RESUMO
A breached nasal epithelial barrier plays an important role in driving allergic rhinitis (AR). Corticosteroids remain the standard of care (SoC) but come with side effects, thus alternative safe and effective treatments able to avoid inflammation and restore barrier integrity are needed. The aim of the present study is to evaluate the barrier-forming capacity of a xyloglucan-based nasal spray (XG) and compare its efficacy to several SoC treatments (corticosteroid spray, oral mast-cell stabilizer and oral antihistamine) in reducing allergic responses in addition to its effect when concomitantly administered with an antihistamine. An ovalbumin (OVA)-induced mouse AR model was used. XG shows a significant efficacy in reducing histological damage in AR mice; improves nasal rubbing and histamine-induced hyper-responsiveness. Total and OVA-specific IgE as well as pro-inflammatory cytokines are significantly reduced compared to OVA challenged-mice, with im-proved efficacy when used as an add-on treatment. However, XG reduces mucous secreting cells (PAS-positive) and mucin mRNA expression similar to the corticosteroid-treated mice. XG-spray maintains tight junction protein expression (ZO-1) and conversely decreases HDAC1 significantly; the latter being highly expressed in AR patients. Moreover, the concomitant treatment showed in all of the endpoints a similar efficacy to the corticosteroids. This innovative approach may represent a novel therapeutic strategy for nasal respiratory diseases like AR, reducing undesirable side effects and improving the quality of life in patients.
Assuntos
Glucanos/farmacologia , Mucosa Nasal/imunologia , Sprays Nasais , Rinite Alérgica/prevenção & controle , Xilanos/farmacologia , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Rinite Alérgica/induzido quimicamente , Rinite Alérgica/imunologia , Proteína da Zônula de Oclusão-1/imunologiaRESUMO
BACKGROUND: Roasted peanut is widely loved as a kind of food with rich taste. However, peanut allergy is one of the major threats to human health, which affects about 5% of children and 1.4-2% of adults in the world. RESULTS: To evaluate the sensitization mechanism of peanut allergen Ara h 3, Caco-2 cells as the model, which has the similar structure and function to differentiated small intestinal epithelial cells. Compared with Ara h 3-raw (purified from raw peanut) group, more significant results such as the inhibited Caco-2 cell viability and proliferation, the increased secretion of reactive oxygen species (ROS) and the decreased transepithelial electrical resistance were obtained in Ara h 3-roasted (purified from roasted peanut) group. Accordingly, oxidative stress and NF-κB signaling pathway were more imbalanced, which lead to the increased of thymic stromal lymphopoietin (TSLP), interleukin 6 (IL-6), IL-8 and monocyte chemotactic protein 1 (MCP-1). Then, the gene expression of tight junction proteins ZO-1, occludin and JAM-1 were reduced, which proved that the integrity of the Caco-2 monolayer barrier is severely damaged. CONCLUSION: These finding identify the mechanisms of the allergenicity of roasted peanut allergy proteins are probably associated with intestinal uptake and cytokine dependent allergies. The aggravated allergic reaction might be caused by the increment of TSLP, IL-6, IL-8 and MCP-1 due to the activated NF-κB signaling pathway, and the enhanced transport of Ara h 3-roasted protein by Caco-2 monolayer. © 2021 Society of Chemical Industry.
Assuntos
Antígenos de Plantas/imunologia , Arachis/imunologia , Células Epiteliais/imunologia , Hipersensibilidade a Amendoim/imunologia , Proteínas de Plantas/imunologia , Alérgenos/química , Alérgenos/imunologia , Antígenos de Plantas/química , Arachis/química , Células CACO-2 , Moléculas de Adesão Celular/imunologia , Quimiocina CCL2/imunologia , Humanos , Interleucina-6/imunologia , Interleucina-8/imunologia , Intestino Delgado/imunologia , NF-kappa B/imunologia , Proteínas de Plantas/química , Receptores de Superfície Celular/imunologia , Sementes/química , Sementes/imunologia , Proteína da Zônula de Oclusão-1/imunologiaRESUMO
BACKGROUND: Protecting the intestinal mucosa from being destroyed helps reduce the inflammation caused by acute pancreatitis (AP). In this study, whether okra pectin (OP) could attenuate the inflammation of AP through protecting the intestinal barrier was investigated. RESULTS: OP was obtained from crude okra pectin (COP) through the purification by DEAE cellulose 52 column. Supplementation with OP or COP in advance reduced the severity of AP, as revealed by lower serum amylase and lipase levels, abated pancreatic edema, attenuated myeloperoxidase activity and pancreas histology. OP or COP inhibited the production of pancreatic proinflammatory cytokines, including tumor necrosis factor-α and interleukin-6. In addition, the upregulation of AP-related proteins including ZO-1, occludin, the antibacterial peptide-defensin-1 (DEFB1) and cathelicidin-related antimicrobial peptide (CRAMP), as well as the histological examination of colon injuries, demonstrated that OP or COP provision could effectively maintain intestinal barrier function. Ultimately, dietary OP or COP supplementation could inhibit AP-induced intestinal inflammation. For the above, the effect of OP was better than COP. CONCLUSION: Dietary OP supplementation could be considered as a preventive method that effectively interferes with intestinal damage and attenuates inflammatory responses trigged by AP. © 2020 Society of Chemical Industry.
Assuntos
Abelmoschus/química , Ceruletídeo/efeitos adversos , Mucosa Intestinal/efeitos dos fármacos , Pancreatite/tratamento farmacológico , Pectinas/administração & dosagem , Extratos Vegetais/administração & dosagem , Animais , Citocinas/genética , Citocinas/imunologia , Frutas/química , Humanos , Mucosa Intestinal/imunologia , Masculino , Camundongos , Ocludina/genética , Ocludina/imunologia , Pancreatite/induzido quimicamente , Pancreatite/genética , Pancreatite/imunologia , Pectinas/química , Extratos Vegetais/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/imunologiaRESUMO
Dendritic cells (DC) are the most important antigen-presenting cells, which guide T cell activation and function, and dysregulated DC function might be one of the crucial causes of inflammatory bowel disease (IBD). It has been well-known that microbiota and their metabolites play an essential role in regulating the biology and function of DC, thus contributing to the pathogenesis of IBD. However, the underlying mechanisms remain largely unknown. Amphiregulin (AREG), a molecule of the epidermal growth factor (EGF) family, is primarily described as an epithelial cell-derived cytokine and recognized as a critical regulator of cell proliferation and tissue repair. Here, we found that DC expression of AREG depended on butyrate (a microbiota-derived short chained fatty acid), which required the interaction between butyrate and G-protein-coupled receptor 43 (GPR43). Furthermore, we found that butyrate-GPR43 interaction failed to induce AREG expression in DC deficient in B lymphocyte induced maturation protein 1 (Blimp-1). Notably, DC-derived AREG was indispensable for the protection against experimental colitis in mice. Additionally, AREG expression was significantly decreased in DC from IBD patients. Our data provide novel evidences to interpret how AREG expression is regulated in DC, and shed new light on the mechanisms whereby microbiota regulate DC function.
Assuntos
Anfirregulina/genética , Butiratos/imunologia , Colite Ulcerativa/genética , Doença de Crohn/genética , Células Dendríticas/imunologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Receptores de Superfície Celular/genética , Anfirregulina/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/microbiologia , Linfócitos B/patologia , Butiratos/metabolismo , Butiratos/farmacologia , Estudos de Casos e Controles , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/imunologia , Colite Ulcerativa/microbiologia , Doença de Crohn/imunologia , Doença de Crohn/microbiologia , Doença de Crohn/patologia , Células Dendríticas/microbiologia , Células Dendríticas/patologia , Feminino , Microbioma Gastrointestinal/imunologia , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas a Pancreatite/deficiência , Proteínas Associadas a Pancreatite/genética , Proteínas Associadas a Pancreatite/imunologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/imunologia , Receptores de Superfície Celular/imunologia , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Transdução de Sinais , Dodecilsulfato de Sódio/administração & dosagem , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/imunologiaRESUMO
Recent studies have revealed that aloe emodin (AE), a natural compound from the root and rhizome of Rheum palmatum L., exhibits significant pharmacologic activities. However, the pharmacologic relevance of the compound, particularly for cardiovascular disease, remains largely unknown. Here, we hypothesized that AE could improve endothelial junction dysfunction through inhibiting the activation of NOD-like receptor family pyrin domain containing-3 (NLRP3) inflammasome regulated by NLRP3 ubiquitination, and ultimately prevent cardiovascular disease. In vivo, we used confocal microscopy to study the expression of tight junction proteins zonula occludens-1/2 (ZO-1/2) and the formation of NLRP3 inflammasome in coronary arteries of hypertension. And the experimental serum was used to detect the activation of NLRP3 inflammasome by ELISA assay. We found that AE could restore the expression of the endothelial connective proteins ZO-1/2 and decrease the release of high mobility group box1 (HMGB1), and also inhibited the formation and activation of NLRP3 inflammasome. Similarly, in vitro, our findings demonstrated that AE could restore the expression of the tight junction proteins ZO-1/2 and decrease monolayer cell permeability that related to endothelial function after stimulation by angiotensin II (Ang II) in microvascular endothelial cells (MECs). We also demonstrated that AE could inhibit Ang II-induced NLRP3 inflammasome formation and activation, which were regulated by NLRP3 ubiquitination in MECs, as shown by fluorescence confocal microscopy and Western blot. Together with these changes, we revealed a new protection mechanism of AE that inhibited NLRP3 inflammasome activation and decreased the release of HMGB1 by promoting NLRP3 ubiquitination. Our findings implicated that AE exhibited immense potential and specific therapeutic value in hypertension-related cardiovascular disease in the early stage and the development of innovative drugs.
Assuntos
Angiotensina II/efeitos adversos , Antraquinonas/farmacologia , Células Endoteliais/imunologia , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Junções Íntimas/imunologia , Ubiquitinação/efeitos dos fármacos , Angiotensina II/farmacologia , Animais , Células Endoteliais/patologia , Proteína HMGB1/imunologia , Masculino , Camundongos , Junções Íntimas/patologia , Ubiquitinação/imunologia , Proteína da Zônula de Oclusão-1/imunologia , Proteína da Zônula de Oclusão-2/imunologiaRESUMO
Previous research has shown that CXCR5-/- mice develop retinal degeneration (RD) with age, a characteristic related to age macular degeneration (AMD). RD in these mice is not well-understood, and in this study, we sought to characterize further the RD phenotype and to gain mechanistic insights into the function of CXCR5 in the retina. CXCR5-/- and WT control mice were used. Fundus images demonstrated a significant (p < 0.001) increase of hypo-pigmented spots in the retina of aged CXCR5-/- mice compared with WT control mice. PAS staining indicated localization of deposits in the sub-retinal pigment epithelia (RPE) layer. AMD-associated proteins Cryab, amyloid beta, and C3d were detected within the RPE/sub-RPE tissues by immunofluorescence (IF). In addition, western blot analysis of COX-2, Arg1, and VEGF-a revealed an increase in the signaling of these molecules within the RPE/choroid complex. Transmission electron microscopy (TEM) indicated a drusen-like structure of sub-RPE deposits with an accumulation of vacuolated cellular debris. Loss of photoreceptors was detected by peanut lectin staining and was corroborated by a reduction in MAP2 signaling. Loss of blood-retinal barrier integrity was demonstrated by a reduction of ZO-1 expression. Inflammatory cells were detected in the sub-RPE space, with an increase in IBA-1 positive microglia cells on the surface of the RPE. Mass spectrometry analysis of CXCR5-/- mouse RPE/choroid proteins extracts, separated by SDS-page and incubated with autologous serum, identified autoantibodies against AMD-associated proteins: Cryaa, Cryab, and Anxa2. In vitro evaluations in BV-2 cell culture indicated a significant increase in production of Arg-1 (p < 0.001) and COX-2 (p < 0.01) in the presence of anti-CXCR5 antibody when compared with Igg-treated control BV-2 cells stimulated with IL-4 and TNFα/IFNγ, respectively. Anti-CXCR5 antibody treatment without stimulating agents did not affect Arg-1 and COX-2 expression; this suggests that CXCR5 may have a regulatory role in microglia cells activation. These results indicate that with age, CXCR5-/- mice develop RD characterized by microglia dysfunction, increased production of CXCL13 in the RPE progressive photoreceptor, neuronal loss, and sub-RPE deposition of cellular debris, resulting in the production of immunogenic proteins and autoimmune-mediated RD.
Assuntos
Autoimunidade/imunologia , Modelos Animais de Doenças , Degeneração Macular/imunologia , Receptores CXCR5/imunologia , Degeneração Retiniana/imunologia , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/metabolismo , Animais , Autoimunidade/genética , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Ciclo-Oxigenase 2/imunologia , Ciclo-Oxigenase 2/metabolismo , Imunofluorescência , Degeneração Macular/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/imunologia , Proteínas dos Microfilamentos/metabolismo , Microglia/citologia , Microglia/imunologia , Microglia/metabolismo , Microscopia Eletrônica de Transmissão , Receptores CXCR5/deficiência , Receptores CXCR5/genética , Degeneração Retiniana/genética , Epitélio Pigmentado da Retina/imunologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/ultraestrutura , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína da Zônula de Oclusão-1/imunologia , Proteína da Zônula de Oclusão-1/metabolismo , Cadeia B de alfa-Cristalina/imunologia , Cadeia B de alfa-Cristalina/metabolismoRESUMO
It is well-known that essential oil thymol exhibits antibacterial activity. The protective effects of thymol on pig intestine during inflammation is yet to be investigated. In this study, an in vitro lipopolysaccharide (LPS)-induced inflammation model using IPEC-J2 cells was established. Cells were pretreated with thymol for 1 h and then exposed to LPS for various assays. Interleukin 8 (IL-8) secretion, the mRNA abundance of cytokines, reactive oxygen species (ROS), nutrient transporters, and tight junction proteins was measured. The results showed that LPS stimulation increased IL-8 secretion, ROS production, and tumor necrosis factor alpha (TNF-α) mRNA abundance ( P < 0.05), but the mRNA abundance of sodium-dependent glucose transporter 1 (SGLT1), excitatory amino acid transporter 1 (EAAC1), and H+/peptide cotransporter 1 (PepT1) were decreased ( P < 0.05). Thymol blocked ROS production ( P < 0.05) and tended to decrease the production of LPS-induced IL-8 secretion ( P = 0.0766). The mRNA abundance of IL-8 and TNF-α was reduced by thymol pretreatment ( P < 0.05), but thymol did not improve the gene expression of nutrient transporters ( P > 0.05). The transepithelial electrical resistance (TEER) was reduced and cell permeability increased by LPS treatment ( P < 0.05), but these effects were attenuated by thymol ( P < 0.05). Moreover, thymol increased zonula occludens-1 (ZO-1) and actin staining in the cells. However, the mRNA abundance of ZO-1 and occludin-3 was not affected by either LPS or thymol treatments. These results indicated that thymol enhances barrier function and reduce ROS production and pro-inflammatory cytokine gene expression in the epithelial cells during inflammation. The regulation of barrier function by thymol and LPS may be at post-transcriptional or post-translational levels.
Assuntos
Células Epiteliais/imunologia , Inflamação/tratamento farmacológico , Intestinos/imunologia , Doenças dos Suínos/tratamento farmacológico , Timol/administração & dosagem , Animais , Células Epiteliais/efeitos dos fármacos , Inflamação/etiologia , Inflamação/genética , Inflamação/imunologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Intestinos/efeitos dos fármacos , Lipopolissacarídeos/efeitos adversos , Ocludina/genética , Ocludina/imunologia , Suínos , Doenças dos Suínos/genética , Doenças dos Suínos/imunologia , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/imunologiaRESUMO
Gut microbiota and bacterial translocation have been implicated as significant contributors to mucosal immune responses and tolerance; alteration of microbial molecules, termed pathogen-associated molecular patterns (PAMP) and bacterial translocation are associated with immune pathology. However, the mechanisms by which dysregulated gut microbiota promotes autoimmunity is unclear. We have taken advantage of a well-characterized murine model of primary biliary cholangitis, dnTGFßRII mice, and an additional unique construct, toll-like receptor 2 (TLR2)-deficient dnTGFßRII mice coined dnTGFßRIITLR2-/- mice to investigate the influences of gut microbiota on autoimmune cholangitis. Firstly, we report that dnTGFßRII mice manifest altered composition of gut microbiota and that alteration of this gut microbiota by administration of antibiotics significantly alleviates T-cell-mediated infiltration and bile duct damage. Second, toll-like receptor 2 (TLR2)-deficient dnTGFßRII mice demonstrate significant exacerbation of autoimmune cholangitis when their epithelial barrier integrity was disrupted. Further, TLR2-deficiency mediates downregulated expression of tight junction-associated protein ZO-1 leading to increased gut permeability and bacterial translocation from gut to liver; use of antibiotics reduces microbiota translocation to liver and also decreases biliary pathology. In conclusion, our data demonstrates the important role of gut microbiota and bacterial translocation in the pathogenesis of murine autoimmune cholangitis.
Assuntos
Doenças Autoimunes/microbiologia , Translocação Bacteriana/imunologia , Ductos Biliares/imunologia , Cirrose Hepática Biliar/imunologia , Receptor do Fator de Crescimento Transformador beta Tipo II/imunologia , Receptor 2 Toll-Like/imunologia , Ampicilina/farmacologia , Animais , Antibacterianos/farmacologia , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/patologia , Translocação Bacteriana/efeitos dos fármacos , Ductos Biliares/efeitos dos fármacos , Ductos Biliares/microbiologia , Ductos Biliares/patologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/microbiologia , Colo/patologia , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/imunologia , Regulação da Expressão Gênica , Imunidade nas Mucosas/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/microbiologia , Fígado/patologia , Cirrose Hepática Biliar/tratamento farmacológico , Cirrose Hepática Biliar/microbiologia , Cirrose Hepática Biliar/patologia , Metronidazol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neomicina/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo II/deficiência , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Transdução de Sinais , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/imunologiaRESUMO
Although UHT heat treatment is being optimized to improve the stability and functional properties of dairy products, its metabolic effects remain scarcely known. As such, we studied the effect of the type of UHT process on lipid metabolism, intestinal barrier, and inflammation in mice. Nine-week-old male C57Bl/6J mice were fed a diet composed of nonlipidic powder mixed with different UHT dairy creams (final: 13% milkfat) for 1 or 4 wk. All creams contained 0.02% of thickener (carrageenan) and were treated via either (1) classical indirect heating process (Th), (2) indirect process at higher temperature (Th+), or (3) direct process by steam injection (ThD). Plasma, epididymal adipose tissue (EAT), and intestine were analyzed. Multivariate principal component analyses were used to identify differential effects of processes. Th+ differed by a globally higher liver damage score compared with that of the other creams. After 4 wk, the duodenal expression of lipid absorption genes fatty acid binding protein 4 (Fatp4) and microsomal triglycerides transfer protein (Mttp) was lower in the Th+ versus Th group. Expression in the colon of tight junction protein zonula occludens 1 (Zo1) and of some endoplasmic reticulum stress markers was lower in both Th+ and ThD versus the Th group. In EAT, ThD had lower gene expression of several inflammatory markers after 4 wk. Some differential effects may be related to heat-induced physicochemical changes of creams. The type of cream UHT process differentially affected metabolic parameters in mice after a 4-wk fat-rich diet, partly due to cream structure. Altogether, direct steam injection process induced the lowest early markers of high-fat-induced metabolic inflammation in EAT.
Assuntos
Tecido Adiposo/imunologia , Laticínios/efeitos adversos , Gorduras/efeitos adversos , Temperatura Alta/efeitos adversos , Leite/química , Tecido Adiposo/metabolismo , Animais , Bovinos , Laticínios/análise , Epididimo/imunologia , Gorduras/química , Gorduras/metabolismo , Intestinos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína da Zônula de Oclusão-1/imunologiaRESUMO
Astragalus polysaccharides (APS) were treated with different gamma irradiation doses (10, 25, 50, 100 and 150â¯kGy) to investigate the effects of gamma radiation processing on structure, physicochemical and immunomodulatory properties. The results revealed both the number-average and weight-average molecular weight of APS significantly decreased with increasing irradiation dose, whereas the solubility was increased after irradiation. A decrease in the apparent viscosity, as well as an increase in amount of small fragments of APS granules was also observed with increasing irradiation dose. FT-IR spectra indicated that gamma irradiation introduced no significant changes into the functional group status of APS. High irradiation dose (>50â¯kGy) caused a significant increase of yellowness and a slightly decrease of thermal stability of APS. Further, the immunomodulatory activity of irradiated APS was evaluated on Caco2 cells. APS irradiated at dose of 25â¯kGy exhibited the highest ability to induce nitric oxide production and up-regulate the mRNA expression of inflammatory cytokines, occludin, zonula occludens protein-1 (ZO-1) and toll-like receptor 4 (TLR4), as well as the protein expression of ZO-1 and TLR4. These findings indicate that gamma irradiation modification with a proper dose enhance immunomodulatory activity of APS by improving physicochemical properties without changing the functional groups.
Assuntos
Astrágalo/química , Raios gama , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores Imunológicos/efeitos da radiação , Polissacarídeos/efeitos da radiação , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Cor , Citocinas/agonistas , Citocinas/genética , Citocinas/imunologia , Relação Dose-Resposta Imunológica , Relação Dose-Resposta à Radiação , Humanos , Fatores Imunológicos/isolamento & purificação , Fatores Imunológicos/farmacologia , Peso Molecular , Óxido Nítrico/agonistas , Óxido Nítrico/biossíntese , Óxido Nítrico/imunologia , Ocludina/agonistas , Ocludina/genética , Ocludina/imunologia , Polissacarídeos/isolamento & purificação , Polissacarídeos/farmacologia , Solubilidade/efeitos da radiação , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Viscosidade/efeitos da radiação , Proteína da Zônula de Oclusão-1/agonistas , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/imunologiaRESUMO
Atopic dermatitis (AD) is a common inflammatory skin disease characterized by a complex, heterogeneous pathogenesis including skin barrier dysfunction, immunology, and pruritus. Although epidermal growth factor (EGF) is essential for epithelial homeostasis and wound healing, the effect of EGF on AD remains to be explored. To develop a new therapy for AD, the anti-AD potential of EGF was investigated by inducing AD-like skin lesions in NC/Nga mice using 2,4-dinitrochlorobenzene (DNCB). EGF was administrated to NC/Nga mice to evaluate its therapeutic effect on DNCB-induced AD. EGF treatment improved dermatitis score, ear thickness, epidermal hyperplasia, serum total immunoglobulin E level, and transepidermal water loss in NC/Nga mice with DNCB-induced AD. In addition, levels of skin barrier-related proteins such as filaggrin, involucrin, loricrin, occludin, and zonula occludens-1 (ZO-1) were increased by EGF treatment. These beneficial effects of EGF on AD may be mediated by EGF regulation of Th1/Th2-mediated cytokines, mast cell hyperplasia, and protease activated receptor-2 (PAR-2) and thymic stromal lymphopoietin (TSLP), which are triggers of AD. Taken together, our findings suggest that EGF may potentially protect against AD lesional skin via regulation of skin barrier function and immune response.
Assuntos
Dermatite Atópica/prevenção & controle , Fator de Crescimento Epidérmico/farmacologia , Mastócitos/efeitos dos fármacos , Pele/efeitos dos fármacos , Administração Tópica , Animais , Citocinas/imunologia , Citocinas/metabolismo , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/imunologia , Dinitroclorobenzeno , Fator de Crescimento Epidérmico/administração & dosagem , Proteínas Filagrinas , Proteínas de Filamentos Intermediários/imunologia , Proteínas de Filamentos Intermediários/metabolismo , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Substâncias Protetoras/administração & dosagem , Substâncias Protetoras/farmacologia , Receptor PAR-2/imunologia , Receptor PAR-2/metabolismo , Pele/imunologia , Pele/metabolismo , Proteína da Zônula de Oclusão-1/imunologia , Proteína da Zônula de Oclusão-1/metabolismo , Linfopoietina do Estroma do TimoRESUMO
Calycosin, a bioactive component derived from Astragali Radix (AR; Huang Qi), has been shown to have an effect of anti-allergic dermatitis with unknown mechanism. This study aims to investigate the mechanism of calycosin related to tight junctions (TJs) and HIF-1α both in FITC-induced mice allergic contact dermatitis and in IL-1ß stimulated HaCaT keratinocytes. Th2 cytokines (IL-4, IL-5 and IL-13) were detected by ELISA. The epithelial TJ proteins (occludin, CLDN1 and ZO-1), initiative key cytokines (TSLP and IL-33) and HIF-1α were assessed by Western blot, real-time PCR, immunohistochemistry or immunofluorescence. Herein, we have demonstrated that allergic inflammation and the Th2 cytokines in ACD mice were reduced significantly by calycosin treatment. Meanwhile, calycosin obviously decreased the expression of HIF-1α and repaired TJs both in vivo and in vitro. In HaCaT keratinocytes, we noted that IL-1ß induced the deterioration of TJs, as well as the increased levels of TSLP and IL-33, which could be reversed by silencing HIF-1α. In addition, administration of 2-methoxyestradiolin (2-ME), a HIF-1α inhibitor,significantly repaired the TJs and alleviated the allergic inflammation in vivo. Furthermore, TJs were destroyed by DMOG or by overexpressing HIF-1α in HaCaT keratinocytes, and simultaneously, calycosin down-regulated the expression of HIF-1α and repaired the TJs in this process. These results revealed that calycosin may act as a potential anti-allergy and barrier-repair agent via regulating HIF-1α in AD and suggested that HIF-1α and TJs might be possible therapy targets for allergic dermatitis.
Assuntos
Dermatite Alérgica de Contato/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Isoflavonas/farmacologia , Junções Íntimas/efeitos dos fármacos , 2-Metoxiestradiol/farmacologia , Animais , Astragalus propinquus , Claudina-1/genética , Claudina-1/imunologia , Citocinas/genética , Citocinas/imunologia , Dermatite Alérgica de Contato/etiologia , Dermatite Alérgica de Contato/genética , Dermatite Alérgica de Contato/imunologia , Medicamentos de Ervas Chinesas/química , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/patologia , Fluoresceína-5-Isotiocianato/administração & dosagem , Regulação da Expressão Gênica , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Interleucina-1beta/farmacologia , Interleucinas/genética , Interleucinas/imunologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ocludina/genética , Ocludina/imunologia , Transdução de Sinais , Pele/efeitos dos fármacos , Pele/imunologia , Pele/patologia , Junções Íntimas/química , Junções Íntimas/imunologia , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/imunologia , Linfopoietina do Estroma do TimoRESUMO
The present work is undertaken to characterize a Granny Smith apple procyanidin extract (AE) and investigate the beneficial effect of the AE in the intestine in vitro. Each AE was characterized via LC-ESI-MS. Caco-2 cells were used to study the preventive actions of the AE against the downregulation of tight junction protein expression, oxidative stress and inflammation induced by lipopolysaccharides (LPS). Phenolic compounds present in the AE, including chlorogenic acid, catechin, epicatechin, proanthocyanidin dimers, and proanthocyanidin trimers, were characterized. The expression of the tight junction protein, including occludin and zona occludens (ZO)-1, increased significantly in LPS + AE treated Caco-2 cells, compared to LPS induced Caco-2 cells. Proanthocyanidin dimers had the most potent effect on increasing tight junction protein expression. The addition of LPS to Caco-2 cells induced oxidative stress and inflammation. However, incubation with proanthocyanidin dimers prevented LPS-mediated oxidative stress, including the increase of SOD, HO-1, CAT, and GSH-Px mRNA expression, and counteracted LPS-mediated inflammation as evidenced by the down-regulation of inflammatory markers (NF-κß, IL-6, and TNF-α mRNA expression). Our findings provide evidence that AE could upregulate tight junction protein expression, probably acting via the reduction of oxidative stress and inflammation.
Assuntos
Biflavonoides/farmacologia , Catequina/farmacologia , Malus/química , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Proantocianidinas/farmacologia , Proteínas de Junções Íntimas/genética , Células CACO-2 , Humanos , Lipopolissacarídeos/efeitos adversos , NF-kappa B/genética , NF-kappa B/imunologia , Ocludina/genética , Ocludina/imunologia , Proteínas de Junções Íntimas/imunologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Regulação para Cima/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/imunologiaRESUMO
Protocols for photoreceptor outer segment (POS) isolation that can be used in phagocytosis assays of retinal pigment epithelium (RPE) cells have routinely used a large number of cow or pig eyes. However, when working with large animal models (e.g., dog, cats, transgenic pigs) of inherited retinal degenerative diseases, access to retinal tissues may be limited. An optimized protocol is presented in this paper to isolate sufficient POS from a single canine retina for use in RPE phagocytosis assays.
Assuntos
Fracionamento Celular/métodos , Fagocitose , Cultura Primária de Células/métodos , Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Animais , Células Cultivadas , Cães , Técnica Direta de Fluorescência para Anticorpo , Corantes Fluorescentes , Rodopsina/análise , Rodopsina/imunologia , Segmento Externo da Célula Bastonete , Coloração e Rotulagem/métodos , Proteína da Zônula de Oclusão-1/análise , Proteína da Zônula de Oclusão-1/imunologiaRESUMO
BACKGROUND: We identified a new variant of endemic pemphigus foliaceus in El Bagre, Colombia, South America, which we term El Bagre-EPF, and observed reactivity to arrector pili muscle (APM), thus we tested for autoimmunity to APM. METHODS: We took skin biopsies from 30 patients with El Bagre-EPF and 30 healthy controls (HCs) matched by age, sex and occupation, who were all from the endemic area, and tested these using direct immunofluorescence (DIF), confocal microscopy, immunohistochemistry and immunoblotting (IB). RESULTS: Of the 30 patients with El Bagre-EPF, 27 had autoantibodies to APM that colocalized with commercial antibodies to myocardium-enriched zonula occludens-1-associated protein (MYZAP), desmoplakin (DP)1 and DP2, plakophilin 4, and Armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF) (P < 0.001, Fisher exact test). The positive staining also colocalized with Junctional Adhesion Molecule 1 (JAM-A), a control antibody for gap cell junctions. No HC samples were positive. In 27 of the 30 patients, serum that was APM-positive also displayed IB colocalization of their autoantibody molecular weights with the Progen antibodies (P < 0.001, Fisher exact test). CONCLUSIONS: Patients affected by El Bagre-EPF have autoantibodies to APM, colocalizing with the antibodies MYZAP, ARVCF, p0071, DP1 and DP2, suggesting that these molecules are El Bagre-EPF antigens. Further, all of these antigens represent components of cell junctions, indicating that the immune response is directed, at least partially, against cell junctions. The immune response in patients affected by El Bagre-EPF is polyclonal, and it includes B and T lymphocytes, mast cells, IgG, IgA, IgM, IgD, IgE, fibrinogen, albumin, complement/C1q, C3c and C4.
Assuntos
Autoanticorpos/sangue , Autoimunidade , Doenças Endêmicas , Músculo Liso/imunologia , Pênfigo/imunologia , Proteínas do Domínio Armadillo/imunologia , Moléculas de Adesão Celular/imunologia , Colômbia , Desmoplaquinas/imunologia , Humanos , Immunoblotting , Imuno-Histoquímica , Pênfigo/patologia , Fosfoproteínas/imunologia , Placofilinas/imunologia , Receptores de Superfície Celular/imunologia , Proteína da Zônula de Oclusão-1/imunologiaRESUMO
Confirmed reports of Zika virus (ZIKV) in human seminal fluid for months after the clearance of viremia suggest the ability of ZIKV to establish persistent infection in the seminiferous tubules, an immune-privileged site in the testis protected by the blood-testis barrier, also called the Sertoli cell (SC) barrier (SCB). However, cellular targets of ZIKV in human testis and mechanisms by which the virus enters seminiferous tubules remain unclear. We demonstrate that primary human SCs were highly susceptible to ZIKV compared to the closely related dengue virus and induced the expression of alpha interferon (IFN-α), key cytokines, and cell adhesion molecules (vascular cell adhesion molecule 1 [VCAM-1] and intracellular adhesion molecule 1 [ICAM-1]). Furthermore, using an in vitro SCB model, we show that ZIKV was released on the adluminal side of the SCB model with a higher efficiency than in the blood-brain barrier model. ZIKV-infected SCs exhibited enhanced adhesion of leukocytes that correlated with decreases in SCB integrity. ZIKV infection did not affect the expression of tight and adherens junction proteins such as ZO-1, claudin, and JAM-A; however, exposure of SCs to inflammatory mediators derived from ZIKV-infected macrophages led to the degradation of the ZO-1 protein, which correlated with increased SCB permeability. Taken together, our data suggest that infection of SCs may be one of the crucial steps by which ZIKV gains access to the site of spermatozoon development and identify SCs as a therapeutic target to clear testicular infections. The SCB model opens up opportunities to assess interactions of SCs with other testicular cells and to test the ability of anti-ZIKV drugs to cross the barrier.IMPORTANCE Recent outbreaks of ZIKV, a neglected mosquito-borne flavivirus, have identified sexual transmission as a new route of disease spread, which has not been reported for other flaviviruses. To be able to sexually transmit for months after the clearance of viremia, ZIKV must establish infection in the seminiferous tubules, the site of spermatozoon development. However, little is known about the cell types that support ZIKV infection in the human testis. Currently, there are no models to study mechanisms of virus persistence in the seminiferous tubules. We provide evidence that ZIKV infection of human Sertoli cells, which are an important component of the seminiferous tubules, is robust and induces a strong antiviral response. The use of an in vitro Sertoli cell barrier to describe how ZIKV or inflammatory mediators derived from ZIKV-infected macrophages compromise barrier integrity will enable studies to explore the interactions of other testicular cells with Sertoli cells and to test novel antivirals for clearing testicular ZIKV infection.
Assuntos
Barreira Hematotesticular/imunologia , Células de Sertoli/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Barreira Hematotesticular/patologia , Barreira Hematotesticular/virologia , Moléculas de Adesão Celular/imunologia , Células Cultivadas , Claudinas/imunologia , Dengue/imunologia , Dengue/patologia , Vírus da Dengue/imunologia , Humanos , Interferon-alfa/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Receptores de Superfície Celular/imunologia , Células de Sertoli/patologia , Células de Sertoli/virologia , Molécula 1 de Adesão de Célula Vascular/imunologia , Infecção por Zika virus/patologia , Proteína da Zônula de Oclusão-1/imunologiaRESUMO
Ocular toxoplasmosis is mediated by monocytes infected with Toxoplasma gondii that are disseminated to target organs. Although infected monocytes can easily access to outer blood-retinal barrier due to leaky choroidal vasculatures, not much is known about the effect of T. gondii-infected monocytes on outer blood-retinal barrier. We prepared human monocytes, THP-1, infected with T. gondii and human retinal pigment epithelial cells, ARPE-19, grown on transwells as an in vitro model of outer blood-retinal barrier. Exposure to infected monocytes resulted in disruption of tight junction protein, ZO-1, and decrease in transepithelial electrical resistance of retinal pigment epithelium. Supernatants alone separated from infected monocytes also decreased transepithelial electrical resistance and disrupted tight junction protein. Further investigation revealed that the supernatants could activate focal adhesion kinase (FAK) signaling in retinal pigment epithelium and the disruption was attenuated by FAK inhibitor. The disrupted barrier was partly restored by blocking CXCL8, a FAK activating factor secreted by infected monocytes. In this study, we demonstrated that monocytes infected with T. gondii can disrupt outer blood-retinal barrier, which is mediated by paracrinely activated FAK signaling. FAK signaling can be a target of therapeutic approach to prevent negative influence of infected monocytes on outer blood-retinal barrier.
Assuntos
Barreira Hematorretiniana , Quinase 1 de Adesão Focal/imunologia , Monócitos , Comunicação Parácrina/imunologia , Transdução de Sinais/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Barreira Hematorretiniana/imunologia , Barreira Hematorretiniana/parasitologia , Linhagem Celular , Humanos , Interleucina-8/imunologia , Monócitos/imunologia , Monócitos/parasitologia , Epitélio Pigmentado da Retina/imunologia , Epitélio Pigmentado da Retina/parasitologia , Proteína da Zônula de Oclusão-1/imunologiaRESUMO
Cerebral malaria claims the lives of over 600,000 African children every year. To better understand the pathogenesis of this devastating disease, we compared the cellular dynamics in the cortical microvasculature between two infection models, Plasmodium berghei ANKA (PbA) infected CBA/CaJ mice, which develop experimental cerebral malaria (ECM), and P. yoelii 17XL (PyXL) infected mice, which succumb to malarial hyperparasitemia without neurological impairment. Using a combination of intravital imaging and flow cytometry, we show that significantly more CD8(+) T cells, neutrophils, and macrophages are recruited to postcapillary venules during ECM compared to hyperparasitemia. ECM correlated with ICAM-1 upregulation on macrophages, while vascular endothelia upregulated ICAM-1 during ECM and hyperparasitemia. The arrest of large numbers of leukocytes in postcapillary and larger venules caused microrheological alterations that significantly restricted the venous blood flow. Treatment with FTY720, which inhibits vascular leakage, neurological signs, and death from ECM, prevented the recruitment of a subpopulation of CD45(hi) CD8(+) T cells, ICAM-1(+) macrophages, and neutrophils to postcapillary venules. FTY720 had no effect on the ECM-associated expression of the pattern recognition receptor CD14 in postcapillary venules suggesting that endothelial activation is insufficient to cause vascular pathology. Expression of the endothelial tight junction proteins claudin-5, occludin, and ZO-1 in the cerebral cortex and cerebellum of PbA-infected mice with ECM was unaltered compared to FTY720-treated PbA-infected mice or PyXL-infected mice with hyperparasitemia. Thus, blood brain barrier opening does not involve endothelial injury and is likely reversible, consistent with the rapid recovery of many patients with CM. We conclude that the ECM-associated recruitment of large numbers of activated leukocytes, in particular CD8(+) T cells and ICAM(+) macrophages, causes a severe restriction in the venous blood efflux from the brain, which exacerbates the vasogenic edema and increases the intracranial pressure. Thus, death from ECM could potentially occur as a consequence of intracranial hypertension.
Assuntos
Barreira Hematoencefálica/imunologia , Córtex Cerebral/imunologia , Malária Cerebral/imunologia , Plasmodium berghei/imunologia , Plasmodium yoelii/imunologia , Animais , Barreira Hematoencefálica/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Córtex Cerebral/parasitologia , Córtex Cerebral/patologia , Claudina-5/imunologia , Modelos Animais de Doenças , Cloridrato de Fingolimode , Humanos , Imunossupressores/farmacologia , Molécula 1 de Adesão Intercelular/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Malária Cerebral/tratamento farmacológico , Malária Cerebral/patologia , Camundongos , Neutrófilos/imunologia , Neutrófilos/patologia , Ocludina/imunologia , Propilenoglicóis/farmacologia , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Proteína da Zônula de Oclusão-1/imunologiaRESUMO
Disruption of the blood-brain barrier (BBB) is a hallmark of acute inflammatory lesions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis. This disruption may precede and facilitate the infiltration of encephalitogenic T cells. The signaling events that lead to this BBB disruption are incompletely understood but appear to involve dysregulation of tight-junction proteins such as claudins. Pharmacological interventions aiming at stabilizing the BBB in MS might have therapeutic potential. Here, we show that the orally available small molecule LY-317615, a synthetic bisindolylmaleimide and inhibitor of protein kinase Cß, which is clinically under investigation for the treatment of cancer, suppresses the transmigration of activated T cells through an inflamed endothelial cell barrier, where it leads to the induction of the tight-junction molecules zona occludens-1, claudin 3, and claudin 5 and other pathways critically involved in transendothelial leukocyte migration. Treatment of mice with ongoing experimental autoimmune encephalomyelitis with LY-317615 ameliorates inflammation, demyelination, axonal damage, and clinical symptoms. Although LY-317615 dose-dependently suppresses T-cell proliferation and cytokine production independent of antigen specificity, its therapeutic effect is abrogated in a mouse model requiring pertussis toxin. This abrogation indicates that the anti-inflammatory and clinical efficacy is mainly mediated by stabilization of the BBB, thus suppressing the transmigration of encephalitogenic T cells. Collectively, our data suggest the involvement of endothelial protein kinase Cß in stabilizing the BBB in autoimmune neuroinflammation and imply a therapeutic potential of BBB-targeting agents such as LY-317615 as therapeutic approaches for MS.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Encefalomielite Autoimune Experimental/prevenção & controle , Indóis/farmacologia , Proteína Quinase C beta/antagonistas & inibidores , Animais , Barreira Hematoencefálica/imunologia , Proliferação de Células/efeitos dos fármacos , Claudina-3/imunologia , Claudina-3/metabolismo , Claudina-5/imunologia , Claudina-5/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Doenças Desmielinizantes/imunologia , Doenças Desmielinizantes/prevenção & controle , Relação Dose-Resposta a Droga , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Imuno-Histoquímica , Indóis/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Microscopia Confocal , Proteína Quinase C beta/imunologia , Proteína Quinase C beta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/imunologia , Junções Íntimas/metabolismo , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Migração Transendotelial e Transepitelial/imunologia , Proteína da Zônula de Oclusão-1/imunologia , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
BACKGROUND: Intestinal barrier failure may lead to systemic inflammation and distant organ injury in patients following severe injury. Enteric glia cells (EGCs) have been shown to play an important role in maintaining gut barrier integrity through secretion of S-Nitrosoglutathione (GSNO). We have recently shown than Vagal Nerve Stimulation (VNS) increases EGC activation, which was associated with improved gut barrier integrity. Thus, we sought to further study the mechanism by which EGCs prevent intestinal barrier breakdown utilizing an in vitro model. We postulated that EGCs, through the secretion of GSNO, would improve intestinal barrier function through improved expression and localization of intestinal tight junction proteins. METHODS: Epithelial cells were co-cultured with EGCs or incubated with GSNO and exposed to Cytomix (TNF-α, INF-γ, IL-1ß) for 24 hours. Barrier function was assessed by permeability to 4kDa FITC-Dextran. Changes in tight junction proteins ZO-1, occludin, and phospho-MLC (P-MLC) were assessed by immunohistochemistry and immunoblot. KEY RESULTS: Co-culture of Cytomix-stimulated epithelial monolayers with EGCs prevented increases in permeability and improved expression and localization of occludin, ZO-1, and P-MLC. Further, treatment of epithelial monolayers with GSNO also prevented Cytomix-induced increases in permeability and exhibited a similar improvement in expression and localization of occludin, ZO-1, and P-MLC. CONCLUSIONS & INFERENCES: The addition of EGCs, or their secreted mediator GSNO, prevents epithelial barrier failure after injury and improved expression of tight junction proteins. Thus, therapies that increase EGC activation, such as VNS, may be a novel strategy to limit barrier failure in patients following severe injury.