RESUMEN
Ribonucleic acid (RNA) therapeutics hold great potential for the advancement of dermatological treatments due to, among other reasons, the possibility of treating previously undruggable targets, high specificity with minimal side effects, and ability to include multiple RNA targets in a single product. Although there have been research relating to RNA therapeutics for decades, there have not been many products translated for clinical use until recently. This may be because of challenges to the application of RNA therapeutics, including the dearth of effective modes of delivery to the target, and rapid degradation of RNA in the human body and environment. This article aims to provide insight on (1) the wide-ranging possibilities of RNA therapeutics in the field of dermatology as well as (2) how key challenges can be addressed, so as to encourage the development of novel dermatological treatments. We also share our experience on how RNA therapeutics have been applied in the management of hypertrophic and keloid scars.
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Queloide , Humanos , Queloide/terapia , Cicatriz Hipertrófica/terapia , Cicatriz Hipertrófica/tratamiento farmacológico , ARN/uso terapéutico , Dermatología/métodos , Enfermedades de la Piel/terapia , Enfermedades de la Piel/tratamiento farmacológico , Terapia Genética/métodosRESUMEN
Surgical management of basal cell carcinoma (BCC) typically involves surgical excision with post-operative margin assessment using the bread-loafing technique; or gold-standard Mohs micrographic surgery (MMS), where margins are iteratively examined for residual cancer after tumour removal, with additional excisions performed upon detecting residual tumour at margins. There is limited sampling of resection margins with bread loafing, with detection of positive margins 44% of the time using 2 mm intervals. To resolve this, we have developed three-dimensional (3D) Tissue Imaging for: (1) complete examination of cancer margins and (2) detection of tumour proximity to nerves and blood vessels. 3D Tissue optical clearing with a light sheet imaging protocol was developed for margin assessment in two datasets assessed by two independent evaluators: (1) 48 samples from 29 patients with varied BCC subtypes, sizes and pigmentation levels; (2) 32 samples with matching Mohs' surgeon reading of tumour margins using two-dimensional haematoxylin & eosin-stained sections. The 3D Tissue Imaging protocol permits a complete examination of deeper and peripheral margins. Two independent evaluators achieved negative predictive values of 92.3% and 88.24% with 3D Tissue Imaging. Images obtained from 3D Tissue Imaging recapitulates histological features of BCC, such as nuclear crowding, palisading and retraction clefting and provides a 3D context for recognising normal skin adnexal structures. Concurrent immunofluorescence labelling of nerves and blood vessels allows visualisation of structures closer to tumour-positive regions, which may have a higher risk for neural and vascular infiltration. Together, this method provides more information in a 3D spatial context, enabling better cancer management by clinicians.
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Carcinoma Basocelular , Imagenología Tridimensional , Márgenes de Escisión , Cirugía de Mohs , Neoplasias Cutáneas , Humanos , Carcinoma Basocelular/diagnóstico por imagen , Carcinoma Basocelular/cirugía , Carcinoma Basocelular/patología , Neoplasias Cutáneas/diagnóstico por imagen , Neoplasias Cutáneas/cirugía , Neoplasias Cutáneas/patologíaRESUMEN
Neutrophils are increasingly recognized as key players in the tumor immune response and are associated with poor clinical outcomes. Despite recent advances characterizing the diversity of neutrophil states in cancer, common trajectories and mechanisms governing the ontogeny and relationship between these neutrophil states remain undefined. Here, we demonstrate that immature and mature neutrophils that enter tumors undergo irreversible epigenetic, transcriptional, and proteomic modifications to converge into a distinct, terminally differentiated dcTRAIL-R1+ state. Reprogrammed dcTRAIL-R1+ neutrophils predominantly localize to a glycolytic and hypoxic niche at the tumor core and exert pro-angiogenic function that favors tumor growth. We found similar trajectories in neutrophils across multiple tumor types and in humans, suggesting that targeting this program may provide a means of enhancing certain cancer immunotherapies.
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Reprogramación Celular , Neoplasias , Neovascularización Patológica , Neutrófilos , Humanos , Neoplasias/irrigación sanguínea , Neoplasias/inmunología , Neutrófilos/inmunología , Proteómica , Reprogramación Celular/genética , Reprogramación Celular/inmunología , Neovascularización Patológica/genética , Neovascularización Patológica/inmunología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Epigénesis Genética , Hipoxia , Transcripción GenéticaRESUMEN
Microglia are specialized brain-resident macrophages that arise from primitive macrophages colonizing the embryonic brain1. Microglia contribute to multiple aspects of brain development, but their precise roles in the early human brain remain poorly understood owing to limited access to relevant tissues2-6. The generation of brain organoids from human induced pluripotent stem cells recapitulates some key features of human embryonic brain development7-10. However, current approaches do not incorporate microglia or address their role in organoid maturation11-21. Here we generated microglia-sufficient brain organoids by coculturing brain organoids with primitive-like macrophages generated from the same human induced pluripotent stem cells (iMac)22. In organoid cocultures, iMac differentiated into cells with microglia-like phenotypes and functions (iMicro) and modulated neuronal progenitor cell (NPC) differentiation, limiting NPC proliferation and promoting axonogenesis. Mechanistically, iMicro contained high levels of PLIN2+ lipid droplets that exported cholesterol and its esters, which were taken up by NPCs in the organoids. We also detected PLIN2+ lipid droplet-loaded microglia in mouse and human embryonic brains. Overall, our approach substantially advances current human brain organoid approaches by incorporating microglial cells, as illustrated by the discovery of a key pathway of lipid-mediated crosstalk between microglia and NPCs that leads to improved neurogenesis.
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Encéfalo , Colesterol , Células Madre Pluripotentes Inducidas , Microglía , Células-Madre Neurales , Neurogénesis , Organoides , Animales , Humanos , Ratones , Encéfalo/citología , Encéfalo/metabolismo , Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Microglía/citología , Microglía/metabolismo , Organoides/citología , Organoides/metabolismo , Colesterol/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Axones , Proliferación Celular , Ésteres/metabolismo , Gotas Lipídicas/metabolismoRESUMEN
Background: Chronic pruritus of unknown origin (CPUO) is poorly understood and lacks effective treatment options. Objectives: We aimed to elucidate abnormalities in the sweat apparatus of patients with CPUO, and to assess efficacy and safety of treatment with systemic retinoids. Methods: An initial case-control study included 20 affected patients and five healthy controls, for whom heat and sweating were induced, either through a standardized exercise protocol or ingestion of hot water. In vivo high-definition optical coherence tomography, whole-body starch-iodine testing, and skin biopsy for immunofluorescence staining were done to evaluate for sweat duct obstruction. A subsequent retrospective cohort analysis included 56 patients with CPUO, seen at an Itch subspecialty clinic of a single tertiary referral centre, who failed conventional treatments and were treated with isotretinoin and/or acitretin from May 2014 to November 2020. Treatment response to retinoids was defined as a sustained reduction in itch score of ≥2/10. Safety was assessed by proportion stopping treatment due to side effects. Results: In vivo imaging in 19 (95%) patients revealed features of partial keratinaceous sweat duct obstruction with statistically significant luminal dilatation compared to controls. Immunofluorescence studies of three patients' paired lesional/non-lesional biopsies revealed dermcidin accumulation within sweat glands coupled with dermcidin leakage in itchy skin. Fifty-six patients (mean [SD] age 55.2 [17.5] years, 69.6% male) were treated with systemic retinoids. Mean (SD) duration of itch was 116.3 (140.4) months and mean (SD) itch score was 8.2 (1.8). Forty-one (73.2%) initially received isotretinoin, and 15 (26.8%) acitretin. At three months, mean itch score reduced by 2.38 (95% CI -3.2 to -1.6, p < 0.0001). Thirty-eight (67.9%) had a sustained response. Eight (14.81%) achieved an itch score of 0 or 1, with four stopping treatment for a mean (SD) of 318.5 (291.2) days without relapse. Eight (14.3%) stopped or switched retinoid due to adverse effects, with similar incidences between both retinoids, the commonest being dryness. Conclusion: Based on novel findings from physiological imaging studies identifying partial keratinaceous sweat duct obstruction in CPUO, we instituted systemic retinoid treatment to address the underlying pathology. In patients who failed conventional therapies, the treatment appears effective and safe.
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Langerhans cell histiocytosis (LCH) is a potentially fatal neoplasm characterized by the aberrant differentiation of mononuclear phagocytes, driven by mitogen-activated protein kinase (MAPK) pathway activation. LCH cells may trigger destructive pathology yet remain in a precarious state finely balanced between apoptosis and survival, supported by a unique inflammatory milieu. The interactions that maintain this state are not well known and may offer targets for intervention. Here, we used single-cell RNA-seq and protein analysis to dissect LCH lesions, assessing LCH cell heterogeneity and comparing LCH cells with normal mononuclear phagocytes within lesions. We found LCH discriminatory signatures pointing to senescence and escape from tumor immune surveillance. We also uncovered two major lineages of LCH with DC2- and DC3/monocyte-like phenotypes and validated them in multiple pathological tissue sites by high-content imaging. Receptor-ligand analyses and lineage tracing in vitro revealed Notch-dependent cooperativity between DC2 and DC3/monocyte lineages during expression of the pathognomonic LCH program. Our results present a convergent dual origin model of LCH with MAPK pathway activation occurring before fate commitment to DC2 and DC3/monocyte lineages and Notch-dependent cooperativity between lineages driving the development of LCH cells.
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Histiocitosis de Células de Langerhans , Neoplasias , Humanos , Linaje de la Célula , Histiocitosis de Células de Langerhans/metabolismo , Histiocitosis de Células de Langerhans/patología , Diferenciación Celular , Monocitos/metabolismoRESUMEN
Circulating Ly6Chi monocytes often undergo cellular death upon exhaustion of their antibacterial effector functions, which limits their capacity for subsequent macrophage differentiation. This shrouds the understanding on how the host replaces the tissue-resident macrophage niche effectively during bacterial invasion to avert infection morbidity. Here, we show that proliferating transitional premonocytes (TpMos), an immediate precursor of mature Ly6Chi monocytes (MatMos), were mobilized into the periphery in response to acute bacterial infection and sepsis. TpMos were less susceptible to apoptosis and served as the main source of macrophage replenishment when MatMos were vulnerable toward bacteria-induced cellular death. Furthermore, TpMo and its derived macrophages contributed to host defense by balancing the proinflammatory cytokine response of MatMos. Consequently, adoptive transfer of TpMos improved the survival outcome of lethal sepsis. Our findings hence highlight a protective role for TpMos during bacterial infections and their contribution toward monocyte-derived macrophage heterogeneity in distinct disease outcomes.
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Infecciones Bacterianas , Sepsis , Animales , Citocinas , Humanos , Macrófagos , Ratones , Ratones Endogámicos C57BL , MonocitosRESUMEN
As the largest organ of the body, the skin is a key barrier tissue with specialized structures where ongoing immune surveillance is critical for protecting the body from external insults. The innate immune system acts as first-responders in a coordinated manner to react to injury or infections, and recent developments in intravital imaging techniques have made it possible to delineate dynamic immune cell responses in a spatiotemporal manner. We review here key studies involved in understanding neutrophil, dendritic cell and macrophage behavior in skin and further discuss how this knowledge collectively highlights the importance of interactions and cellular functions in a systems biology manner. Furthermore, we will review emerging imaging technologies such as high-content proteomic screening, spatial transcriptomics and three-dimensional volumetric imaging and how these techniques can be integrated to provide a systems overview of the immune system that will further our current knowledge and lead to potential exciting discoveries in the upcoming decades.
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Microscopía Intravital , Proteómica , Humanos , Microscopía Intravital/métodos , Macrófagos , Neutrófilos , PielRESUMEN
Tissue-resident macrophages in white adipose tissue (WAT) dynamically adapt to the metabolic changes of their microenvironment that are often induced by excess energy intake. Currently, the exact contribution of these macrophages in obesity-driven WAT remodeling remains controversial. Here, using a transgenic CD169-DTR mouse strain, we provide new insights into the interplay between CD169+ adipose tissue macrophages (ATMs) and their surrounding WAT microenvironment. Using targeted in vivo ATM ablation followed by transcriptional and metabolic WAT profiling, we found that ATMs protect WAT from the excessive pathological remodeling that occurs during obesity. As obesity progresses, ATMs control not only vascular integrity, adipocyte function, and lipid and metabolic derangements but also extracellular matrix accumulation and resultant fibrosis in the WAT. The protective role of ATMs during obesity-driven WAT dysfunction supports the notion that ATMs represent friends, rather than foes, as has previously assumed.
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Tejido Adiposo , Macrófagos , Tejido Adiposo Blanco , Animales , Ratones , Ratones Endogámicos C57BL , Ratones ObesosRESUMEN
The complex bone marrow microenvironment or niche is an important anatomical structure responsible for hematopoiesis and providing support to the immune cells function. Being the source of immune and blood cells, the interaction of these hematopoietic stem and progenitor cells with the cellular niches regulates their ability for self-renewal, proliferation, and differentiation. Dynamic imaging not only provides spatiotemporal information of cell motility but also the morphological changes due to cell-cell interactions in the bone marrow, providing insights into the ongoing physiological activities within the tissue. Here, we describe customized stages with compatible equipment best suited for the upright two-photon microscope, accompanied by detailed methods for both calvarial and tibial intravital imaging. We demonstrate a general protocol for calvarial imaging using a minimally invasive surgical approach, and introduce a bone shaving-based tibial imaging as a complementary method. To demonstrate the applicability of our method we used Lyz2-EGFP transgenic mice to track bone marrow neutrophil activities as an example.
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Médula Ósea/fisiología , Rastreo Celular , Células Madre Hematopoyéticas/fisiología , Microscopía Intravital , Microscopía de Fluorescencia por Excitación Multifotónica , Neutrófilos/fisiología , Cráneo/fisiología , Nicho de Células Madre , Tibia/fisiología , Animales , Médula Ósea/metabolismo , Movimiento Celular , Femenino , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Muramidasa/genética , Muramidasa/metabolismo , Neutrófilos/metabolismo , Cráneo/citología , Cráneo/metabolismo , Tibia/citología , Tibia/metabolismoRESUMEN
A functional lymphatic vasculature is essential for tissue fluid homeostasis, immunity, and lipid clearance. Although atherosclerosis has been linked to adventitial lymphangiogenesis, the functionality of aortic lymphatic vessels draining the diseased aorta has never been assessed and the role of lymphatic drainage in atherogenesis is not well understood. We develop a method to measure aortic lymphatic transport of macromolecules and show that it is impaired during atherosclerosis progression, whereas it is ameliorated during lesion regression induced by ezetimibe. Disruption of aortic lymph flow by lymphatic ligation promotes adventitial inflammation and development of atherosclerotic plaque in hypercholesterolemic mice and inhibits ezetimibe-induced atherosclerosis regression. Thus, progression of atherosclerotic plaques may result not only from increased entry of atherogenic factors into the arterial wall but also from reduced lymphatic clearance of these factors as a result of aortic lymph stasis. Our findings suggest that promoting lymphatic drainage might be effective for treating atherosclerosis.
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Aterosclerosis , Placa Aterosclerótica , Animales , Aorta/patología , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/patología , Ezetimiba/farmacología , Ezetimiba/uso terapéutico , Linfangiogénesis , Ratones , Placa Aterosclerótica/tratamiento farmacológico , Placa Aterosclerótica/patologíaRESUMEN
Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs.
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Células Precursoras de Granulocitos/citología , Monocitos/citología , Mielopoyesis/fisiología , Neutrófilos/citología , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de la Célula IndividualRESUMEN
Skin histology is traditionally carried out using two-dimensional tissue sections, which allows for rapid staining, but these sections cannot accurately represent three-dimensional structures in skin such as nerves, vasculature, hair follicles, and sebaceous glands. Although it may be ideal to image skin in a three-dimensional manner, it is technically challenging to image deep into tissue because of light scattering from collagen fibrils in the dermis and refractive index mismatch owing to the presence of differing biological materials such as cytoplasm, and lipids in the skin. Different optical clearing methods have been developed recently, making it possible to render tissues transparent using different approaches. Here, we discuss the steps involved in tissue preparation for three-dimensional volumetric imaging and provide a brief overview of the different optical clearing methods as well as different imaging modalities for three-dimensional imaging.
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Biopsia/métodos , Folículo Piloso/diagnóstico por imagen , Imagenología Tridimensional , Imagen Óptica/métodos , Glándulas Sebáceas/diagnóstico por imagen , Enfermedades de la Piel/diagnóstico por imagen , Animales , Colágeno/química , Citoplasma/metabolismo , Dermis/diagnóstico por imagen , Humanos , Luz , Lípidos/química , Ratones , Ratones Endogámicos BALB C , Permeabilidad , Refractometría , Dispersión de Radiación , SolventesRESUMEN
Optical imaging is a valuable tool to visualise biological processes in the context of the tissue. Each imaging modality provides the biologist with different types of information - cell dynamics and migration over time can be tracked with time-lapse imaging (e.g. intra-vital imaging); an overview of whole tissues can be acquired using optical clearing in conjunction with light sheet microscopy; finer details such as cellular morphology and fine nerve tortuosity can be imaged at higher resolution using the confocal microscope. Multi-modal imaging combined with image cytometry - a form of quantitative analysis of image datasets - provides an objective basis for comparing between sample groups. Here, we provide an overview of technical aspects to look out for in an image cytometry workflow, and discuss issues related to sample preparation, image post-processing and analysis for intra-vital and whole organ imaging.
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Optical imaging is a valuable tool to visualise biological processes in the context of the tissue. Each imaging modality provides the biologist with different types of information - cell dynamics and migration over time can be tracked with time-lapse imaging (e.g. intra-vital imaging); an overview of whole tissues can be acquired using optical clearing in conjunction with light sheet microscopy; finer details such as cellular morphology and fine nerve tortuosity can be imaged at higher resolution using the confocal microscope. Multi-modal imaging combined with image cytometry - a form of quantitative analysis of image datasets - provides an objective basis for comparing between sample groups. Here, we provide an overview of technical aspects to look out for in an image cytometry workflow, and discuss issues related to sample preparation, image post-processing and analysis for intra-vital and whole organ imaging.
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Citometría de Imagen , Animales , Encéfalo/citología , Conjuntos de Datos como Asunto , Predicción , Humanos , Citometría de Imagen/métodos , Citometría de Imagen/tendencias , Procesamiento de Imagen Asistido por Computador , Ratones , Microscopía Confocal , Programas InformáticosAsunto(s)
Dermatitis Atópica/diagnóstico por imagen , Epidermis/inervación , Imagenología Tridimensional , Terminaciones Nerviosas/patología , Psoriasis/diagnóstico por imagen , Biopsia con Aguja , Estudios de Casos y Controles , Dermatitis Atópica/metabolismo , Dermatitis Atópica/patología , Epidermis/diagnóstico por imagen , Epidermis/patología , Femenino , Humanos , Inmunohistoquímica , Masculino , Prurito/diagnóstico por imagen , Prurito/metabolismo , Prurito/patología , Psoriasis/metabolismo , Psoriasis/patología , Valores de Referencia , Índice de Severidad de la Enfermedad , Imagen de Colorante Sensible al VoltajeRESUMEN
Image cytometry is the process of converting image data to flow cytometry-style plots, and it usually requires computer-aided surface creation to extract out statistics for cells or structures. One way of dealing with structures stained with multiple markers in three-dimensional images, is carrying out multiple rounds of channel co-localization and image masking before surface creation, which is cumbersome and laborious. We propose the application of the hue-saturation-brightness color space to streamline this process, which produces complete surfaces, and allows the user to have a global view of the data before flexibly defining cell subsets. Spectral compensation can also be performed after surface creation to accurately resolve different signals. We demonstrate the utility of this workflow in static and dynamic imaging datasets of a needlestick injury on the mouse ear, and we believe this scalable and intuitive approach will improve the ease of performing histocytometry on biological samples.
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The maintenance of appropriate arterial tone is critically important for normal physiological arterial function. However, the cellular and molecular mechanisms remain poorly defined. Here, we have shown that in the mouse aorta, resident macrophages prevented arterial stiffness and collagen deposition in the steady state. Using phenotyping, transcriptional profiling, and targeted deletion of Csf1r, we have demonstrated that these macrophages-which are a feature of blood vessels invested with smooth muscle cells (SMCs) in both mouse and human tissues-expressed the hyaluronan (HA) receptor LYVE-l. Furthermore, we have shown they possessed the unique ability to modulate collagen expression in SMCs by matrix metalloproteinase MMP-9-dependent proteolysis through engagement of LYVE-1 with the HA pericellular matrix of SMCs. Our study has unveiled a hitherto unknown homeostatic contribution of arterial LYVE-1+ macrophages through the control of collagen production by SMCs and has identified a function of LYVE-1 in leukocytes.