Vaginal estrogen therapy is associated with increased Lactobacillus in the urine of postmenopausal women with overactive bladder symptoms.

BACKGROUND: Previous work has shown that the vaginal microbiome decreases in Lactobacillus predominance and becomes more diverse after menopause. It has also been shown that estrogen therapy restores Lactobacillus dominance in the vagina and that topical estrogen is associated with overactive bladder symptom improvement. We now know that the bladder contains a unique microbiome and that increased bladder microbiome diversity is associated with overactive bladder. However, there is no understanding of how quickly each pelvic floor microbiome responds to estrogen or if those changes are associated with symptom improvement. OBJECTIVE: This study aimed to determine if estrogen treatment of postmenopausal women with overactive bladder decreases urobiome diversity. STUDY DESIGN: We analyzed data from postmenopausal participants in 2 trials (NCT02524769 and NCT02835846) who chose vaginal estrogen as the primary overactive bladder treatment and used 0.5 g of conjugated estrogen (Premarin cream; Pfizer, New York City, NY) twice weekly for 12 weeks. Baseline and 12-week follow-up data included the Overactive Bladder questionnaire, and participants provided urine samples via catheter, vaginal swabs, perineal swabs, and voided urine samples. Microbes were detected by an enhanced culture protocol. Linear mixed models were used to estimate microbiome changes over time. Urinary antimicrobial peptide activity was assessed by a bacterial growth inhibition assay and correlated with relative abundance of members of the urobiome. RESULTS: In this study, 12 weeks of estrogen treatment resulted in decreased microbial diversity within the vagina (Shannon, P=.047; Richness, P=.043) but not in the other niches. A significant increase in Lactobacillus was detected in the bladder (P=.037) but not in the vagina (P=.33), perineum (P=.56), or voided urine (P=.28). The change in Lactobacillus levels in the bladder was associated with modest changes in urgency incontinence symptoms (P=.02). The relative abundance of the genus Corynebacterium correlated positively with urinary antimicrobial peptide activity after estrogen treatment. CONCLUSION: Estrogen therapy may change the microbiome of different pelvic floor niches. The vagina begins to decrease in diversity, and the bladder experiences a significant increase in Lactobacillus levels; the latter is correlated with a modest improvement in the symptom severity subscale of the Overactive Bladder questionnaire.

Topical nicotinic receptor activation improves wound bacterial infection outcomes and TLR2-mediated inflammation in diabetic mouse wounds.

The cholinergic anti-inflammatory pathway can directly affect skin antibacterial responses via nicotinic acetylcholine receptors (nAChRs). In particular, α7 nAChR (CHRNA7) present in the epidermis modulates the host response to wounding and/or wound bacterial infection. While physiologic inflammation is required to initiate normal wound repair and can be triggered by Toll-like receptor (TLR) activation, chronic inflammation is frequently observed in diabetic wounds and can occur, in part, via excessive TLR2 activation or production. Consequently, this can delay physiologic wound healing responses and increase diabetic host susceptibility to bacterial infection. In this study, we demonstrate that topical nAChR activation diminishes bacterial survival and systemic dissemination in a mouse model of diabetic wound infection, while reducing wound TLR2 production, relative to control mice. We further determined that the antimicrobial peptide activity of diabetic mouse wounds is increased compared to control mice, but this effect is lost following topical nAChR activation. Finally, we observed that human diabetic wounds exhibit impaired α7 nAChR (CHRNA7) abundance and localization relative to human control (nondiabetic) skin. These findings suggest that topical administration of nAChR agonists may improve wound healing and infection outcomes in diabetic wounds by dampening TLR2-mediated inflammation and antimicrobial peptide response, and that the diabetic microenvironment may promote aberrant CHRNA7 production/activation that likely contributes to diabetic wound pathogenesis.

Cutaneous Burn Injury Promotes Shifts in the Bacterial Microbiome in Autologous Donor Skin: Implications for Skin Grafting Outcomes.

INTRODUCTION: The cutaneous microbiome maintains skin barrier function, regulates inflammation, and stimulates wound-healing responses. Burn injury promotes an excessive activation of the cutaneous and systemic immune response directed against commensal and invading pathogens. Skin grafting is the primary method of reconstructing full-thickness burns, and wound infection continues to be a significant complication. METHODS: In this study, the cutaneous bacterial microbiome was evaluated and subsequently compared to patient outcomes. Three different full-thickness skin specimens were assessed: control skin from non-burned subjects; burn margin from burn patients; and autologous donor skin from the same cohort of burn patients. RESULTS: We observed that skin bacterial community structure of burn patients was significantly altered compared with control patients. We determined that the unburned autologous donor skin from burn patients exhibits a microbiome similar to that of the burn margin, rather than unburned controls, and that changes in the cutaneous microbiome statistically correlate with several post-burn complications. We established that Corynebacterium positively correlated with burn wound infection, while Staphylococcus and Propionibacterium negatively correlated with burn wound infection. Both Corynebacterium and Enterococcus negatively correlated with the development of sepsis. CONCLUSIONS: This study identifies distinct differences in the cutaneous microbiome between burn subjects and unburned controls, and ascertains that select bacterial taxa significantly correlate with several comorbid complications of burn injury. These preliminary data suggest that grafting donor skin exhibiting bacterial dysbiosis may augment infection and/or graft failure and sets the foundation for more in-depth and mechanistic analyses in presumably "healthy" donor skin from patients requiring skin grafting procedures.

Cutaneous Burn Injury Modulates Urinary Antimicrobial Peptide Responses and the Urinary Microbiome.

OBJECTIVES: Characterization of urinary bacterial microbiome and antimicrobial peptides after burn injury to identify potential mechanisms leading to urinary tract infections and associated morbidities in burn patients. DESIGN: Retrospective cohort study using human urine from control and burn subjects. SETTING: University research laboratory. PATIENTS: Burn patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Urine samples from catheterized burn patients were collected hourly for up to 40 hours. Control urine was collected from "healthy" volunteers. The urinary bacterial microbiome and antimicrobial peptide levels and activity were compared with patient outcomes. We observed a significant increase in urinary microbial diversity in burn patients versus controls, which positively correlated with a larger percent burn and with the development of urinary tract infection and sepsis postadmission, regardless of age or gender. Urinary psoriasin and ß-defensin antimicrobial peptide levels were significantly reduced in burn patients at 1 and 40 hours postadmission. We observed a shift in antimicrobial peptide hydrophobicity and activity between control and burn patients when urinary fractions were tested against Escherichia coli and Enterococcus faecalis urinary tract infection isolates. Furthermore, the antimicrobial peptide activity in burn patients was more effective against E. coli than E. faecalis. Urinary tract infection-positive burn patients with altered urinary antimicrobial peptide activity developed either an E. faecalis or Pseudomonas aeruginosa urinary tract infection, suggesting a role for urinary antimicrobial peptides in susceptibility to select uropathogens. CONCLUSIONS: Our data reveal potential links for urinary tract infection development and several morbidities in burn patients through alterations in the urinary microbiome and antimicrobial peptides. Overall, this study supports the concept that early assessment of urinary antimicrobial peptide responses and the bacterial microbiome may be used to predict susceptibility to urinary tract infections and sepsis in burn patients.

Burn Injury Alters Epidermal Cholinergic Mediators and Increases HMGB1 and Caspase 3 in Autologous Donor Skin and Burn Margin.

Burn wound healing complications, such as graft failure or infection, are a major source of morbidity and mortality in burn patients. The mechanisms by which local burn injury alters epidermal barrier function in autologous donor skin and surrounding burn margin are largely undefined. We hypothesized that defects in the epidermal cholinergic system may impair epidermal barrier function and innate immune responses. The objective was to identify alterations in the epidermal cholinergic pathway, and their downstream targets, associated with inflammation and cell death. We established that protein levels, but not gene expression, of the α7 nicotinic acetylcholine receptor (CHRNA7) were significantly reduced in both donor and burn margin skin. Furthermore, the gene and protein levels of an endogenous allosteric modulator of CHRNA7, secreted mammalian Ly-6/ urokinase-type plasminogen activator receptor-related protein-1, and acetylcholine were significantly elevated in donor and burn margin skin. As downstream proteins of inflammatory and cell death targets of nAChR activation, we found significant elevations in epidermal High Mobility Group Box Protein 1 and caspase 3 in donor and burn margin skin. Lastly, we employed a novel in vitro keratinocyte burn model to establish that burn injury influences the gene expression of these cholinergic mediators and their downstream targets. These results indicate that defects in cholinergic mediators and inflammatory/apoptotic molecules in donor and burn margin skin may directly contribute to graft failure or infection in burn patients.

Local Burn Injury Promotes Defects in the Epidermal Lipid and Antimicrobial Peptide Barriers in Human Autograft Skin and Burn Margin: Implications for Burn Wound Healing and Graft Survival.

Burn injury increases the risk of morbidity and mortality by promoting severe hemodynamic shock and risk for local or systemic infection. Graft failure due to poor wound healing or infection remains a significant problem for burn subjects. The mechanisms by which local burn injury compromises the epithelial antimicrobial barrier function in the burn margin, containing the elements necessary for healing of the burn site, and in distal unburned skin, which serves as potential donor tissue, are largely unknown. The objective of this study was to establish defects in epidermal barrier function in human donor skin and burn margin, to identify potential mechanisms that may lead to graft failure and/or impaired burn wound healing. In this study, we established that epidermal lipids and respective lipid synthesis enzymes were significantly reduced in both donor skin and burn margin. We further identified diverse changes in the gene expression and protein production of several candidate skin antimicrobial peptides (AMPs) in both donor skin and burn margin. These results also parallel changes in cutaneous AMP activity against common burn wound pathogens, aberrant production of epidermal proteases known to regulate barrier permeability and AMP activity, and greater production of proinflammatory cytokines known to be induced by AMPs. These findings suggest that impaired epidermal lipid and AMP regulation could contribute to graft failure and infectious complications in subjects with burn or other traumatic injury.

Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes.

Dysregulated inflammatory responses are known to impair wound healing in diabetes, but the underlying mechanisms are poorly understood. Here we show that the antimicrobial protein REG3A controls TLR3-mediated inflammation after skin injury. This control is mediated by REG3A-induced SHP-1 protein, and acts selectively on TLR3-activated JNK2. In diabetic mouse skin, hyperglycaemia inhibits the expression of IL-17-induced IL-33 via glucose glycation. The decrease in cutaneous IL-33 reduces REG3A expression in epidermal keratinocytes. The reduction in REG3A is associated with lower levels of SHP-1, which normally inhibits TLR3-induced JNK2 phosphorylation, thereby increasing inflammation in skin wounds. To our knowledge, these findings show for the first time that REG3A can modulate specific cutaneous inflammatory responses and that the decrease in cutaneous REG3A exacerbates inflammation in diabetic skin wounds.

Keratinocyte nicotinic acetylcholine receptor activation modulates early TLR2-mediated wound healing responses.

The cholinergic anti-inflammatory pathway spans several macro- and micro-environments to control inflammation via α7 nicotinic acetylcholine receptors (nAChRs). Physiologic inflammation is necessary for normal wound repair and is triggered, in part, via Toll-like receptors (TLRs). Here, we demonstrate that keratinocyte nAChR activation dampens TLR2-mediated migration and pro-inflammatory cytokine and antimicrobial peptide (AMP) production, which is restored by a α7-selective nAChR antagonist. The mechanism of this response occurs by blocking the NF-κB and Erk1/2 pathway during early and late wound healing. In a mouse model of Staphylococcus aureus wound infection, topical nAChR activation reduces wound AMP and TLR2 production to augment bacterial survival in wild-type mice. These findings suggest that aberrant α7 nAChR activation may impair normal wound healing responses, and that pharmacologic administration of topical nAChR antagonists may improve wound healing outcomes in wounds necessitating a more robust inflammatory response.

Dynamic Role of Host Stress Responses in Modulating the Cutaneous Microbiome: Implications for Wound Healing and Infection.

Significance: Humans are under constant bombardment by various stressors, including psychological anxiety and physiologic injury. Understanding how these stress responses influence the innate immune system and the skin microbiome remains elusive due to the complexity of the neuroimmune and stress response pathways. Both animal and human studies have provided critical information upon which to further elucidate the mechanisms by which mammalian stressors impair normal wound healing and/or promote chronic wound progression. Recent Advances: Development of high-throughput genomic and bioinformatic approaches has led to the discovery of both an epidermal and dermal microbiome with distinct characteristics. This technology is now being used to identify statistical correlations between specific microbiota profiles and clinical outcomes related to cutaneous wound healing and the response to pathogenic infection. Studies have also identified more prominent roles for typical skin commensal organisms in maintaining homeostasis and modulating inflammatory responses. Critical Issues: It is well-established that stress-induced factors, including catecholamines, acetylcholine, and glucocorticoids, increase the risk of impaired wound healing and susceptibility to infection. Despite the characterization of the cutaneous microbiome, little is known regarding the impact of these stress-induced molecules on the development and evolution of the cutaneous microbiome during wound healing. Future Directions: Further characterization of the mechanisms by which stress-induced molecules influence microbial proliferation and metabolism in wounds is necessary to identify altered microbial phenotypes that differentially influence host innate immune responses required for optimal healing. These mechanisms may yield beneficial as targets for manipulation of the microbiome to further benefit the host after cutaneous injury.

Interplay between bladder microbiota and urinary antimicrobial peptides: mechanisms for human urinary tract infection risk and symptom severity.

Resident bacterial communities (microbiota) and host antimicrobial peptides (AMPs) are both essential components of normal host innate immune responses that limit infection and pathogen induced inflammation. However, their interdependence has not been investigated in the context of urinary tract infection (UTI) susceptibility. Here, we explored the interrelationship between the urinary microbiota and host AMP responses as mechanisms for UTI risk. Using prospectively collected day of surgery (DOS) urine specimens from female pelvic floor surgery participants, we report that the relative abundance and/or frequency of specific urinary microbiota distinguished between participants who did or did not develop a post-operative UTI. Furthermore, UTI risk significantly correlated with both specific urinary microbiota and ß-defensin AMP levels. Finally, urinary AMP hydrophobicity and protease activity were greater in participants who developed UTI, and correlated positively with both UTI risk and pelvic floor symptoms. These data demonstrate an interdependency between the urinary microbiota, AMP responses and symptoms, and identify a potential mechanism for UTI risk. Assessment of bacterial microbiota and host innate immune AMP responses in parallel may identify increased risk of UTI in certain populations.

Episodic binge ethanol exposure impairs murine macrophage infiltration and delays wound closure by promoting defects in early innate immune responses.

BACKGROUND: Exacerbation of cutaneous wound infections and delayed wound closure are frequent complications seen in alcohol exposed subjects who sustain injuries. We previously reported that acute alcohol exposure alters the early dermal inflammatory phase of wound healing and also several parameters of the proliferative wound healing phase in wounds from ethanol (EtOH)-treated mice for several days or weeks after EtOH exposure. Hence, it is likely that the cumulative defects arising in the early phases of the wound healing process directly contribute to the increased complications observed in intoxicated patients at the time of injury. METHODS: C57BL/6 mice were given intraperitoneal EtOH (2.2 g/kg body weight) or vehicle (saline) EtOH using our episodic binge EtOH exposure protocol (3 days EtOH, 4 days off, 3 days EtOH) to yield a blood alcohol concentration (BAC) of 300 mg/dl at the time of wounding. Mice were subjected to six 3 mm full-thickness dorsal wounds and immediately treated topically with 10 µl of sterile saline (control) or diluted Staphylococcus aureus corresponding to 1 × 10(4) CFU/wound. Wounds were harvested at 24 hours post injury to evaluate wound area, neutrophil and macrophage accumulation, and the protein levels of cytokines, interleukin-6 (IL-6), IL-1ß, and IL-10, and chemokines, macrophage inflammatory protein-2 (MIP-2) and MIP-1α, monocyte chemotactic protein-1 (MCP-1), and keratinocyte-derived chemokine (KC). The abundance and localization of cathelicidin-related antimicrobial peptide (CRAMP) and the kallikrein epidermal proteases (KLK5 and KLK7) were also determined. RESULTS: Compared to control mice, EtOH-treated mice exhibited delayed wound closure, decreased macrophage accumulation, and impaired production of MIP-1α. Furthermore, skin from EtOH-treated mice demonstrated a reduction in the abundance of epidermal CRAMP and KLK7. CONCLUSIONS: These findings suggest that EtOH exposure hinders several distinct components of the innate immune response, including phagocyte recruitment and chemokine/cytokine and AMP production. Together, these effects likely contribute to delayed wound closure and enhanced infection severity observed in intoxicated patients.

Local burn injury impairs epithelial permeability and antimicrobial peptide barrier function in distal unburned skin.

OBJECTIVES: Our objective was to characterize the mechanisms by which local burn injury compromises epithelial barrier function in burn margin, containing the elements necessary for healing of the burn site, and in distal unburned skin, which serves as potential donor tissue. DESIGN: Experimental mouse scald burn injury. SETTING: University Research Laboratory. SUBJECTS: C57/Bl6 Male mice, 8-12 weeks old. INTERVENTIONS: To confirm that dehydration was not contributing to our observed barrier defects, in some experiments mice received 1 mL of saline fluid immediately after burn, while a subgroup received an additional 0.5 mL at 4 hours and 1 mL at 24 hours following burn. We then assessed skin pH and transepidermal water loss every 12 hours on the burn wounds for 72 hours postburn. MEASUREMENTS AND MAIN RESULTS: Burn margin exhibited increased epidermal barrier permeability indicated by higher pH, greater transepidermal water loss, and reduced lipid synthesis enzyme expression and structural protein production up to 96 hours postburn. By contrast, antimicrobial peptide production and protease activity were elevated in burn margin. Skin extracts from burn margin did not exhibit changes in the ability to inhibit bacterial growth. However, distal unburned skin from burned mice also demonstrated an impaired response to barrier disruption, indicated by elevated transepidermal water loss and reduced lipid synthesis enzyme and structural protein expression up to 96 hours postburn. Furthermore, skin extracts from distal unburned skin exhibited greater protease activity and a reduced capacity to inhibit bacterial growth of several skin pathogens. Finally, we established that antimicrobial peptide levels were also altered in the lung and bladder, which are common sites of secondary infection in burn-injured patients. CONCLUSIONS: These findings reveal several undefined deficiencies in epithelial barrier function at the burn margin, potential donor skin sites, and organs susceptible to secondary infection. These functional and biochemical data provide novel insights into the mechanisms for graft failure and secondary infection after burn injury.

Interleukin-33 increases antibacterial defense by activation of inducible nitric oxide synthase in skin.

Interleukin-33 (IL-33) is associated with multiple diseases, including asthma, rheumatoid arthritis, tissue injuries and infections. Although IL-33 has been indicated to be involved in Staphylococcus aureus (S. aureus) wound infection, little is known about how IL-33 is regulated as a mechanism to increase host defense against skin bacterial infections. To explore the underlying intricate mechanism we first evaluated the expression of IL-33 in skin from S. aureus-infected human patients. Compared to normal controls, IL-33 was abundantly increased in skin of S. aureus-infected patients. We next developed a S. aureus cutaneous infection mouse model and found that IL-33 was significantly increased in dermal macrophages of infected mouse skin. The expression of IL-33 by macrophages was induced by staphylococcal peptidoglycan (PGN) and lipoteichoic acid (LTA) via activation of toll-like receptor 2(TLR2)-mitogen-activated protein kinase (MAPK)-AKT-signal transducer and activator of transcription 3(STAT3) signaling pathway as PGN and LTA failed to induce IL-33 in Tlr2-deficient peritoneal macrophages, and MAPK,AKT, STAT3 inhibitors significantly decreased PGN- or LTA-induced IL-33. IL-33, in turn, acted on macrophages to induce microbicidal nitric oxygen (NO) release. This induction was dependent on inducible nitric oxide synthase (iNOS) activation, as treatment of macrophages with an inhibitor of iNOS, aminoguanidine, significantly decreased IL-33-induced NO release. Moreover, aminoguanidine significantly blocked the capacity of IL-33 to inhibit the growth of S. aureus, and IL-33 silencing in macrophages significantly increased the survival of S. aureus in macrophages. Furthermore, the administration of IL-33-neutralizing antibody into mouse skin decreased iNOS production but increased the survival of S. aureus in skin. These findings reveal that IL-33 can promote antimicrobial capacity of dermal macrophages, thus enhancing antimicrobial defense against skin bacterial infections.

Chronic alcohol exposure renders epithelial cells vulnerable to bacterial infection.

Despite two centuries of reports linking alcohol consumption with enhanced susceptibility to bacterial infections and in particular gut-derived bacteria, there have been no studies or model systems to assess the impact of long-term alcohol exposure on the ability of the epithelial barrier to withstand bacterial infection. It is well established that acute alcohol exposure leads to reduction in tight and adherens junctions, which in turn leads to increases in epithelial cellular permeability to bacterial products, leading to endotoxemia and a variety of deleterious effects in both rodents and human. We hypothesized that reduced fortification at junctional structures should also reduce the epithelial barrier's capacity to maintain its integrity in the face of bacterial challenge thus rendering epithelial cells more vulnerable to infection. In this study, we established a cell-culture based model system for long-term alcohol exposure to assess the impact of chronic alcohol exposure on the ability of Caco-2 intestinal epithelial cells to withstand infection when facing pathogenic bacteria under the intact or wounded conditions. We report that daily treatment with 0.2% ethanol for two months rendered Caco-2 cells far more susceptible to wound damage and cytotoxicity caused by most but not all bacterial pathogens tested in our studies. Consistent with acute alcohol exposure, long-term ethanol exposure also adversely impacted tight junction structures, but in contrast, it did not affect the adherens junction. Finally, alcohol-treated cells partially regained their ability to withstand infection when ethanol treatment was ceased for two weeks, indicating that alcohol's deleterious effects on cells may be reversible.

Nicotinic acetylcholine receptor stimulation impairs epidermal permeability barrier function and recovery and modulates cornified envelope proteins.

AIM: To characterize how nicotinic acetylcholine receptors (nAChRs) influence epidermal barrier function and recovery following prolonged stress or direct nAChR activation or antagonism. MAIN METHODS: Mice were subjected to psychological stress or treated topically with nAChR agonist or antagonist for 3 days. We assessed barrier permeability and recovery by measuring transepidermal water loss (TEWL) before and after barrier disruption. In parallel, we analyzed the production and localization of several epidermal cornified envelope proteins in mouse skin and in human EpiDerm™ organotypic constructs stimulated with a nAChR agonist (nicotine) and/or a nAChR selective antagonist (α-bungarotoxin). KEY FINDINGS: We determined that psychological stress in mice impairs barrier permeability function and recovery, an effect that is reversed by application of the α7 selective nAChR antagonist, α-bungarotoxin (Bung). In the absence of stress, both topical nicotine or Bung treatment alone impaired barrier permeability. We further observed that stress, topical nicotine, or topical Bung treatment in mice influenced the abundance and/or localization of filaggrin, loricrin, and involucrin. Similar alterations in these three major cornified envelope proteins were observed in human EpiDerm™ cultures. SIGNIFICANCE: Perceived psychological stress and nicotine usage can both initiate or exacerbate several dermatoses by altering the cutaneous permeability barrier. Modulation of nAChRs by topical agonists or antagonists may be used to improve epidermal barrier function in skin diseases associated with defects in epidermal barrier permeability.

The antimicrobial protein REG3A regulates keratinocyte proliferation and differentiation after skin injury.

Epithelial keratinocyte proliferation is an essential element of wound repair, and abnormal epithelial proliferation is an intrinsic element in the skin disorder psoriasis. The factors that trigger epithelial proliferation in these inflammatory processes are incompletely understood. Here we have shown that regenerating islet-derived protein 3-alpha (REG3A) is highly expressed in keratinocytes during psoriasis and wound repair and in imiquimod-induced psoriatic skin lesions. The expression of REG3A by keratinocytes is induced by interleukin-17 (IL-17) via activation of keratinocyte-encoded IL-17 receptor A (IL-17RA) and feeds back on keratinocytes to inhibit terminal differentiation and increase cell proliferation by binding to exostosin-like 3 (EXTL3) followed by activation of phosphatidylinositol 3 kinase (PI3K) and the kinase AKT. These findings reveal that REG3A, a secreted intestinal antimicrobial protein, can promote skin keratinocyte proliferation and can be induced by IL-17. This observation suggests that REG3A may mediate the epidermal hyperproliferation observed in normal wound repair and in psoriasis.

Sugar-coating wound repair: a review of FGF-10 and dermatan sulfate in wound healing and their potential application in burn wounds.

Thousands of patients suffer from burn injuries each year, yet few therapies have been developed to accelerate the wound healing process. Most fibroblast growth factors (FGFs) have been extensively evaluated but only a few have been found to participate in the wound healing process. In particular, FGF-10 is robustly increased in the wound microenvironment after injury and has demonstrated some ability to promote wound healing in vitro and in vivo. Glycosaminoglycans are linear carbohydrates that participate in wound repair by influencing cytokine/growth factor localization and interaction with cognate receptors. Dermatan sulfate (DS) is the most abundant glycosaminoglycan in human wound fluid and has been postulated to be directly involved in the healing process. Recently, the combination of FGF-10 and DS demonstrated the potential to accelerate wound healing via increased keratinocyte proliferation and migration. Based on these preliminary studies, DS may serve as a cofactor for FGF-10, and together they are likely to expedite the healing process by stimulating keratinocyte activity. As a specific subtype of wounds, the overall healing process of burn injuries does not significantly differ from other types of wounds, where optimal repair results in matrix regeneration and complete reepithelialization. At present, standard burn treatment primarily involves topical application of antimicrobial agents, while no routine therapies target acceleration of reepithelialization, the key to wound closure. Thus, this novel therapeutic combination could be used in conjunction with some of the current therapies, but it would have the unique ability to initiate wound healing by stimulating keratinocyte epithelialization.

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.

Expression of epidermal CAMP changes in parallel with permeability barrier status.

Two critical defensive functions of the outer epidermis, the permeability barrier and antimicrobial defense, share certain structural and biochemical features. Moreover, three antimicrobial peptides (AMPs), i.e., mouse ß-defensin 3 (mBD3), mouse cathelicidin antimicrobial peptide (mCAMP), and the neuroendocrine peptide, catestatin (Cst), all localize to the outer epidermis, and both mBD3 and mCAMP are secreted from the epidermal lamellar bodies with other organelle contents that subserve the permeability barrier. These three AMPs are upregulated in response to acute permeability barrier disruption, whereas conversely, mCAMP-/- mice (unable to combat Gram-positive pathogens) also display abnormal barrier homeostasis. To determine further whether these two functions are co-regulated, we investigated changes in immunostaining for these three AMPs in skin samples in which the permeability barrier function in mice had been either compromised or enhanced. Compromised or enhanced barrier function correlated with reduced or enhanced immunohistochemical expression of mCAMP, respectively, but conversely with Cst expression, likely due to the role of this AMP as an endogenous inhibitor of cathelicidin expression. mBD3 expression correlated with experimental barrier perturbations, but poorly with developmental changes in barrier function. These studies show that changes in cathelicidin and Cst expression parallel changes in permeability barrier status, with a less clear relationship with mBD3 expression.