Engineering selectivity of Cutibacterium acnes phages by epigenetic imprinting.

Cutibacterium acnes (C. acnes) is a gram-positive bacterium and a member of the human skin microbiome. Despite being the most abundant skin commensal, certain members have been associated with common inflammatory disorders such as acne vulgaris. The availability of the complete genome sequences from various C. acnes clades have enabled the identification of putative methyltransferases, some of them potentially belonging to restriction-modification (R-M) systems which protect the host of invading DNA. However, little is known on whether these systems are functional in the different C. acnes strains. To investigate the activity of these putative R-M and their relevance in host protective mechanisms, we analyzed the methylome of six representative C. acnes strains by Oxford Nanopore Technologies (ONT) sequencing. We detected the presence of a 6-methyladenine modification at a defined DNA consensus sequence in strain KPA171202 and recombinant expression of this R-M system confirmed its methylation activity. Additionally, a R-M knockout mutant verified the loss of methylation properties of the strain. We studied the potential of one C. acnes bacteriophage (PAD20) in killing various C. acnes strains and linked an increase in its specificity to phage DNA methylation acquired upon infection of a methylation competent strain. We demonstrate a therapeutic application of this mechanism where phages propagated in R-M deficient strains selectively kill R-M deficient acne-prone clades while probiotic ones remain resistant to phage infection.

Therapeutic Applications of Essential Oils from Native and Cultivated Ecuadorian Plants: Cutaneous Candidiasis and Dermal Anti-Inflammatory Activity.

Essential oils are a complex mixture of aromatic substances whose pharmacological actions, including antimicrobial, antioxidant, anticancer, and anti-inflammatory activities, have been widely reported. This study aimed to evaluate the anti-Candida and dermal anti-inflammatory activity of essential oils from native and cultivated Ecuadorian plants. Essential oils from Bursera graveolens, Dacryodes peruviana, Mespilodaphne quixos, and Melaleuca armillaris were isolated by hydrodistillation and were characterized physically and chemically. Its tolerance was analyzed by in vitro and in vivo studies. The antifungal activity was studied against Candida albicans, Candida glabrata, and Candida parapsilosis, whereas the anti-inflammatory effect was evaluated by a mouse ear edema model. The main compounds were limonene, α-phellandrene, (E)-methyl cinnamate, and 1,8-cineole, respectively. All essential oils showed high tolerability for skin application, antifungal activity against the three Candida strains, and anti-inflammatory efficacy by decreasing edema and overexpression of pro-inflammatory cytokines. Dacryodes peruviana essential oil showed the highest antifungal activity. On the other hand, Dacryodes peruviana and Melaleuca armillaris showed the greatest anti-inflammatory potential, decreasing edema by 53.3% and 65.25%, respectively, and inhibiting the overexpression of TNF-α, IL-8, IL-17A, and IL-23. The results suggest that these essential oils could be used as alternative therapies in the treatment of both cutaneous candidiasis and dermal inflammation.

Development of Halobetasol-loaded nanostructured lipid carrier for dermal administration: Optimization, physicochemical and biopharmaceutical behavior, and therapeutic efficacy.

Halobetasol propionate (HB) is considered a super potent drug in the group of topical corticosteroids. HB has anti-inflammatory activity, vasoconstriction properties, and due to its high skin penetration, it can cause systemic side effects. To improve its characteristics, enhance topical effectiveness and reduce penetration to systemic circulation, a study to optimize and characterize a HB-loaded lipid nanocarrier (HB-NLC) has been made by high-pressure homogenization method. The formulation is composed by HB, surfactant, glyceryl distearate and capric glycerides. The optimized HB-NLC containing 0.01% of HB and 3% of total lipid shows an average size below 200 nm with a polydispersity index ≪0.2 and an encapsulation efficiency ≫90%. The in vitro and in vivo tests indicate that the HB-NLC is not toxic, is well tolerated and has an anti-inflammatory effect because they decrease the production of Interleukins in keratinocytes and monocytes. HB-NLC is considered an alternative treatment for skin inflammatory disorders.

Nanoemulsion strategy of pioglitazone for the treatment of skin inflammatory diseases.

Pioglitazone (PGZ) is a peroxisome proliferator-activated receptor agonist. Its role in the inflammatory response modulation paves the way for additional therapeutic applications. The purpose of this study was to develop a pioglitazone nanoemulsion (PGZ-NE) in order to investigate its anti-inflammatory efficacy on the skin. To that end, an NE vehicle developed for skin delivery was optimized and characterized. The resulting PGZ-NE showed good anti-inflammatory efficacy by decreasing the expression of inflammatory cytokines IL-6, IL-1ß and TNF-α. The properties of the developed nanocarrier allowed achievement of a high permeation flux of PGZ through the skin as well as a high retained amount in the skin, likely due to the depot effect of ingredients, which assured a prolonged local action, with good skin tolerability among participating individuals. Consequently, these results suggest that PGZ-NE may be used as an alternative treatment for inflammatory skin diseases such as rosacea, atopic dermatitis or psoriasis.

Multifunctional Serine Protease Inhibitor-Coated Water-Soluble Gold Nanoparticles as a Novel Targeted Approach for the Treatment of Inflammatory Skin Diseases.

The overexpression and increased activity of the serine protease Kallikrein 5 (KLK5) is characteristic of inflammatory skin diseases such as Rosacea. The use of inhibitors of this enzyme-such as 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF·HCl) or the anti-human recombinant Kallikrein 5 (anti-KLK5) antibody-in the treatment of the disease has been limited due to their low bioavailability, for which their immobilization in drug delivery agents can contribute to making serine protease inhibitors clinically useful. In this work, we synthesized gold nanoparticles (GNP) coated with a mixture of hydroxyl- and carboxyl-terminated thiolates (GNP.OH/COOH), whose carboxyl groups were used to further functionalize the nanoparticles with the serine protease inhibitor AEBSF·HCl either electrostatically or covalently (GNP.COOH AEBSF and GNP.AEBSF, respectively), or with the anti-KLK5 antibody (GNP.antiKLK5). The synthesized and functionalized GNP were highly water-soluble, and they were extensively characterized using UV-vis absorption spectroscopy, Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), and Thermogravimetric Analysis (TGA). GNP.OH/COOH and their subsequent functionalizations effectively inhibited KLK5 in vitro. Internalization of fluorophore-coated GNP.OH/COOH in human keratinocytes (HaCaT cells) was proven using confocal fluorescence microscopy. Cell viability assays revealed that the cytotoxicity of free AEBSF is importantly decreased when it is incorporated in the nanoparticles, either ionically (GNP.COOH AEBSF) or, most importantly, covalently (GNP.AEBSF). The functionalized nanoparticles GNP.AEBSF and GNP.antiKLK5 inhibited intracellular KLK5 activity in HaCaT cells and diminished secretion of IL-8 under inflammatory conditions triggered by TLR-2 ligands. This study points to the great potential of these GNP as a new intracellular delivery strategy for both small drugs and antibodies in the treatment of skin diseases such as Rosacea.

Human Skin Permeation Studies with PPARγ Agonist to Improve Its Permeability and Efficacy in Inflammatory Processes.

Rosacea is the most common inflammatory skin disease. It is characterized by erythema, inflammatory papules and pustules, visible blood vessels, and telangiectasia. The current treatment has limitations and unsatisfactory results. Pioglitazone (PGZ) is an agonist of peroxisome proliferator-activated receptors (PPARs), a nuclear receptor that regulates important cellular functions, including inflammatory responses. The purpose of this study was to evaluate the permeation of PGZ with a selection of penetration enhancers and to analyze its effectiveness for treating rosacea. The high-performance liquid chromatography (HPLC) method was validated for the quantitative determination of PGZ. Ex vivo permeation experiments were realized in Franz diffusion cells using human skin, in which PGZ with different penetration enhancers were assayed. The results showed that the limonene was the most effective penetration enhancer that promotes the permeation of PGZ through the skin. The cytotoxicity studies and the Draize test detected cell viability and the absence of skin irritation, respectively. The determination of the skin color using a skin colorimetric probe and the results of histopathological studies confirmed the ability of PGZ-limonene to reduce erythema and vasodilation. This study suggests new pharmacological indications of PGZ and its possible application in the treatment of skin diseases, namely rosacea.

The secreted autotransporter toxin (Sat) does not act as a virulence factor in the probiotic Escherichia coli strain Nissle 1917.

BACKGROUND: Escherichia coli Nissle 1917 (EcN) is a probiotic used in the treatment of intestinal diseases. Although it is considered safe, EcN is closely related to the uropathogenic E. coli strain CFT073 and contains many of its predicted virulence elements. Thus, it is relevant to assess whether virulence-associated genes are functional in EcN. One of these genes encodes the secreted autotransporter toxin (Sat), a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs) that are secreted following the type V autotransporter pathway. Sat is highly prevalent in certain E. coli pathogenic groups responsible for urinary and intestinal infections. In these pathogens Sat promotes cytotoxic effects in several lines of undifferentiated epithelial cells, but not in differentiated Caco-2 cells. RESULTS: Here we provide evidence that sat is expressed by EcN during the colonization of mouse intestine. The EcN protein is secreted as an active serine protease, with its 107 kDa-passenger domain released into the medium as a soluble protein. Expression of recombinant EcN Sat protein in strain HB101 increases paracellular permeability to mannitol in polarized Caco-2 monolayers. This effect, also reported for the Sat protein of diffusely adherent E. coli, is not observed when this protein is expressed in the EcN background. In addition, we show that EcN supernatants confer protection against Sat-mediated effects on paracellular permeability, thus indicating that other secreted EcN factors are able to prevent barrier disruption caused by pathogen-related factors. Sat is not required for intestinal colonization, but the EcNsat::cat mutant outcompetes wild-type EcN in the streptomycin-treated mouse model. Analysis of the presence of sat in 29 strains of the ECOR collection isolated from stools of healthy humans shows 34.8 % positives, with high prevalence of strains of the phylogenetic groups D and B2, related with extra-intestinal infections. CONCLUSIONS: Sat does not act as a virulence factor in EcN. The role of Sat in intestinal pathogenesis relies on other genetic determinants responsible for the bacterial pathotype.

Delivery of a sebum modulator by an engineered skin microbe in mice.

Microorganisms can be equipped with synthetic genetic programs for the production of targeted therapeutic molecules. Cutibacterium acnes is the most abundant commensal of the human skin, making it an attractive chassis to create skin-delivered therapeutics. Here, we report the engineering of this bacterium to produce and secrete the therapeutic molecule neutrophil gelatinase-associated lipocalin, in vivo, for the modulation of cutaneous sebum production.

Establishing a Cell-Free Transcription-Translation Platform for Cutibacterium acnes to Prototype Engineered Metabolic and Synthetic Biology.

In the past few years, new bacterial-cell-free transcription-translation systems have emerged as potent and quick platforms for protein production as well as for prototyping of DNA regulatory elements, genetic circuits, and metabolic pathways. The Gram-positive commensal Cutibacterium acnes is one of the most abundant bacteria present in the human skin microbiome. However, it has recently been reported that some C. acnes phylotypes can be associated with common inflammatory skin conditions, such as acne vulgaris, whereas others seem to play a protective role, acting as possible "skin probiotics". This fact has made C. acnes become a bacterial model of interest for the cosmetic industry. In the present study we report for the first time the development and optimization of a C. acnes-based cell-free system (CFS) that is able to produce 85 µg/mL firefly luciferase. We highlight the importance of harvesting the bacterial pellet in mid log phase and maintaining CFS reactions at 30 °C and physiological pH to obtain the optimal yield. Additionally, a C. acnes promoter library was engineered to compare coupled in vitro TX-TL activities, and a temperature biosensor was tested, demonstrating the wide range of applications of this toolkit in the synthetic biology field.

New insights into the role of Cutibacterium acnes-derived extracellular vesicles in inflammatory skin disorders.

Cutibacterium acnes (C. acnes) is one of the most prevalent bacteria that forms the human skin microbiota. Specific phylotypes of C. acnes have been associated with the development of acne vulgaris, while other phylotypes have been linked to healthy skin. In this scenario, bacterial extracellular vesicles (EVs) play a role in the interkingdom communication role with the human host. The purpose of this study was to examine the impact of EVs generated by various phylotypes of C. acnes on inflammation and sebum production using different in vitro skin cell types. The main findings of this study reveal that the proteomic profile of the cargo embodied in the EVs reflects distinct characteristics of the different C. acnes phylotypes in terms of life cycle, survival, and virulence. The in vitro skin cell types showed an extended pro-inflammatory modulation of SLST A1 EVs consistently triggering the activation of the inflammation-related factors IL-8, IL-6, TNFα and GM-CSF, in comparison to SLST H1 and SLST H2. Additionally, an acne-prone skin model utilizing PCi-SEB and arachidonic acid as a sebum inducer, was employed to investigate the impact of C. acnes EVs on sebum regulation. Our findings indicated that all three types of EVs significantly inhibited sebum production after a 24-h treatment period, with SLST H1 EVs exhibiting the most pronounced inhibitory effect when compared to the positive control. The results of this study highlight the protective nature of C. acnes SLST H1 EVs and their potential use as a natural treatment option for alleviating symptoms associated with inflammation and oily skin.

Efficacy of Apremilast Gels in Mouse Model of Imiquimod-Induced Psoriasis Skin Inflammation.

Apremilast (APM) is a novel drug for the treatment of psoriasis and psoriatic arthritis. APM is a phosphodiesterase 4 (PDE4) inhibitor, raising intracellular cAMP levels and thereby decreasing the inflammatory response by modulating the expression of TNF-α, IL-17, IL-23, and other inflammatory cytokines. The goal of this study is to develop APM gels as a new pharmaceutical formulation for the treatment of topical psoriasis. APM was solubilized in Transcutol-P and incorporated into Pluronic F127, Sepigel, and carbomer bases at different proportions. All formulations were characterized physiochemically. A biopharmaceutical study (release profile) was performed, and ex vivo permeation was evaluated using a human skin model. A toxicity assay was carried out on the HaCaT cell line. A mouse model of imiquimod-induced psoriasis skin inflammation was carried out to determine its efficacy by histological analysis, RNA extraction, and RT-qPCR assays. APM gel formulations showed good physicochemical characteristics and a sustained release profile. There was no permeation of any gel measured through human skin, indicating a high retained amount of APM on the skin. Cell viability was greater than 80% at most dilution concentrations. APM gels treated the psoriasis mouse model, and it shows a reduction in the proinflammatory cytokines (IL-8, IL-17A, IL-17F, and IL-23). APM gels could be a new approach for the treatment of topical psoriasis.

Characterization and In Vivo Anti-Inflammatory Efficacy of Copal (Dacryodes peruviana (Loes.) H.J. Lam) Essential Oil.

Essential oils are natural aromatic substances that contain complex mixtures of many volatile compounds frequently used in pharmaceutical and cosmetic industries. Dacryodes peruviana (Loes.) H.J. Lam is a native species from Ecuador whose anti-inflammatory activity has not been previously reported, thus the aim of this study was to evaluate the anti-inflammatory activity of D. peruviana essential oil. To that end, essential oil from D. peruviana fruits was isolated by hydrodistillation and characterized physically and chemically. The tolerance of the essential oil was analyzed by cytotoxicity studies using human keratinocytes. The anti-inflammatory activity was evaluated by an arachidonic acid-induced edema model in mouse ear. The predominant compounds in D. peruviana essential oil were α-phellandrene, limonene, and α-pinene, with the three compounds reaching approximately 83% of the total composition. Tolerance studies showed high biocompatibility of this essential oil with human keratinocytes. In vivo studies demonstrated a moisturizing effect and an alleviation of several events occurred during the inflammatory process after topical treatment with D. peruviana essential oil such as decline in skin edema; reduction in leukocytic infiltrate; and decrease in inflammatory cytokines TNFα, IL-8, IL-17A, and IL-23. Therefore, this essential oil could be an attractive treatment for skin inflammation.

From Dysbiosis to Healthy Skin: Major Contributions of Cutibacterium acnes to Skin Homeostasis.

Cutibacterium acnes is the most abundant bacterium living in human, healthy and sebum-rich skin sites, such as the face and the back. This bacterium is adapted to this specific environment and therefore could have a major role in local skin homeostasis. To assess the role of this bacterium in healthy skin, this review focused on (i) the abundance of C. acnes in the skin microbiome of healthy skin and skin disorders, (ii) its major contributions to human skin health, and (iii) skin commensals used as probiotics to alleviate skin disorders. The loss of C. acnes relative abundance and/or clonal diversity is frequently associated with skin disorders such as acne, atopic dermatitis, rosacea, and psoriasis. C. acnes, and the diversity of its clonal population, contributes actively to the normal biophysiological skin functions through, for example, lipid modulation, niche competition and oxidative stress mitigation. Compared to gut probiotics, limited dermatological studies have investigated skin probiotics with skin commensal strains, highlighting their unexplored potential.

Apremilast Microemulsion as Topical Therapy for Local Inflammation: Design, Characterization and Efficacy Evaluation.

Apremilast (APR) is a selective phosphodiesterase 4 inhibitor administered orally in the treatment of moderate-to-severe plaque psoriasis and active psoriatic arthritis. The low solubility and permeability of this drug hinder its dermal administration. The purpose of this study was to design and characterize an apremilast-loaded microemulsion (APR-ME) as topical therapy for local skin inflammation. Its composition was determined using pseudo-ternary diagrams. Physical, chemical and biopharmaceutical characterization were performed. Stability of this formulation was studied for 90 days. Tolerability of APR-ME was evaluated in healthy volunteers while its anti-inflammatory potential was studied using in vitro and in vivo models. A homogeneous formulation with Newtonian behavior and droplets of nanometric size and spherical shape was obtained. APR-ME released the incorporated drug following a first-order kinetic and facilitated drug retention into the skin, ensuring a local effect. Anti-inflammatory potential was observed for its ability to decrease the production of IL-6 and IL-8 in the in vitro model. This effect was confirmed in the in vivo model histologically by reduction in infiltration of inflammatory cells and immunologically by decrease of inflammatory cytokines IL-8, IL-17A and TNFα. Consequently, these results suggest that this formulation could be used as an attractive topical treatment for skin inflammation.

Outer Membrane Vesicles From Probiotic and Commensal Escherichia coli Activate NOD1-Mediated Immune Responses in Intestinal Epithelial Cells.

Gut microbiota plays a critical role in maintaining human intestinal homeostasis and host health. Bacterial extracellular vesicles are key players in bacteria-host communication, as they allow delivery of effector molecules into the host cells. Outer membrane vesicles (OMVs) released by Gram-negative bacteria carry many ligands of pattern recognition receptors that are key components of innate immunity. NOD1 and NOD2 cytosolic receptors specifically recognize peptidoglycans present within the bacterial cell wall. These intracellular immune receptors are essential in host defense against bacterial infections and in the regulation of inflammatory responses. Recent contributions show that NODs are also fundamental to maintain intestinal homeostasis and microbiota balance. Peptidoglycan from non-invasive pathogens is delivered to cytosolic NODs through OMVs, which are internalized via endocytosis. Whether this pathway could be used by microbiota to activate NOD receptors remains unexplored. Here, we report that OMVs isolated from the probiotic Escherichia coli Nissle 1917 and the commensal ECOR12 activate NOD1 signaling pathways in intestinal epithelial cells. NOD1 silencing and RIP2 inhibition significantly abolished OMV-mediated activation of NF-κB and subsequent IL-6 and IL-8 expression. Confocal fluorescence microscopy analysis confirmed that endocytosed OMVs colocalize with NOD1, trigger the formation of NOD1 aggregates, and promote NOD1 association with early endosomes. This study shows for the first time the activation of NOD1-signaling pathways by extracellular vesicles released by gut microbiota.

Development of a Nasal Donepezil-loaded Microemulsion for the Treatment of Alzheimer's Disease: in vitro and ex vivo Characterization.

BACKGROUND: Donepezil (DPZ) is widely prescribed as a specific and reversible acetylcholinesterase inhibitor for the symptomatic treatment of mild to moderate Alzheimer's disease (AD). OBJECTIVE: Considering the therapeutic potential of DPZ and the advantages offered by the intranasal route as an alternative for drug administration, the aim of this study was the development and characterization of a DPZ microemulsion (ME) for nose-to-brain delivery. METHOD: The ME was developed by construction of pseudoternary phase diagrams and characterized by dynamic light scattering and transmission electron microscopy. Flow properties and viscosity, as well as optical stability and stability under storage at different temperatures were evaluated. Finally, in vitro release and ex vivo permeation studies through porcine nasal mucosa were accomplished. RESULTS: A transparent and homogeneous DPZ-ME (12.5 mg/ml) was obtained. The pH and viscosity were 6.38 and 44.69 mPa·s, respectively, indicating nasal irritation prevention and low viscosity. The mean droplet size was 58.9±3.2 nm with a polydispersity index of 0.19±0.04. The morphological analysis revealed the spherical shape of droplets, as well as their smooth and regular surface. Optical stability evidenced no destabilization processes. DPZ release profile indicated that the ME followed a hyperbolic kinetic model while the ex vivo permeation profile showed that the highest permeation occurred during initial 4 h and the maximum permeated amount was approximately 2000 µg, which corresponds to 80% of the starting amount of drug. CONCLUSION: We conclude that our nasal ME could be considered as a new potential tool for further investigation in the AD.

Development of Pranoprofen Loaded Nanostructured Lipid Carriers to Improve Its Release and Therapeutic Efficacy in Skin Inflammatory Disorders.

Pranoprofen (PF)-loaded nanostructured lipid carriers (NLCs), prepared using a high-pressure homogenization method, have been optimized and characterized to improve the biopharmaceutical profile of the drug. The optimized PF-NLCs exhibited physicochemical characteristics and morphological properties that were suitable for dermal application. Stability assays revealed good physical stability, and the release behavior of PF from these NLCs showed a sustained release pattern. Cell viability results revealed no toxicity. Ex vivo human skin permeation studies in Franz diffusion cells were performed to determine the influence of different skin penetration enhancers (pyrrolidone, decanol, octanoic acid, nonane, menthone, squalene, linoleic acid, and cineol) on skin penetration and retention of PF, being the highest dermal retention in the presence of linoleic acid. The selected formulations of NLCs exhibited a high retained amount of PF in the skin and no systemic effects. In vivo mice anti-inflammatory efficacy studies showed a significant reduction in dermal oedema. NLCs containing linoleic acid presented better anti-inflammatory efficacy by decreasing the production of interleukins in keratinocytes and monocytes. The biomechanical properties of skin revealed an occlusive effect and no hydration power. No signs of skin irritancy in vivo were detected. According to these results, dermal PF-NLCs could be an effective system for the delivery and controlled release of PF, improving its dermal retention, with reduced dermal oedema as a possible effect of this drug.

Intestinal Anti-inflammatory Effects of Outer Membrane Vesicles from Escherichia coli Nissle 1917 in DSS-Experimental Colitis in Mice.

Escherichia coli Nissle 1917 (EcN) is a probiotic strain with proven efficacy in inducing and maintaining remission of ulcerative colitis. However, the microbial factors that mediate these beneficial effects are not fully known. Gram-negative bacteria release outer membrane vesicles (OMVs) as a direct pathway for delivering selected bacterial proteins and active compounds to the host. In fact, vesicles released by gut microbiota are emerging as key players in signaling processes in the intestinal mucosa. In the present study, the dextran sodium sulfate (DSS)-induced colitis mouse model was used to investigate the potential of EcN OMVs to ameliorate mucosal injury and inflammation in the gut. The experimental protocol involved pre-treatment with OMVs for 10 days before DSS intake, and a 5-day recovery period. Oral administration of purified EcN OMVs (5 µg/day) significantly reduced DSS-induced weight loss and ameliorated clinical symptoms and histological scores. OMVs treatment counteracted altered expression of cytokines and markers of intestinal barrier function. This study shows for the first time that EcN OMVs can mediate the anti-inflammatory and barrier protection effects previously reported for this probiotic in experimental colitis. Remarkably, translation of probiotics to human healthcare requires knowledge of the molecular mechanisms involved in probiotic-host interactions. Thus, OMVs, as a non-replicative bacterial form, could be explored as a new probiotic-derived therapeutic approach, with even lower risk of adverse events than probiotic administration.