Evidence that keratinocyte microvesicle particles carrying Platelet-activating factor mediate the widespread multi-organ damage associated with intoxicated thermal burn injury.

Thermal burn injuries can result in significant morbidity and mortality. The combination of ethanol intoxication with thermal burn injury results in increased morbidity through an exaggerated inflammatory response involving many organs. Recent studies have linked involvement of the lipid mediator Platelet-activating factor (PAF) in the pathology associated with intoxicated thermal burn injury (ITBI). The present studies tested the roles of PAF and the elevated levels of subcellular microvesicle particles (MVP) generated in response to ITBI in the subsequent multi-organ toxicity. First, thermal burn injury of HaCaT keratinocytes preincubated with ethanol resulted in augmented MVP release, which was blocked by inhibiting the PAF-generating enzyme cytosolic phospholipase A2 and the PAF receptor (PAFR). Second, ITBI of mice resulted in increased pro-inflammatory cytokine production and neutrophilic inflammation in multiple organs which were not present in mice deficient in PAFRs nor the MVP-generating enzyme acid sphingomyelinase (aSMase). Moreover, the increased bacterial translocation from the gut to mesenteric lymph nodes previously reported in murine ITBI was also dependent upon PAFR and aSMase. MVP released from ITBI-treated keratinocytes contained high levels of PAFR agonistic activity. Finally, use of topical aSMase inhibitor imipramine following ITBI attenuated the widespread organ inflammatory response of ITBI, suggesting a potential therapeutic for this condition. These studies provide evidence for PAF-enriched MVP generated in skin, which then act upon the gut PAFR resulting in bacterial translocation as the mechanism for the multi-organ dysfunction associated with ITBI. Inasmuch as aSMase inhibitors are widely available, these studies could result in effective treatments for ITBI.

Evidence for the involvement of keratinocyte-derived microvesicle particles in the photosensitivity associated with xeroderma pigmentosum type A deficiency.

Photosensitivity can be due to numerous causes. The photosensitivity associated with deficiency of xeroderma pigmentosum type A (XPA) has been previously shown to be associated with excess levels of the lipid mediator platelet-activating factor (PAF) generated by the keratinocyte. As PAF has been reported to trigger the production of subcellular microvesicle particles (MVP) due to the enzyme acid sphingomyelinase (aSMase), the goal of these studies was to discern if PAF and aSMase could serve as therapeutic targets for the XPA deficiency photosensitivity. HaCaT keratinocytes lacking XPA generated greater levels of MVP in comparison to control cells. Mice deficient in XPA also generated enhanced MVP levels in skin and in plasma in response to UV radiation. Use of a genetic strategy with mice deficient in both XPA and PAF receptors revealed that these mice generated less MVP release as well as decreased skin erythema and cytokine release compared to XPA knockout mice alone. Finally, the aSMase inhibitor imipramine blocked UV-induced MVP release in HaCaT keratinocytes, as well as XPA knockout mice. These studies support the concept that the photosensitivity associated with XPA involves PAF- and aSMase-mediated MVP release and provides a potential pharmacologic target in treating this form of photosensitivity.

Topical Photodynamic Therapy Generates Bioactive Microvesicle Particles: Evidence for a Pathway Involved in Immunosuppressive Effects.

Although effective in treating actinic damage, topical photodynamic therapy (PDT) has been shown to be immunosuppressive through unknown mechanisms, which could potentially limit its effectiveness. Multiple types of environmental stressors, including PDT, can produce the immunosuppressive lipid mediator platelet-activating factor (PAF). Because PAF can produce subcellular microvesicle particles (MVPs), these studies tested whether PDT can generate PAF and MVP release and whether these are involved in PDT-induced immunosuppression. Previously, topical PDT using blue light and 5-aminolevulinic acid was found to be a potent stimulus for PAF production in mice and human skin explants and human patients, and we show that experimental PDT also generates high levels of MVP. PDT-generated MVPs were independent of the PAF receptor but were dependent on the MVP-generating enzyme acid sphingomyelinase. Patients undergoing topical PDT treatment to at least 10% of body surface area showed local and systemic immunosuppression as measured by inhibition of delayed-type hypersensitivity reactions. Finally, using a murine model of contact hypersensitivity, PDT immunosuppression was blocked by genetic and pharmacologic inhibition of acid sphingomyelinase and genetic inhibition of PAF receptor signaling. These studies describe a mechanism involving MVP through which PDT exerts immunomodulatory effects, providing a potential target to improve its effectiveness.

Topical application of gemcitabine generates microvesicle particles in human and murine skin.

Chemotherapy has remained the mainstay for the treatment of multiple types of cancers. In particular, topical use of chemotherapy has been used for skin cancers. Though effective, topical chemotherapy has been limited due to adverse effects such as local and even systemic toxicities. Our recent studies demonstrated that exposure to pro-oxidative stressors, including therapeutic agents induces the generation of extracellular vesicles known as microvesicle particles (MVP) which are dependent on activation of the Platelet-activating factor-receptor (PAFR), a G-protein coupled receptor present on various cell types, and acid sphingomyelinase (aSMase), an enzyme required for MVP biogenesis. Based upon this premise, we tested the hypothesis that topical application of gemcitabine will induce MVP generation in human and murine skin. Our ex vivo studies using human skin explants demonstrate that gemcitabine treatment results in MVP generation in a dose-dependent manner in a process blocked by PAFR antagonist and aSMase inhibitor. Importantly, gemcitabine-induced MVPs carry PAFR agonists. To confirm the mechanisms, we employed PAFR-expressing and deficient (Ptafr-/- ) mouse models as well as mice deficient in aSMase enzyme (Spmd1-/- ). Similar to the findings using pharmacologic tools, genetic-based approaches demonstrate that gemcitabine-induced MVP release in WT mice was blunted in Ptafr-/- and Spmd1-/- mice. These findings demonstrate a novel mechanism by which local chemotherapy can generate bioactive components as a bystander effect in a process that is dependent upon the PAFR-aSMase pathway.

Insights into Lichen Planus Pigmentosus Inversus using Minimally Invasive Dermal Patch and Whole Transcriptome Analysis.

Lichen Planus Pigmentosus inversus (LPPi) is a rare interface and lichenoid dermatitis (ILD) and supposed variant of lichen planus (LP) that presents as well-demarcated brown to grey macules in flexural and intertriginous areas. LPPi is deemed 'inversus' because its anatomical distribution in skin folds is opposite that seen in lichen planus pigmentosus (LPP) whose pigmented lesions arise on sun-exposed skin. Biopsy is required for the clinical diagnosis of all ILDs. Though multiple clinically-oriented studies have reported differences between LPP, LPPi, and LP, few molecular studies have been performed. In this case study, 3 patients, 2 with LPPi and one with LP, provided samples using minimally invasive whole transcriptome analysis using a dermal biomarker patch. This study confirms the involvement of interferon signaling and T-cell activation in LPPi and suggests an expression profile distinct from LP. Specific genes significantly upregulated in LPPi vs LP include an intergenic splice variant of the primary pigmentation determining receptor in humans and dysregulation of genes essential for ceramide synthesis and construction of the cornified envelope. This work expands upon our knowledge of the pathogenesis of LPPi vs LP, and supports the potential use of this technology in the diagnostic clinical setting to mitigate the need for invasive procedures.

Low UVB Fluences Augment Microvesicle Particle Generation in Keratinocytes.

Microvesicle particles (MVP) are bioactive subcellular particles which have been recently implicated in the keratinocyte response to many environmental stressors including ultraviolet B radiation (UVB). Previous studies have demonstrated that UVB generates high levels of MVP in a process involving the platelet-activating factor receptor (PAFR) and the enzyme acid sphingomyelinase (aSMase). Yet the fluences of UVB needed to generate MVP are usually above those commonly encountered. Using models including human epithelial cell lines in vitro, human skin explants ex vivo and murine studies in vivo, the present studies indicate that pretreatment of epithelial cells/skin with PAFR agonist/phorbol ester can synergize with low fluences of UVB to generate high levels of MVP. These studies indicate the possibility that MVP could play a role in combinatorial pathologic processes involving UVB.

Evidence for Systemic Reactive Oxygen Species in UVB-mediated Microvesicle Formation.

Recent studies have implicated subcellular microvesicle particles (MVP) in the ability of ultraviolet B radiation to exert both local and systemic effects. Indeed, UVB generates MVP (UVB-MVP) in human skin and systemically following phototherapy. The current studies were designed to test the hypothesis that the ability of UVB to generate MVP was dependent upon reactive oxygen species (ROS). To that end, we tested urine samples from subjects undergoing UVB phototherapy for the presence of isoprostanes as well as the oxidized guanosine derivative 8OHdG. We also conducted a clinical study in which volar forearms of subjects were treated with localized UVB and erythema/MVP measured. The same cohort was then treated with 7 days of vitamin C (2 g day-1 ) and vitamin E (1000 IU day-1 ), and UVB-induced MVPs tested on the contralateral forearm. Urine specimens from subjects undergoing phototherapy were found to have increased levels of isoprostanes and 8OHdG, with maximal levels noted 8-16 h post-treatment. Treatment with antioxidant vitamins resulted in diminished UVB-generated skin MVP to baseline levels. These studies suggest that whole-body UVB generates a systemic pro-oxidative response, and that antioxidants can attenuate localized skin UVB-MVPs.

Acute ethanol exposure stimulates microvesicle particle generation in keratinocytes.

Ethanol has been demonstrated to exert profound effects upon cells and tissues via multiple mechanisms. One recently appreciated means by which cells can communicate with other cells is via the production and release of extracellular vesicles. Though smaller exosomes have been demonstrated to be released in response to ethanol exposure, the ability of ethanol to modulate the generation and release of larger microvesicle particles (MVP) is lesser studied. The present studies examined the ability of exogenous ethanol to generate MVP with a focus on skin cells. Acute ethanol exposure resulted in augmented MVP release in keratinocytes and in the skin and blood of mice. Unlike other stimuli such as ultraviolet B radiation or thermal burn injury, ethanol-mediated MVP release was independent of the Platelet-activating Factor receptor (PAFR). However, ethanol pretreatment was found to augment thermal burn injury-induced MVP in a PAFR-dependent manner. These studies provide a novel mechanism for ethanol-mediated effects, that could be relevant in the significant toxicity associated with thermal burn injury in the setting of alcohol intoxication.

Evidence for microvesicle particles in UVB-mediated IL-8 generation in keratinocytes.

Recent studies have implicated bioactive microvesicle particles (MVP) in the keratinocyte response to many environmental stressors, in partricular ultraviolet B radiation (UVB). The generation of MVP in response to UVB involves the Platelet-activating factor receptor (PAFR) and the enzyme acid sphingomyelinase (aSMase). As UVB generates some cytokines such as interleukin-8 (IL-8) in a PAFR-dependent manner, one question is if the production and release of IL-8 and MVP could be linked. Using the human keratinocyte-derived cell line HaCaT, the present in vitro studies indicate that pretreatment of HaCaT keratinocytes with PAFR agonist ester can synergize with low fluences of UVB to generate high levels of MVP as well as IL-8 protein. Treatment of cells with an aSMase pharmacologic inhibitor blocked both processes. These studies indicate the possibility that MVP could be involved in pathologic processes involving UVB-generated production of pro-inflammatory cytokines such as IL-8.

Evaluation of SARS-CoV-2 Spike S1 Protein Response on PI3K-Mediated IL-8 Release.

A novel coronavirus related to a condition known as a severe acute respiratory syndrome (SARS) was termed as SARS Coronavirus-19 (SARS-CoV-2 or COVID-19), which has caused an unprecedented global pandemic. Extensive efforts have been dedicated worldwide towards determining the mechanisms of COVID-19 associated pathogenesis with the goals of devising potential therapeutic approaches to mitigate or overcome comorbidities and mortalities. While the mode of SARS-CoV-2 infection, its structural configuration, and mechanisms of action, including the critical roles of the Spike protein have been substantially explored, elucidation of signaling pathways regulating its cellular responses is yet to be fully determined. Notably, phosphoinositide 3-kinases (PI3K) and its downstream pathway have been exploited among potential therapeutic targets for SARS-CoV-2, and its activation modulates the release of cytokines such as IL-8. To that end, the current studies were sought to determine the response of the SARS-CoV-2 Spike S1 protein on PI3K-mediated IL-8 release using relevant and widely used cellular models. Overall, these studies indicate that PI3K signaling does not directly mediate Spike S1 protein-induced IL-8 release in these cellular models.

Keratinocyte-derived microvesicle particles mediate ultraviolet B radiation-induced systemic immunosuppression.

A complete carcinogen, ultraviolet B (UVB) radiation (290-320 nm), is the major cause of skin cancer. UVB-induced systemic immunosuppression that contributes to photocarcinogenesis is due to the glycerophosphocholine-derived lipid mediator platelet-activating factor (PAF). A major question in photobiology is how UVB radiation, which only absorbs appreciably in the epidermal layers of skin, can generate systemic effects. UVB exposure and PAF receptor (PAFR) activation in keratinocytes induce the release of large numbers of microvesicle particles (MVPs; extracellular vesicles ranging from 100 to 1000 nm in size). MVPs released from skin keratinocytes in vitro in response to UVB (UVB-MVPs) are dependent on the keratinocyte PAFR. Here, we used both pharmacologic and genetic approaches in cells and mice to show that both the PAFR and enzyme acid sphingomyelinase (aSMase) were necessary for UVB-MVP generation. Our discovery that the calcium-sensing receptor is a keratinocyte-selective MVP marker allowed us to determine that UVB-MVPs leaving the keratinocyte can be found systemically in mice and humans following UVB exposure. Moreover, we found that UVB-MVPs contained bioactive contents including PAFR agonists that allowed them to serve as effectors for UVB downstream effects, in particular UVB-mediated systemic immunosuppression.

Thermal Burn Injury Generates Bioactive Microvesicles: Evidence for a Novel Transport Mechanism for the Lipid Mediator Platelet-Activating Factor (PAF) That Involves Subcellular Particles and the PAF Receptor.

Thermal burn injuries are an important environmental stressor that can result in considerable morbidity and mortality. The exact mechanism by which an environmental stimulus to skin results in local and systemic effects is an area of active research. One potential mechanism to allow skin keratinocytes to disperse bioactive substances is via microvesicle particles, which are subcellular bodies released directly from cellular membranes. Our previous studies have indicated that thermal burn injury of the skin keratinocyte in vitro results in the production of the lipid mediator platelet-activating factor (PAF). The present studies demonstrate that thermal burn injury to keratinocytes in vitro and human skin explants ex vivo, and mice in vivo generate microvesicle particles. Use of pharmacologic and genetic tools indicates that the optimal release of microvesicles is dependent upon the PAF receptor. Of note, burn injury-stimulated microvesicle particles do not carry appreciable protein cytokines yet contain high levels of PAF. These studies describe a novel mechanism involving microvesicle particles by which a metabolically labile bioactive lipid can travel from cells in response to environmental stimuli.

Gemcitabine Induces Microvesicle Particle Release in a Platelet-Activating Factor-Receptor-Dependent Manner via Modulation of the MAPK Pathway in Pancreatic Cancer Cells.

Studies, including ours, have shown that pro-oxidative stressors, such as chemotherapeutic agents, generate oxidized lipids with agonistic platelet-activating factor (PAF) activity. Importantly, recent reports have implicated that these PAF-agonists are transported extracellularly via microvesicle particles (MVPs). While the role of PAF-receptor (PAF-R) has been implicated in mediating chemotherapy effects, its significance in chemotherapy-mediated MVP release in pancreatic cancer has not been studied. The current studies determined the functional significance of PAF-R in gemcitabine chemotherapy-mediated MVP release in human pancreatic cancer cells. Using PAF-R-expressing (PANC-1) and PAF-R-deficient (Hs766T) cells, we demonstrate that gemcitabine induces MVP release in a PAF-R-dependent manner. Blocking of PAF-R via PAF-R antagonist or inhibition of MVP generation via inhibitor of acid sphingomyelinase (aSMase) enzyme, significantly attenuated gemcitabine-mediated MVP release from PANC-1 cells, however, exerted no effects in Hs766T cells. Notably, MVPs from gemcitabine-treated PANC-1 cells, contained a measurable amount of PAF-agonists. Mechanistically, pretreatment with ERK1/2 or p38 inhibitors significantly abrogated gemcitabine-mediated MVP release, indicating the involvement of mitogen-activated protein kinase (MAPK) pathway in PAF-R-dependent gemcitabine-mediated MVP release. These findings demonstrate the significance of PAF-R in gemcitabine-mediated MVP release, as well as the rationale of evaluating PAF-R targeting agents with gemcitabine against pancreatic cancer.

Systemic Platelet-Activating Factor-Receptor Agonism Enhances Non-Melanoma Skin Cancer Growth.

Platelet-activating factor-receptor (PAF-R) agonists are pleiotropic lipid factors that influence multiple biological processes, including the induction and resolution of inflammation as well as immunosuppression. PAF-R agonists have been shown to modulate tumorigenesis and/or tumor growth in various skin cancer models by suppressing either cutaneous inflammation and/or anti-tumoral adaptive immunity. We have previously shown that a chronic systemic PAF-R agonist administration of mice enhances the growth of subcutaneously implanted melanoma tumors. Conversely, chronic topical applications of a PAF-R agonist suppressed non-melanoma skin cancer (NMSC) in a topical chemical carcinogenesis model (dimethylbenz[a]anthracene/phorbol 12-myristate 13-acetate (DMBA/PMA)) in-part via anti-inflammatory effects. These results indicate that the context of PAF-R agonist exposure via either chronic cutaneous or systemic administration, result in seemingly disparate effects on tumor promotion. To further dissect the contextual role of PAF-R agonism on tumorigenesis, we chronically administered systemic PAF-R agonist, carbamoyl-PAF (CPAF) to mice under a cutaneous chemical carcinogenesis protocol, recently characterized to initiate both NMSC and melanocytic nevus formation that can progress to malignant melanoma. Our results showed that while systemic CPAF did not modulate melanocytic nevus formation, it enhanced the growth of NMSC tumors.

Enhanced Platelet-Activating Factor Synthesis Facilitates Acute and Delayed Effects of Ethanol-Intoxicated Thermal Burn Injury.

Thermal burn injuries in patients who are alcohol-intoxicated result in greater morbidity and mortality. Murine models combining ethanol and localized thermal burn injury reproduce the systemic toxicity seen in human subjects, which consists of both acute systemic cytokine production with multiple organ dysfunction, as well as a delayed systemic immunosuppression. However, the exact mechanisms for these acute and delayed effects are unclear. These studies sought to define the role of the lipid mediator platelet-activating factor in the acute and delayed effects of intoxicated burn injury. Combining ethanol and thermal burn injury resulted in increased enzymatic platelet-activating factor generation in a keratinocyte cell line in vitro, human skin explants ex vivo, as well as in murine skin in vivo. Further, the acute increase in inflammatory cytokines, such as IL-6, and the systemic immunosuppressive effects of intoxicated thermal burn injury were suppressed in mice lacking platelet-activating factor receptors. Together, these studies provide a potential mechanism and treatment strategies for the augmented toxicity and immunosuppressive effects of thermal burn injury in the setting of acute ethanol exposure, which involves the pleotropic lipid mediator platelet-activating factor.

UVB-generated Microvesicle Particles: A Novel Pathway by Which a Skin-specific Stimulus Could Exert Systemic Effects.

Ultraviolet B radiation (UVB) exerts profound effects on human skin. Much is known regarding the ability of UVB to generate a plethora of bioactive agents ranging from cytokines and other bioactive proteins, lipid mediators and microRNAs. It is presumed that these agents are in large part responsible for the effects of UVB, which is only absorbed appreciably in the epidermis. However, the exact mechanism by which these bioactive agents can leave the epidermis are as yet unclear. This review addresses the potential role of microvesicle particles (MVP) as UVB signaling agents through transmitting biologic mediators. New data are provided that UVB treatment of human skin explants also generates MVP production. We hypothesize that UVB production of MVPs (UVB-MVP) could serve this important function of transmitting keratinocyte-derived bioactive agents. Moreover, we propose that UVB-MVP formation involves the lipid mediator platelet-activating factor. This novel pathway has the potential to be exploited pharmacologically to modulate UVB effects.