholinergic signaling believed totrigger stress-induced skin disorders by modulating keratino-cyte innate immune and barrier function, with a focus onAMP regulation.

THE HPA AXIS, ADRENERGIC PATHWAY, AND THECHOLINERGIC PATHWAY OF THE STRESS RESPONSEWithin the last few decades, a significant amount of evidencesuggests that an elaborate communication network existsbetween the nervous, endocrine, and innate immune systemsto delicately balance the response to microbial invasion and/or injury. The immediate host reaction to physical insult, suchas wounding or pathogen challenge, or to psychologicalstress is the rapid activation of the evolutionarily conserved‘‘fight or flight’’ response. This initial danger-associatedresponse promotes inflammation and immune cell infiltrationto combat an infectious threat or injury, which is thenfollowed by immunosuppression via neuroendocrine path-ways to return to homeostasis (Tracey, 2002). Once thestressors are eliminated, the biochemical profile of theorganism is expected to return to normal physiologicalhomeostasis. However, prolonged activation of neuroendo-crine responses can directly promote systemic immunosup-pression (reviewed in Dhabhar, 2000).

Stress leads to activation of the parasympathetic andsympathetic nervous systems, as well as the hypothalamic–-pituitary–adrenal axis (HPA). Activation of the parasympa-thetic nervous system results in the release of ACh from nervefibers innervating the organs, resulting in activation of nAChRand/or muscarinic ACh receptors (mAChRs). Activation ofthe sympathetic nervous system results in the release ofepinephrine/norepinephrine, activating a- or b-adrenergicreceptors. Parasympathetic nervous system and sympatheticnervous system activation mediates a variety of immuno-logical and homeostatic functions, including changes incellular proliferation, differentiation, and migration (Maes-troni, 2006; reviewed in Slominski and Wortsman, 2000).Activation of the hypothalamic–pituitary–adrenal axis stimu-lates the release of several neuroendocrine mediators, such asGCs (e.g., cortisol), into the bloodstream, which functionupon specific cell populations and tissues to generate animmunosuppressive effect and return proinflammatorymediators to normal, baseline levels (Herman and Cullinan,1997). However, prolonged or chronic stress tends to beimmunosuppressive, in part, by dampening cytokine produc-tion and immune cell phagocytic capacity (Abang et al.,2006; Ashcraft and Bonneau, 2008; Dhabhar et al., 2010).This pathway was later coined the ‘‘cholinergic anti-inflammatory pathway’’ after efferent activity in the vagusnerve was found to inhibit local proinflammatory productionof cytokines by signaling through the innervated regionsof major organs involved in the response to endotoxin,without affecting the production of anti-inflammatory

cytokines (Tajima et al., 1996; Bernik et al., 2002a; Saeedet al., 2005; Pavlov et al., 2006). Our studies recentlyrevealed that cholinergic activation in non-neuronal kerati-nocytes is a major pathway involved in the suppression ofkeratinocyte antimicrobial activity, and is the focus of thisreview.

LINKING STRESS RESPONSES TO EPIDERMAL INNATEIMMUNE FUNCTION DURING INJURY OR INFECTIONDynamic cross talk is required between the nervous,endocrine, and immune systems to regulate inflammatoryprocesses across a vast array of cell types and tissues(Elenkov et al., 2000; Tracey, 2002). Many of the neuroen-docrine ligands and ligand receptors derived from all threebranches of the stress-response pathways participate incellular communication and are expressed by immune,nerve, and epidermal cells. For example, macrophagesrelease tumor necrosis factor (TNF)-a during injury orinfection to amplify the inflammatory response by stimulat-ing the release of proinflammatory mediators, including IL-1,reactive oxygen species, nitric oxide, and eicosanoids(Wood et al., 1992; Nickoloff and Naidu, 1994). Duringthe initial fight or flight response, all three pathways of thestress response are coordinately activated to modulateinflammation. For example, epinephrine/norepinephrinecan block macrophage activation and dampen productionof TNF-a and other proinflammatory cytokines (Chrousos,2000; Molina, 2001; Molina et al., 2001). Alternatively,epinephrine/norepinephrine can induce the release of theanti-inflammatory cytokine, IL-10, from monocytes (van derPoll et al., 1996; Woiciechowsky et al., 1998). In parallel,ACh has the capacity to inhibit macrophage activation andrelease of proinflammatory cytokines, such as TNF-a, IL-1,and high-mobility group protein B1 (Borovikova et al.,2000), ultimately limiting tissue damage caused by excessiveinflammation.

Psychological stress was initially found to delay woundhealing and diminish epidermal barrier homeostasis (Kiecolt-Glaser et al., 1995; Denda et al., 1998; Marucha et al.,1998; Garg et al., 2001; Rojas et al., 2002), mediated throughthe activation of both GC and adrenergic signaling pathwaysin the skin. Mice subjected to insomnia stress for 3 daysdemonstrated a decrease in the protein abundance oftwo major skin AMPs, cathelicidin antimicrobial peptide(human; CAMP) and mouse beta defensin-3, compared withunstressed mice. Moreover, these mice were more suscep-tible to cutaneous group A Streptococcus infection, asindicated by larger skin lesions compared with unstressedmice. The adverse affect of stress on AMP expression andlesion size was reversed by systemic administration ofRU-486, a GC/progesterone antagonist, suggesting that GC-receptor-mediated activation was involved in the suppressionof AMP expression (Aberg et al., 2007). These studies werethe first to establish a direct connection between stressand AMP expression in the context of cutaneous infection.Earlier studies showed that catecholamines promote therelease of fully processed active AMPs from the skinsurface (Benson and Hadley, 1969; Zasloff, 1987). More

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BJ Curtis and KA RadekCholinergic Regulation of the Skin Barrier

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recent observations in keratinocytes demonstrated thatb-adrenergic activation impairs cell motility and woundclosure, indicating a pathway by which stress impairscutaneous healing (Pullar et al., 2006; Sivamani et al.,2009). Together, mediators of the stress response can directlyor indirectly influence several key components of epidermaldefense.

THE EVOLUTION OF THE CHOLINERGIC‘‘INFLAMMATORY REFLEX’’During embryonic development, the skin and nervoussystem are both derived from the neuroectoderm. Conse-quently, many key factors that influence neuronal centralnervous system activities also contribute to non-neuronalcellular function, as most immune and epidermal cellsexpress several components of the cholinergic system(reviewed in Radek, 2010). ACh can facilitate the commu-nication between immune and epidermal cells to mount aphysiological response to injury or infection. Langerhanscells, mast cells, neutrophils, and macrophages all expressvarious components required for cholinergic signalingvia ACh, including the enzymes mediating ACh synthesisand degradation, as well as nAChR and mAChR subunits(reviewed in Kawashima and Fujii, 2004). Previous workhas identified that the a7nAChR is the primary receptorinvolved in the cholinergic anti-inflammatory pathway inbone marrow-derived immune cells (Wang et al., 2003).During bacterial infection or following injury, TNF-afunctions as an inflammatory input signal to the autonomicnervous system, which reflexively responds by stimu-lating the vagus nerve, resulting in activation of humoralanti-inflammatory responses (Tracey, 2002). Preclinicalanimal studies have shown that vagus nerve stimu-lators designed to promote release of ACh are extremelyeffective in suppressing cytokine-mediated inflammationand damage through a7nAChRs expressed on cytokine-producing cells (e.g., macrophages and dendritic cells).These observations exposed a central regulatory role forvagus nerve-mediated ACh activity in suppressing systemiccytokine release in organs and in serum, and attenuation ofdisease severity in response to a variety of inflammatorystimuli.

Keratinocytes comprise the epidermal non-neuronalcholinergic system, where the ‘‘non-neuronal cholinergicsystem’’ refers to cholinergic signaling pertaining to orcomposed of nonconducting cells of the nervous system(e.g., cells other than neurons; Grando et al., 1993, 2007).ACh released from keratinocytes in response to pathogenchallenge or wounding likely contributes to the regulation oflocal immune responses, and potentially that of infiltratingimmune cells. Keratinocytes express different nAChR sub-types during the course of epidermal differentiation, andknockout animals for several nAChR subtypes display diversephenotypes (reviewed in Grando et al., 2006). Although therole of cholinergic signaling in keratinocyte proliferation,differentiation, and migration has been well described(reviewed inGrando et al., 2006), the role in keratinocyteinnate immunity remains elusive.

Our new observations identifying cholinergic activation asa mechanism for keratinocyte AMP regulation supports theparadigm upon which the inflammatory reflex is based todescribe a global response involving a biochemical triad ofthe nervous, immune, and cutaneous systems to orchestrateanti-inflammatory pathways. The cholinergic pathway inboth neuronal and non-neuronal cells is likely activated bythe prolonged stress response in an attempt to restrainexcessive pathological inflammation (Tracey, 2007). Ulti-mately, this creates a detrimental negative feedback loop inthe epidermis that results in immunosuppresion of AMPproduction and activity, and likely other proinflammatorycytokines, to diminish the capacity of the epidermis to resistinfection (Figure 1). Therefore, it is of critical importance toidentify which nAChR subtype(s) is/are primarily involved inAMP expression. Our work in both keratinocytes and mousemodels of nAChR activation determined that the a7nAChRsubtype is a major factor involved in AMP suppression in theepidermis (Radek et al., 2010), which parallels the work byKevin Tracey and colleagues (Borovikova et al., 2000; Berniket al., 2002a, b; Tracey, 2002, 2007). Current studies in ourlaboratory are further exploring the mechanism behindcholinergic regulation of AMPs in keratinocytes. The poten-tial involvement of aberrant cholinergic activation in thedevelopment of cutaneous disease in the context of AMPsand barrier function is later discussed and is summarized inTable 1.

Stress

Efferentvagus nervestimulation

Non-neuronal

ACh

?

ACh

nAChRAMP

production

Lipidsynthesis

Local inflammationand

disease progression

Cholinergic feedback loop

NeuronalACh

Injury,infection

Figure 1. Elements of the systemic and local cholinergic anti-inflammatory

pathway proposed to augment cutaneous disease progression. Stress can

initiate a systemic response to activate the systemic cholinergic anti-

inflammatory pathway through an efferent relay and stimulate acetylcholine

(ACh) release from neuronal cells in the skin (e.g., nerve cells) to potentially

function on keratinocytes. The non-neuronal cholinergic system in

keratinocytes is capable of producing ACh that can be released into the

extracellular space to activate nicotinic ACh receptors (nAChRs).

Consequently, nAChR activation dampens antimicrobial peptide (AMP)

production and epidermal barrier integrity. Injury or infection during stress

can either initiate or exacerbate existing skin pathologies. Episodes of more

frequent or severe outbreaks of skin disease can signal a negative feedback

loop back to the brain to provoke a secondary stress response to further

suppress keratinocyte AMP and barrier function.

30 Journal of Investigative Dermatology (2012), Volume 132


EVIDENCE FOR A CUTANEOUS NON-NEURONALCHOLINERGIC SYSTEMThe non-neuronal cholinergic system in keratinocytes isprimarily regulated through the binding of the neurotrans-mitter, ACh, to nAChRs and mAChRs, classified on the basisof their respective agonists, nicotine and muscarine (Grandoet al., 2006). ACh is synthesized from coenzyme A andcholine by choline acetyltransferase and is degraded byacetylcholinesterase. Keratinocytes have the capacity tosynthesize, process, store, and release ACh that can bindto nAChRs or mAChRs expressed on their cell surface toregulate an array of cellular processes, including migration,differentiation, proliferation, and apoptosis (Grando, 1993;Denda et al., 2000) Keratinocyte cells can synthesize anaverage of 2�10�17 mol and secrete 7�10�19 mol of AChper minute (Grando et al., 1993). However, a gradient of ACh

expression throughout the epidermis is observed, with highestACh levels present in the uppermost epidermal compartment(Nguyen et al., 2001), likely because the highest amounts ofcholine acetyltransferase, the ACh-degrading enzyme, islocated predominantly in the basal layer (Johansson andWang, 1993, 2006; Klapproth et al., 1997). This intricateACh-dependent network is referred to as the non-neuronalcholinergic system (Klapproth et al., 1997; Wessler et al.,1998, 1999).

The nAChR superfamily of ligand-gated ionotropic recep-tors comprise homo- or heterotypic combinations of nine asubunits (a2–a10) and three b subunits (b2–b4; Steinbach,1990). During the course of keratinocyte differentiation, theexpression of nAChR subunits and ACh regulatory enzymes ismodified accordingly to support essential functions requiredfor the specific microenvironment of each stratified layer of

Table 1. A select list of cutaneous diseases involving aberrant cholinergic signaling

Skin disease

Factors contributing to disease pathology andcomplementary evidence to indicate involvement ofcholinergic signaling References

Atopic dermatitis kExpression of AMPs, including hbD2, hbD3, and

cathelicidin, compared with psoriatic skin

kExpression of dermcidin, compared with normal patients

Psychological stress correlated with onset/exacerbation of

disease; can be partially restored with topical

glucocorticoid receptor antagonists; glucocorticoids

suggested to modulate nAChR activation

High variability of nAChR subtype expression throughout

the stratum corneum, suggestive of erroneous cholinergic

signaling

Ellison et al., 2000; Ong et al., 2002; Raap et al., 2003;

Kurzen and Schallreuter, 2004; Rieg et al., 2005; Choi et al.,

2006; Howell et al., 2006b

Psoriasis High expression of AMPs (LL-37, hbD2, psoriasin, and

elafin/SKALP) in skin lesions; greater resistance to infection

Elevated SLURP-2, an a3nAChR signaling cofactor

Stress triggers disease onset/exacerbation; patients who

believe their psoriasis can be induced by stress exhibit an

altered HPA response, resulting in decreased cortisol levels

Autoimmune feedback loop via LL-37 binding to self DNA;

enhanced T-cell response

Smoking is the most prominent environmental risk factor

thus far described

Arnetz et al., 1985; Madsen et al., 1992; Nonomura et al.,

1994; Harder et al., 1997; Tsuji et al., 2003; O’Leary et al.,

2004; Fortune et al., 2005; Naldi et al., 2005; Richards

et al., 2005; Arredondo et al., 2006; Lande et al., 2007;

Evers et al., 2010

Mal de Meleda Genetic mutation in SLURP-1, an a7nAChR cofactor;

a7nAChR signaling indicated in disease pathology

Chimienti et al., 2003, 2007

Rosacea Hyperactive cathelicidin processing; enhanced proteolytic

processing of peptides; increased inflammation; and

ineffective microbe-killing capacity

Clinical onset/exacerbation triggered by stress

Smoking (i.e., nicotine) decreases rosacea severity

Yamasaki et al., 2006, 2007

Pemphigus Autoantibodies against cell–cell junction proteins, leading

to barrier disruption

Autoantibodies against nAChR; m cholinergic signaling

disrupts normal barrier integrity

Psychological stress correlated with disease onset/

exacerbation

Nguyen et al., 2000a,b, 2001

Palmoplantar pustulosis Irregular distribution/expression patterns of a3nAChR and

a7nAChR throughout the stratum corneum

Strongly correlated with smoking status (i.e., nicotine

exposure)

Hagforsen et al., 2002

Abbreviations: AMP, antimicrobial peptide; hbD, human beta-defensin; HPA, hypothalamic–pituitary–adrenal axis; nAChR, nicotinic acetylcholine receptor;SLURP-1, secreted mammalian Ly-6/uPAR-related protein-1; SLURP-2, secreted mammalian Ly-6/uPAR-related protein-2.




the epidermis (Kurzen et al., 2004; Figure 2). Keratinocytetransition from the granular cell to the flattened corneocyte isfacilitated by ACh to induce apoptotic secretion, which refersto the sporadic extrusion of cytoplasmic components and therelease of humectants to prevent epidermal water loss (Nguyenet al., 2001). Knockdown of a7nAChR in cultured keratino-cytes blocked terminal differentiation, which was coupled tothe increased expression of proapoptotic signaling factors.Furthermore, the inhibition of a7nAChR signaling using thepharmacological antagonist a-bungarotoxin promoted cellcycle progression (Arredondo et al., 2003). This suggests thata7nAChR signaling facilitates normal apoptotic signals togenerate the stratum corneum. Combined pharmacologicalnAChR agonist and mAChR antagonist was also found toincrease intracellular Ca2þ and trigger apoptotic secretionin vitro (Nguyen et al., 2001). Collectively, ACh signaling ismediated by distinct combinations of nAChRs as keratinocytesmature, where each subunit regulates a particular cell functionat a specific point in epidermal differentiation. Our recentobservations identifying a novel pathway for keratinocyte AMPregulation through cholinergic signaling further extends thefunctional contribution of ACh to include epidermal barrierfunction through elements of the innate immune system.

KERATINOCYTES AS KEY MEDIATORS OF CUTANEOUSBARRIER FUNCTIONPhysical maintenance of skin permeability barrier function bythe stratum corneum

The fundamental purpose of the stratum corneum is toprevent external water loss and maintain a homeostatic

microenvironment within the epidermis. The stratum cor-neum defends against pathogen invasion by creating both aphysical and chemical shield mainly comprising lipids andAMPs, respectively. Corneocytes are terminally differentiatedkeratinocyte cells located in the stratum corneum, whichcomprise both the physical and chemical barrier to protectthe skin against physical insult, ultraviolet light, chemicalexposure, and microbial invasion, as well as prevent waterand electrolyte loss into the external environment (Feingold,2009). The physical permeability barrier is created by thepresence of a hydrophobic extracellular matrix composed offree fatty acids (FFAs), cholesterol, and ceramides secretedfrom lamellar bodies, which are secretory organelles uniqueto keratinocytes (Rassner et al., 1999).

An effective permeability barrier depends on tight regula-tion and organization of cholesterols, ceramides, and FFAswithin the stratum corneum interstices (reviewed in Elias,2007; Cork et al., 2009; Ahrens et al., 2011; Borkowski andGallo, 2011). Strong cell–cell physical associations are createdby cellular desmosomal junctions and secretion of lipidspecies from lamellar bodies, which covalently bind cellmembranes and form the corneocyte lipid envelope (Behneet al., 2000). Experimentally, transepidermal water lossmeasurements are a useful biomarker for determining theintegrity and health of the stratum corneum (Kalia et al., 2000).Barrier recovery can be determined by measuring changes intransepidermal water loss values following barrier disruptionvia tape stripping or acetone treatment. Tape stripping is a typeof superficial wounding that physically removes the corneo-cyte layer (Pinkus, 1951). Contradictory to earlier reportssuggesting that acetone treatment extracted epidermal lipidswhile leaving the corneocyte layer mostly intact, it wasrecently demonstrated that acetone treatment also removes thecorneocyte layer, but only mildly extracts lipids (Rissmannet al., 2009). Elevated transepidermal water loss values can beobserved following chemical exposure, physical disruption, orseveral disease states, including but not limited to AD (Guptaet al., 2008), perioral dermatitis (Dirschka et al., 2004), andpsoriasis (Motta et al., 1994).

Initial studies aimed at determining factors that negativelyregulate epidermal lipid synthesis began by investigating theinfluence of psychological stress created by either immobi-lization or crowding of mice (Denda et al., 1998). Stressedanimals exhibited delayed barrier recovery following barrierdisruption via tape stripping indicated by higher transepider-mal water loss measurements. Conversely, pretreatment witha sedative or GC receptor antagonist prevented the alteredbarrier recovery observed (Denda et al., 1998, 2000).Psychological stress in mice was also shown to decreaseepidermal cell proliferation and differentiation while simul-taneously reducing the production and secretion of lamellarbodies (Choi et al., 2005). This effect was reversed by topicallipid application, or systemic GC receptor antagonist admin-istration (Choi et al., 2006), establishing a direct effect ofneuroendocrine mediators on biochemical pathways re-quired for normal skin barrier homeostasis. Preliminarystudies in healthy humans using two validated measures,mood states and the perceived stress scale, demonstrated the

nAChR expression

Epidermis(keratinocytes)

Dermis

α7, α9

Diff

eren

tiatio

n

AC

h

Ca2+

α3(β2/β4)α5, α9

α3β2, α9,and

α3β4, α9

Figure 2. Expression of nicotinic acetylcholine receptor (nAChR) subtypes is

dependent upon differentiation, free ACh concentration, and free Ca2þ

concentration gradients. The nAChR superfamily of ligand-gated ionotropic

receptors are differentially expressed during the course of keratinocyte

differentiation. Corneocyte cells comprising the top layer of the epidermis

(stratum corneum) express primarily a7nAChR. Transitional (prickle)

keratinocytes located in the middle layer of the epidermis express primarily

a3(b2/b4)a5nAChR. Immature (basal) keratinocytes located within the base

layer of the epidermis express primarily a3b2 or nAChRa3b4nAChR. All

keratinocyte cells appear to express a9nAChR. Activation of each nAChR

subtype is associated with a different cellular response, including apoptosis,

cornification, proliferation, migration, and differentiation. Atypical expression

of specific nAChR subtypes throughout the epidermis has been correlated

with disease. The gradients on the right represent the state of differentiation

and the concentrations of ACh and free calcium, with the greatest degree of

differentiation and highest ACh and free calcium concentrations occurring at

the interface with the external environment (e.g., stratum corneum).



first correlation between perceived psychological stress anddeteriorated cutaneous barriers, providing the first evidencethat stress-induced alterations in the epithelial barrier maytrigger the onset of clinical manifestations of skin diseasepathogenesis (Garg et al., 2001).

AMPs are multifunctional peptides that not only protectagainst microbial invasion and insult through direct pathogenkilling, but also signal to the innate immune system andcontribute to the generation and maintenance of thepermeability barrier. In addition to its antimicrobial function,one AMP in particular, murine cathelicidin (CAMP), wasfound to have a crucial role in upholding epidermal barrierhomeostasis. Mice deficient in cathelicidin (Camp�/�) have adiminished capacity to restrict barrier permeability, as theyexhibit delayed barrier recovery following tape stripping(Aberg et al., 2008). Camp�/� mice also display decreasedlamellar body formation and secretion, and profound lipidcomposition defects compared with wild-type mice (Aberget al., 2008). The role of AMPs in protection againstcutaneous infection is discussed below.

Chemical maintenance of the skin barrier by lipids and AMPs

Apart from the physical barrier comprising keratinocytes andlipids, the skin contains a chemical barrier partially consistingof lipids, but largely consisting of AMPs to restrict microbialgrowth on the surface of the skin and help maintain barrierintegrity (reviewed in Braff et al., 2005). Long-chain bases,such as sphingosines, and FFAs, such as lauric acid andsapienic acid, are derived from the partial hydrolysis ofceramide by ceramidases (Houben et al., 2006), and are themost potent antimicrobial lipid species in the stratumcorneum (Miller et al., 1988; Bibel et al., 1992). Sphingoidbases have broad antifungal and antimicrobial propertiesagainst a wide range of skin pathogens, including Gram-positive bacteria (e.g., Staphylococcus aureus, S. epidermidis,and S.s pyogenes), Gram-negative bacteria (e.g., Escherichiacoli), and yeast (e.g., Candida albicans; Drake et al., 2008).FFAs are selective against Gram-positive bacteria (e.g.,S. aureus), but not Gram-negative bacteria and yeast (e.g.,C. albicans; Bibel et al., 1989, 1992, 1995; Aly et al., 1994).The importance of understanding the contribution of FFAs tohost defense is highlighted by the fact that different combina-tions of FFAs may exert different antimicrobial potenciesagainst microbial species. For example, sapienic acid is moreeffective for the treatment of methicillin-resistant S. aureusinfection than mupirocin, the current ‘‘gold standard’’ oftreatment of methicillin-resistant S. aureus infections (Drakeet al., 2008). Overall, the lipid component of the skinpermeability barrier contributes to antimicrobial defense inparallel with AMPs.

AMPs are an evolutionarily conserved component of theinnate immune system that function at the external peripheryas the initial line of defense against invasion by pathogens(Gallo and Nizet, 2003; Ganz, 2003; Nizet and Gallo, 2003).Several families of AMPs, including cathelicidins (LL-37),defensins, and chromogranins, are expressed in immune cells(e.g., neutrophils, natural killer cells, mast cells, andmacrophages), as well as epithelial cells such as sebocytes

and keratinocytes (Agerberth et al., 2000; Di Nardo et al.,2003; Braff and Gallo, 2006; Lee et al., 2008). To date, thereexist over 20 epidermal AMPs (reviewed in Schauber andGallo, 2009). Several key epidermal AMPs, ribonuclease 7,and lysozyme are constitutively active and expressed at lowbasal levels (Harder et al., 2007). Other AMPs, such ashuman beta-defensin (hbD)-2 and hbD-3, and the CAMPcleavage product, LL-37, are only expressed in response toinflammation (Gudmundsson et al., 1996; Harder et al.,1997, 2001; Schroder and Harder, 1999; Dorschner et al.,2001; Nizet et al., 2001; Harder and Schroder, 2002; Glaseret al., 2005; Pazgier et al., 2006). Other epidermal AMPs,such as dermcidin, are sweat-gland derived (Schittek et al.,2001), whereas the S100 protein psoriasin is focally presentat varying levels throughout healthy skin (Glaser et al., 2005)and induced upon wounding (Lee and Eckert, 2007) or skininflammation (Madsen et al., 1991).

Cathelicidin is proteolytically cleaved from the largerhCAP18 pro-protein into the catalytically active peptide(human LL-37, murine CAMP) by kallikrein proteasesproduced by keratinocytes, or by neutrophil-derived protein-ase-3 (Hansson et al., 1994; Ekholm et al., 2000). In humankeratinocytes, cathelicidin expression can be augmented inresponse to pathogen challenge, vitamin D3 exposure, andwounding (Dorschner et al., 2001; Butmarc et al., 2004;Wang et al., 2004; Schauber et al., 2008), where it functionsas natural antibiotics to not only directly eradicate microbialpathogens, but also coordinate multiple components of theinnate and adaptive immune responses (Brogden et al., 2003;Kandler et al., 2006). For example, LL-37 can directlypromote the secretion of chemokines from keratinocytes(Niyonsaba et al., 2007). Furthermore, LL-37 has the capacityto enhance Toll-like receptor signaling in immune cells andinitiate a cytokine cascade responsible for bacterial recogni-tion (Niyonsaba et al., 2001), while stimulating the release ofreactive oxygen species from activated neutrophils (Zhenget al., 2007) to enhance the overall response to infection.Conflicting reports of LL-37 immunoreactivity throughout theepidermis have been described, with some demonstratinglocalization in the extracellular space between the stratumgranulosum and stratum corneum (Goo et al., 2010) andothers to the stratum basale ((Heilborn et al., 2003; Mallbriset al., 2010). As the expression of specific nAChR andmAChR subtypes varies throughout the epidermis, under-standing how ACh contributes to LL-37 expression/activitywill require further investigation into its localization.

The role of AMPs in keratinocyte innate defense has beenprimarily described using murine model systems. However, itshould be noted that although most AMPs appear to beconserved between mice and humans, several importantdifferences between expression patterns and regulatorymechanisms have been described. For example, humanshave both epidermal and neutrophil-derived defensins,whereas mice only express defensins in epithelial cells,suggesting that murine and human innate immune responsesmay be significantly different (Eisenhauer and Lehrer, 1992;Harder et al., 1997). The mechanisms regulating humanCAMP and murine Camp also vary considerable with respect




to the cathelicidin promoter. In humans, CAMP transcriptionrequires binding of the vitamin D receptor to the vitamin Dresponse element consensus sequence in the cathelicidinpromoter (Gombart et al., 2005). However, the nocturnalnature of mice likely explains the absence of the vitamin Dresponse element consensus sequence in the murine Camppromoter (Gombart et al., 2005). Recent studies in mice witha keratinocyte-specific deletion of hypoxia-inducible factor-1a indicates that Camp expression in mice is partiallyregulated by hypoxia-inducible factor-1a. Hypoxia-induciblefactor-1a-null neutrophils from these mice presented withreduced levels of cathelicidin and exhibited a decreasedphagocytic capacity of skin pathogens (Peyssonnaux et al.,2005). Together, the variability in AMP regulation betweenspecies is of critical importance and should be taken intoconsideration when interpreting data acquired from humanand mouse experiments. A more relevant model systemwould be the rhesus monkey, as defensin and cathelicidinAMPs are expressed in the same cell types and tissues as inhumans (Bals et al., 2001), and the cathelicidin promotercontains vitamin D response elements (Gombart et al., 2005),suggesting that similar mechanisms of AMP transcriptionalregulation occur between humans and nonhuman primates.

THE ROLE OF a7nAChR IN KERATINOCYE BARRIERFUNCTION: LESSONS FROM MURINE KNOCKOUTSAlthough numerous nAChR subtypes exist in the epidermis,there is evidence to suggest that similar to the systemic,neuronal cholinergic anti-inflammatory pathway, thea7nAChR subtype is likely to have a significant role in theregulation of the local cutaneous innate immune and barrierfunction. Loss of a7nAChR expression in Chrna7�/� miceresulted in profound decreases (34–56%) in genes related toterminal differentiation, including murine filaggrin, cytoker-atin-1, and cytokeratin-10. Furthermore, these mice alsodisplayed a reduction in the production of extracellularmatrix proteins collagen-1a1 and elastin, and metallopro-teinase-1 (Arredondo et al., 2002). Significant differencesbetween the histology of Chrna7�/� and Chrna7þ /þ wereobserved in 1- to 3-week-old mice, where Chrna7þ /þ skinexhibited the normal, upper compact horny layer derivedfrom dead corneocytes, 1–2 layers of live, nucleatedkeratinocytes, and a single row of suprabasal keratinocytes.In contrast, Chrna7�/� skin had a loose, widened corneocytelayer, 1–3 layers of granulated keratinocytes, and 1–3 extrarows of suprabasilar enlarged keratinocytes, a phenotypeconsistent with retention hyperkeratosis (Arredondo et al.,2002). Interestingly, Chrna7�/� mice also show a differentialgene expression of the other nAChR subunits, suggesting thata compensatory mechanism may have developed to regulatekeratinocyte cell cycle progression, apoptosis, and terminaldifferentiation genes in the absence of a7nAChR signaling(Arredondo et al., 2002).

LINKING CHOLINERGIC SIGNALING WITH EPIDERMALAMP ACTIVITY AND SUSCEPTIBILITY TO INFECTIONFor decades, it has been well documented that stressstrongly correlates with an increased incidence of infection

(Locke, 1982). Only recently have we begun to elucidatethe neuroendrocrine contribution of epithelial immunity.Stressed individuals have increased epithelial ACh levels(Schlereth et al., 2007) and are more susceptible toopportunistic infections (Wu et al., 2005; Ashcraft et al.,2008). Overactivation of the stress-response pathways, asobserved during chronic stress, results in a relative state ofimmunosuppression that can be partly attributed to activationof the cholinergic anti-inflammatory pathway describedearlier.

As the cholinergic system is directly involved in systemicimmunosuppression, it was likely that non-neuronal choli-nergic activation could suppress the local cutaneous AMPresponse in keratinocytes. Normal human epidermal kerati-nocytes (NHEKs) stimulated with the bioactive form ofvitamin D, 1,25-dihydroxy vitamin D3 (VD3), and the Toll-like receptor 2/6 agonist, macrophage-activating lipoprotein-2 (MALP-2), allows for maximal gene and protein expressionof both cathelicidin and b-defensin-2 through differentiationand bacterial stimuli, respectively (Weber et al., 2005;Schauber et al., 2006). Our initial results identified that thein vitro expression of CAMP in NHEKs was induced B100-fold over baseline levels by VD3þMALP-2, whereas thepresence of 0.01 nM ACh resulted in an B50% reduction inCAMP (Radek et al., 2010). We observed a similar suppres-sion of hbD-2 gene and protein expression by ACh comparedwith the MALP-2 alone. To further elucidate the specificreceptor subtype mediating the dampening response by ACh,additional experiments were conducted using two pharma-cological antagonists, a-bungarotoxin, which is selective forthe a7nAChR subunit, and mecamylamine, with knownselectivity for a3b4nAChR subtypes (Grando et al., 2006), oran endogenous epidermal nAChR antagonist, catestatin (Cst;Radek et al., 2008). CAMP suppression by ACh was restoredby the presence of Cst and mecamylamine, with maximalrestoration observed with a-bungarotoxin. The induction ofCAMP and b-defensin expression paralleled the capacity ofNHEKs to restrict the growth of the major skin pathogenS. aureus. Lysates of NHEKs treated with VD3þMALP-2restricted staphylococcal growth by 90% as compared withlysates from untreated cells. Treatment with 0.01 nM AChsuppressed the antimicrobial activity of VD3þMALP-2lysates to that of untreated cell lysates. This effect was againrestored by the presence of Cst or a-bungarotoxin. Thisstrongly indicates that nAChR signaling dampens the directextractable antibacterial activity in NHEKs consistent with thecritical role of AMPs in this response (Radek et al., 2010).Treatment of NHEKs with muscarine, a mAChR-specificagonist, or propranolol, a b-adrenergic pathway agonist, wasunable to suppress the induced AMP expression observedwith VD3/MALP-2 treatment, suggesting that ACh regulationof AMP expression in keratinocytes is through directactivation of nAChRs (Radek et al., 2010). The observationthat AMP suppression by ACh could be fully restored by thepresence of a-bungarotoxin, and mostly restored by theaddition of mecamylamine or Cst, suggests that ACh likelyfacilitates AMP suppression via a7nAChR, similar to theneuronal cholinergic response to infection (Tracey, 2007).



Our results indicate that the changes in direct extractableantibacterial activity in these NHEKs lysates was likely dueto, in part, decreased cathelicidin and hbD-2 expression.However, it is possible that the levels of other inducibleAMPs (e.g., ribonuclease 7 and hbD-3) or constitutive AMPs(e.g., psoriasin), normally increased by the presence ofbacterial membrane components, were also suppressed bythe presence of ACh (Harder et al., 2001; Harder andSchroder, 2002). Similar studies designed to identify changesin specific AMPs are currently in progress.

Cst was initially identified as an AMP, as it exhibited abroad-spectrum killing against several pathogens includingS. aureus, group A Streptococcus, E. coli, Pseudomonasaeruginosa, and C. albicans, as well as several varieties offilamentous fungi in vitro (Briolat et al., 2005; Radek et al.,2008). As the concentration of Cst required for microbicidalkilling was much higher than levels observed in normal skin(Radek et al., 2008), we proposed that the antimicrobialproperties may be secondary to its primary role as a natural,endogenous nAChR antagonist. Similar to several neuroen-docrine-derived molecules, such as a-melanocyte-stimulat-ing hormone, neurokinin-1, and adrenomedullin, Cst mayexhibit microbicidal activity due to their inherently small,cationic, amphipathic structure (reviewed in Radek andGallo, 2007). In our studies, we found that AMP suppressionby ACh was mostly restored by the presence of Cst, indicatingthat Cst can block the effects of ACh by functioning primarilyas an endogenous nAChR antagonist (Radek et al., 2010).

To verify the functional relevance of cholinergic activationand keratinocyte AMP regulation in vivo, we used apharmacological and genetic approach using two murinemodels of cholinergic activation (Radek et al., 2010). For thepharmacological approach, we applied a-bungarotoxin orvehicle topically to psychologically stressed mice (e.g.,insomnia stress) and assessed their susceptibility to infection.We found that stressed mice treated with a-bungarotoxinwere less susceptible to group A Streptococcus infectioncompared with vehicle-treated mice, as indicated by smallerlesion size, less surviving bacteria in lesions, and lowerdissemination into distal organs (Radek et al., 2010). Micetreated topically with nicotine (in the absence of stress)demonstrated a significant reduction in extractable AMPactivity, indicating that direct activation of nAChRs nega-tively regulates AMP expression (Radek et al., 2010). We laterdefined the role of nAChR activation in regulating AMPactivity in the skin by employing a genetic approach by usingCHGA�/� mice, which lack the endogenous nAChR antago-nist Cst and hence exhibit unopposed nAChR activation.CHGA�/� mice were significantly more susceptible to bothS. aureus and group A Streptococcus skin infection. Thesemice also exhibited a significant reduction in extractableAMP activity, which was restored by the presence of topicala-bungarotoxin. Together, these data suggested that theregulation of epidermal AMP expression and activity byACh is highly dependent on the a7nAChR subtype, but mayalso involve other subtypes to a lesser degree (Radek et al.,2010). In addition, the increase in bacterial survival followingprolonged nAChR activation was likely the result of several

immunosuppressive responses, including AMP-dependentproduction of proinflammatory cytokines and enhancementof bacterial survival and proliferation through changes in theepidermal microenvironment (Freestone and Lyte, 2008). Inhumans, a likely detrimental consequence of a diminishedepidermal immune response following nAChR activationwould lead to bacterial dissemination from initial sites ofinfection and colonization in distal tissues and organs.

Our previous investigations also provided a critical pieceof data that nAChR-mediated cathelicidin dysregulation is apotential mechanism for increased susceptibility to infection.We found that stress did not augment the susceptibility toinfection in cathelicidin-deficient (Camp�/�) or CHGA�/�

mice. This indicates that nAChR activation is a majorcontributing factor to the suppression of the AMP responseto infection during prolonged nAChR activation by stress, andthat cathelicidin is a key AMP influenced by cholinergicactivation of nAChRs (Radek et al., 2010). If other AMPs orneuroendocrine mediators were more involved in AMPsuppression, we would expect larger lesions and greaterbacterial burden in both Camp�/� and CHGA�/� micefollowing stress. In addition, cathelicidin is an autocrineregulator of the response to purinergic agonists throughmodification of receptors involved in the tissue response toinflammation or injury, indicating a direct role for cathe-licidin in cholinergic-mediated immunosuppression (Pochetet al., 2006). Thus, the global suppression of AMP activityand cathelicidin seen with nAChR activation may exacerbateimmunosuppression by blocking necessary stimulation ofdownstream immune responses that are AMP dependent.

IMPLICATIONS FOR THE ROLE OF CHOLINERGICACTIVATION IN THE DEVELOPMENT OF SKINPATHOLOGIESDelayed wound healing

AMPs have an important role in modulating wound healingresponses by increasing the expression of chemokines andchemokine receptors, stimulating epithelial proliferation, andpromoting adaptive immune responses and angiogenesis(Scott et al., 2002; Mookherjee and Hancock, 2007; Laiand Gallo, 2009). Therefore, a defective permeability barrier,arising from altered skin pH, lipid composition, or flawedcell–cell junctions, in addition to an insufficient AMPrepertoire, can result in overactive cytokine/chemokinecascades and can foster a destructive inflammatory milieu.As AMPs have a multifunctional role in regulating innateimmune responses, inflammation, and barrier function, it iscritical to maintain their homeostatic balance to allow formaximal wound repair processes.

Cholinergic signaling has been implicated in the regula-tion of re-epithelialization during tissue repair. Immediatelyfollowing barrier disruption and wounding, proinflammatorycytokines, such as TNF-a, IL-1-a, and IL-1b, are released byimmune and keratinocytic cells to stimulate cellular prolif-eration required for epidermal regeneration (e.g., re-epithe-lialization; Wood et al., 1992; Nickoloff and Naidu, 1994). Atthe onset of re-epithelialization, keratinocyte cell–cell des-mosomal and hemidesmosomal connections are dissolved,




followed by the development of peripheral cytoplasmic actinfilaments (Gabbiani et al., 1978; Goliger and Paul, 1995).Concurrently, keratinocyte integrin expression increases toallow for lateral migration across the wound bed (Clark,1990; Clark et al., 1996), which ceases when keratinocytescollide to form new cell–cell junctions and ensue withterminal differentiation (Adams and Watt, 1990; Martin,1997; Singer and Clark, 1999). Cholinergic signaling byseveral nAChRs was found to be directly involved inkeratinocyte chemotaxis and chemokinesis, respectively. Inin vitro migration assays, reorganization of a7nAChRs alongthe leading edge of keratinocytes occurred before theconventional crescent-shaped formation and directionalmigration of human keratinocytes in the presence of achemoattractant, whereas several nAChRs were found tolocalize with integrin-b1 expressed on the keratinocyte cellmembrane (Chernyavsky et al., 2004). Experimental evidencealso suggests that a9nAChR activation leads to downstreamphosphorylation of cytoskelatal proteins required for cellmotility (Szonyi et al., 1999) and keratinocyte cell–celladhesion (Nguyen et al., 2000a). Collectively, this suggeststhat ACh likely contributes to both the initiation andtermination of re-epithelialization during wound repair tore-establish the epidermal permeability barrier. (Grandoet al., 2006).

AD and psoriasis

A key factor in a myriad of cutaneous inflammatory diseasesincludes AMPs. Up to 90% of AD skin lesions are typicallycolonized by S. aureus (Leung, 2003). In the past, greatersusceptibility was attributed to an overall decrease in AMPexpression, as it was demonstrated that cathelicidin (LL-37),hbD-2, and hbD-3 levels were all decreased in AD skin whencompared with psoriatic skin (Ong et al., 2002). Therefore, itwas presumed that the overall decrease in AMP expressiondirectly resulted in suppressed antimicrobial potential and acompromised barrier. More recently, a study with a largepatient cohort of atopic excema, psoriatic, and normalcontrols demonstrated that atopic excema lesions have LL-37 levels similar to that of normal, non-lesional skin, andhealthy skin from normal donors (Gambichler et al., 2008).Psoriasin secretion was also profoundly increased (up to1,500-fold) when comparing AD skin with healthy skin fromthe same individual (Glaser et al., 2009). These conflictingreports highlight the importance of understanding the factorsthat contribute to AMP regulation. Although the baselinelevels of AMP expression in AD patients may be equal to orhigher than normal, uninjured skin, the antimicrobialpotential of these AMPs remains to be determined. It hasbeen demonstrated that the increased LL-37 expressionfollowing injury observed in healthy volunteers was notobserved in AD lesions (Mallbris et al., 2010), suggesting thatalthough the baseline level of LL-37 among normal, atopic,and psoriatic skin appears about the same, increasedmicrobial colonization likely arises from the inability toinduce LL-37 following wounding or in the presence of activeinfection. Furthermore, it has been shown that Th2 cytokines,IL-4 and IL-13, directly downregulate AMP expression in

cultured human keratinocytes (Ong et al., 2002; Nomuraet al., 2003). In parallel, AD patients have an elevated Th2cytokine milieu, at least in comparison with psoriatic skin(Nomura et al., 2003). This altered cytokine milieu isbelieved to directly suppress the AMP response to injuryand infection in the skin, providing further evidence that theincreased susceptibility to infection observed in AD patientsis multifaceted, arising from defects in innate antimicrobialresponses, as well as adaptive immunity and cytokineresponses. These observations emphasize the importance ofidentifying common mechanisms behind AMP induction andsuppression that may yield unconventional therapeuticinterventions that can be customized to treat cutaneousdisorders with AMP dysregulation as a major contributingfactor.

AD is a common skin disease in which clinical onset isusually triggered by environmental or psychological stress(Raap et al., 2003). Variable differences in expression of AChreceptor subunits have been observed in patients with AD, aswell as in response to minimal trauma (Kurzen andSchallreuter, 2004). Patients with AD have a significantincrease (14-fold) in ACh expression in the skin, as comparedwith normal individuals (Wessler et al., 2003). Moreover,ACh contributes to the pathophysiological characteristics ofAD, such as increased pruritis, or itch, due to its ability tocause vasodilatation and the wheal and flare reaction (Heyeret al., 1997; Rukwied and Heyer, 1999; Wessler et al., 2003).

NC/Nga mice exhibit increased serum IgE, scaly AD-likelesions, and mast cell accumulation similar to serum and skinlesions of AD patients (Suto et al., 1999), and these mice arefrequently used as a murine model of AD. Psychologicalstress was shown to trigger the development of thesehistopathologies in NC/Nga mice, where pretreatment withcorticotropin-releasing hormone (CRH), a neurogenic peptidereleased in response to cholinergic stimulation, completelyprevented the onset of AD lesions in these animals (Amanoet al., 2008). Epidermal cells expressing CRH receptors mayhave a role in the mechanism by which stress-inducedcholinergic activation triggers the onset and exacerbation ofAD (Amano et al., 2008). These data strongly suggest a directrole for ACh and cholinergic signaling in the development ofAD pathophysiology.

Lesions from patients with psoriasis show epidermalhyperplasia and increased AMP expression, excess inflam-mation, and a diminished susceptibility to infection (Madsenet al., 1991; Ong et al., 2002; Rieg et al., 2005; Howell et al.,2006a). Extensive studies in humans indicate a directinteraction between smoking (e.g., exposure to nicotine)and the development of psoriatic arthritis through geneticpolymorphisms in the gene encoding for the Th2 cytokine, IL-13 (Duffin et al., 2009). The clinical onset and exacerbationof psoriasis is associated with environmental and psycholo-gical stress, as well as increased CRH levels (Kim et al.,2007). As CRH release is dependent upon cholinergicactivation in the hypothalamus (Karanth et al., 1999), it ispossible that the increased CRH levels are a result ofenhanced cholinergic regulation in the skin. Furthermore,it was recently demonstrated that secreted mammalian



Ly-6/uPAR-related protein-1 is upregulated 3-fold in hyper-proliferative skin from patients with psoriasis (Tsuji et al.,2003; Arredondo et al., 2006). Secreted mammalian Ly-6/uPAR-related protein-2 appears to preferentially bind thea3nAChR subtype, which indicates a potential role for thisnAChR subtype in the progression of psoriasis (Arredondoet al., 2006).

Pemphigus

Pemphigus is a rare, blistering autoimmune skin disorder thattargets the skin and mucous membranes, and is mediated bythe production of autoantibodies against keratinocyte anti-gens, including desmogleins, proteins important for cell–celljunctions, and also several nAChRs (reviewed in Grando,2000). Pemphigus patients have reported that an emotional,stressful event is typically a worsening or precipitating factorof pemphigus (Morell-Dubois et al., 2008). In neonatal mice,nAChR antibodies can induce pemphigus-like lesions,suggesting that these receptors may contribute to thepathogenesis of Pemphigus vulgaris (Nguyen et al., 2000b).The role of AMPs in this disease has yet to be elucidated.However, the experimental evidence described here suggeststhat it is highly likely that the result of desensitized nAChRsignaling in keratinocytes could potentially augment cuta-neous inflammation and/or AMP expression to furtheraggravate this disease.

Mal de Meleda

Mal de Meleda is an autosomal recessive inflammatory andkeratosis palmoplantar disorder, resulting from mutation ofsecreted mammalian Ly-6/uPAR-related protein-1 (Arredondoet al., 2005), which functions as a cofactor to fine-tunecholinergic signaling in keratinocytes. Secreted mammalianLy-6/uPAR-related protein-1 facilitates keratinization andapoptosis (Mastrangeli et al., 2003) and shows high amino-acid composition homology with a-bungarotoxin to functionas an allosteric agonist to potentiate acetlycholine-mediatedsignaling via the a7nAChR subtype (Arredondo et al., 2005;Grando, 2008).

Rosacea

Patients with rosacea present with excessive facial skininflammation determined to be triggered, in part, byincreased expression of cutaneous serine proteases, kallikrein5 and 7. Overactive serine protease activity in the stratumcorneum results in shorter LL-37 peptides possessing im-munostimulatory activity due to additional posttranslationalcleavage (Yamasaki et al., 2007). Clinical observation hasindicated that rosacea symptoms improve when patients areactively smoking (Mills, 1998), whereas symptoms appear toworsen upon cessation of nicotine exposure. This insinuatesthat nAChR activation, at least in some cases, may serve aprotective role to prevent inflammation.

Netherton’s syndrome

Patients with Netherton’s syndrome, a genetically conferreddisease, are predisposed to AD and demonstrate a higher riskfor cutaneous infection (Stryk et al., 1999; Chao et al., 2005).

Patients with Netherton’s syndrome carry a mutation in theserine protease inhibitor Kazal-type 5 gene, which codes forlymphoepithelial Kazal-type-related inhibitor (Magert et al.,1999), a serine proteinase required for the processing ofkallikrein proteases into active form (Schechter et al., 2005).Loss of lymphoepithelial Kazal-type-related inhibitor maycontribute to AD predisposition by altering expression of bothstructural/cell–cell junction proteins, therefore destabilizingthe epidermal barrier. In parallel, the observed increase in theexpression of kallikrein proteases in this disease likelyaugments the proteolytic processing of AMPs and, conse-quently, inflammation (as indicated for rosacea above).

Vitiligo

Vitiligo is a chronic skin disorder in which loss of pigmentresults in irregular white patches. The abundance ofacetylcholinesterase, the enzyme responsible for degradingACh, is considerably reduced in vitiliginous skin duringdepigmentation, resulting in elevated ACh levels and con-comitant pruritis (Iyengar, 1989). Although cholinergicsignaling has not been directly correlated with this disease,high ACh levels make it likely that excessive nAChR signalingcontributes to disease pathology.

BOTULINUM TOXIN-A AS A POTENTIALANTI-CHOLINERGIC THERAPEUTIC FOR TREATINGSKIN DERMATOSESBotulinum toxin-A inhibits the release of ACh from pre-synaptic vesicles (Hallett, 2000; Huang et al., 2000), makingit a potential therapy for inflammatory diseases correlatedwith hyperactive nAChR signaling. Several clinical trials havealready reported positive outcomes. Subcutaneous injectionof botulinum toxin-A in numerous AD clinical trials improvedpruritis symptoms (Swartling et al., 2002; Wollina andKaramfilov, 2002). Successful treatments of acne, notalgiaparesthetica, and inverse psoriasis have also been described(Diamond and Jankovic, 2006). In a double-blind, placebo-controlled study of human patients with normal skin,subcutaneous injection of botulinum toxin-A decreased theitch intensity, blood flow, and neurogenic inflammation inresponse to the histamine prick test (Gazerani et al., 2009).Thus, blockade of cholinergic signaling in targeted regions ofthe skin may help alleviate clinical manifestations ofcutaneous diseases associated with ACh-mediated symptoms.

FUTURE DIRECTIONSOn the basis of the clinical and experimental evidencepresented in this review, it is evident that the cholinergicanti-inflammatory pathway spans several macro- and micro-environments to provide a universal network to controlinflammation. In the skin, it remains to be determinedwhether neuronal ACh released from neural components inthe skin is functioning in parallel with or independently fromnon-neuronal ACh released from keratinocytes to regulateAMP expression and activity in the epidermis. Our in vitroresults indicate that the epidermis comprises all of thenecessary components to autonomously dampen the epider-mal AMP response to infection. The local coordination of ACh




signaling within the epidermis makes topical therapeutics apromising solution for directly targeting the cells responsiblefor ACh-dependent downregulation of AMP expression, andeliminates the need to systemically target the vagus nerve.However, it cannot be ruled out that the immunosuppressiveeffects of nAChR activation are not multifaceted, wheretopical agonists or antagonists may activate other resident skincells, including Langerhans and gd-T cells, or sensory nervecells to further inhibit inflammatory pathways in the skin.Elucidating the contribution of each particular nAChR subtypeto keratinocyte AMP production and synthesis of structuralelements necessary for barrier function is critical for devel-oping unique therapeutic strategies for cutaneous diseaseswith AMP function as a central component. Moreover, a highdegree of cross talk likely exists between the three branches ofthe stress response. Dissecting the contributions of eachparticular stress pathway will be a vital factor in thedevelopment of therapies. A possibility exists that specificconcentrations of ACh must be appropriately maintainedwithin the stratified epidermis to maintain AMP expressionand activity during periods of acute stress, which is likelyskewed during periods of prolonged stress. This imbalancewill consequently augment nAChR activation, resulting inimmunosuppression and compromised barrier function. Col-lectively, a complex network of cellular and biochemicalmechanisms facilitate the pathogenesis of skin disease uponactivation of the stress response. Activation of the hypotha-lamic–pituitary–adrenal axis and the adrenergic system are themost extensively studied pathways to date, but a greateremphasis must be made on the role of the cholinergic systemin maintaining the chemical and physical skin barrier.Ultimately, these investigations will yield new strategies inthe treatment of inflammatory skin disorders.

CONFLICT OF INTERESTThe authors state no conflict of interest.

ACKNOWLEDGMENTSData from Radek et al. presented in this article were supported by thefollowing VA and NIH/NIAID funds: HHSN26620040029C, ADB contractsN01-AI-40029AI48176, AR45676, AI052453 (awarded to RL Gallo), andNIH F32-AR054220-01A2 (awarded to KAR). Current Loyola UniversityMedical Center research support is provided by NIH/NIAAA 1P30AA019373-01 and 5T32AA013527-09.

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