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Cholinergic Regulation of Keratinocyte Innate Immunity and Permeability Barrier Integrity: New Perspectives in Epidermal Immunity and Disease

      Several cutaneous inflammatory diseases and their clinical phenotypes are recapitulated in animal models of skin disease. However, the identification of shared pathways for disease progression is limited by the ability to delineate the complex biochemical processes fundamental for development of the disease. Identifying common signaling pathways that contribute to cutaneous inflammation and immune function will facilitate better scientific and therapeutic strategies to span a variety of inflammatory skin diseases. Aberrant antimicrobial peptide (AMP) expression and activity is one mechanism behind the development and severity of several inflammatory skin diseases and directly influences the susceptibility of skin to microbial infections. Our studies have recently exposed a newly identified pathway for negative regulation of AMPs in the skin by the cholinergic anti-inflammatory pathway via acetylcholine (ACh). The role of ACh in AMP regulation of immune and permeability barrier function in keratinocytes is reviewed, and the importance for a better comprehension of cutaneous disease progression by cholinergic signaling is discussed.

      Abbreviations

      ACh
      acetylcholine
      AD
      atopic dermatitis
      AMP
      antimicrobial peptide
      CAMP
      cathelicidin antimicrobial peptide (human)
      CRH
      corticotropin-releasing hormone
      Cst
      catestatin
      FFA
      free fatty acid
      GC
      glucocorticoid
      hβD-2
      human beta-defensin
      mAChR
      muscarinic ACh receptor
      MALP-2
      macrophage-activating lipoprotein-2
      nAChR
      nicotinic ACh receptor
      NHEK
      normal human epidermal keratinocyte
      TNF-α
      tumor necrosis factor-α
      VD3
      1,25-dihydroxy vitamin D3

      Introduction and Scope of the Problem

      Inflammatory skin diseases are a major cause of morbidity and mortality for patients, where the atypical cytokine and cellular milieu creates a reservoir for cutaneous infection and diminished barrier function. Loss of skin permeability barrier function leads to excessive water loss and penetration of irritants, allergens, and microbes. Moreover, barrier function studies demonstrate that physical cutaneous permeability barrier (e.g., electrolyte and water movement) and the chemical antimicrobial barrier (e.g., antimicrobial peptide (AMP)) are codependent (
      • Ahrens K.
      • Schunck M.
      • Podda G.F.
      • et al.
      Mechanical and metabolic injury to the skin barrier leads to increased expression of murine beta-defensin-1, -3, and -14.
      ). Studies have shown that a diminished permeability barrier function is associated with reduced lipid synthesis and AMP production (
      • Aberg K.M.
      • Man M.Q.
      • Gallo R.L.
      • et al.
      Co-regulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers.
      ). Injury and infection function as a trigger for the development of numerous skin diseases by providing a stimulus for activation of distinct inflammatory pathways. Infections caused by viral, bacterial, or fungal agents can not only initiate disease directly, but also further exacerbate inflammatory skin diseases. Although staphylococcal and streptococcal infections often precede the development of psoriasis, which is generally considered the most stress-responsive skin disease compared with atopic dermatitis (AD) and acne vulgaris (
      • Griffiths C.E.
      • Richards H.L.
      Psychological influences in psoriasis.
      ), psoriatic plaques are generally resistant to infection (
      • Raychaudhuri S.P.
      • Raychaudhuri S.K.
      Relationship between kinetics of lesional cytokines and secondary infection in inflammatory skin disorders: a hypothesis.
      ). AD is also initiated and worsened by prolonged psychological stress and injury or infection; however, lesions from these patients tend to be more susceptible to colonization and infection following development of the disease (
      • Ong P.Y.
      • Ohtake T.
      • Brandt C.
      • et al.
      Endogenous antimicrobial peptides and skin infections in atopic dermatitis.
      ;
      • Nomura I.
      • Goleva E.
      • Howell M.D.
      • et al.
      Cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes.
      ). Thus, the inflammatory pathways activated in response to primary injury or infection further exacerbate the barrier defect to facilitate disease progression. The current paradigm tends to categorize inflammatory responses as either pro- or anti-inflammatory, but fails to recognize the unifying concepts that initiate these injury and infection response patterns.
      Both anecdotal and clinical evidence for the role of perceived stress in the pathophysiology of skin diseases has existed for decades (
      • Kimyai-Asadi A.
      • Usman A.
      The role of psychological stress in skin disease.
      ;
      • Picardi A.
      • Abeni D.
      Stressful life events and skin diseases: disentangling evidence from myth.
      ). This effect is 2-fold, where stress can initiate the onset of symptoms and, at a later time, the disease manifestations can become a major source of stress for the patients to further exacerbate the clinical manifestations of their disease. Although the influence of glucocorticoids (GCs) and catecholamines on skin barrier and innate immune function have been more thoroughly studied in the context of skin infection and disease progression, less attention has been given to the role of the cholinergic system on epidermal permeability barrier and innate immune function. Our recent investigations into how the neuroendocrine system influences epidermal AMP function has uncovered a newly recognized pathway for the negative regulation of AMPs in the skin through the activation of nicotinic acetylcholine (ACh) receptors (nAChRs) by ACh. Here, we will review the current mechanisms related to cholinergic signaling believed to trigger stress-induced skin disorders by modulating keratinocyte innate immune and barrier function, with a focus on AMP regulation.

      The HPA Axis, Adrenergic Pathway, and the Cholinergic Pathway of the Stress Response

      Within the last few decades, a significant amount of evidence suggests that an elaborate communication network exists between the nervous, endocrine, and innate immune systems to delicately balance the response to microbial invasion and/or injury. The immediate host reaction to physical insult, such as wounding or pathogen challenge, or to psychological stress is the rapid activation of the evolutionarily conserved “fight or flight” response. This initial danger-associated response promotes inflammation and immune cell infiltration to combat an infectious threat or injury, which is then followed by immunosuppression via neuroendocrine pathways to return to homeostasis (
      • Tracey K.J.
      The inflammatory reflex.
      ). Once the stressors are eliminated, the biochemical profile of the organism is expected to return to normal physiological homeostasis. However, prolonged activation of neuroendocrine responses can directly promote systemic immunosuppression (reviewed in
      • Dhabhar F.S.
      Acute stress enhances while chronic stress suppresses skin immunity. The role of stress hormones and leukocyte trafficking.
      ).
      Stress leads to activation of the parasympathetic and sympathetic nervous systems, as well as the hypothalamic–pituitary–adrenal axis (HPA). Activation of the parasympathetic nervous system results in the release of ACh from nerve fibers innervating the organs, resulting in activation of nAChR and/or muscarinic ACh receptors (mAChRs). Activation of the sympathetic nervous system results in the release of epinephrine/norepinephrine, activating α- or β-adrenergic receptors. Parasympathetic nervous system and sympathetic nervous system activation mediates a variety of immunological and homeostatic functions, including changes in cellular proliferation, differentiation, and migration (
      • Maestroni G.J.
      Sympathetic nervous system influence on the innate immune response.
      ; reviewed in
      • Slominski A.
      • Wortsman J.
      Neuroendocrinology of the skin.
      ). Activation of the hypothalamic–pituitary–adrenal axis stimulates the release of several neuroendocrine mediators, such as GCs (e.g., cortisol), into the bloodstream, which function upon specific cell populations and tissues to generate an immunosuppressive effect and return proinflammatory mediators to normal, baseline levels (
      • Herman J.P.
      • Cullinan W.E.
      Neurocircuitry of stress: central control of the hypothalamo-pituitary-adrenocortical axis.
      ). However, prolonged or chronic stress tends to be immunosuppressive, in part, by dampening cytokine production and immune cell phagocytic capacity (
      • Abang M.M.
      • Baum M.
      • Ceccarelli S.
      • et al.
      Differential selection on rhynchosporium secalis during parasitic and saprophytic phases in the barley scald disease cycle.
      ;
      • Ashcraft K.A.
      • Bonneau R.H.
      Psychological stress exacerbates primary vaginal herpes simplex virus type 1 (HSV-1) infection by impairing both innate and adaptive immune responses.
      ;
      • Dhabhar F.S.
      • Saul A.N.
      • Daugherty C.
      • et al.
      Short-term stress enhances cellular immunity and increases early resistance to squamous cell carcinoma.
      ). This pathway was later coined the “cholinergic anti-inflammatory pathway” after efferent activity in the vagus nerve was found to inhibit local proinflammatory production of cytokines by signaling through the innervated regions of major organs involved in the response to endotoxin, without affecting the production of anti-inflammatory cytokines (
      • Tajima T.
      • Endo H.
      • Suzuki Y.
      • et al.
      Immobilization stress-induced increase of hippocampal acetylcholine and of plasma epinephrine, norepinephrine and glucose in rats.
      ;
      • Bernik T.R.
      • Friedman S.G.
      • Ochani M.
      • et al.
      Cholinergic antiinflammatory pathway inhibition of tumor necrosis factor during ischemia reperfusion.
      ;
      • Saeed R.W.
      • Varma S.
      • Peng-Nemeroff T.
      • et al.
      Cholinergic stimulation blocks endothelial cell activation and leukocyte recruitment during inflammation.
      ;
      • Pavlov V.A.
      • Ochani M.
      • Gallowitsch-Puerta M.
      • et al.
      Central muscarinic cholinergic regulation of the systemic inflammatory response during endotoxemia.
      ). Our studies recently revealed that cholinergic activation in non-neuronal keratinocytes is a major pathway involved in the suppression of keratinocyte antimicrobial activity, and is the focus of this review.

      Linking Stress Responses to Epidermal Innate Immune Function During Injury or Infection

      Dynamic cross talk is required between the nervous, endocrine, and immune systems to regulate inflammatory processes across a vast array of cell types and tissues (
      • Elenkov I.J.
      • Wilder R.L.
      • Chrousos G.P.
      • et al.
      The sympathetic nerve—an integrative interface between two supersystems: the brain and the immune system.
      ;
      • Tracey K.J.
      The inflammatory reflex.
      ). Many of the neuroendocrine ligands and ligand receptors derived from all three branches of the stress-response pathways participate in cellular communication and are expressed by immune, nerve, and epidermal cells. For example, macrophages release tumor necrosis factor (TNF)-α during injury or infection to amplify the inflammatory response by stimulating the release of proinflammatory mediators, including IL-1, reactive oxygen species, nitric oxide, and eicosanoids (
      • Wood L.C.
      • Jackson S.M.
      • Elias P.M.
      • et al.
      Cutaneous barrier perturbation stimulates cytokine production in the epidermis of mice.
      ;
      • Nickoloff B.J.
      • Naidu Y.
      Perturbation of epidermal barrier function correlates with initiation of cytokine cascade in human skin.
      ). During the initial fight or flight response, all three pathways of the stress response are coordinately activated to modulate inflammation. For example, epinephrine/norepinephrine can block macrophage activation and dampen production of TNF-α and other proinflammatory cytokines (
      • Chrousos G.P.
      The stress response and immune function: clinical implications. The 1999 Novera H. Spector Lecture.
      ;
      • Molina P.E.
      Noradrenergic inhibition of TNF upregulation in hemorrhagic shock.
      ;
      • Molina P.E.
      • Bagby G.J.
      • Stahls P.
      Hemorrhage alters neuroendocrine, hemodynamic, and compartment-specific TNF responses to LPS.
      ). Alternatively, epinephrine/norepinephrine can induce the release of the anti-inflammatory cytokine, IL-10, from monocytes (
      • van der Poll T.
      • Coyle S.M.
      • Barbosa K.
      • et al.
      Epinephrine inhibits tumor necrosis factor-alpha and potentiates interleukin 10 production during human endotoxemia.
      ;
      • Woiciechowsky C.
      • Asadullah K.
      • Nestler D.
      • et al.
      Sympathetic activation triggers systemic interleukin-10 release in immunodepression induced by brain injury.
      ). In parallel, ACh has the capacity to inhibit macrophage activation and release of proinflammatory cytokines, such as TNF-α, IL-1, and high-mobility group protein B1 (
      • Borovikova L.V.
      • Ivanova S.
      • Zhang M.
      • et al.
      Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin.
      ), ultimately limiting tissue damage caused by excessive inflammation.
      Psychological stress was initially found to delay wound healing and diminish epidermal barrier homeostasis (
      • Kiecolt-Glaser J.K.
      • Marucha P.T.
      • Malarkey W.B.
      • et al.
      Slowing of wound healing by psychological stress.
      ;
      • Denda M.
      • Tsuchiya T.
      • Hosoi J.
      • et al.
      Immobilization-induced and crowded environment-induced stress delay barrier recovery in murine skin.
      ;
      • Marucha P.T.
      • Kiecolt-Glaser J.K.
      • Favagehi M.
      Mucosal wound healing is impaired by examination stress.
      ;
      • Garg A.
      • Chren M.M.
      • Sands L.P.
      • et al.
      Psychological stress perturbs epidermal permeability barrier homeostasis: implications for the pathogenesis of stress-associated skin disorders.
      ;
      • Rojas I.G.
      • Padgett D.A.
      • Sheridan J.F.
      • et al.
      Stress-induced susceptibility to bacterial infection during cutaneous wound healing.
      ), mediated through the activation of both GC and adrenergic signaling pathways in the skin. Mice subjected to insomnia stress for 3 days demonstrated a decrease in the protein abundance of two major skin AMPs, cathelicidin antimicrobial peptide (human; CAMP) and mouse beta defensin-3, compared with unstressed mice. Moreover, these mice were more susceptible to cutaneous group A Streptococcus infection, as indicated by larger skin lesions compared with unstressed mice. The adverse affect of stress on AMP expression and lesion size was reversed by systemic administration of RU-486, a GC/progesterone antagonist, suggesting that GC-receptor-mediated activation was involved in the suppression of AMP expression (
      • Aberg K.M.
      • Radek K.A.
      • Choi E.H.
      • et al.
      Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice.
      ). These studies were the first to establish a direct connection between stress and AMP expression in the context of cutaneous infection. Earlier studies showed that catecholamines promote the release of fully processed active AMPs from the skin surface (
      • Benson B.J.
      • Hadley M.E.
      In vitro characterization of adrenergic receptors controlling skin gland secretion in two anurans Rana pipiens and Xenopus laevis.
      ;
      • Zasloff M.
      Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor.
      ). More recent observations in keratinocytes demonstrated that β-adrenergic activation impairs cell motility and wound closure, indicating a pathway by which stress impairs cutaneous healing (
      • Pullar C.E.
      • Rizzo A.
      • Isseroff R.R.
      beta-Adrenergic receptor antagonists accelerate skin wound healing: evidence for a catecholamine synthesis network in the epidermis.
      ;
      • Sivamani R.K.
      • Pullar C.E.
      • Manabat-Hidalgo C.G.
      • et al.
      Stress-mediated increases in systemic and local epinephrine impair skin wound healing: potential new indication for beta blockers.
      ). Together, mediators of the stress response can directly or indirectly influence several key components of epidermal defense.

      The Evolution of the Cholinergic “Inflammatory Reflex”

      During embryonic development, the skin and nervous system are both derived from the neuroectoderm. Consequently, many key factors that influence neuronal central nervous system activities also contribute to non-neuronal cellular function, as most immune and epidermal cells express several components of the cholinergic system (reviewed in
      • Radek K.A.
      Antimicrobial anxiety: the impact of stress on antimicrobial immunity.
      ). ACh can facilitate the communication between immune and epidermal cells to mount a physiological response to injury or infection. Langerhans cells, mast cells, neutrophils, and macrophages all express various components required for cholinergic signaling via ACh, including the enzymes mediating ACh synthesis and degradation, as well as nAChR and mAChR subunits (reviewed in
      • Kawashima K.
      • Fujii T.
      Expression of non-neuronal acetylcholine in lymphocytes and its contribution to the regulation of immune function.
      ). Previous work has identified that the α7nAChR is the primary receptor involved in the cholinergic anti-inflammatory pathway in bone marrow-derived immune cells (
      • Wang H.
      • Yu M.
      • Ochani M.
      • et al.
      Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation.
      ). During bacterial infection or following injury, TNF-α functions as an inflammatory input signal to the autonomic nervous system, which reflexively responds by stimulating the vagus nerve, resulting in activation of humoral anti-inflammatory responses (
      • Tracey K.J.
      The inflammatory reflex.
      ). Preclinical animal studies have shown that vagus nerve stimulators designed to promote release of ACh are extremely effective in suppressing cytokine-mediated inflammation and damage through α7nAChRs expressed on cytokine-producing cells (e.g., macrophages and dendritic cells). These observations exposed a central regulatory role for vagus nerve-mediated ACh activity in suppressing systemic cytokine release in organs and in serum, and attenuation of disease severity in response to a variety of inflammatory stimuli.
      Keratinocytes comprise the epidermal non-neuronal cholinergic system, where the “non-neuronal cholinergic system” refers to cholinergic signaling pertaining to or composed of nonconducting cells of the nervous system (e.g., cells other than neurons;
      • Grando S.A.
      • Kist D.A.
      • Qi M.
      • et al.
      Human keratinocytes synthesize, secrete, and degrade acetylcholine.
      ,
      • Grando S.A.
      • Kawashima K.
      • Kirkpatrick C.J.
      • et al.
      Recent progress in understanding the non-neuronal cholinergic system in humans.
      ). ACh released from keratinocytes in response to pathogen challenge or wounding likely contributes to the regulation of local immune responses, and potentially that of infiltrating immune cells. Keratinocytes express different nAChR subtypes during the course of epidermal differentiation, and knockout animals for several nAChR subtypes display diverse phenotypes (reviewed in
      • Grando S.A.
      • Pittelkow M.R.
      • Schallreuter K.U.
      Adrenergic and cholinergic control in the biology of epidermis: physiological and clinical significance.
      ). Although the role of cholinergic signaling in keratinocyte proliferation, differentiation, and migration has been well described (reviewed in
      • Grando S.A.
      • Pittelkow M.R.
      • Schallreuter K.U.
      Adrenergic and cholinergic control in the biology of epidermis: physiological and clinical significance.
      ), the role in keratinocyte innate immunity remains elusive.
      Our new observations identifying cholinergic activation as a mechanism for keratinocyte AMP regulation supports the paradigm upon which the inflammatory reflex is based to describe a global response involving a biochemical triad of the nervous, immune, and cutaneous systems to orchestrate anti-inflammatory pathways. The cholinergic pathway in both neuronal and non-neuronal cells is likely activated by the prolonged stress response in an attempt to restrain excessive pathological inflammation (
      • Tracey K.J.
      Physiology and immunology of the cholinergic antiinflammatory pathway.
      ). Ultimately, this creates a detrimental negative feedback loop in the epidermis that results in immunosuppresion of AMP production and activity, and likely other proinflammatory cytokines, to diminish the capacity of the epidermis to resist infection (Figure 1). Therefore, it is of critical importance to identify which nAChR subtype(s) is/are primarily involved in AMP expression. Our work in both keratinocytes and mouse models of nAChR activation determined that the α7nAChR subtype is a major factor involved in AMP suppression in the epidermis (
      • Radek K.A.
      • Elias P.M.
      • Taupenot L.
      • et al.
      Neuroendocrine nicotinic receptor activation increases susceptibility to bacterial infections by suppressing antimicrobial peptide production.
      ), which parallels the work by Kevin Tracey and colleagues (
      • Borovikova L.V.
      • Ivanova S.
      • Zhang M.
      • et al.
      Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin.
      ;
      • Bernik T.R.
      • Friedman S.G.
      • Ochani M.
      • et al.
      Cholinergic antiinflammatory pathway inhibition of tumor necrosis factor during ischemia reperfusion.
      ,
      • Bernik T.R.
      • Friedman S.G.
      • Ochani M.
      • et al.
      Pharmacological stimulation of the cholinergic antiinflammatory pathway.
      ;
      • Tracey K.J.
      The inflammatory reflex.
      ,
      • Tracey K.J.
      Physiology and immunology of the cholinergic antiinflammatory pathway.
      ). Current studies in our laboratory are further exploring the mechanism behind cholinergic regulation of AMPs in keratinocytes. The potential involvement of aberrant cholinergic activation in the development of cutaneous disease in the context of AMPs and barrier function is later discussed and is summarized in Table 1.
      Figure thumbnail gr1
      Figure 1Elements 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.
      Table 1A select list of cutaneous diseases involving aberrant cholinergic signaling
      Skin diseaseFactors contributing to disease pathology and complementary evidence to indicate involvement of cholinergic signalingReferences
      Atopic dermatitis↓Expression of AMPs, including hβD2, hβD3, and cathelicidin, compared with psoriatic skin

      ↓Expression 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 J.A.
      • Patel L.
      • Ray D.W.
      • et al.
      Hypothalamic-pituitary-adrenal function and glucocorticoid sensitivity in atopic dermatitis.
      ;
      • Ong P.Y.
      • Ohtake T.
      • Brandt C.
      • et al.
      Endogenous antimicrobial peptides and skin infections in atopic dermatitis.
      ;
      • Raap U.
      • Werfel T.
      • Jaeger B.
      • et al.
      [Atopic dermatitis and psychological stress].
      ;
      • Kurzen H.
      • Schallreuter K.U.
      Novel aspects in cutaneous biology of acetylcholine synthesis and acetylcholine receptors.
      ;
      • Rieg S.
      • Steffen H.
      • Seeber S.
      • et al.
      Deficiency of dermcidin-derived antimicrobial peptides in sweat of patients with atopic dermatitis correlates with an impaired innate defense of human skin in vivo.
      ;
      • Choi E.H.
      • Demerjian M.
      • Crumrine D.
      • et al.
      Glucocorticoid blockade reverses psychological stress-induced abnormalities in epidermal structure and function.
      ;
      • Howell M.D.
      • Wollenberg A.
      • Gallo R.L.
      • et al.
      Cathelicidin deficiency predisposes to eczema herpeticum.
      PsoriasisHigh expression of AMPs (LL-37, hβD2, psoriasin, and elafin/SKALP) in skin lesions; greater resistance to infection

      Elevated SLURP-2, an α3nAChR 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 B.B.
      • Fjellner B.
      • Eneroth P.
      • et al.
      Stress and psoriasis: psychoendocrine and metabolic reactions in psoriatic patients during standardized stressor exposure.
      ;
      • Madsen P.
      • Rasmussen H.H.
      • Leffers H.
      • et al.
      Molecular cloning and expression of a novel keratinocyte protein (psoriasis-associated fatty acid-binding protein [PA-FABP]) that is highly up-regulated in psoriatic skin and that shares similarity to fatty acid-binding proteins.
      ;
      • Nonomura K.
      • Yamanishi K.
      • Yasuno H.
      • et al.
      Up-regulation of elafin/SKALP gene expression in psoriatic epidermis.
      ;
      • Harder J.
      • Bartels J.
      • Christophers E.
      • et al.
      A peptide antibiotic from human skin.
      ;
      • Tsuji H.
      • Okamoto K.
      • Matsuzaka Y.
      • et al.
      SLURP-2, a novel member of the human Ly-6 superfamily that is up-regulated in psoriasis vulgaris.
      ;
      • O’Leary C.J.
      • Creamer D.
      • Higgins E.
      • et al.
      Perceived stress, stress attributions and psychological distress in psoriasis.
      ;
      • Fortune D.G.
      • Richards H.L.
      • Griffiths C.E.
      Psychologic factors in psoriasis: consequences, mechanisms, and interventions.
      ;
      • Naldi L.
      • Chatenoud L.
      • Linder D.
      • et al.
      Cigarette smoking, body mass index, and stressful life events as risk factors for psoriasis: results from an Italian case-control study.
      ;
      • Richards H.L.
      • Ray D.W.
      • Kirby B.
      • et al.
      Response of the hypothalamic-pituitary-adrenal axis to psychological stress in patients with psoriasis.
      ;
      • Arredondo J.
      • Chernyavsky A.I.
      • Jolkovsky D.L.
      • et al.
      SLURP-2: a novel cholinergic signaling peptide in human mucocutaneous epithelium.
      ;
      • Lande R.
      • Gregorio J.
      • Facchinetti V.
      • et al.
      Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide.
      ;
      • Evers A.W.
      • Verhoeven E.W.
      • Kraaimaat F.W.
      • et al.
      How stress gets under the skin: cortisol and stress reactivity in psoriasis.
      Mal de MeledaGenetic mutation in SLURP-1, an α7nAChR cofactor; α7nAChR signaling indicated in disease pathology
      • Chimienti F.
      • Hogg R.C.
      • Plantard L.
      • et al.
      Identification of SLURP-1 as an epidermal neuromodulator explains the clinical phenotype of Mal de Meleda.
      ,
      • Yamasaki K.
      • Di Nardo A.
      • Bardan A.
      • et al.
      Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea.
      RosaceaHyperactive 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 K.
      • Schauber J.
      • Coda A.
      • et al.
      Kallikrein-mediated proteolysis regulates the antimicrobial effects of cathelicidins in skin.
      ,
      • Yamasaki K.
      • Di Nardo A.
      • Bardan A.
      • et al.
      Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea.
      PemphigusAutoantibodies against cell–cell junction proteins, leading to barrier disruption

      Autoantibodies against nAChR; ↑ cholinergic signaling disrupts normal barrier integrity

      Psychological stress correlated with disease onset/exacerbation
      • Nguyen V.T.
      • Ndoye A.
      • Grando S.A.
      Novel human alpha9 acetylcholine receptor regulating keratinocyte adhesion is targeted by Pemphigus vulgaris autoimmunity.
      ,
      • Nguyen V.T.
      • Ndoye A.
      • Shultz L.D.
      • et al.
      Antibodies against keratinocyte antigens other than desmogleins 1 and 3 can induce pemphigus vulgaris-like lesions.
      ,
      • Niyonsaba F.
      • Someya A.
      • Hirata M.
      • et al.
      Evaluation of the effects of peptide antibiotics human beta-defensins-1/-2 and LL-37 on histamine release and prostaglandin D(2) production from mast cells.
      Palmoplantar pustulosisIrregular distribution/expression patterns of α3nAChR and α7nAChR throughout the stratum corneum

      Strongly correlated with smoking status (i.e., nicotine exposure)
      • Hagforsen E.
      • Edvinsson M.
      • Nordlind K.
      • et al.
      Expression of nicotinic receptors in the skin of patients with palmoplantar pustulosis.
      Abbreviations: AMP, antimicrobial peptide; hβD, 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.

      Evidence for a Cutaneous Non-Neuronal Cholinergic System

      The non-neuronal cholinergic system in keratinocytes is primarily regulated through the binding of the neurotransmitter, ACh, to nAChRs and mAChRs, classified on the basis of their respective agonists, nicotine and muscarine (
      • Grando S.A.
      • Pittelkow M.R.
      • Schallreuter K.U.
      Adrenergic and cholinergic control in the biology of epidermis: physiological and clinical significance.
      ). ACh is synthesized from coenzyme A and choline by choline acetyltransferase and is degraded by acetylcholinesterase. Keratinocytes have the capacity to synthesize, process, store, and release ACh that can bind to nAChRs or mAChRs expressed on their cell surface to regulate an array of cellular processes, including migration, differentiation, proliferation, and apoptosis (
      • Grando S.A.
      Physiology of endocrine skin interrelations.
      ;
      • Denda M.
      • Tsuchiya T.
      • Elias P.M.
      • et al.
      Stress alters cutaneous permeability barrier homeostasis.
      ) Keratinocyte cells can synthesize an average of 2 × 10−17 mol and secrete 7 × 10−19 mol of ACh per minute (
      • Grando S.A.
      • Kist D.A.
      • Qi M.
      • et al.
      Human keratinocytes synthesize, secrete, and degrade acetylcholine.
      ). However, a gradient of ACh expression throughout the epidermis is observed, with highest ACh levels present in the uppermost epidermal compartment (
      • Nguyen V.T.
      • Ndoye A.
      • Hall L.L.
      • et al.
      Programmed cell death of keratinocytes culminates in apoptotic secretion of a humectant upon secretagogue action of acetylcholine.
      ), likely because the highest amounts of choline acetyltransferase, the ACh-degrading enzyme, is located predominantly in the basal layer (
      • Johansson O.
      • Wang L.
      Choline acetyltransferase-like immunofluorescence in epidermis of human skin.
      ,
      • Grando S.A.
      • Pittelkow M.R.
      • Schallreuter K.U.
      Adrenergic and cholinergic control in the biology of epidermis: physiological and clinical significance.
      ;
      • Klapproth H.
      • Reinheimer T.
      • Metzen J.
      • et al.
      Non-neuronal acetylcholine, a signalling molecule synthesized by surface cells of rat and man.
      ). This intricate ACh-dependent network is referred to as the non-neuronal cholinergic system (
      • Klapproth H.
      • Reinheimer T.
      • Metzen J.
      • et al.
      Non-neuronal acetylcholine, a signalling molecule synthesized by surface cells of rat and man.
      ;
      • Wessler I.
      • Kirkpatrick C.J.
      • Racke K.
      Non-neuronal acetylcholine, a locally acting molecule, widely distributed in biological systems: expression and function in humans.
      ,
      • Wessler I.
      • Kirkpatrick C.J.
      • Racke K.
      The cholinergic ‘pitfall’: acetylcholine, a universal cell molecule in biological systems, including humans.
      ).
      The nAChR superfamily of ligand-gated ionotropic receptors comprise homo- or heterotypic combinations of nine α subunits (α2–α10) and three β subunits (β2–β4;
      • Steinbach J.H.
      Mechanism of action of the nicotinic acetylcholine receptor.
      ). During the course of keratinocyte differentiation, the expression of nAChR subunits and ACh regulatory enzymes is modified accordingly to support essential functions required for the specific microenvironment of each stratified layer of the epidermis (
      • Kurzen H.
      • Berger H.
      • Jager C.
      • et al.
      Phenotypical and molecular profiling of the extraneuronal cholinergic system of the skin.
      ; Figure 2). Keratinocyte transition from the granular cell to the flattened corneocyte is facilitated by ACh to induce apoptotic secretion, which refers to the sporadic extrusion of cytoplasmic components and the release of humectants to prevent epidermal water loss (
      • Nguyen V.T.
      • Ndoye A.
      • Hall L.L.
      • et al.
      Programmed cell death of keratinocytes culminates in apoptotic secretion of a humectant upon secretagogue action of acetylcholine.
      ). Knockdown of α7nAChR in cultured keratinocytes blocked terminal differentiation, which was coupled to the increased expression of proapoptotic signaling factors. Furthermore, the inhibition of α7nAChR signaling using the pharmacological antagonist α-bungarotoxin promoted cell cycle progression (
      • Arredondo J.
      • Nguyen V.T.
      • Chernyavsky A.I.
      • et al.
      Functional role of alpha7 nicotinic receptor in physiological control of cutaneous homeostasis.
      ). This suggests that α7nAChR signaling facilitates normal apoptotic signals to generate the stratum corneum. Combined pharmacological nAChR agonist and mAChR antagonist was also found to increase intracellular Ca2+ and trigger apoptotic secretion in vitro (
      • Nguyen V.T.
      • Ndoye A.
      • Hall L.L.
      • et al.
      Programmed cell death of keratinocytes culminates in apoptotic secretion of a humectant upon secretagogue action of acetylcholine.
      ). Collectively, ACh signaling is mediated by distinct combinations of nAChRs as keratinocytes mature, where each subunit regulates a particular cell function at a specific point in epidermal differentiation. Our recent observations identifying a novel pathway for keratinocyte AMP regulation through cholinergic signaling further extends the functional contribution of ACh to include epidermal barrier function through elements of the innate immune system.
      Figure thumbnail gr2
      Figure 2Expression of nicotinic acetylcholine receptor (nAChR) subtypes is dependent upon differentiation, free ACh concentration, and free Ca2+ 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 α7nAChR. Transitional (prickle) keratinocytes located in the middle layer of the epidermis express primarily α3(β2/β4)α5nAChR. Immature (basal) keratinocytes located within the base layer of the epidermis express primarily α3β2 or nAChRα3β4nAChR. All keratinocyte cells appear to express α9nAChR. 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).

      Keratinocytes as Key Mediators of Cutaneous Barrier Function

      Physical maintenance of skin permeability barrier function by the stratum corneum

      The fundamental purpose of the stratum corneum is to prevent external water loss and maintain a homeostatic microenvironment within the epidermis. The stratum corneum defends against pathogen invasion by creating both a physical and chemical shield mainly comprising lipids and AMPs, respectively. Corneocytes are terminally differentiated keratinocyte cells located in the stratum corneum, which comprise both the physical and chemical barrier to protect the skin against physical insult, ultraviolet light, chemical exposure, and microbial invasion, as well as prevent water and electrolyte loss into the external environment (
      • Feingold K.R.
      The outer frontier: the importance of lipid metabolism in the skin.
      ). The physical permeability barrier is created by the presence of a hydrophobic extracellular matrix composed of free fatty acids (FFAs), cholesterol, and ceramides secreted from lamellar bodies, which are secretory organelles unique to keratinocytes (
      • Rassner U.
      • Feingold K.R.
      • Crumrine D.A.
      • et al.
      Coordinate assembly of lipids and enzyme proteins into epidermal lamellar bodies.
      ).
      An effective permeability barrier depends on tight regulation and organization of cholesterols, ceramides, and FFAs within the stratum corneum interstices (reviewed in
      • Elias P.M.
      The skin barrier as an innate immune element.
      ;
      • Cork M.J.
      • Danby S.G.
      • Vasilopoulos Y.
      • et al.
      Epidermal barrier dysfunction in atopic dermatitis.
      ;
      • Ahrens K.
      • Schunck M.
      • Podda G.F.
      • et al.
      Mechanical and metabolic injury to the skin barrier leads to increased expression of murine beta-defensin-1, -3, and -14.
      ;
      • Borkowski A.W.
      • Gallo R.L.
      The coordinated response of the physical and antimicrobial peptide barriers of the skin.
      ). Strong cell–cell physical associations are created by cellular desmosomal junctions and secretion of lipid species from lamellar bodies, which covalently bind cell membranes and form the corneocyte lipid envelope (
      • Behne M.
      • Uchida Y.
      • Seki T.
      • et al.
      Omega-hydroxyceramides are required for corneocyte lipid envelope (CLE) formation and normal epidermal permeability barrier function.
      ). Experimentally, transepidermal water loss measurements are a useful biomarker for determining the integrity and health of the stratum corneum (
      • Kalia Y.N.
      • Alberti I.
      • Sekkat N.
      • et al.
      Normalization of stratum corneum barrier function and transepidermal water loss in vivo.
      ). Barrier recovery can be determined by measuring changes in transepidermal water loss values following barrier disruption via tape stripping or acetone treatment. Tape stripping is a type of superficial wounding that physically removes the corneocyte layer (
      • Pinkus H.
      Examination of the epidermis by the strip method of removing horny layers. I. Observations on thickness of the horny layer, and on mitotic activity after stripping.
      ). Contradictory to earlier reports suggesting that acetone treatment extracted epidermal lipids while leaving the corneocyte layer mostly intact, it was recently demonstrated that acetone treatment also removes the corneocyte layer, but only mildly extracts lipids (
      • Rissmann R.
      • Oudshoorn M.H.
      • Hennink W.E.
      • et al.
      Skin barrier disruption by acetone: observations in a hairless mouse skin model.
      ). Elevated transepidermal water loss values can be observed following chemical exposure, physical disruption, or several disease states, including but not limited to AD (
      • Gupta J.
      • Grube E.
      • Ericksen M.B.
      • et al.
      Intrinsically defective skin barrier function in children with atopic dermatitis correlates with disease severity.
      ), perioral dermatitis (
      • Dirschka T.
      • Tronnier H.
      • Folster-Holst R.
      Epithelial barrier function and atopic diathesis in rosacea and perioral dermatitis.
      ), and psoriasis (
      • Motta S.
      • Monti M.
      • Sesana S.
      • et al.
      Abnormality of water barrier function in psoriasis. Role of ceramide fractions.
      ).
      Initial studies aimed at determining factors that negatively regulate epidermal lipid synthesis began by investigating the influence of psychological stress created by either immobilization or crowding of mice (
      • Denda M.
      • Tsuchiya T.
      • Hosoi J.
      • et al.
      Immobilization-induced and crowded environment-induced stress delay barrier recovery in murine skin.
      ). Stressed animals exhibited delayed barrier recovery following barrier disruption via tape stripping indicated by higher transepidermal water loss measurements. Conversely, pretreatment with a sedative or GC receptor antagonist prevented the altered barrier recovery observed (
      • Denda M.
      • Tsuchiya T.
      • Hosoi J.
      • et al.
      Immobilization-induced and crowded environment-induced stress delay barrier recovery in murine skin.
      ,
      • Denda M.
      • Tsuchiya T.
      • Elias P.M.
      • et al.
      Stress alters cutaneous permeability barrier homeostasis.
      ). Psychological stress in mice was also shown to decrease epidermal cell proliferation and differentiation while simultaneously reducing the production and secretion of lamellar bodies (
      • Choi E.H.
      • Brown B.E.
      • Crumrine D.
      • et al.
      Mechanisms by which psychologic stress alters cutaneous permeability barrier homeostasis and stratum corneum integrity.
      ). This effect was reversed by topical lipid application, or systemic GC receptor antagonist administration (
      • Choi E.H.
      • Demerjian M.
      • Crumrine D.
      • et al.
      Glucocorticoid blockade reverses psychological stress-induced abnormalities in epidermal structure and function.
      ), establishing a direct effect of neuroendocrine mediators on biochemical pathways required for normal skin barrier homeostasis. Preliminary studies in healthy humans using two validated measures, mood states and the perceived stress scale, demonstrated the first correlation between perceived psychological stress and deteriorated cutaneous barriers, providing the first evidence that stress-induced alterations in the epithelial barrier may trigger the onset of clinical manifestations of skin disease pathogenesis (
      • Garg A.
      • Chren M.M.
      • Sands L.P.
      • et al.
      Psychological stress perturbs epidermal permeability barrier homeostasis: implications for the pathogenesis of stress-associated skin disorders.
      ).
      AMPs are multifunctional peptides that not only protect against microbial invasion and insult through direct pathogen killing, but also signal to the innate immune system and contribute to the generation and maintenance of the permeability barrier. In addition to its antimicrobial function, one AMP in particular, murine cathelicidin (CAMP), was found to have a crucial role in upholding epidermal barrier homeostasis. Mice deficient in cathelicidin (Camp−/−) have a diminished capacity to restrict barrier permeability, as they exhibit delayed barrier recovery following tape stripping (
      • Aberg K.M.
      • Man M.Q.
      • Gallo R.L.
      • et al.
      Co-regulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers.
      ). Camp−/− mice also display decreased lamellar body formation and secretion, and profound lipid composition defects compared with wild-type mice (
      • Aberg K.M.
      • Man M.Q.
      • Gallo R.L.
      • et al.
      Co-regulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers.
      ). The role of AMPs in protection against cutaneous infection is discussed below.

      Chemical maintenance of the skin barrier by lipids and AMPs

      Apart from the physical barrier comprising keratinocytes and lipids, the skin contains a chemical barrier partially consisting of lipids, but largely consisting of AMPs to restrict microbial growth on the surface of the skin and help maintain barrier integrity (reviewed in
      • Braff M.H.
      • Bardan A.
      • Nizet V.
      • et al.
      Cutaneous defense mechanisms by antimicrobial peptides.
      ). Long-chain bases, such as sphingosines, and FFAs, such as lauric acid and sapienic acid, are derived from the partial hydrolysis of ceramide by ceramidases (
      • Houben E.
      • Holleran W.M.
      • Yaginuma T.
      • et al.
      Differentiation-associated expression of ceramidase isoforms in cultured keratinocytes and epidermis.
      ), and are the most potent antimicrobial lipid species in the stratum corneum (
      • Miller S.J.
      • Aly R.
      • Shinefeld H.R.
      • et al.
      In vitro and in vivo antistaphylococcal activity of human stratum corneum lipids.
      ;
      • Bibel D.J.
      • Aly R.
      • Shinefield H.R.
      Antimicrobial activity of sphingosines.
      ). Sphingoid bases have broad antifungal and antimicrobial properties against 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., Escherichia coli), and yeast (e.g., Candida albicans;
      • Drake D.R.
      • Brogden K.A.
      • Dawson D.V.
      • et al.
      Thematic review series: skin lipids. Antimicrobial lipids at the skin surface.
      ). FFAs are selective against Gram-positive bacteria (e.g., S. aureus), but not Gram-negative bacteria and yeast (e.g., C. albicans;
      • Bibel D.J.
      • Miller S.J.
      • Brown B.E.
      • et al.
      Antimicrobial activity of stratum corneum lipids from normal and essential fatty acid-deficient mice.
      ,
      • Bibel D.J.
      • Aly R.
      • Shinefield H.R.
      Antimicrobial activity of sphingosines.
      ,
      • Bibel D.J.
      • Aly R.
      • Shinefield H.R.
      Topical sphingolipids in antisepsis and antifungal therapy.
      ;
      • Aly R.
      • Bayles C.I.
      • Oakes R.A.
      • et al.
      Topical griseofulvin in the treatment of dermatophytoses.
      ). The importance of understanding the contribution of FFAs to host defense is highlighted by the fact that different combinations of FFAs may exert different antimicrobial potencies against microbial species. For example, sapienic acid is more effective for the treatment of methicillin-resistant S. aureus infection than mupirocin, the current “gold standard” of treatment of methicillin-resistant S. aureus infections (
      • Drake D.R.
      • Brogden K.A.
      • Dawson D.V.
      • et al.
      Thematic review series: skin lipids. Antimicrobial lipids at the skin surface.
      ). Overall, the lipid component of the skin permeability barrier contributes to antimicrobial defense in parallel with AMPs.
      AMPs are an evolutionarily conserved component of the innate immune system that function at the external periphery as the initial line of defense against invasion by pathogens (
      • Gallo R.L.
      • Nizet V.
      Endogenous production of antimicrobial peptides in innate immunity and human disease.
      ;
      • Ganz T.
      Defensins: antimicrobial peptides of innate immunity.
      ;
      • Nizet V.
      • Gallo R.L.
      Cathelicidins and innate defense against invasive bacterial infection.
      ). Several families of AMPs, including cathelicidins (LL-37), defensins, and chromogranins, are expressed in immune cells (e.g., neutrophils, natural killer cells, mast cells, and macrophages), as well as epithelial cells such as sebocytes and keratinocytes (
      • Agerberth B.
      • Charo J.
      • Werr J.
      • et al.
      The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations.
      ;
      • Di Nardo A.
      • Vitiello A.
      • Gallo R.L.
      Cutting edge: mast cell antimicrobial activity is mediated by expression of cathelicidin antimicrobial peptide.
      ;
      • Braff M.H.
      • Gallo R.L.
      Antimicrobial peptides: an essential component of the skin defensive barrier.
      ;
      • Lee D.Y.
      • Yamasaki K.
      • Rudsil J.
      • et al.
      Sebocytes express functional cathelicidin antimicrobial peptides and can act to kill propionibacterium acnes.
      ). To date, there exist over 20 epidermal AMPs (reviewed in
      • Schauber J.
      • Gallo R.L.
      Antimicrobial peptides and the skin immune defense system.
      ). Several key epidermal AMPs, ribonuclease 7, and lysozyme are constitutively active and expressed at low basal levels (
      • Harder J.
      • Glaser R.
      • Schroder J.M.
      Human antimicrobial proteins effectors of innate immunity.
      ). Other AMPs, such as human beta-defensin (hβD)-2 and hβD-3, and the CAMP cleavage product, LL-37, are only expressed in response to inflammation (
      • Gudmundsson G.H.
      • Agerberth B.
      • Odeberg J.
      • et al.
      The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes.
      ;
      • Harder J.
      • Bartels J.
      • Christophers E.
      • et al.
      A peptide antibiotic from human skin.
      ,
      • Harder J.
      • Bartels J.
      • Christophers E.
      • et al.
      Isolation and characterization of human beta -defensin-3, a novel human inducible peptide antibiotic.
      ;
      • Schroder J.M.
      • Harder J.
      Human beta-defensin-2.
      ;
      • Dorschner R.A.
      • Pestonjamasp V.K.
      • Tamakuwala S.
      • et al.
      Cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group A Streptococcus.
      ;
      • Nizet V.
      • Ohtake T.
      • Lauth X.
      • et al.
      Innate antimicrobial peptide protects the skin from invasive bacterial infection.
      ;
      • Harder J.
      • Schroder J.M.
      RNase 7, a novel innate immune defense antimicrobial protein of healthy human skin.
      ;
      • Glaser R.
      • Harder J.
      • Lange H.
      • et al.
      Antimicrobial psoriasin (S100A7) protects human skin from Escherichia coli infection.
      ;
      • Pazgier M.
      • Hoover D.M.
      • Yang D.
      • et al.
      Human beta-defensins.
      ). Other epidermal AMPs, such as dermcidin, are sweat-gland derived (
      • Schittek B.
      • Hipfel R.
      • Sauer B.
      • et al.
      Dermcidin: a novel human antibiotic peptide secreted by sweat glands.
      ), whereas the S100 protein psoriasin is focally present at varying levels throughout healthy skin (
      • Glaser R.
      • Harder J.
      • Lange H.
      • et al.
      Antimicrobial psoriasin (S100A7) protects human skin from Escherichia coli infection.
      ) and induced upon wounding (
      • Lee K.C.
      • Eckert R.L.
      S100A7 (Psoriasin)—mechanism of antibacterial action in wounds.
      ) or skin inflammation (
      • Madsen P.
      • Rasmussen H.H.
      • Leffers H.
      • et al.
      Molecular cloning, occurrence, and expression of a novel partially secreted protein “psoriasin” that is highly up-regulated in psoriatic skin.
      ).
      Cathelicidin is proteolytically cleaved from the larger hCAP18 pro-protein into the catalytically active peptide (human LL-37, murine CAMP) by kallikrein proteases produced by keratinocytes, or by neutrophil-derived proteinase-3 (
      • Hansson L.
      • Stromqvist M.
      • Backman A.
      • et al.
      Cloning, expression, and characterization of stratum corneum chymotryptic enzyme. A skin-specific human serine proteinase.
      ;
      • Ekholm I.E.
      • Brattsand M.
      • Egelrud T.
      Stratum corneum tryptic enzyme in normal epidermis: a missing link in the desquamation process?.
      ). In human keratinocytes, cathelicidin expression can be augmented in response to pathogen challenge, vitamin D3 exposure, and wounding (
      • Dorschner R.A.
      • Pestonjamasp V.K.
      • Tamakuwala S.
      • et al.
      Cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group A Streptococcus.
      ;
      • Butmarc J.
      • Yufit T.
      • Carson P.
      • et al.
      Human beta-defensin-2 expression is increased in chronic wounds.
      ;
      • Wang T.T.
      • Nestel F.P.
      • Bourdeau V.
      • et al.
      Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression.
      ;
      • Schauber J.
      • Oda Y.
      • Buchau A.S.
      • et al.
      Histone acetylation in keratinocytes enables control of the expression of cathelicidin and CD14 by 1,25-dihydroxyvitamin D3.
      ), where it functions as natural antibiotics to not only directly eradicate microbial pathogens, but also coordinate multiple components of the innate and adaptive immune responses (
      • Brogden K.A.
      • Ackermann M.
      • McCray Jr., P.B.
      • et al.
      Antimicrobial peptides in animals and their role in host defences.
      ;
      • Kandler K.
      • Shaykhiev R.
      • Kleemann P.
      • et al.
      The anti-microbial peptide LL-37 inhibits the activation of dendritic cells by TLR ligands.
      ). For example, LL-37 can directly promote the secretion of chemokines from keratinocytes (
      • Niyonsaba F.
      • Ushio H.
      • Nakano N.
      • et al.
      Antimicrobial peptides human beta-defensins stimulate epidermal keratinocyte migration, proliferation and production of proinflammatory cytokines and chemokines.
      ). Furthermore, LL-37 has the capacity to enhance Toll-like receptor signaling in immune cells and initiate a cytokine cascade responsible for bacterial recognition (
      • Niyonsaba F.
      • Someya A.
      • Hirata M.
      • et al.
      Evaluation of the effects of peptide antibiotics human beta-defensins-1/-2 and LL-37 on histamine release and prostaglandin D(2) production from mast cells.
      ), while stimulating the release of reactive oxygen species from activated neutrophils (
      • Zheng Y.
      • Niyonsaba F.
      • Ushio H.
      • et al.
      Cathelicidin LL-37 induces the generation of reactive oxygen species and release of human alpha-defensins from neutrophils.
      ) to enhance the overall response to infection. Conflicting reports of LL-37 immunoreactivity throughout the epidermis have been described, with some demonstrating localization in the extracellular space between the stratum granulosum and stratum corneum (
      • Goo J.
      • Ji J.H.
      • Jeon H.
      • et al.
      Expression of antimicrobial peptides such as LL-37 and hBD-2 in nonlesional skin of atopic individuals.
      ) and others to the stratum basale ((
      • Heilborn J.D.
      • Nilsson M.F.
      • Kratz G.
      • et al.
      The cathelicidin anti-microbial peptide LL-37 is involved in re-epithelialization of human skin wounds and is lacking in chronic ulcer epithelium.
      ;
      • Mallbris L.
      • Carlen L.
      • Wei T.
      • et al.
      Injury downregulates the expression of the human cathelicidin protein hCAP18/LL-37 in atopic dermatitis.
      ). As the expression of specific nAChR and mAChR subtypes varies throughout the epidermis, understanding how ACh contributes to LL-37 expression/activity will require further investigation into its localization.
      The role of AMPs in keratinocyte innate defense has been primarily described using murine model systems. However, it should be noted that although most AMPs appear to be conserved between mice and humans, several important differences between expression patterns and regulatory mechanisms have been described. For example, humans have both epidermal and neutrophil-derived defensins, whereas mice only express defensins in epithelial cells, suggesting that murine and human innate immune responses may be significantly different (
      • Eisenhauer P.B.
      • Lehrer R.I.
      Mouse neutrophils lack defensins.
      ;
      • Harder J.
      • Bartels J.
      • Christophers E.
      • et al.
      A peptide antibiotic from human skin.
      ). The mechanisms regulating human CAMP and murine Camp also vary considerable with respect to the cathelicidin promoter. In humans, CAMP transcription requires binding of the vitamin D receptor to the vitamin D response element consensus sequence in the cathelicidin promoter (
      • Gombart A.F.
      • Borregaard N.
      • Koeffler H.P.
      Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3.
      ). However, the nocturnal nature of mice likely explains the absence of the vitamin D response element consensus sequence in the murine Camp promoter (
      • Gombart A.F.
      • Borregaard N.
      • Koeffler H.P.
      Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3.
      ). Recent studies in mice with a keratinocyte-specific deletion of hypoxia-inducible factor-1α indicates that Camp expression in mice is partially regulated by hypoxia-inducible factor-1α. Hypoxia-inducible factor-1α-null neutrophils from these mice presented with reduced levels of cathelicidin and exhibited a decreased phagocytic capacity of skin pathogens (
      • Peyssonnaux C.
      • Datta V.
      • Cramer T.
      • et al.
      HIF-1alpha expression regulates the bactericidal capacity of phagocytes.
      ). Together, the variability in AMP regulation between species is of critical importance and should be taken into consideration when interpreting data acquired from human and mouse experiments. A more relevant model system would be the rhesus monkey, as defensin and cathelicidin AMPs are expressed in the same cell types and tissues as in humans (
      • Bals R.
      • Lang C.
      • Weiner D.J.
      • et al.
      Rhesus monkey (Macaca mulatta) mucosal antimicrobial peptides are close homologues of human molecules.
      ), and the cathelicidin promoter contains vitamin D response elements (
      • Gombart A.F.
      • Borregaard N.
      • Koeffler H.P.
      Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3.
      ), suggesting that similar mechanisms of AMP transcriptional regulation occur between humans and nonhuman primates.

      The Role of α7nAChR in Keratinocye Barrier Function: Lessons from Murine Knockouts

      Although numerous nAChR subtypes exist in the epidermis, there is evidence to suggest that similar to the systemic, neuronal cholinergic anti-inflammatory pathway, the α7nAChR subtype is likely to have a significant role in the regulation of the local cutaneous innate immune and barrier function. Loss of α7nAChR expression in Chrna7−/− mice resulted in profound decreases (34–56%) in genes related to terminal differentiation, including murine filaggrin, cytokeratin-1, and cytokeratin-10. Furthermore, these mice also displayed a reduction in the production of extracellular matrix proteins collagen-1α1 and elastin, and metalloproteinase-1 (
      • Arredondo J.
      • Nguyen V.T.
      • Chernyavsky A.I.
      • et al.
      Central role of alpha7 nicotinic receptor in differentiation of the stratified squamous epithelium.
      ). Significant differences between the histology of Chrna7−/− and Chrna7+/+ were observed in 1- to 3-week-old mice, where Chrna7+/+ skin exhibited the normal, upper compact horny layer derived from dead corneocytes, 1–2 layers of live, nucleated keratinocytes, and a single row of suprabasal keratinocytes. In contrast, Chrna7−/− skin had a loose, widened corneocyte layer, 1–3 layers of granulated keratinocytes, and 1–3 extra rows of suprabasilar enlarged keratinocytes, a phenotype consistent with retention hyperkeratosis (
      • Arredondo J.
      • Nguyen V.T.
      • Chernyavsky A.I.
      • et al.
      Central role of alpha7 nicotinic receptor in differentiation of the stratified squamous epithelium.
      ). Interestingly, Chrna7−/− mice also show a differential gene expression of the other nAChR subunits, suggesting that a compensatory mechanism may have developed to regulate keratinocyte cell cycle progression, apoptosis, and terminal differentiation genes in the absence of α7nAChR signaling (
      • Arredondo J.
      • Nguyen V.T.
      • Chernyavsky A.I.
      • et al.
      Central role of alpha7 nicotinic receptor in differentiation of the stratified squamous epithelium.
      ).

      Linking Cholinergic Signaling with Epidermal Amp Activity and Susceptibility to Infection

      For decades, it has been well documented that stress strongly correlates with an increased incidence of infection (
      • Locke S.E.
      Stress, adaptation, and immunity: studies in humans.
      ). Only recently have we begun to elucidate the neuroendrocrine contribution of epithelial immunity. Stressed individuals have increased epithelial ACh levels (
      • Schlereth T.
      • Schonefeld S.
      • Birklein F.
      • et al.
      In vivo release of non-neuronal acetylcholine from human skin by dermal microdialysis: effects of sunlight, UV-A and tactile stimulus.
      ) and are more susceptible to opportunistic infections (
      • Wu L.R.
      • Zaborina O.
      • Zaborin A.
      • et al.
      Surgical injury and metabolic stress enhance the virulence of the human opportunistic pathogen Pseudomonas aeruginosa.
      ;
      • Ashcraft K.A.
      • Hunzeker J.
      • Bonneau R.H.
      Psychological stress impairs the local CD8+ T cell response to mucosal HSV-1 infection and allows for increased pathogenicity via a glucocorticoid receptor-mediated mechanism.
      ). Overactivation of the stress-response pathways, as observed during chronic stress, results in a relative state of immunosuppression that can be partly attributed to activation of the cholinergic anti-inflammatory pathway described earlier.
      As the cholinergic system is directly involved in systemic immunosuppression, it was likely that non-neuronal cholinergic activation could suppress the local cutaneous AMP response in keratinocytes. Normal human epidermal keratinocytes (NHEKs) stimulated with the bioactive form of vitamin 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 expression of both cathelicidin and β-defensin-2 through differentiation and bacterial stimuli, respectively (
      • Weber G.
      • Heilborn J.D.
      • Chamorro Jimenez C.I.
      • et al.
      Vitamin D induces the antimicrobial protein hCAP18 in human skin.
      ;
      • Schauber J.
      • Dorschner R.A.
      • Yamasaki K.
      • et al.
      Control of the innate epithelial antimicrobial response is cell-type specific and dependent on relevant microenvironmental stimuli.
      ). Our initial results identified that the in vitro expression of CAMP in NHEKs was induced ∼100-fold over baseline levels by VD3+MALP-2, whereas the presence of 0.01nM ACh resulted in an ∼50% reduction in CAMP (
      • Radek K.A.
      • Elias P.M.
      • Taupenot L.
      • et al.
      Neuroendocrine nicotinic receptor activation increases susceptibility to bacterial infections by suppressing antimicrobial peptide production.
      ). We observed a similar suppression of hβD-2 gene and protein expression by ACh compared with the MALP-2 alone. To further elucidate the specific receptor subtype mediating the dampening response by ACh, additional experiments were conducted using two pharmacological antagonists, α-bungarotoxin, which is selective for the α7nAChR subunit, and mecamylamine, with known selectivity for α3β4nAChR subtypes (
      • Grando S.A.
      • Pittelkow M.R.
      • Schallreuter K.U.
      Adrenergic and cholinergic control in the biology of epidermis: physiological and clinical significance.
      ), or an endogenous epidermal nAChR antagonist, catestatin (Cst;
      • Radek K.A.
      • Lopez-Garcia B.
      • Hupe M.
      • et al.
      The neuroendocrine peptide catestatin is a cutaneous antimicrobial and induced in the skin after injury.
      ). CAMP suppression by ACh was restored by the presence of Cst and mecamylamine, with maximal restoration observed with α-bungarotoxin. The induction of CAMP and β-defensin expression paralleled the capacity of NHEKs to restrict the growth of the major skin pathogen S. aureus. Lysates of NHEKs treated with VD3+MALP-2 restricted staphylococcal growth by 90% as compared with lysates from untreated cells. Treatment with 0.01nM ACh suppressed the antimicrobial activity of VD3+MALP-2 lysates to that of untreated cell lysates. This effect was again restored by the presence of Cst or α-bungarotoxin. This strongly indicates that nAChR signaling dampens the direct extractable antibacterial activity in NHEKs consistent with the critical role of AMPs in this response (
      • Radek K.A.
      • Elias P.M.
      • Taupenot L.
      • et al.
      Neuroendocrine nicotinic receptor activation increases susceptibility to bacterial infections by suppressing antimicrobial peptide production.
      ). Treatment of NHEKs with muscarine, a mAChR-specific agonist, or propranolol, a β-adrenergic pathway agonist, was unable to suppress the induced AMP expression observed with VD3/MALP-2 treatment, suggesting that ACh regulation of AMP expression in keratinocytes is through direct activation of nAChRs (
      • Radek K.A.
      • Elias P.M.
      • Taupenot L.
      • et al.
      Neuroendocrine nicotinic receptor activation increases susceptibility to bacterial infections by suppressing antimicrobial peptide production.
      ). The observation that AMP suppression by ACh could be fully restored by the presence of α-bungarotoxin, and mostly restored by the addition of mecamylamine or Cst, suggests that ACh likely facilitates AMP suppression via α7nAChR, similar to the neuronal cholinergic response to infection (
      • Tracey K.J.
      Physiology and immunology of the cholinergic antiinflammatory pathway.
      ). Our results indicate that the changes in direct extractable antibacterial activity in these NHEKs lysates was likely due to, in part, decreased cathelicidin and hβD-2 expression. However, it is possible that the levels of other inducible AMPs (e.g., ribonuclease 7 and hβD-3) or constitutive AMPs (e.g., psoriasin), normally increased by the presence of bacterial membrane components, were also suppressed by the presence of ACh (
      • Harder J.
      • Bartels J.
      • Christophers E.
      • et al.
      Isolation and characterization of human beta -defensin-3, a novel human inducible peptide antibiotic.
      ;
      • Harder J.
      • Schroder J.M.
      RNase 7, a novel innate immune defense antimicrobial protein of healthy human skin.
      ). Similar studies designed to identify changes in specific AMPs are currently in progress.
      Cst was initially identified as an AMP, as it exhibited a broad-spectrum killing against several pathogens including S. aureus, group A Streptococcus, E. coli, Pseudomonas aeruginosa, and C. albicans, as well as several varieties of filamentous fungi in vitro (
      • Briolat J.
      • Wu S.D.
      • Mahata S.K.
      • et al.
      New antimicrobial activity for the catecholamine release-inhibitory peptide from chromogranin A.
      ;
      • Radek K.A.
      • Lopez-Garcia B.
      • Hupe M.
      • et al.
      The neuroendocrine peptide catestatin is a cutaneous antimicrobial and induced in the skin after injury.
      ). As the concentration of Cst required for microbicidal killing was much higher than levels observed in normal skin (
      • Radek K.A.
      • Lopez-Garcia B.
      • Hupe M.
      • et al.
      The neuroendocrine peptide catestatin is a cutaneous antimicrobial and induced in the skin after injury.
      ), we proposed that the antimicrobial properties may be secondary to its primary role as a natural, endogenous nAChR antagonist. Similar to several neuroendocrine-derived molecules, such as α-melanocyte-stimulating hormone, neurokinin-1, and adrenomedullin, Cst may exhibit microbicidal activity due to their inherently small, cationic, amphipathic structure (reviewed in
      • Radek K.
      • Gallo R.
      Antimicrobial peptides: natural effectors of the innate immune system.
      ). In our studies, we found that AMP suppression by ACh was mostly restored by the presence of Cst, indicating that Cst can block the effects of ACh by functioning primarily as an endogenous nAChR antagonist (
      • Radek K.A.
      • Elias P.M.
      • Taupenot L.
      • et al.
      Neuroendocrine nicotinic receptor activation increases susceptibility to bacterial infections by suppressing antimicrobial peptide production.
      ).
      To verify the functional relevance of cholinergic activation and keratinocyte AMP regulation in vivo, we used a pharmacological and genetic approach using two murine models of cholinergic activation (
      • Radek K.A.
      • Elias P.M.
      • Taupenot L.
      • et al.
      Neuroendocrine nicotinic receptor activation increases susceptibility to bacterial infections by suppressing antimicrobial peptide production.
      ). For the pharmacological approach, we applied α-bungarotoxin or vehicle topically to psychologically stressed mice (e.g., insomnia stress) and assessed their susceptibility to infection. We found that stressed mice treated with α-bungarotoxin were less susceptible to group A Streptococcus infection compared with vehicle-treated mice, as indicated by smaller lesion size, less surviving bacteria in lesions, and lower dissemination into distal organs (
      • Radek K.A.
      • Elias P.M.
      • Taupenot L.
      • et al.
      Neuroendocrine nicotinic receptor activation increases susceptibility to bacterial infections by suppressing antimicrobial peptide production.
      ). Mice treated topically with nicotine (in the absence of stress) demonstrated a significant reduction in extractable AMP activity, indicating that direct activation of nAChRs negatively regulates AMP expression (
      • Radek K.A.
      • Elias P.M.
      • Taupenot L.
      • et al.
      Neuroendocrine nicotinic receptor activation increases susceptibility to bacterial infections by suppressing antimicrobial peptide production.
      ). We later defined the role of nAChR activation in regulating AMP activity in the skin by employing a genetic approach by using CHGA−/− mice, which lack the endogenous nAChR antagonist Cst and hence exhibit unopposed nAChR activation. CHGA−/− mice were significantly more susceptible to both S. aureus and group A Streptococcus skin infection. These mice also exhibited a significant reduction in extractable AMP activity, which was restored by the presence of topical α-bungarotoxin. Together, these data suggested that the regulation of epidermal AMP expression and activity by ACh is highly dependent on the α7nAChR subtype, but may also involve other subtypes to a lesser degree (
      • Radek K.A.
      • Elias P.M.
      • Taupenot L.
      • et al.
      Neuroendocrine nicotinic receptor activation increases susceptibility to bacterial infections by suppressing antimicrobial peptide production.
      ). In addition, the increase in bacterial survival following prolonged nAChR activation was likely the result of several immunosuppressive responses, including AMP-dependent production of proinflammatory cytokines and enhancement of bacterial survival and proliferation through changes in the epidermal microenvironment (
      • Freestone P.P.
      • Lyte M.
      Microbial endocrinology: experimental design issues in the study of interkingdom signalling in infectious disease.
      ). In humans, a likely detrimental consequence of a diminished epidermal immune response following nAChR activation would lead to bacterial dissemination from initial sites of infection and colonization in distal tissues and organs.
      Our previous investigations also provided a critical piece of data that nAChR-mediated cathelicidin dysregulation is a potential mechanism for increased susceptibility to infection. We found that stress did not augment the susceptibility to infection in cathelicidin-deficient (Camp−/−) or CHGA−/− mice. This indicates that nAChR activation is a major contributing factor to the suppression of the AMP response to infection during prolonged nAChR activation by stress, and that cathelicidin is a key AMP influenced by cholinergic activation of nAChRs (
      • Radek K.A.
      • Elias P.M.
      • Taupenot L.
      • et al.
      Neuroendocrine nicotinic receptor activation increases susceptibility to bacterial infections by suppressing antimicrobial peptide production.
      ). If other AMPs or neuroendocrine mediators were more involved in AMP suppression, we would expect larger lesions and greater bacterial burden in both Camp−/− and CHGA−/− mice following stress. In addition, cathelicidin is an autocrine regulator of the response to purinergic agonists through modification of receptors involved in the tissue response to inflammation or injury, indicating a direct role for cathelicidin in cholinergic-mediated immunosuppression (
      • Pochet S.
      • Tandel S.
      • Querriere S.
      • et al.
      Modulation by LL-37 of the responses of salivary glands to purinergic agonists.
      ). Thus, the global suppression of AMP activity and cathelicidin seen with nAChR activation may exacerbate immunosuppression by blocking necessary stimulation of downstream immune responses that are AMP dependent.

      Implications for the Role of Cholinergic Activation in the Development of Skin Pathologies

      Delayed wound healing

      AMPs have an important role in modulating wound healing responses by increasing the expression of chemokines and chemokine receptors, stimulating epithelial proliferation, and promoting adaptive immune responses and angiogenesis (
      • Scott M.G.
      • Davidson D.J.
      • Gold M.R.
      • et al.
      The human antimicrobial peptide LL-37 is a multifunctional modulator of innate immune responses.
      ;
      • Mookherjee N.
      • Hancock R.E.
      Cationic host defence peptides: innate immune regulatory peptides as a novel approach for treating infections.
      ;
      • Lai Y.
      • Gallo R.L.
      AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense.
      ). Therefore, a defective permeability barrier, arising from altered skin pH, lipid composition, or flawed cell–cell junctions, in addition to an insufficient AMP repertoire, can result in overactive cytokine/chemokine cascades and can foster a destructive inflammatory milieu. As AMPs have a multifunctional role in regulating innate immune responses, inflammation, and barrier function, it is critical to maintain their homeostatic balance to allow for maximal wound repair processes.
      Cholinergic signaling has been implicated in the regulation of re-epithelialization during tissue repair. Immediately following barrier disruption and wounding, proinflammatory cytokines, such as TNF-α, IL-1-α, and IL-1β, are released by immune and keratinocytic cells to stimulate cellular proliferation required for epidermal regeneration (e.g., re-epithelialization;
      • Wood L.C.
      • Jackson S.M.
      • Elias P.M.
      • et al.
      Cutaneous barrier perturbation stimulates cytokine production in the epidermis of mice.
      ;
      • Nickoloff B.J.
      • Naidu Y.
      Perturbation of epidermal barrier function correlates with initiation of cytokine cascade in human skin.
      ). At the onset of re-epithelialization, keratinocyte cell–cell desmosomal and hemidesmosomal connections are dissolved, followed by the development of peripheral cytoplasmic actin filaments (
      • Gabbiani G.
      • Chaponnier C.
      • Huttner I.
      Cytoplasmic filaments and gap junctions in epithelial cells and myofibroblasts during wound healing.
      ;
      • Goliger J.A.
      • Paul D.L.
      Wounding alters epidermal connexin expression and gap junction-mediated intercellular communication.
      ). Concurrently, keratinocyte integrin expression increases to allow for lateral migration across the wound bed (
      • Clark R.A.
      Fibronectin matrix deposition and fibronectin receptor expression in healing and normal skin.
      ;
      • Clark R.A.
      • Ashcroft G.S.
      • Spencer M.J.
      • et al.
      Re-epithelialization of normal human excisional wounds is associated with a switch from alpha v beta 5 to alpha v beta 6 integrins.
      ), which ceases when keratinocytes collide to form new cell–cell junctions and ensue with terminal differentiation (
      • Adams J.C.
      • Watt F.M.
      Changes in keratinocyte adhesion during terminal differentiation: reduction in fibronectin binding precedes alpha 5 beta 1 integrin loss from the cell surface.
      ;
      • Martin P.
      Wound healing—aiming for perfect skin regeneration.
      ;
      • Singer A.J.
      • Clark R.A.
      Cutaneous wound healing.
      ). Cholinergic signaling by several nAChRs was found to be directly involved in keratinocyte chemotaxis and chemokinesis, respectively. In in vitro migration assays, reorganization of α7nAChRs along the leading edge of keratinocytes occurred before the conventional crescent-shaped formation and directional migration of human keratinocytes in the presence of a chemoattractant, whereas several nAChRs were found to localize with integrin-β1 expressed on the keratinocyte cell membrane (
      • Chernyavsky A.I.
      • Arredondo J.
      • Marubio L.M.
      • et al.
      Differential regulation of keratinocyte chemokinesis and chemotaxis through distinct nicotinic receptor subtypes.
      ). Experimental evidence also suggests that α9nAChR activation leads to downstream phosphorylation of cytoskelatal proteins required for cell motility (
      • Szonyi M.
      • Csermely P.
      • Sziklai I.
      Acetylcholine-induced phosphorylation in isolated outer hair cells.
      ) and keratinocyte cell–cell adhesion (
      • Nguyen V.T.
      • Ndoye A.
      • Grando S.A.
      Novel human alpha9 acetylcholine receptor regulating keratinocyte adhesion is targeted by Pemphigus vulgaris autoimmunity.
      ). Collectively, this suggests that ACh likely contributes to both the initiation and termination of re-epithelialization during wound repair to re-establish the epidermal permeability barrier. (
      • Grando S.A.
      • Pittelkow M.R.
      • Schallreuter K.U.
      Adrenergic and cholinergic control in the biology of epidermis: physiological and clinical significance.
      ).

      AD and psoriasis

      A key factor in a myriad of cutaneous inflammatory diseases includes AMPs. Up to 90% of AD skin lesions are typically colonized by S. aureus (
      • Leung D.Y.
      Infection in atopic dermatitis.
      ). In the past, greater susceptibility was attributed to an overall decrease in AMP expression, as it was demonstrated that cathelicidin (LL-37), hβD-2, and hβD-3 levels were all decreased in AD skin when compared with psoriatic skin (
      • Ong P.Y.
      • Ohtake T.
      • Brandt C.
      • et al.
      Endogenous antimicrobial peptides and skin infections in atopic dermatitis.
      ). Therefore, it was presumed that the overall decrease in AMP expression directly resulted in suppressed antimicrobial potential and a compromised barrier. More recently, a study with a large patient cohort of atopic excema, psoriatic, and normal controls demonstrated that atopic excema lesions have LL-37 levels similar to that of normal, non-lesional skin, and healthy skin from normal donors (
      • Gambichler T.
      • Skrygan M.
      • Tomi N.S.
      • et al.
      Differential mRNA expression of antimicrobial peptides and proteins in atopic dermatitis as compared to psoriasis vulgaris and healthy skin.
      ). Psoriasin secretion was also profoundly increased (up to 1,500-fold) when comparing AD skin with healthy skin from the same individual (
      • Glaser R.
      • Meyer-Hoffert U.
      • Harder J.
      • et al.
      The antimicrobial protein psoriasin (S100A7) is upregulated in atopic dermatitis and after experimental skin barrier disruption.
      ). These conflicting reports highlight the importance of understanding the factors that contribute to AMP regulation. Although the baseline levels of AMP expression in AD patients may be equal to or higher than normal, uninjured skin, the antimicrobial potential of these AMPs remains to be determined. It has been demonstrated that the increased LL-37 expression following injury observed in healthy volunteers was not observed in AD lesions (
      • Mallbris L.
      • Carlen L.
      • Wei T.
      • et al.
      Injury downregulates the expression of the human cathelicidin protein hCAP18/LL-37 in atopic dermatitis.
      ), suggesting that although the baseline level of LL-37 among normal, atopic, and psoriatic skin appears about the same, increased microbial colonization likely arises from the inability to induce LL-37 following wounding or in the presence of active infection. Furthermore, it has been shown that Th2 cytokines, IL-4 and IL-13, directly downregulate AMP expression in cultured human keratinocytes (
      • Ong P.Y.
      • Ohtake T.
      • Brandt C.
      • et al.
      Endogenous antimicrobial peptides and skin infections in atopic dermatitis.
      ;
      • Nomura I.
      • Goleva E.
      • Howell M.D.
      • et al.
      Cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes.
      ). In parallel, AD patients have an elevated Th2 cytokine milieu, at least in comparison with psoriatic skin (
      • Nomura I.
      • Goleva E.
      • Howell M.D.
      • et al.
      Cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes.
      ). This altered cytokine milieu is believed to directly suppress the AMP response to injury and infection in the skin, providing further evidence that the increased susceptibility to infection observed in AD patients is multifaceted, arising from defects in innate antimicrobial responses, as well as adaptive immunity and cytokine responses. These observations emphasize the importance of identifying common mechanisms behind AMP induction and suppression that may yield unconventional therapeutic interventions that can be customized to treat cutaneous disorders with AMP dysregulation as a major contributing factor.
      AD is a common skin disease in which clinical onset is usually triggered by environmental or psychological stress (
      • Raap U.
      • Werfel T.
      • Jaeger B.
      • et al.
      [Atopic dermatitis and psychological stress].
      ). Variable differences in expression of ACh receptor subunits have been observed in patients with AD, as well as in response to minimal trauma (
      • Kurzen H.
      • Schallreuter K.U.
      Novel aspects in cutaneous biology of acetylcholine synthesis and acetylcholine receptors.
      ). Patients with AD have a significant increase (14-fold) in ACh expression in the skin, as compared with normal individuals (
      • Wessler I.
      • Reinheimer T.
      • Kilbinger H.
      • et al.
      Increased acetylcholine levels in skin biopsies of patients with atopic dermatitis.
      ). Moreover, ACh contributes to the pathophysiological characteristics of AD, such as increased pruritis, or itch, due to its ability to cause vasodilatation and the wheal and flare reaction (
      • Heyer G.
      • Vogelgsang M.
      • Hornstein O.P.
      Acetylcholine is an inducer of itching in patients with atopic eczema.
      ;
      • Rukwied R.
      • Heyer G.
      Administration of acetylcholine and vasoactive intestinal polypeptide to atopic eczema patients.
      ;
      • Wessler I.
      • Reinheimer T.
      • Kilbinger H.
      • et al.
      Increased acetylcholine levels in skin biopsies of patients with atopic dermatitis.
      ).
      NC/Nga mice exhibit increased serum IgE, scaly AD-like lesions, and mast cell accumulation similar to serum and skin lesions of AD patients (
      • Suto H.
      • Matsuda H.
      • Mitsuishi K.
      • et al.
      NC/Nga mice: a mouse model for atopic dermatitis.
      ), and these mice are frequently used as a murine model of AD. Psychological stress was shown to trigger the development of these histopathologies in NC/Nga mice, where pretreatment with corticotropin-releasing hormone (CRH), a neurogenic peptide released in response to cholinergic stimulation, completely prevented the onset of AD lesions in these animals (
      • Amano H.
      • Negishi I.
      • Akiyama H.
      • et al.
      Psychological stress can trigger atopic dermatitis in NC/Nga mice: an inhibitory effect of corticotropin-releasing factor.
      ). Epidermal cells expressing CRH receptors may have a role in the mechanism by which stress-induced cholinergic activation triggers the onset and exacerbation of AD (
      • Amano H.
      • Negishi I.
      • Akiyama H.
      • et al.
      Psychological stress can trigger atopic dermatitis in NC/Nga mice: an inhibitory effect of corticotropin-releasing factor.
      ). These data strongly suggest a direct role for ACh and cholinergic signaling in the development of AD pathophysiology.
      Lesions from patients with psoriasis show epidermal hyperplasia and increased AMP expression, excess inflammation, and a diminished susceptibility to infection (
      • Madsen P.
      • Rasmussen H.H.
      • Leffers H.
      • et al.
      Molecular cloning, occurrence, and expression of a novel partially secreted protein “psoriasin” that is highly up-regulated in psoriatic skin.
      ;
      • Ong P.Y.
      • Ohtake T.
      • Brandt C.
      • et al.
      Endogenous antimicrobial peptides and skin infections in atopic dermatitis.
      ;
      • Rieg S.
      • Steffen H.
      • Seeber S.
      • et al.
      Deficiency of dermcidin-derived antimicrobial peptides in sweat of patients with atopic dermatitis correlates with an impaired innate defense of human skin in vivo.
      ;
      • Howell M.D.
      • Boguniewicz M.
      • Pastore S.
      • et al.
      Mechanism of HBD-3 deficiency in atopic dermatitis.
      ). Extensive studies in humans indicate a direct interaction between smoking (e.g., exposure to nicotine) and the development of psoriatic arthritis through genetic polymorphisms in the gene encoding for the Th2 cytokine, IL-13 (
      • Duffin K.C.
      • Freeny I.C.
      • Schrodi S.J.
      • et al.
      Association between IL13 polymorphisms and psoriatic arthritis is modified by smoking.
      ). The clinical onset and exacerbation of psoriasis is associated with environmental and psychological stress, as well as increased CRH levels (
      • Kim J.E.
      • Cho D.H.
      • Kim H.S.
      • et al.
      Expression of the corticotropin-releasing hormone-proopiomelanocortin axis in the various clinical types of psoriasis.
      ). As CRH release is dependent upon cholinergic activation in the hypothalamus (
      • Karanth S.
      • Lyson K.
      • McCann S.M.
      Effects of cholinergic agonists and antagonists on interleukin-2-induced corticotropin-releasing hormone release from the mediobasal hypothalamus.
      ), it is possible that the increased CRH levels are a result of enhanced cholinergic regulation in the skin. Furthermore, it was recently demonstrated that secreted mammalian Ly-6/uPAR-related protein-1 is upregulated 3-fold in hyperproliferative skin from patients with psoriasis (
      • Tsuji H.
      • Okamoto K.
      • Matsuzaka Y.
      • et al.
      SLURP-2, a novel member of the human Ly-6 superfamily that is up-regulated in psoriasis vulgaris.
      ;
      • Arredondo J.
      • Chernyavsky A.I.
      • Jolkovsky D.L.
      • et al.
      SLURP-2: a novel cholinergic signaling peptide in human mucocutaneous epithelium.
      ). Secreted mammalian Ly-6/uPAR-related protein-2 appears to preferentially bind the α3nAChR subtype, which indicates a potential role for this nAChR subtype in the progression of psoriasis (
      • Arredondo J.
      • Chernyavsky A.I.
      • Jolkovsky D.L.
      • et al.
      SLURP-2: a novel cholinergic signaling peptide in human mucocutaneous epithelium.
      ).

      Pemphigus

      Pemphigus is a rare, blistering autoimmune skin disorder that targets the skin and mucous membranes, and is mediated by the production of autoantibodies against keratinocyte antigens, including desmogleins, proteins important for cell–cell junctions, and also several nAChRs (reviewed in
      • Grando S.A.
      Autoimmunity to keratinocyte acetylcholine receptors in pemphigus.
      ). Pemphigus patients have reported that an emotional, stressful event is typically a worsening or precipitating factor of pemphigus (
      • Morell-Dubois S.
      • Carpentier O.
      • Cottencin O.
      • et al.
      Stressful life events and pemphigus.
      ). In neonatal mice, nAChR antibodies can induce pemphigus-like lesions, suggesting that these receptors may contribute to the pathogenesis of Pemphigus vulgaris (
      • Nguyen V.T.
      • Ndoye A.
      • Shultz L.D.
      • et al.
      Antibodies against keratinocyte antigens other than desmogleins 1 and 3 can induce pemphigus vulgaris-like lesions.
      ). The role of AMPs in this disease has yet to be elucidated. However, the experimental evidence described here suggests that it is highly likely that the result of desensitized nAChR signaling in keratinocytes could potentially augment cutaneous inflammation and/or AMP expression to further aggravate this disease.

      Mal de Meleda

      Mal de Meleda is an autosomal recessive inflammatory and keratosis palmoplantar disorder, resulting from mutation of secreted mammalian Ly-6/uPAR-related protein-1 (
      • Arredondo J.
      • Chernyavsky A.I.
      • Webber R.J.
      • et al.
      Biological effects of SLURP-1 on human keratinocytes.
      ), which functions as a cofactor to fine-tune cholinergic signaling in keratinocytes. Secreted mammalian Ly-6/uPAR-related protein-1 facilitates keratinization and apoptosis (
      • Mastrangeli R.
      • Donini S.
      • Kelton C.A.
      • et al.
      ARS component B: structural characterization, tissue expression and regulation of the gene and protein (SLURP-1) associated with Mal de Meleda.
      ) and shows high amino-acid composition homology with α-bungarotoxin to function as an allosteric agonist to potentiate acetlycholine-mediated signaling via the α7nAChR subtype (
      • Arredondo J.
      • Chernyavsky A.I.
      • Webber R.J.
      • et al.
      Biological effects of SLURP-1 on human keratinocytes.
      ;
      • Grando S.A.
      Basic and clinical aspects of non-neuronal acetylcholine: biological and clinical significance of non-canonical ligands of epithelial nicotinic acetylcholine receptors.
      ).

      Rosacea

      Patients with rosacea present with excessive facial skin inflammation determined to be triggered, in part, by increased expression of cutaneous serine proteases, kallikrein 5 and 7. Overactive serine protease activity in the stratum corneum results in shorter LL-37 peptides possessing immunostimulatory activity due to additional posttranslational cleavage (
      • Yamasaki K.
      • Di Nardo A.
      • Bardan A.
      • et al.
      Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea.
      ). Clinical observation has indicated that rosacea symptoms improve when patients are actively smoking (
      • Mills C.M.
      Cigarette smoking, cutaneous immunity, and inflammatory response.
      ), whereas symptoms appear to worsen upon cessation of nicotine exposure. This insinuates that nAChR activation, at least in some cases, may serve a protective role to prevent inflammation.

      Netherton's syndrome

      Patients with Netherton's syndrome, a genetically conferred disease, are predisposed to AD and demonstrate a higher risk for cutaneous infection (
      • Stryk S.
      • Siegfried E.C.
      • Knutsen A.P.
      Selective antibody deficiency to bacterial polysaccharide antigens in patients with Netherton syndrome.
      ;
      • Chao S.C.
      • Richard G.
      • Lee J.Y.
      Netherton syndrome: report of two Taiwanese siblings with staphylococcal scalded skin syndrome and mutation of SPINK5.
      ). Patients with Netherton's syndrome carry a mutation in the serine protease inhibitor Kazal-type 5 gene, which codes for lymphoepithelial Kazal-type-related inhibitor (
      • Magert H.J.
      • Standker L.
      • Kreutzmann P.
      • et al.
      LEKTI, a novel 15-domain type of human serine proteinase inhibitor.
      ), a serine proteinase required for the processing of kallikrein proteases into active form (
      • Schechter N.M.
      • Choi E.J.
      • Wang Z.M.
      • et al.
      Inhibition of human kallikreins 5 and 7 by the serine protease inhibitor lympho-epithelial Kazal-type inhibitor (LEKTI).
      ). Loss of lymphoepithelial Kazal-type-related inhibitor may contribute to AD predisposition by altering expression of both structural/cell–cell junction proteins, therefore destabilizing the epidermal barrier. In parallel, the observed increase in the expression of kallikrein proteases in this disease likely augments the proteolytic processing of AMPs and, consequently, inflammation (as indicated for rosacea above).

      Vitiligo

      Vitiligo is a chronic skin disorder in which loss of pigment results in irregular white patches. The abundance of acetylcholinesterase, the enzyme responsible for degrading ACh, is considerably reduced in vitiliginous skin during depigmentation, resulting in elevated ACh levels and concomitant pruritis (
      • Iyengar B.
      Modulation of melanocytic activity by acetylcholine.
      ). Although cholinergic signaling has not been directly correlated with this disease, high ACh levels make it likely that excessive nAChR signaling contributes to disease pathology.

      Botulinum Toxin-A as a Potential Anti-Cholinergic Therapeutic for Treating Skin Dermatoses

      Botulinum toxin-A inhibits the release of ACh from presynaptic vesicles (
      • Hallett M.
      How does botulinum toxin work?.
      ;
      • Huang W.
      • Foster J.A.
      • Rogachefsky A.S.
      Pharmacology of botulinum toxin.
      ), making it a potential therapy for inflammatory diseases correlated with hyperactive nAChR signaling. Several clinical trials have already reported positive outcomes. Subcutaneous injection of botulinum toxin-A in numerous AD clinical trials improved pruritis symptoms (
      • Swartling C.
      • Naver H.
      • Lindberg M.
      • et al.
      Treatment of dyshidrotic hand dermatitis with intradermal botulinum toxin.
      ;
      • Wollina U.
      • Karamfilov T.
      Adjuvant botulinum toxin A in dyshidrotic hand eczema: a controlled prospective pilot study with left-right comparison.
      ). Successful treatments of acne, notalgia paresthetica, and inverse psoriasis have also been described (
      • Diamond A.
      • Jankovic J.
      Botulinum toxin in dermatology - beyond wrinkles and sweat.
      ). In a double-blind, placebo-controlled study of human patients with normal skin, subcutaneous injection of botulinum toxin-A decreased the itch intensity, blood flow, and neurogenic inflammation in response to the histamine prick test (
      • Gazerani P.
      • Pedersen N.S.
      • Drewes A.M.
      • et al.
      Botulinum toxin type A reduces histamine-induced itch and vasomotor responses in human skin.
      ). Thus, blockade of cholinergic signaling in targeted regions of the skin may help alleviate clinical manifestations of cutaneous diseases associated with ACh-mediated symptoms.

      Future Directions

      On the basis of the clinical and experimental evidence presented in this review, it is evident that the cholinergic anti-inflammatory pathway spans several macro- and microenvironments to provide a universal network to control inflammation. In the skin, it remains to be determined whether neuronal ACh released from neural components in the skin is functioning in parallel with or independently from non-neuronal ACh released from keratinocytes to regulate AMP expression and activity in the epidermis. Our in vitro results indicate that the epidermis comprises all of the necessary components to autonomously dampen the epidermal AMP response to infection. The local coordination of ACh signaling within the epidermis makes topical therapeutics a promising solution for directly targeting the cells responsible for ACh-dependent downregulation of AMP expression, and eliminates the need to systemically target the vagus nerve. However, it cannot be ruled out that the immunosuppressive effects of nAChR activation are not multifaceted, where topical agonists or antagonists may activate other resident skin cells, including Langerhans and γδ-T cells, or sensory nerve cells to further inhibit inflammatory pathways in the skin. Elucidating the contribution of each particular nAChR subtype to keratinocyte AMP production and synthesis of structural elements necessary for barrier function is critical for developing unique therapeutic strategies for cutaneous diseases with AMP function as a central component. Moreover, a high degree of cross talk likely exists between the three branches of the stress response. Dissecting the contributions of each particular stress pathway will be a vital factor in the development of therapies. A possibility exists that specific concentrations of ACh must be appropriately maintained within the stratified epidermis to maintain AMP expression and activity during periods of acute stress, which is likely skewed during periods of prolonged stress. This imbalance will consequently augment nAChR activation, resulting in immunosuppression and compromised barrier function. Collectively, a complex network of cellular and biochemical mechanisms facilitate the pathogenesis of skin disease upon activation of the stress response. Activation of the hypothalamic–pituitary–adrenal axis and the adrenergic system are the most extensively studied pathways to date, but a greater emphasis must be made on the role of the cholinergic system in maintaining the chemical and physical skin barrier. Ultimately, these investigations will yield new strategies in the treatment of inflammatory skin disorders.

      ACKNOWLEDGMENTS

      Data from Radek et al. presented in this article were supported by the following VA and NIH/NIAID funds: HHSN26620040029C, ADB contracts N01-AI-40029AI48176, AR45676, AI052453 (awarded to RL Gallo), and NIH F32-AR054220-01A2 (awarded to KAR). Current Loyola University Medical Center research support is provided by NIH/NIAAA 1P30AA019373-01 and 5T32AA013527-09.

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