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BACKGROUND: Rosacea, a common chronic inflammatory skin disease worldwide, is currently incurable with complex pathogenesis. Dendrobium polysaccharide (DOP) may exert therapeutic effects on rosacea via acting on the NF-κB-related inflammatory and oxidative processes. MATERIALS AND METHODS: In this study, an LL-37-induced rosacea-like mouse model was established. HE staining was used to assess the skin lesions, erythema severity scores, pathological symptoms, and inflammatory cell numbers of mice in each group. The inflammation level was quantitatively analyzed using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). The expression levels of TLR4 and p-NF-κB were finally detected. RESULTS: DOP improved skin pathological symptoms of rosacea mice. DOP also alleviated the inflammation of rosacea mice. Moreover, the TLR4/NF-κB pathway was observed to be inhibited in the skin of mice after DOP application. These findings evidenced the anti-inflammatory effects of DOP on the LL-37-induced rosacea mouse model. DOP could inhibit NF-κB activation, suppress neutrophil infiltration, and reduce pro-inflammatory cytokines production, which may be the reason for DOP protecting against rosacea. CONCLUSION: This study may propose an active candidate with great potential for rosacea drug development and lay a solid experimental foundation for promoting DOP application in rosacea therapy.
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Dendrobium , Rosácea , Animais , Camundongos , NF-kappa B , Receptor 4 Toll-Like , Rosácea/induzido quimicamente , Rosácea/tratamento farmacológico , Modelos Animais de Doenças , Inflamação , Polissacarídeos/farmacologia , Polissacarídeos/uso terapêuticoRESUMO
BACKGROUND: Androgenetic alopecia can affect up to 70% of males and 40% of females; however, certain therapeutic medications offer partial and transitory improvement but with major side effects. Dendrobium officinale polysaccharide (DOP) has been reported to improve androgen-related hair loss in mice, but the molecular mechanism remains unclear. OBJECTIVES: To explore the effects of DOP on androgenetic alopecia. METHODS: In this study, testosterone was subcutaneously administered to shave dorsa skin of mice to establish androgenetic alopecia; the effects of DOP in androgenetic alopecia were explored by DOP administration. RESULTS: Testosterone treatment extended the time of skin growing dark and hair growing, decreased the mean numbers of follicles in skin tissues, decreased ß-catenin and cyclin D1 levels, and elevated testosterone, DHT (dihydrotestosterone), and 5α-reductase levels. In contrast, DOP administration shortened skin growing dark and hair growing times, promoted follicle cell proliferation, increased follicle numbers, increased ß-catenin and cyclin D1 levels, and decreased testosterone, DHT, and 5α-reductase levels. CONCLUSION: DOP application significantly improved testosterone-induced hair follicle miniaturization and hair loss, possibly through affecting the Wnt signaling and hair follicle stem cell functions. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .
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Dendrobium , Testosterona , Masculino , Feminino , Camundongos , Animais , Testosterona/farmacologia , beta Catenina/farmacologia , Ciclina D1/farmacologia , Cabelo , Alopecia/induzido quimicamente , Alopecia/tratamento farmacológico , Polissacarídeos/farmacologiaRESUMO
Melanin metabolism disorders may cause severe impacts on the psychological and social activities of patients. Different from the other two steps of melanin metabolism, namely synthesis and transport, little has been known about the mechanism of melanin degradation. Isoimperatorin (ISO) suppressed the activity of tyrosinase, an essential enzyme in melanin biosynthesis, hence, we investigated the effects and mechanism of ISO in melanin reduction. ISO stimulation significantly reduces the melanin contents and PMEL 17 protein levels; meanwhile, the activity and the protein levels of two critical lysosomal enzymes, Cathepsin B and Cathepsin D, can be significantly increased by ISO treatment. MiR-3619 inhibited the expression of CSTB and CSTD, therefore affecting ISO-induced degradation of melanin. In summary, ISO reduces the melanin content via miR-3619/CSTB and miR-3619/CSTD axes. ISO could be a potent skin-whitening agent, which needs further in vivo and clinical investigation.
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Catepsina B/metabolismo , Catepsina D/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Furocumarinas/farmacologia , Queratinócitos/metabolismo , Melaninas/metabolismo , MicroRNAs/metabolismo , Transdução de Sinais/efeitos dos fármacos , Preparações Clareadoras de Pele/farmacologia , Catepsina B/genética , Catepsina D/genética , Técnicas de Silenciamento de Genes , Células HaCaT , Humanos , MicroRNAs/genética , Monofenol Mono-Oxigenase/antagonistas & inibidores , Transdução de Sinais/genética , Transfecção , Antígeno gp100 de Melanoma/metabolismoRESUMO
Chronic skin inflammation in atopic eczema is associated with elevated expression of proinflammatory genes and activation of innate immune responses in keratinocytes. MicroRNAs (miRNAs) are short, single-stranded RNA molecules that silence genes via the degradation of target mRNAs or inhibition of translation. Recent studies have demonstrated that miR-124 is associated with regulation of inflammation factors in several diseases. The aim of this study was to investigate the role of miR-124 in skin inflammation of atopic eczema. We showed that miR-124 expression is decreased in chronic lesional skin of patients with atopic eczema, and could be strongly inhibited by IFN-γ and TNF-α. Through Western blot, real-time PCR and luciferase assays, we revealed that miR-124 inhibited the expression of p65, a member of NF-κB family which can regulate many factors involved in the immune response and inflammatory reactions, through direct targeting. Further, upon IFN-γ or TNF-α stimulation, IL8, CCL5 and CCL8 showed to be significantly upregulated by IFN-γ or TNF-α, downregulated by miR-124; the promotive effect of IFN-γ and TNF-α could be partially reversed by miR-124. The levels of IL8, CCL5 and CCL8 could be significantly downregulated by p65 knockdown, upregulated by miR-124 inhibition; the suppressive effect of p65 knockdown could be partially reversed by miR-124. Moreover, contrary to miR-124, p65, IL8, CCL5 and CCL8 mRNA expression was upregulated in chronic lesional skin of patients with atopic eczema, and all inversely correlated with miR-124. Taken together, our data demonstrate that miR-124 controls NF-κB-dependent inflammatory responses in keratinocytes and chronic skin inflammation in atopic eczema; rescuing miR-124 expression presents a promising strategy for atopic eczema treatment.
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Dermatite Atópica/imunologia , Queratinócitos/imunologia , MicroRNAs/imunologia , Fator de Transcrição RelA/imunologia , Sequência de Bases , Quimiocina CCL5/genética , Quimiocina CCL5/imunologia , Quimiocina CCL8/genética , Quimiocina CCL8/imunologia , Dermatite Atópica/genética , Dermatite Atópica/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Interferon gama/farmacologia , Interleucina-8/genética , Interleucina-8/imunologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , MicroRNAs/genética , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Transdução de Sinais , Pele/efeitos dos fármacos , Pele/imunologia , Pele/patologia , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
This study aims to explore the impact of the herbal ointment Chushi Zhiyang Ruangao (CSZYRG) on the skin's microecological environment in a mouse model of atopic dermatitis (AD) and to understand the underlying mechanisms involved. The AD model was established in C57 mice using calpolitol (a hypocalcemic analog of vitamin D3; MC903). Medication-free matrix ointment, CSZYRG, and mometasone furoate cream (positive control group) were applied to the injured areas. The skin lesions of AD model mice were photographed. Skin lesions were applied for the hematoxylin and eosin (H&E) staining to observe any pathological changes. Serum immunoglobulin IgE was detected by enzyme-linked immunosorbent assay (ELISA). The changes in the expression of inflammation-related factors TNF-α, IL-1ß, and IL-6 in mice were detected using ELISA and qRT-PCR. Skin microflora samples were taken for 16S rDNA sequencing and analyzed for changes in the skin flora diversity, abundance, and dominant flora in mice. It was concluded that CSZYRG effectively alleviates skin lesions, serum IgE, and levels of TNF-α, IL-1ß, and IL-6 in AD model mice. However, CSZYRG did not affect the skin microbial diversity of AD model mice but could exert an effect on the skin microbial community in AD mice and the relative abundance of the dominant microflora. CSZYRG may play a therapeutic role in AD by affecting the skin microbial community and relative abundance of dominant microflora in AD mice.
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Introduction: Hair loss is one of the common clinical conditions in modern society. Although it is not a serious disease that threatens human life, it brings great mental stress and psychological burden to patients. This study investigated the role of dendrobium officinale polysaccharide (DOP) in hair follicle regeneration and hair growth and its related mechanisms. Methods: After in vitro culture of mouse antennal hair follicles and mouse dermal papilla cells (DPCs), and mouse vascular endothelial cells (MVECs), the effects of DOP upon hair follicles and cells were evaluated using multiple methods. DOP effects were evaluated by measuring tentacle growth, HE staining, immunofluorescence, Western blot, CCK-8, ALP staining, tube formation, scratch test, and Transwell. LDH levels, WNT signaling proteins, and therapeutic mechanisms were also analyzed. Results: DOP promoted tentacle hair follicle and DPCs growth in mice and the angiogenic, migratory and invasive capacities of MVECs. Meanwhile, DOP was also capable of enhancing angiogenesis and proliferation-related protein expression. Mechanistically, DOP activated the WNT signaling and promoted the expression level of ß-catenin, a pivotal protein of the pathway, and the pathway target proteins Cyclin D1, C-Myc, and LDH activity. The promotional effects of DOP on the biological functions of DPCs and MVECs could be effectively reversed by the WNT signaling pathway inhibitor IWR-1. Conclusion: DOP advances hair follicle and hair growth via the activation of the WNT signaling. This finding provides a mechanistic reference and theoretical basis for the clinical use of DOP in treating hair loss.
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Psoriasis, a chronic and easily recurring inflammatory skin disease, causes a great economic burden to the patient's family because the etiology and mechanism are still unclear and the treatment cycle is long. In this study, the function and related mechanisms of Momordin Ic in psoriasis were investigated. The IMQ-induced mouse psoriasis model was constructed. The protective effects of different doses of Momordin Ic on psoriasis skin damage in mice were detected by PASI score, HE staining and Ki-67 staining. A psoriasis-like keratinocyte model was established at the cellular level using M5 (IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α) triggered HaCaT. The effects of Momordin Ic upon HaCaT cell biological behavior were examined using MTT and CCK-8 assays. In terms of mechanism, the expression level of each inflammatory factor was assessed using IHC staining and/or ELISA, qRT-PCR, the expression of oxidative stress-related indicators was detected biochemically, and western blot was performed to detect the levels of key proteins of the Wnt signaling and VEGF. As the results shown, at the in vivo level, Momordin Ic significantly alleviated skin damage, reduced PASI score and inhibited hyperproliferation of keratinized cells in psoriasis mice. At the cellular level, Momordin Ic also significantly reversed M5-induced hyperproliferation of HaCaT keratinocytes. In terms of mechanism, Momordin Ic significantly inhibited the IL-23/IL-17 axis, dramatically elevated the levels of intracellular antioxidants including SOD, GSH-Px, and CAT, and significantly down-regulated the levels of the indicator of oxidative damage, malondialdehyde (MDA). In addition, Momordin Ic also significantly inhibited the level of ß-catenin, a pivotal protein of the Wnt signaling, C-Myc, a target gene of the Wnt signaling, and VEGF, a critical protein of angiogenesis. In conclusion, Momordin Ic can be involved in the skin-protective effects of psoriasis by multiple mechanisms, including inhibition of the Wnt signaling pathway and the IL-23/IL-17 axis, and suppression of oxidative damageand VEGF expression. Momordin Ic has been proven to be an underlying therapeutic drug for the treatment of psoriasis.
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Modelos Animais de Doenças , Interleucina-17 , Interleucina-23 , Queratinócitos , Psoríase , Pele , Via de Sinalização Wnt , Animais , Humanos , Camundongos , Proliferação de Células/efeitos dos fármacos , Células HaCaT , Imiquimode , Interleucina-17/metabolismo , Interleucina-23/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos Endogâmicos BALB C , Estresse Oxidativo/efeitos dos fármacos , Psoríase/tratamento farmacológico , Psoríase/patologia , Psoríase/induzido quimicamente , Psoríase/imunologia , Pele/patologia , Pele/efeitos dos fármacos , Pele/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
PURPOSE: Psoriasis is a skin disease characterized by excessive proliferation, inflammation and oxidative stress in keratinocytes. The present study aimed to investigate the therapeutic effects of Dendrobium officinale polysaccharide (DOP) on keratinocyte psoriasis-like models. METHODS: The HaCaT keratinocyte inflammation models were induced by interleukin (IL)-22 or lipopolysaccharide (LPS), respectively, and oxidative stress damage within cells was elicited by H2O2 and treated using DOP. CCK-8 and EdU were carried out to detect cell proliferation. ELISA, qRT-PCR, and Western blot were conducted to measure the expression of pro-inflammatory cytokines IL17A, IL-23, IL1ß, tumor necrosis factor alpha (TNF-α), and IL-6. Reactive oxygen species (ROS) level in keratinocytes was detected by flow cytometry. Cell proliferation-associated proteins (PCNA, Ki67, Cyclin D1) and pathway proteins (p-AKT and AKT), and oxidative stress marker proteins (Nrf-2, CAT, SOD1) were detected by Western blot. RESULT: DOP did not affect the proliferation of normal keratinocytes, but DOP was able to inhibit the proliferative activity of IL-22-induced overproliferating keratinocytes and suppress the expression of proliferation-related factors PCNA, Ki67, and Cyclin D1 as well as the proliferation pathway p-AKT. In addition, DOP treatment was able to inhibit IL-22 and LPS-induced inflammation and H2O2-induced oxidative stress, including the expression of IL17A, IL-23, IL1ß, TNF-α, IL-6, and IL1ß, as well as the expression levels of intracellular ROS levels and cellular oxidative stress-related indicators SOD, MDA, CAT, Nrf-2 and SOD1. CONCLUSION: DOP inhibits keratinocyte hyperproliferation, inflammation and oxidative stress to improve the keratinocyte psoriasis-like state.
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Proliferação de Células , Dendrobium , Inflamação , Queratinócitos , Estresse Oxidativo , Polissacarídeos , Psoríase , Estresse Oxidativo/efeitos dos fármacos , Dendrobium/química , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Proliferação de Células/efeitos dos fármacos , Polissacarídeos/farmacologia , Inflamação/tratamento farmacológico , Inflamação/patologia , Inflamação/metabolismo , Psoríase/tratamento farmacológico , Psoríase/patologia , Psoríase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Citocinas/metabolismoRESUMO
Suitable methods to assess in vivo immunogenicity and therapeutic efficacy of cancer vaccines in preclinical cancer models are critical to overcome current limitations of cancer vaccines and enhance the clinical applicability of this promising immunotherapeutic strategy. In particular, availability of methods allowing the characterization of T cell responses to endogenous tumor antigens is required to assess vaccine potency and improve the antigen formulation. Moreover, multiparametric assays to deeply characterize tumor-induced and therapy-induced immune modulation are relevant to design mechanism-based combination immunotherapies. Here we describe a versatile multiparametric flow cytometry method to assess the polyfunctionality of tumor antigen-specific CD4+ and CD8+ T cell responses based on their production of multiple cytokines after short-term ex vivo restimulation with relevant tumor epitopes of the most common mouse strains. We also report the development and application of two 21-color flow cytometry panels allowing a comprehensive characterization of T cell and natural killer cell exhaustion and memory phenotypes in mice with a particular focus on preclinical cancer models.
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Vacinas Anticâncer , Neoplasias , Animais , Camundongos , Citometria de Fluxo , Células Matadoras Naturais , Neoplasias/terapia , Fenótipo , Antígenos de NeoplasiasRESUMO
Vaccines have been hailed as one of the most remarkable medical advancements in human history, and their potential for treating cancer by generating or expanding anti-tumor T cells has garnered significant interest in recent years. However, the limited efficacy of therapeutic cancer vaccines in clinical trials can be partially attributed to the inadequacy of current preclinical mouse models in recapitulating the complexities of the human immune system. In this study, we developed two innovative humanized mouse models to assess the immunogenicity and therapeutic effectiveness of vaccines targeting human papillomavirus (HPV16) antigens and delivering tumor antigens to human CD141+ dendritic cells (DCs). Both models were based on the transference of human peripheral blood mononuclear cells (PBMCs) into immunocompromised HLA-A*02-NSG mice (NSG-A2), where the use of fresh PBMCs boosted the engraftment of human cells up to 80%. The dynamics of immune cells in the PBMC-hu-NSG-A2 mice demonstrated that T cells constituted the vast majority of engrafted cells, which progressively expanded over time and retained their responsiveness to ex vivo stimulation. Using the PBMC-hu-NSG-A2 system, we generated a hyperplastic skin graft model expressing the HPV16-E7 oncogene. Remarkably, human cells populated the skin grafts, and upon vaccination with a DNA vaccine encoding an HPV16-E6/E7 protein, rapid rejection targeted to the E7-expressing skin was detected, underscoring the capacity of the model to mount a vaccine-specific response. To overcome the decline in DC numbers observed over time in PBMC-hu-NSG-A2 animals, we augmented the abundance of CD141+ DCs, the specific targets of our tailored nanoemulsions (TNEs), by transferring additional autologous PBMCs pre-treated in vitro with the growth factor Flt3-L. The Flt3-L treatment bolstered CD141+ DC numbers, leading to potent antigen-specific CD4+ and CD8+ T cell responses in vivo, which caused the regression of pre-established triple-negative breast cancer and melanoma tumors following CD141+ DC-targeting TNE vaccination. Notably, using HLA-A*02-matching PBMCs for humanizing NSG-A2 mice resulted in a delayed onset of graft-versus-host disease and enhanced the efficacy of the TNE vaccination compared with the parental NSG strain. In conclusion, we successfully established two humanized mouse models that exhibited strong antigen-specific responses and demonstrated tumor regression following vaccination. These models serve as valuable platforms for assessing the efficacy of therapeutic cancer vaccines targeting HPV16-dysplastic skin and diverse tumor antigens specifically delivered to CD141+ DCs.
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Vacinas Anticâncer , Melanoma , Humanos , Animais , Camundongos , Transplante de Pele , Leucócitos Mononucleares , Hiperplasia , Anticorpos , Modelos Animais de Doenças , Antígenos de Neoplasias , Células Dendríticas , Antígenos HLA-ARESUMO
The extracellular matrix (ECM) is an essential component of the tumor microenvironment. It plays a critical role in regulating cell-cell and cell-matrix interactions. However, there is lack of systematic and comparative studies on different widely-used ECM mimicking hydrogels and their properties, making the selection of suitable hydrogels for mimicking different in vivo conditions quite random. This study systematically evaluates the biophysical attributes of three widely used natural hydrogels (Matrigel, collagen gel and agarose gel) including complex modulus, loss tangent, diffusive permeability and pore size. A new and facile method was developed combining Critical Point Drying, Scanning Electron Microscopy imaging and a MATLAB image processing program (CSM method) for the characterization of hydrogel microstructures. This CSM method allows accurate measurement of the hydrogel pore size down to nanometer resolution. Furthermore, a microfluidic device was implemented to measure the hydrogel permeability (Pd) as a function of particle size and gel concentration. Among the three gels, collagen gel has the lowest complex modulus, medium pore size, and the highest loss tangent. Agarose gel exhibits the highest complex modulus, the lowest loss tangent and the smallest pore size. Collagen gel and Matrigel produced complex moduli close to that estimated for cancer ECM. The Pd of these hydrogels decreases significantly with the increase of particle size. By assessing different hydrogels' biophysical characteristics, this study provides valuable insights for tailoring their properties for various three-dimensional cancer models.
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Matriz Extracelular , Hidrogéis , Neoplasias , Fenômenos Biofísicos , Colágeno/análise , Matriz Extracelular/química , Humanos , Hidrogéis/análise , Sefarose/análise , Microambiente TumoralRESUMO
Psoriasis is a chronic, recurrent, immunoinflammatory disease. For a long period, Traditional Chinese Medicine (TCM) is considered a reliable alternative therapy for patients with psoriasis. Fructus Kochiae (or Kochia scoparia) and its principle saponin, Momordin Ic, have been reported to protect against inflammation. Herein, we demonstrated that Momordin Ic could inhibit HaCaT cell proliferation and enhance cell apoptosis. In the meantime, Momordin Ic alters Wnt/ß-catenin pathway activation by affecting ß-catenin nuclear distribution. The Wnt/ß-catenin signaling activator LiCl partially reversed the effects of Momordin Ic on HaCaT phenotypes and the Wnt/ß-catenin pathway factors. Altogether, we demonstrate the inhibitory effects of Momordin Ic, one of the major saponin constituents of Fructus Kochiae, on HaCaT cell proliferation and Momordin Ic-induced alteration within the Wnt/ß-catenin pathway. Momordin Ic might act on HaCaT cells by modulating the Wnt/ß-catenin pathway.
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Drugs selectively targeting replication stress have demonstrated significant preclinical activity, but this has not yet translated into an effective clinical treatment. Here we report that targeting increased replication stress with a combination of Checkpoint kinase 1 inhibitor (CHK1i) with a subclinical dose of hydroxyurea targets also promotes pro-inflammatory cytokine/chemokine expression that is independent of cGAS-STING pathway activation and immunogenic cell death in human and murine melanoma cells. In vivo, this drug combination induces tumour regression which is dependent on an adaptive immune response. It increases cytotoxic CD8+ T cell activity, but the major adaptive immune response is a pronounced NKT cell tumour infiltration. Treatment also promotes an immunosuppressive tumour microenvironment through CD4+ Treg and FoxP3+ NKT cells. The number of these accumulated during treatment, the increase in FoxP3+ NKT cells numbers correlates with the decrease in activated NKT cells, suggesting they are a consequence of the conversion of effector to suppressive NKT cells. Whereas tumour infiltrating CD8+ T cell PD-1 and tumour PD-L1 expression was increased with treatment, peripheral CD4+ and CD8+ T cells retained strong anti-tumour activity. Despite increased CD8+ T cell PD-1, combination with anti-PD-1 did not improve response, indicating that immunosuppression from Tregs and FoxP3+ NKT cells are major contributors to the immunosuppressive tumour microenvironment. This demonstrates that therapies targeting replication stress can be well tolerated, not adversely affect immune responses, and trigger an effective anti-tumour immune response.
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Targeted drug delivery remains attractive but challenging for cancer therapy. Cell-mediated drug delivery has emerged as a promising strategy to improve targeted drug delivery to tumors due to the intrinsic ability of certain types of cells (e.g., macrophage) to pass through physiological barriers and specifically home to tumors. To fundamentally understand how macrophage-based drug carriers transport and interact with the tumor microenvironment, we developed a tumor-microenvironment-on-a-chip (TMOC) model that enables the coculture of tumor spheroids and macrophages in a three-dimensional (3D) gel matrix. By introducing drug-loaded macrophages, the TMOC model allows real-time observation of macrophage migration toward the tumor, infiltration into tumor spheroids, and subsequent response of tumor to drugs. Our results demonstrated the superior capability of macrophages migrating toward the tumor and infiltrate tumor spheroids. Drug loading in macrophages had minimum effect on their cell viability, and drug-carrying macrophages exhibited greater tumor cell cytotoxicity compared to their nanoparticle counterparts. Our work highlighted the great potential of macrophages as novel drug carriers for targeted drug delivery, and the TMOC model serves as a versatile platform to enable quick evaluation of such cell-mediated drug delivery systems.
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Nanopartículas , Microambiente Tumoral , Sistemas de Liberação de Medicamentos , Dispositivos Lab-On-A-Chip , MacrófagosRESUMO
Psoriasis is an immune-mediated chronic inflammatory skin disease. Keratinocyte hyperproliferation has been regarded as a significant event in psoriasis pathogenesis. Considering the vital role of miRNA-mediated mRNA repression in psoriasis pathogenesis, in the present study, we attempted to investigate the mechanism of keratinocyte overproliferation from the point of miRNA-mRNA regulation. Both online microarray expression profiles and experimental results indicated that the expression of LXR-α and PPAR-γ was downregulated in psoriasis lesion skin. LXR-α or PPAR-γ overexpression alone was sufficient to inhibit keratinocyte proliferation, decrease KRT5 and KRT14 protein levels and increase KRT1 and KRT10 protein levels. miR-203 negatively regulated LXR-α and PPAR-γ expression through direct targeting. miR-203 inhibition exerted the opposite effects to LXR-α or PPAR-γ overexpression on HaCaT cells. More importantly, LXR-α or PPAR-γ overexpression could markedly remarkably attenuate the effects of miR-203 overexpression in keratinocytes, indicating that miR-203 promotes keratinocyte proliferation by targeting LXR-α and PPAR-γ. In conclusion, the miR-203-LXR-α/PPAR-γ axis modulates the proliferation of keratinocytes and might be a novel target for psoriasis treatment, which needs further in vivo investigation.
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Células HaCaT/citologia , Células HaCaT/metabolismo , Receptores X do Fígado/metabolismo , MicroRNAs/metabolismo , PPAR gama/metabolismo , Proliferação de Células/genética , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , MicroRNAs/genética , Psoríase/genética , Psoríase/patologiaRESUMO
Keratinocyte hyperproliferation has been regarded as a central event in psoriasis pathogenesis. Investigating the mechanisms of keratinocyte hyperproliferation might provide novel strategies for psoriasis treatment. we demonstrated that fibroblast growth factor receptor 2 (FGFR2) expression was abnormally upregulated within psoriatic lesion tissues and HaCaT cells under rIL-22 stimulation. FGFR2 silence within HaCaT cells under rIL-22 stimulation significantly inhibited the capacity of cells to proliferate and to migrate, reduced IL-17A and TNFα mRNA expression, and decreased the protein levels of FGFR2, keratin 6, keratin 16, MMP1, MMP9, p-PI3K, p-AKT and p-ERK. In contrast to FGFR2, the expression of miR-124-3p showed to be remarkably downregulated within psoriasis lesion tissue samples and rIL-22-stimulated HaCaT cells. miR-124-3p inhibited the expression of FGFR2 via direct binding to its 3'UTR. Within HaCaT cells under rIL-22 stimulation, the overexpression of miR-124-3p also suppressed the capacity of cells to proliferate and to migrate, reduced IL-17A and TNFα mRNA expression, and decreased the protein levels of FGFR2, keratin 6, keratin 16, MMP1, MMP9 and p-PI3K, p-AKT and p-ERK. More importantly, when co-transfected to HaCaT cells, FGFR2-overexpressing vector significantly attenuated the effects of miR-124-3p mimics on HaCaT cells. In conclusion, we demonstrated an miR124-3p/FGFR2 axis that might inhibit human keratinocyte proliferation, migration, and improve the inflammatory microenvironment in psoriasis. miR124-3p/FGFR2 axis could be an underlying target for psoriasis therapy, which requires further in vivo and clinical investigation.
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OBJECTIVES: Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells is a form of cancer immunotherapy that has achieved remarkable efficacy in patients with some haematological cancers. However, challenges remain in CAR T-cell treatment of solid tumours because of tumour-mediated immunosuppression. METHODS: We have demonstrated that CAR T-cell stimulation through T-cell receptors (TCRs) in vivo can generate durable responses against solid tumours in a variety of murine models. Since Clec9A-targeting tailored nanoemulsion (Clec9A-TNE) vaccine enhances antitumour immune responses through selective activation of Clec9A+ cross-presenting dendritic cells (DCs), we hypothesised that Clec9A-TNE could prime DCs for antigen presentation to CAR T cells through TCRs and thus improve CAR T-cell responses against solid tumours. To test this hypothesis, we used CAR T cells expressing transgenic TCRs specific for ovalbumin (OVA) peptides SIINFEKL (CAROTI) or OVA323-339 (CAROTII). RESULTS: We demonstrated that the Clec9A-TNEs encapsulating full-length recombinant OVA protein (OVA-Clec9A-TNE) improved CAROT T-cell proliferation and inflammatory cytokine secretion in vitro. Combined treatment using the OVA-Clec9A-TNE and CAROT cells resulted in durable responses and some rejections of tumours in immunocompetent mice. Tumour regression was accompanied by enhanced CAROT cell proliferation and infiltration into the tumours. CONCLUSION: Our study presents Clec9A-TNE as a prospective avenue to enhance CAR T-cell efficacy for solid cancers.
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Invariant natural killer T (iNKT) cells are a subset of lymphocytes with immune regulatory activity. Their ability to bridge the innate and adaptive immune systems has been studied using the glycolipid ligand α-galactosylceramide (αGC). To better harness the immune adjuvant properties of iNKT cells to enhance priming of antigen-specific CD8+ T cells, we encapsulated both αGC and antigen in a Clec9a-targeted nanoemulsion (TNE) to deliver these molecules to cross-presenting CD8+ dendritic cells (DC). We demonstrate that, even in the absence of exogenous glycolipid, iNKT cells supported the maturation of CD8α+ DCs to drive efficient cross-priming of antigen-specific CD8+ T cells upon delivery of Clec9a/OVA-TNE. The addition of αGC to the TNE (Clec9a/OVA/αGC) further enhanced activation of iNKT cells, NK cells, CD8α+ DCs, and polyfunctional CD8+ T cells. When tested therapeutically against HPVE7-expressing TC-1 tumors, long-term tumor suppression was achieved with a single administration of Clec9a/E7 peptide/αGC TNE. Antitumor activity was correlated with the recruitment of mature DCs, NK cells, and tumor-specific effector CD8+ T cells to the tumor-draining lymph node and tumor tissue. Thus, Clec9a-TNE codelivery of CD8+ T-cell epitopes with αGC induces alternative helper signals from activated iNKT cells, elicits innate (iNKT, NK) immunity, and enhances antitumor CD8+ T-cell responses for control of solid tumors.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Imunidade/efeitos dos fármacos , Lectinas Tipo C/antagonistas & inibidores , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Receptores Mitogênicos/antagonistas & inibidores , Animais , Antineoplásicos Imunológicos/administração & dosagem , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citotoxicidade Imunológica , Emulsões , Galactosilceramidas/metabolismo , Imunidade Inata , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Terapia de Alvo Molecular , NanopartículasRESUMO
Autoimmune diseases resulting from MHC class II-restricted autoantigen-specific T cell immunity include the systemic inflammatory autoimmune conditions rheumatoid arthritis and vasculitis. While currently treated with broad-acting immunosuppressive drugs, a preferable strategy is to regulate antigen-specific effector T cells (Teffs) to restore tolerance by exploiting DC antigen presentation. We targeted draining lymph node (dLN) phagocytic DCs using liposomes encapsulating 1α,25-dihydroxyvitamin D3 (calcitriol) and antigenic peptide to elucidate mechanisms of tolerance used by DCs and responding T cells under resting and immunized conditions. PD-L1 expression was upregulated in dLNs of immunized relative to naive mice. Subcutaneous administration of liposomes encapsulating OVA323-339 and calcitriol targeted dLN PD-L1hi DCs of immunized mice and reduced their MHC class II expression. OVA323-339/calcitriol liposomes suppressed expansion, differentiation, and function of Teffs and induced Foxp3+ and IL-10+ peripheral Tregs in an antigen-specific manner, which was dependent on PD-L1. Peptide/calcitriol liposomes modulated CD40 expression by human DCs and promoted Treg induction in vitro. Liposomes encapsulating calcitriol and disease-associated peptides suppressed the severity of rheumatoid arthritis and Goodpasture's vasculitis models with suppression of antigen-specific memory T cell differentiation and function. Accordingly, peptide/calcitriol liposomes leverage DC PD-L1 for antigen-specific T cell regulation and induce antigen-specific tolerance in inflammatory autoimmune diseases.
Assuntos
Doença Antimembrana Basal Glomerular/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Calcitriol/administração & dosagem , Células Dendríticas/imunologia , Epitopos Imunodominantes/administração & dosagem , Transferência Adotiva , Animais , Doença Antimembrana Basal Glomerular/diagnóstico , Doença Antimembrana Basal Glomerular/imunologia , Apresentação de Antígeno/efeitos dos fármacos , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/imunologia , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Células CHO , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Cricetulus , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/transplante , Modelos Animais de Doenças , Feminino , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Humanos , Tolerância Imunológica/efeitos dos fármacos , Epitopos Imunodominantes/imunologia , Memória Imunológica/efeitos dos fármacos , Injeções Subcutâneas , Lipossomos , Linfonodos/citologia , Camundongos , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Índice de Gravidade de Doença , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Nanoparticle tumor accumulation relies on a key mechanism, the enhanced permeability and retention (EPR) effect, but it remains challenging to decipher the exact impact of the EPR effect. Animal models in combination with imaging modalities are useful, but it is impossible to delineate the roles of multiple biological barriers involved in nanoparticle tumor accumulation. Here we report a microfluidic tumor-vasculature-on-a-chip (TVOC) mimicking two key biological barriers, namely, tumor leaky vasculature and 3D tumor tissue with dense extracellular matrix (ECM), to study nanoparticle extravasation through leaky vasculature and the following accumulation in tumor tissues. Intact 3D tumor vasculature was developed with selective permeability of small molecules (20 kDa) but not large ones (70 kDa). The permeability was further tuned by cytokine stimulation, demonstrating the independent control of the leaky tumor vasculature. Combined with tumor spheroids in dense ECM, our TVOC model is capable of predicting nanoparticles' in vivo tumor accumulation, thus providing a powerful platform for nanoparticle evaluation.