Defining the activity of pro-reparative extracellular vesicles in wound healing based on miRNA payloads and cell type-specific lineage mapping.

Small extracellular vesicles (EVs) are released by cells and deliver biologically active payloads to coordinate the response of multiple cell types in cutaneous wound healing. Here we used a cutaneous injury model as a donor of pro-reparative EVs to treat recipient diabetic obese mice, a model of impaired wound healing. We established a functional screen for microRNAs (miRNAs) that increased the pro-reparative activity of EVs and identified a down-regulation of miR-425-5p in EVs in vivo and in vitro associated with the regulation of adiponectin. We tested a cell type-specific reporter of a tetraspanin CD9 fusion with GFP to lineage map the release of EVs from macrophages in the wound bed, based on the expression of miR-425-5p in macrophage-derived EVs and the abundance of macrophages in EV donor sites. Analysis of different promoters demonstrated that EV release under the control of a macrophage-specific promoter was most abundant and that these EVs were internalized by dermal fibroblasts. These findings suggested that pro-reparative EVs deliver miRNAs, such as miR-425-5p, that stimulate the expression of adiponectin that has insulin-sensitizing properties. We propose that EVs promote intercellular signaling between cell layers in the skin to resolve inflammation, induce proliferation of basal keratinocytes, and accelerate wound closure.

Serpin-loaded extracellular vesicles promote tissue repair in a mouse model of impaired wound healing.

Chronic metabolic diseases such as diabetes are characterized by delayed wound healing and a dysregulation of the inflammatory phase of wound repair. Our study focuses on changes in the payload of extracellular vesicles (EVs) communicating between immune cells and stromal cells in the wound bed, which regulate the rate of wound closure. Adoptive transfer of EVs from genetically defined mouse models are used here to demonstrate a functional and molecular basis for differences in the pro-reparative biological activity of diabetic (db/db) vs. wildtype EVs in wound healing. We identify several members of the Serpin family of serine protease inhibitors that are absent in db/db EVs, then we overexpress Serpin A1, F2 and G1 in EVs to evaluate their effect on wound healing in db/db mice. Serpins have an important role in regulating levels of elastase, plasmin and complement factors that coordinate immune cell signaling in full thickness wounds in a diabetic model. Here, we establish a novel therapeutic approach by engineering the payload of EVs based on proteomic analysis. Serpin-loaded EVs were used to rescue the Serpin deficiency identified by proteomics and promote wound healing in db/db mice, as well as evaluated how EVs affected extracellular matrix remodeling and the resolution of tissue injury. Therefore, we propose that the identification of EV payloads that are downregulated in diabetic wounds can be systematically analyzed for their functional activity and potential as a therapeutic, based on whether their re-expression in engineered EVs restores normal kinetics of tissue repair in chronic wounds.

ECRG4 regulates neutrophil recruitment and CD44 expression during the inflammatory response to injury.

The complex molecular microenvironment of the wound bed regulates the duration and degree of inflammation in the wound repair process, while its dysregulation leads to impaired healing. Understanding factors controlling this response provides therapeutic targets for inflammatory disease. Esophageal cancer-related gene 4 (ECRG4) is a candidate chemokine that is highly expressed on leukocytes. We used ECRG4 knockout (KO) mice to establish that the absence of ECRG4 leads to defective neutrophil recruitment with a delay in wound healing. An in vitro human promyelocyte model identified an ECRG4-mediated suppression of the hyaluronic acid receptor, CD44, a key receptor mediating inflammation resolution. In ECRG4 KO mouse leukocytes, there was an increase in CD44 expression, consistent with a model in which ECRG4 negatively regulates CD44 levels. Therefore, we propose a previously unidentified mechanism in which ECRG4 regulates early neutrophil recruitment and subsequent CD44-mediated resolution of inflammation.

TBC1D3 regulates the payload and biological activity of extracellular vesicles that mediate tissue repair.

Healthy repair of cutaneous injury is a coordinated response of inflammatory cells, secreted factors, and biologically active extracellular vesicles (EVs). Although constitutive release of EVs into biologic fluids is a hallmark of cultured cells and tumors, their payload and biologic activity appears to be tightly regulated. We show that Tre-2/Bub2/Cdc16 (TBC1) domain family member 3 (TBC1D3) drives the release of an EV population that causes a decrease in phosphorylation of the transcription factor signal transducer and activator of transcription 3 in naive recipient cells. To explore the biologic activity of EVs in vivo, we used a mouse model of sterile subcutaneous inflammation to determine the payload and biologic activity of EVs released into the microenvironment by committed myeloid lineages and stroma. Expression of TBC1D3 in macrophages altered the payload of their released EVs, including RNA-binding proteins, molecular motors, and proteins regulating secretory pathways. A wound-healing model demonstrated that closure was delayed by EVs released under the control of TBC1D3. We show that modulating the secretory repertoire of a cell regulates EV payload and biologic activity that affects outcomes in tissue repair and establishes a strategy for modifying EVs mediating specific biologic responses.-Qin, S., Dorschner, R. A., Masini, I., Lavoie-Gagne, O., Stahl, P. D., Costantini, T. W., Baird, A., Eliceiri, B. P. TBC1D3 regulates the payload and biological activity of extracellular vesicles that mediate tissue repair.

Activation of Parathyroid Hormone 2 Receptor Induces Decorin Expression and Promotes Wound Repair.

In this study, we report that TIP39, a parathyroid hormone ligand family member that was recently identified to be expressed in the skin, can induce decorin expression and enhance wound repair. Topical treatment of mice with TIP39 accelerated wound repair, whereas TIP39-deficient mice had delayed repair that was associated with formation of abnormal collagen bundles. To study the potential mechanism responsible for the action of TIP39 in the dermis, fibroblasts were cultured in three-dimensional collagen gels, a process that results in enhanced decorin expression unless activated to differentiate to adipocytes, whereupon these cells reduce expression of several proteoglycans, including decorin. Small interfering RNA-mediated silencing of parathyroid hormone 2 receptor (PTH2R), the receptor for TIP39, suppressed the expression of extracellular matrix-related genes, including decorin, collagens, fibronectin, and matrix metalloproteases. Skin wounds in TIP39-/- mice had decreased decorin expression, and addition of TIP39 to cultured fibroblasts induced decorin and increased phosphorylation and nuclear translocation of CREB. Fibroblasts differentiated to adipocytes and treated with TIP39 also showed increased decorin and production of chondroitin sulfate. Furthermore, the skin of PTH2R-/- mice showed abnormal extracellular matrix structure, decreased decorin expression, and skin hardness. Thus, the TIP39-PTH2R system appears to be a previously unrecognized mechanism for regulation of extracellular matrix formation and wound repair.

Selective antimicrobial action is provided by phenol-soluble modulins derived from Staphylococcus epidermidis, a normal resident of the skin.

Antimicrobial peptides serve as a first line of innate immune defense against invading organisms such as bacteria and viruses. In this study, we hypothesized that peptides produced by a normal microbial resident of human skin, Staphylococcus epidermidis, might also act as an antimicrobial shield and contribute to normal defense at the epidermal interface. We show by circular dichroism and tryptophan spectroscopy that phenol-soluble modulins (PSMs) gamma and delta produced by S. epidermidis have an alpha-helical character and a strong lipid membrane interaction similar to mammalian AMPs such as LL-37. Both PSMs directly induced lipid vesicle leakage and exerted selective antimicrobial action against skin pathogens such as Staphylococcus aureus. PSMs functionally cooperated with each other and LL-37 to enhance antimicrobial action. Moreover, PSMs reduced Group A Streptococcus (GAS) but not the survival of S. epidermidis on mouse skin. Thus, these data suggest that the production of PSMgamma and PSMdelta by S. epidermidis can benefit cutaneous immune defense by selectively inhibiting the survival of skin pathogens while maintaining the normal skin microbiome.

Mast cell cathelicidin antimicrobial peptide prevents invasive group A Streptococcus infection of the skin.

Mast cells (MC) express cathelicidin antimicrobial peptides that act as broad-spectrum antibiotics and influence the immune defense of multiple epithelial surfaces. We hypothesized that MC help protect against skin infection through the expression of cathelicidin. The susceptibility of MC-deficient mice (Kit Wsh(-/-)) to invasive group A streptococcus (GAS) was compared with control mice. Following s.c. injection of GAS, MC-deficient mice had 30% larger skin lesions, 80% more lesional bacteria, and 30% more spleens positive for bacteria. In contrast to results obtained when GAS was injected into skin, no significant differences were noted between MC-deficient mice and control mice after GAS was applied topically, indicating that MC activity is most important after barrier penetration. To determine whether these differences were due to MC expression of cathelicidin, MC-deficient mice were reconstituted with MC derived from either wild-type or cathelicidin-deficient (Camp(-/-)) mice and challenged with GAS. Forty-eight hours after bacterial injection, mice that did not receive MC had an average lesion size of 200 mm(2), mice reconstituted with wild-type MC showed lesions comparable to control mice (25 mm(2)), while mice reconstituted with Camp(-/-) MC showed an average lesion size of 120 mm(2). Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) analysis of cathelicidin peptide purified from mast cells defined this as a unique 28-aa peptide. Combined, these results show that MC confer defense against Gram-positive bacterial infection in the skin, a function mediated in part by the expression of a unique cathelicidin peptide.

Expression of antimicrobial peptides in the normal and involved skin of patients with infective cellulitis.

BACKGROUND: Endogenous antimicrobial peptides participate in the innate defense of skin against a variety of pathogens. The systemic expression of these peptides in normal-appearing skin of patients with infective cellulitis is unknown. METHODS: Study patients were adults with infective cellulitis and signs of systemic inflammation. Skin biopsy and serum specimens were obtained from patients and from control subjects with no active infection. Cathelicidin and human beta-defensin 2 mRNA expression were determined by real-time polymerase chain reaction. RESULTS: Skin biopsy specimens from 11 patients and 4 uninfected control subjects were analyzed. The relative expression level for cathelicidin mRNA was elevated in both the involved and the distal normal-appearing skin of patients with cellulitis, compared with expression in the skin of control subjects (mean ratios, 39.46 vs. 1.32, P=.0059; and 21.41 vs. 1.32, P=.0059). Similarly, the relative expression level of human beta -defensin 2 mRNA was elevated in both the involved skin (mean ratios, 20,844 vs. 11.65; P=.0015) and in distal normal-appearing skin of patients with cellulitis (mean ratios, 201.1 vs. 11.65; P=.0103). DISCUSSION: In response to cutaneous infection there is a local and distal increase in endogenous antimicrobial peptide mRNA in both involved and normal-appearing skin. These observations show, for the first time to our knowledge, that after infection the human body responds by increasing systemic innate immunity.

Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea.

Acne rosacea is an inflammatory skin disease that affects 3% of the US population over 30 years of age and is characterized by erythema, papulopustules and telangiectasia. The etiology of this disorder is unknown, although symptoms are exacerbated by factors that trigger innate immune responses, such as the release of cathelicidin antimicrobial peptides. Here we show that individuals with rosacea express abnormally high levels of cathelicidin in their facial skin and that the proteolytically processed forms of cathelicidin peptides found in rosacea are different from those present in normal individuals. These cathelicidin peptides are a result of a post-translational processing abnormality associated with an increase in stratum corneum tryptic enzyme (SCTE) in the epidermis. In mice, injection of the cathelicidin peptides found in rosacea, addition of SCTE, and increasing protease activity by targeted deletion of the serine protease inhibitor gene Spink5 each increases inflammation in mouse skin. The role of cathelicidin in enabling SCTE-mediated inflammation is verified in mice with a targeted deletion of Camp, the gene encoding cathelicidin. These findings confirm the role of cathelicidin in skin inflammatory responses and suggest an explanation for the pathogenesis of rosacea by demonstrating that an exacerbated innate immune response can reproduce elements of this disease.

Injury enhances TLR2 function and antimicrobial peptide expression through a vitamin D-dependent mechanism.

An essential element of the innate immune response to injury is the capacity to recognize microbial invasion and stimulate production of antimicrobial peptides. We investigated how this process is controlled in the epidermis. Keratinocytes surrounding a wound increased expression of the genes coding for the microbial pattern recognition receptors CD14 and TLR2, complementing an increase in cathelicidin antimicrobial peptide expression. These genes were induced by 1,25(OH)2 vitamin D3 (1,25D3; its active form), suggesting a role for vitamin D3 in this process. How 1,25D3 could participate in the injury response was explained by findings that the levels of CYP27B1, which converts 25OH vitamin D3 (25D3) to active 1,25D3, were increased in wounds and induced in keratinocytes in response to TGF-beta1. Blocking the vitamin D receptor, inhibiting CYP27B1, or limiting 25D3 availability prevented TGF-beta1 from inducing cathelicidin, CD14, or TLR2 in human keratinocytes, while CYP27B1-deficient mice failed to increase CD14 expression following wounding. The functional consequence of these observations was confirmed by demonstrating that 1,25D3 enabled keratinocytes to recognize microbial components through TLR2 and respond by cathelicidin production. Thus, we demonstrate what we believe to be a previously unexpected role for vitamin D3 in innate immunity, enabling keratinocytes to recognize and respond to microbes and to protect wounds against infection.

Kallikrein-mediated proteolysis regulates the antimicrobial effects of cathelicidins in skin.

The presence of cathelicidin antimicrobial peptides provides an important mechanism for prevention of infection against a wide variety of microbial pathogens. The activity of cathelicidin is controlled by enzymatic processing of the proform (hCAP18 in humans) to a mature peptide (LL-37 in human neutrophils). In this study, elements important to the processing of cathelicidin in the skin were examined. Unique cathelicidin peptides distinct from LL-37 were identified in normal skin. Through the use of selective inhibitors, SELDI-TOF-MS, Western blot, and siRNA, the serine proteases stratum corneum tryptic enzyme (SCTE, kallikrein 5) and stratum corneum chymotryptic protease (SCCE, kallikrein 7) were shown to control activation of the human cathelicidin precursor protein hCAP18 and also influence further processing to smaller peptides with alternate biological activity. The importance of this serine protease activity to antimicrobial activity in vivo was illustrated in SPINK5-deficient mice that lack the serine protease inhibitor LEKTI. Epidermal extracts of these animals show a significant increase in antimicrobial activity compared with controls, and immunoabsorption of cathelicidin diminished antimicrobial activity. These observations demonstrate that the balance of proteolytic activity at an epithelial interface will control innate immune defense.

Control of the innate epithelial antimicrobial response is cell-type specific and dependent on relevant microenvironmental stimuli.

Immune defence against microbes depends in part on the production of antimicrobial peptides, a process that occurs in a variety of cell types but is incompletely understood. In this study, the mechanisms responsible for the induction of cathelicidin and beta-defensin antimicrobial peptides were found to be independent and specific to the cell type and stimulus. Vitamin D3 induced cathelicidin expression in keratinocytes and monocytes but not in colonic epithelial cells. Conversely, butyrate induced cathelicidin in colonic epithelia but not in keratinocytes or monocytes. Distinct factors induced beta-defensin expression. In all cell types, vitamin D3 activated the cathelicidin promoter and was dependent on a functional vitamin D responsive element. However, in colonic epithelia butyrate induced cathelicidin expression without increasing promoter activity and vitamin D3 activated the cathelicidin promoter without a subsequent increase in transcript accumulation. Induction of cathelicidin transcript correlated with increased processed mature peptide and enhanced antimicrobial activity against Staphylococcus aureus. However, induction of beta-defensin-2 expression did not alter the innate antimicrobial capacity of cells in culture. These data suggest that antimicrobial peptide expression is regulated in a tissue-specific manner at transcriptional, post-transcriptional and post-translational levels. Furthermore, these data show for the first time that innate antimicrobial activity can be triggered independently of the release of other pro-inflammatory molecules, and suggest strategies for augmenting innate immune defence without increasing inflammation.

The mammalian ionic environment dictates microbial susceptibility to antimicrobial defense peptides.

Antimicrobial peptides (AMPs) have been shown in animal and human systems to be effective natural antibiotics. However, it is unclear how they convey protection; they often appear inactive when assayed under culture conditions applied to synthetic antibiotics. This inactivation has been associated with loss of function in physiological concentrations of NaCl or serum. In this study we show that the balance of host ionic conditions dictate microbial sensitivity to AMPs. Carbonate is identified as the critical ionic factor present in mammalian tissues that imparts the ability of AMPs such as cathelicidins and defensins to kill at physiological NaCl concentrations. After adapting to carbonate-containing solutions, global changes occur in Staphylococcus aureus and Escherichia coli structure and gene expression despite no change in growth rate. Our findings show that changes in cell wall thickness and Sigma factor B expression correspond to the increased susceptibility to the AMP LL-37. These observations provide new insight into the factors involved in enabling function of innate immune effector molecules, and suggest that discovery of new antimicrobials should specifically target pathogens as they exist in the host and not the distinctly different phenotype of bacteria grown in culture broth.

Expression and secretion of cathelicidin antimicrobial peptides in murine mammary glands and human milk.

Mammalian milk possesses inherent antimicrobial properties that have been attributed to several diverse molecules. Recently, antimicrobial peptides that belong to the cathelicidin gene family have been found to be important to the mammalian immune response. This antimicrobial is expressed in several tissues and increased in neonatal skin, possibly to compensate for an immature adaptive immune response. We hypothesized that the mammary gland could produce and secrete cathelicidin onto the epithelial surface and into milk. Human cathelicidin hCAP18/LL-37 mRNA was detected in human milk cells by PCR. Quantitative real-time PCR demonstrated an increase in relative expression levels at 30 and 60 d after parturition. Immunohistochemistry of mouse breast tissue identified the murine cathelicidin-related antimicrobial peptide in lobuloacinar and ductules. Western blot analysis of human milk showed that LL-37 was secreted and present in the mature peptide form. The antimicrobial activity of LL-37 against Staphylococcus aureus, group A Streptococcus, and enteroinvasive Escherichia coli O29 in the human milk ionic environment was confirmed by solution colony-forming assay using synthetic peptide. These results indicate that cathelicidin is secreted in mammary gland and human milk, has antimicrobial activity against both Gram-positive and Gram-negative bacteria, and can contribute to the anti-infectious properties of milk.

Innate immune defense of the nail unit by antimicrobial peptides.

BACKGROUND: The nail is susceptible to microbial invasion, yet is usually able to defend itself from infection. This occurs despite isolation from cell-mediated immunity. OBJECTIVE: The aim of this study was to determine whether soluble innate immune molecules are present in the nail environment that can protect against microbial colonization. METHODS: Chromatographic techniques were used to purify cationic antimicrobial molecules from porcine hoof extracts. Sections of human and mouse digits were immunostained with antibodies to each species' cathelicidin antimicrobial peptide. Liquid antimicrobial assays were used to determine the activity of these molecules against relevant pathogens. RESULTS: Human, porcine, and murine nails contain antimicrobial molecules, and the human cathelicidin LL-37 can kill Candida albicans. CONCLUSION: The presence of antimicrobial peptides in nails with activity against relevant nail pathogens may account for the ability of the nail unit to resist infection in the absence of direct access to the cellular immune system.

Postsecretory processing generates multiple cathelicidins for enhanced topical antimicrobial defense.

The production of antimicrobial peptides and proteins is essential for defense against infection. Many of the known human antimicrobial peptides are multifunctional, with stimulatory activities such as chemotaxis while simultaneously acting as natural antibiotics. In humans, eccrine appendages express DCD and CAMP, genes encoding proteins processed into the antimicrobial peptides dermcidin and LL-37. In this study we show that after secretion onto the skin surface, the CAMP gene product is processed by a serine protease-dependent mechanism into multiple novel antimicrobial peptides distinct from the cathelicidin LL-37. These peptides show enhanced antimicrobial action, acquiring the ability to kill skin pathogens such as Staphylococcus aureus and Candida albicans. Furthermore, although LL-37 may influence the host inflammatory response by stimulating IL-8 release from keratinocytes, this activity is lost in subsequently processed peptides. Thus, a single gene product encoding an important defense molecule alters structure and function in the topical environment to shift the balance of activity toward direct inhibition of microbial colonization.

Neonatal skin in mice and humans expresses increased levels of antimicrobial peptides: innate immunity during development of the adaptive response.

The expression of antimicrobial peptides and proteins is an important innate immune defense mechanism that has recently been shown to be essential for cutaneous defense against invasive bacterial disease. Newborns have an immature cellular immune defense system that leads to increased susceptibility to infections. Here we show that skin from embryonic and newborn mice, as well as human newborn foreskin, express antimicrobial peptides of the cathelicidin and beta-defensin gene families. Immunohistochemistry and in situ hybridization demonstrated abundant cathelicidin protein and mRNA is present in normal skin during the perinatal period. Quantitative real-time PCR showed mouse cathelicidin expression (CRAMP) is 10- to 100-fold greater in the perinatal period than adult. Murine beta-defensins-1 and -4 and human beta-defensin-2 were also present in newborn skin. Combined, human cathelicidin (LL-37/hCAP/18) and beta-defensin-2 demonstrated synergistic antimicrobial activity and efficiently killed group B Streptococcus, an important neonatal pathogen. Antimicrobial peptides may therefore provide a compensatory innate defense mechanism during development of cellular immune response mechanisms in the newborn period.

Cathelicidin anti-microbial peptide expression in sweat, an innate defense system for the skin.

The eccrine gland is one of the major cutaneous appendages and secretes sweat. Its principal function is thermoregulation during exposure to a hot environment or physical exercise. In addition to this function, we show that LL-37, a member of cathelicidin family of anti-microbial peptides, is expressed in sweat. LL-37 protein and mRNA was seen in the eccrine structures of normal human skin by immunohistochemistry and in situ hybridization. LL-37 was localized to both the eccrine gland and sweat ductal epithelial cells, whereas dermcidin, a previously described natural antibiotic in sweat, was expressed only in the gland itself. The anti-microbial activity of LL-37 and dermcidin against various bacteria in the sweat ionic environment was demonstrated by solution colony forming assay using synthetic peptides, and in sweat obtained from normal volunteers. These results indicate that cathelicidin is secreted in human sweat, has potent anti-microbial activity against both gram-positive and gram-negative bacteria, and can, after processing from the preproform, provide a barrier for protection against infection. Thus, sweat represents a unique mode of delivery for potent innate immune effector molecules in the absence of inflammation.