CHOLINERGIC REGULATION OF KERATINOCYTE INNATE IMMUNITY AND PERMEABILITY BARRIER INTEGRITY: NEW PERSPECTIVES IN EPIDERMAL IMMUNITY AND DISEASE

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. AMP) are co-dependent (Ahrens, Schunck et al. 2011). Studies have shown that a diminished permeability barrier function is associated with reduced lipid synthesis and AMP production (Aberg, Man et al. 2008). Injury and infection act 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 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 to atopic dermatitis (AD) and acne vulgaris (Griffiths and Richards 2001), psoriatic plaques are generally resistant to infection (Raychaudhuri and Raychaudhuri 1993). 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, Ohtake et al. 2002; Nomura, Goleva et al. 2003). 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 and Usman 2001; Picardi and Abeni 2001). This effect is twofold, 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 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 2002). Once the stressors are eliminated, the biochemical profile of the organism is expected to return to normal physiologic homeostasis. However, prolonged activation of neuroendocrine responses can directly promote systemic immunosuppression (reviewed in (Dhabhar 2000)).

Stress leads to activation of the parasympathetic (PSNS) and sympathetic nervous systems (SNS), as well as the hypothalamic-pituitary-adrenal axis (HPA). Activation of the PSNS results in the release of ACh from nerve fibers innervating the organs, resulting in activation of nAChR and/or muscarinic acetylcholine receptors (mAChRs). Activation of the SNS results in the release of epinephrine/norepinephrine (E/NE), activating α- or β-adrenergic receptors. PSNS and SNS activation mediates a variety of immunological and homeostatic functions, including changes in cellular proliferation, differentiation, and migration ((Maestroni 2006) (reviewed in (Slominski and Wortsman 2000)). Activation of the HPA axis stimulates the release of several neuroendocrine mediators, such as GCs (e.g. cortisol), into the bloodstream that act upon specific cell populations and tissues to generate an immunosuppressive effect and return proinflammatory mediators to normal, baseline levels (Herman and Cullinan 1997). However, prolonged or chronic stress tends to be immunosuppressive, in part, by dampening cytokine production and immune cell phagocytic capacity ((Abang, Baum et al. 2006); (Ashcraft and Bonneau 2008); (Dhabhar, Saul et al. 2010)). This feedback loop was discovered over a decade ago by Kevin Tracey and colleagues. This pathway was later coined the “Cholinergic anti-inflammatory pathway” after efferent activity in the vagus nerve was found to inhibit local pro-inflammatory 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, Endo et al. 1996; Bernik, Friedman et al. 2002; Saeed, Varma et al. 2005; Pavlov, Ochani et al. 2006). 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, Wilder et al. 2000; Tracey 2002). 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 TNF-α during injury or infection to amplify the inflammatory response by stimulating the release of pro-inflammatory mediators, including IL-1, reactive oxygen species (ROS), nitric oxide (NO), and eicosanoids (Wood, Jackson et al. 1992; Nickoloff and Naidu 1994). During the initial fight-or-flight response, all three pathways of the stress response are coordinately activated to modulate inflammation. For example, E/NE can block macrophage activation and dampen production of TNF-α and other proinflammatory cytokines (Chrousos 2000; Molina 2001; Molina, Bagby et al. 2001). Alternatively, E/NE can induce the release of the anti-inflammatory cytokine, interleukin-10 (IL-10) from monocytes (van der Poll, Coyle et al. 1996; Woiciechowsky, Asadullah et al. 1998). In parallel, ACh has the capacity to inhibit macrophage activation and release of pro-inflammatory cytokines, such as TNF-α, IL-1, and high mobility group protein B1 (HMBG1) (Borovikova, Ivanova et al. 2000), ultimately limiting tissue damage caused by excessive inflammation.

Psychological stress was initially found to delay wound healing and diminish epidermal barrier homeostasis (Kiecolt-Glaser, Marucha et al. 1995; Denda, Tsuchiya et al. 1998; Marucha, Kiecolt-Glaser et al. 1998; Garg, Chren et al. 2001; Rojas, Padgett et al. 2002), 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, CAMP and mBD-3, compared to unstressed mice. Moreover, these mice were more susceptible to cutaneous Group A Streptococcus (GAS) infection, as indicated by larger skin lesions compared to 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 GC-receptor mediated activation was involved in the suppression of AMP expression (Aberg, Radek et al. 2007). 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 and Hadley 1969; Zasloff 1987). 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, Rizzo et al. 2006; Sivamani, Pullar et al. 2009). 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 the neuroectoderm. Consequently, many key factors that influence neuronal CNS activities also contribute to non-neuronal cellular function, since most immune and epidermal cells express several components of the cholinergic system ((reviewed in (Radek 2010). ACh can facilitate the communication between immune and epidermal cells to mount a physiologic 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 and Fujii 2004)). Previous work by Kevin Tracey and colleagues identified that the α7 nAChR is the primary receptor involved in the cholinergic anti-inflammatory pathway in bone-marrow derived immune cells (Wang, Yu et al. 2003). During bacterial infection or following injury, TNF-α acts as an inflammatory input signal to the autonomic nervous system (ANS), which reflexively responds by stimulating the vagus nerve, resulting in activation of humoral anti-inflammatory responses (Tracey 2002). 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 α7 nAChRs expressed on cytokine-producing cells (e.g. macrophages, 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, Kist et al. 1993; Grando, Kawashima et al. 2007). 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 knock-out animals for several nAChR subtypes display diverse phenotypes (reviewed in (Grando, Pittelkow et al. 2006)). While the role of cholinergic signaling in keratinocyte proliferation, differentiation, and migration has been well described (reviewed in(Grando, Pittelkow et al. 2006)), 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 2007). Ultimately, this creates a detrimental negative feedback loop in the epidermis that results in immunosuppresion of AMP production and activity, and likely other pro-inflammatory 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 AMP expression. Our work in both keratinocytes and mouse models of nAChR activation determined that the α7 nAChR subtype is a major player involved in AMP suppression in the epidermis (Radek, Elias et al. 2010), which parallels the work by Kevin Tracey and colleagues (Borovikova, Ivanova et al. 2000; Bernik, Friedman et al. 2002; Bernik, Friedman et al. 2002; Tracey 2002; Tracey 2007). 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.

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 based on their respective agonists, nicotine and muscarine (Grando, Pittelkow et al. 2006). ACh is synthesized from coenzyme A and choline by cholineacetyltransferase (ChAT) and is degraded by Acetylcholinesterase (AChE). 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 1993; Denda, Tsuchiya et al. 2000) Keratinocyte cells can synthesize a average of 2 × 10⁻¹⁷ mol and secrete 7 × 10⁻¹⁹ mol of ACh per minute ((Grando, Kist et al. 1993). However, a gradient of ACh expression throughout the epidermis is observed, with highest ACh levels present in the uppermost epidermal compartment (Nguyen, Ndoye et al. 2001), likely because the highest amounts of ChAT, the ACh degrading enzyme is located predominantly in the basal layer (Johansson and Wang 1993; Klapproth, Reinheimer et al. 1997) . This intricate ACh-dependent network is referred to as the non-neuronal cholinergic system (Klapproth, Reinheimer et al. 1997; Wessler, Kirkpatrick et al. 1998; Wessler, Kirkpatrick et al. 1999).

The nAChR superfamily of ligand-gated ionotropic receptors are comprised of homo-or heterotypic combinations of nine α subunits (α₂–α₁₀) and three β subunits (β₂–β₄) (Steinbach 1990). 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, Berger et al. 2004) ( 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, Ndoye et al. 2001). Knockdown of α7 nAChR in cultured keratinocytes blocked terminal differentiation, which was coupled to the increased expression of pro-apoptotic signaling factors. Furthermore, the inhibition of α7 nAChR signaling using the pharmacologic antagonist α-Bungarotoxin promoted cell cycle progression (Arredondo, Nguyen et al. 2003). This suggests that α7 nAChR signaling facilitates normal apoptotic signals to generate the stratum corneum. Combined pharmacologic nAChR agonist and mAChR antagonist was also found to increase intracellular Ca²⁺ and trigger apoptotic secretion in vitro (Nguyen, Ndoye et al. 2001). 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.

KERATINOCYTES AS KEY MEDIATORS OF CUTANEOUS BARRIER FUNCTION

THE ROLE OF α7 nAChR IN KERATINOCYE BARRIER FUNCTION: LESSONS FROM MURINE KNOCKOUTS

Although numerous nAChR subtypes exist in the epidermis, there is evidence to suggest that similarly to the systemic, neuronal cholinergic anti-inflammatory pathway, the α7 nAChR subtype is likely to play a significant role in the regulation of the local cutaneous innate immune and barrier function. Loss of α7 nAChR 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, Nguyen et al. 2002). Dramatic differences between the histology of Chrna7 ^−/− and Chrna7^+/+ were observed in 1–3 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, Nguyen et al. 2002). 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 α7 nAChR signaling (Arredondo, Nguyen et al. 2002).

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 1982). Only recently have we begun to elucidate the neuroendrocrine contribution of epithelial immunity. Stressed individuals have increased epithelial ACh levels (Schlereth, Schonefeld et al. 2007) and are more susceptible to opportunistic infections (Wu, Zaborina et al. 2005; Ashcraft, Hunzeker et al. 2008). 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.

Since 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 VitaminD₃ (VD3), and the TLR2/6 agonist, macrophage activating lipoprotein-2 (MALP2) allows for maximal gene and protein expression of both cathelicidin and β-defensin-2 through differentiation and bacterial stimuli, respectively (Weber, Heilborn et al. 2005; Schauber, Dorschner et al. 2006). 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 a ~50% reduction in CAMP (Radek, Elias et al. 2010). We observed a similar suppression of hBD-2 gene and protein expression by ACh compared the MALP-2 alone. To further elucidate the specific receptor subtype mediating the dampening response by ACh, additional experiments were performed using two pharmacologic antagonists, α-Bungarotoxin, which is selective for the α7 nAChR subunit, and mecamylamine, with known selectivity for α3β4 nAChR subtypes (Grando, Pittelkow et al. 2006), or an endogenous epidermal nAChR antagonist, Cst (Radek, Lopez-Garcia et al. 2008). 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 to 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 anti-bacterial activity in NHEKs consistent with the critical role of AMPs in this response (Radek, Elias et al. 2010). Treatment of NHEKs with muscarine, a mAChR-specific agonist or propranolol, a β-adrenergic pathway agonist, were unable to suppress the induced AMP expression observed with VD3/MALP2 treatment, suggesting ACh regulation of AMP expression in keratinocytes is through direct activation of nAChRs (Radek, Elias et al. 2010). 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 α7 nAChR, similarly to the neuronal cholinergic response to infection (Tracey 2007). Our results indicate that the changes in direct extractable anti-bacterial activity in these NHEKs lysates was likely due to, in part, decreased cathelicidin and hBD-2 expression However, it is possible that the levels of other inducible AMPs, (e.g. RNAse7 and hBD-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, Bartels et al. 2001; Harder and Schroder 2002). 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 (GAS), E. coli, Pseudomonas aeruginosa, and C. albicans, as well as several varieties of filamentous fungi in vitro (Briolat, Wu et al. 2005; Radek, Lopez-Garcia et al. 2008). Since the concentration of Cst required for microbicidal killing was much higher than levels observed in normal skin (Radek, Lopez-Garcia et al. 2008), we proposed that the antimicrobial properties may be secondary to its primary role as a natural, endogenous nAChR antagonist. Like several neuroendocrine-derived molecules, such as α-Melanocyte Stimulating Hormone (α-MSH), Neurokinin-1 (NK-1), and Adrenomedullin (AM), Cst may exhibit microbicidal activity due to their inherently small, cationic, amphipathic structure (Reviewed in (Radek and Gallo 2007). 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 acting primarily as an endogenous nAChR antagonist (Radek, Elias et al. 2010).

To verify the functional relevance of cholinergic activation and keratinocyte AMP regulation in vivo, we used a pharmacologic and genetic approach using two murine models of cholinergic activation (Radek, Elias et al. 2010). For the pharmacologic 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 GAS infection compared to vehicle treated mice, as indicated by smaller lesion size, less surviving bacteria in lesions, and lower dissemination into distal organs (Radek, Elias et al. 2010). 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, Elias et al. 2010). We later defined the role of nAChR activation in regulating AMP activity in the skin by employing a genetic approach by utilizing 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 GAS skin infection. These mice also exhibited a significant reduction in extractable AMP activity, which was restored by the presence of topical of α-Bungarotoxin. Together, these data suggested that the regulation of epidermal AMP expression and activity by ACh is highly dependent on the α7 nAChR subtype, but may also involve other subtypes to a lesser degree (Radek, Elias et al. 2010). Additionally, 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 and Lyte 2008). 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, Elias et al. 2010). 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, Tandel et al. 2006). 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

BOTULINUM TOXIN-A AS A POTENTIAL ANTI-CHOLINERGIC THERAPEUTIC FOR TREATING SKIN DERMATOSES

Botulinum toxin-A (BoNT/A) inhibits the release of ACh from presynaptic vesicles (Hallett 2000; Huang, Foster et al. 2000), making it a potential therapy for inflammatory diseases correlated with hyperactive nAChR signaling. Several clinical trials have already reported positive outcomes. Subcutaneous injection of BoNT/A in numerous AD clinical trials improved pruritis symptoms (Swartling, Naver et al. 2002; Wollina and Karamfilov 2002). Successful treatments of acne, notalgia paresthetica, and inverse psoriasis has also been described (Diamond and Jankovic 2006). In a double-blind, placebo controlled study of human patients with normal skin, subcutaneous injection of BoNT/A decreased the itch intensity, blood flow and neurogenic inflammation in response to the histamine prick test (Gazerani, Pedersen et al. 2009). 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

Based on 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 if neuronal ACh released from neural components in the skin is acting in parallel with or independently from non-neuronal ACh released from keratinocytes to regulation AMP expression and activity in the epidermis. Our in vitro results indicate that the epidermis is comprised of 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 of 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 skin disease upon activation of the stress response. Activation of the HPA 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.