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BACKGROUND: Approximately 70% of individuals allergic to birch pollen (Bet v 1.01 [Bet]) develop a secondary food allergy (e.g., hazelnut: Cor a 1.04 [Cor]), due to allergen cross-reactivity. However, standard immunotherapy for type I allergies often does not improve the food allergy sufficiently. We analyzed the allergen-specific and cross-reactive suppressive capacity of primary human regulatory T cells (Treg) induced by autologous IL-10-modulated dendritic cells (IL-10 DC) in vitro and in vivo. METHODS: CD4+ T cells of patients with birch pollen and associated hazelnut allergies were differentiated into Bet-specific or non-specific induced Treg (iTreg). After Bet- or Cor-specific restimulation the phenotype, proliferation, and suppressive capacity of iTreg subsets were analyzed. iTreg function was further investigated in humanized mouse models of airway and intestinal allergy, generated by engraftment of peripheral blood mononuclear cells from allergic donors into immunodeficient animals. RESULTS: After IL-10 DC priming and allergen-specific restimulation (Bet or Cor), non-specific control iTreg remained anergic, whereas Bet-specific iTreg proliferated extensively and exhibited a regulatory phenotype (enhanced expression of CTLA-4, PD-1, TNFR2, IL-10). Accordingly, activated Bet-specific iTreg displayed a high capacity to suppress Bet- and Cor-induced responder Th2 cell responses in vitro, indicating induction of both allergen-specific (birch) and cross-reactive tolerance (hazelnut). In vivo, the beneficial effect of Bet-specific iTreg was verified in humanized mouse models of allergic airway and intestinal inflammation, resulting in reduced allergen-induced clinical symptoms, and immune responses. CONCLUSION: Human IL-10 DC-induced iTreg facilitate allergen-specific and cross-reactive tolerance. Therefore, they are potential candidates for regulatory cell therapy in allergic and autoimmune diseases.
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The immune microenvironment plays an important role in the regulation of diseases. The characterization of the cellular composition of immune cell infiltrates in diseases and respective models is a major task in pathogenesis research and diagnostics. For the assessment of immune cell populations in tissues, fluorescence-activated cell sorting (FACS) or immunohistochemistry (IHC) are the two most common techniques presently applied, but they are cost intensive, laborious, and sometimes limited by the availability of suitable antibodies. Complementary rapid qPCR-based approaches exist for the human situation but are lacking for experimental mouse models. Accordingly, we developed a robust, rapid RT-qPCR-based approach to determine and quantify the abundance of prominent immune cell populations such as T cells, helper T (Th) cells, cytotoxic T cells, Th1 cells, B cells, and macrophages in mouse tissues. The results were independently validated by the gold standards IHC and FACS in corresponding tissues and showed high concordance.
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Macrófagos , Linfócitos T Auxiliares-Indutores , Humanos , Camundongos , AnimaisRESUMO
BACKGROUND: Tofacitinib, as inhibitor of Janus kinases (JAK), interrupts the transmission of numerous pro-inflammatory cytokines involved in the pathogenesis of inflammatory bowel diseases (IBD). Therefore, tofacitinib provides a potent option to treat ulcerative colitis (UC). Besides the anti-inflammatory potential, inhibition of widespread JAKs carries the risk of side effects. Macrophages are involved in the form of different subtypes in inflammation, wound healing, and even coagulation. This study aimed to explore the balanced use of tofacitinib in M1-like as well as M2-like macrophages of healthy donors and patients with IBD. METHODS: Monocytes of healthy donors and patients with chronic courses of IBD were obtained from blood samples. Macrophage colony-stimulating factor (M-CSF)-derived macrophages were treated with tofacitinib (1 µM, 5 µM, 10 µM) and polarized with either lipopolysaccharide and interferon (IFN)-γ towards M1-like-phenotype or with interleukin (IL)-4 towards M2-like-phenotype. ELISA and flow cytometry were used to evaluate cytokine levels and surface molecules. RESULTS: Tofacitinib had a modulating effect on M1-like macrophages whereby the effect on pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß, IL-12, IL-23) was less pronounced than the induction of anti-inflammatory IL-10. However, during M2-like polarization tofacitinib impaired the development of the corresponding phenotype becoming evident through decreased IL-10 levels and CD206 expression in treated macrophages. In both phenotypes, tofacitinib strongly downregulated the expression of immunostimulatory molecules (CD80, CD86, CD83, CD40). Furthermore, a dose-dependent correlation between treatment with tofacitinib and expressed tissue factor was noticed. CONCLUSIONS: Tofacitinib influences both polarizations (M1/M2) and the expression of tissue factor in a dose-dependent manner.
This study revealed a dose-dependent effect of tofacitinib on both M1-like and M2-like polarization, resulting in a decreased development of the corresponding phenotype. Furthermore, the applied dose of tofacitinib correlated with the expressed tissue factor in M1-like macrophages.
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Macrófagos , Piperidinas , Pirimidinas , Pirróis , Tromboplastina , Humanos , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Pirimidinas/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Pirróis/farmacologia , Tromboplastina/metabolismo , Citocinas/metabolismo , Adulto , Voluntários Saudáveis , Masculino , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/metabolismo , Feminino , Inibidores de Proteínas Quinases/farmacologia , Estudos de Casos e Controles , Células Cultivadas , Pessoa de Meia-IdadeRESUMO
Inflammatory bowel disease (IBD) is an autoimmune disease that leads to severe bowel symptoms and complications. Currently, there is no effective treatment, and the exact cause of IBD remains unclear. In the last decades, numerous studies have confirmed that flavonoids can have a positive impact on the treatment of IBD. Therefore, this study investigated the protective effect of a flavonoid combination of apigenin and epigallocatechin-3-gallate (EGCG) on IBD. In vitro studies in which Caco-2 cell monolayers were incubated with different concentrations of flavonoids found that the flavonoid-treated group exhibited increased transepithelial electrical resistance (TEER) at high concentrations, indicating a protective effect on the barrier function of the intestinal epithelium. In vivo studies showed that flavonoids significantly attenuated inflammatory levels in both chronic and acute hapten-mediated experimental colitis models in a time- and dose-dependent manner. In addition, the activity of myeloperoxidase (MPO) and the level of proinflammatory cytokines in the colon tissue were significantly reduced. Interestingly, the levels of anti-inflammatory cytokines were also dramatically increased. Finally, flavonoids were found to positively modulate the composition of the gut microbiota in the colon. Therefore, a combination of flavonoids could be a promising therapeutic agent for the future adjunctive treatment of IBD.
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Colite , Doenças Inflamatórias Intestinais , Humanos , Animais , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Apigenina/farmacologia , Apigenina/uso terapêutico , Células CACO-2 , Colite/induzido quimicamente , Colite/tratamento farmacológico , Doenças Inflamatórias Intestinais/tratamento farmacológico , Citocinas , Inflamação/tratamento farmacológico , Sulfato de Dextrana , Modelos Animais de DoençasRESUMO
IL-3 has been reported to be involved in various inflammatory disorders, but its role in inflammatory bowel disease (IBD) has not been addressed so far. Here, we determined IL-3 expression in samples from patients with IBD and studied the impact of Il3 or Il3r deficiency on T cell-dependent experimental colitis. We explored the mechanical, cytoskeletal and migratory properties of Il3r -/- and Il3r +/+ T cells using real-time deformability cytometry, atomic force microscopy, scanning electron microscopy, fluorescence recovery after photobleaching and in vitro and in vivo cell trafficking assays. We observed that, in patients with IBD, the levels of IL-3 in the inflamed mucosa were increased. In vivo, experimental chronic colitis on T cell transfer was exacerbated in the absence of Il-3 or Il-3r signalling. This was attributable to Il-3r signalling-induced changes in kinase phosphorylation and actin cytoskeleton structure, resulting in increased mechanical deformability and enhanced egress of Tregs from the inflamed colon mucosa. Similarly, IL-3 controlled mechanobiology in human Tregs and was associated with increased mucosal Treg abundance in patients with IBD. Collectively, our data reveal that IL-3 signaling exerts an important regulatory role at the interface of biophysical and migratory T cell features in intestinal inflammation and suggest that this might be an interesting target for future intervention.
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Colite , Doenças Inflamatórias Intestinais , Humanos , Linfócitos T Reguladores , Receptores de Interleucina-3/metabolismo , Interleucina-3/metabolismo , Inflamação/metabolismo , Colite/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismoRESUMO
Ulcerative colitis (UC) is a type of inflammatory bowel disease characterized by inflammation, ulcers and irritation of the mucosal lining. Oral drug delivery in UC encounters challenges because of multifaceted barriers. Dexamethasone-loaded galactosylated-PLGA/Eudragit S100/pullulan nanocargoes (Dexa-GP/ES/Pu NCs) have been developed with a dual stimuli-sensitive coating responsive to both colonic pH and microbiota, and an underneath galactosylated-PLGA core (GP). The galactose ligand of the GP preferentially binds to the macrophage galactose type-lectin-C (MGL-2) surface receptor. Therefore, both stimuli and ligand-mediated targeting facilitate nanocargoes to deliver Dexa specifically to the colon with enhanced macrophage uptake. Modified emulsion method coupled with a solvent evaporation coating technique was employed to prepare Dexa-GP/ES/Pu NCs. The nanocargoes were tested using in vitro, ex vivo techniques and dextran sodium sulfate (DSS) induced UC model. Prepared nanocargoes had desired physicochemical properties, drug release, cell uptake and cellular viability. Investigations using a DSS-colitis model showed high localization and mitigation of colitis with downregulation of NF-ĸB and COX-2, and restoration of clinical, histopathological, biochemical indices, antioxidant balance, microbial alterations, FTIR spectra, and epithelial junctions' integrity. Thus, Dexa-GP/ES/Pu NCs found to be biocompatible nanocargoes capable of delivering drugs to the inflamed colon with unique targeting properties for prolonged duration.
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Inflammatory bowel disease (IBD) is a group of disorders that cause chronic non-specific inflammation in the gastrointestinal (GI) tract, primarily affecting the ileum and colon. The incidence of IBD has risen sharply in recent years. Despite continuous research efforts over the past decades, the aetiology of IBD is still not fully understood and only a limited number of drugs are available for its treatment. Flavonoids, a ubiquitous class of natural chemicals found in plants, have been widely used in the prevention and treatment of IBD. However, their therapeutic efficacy is unsatisfactory due to poor solubility, instability, rapid metabolism, and rapid systemic elimination. With the development of nanomedicine, nanocarriers can efficiently encapsulate various flavonoids and subsequently form nanoparticles (NPs), which greatly improves the stability and bioavailability of flavonoids. Recently, progress has also been made in the methodology of biodegradable polymers that can be used to fabricate NPs. As a result, NPs can significantly enhance the preventive or therapeutic effects of flavonoids on IBD. In this review, we aim to evaluate the therapeutic effect of flavonoid NPs on IBD. Furthermore, we discuss possible challenges and future perspectives.
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Doenças Inflamatórias Intestinais , Nanopartículas , Humanos , Flavonoides/farmacologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Colo , Polímeros/farmacologiaRESUMO
Colon mucosal inflammation attracts a plethora of immune cells with overexpressed surface receptors. Colon drug targeting can be aided by exploiting overexpressed cell surface receptors which improve drug site retention for an extended period. We developed Tofacitinib citrate (Tofa) loaded transferrin anchored PLGA nanocarriers (Tofa-P/tfr NCs) via the quality by design (QbD) approach for specific binding to the transferrin receptor (TFR-1/CD71) overexpressed on macrophages and colon epithelial cells. Nanocarriers were produced using a modified emulsion-evaporation method with a protein adsorption technique. The QbD-risk assessment method was adopted to screen the variables impacting the quality of nanocarriers, which were then optimized using the 33 Box-Behnken design of experiment (DOE). The obtained nanocarriers have the desired physicochemical properties, drug entrapment, tfr adsorption, stability, mucoadhesion, and sustained drug release pattern at pH 7.4 (colon pH). In vitro cell-based studies confirmed the cellular biocompatibility and considerable uptake of nanocarriers by colon and macrophage cells; the uptake was diminished by anti-CD71/TFR1 antibodies. Tofa-P/tfr NCs demonstrated good colon targeting potential in the dextran sulfate sodium (DSS) induced ulcerative colitis (UC) model. In vivo therapeutic efficacy against UC was established through restored morphological and histopathological scores, vascular integrity, antioxidant levels, hematological parameters, pro-inflammatory cytokine/marker levels, and microbial indices. Tofa-P/tfr NCs shut down the elevated STAT-1 and TFR-1 levels, demonstrating the enhanced efficacy of the encapsulated drug. Thus, the QbD-driven approach successfully developed Tofa-P/tfr NCs with good potential to mitigate mucosal inflammation by targeting colon and macrophage surface receptors.
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Colite Ulcerativa , Colite , Humanos , Animais , Transferrina , Sistemas de Liberação de Medicamentos , Colite Ulcerativa/induzido quimicamente , Colite/induzido quimicamente , Colite/tratamento farmacológico , Macrófagos/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Modelos Animais de DoençasRESUMO
OBJECTIVE: Increased apoptotic shedding has been linked to intestinal barrier dysfunction and development of inflammatory bowel diseases (IBD). In contrast, physiological cell shedding allows the renewal of the epithelial monolayer without compromising the barrier function. Here, we investigated the role of live cell extrusion in epithelial barrier alterations in IBD. DESIGN: Taking advantage of conditional GGTase and RAC1 knockout mice in intestinal epithelial cells (Pggt1b iΔIEC and Rac1 iΔIEC mice), intravital microscopy, immunostaining, mechanobiology, organoid techniques and RNA sequencing, we analysed cell shedding alterations within the intestinal epithelium. Moreover, we examined human gut tissue and intestinal organoids from patients with IBD for cell shedding alterations and RAC1 function. RESULTS: Epithelial Pggt1b deletion led to cytoskeleton rearrangement and tight junction redistribution, causing cell overcrowding due to arresting of cell shedding that finally resulted in epithelial leakage and spontaneous mucosal inflammation in the small and to a lesser extent in the large intestine. Both in vivo and in vitro studies (knockout mice, organoids) identified RAC1 as a GGTase target critically involved in prenylation-dependent cytoskeleton dynamics, cell mechanics and epithelial cell shedding. Moreover, inflamed areas of gut tissue from patients with IBD exhibited funnel-like structures, signs of arrested cell shedding and impaired RAC1 function. RAC1 inhibition in human intestinal organoids caused actin alterations compatible with arresting of cell shedding. CONCLUSION: Impaired epithelial RAC1 function causes cell overcrowding and epithelial leakage thus inducing chronic intestinal inflammation. Epithelial RAC1 emerges as key regulator of cytoskeletal dynamics, cell mechanics and intestinal cell shedding. Modulation of RAC1 might be exploited for restoration of epithelial integrity in the gut of patients with IBD.
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Citoesqueleto , Doenças Inflamatórias Intestinais , Animais , Humanos , Camundongos , Células Epiteliais , Inflamação , Doenças Inflamatórias Intestinais/genética , Mucosa Intestinal/fisiologia , Camundongos Knockout , Proteínas rac1 de Ligação ao GTPRESUMO
The mechanism of RNA interference (RNAi) could represent a breakthrough in the therapy of all diseases that arise from a gene defect or require the inhibition of a specific gene expression. In particular, small interfering RNA (siRNA) offers an attractive opportunity to achieve a new milestone in the therapy of human diseases. The limitations of siRNA, such as poor stability, inefficient cell uptake, and undesired immune activation, as well as the inability to specifically reach the target tissue in the body, can be overcome by further developments in the field of nanoparticulate drug delivery. Therefore, types of surface modified siRNA nanoparticles are presented and illustrate how a more efficient and safer distribution of siRNA at the target site is possible by modifying the surface properties of nanoparticles with antibodies. However, the development of such efficient and safe delivery strategies is currently still a major challenge. In consideration of that, this review article aims to demonstrate the function and targeted delivery of siRNA nanoparticles, focusing on the surface modification via antibodies, various lipid- and polymer-components, and the therapeutic effects of these delivery systems.
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Nanopartículas , Polímeros , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Sistemas de Liberação de Medicamentos , Anticorpos , LipídeosRESUMO
Gut-related diseases like ulcerative colitis, Crohn's disease, or colorectal cancer affect millions of people worldwide. It is an ongoing process finding causes leading to the development and manifestation of those disorders. This is highly relevant since understanding molecular processes and signalling pathways offers new opportunities in finding novel ways to interfere with and apply new pharmaceuticals. Memory T cells (mT cells) and their pro-inflammatory properties have been proven to play an important role in gastrointestinal diseases and are therefore increasingly spotlighted. This review focuses on mT cells and their subsets in the context of disease pathogenesis and maintenance. It illustrates the network of regulatory proteins and metabolites connecting mT cells with other cell types and tissue compartments. Furthermore, the crosstalk with various microbes will be a subject of discussion. Characterizing mT cell interactions will help to further elucidate the sophisticated molecular and cellular networking system in the intestine and may present new ideas for future research approaches to control gut-related diseases.
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Colite Ulcerativa , Gastroenteropatias , Colite Ulcerativa/patologia , Humanos , Células T de Memória , Preparações FarmacêuticasRESUMO
BACKGROUND AND AIMS: Colorectal cancer [CRC] is one of the most frequent malignancies, but the molecular mechanisms driving cancer growth are incompletely understood. We characterised the roles of the cytokine IL-9 and Th9 cells in regulating CRC development. METHODS: CRC patient samples and samples from AOM/DSS treated mice were analysed for expression of IL-9, CD3, and PU.1 by FACS analysis and immunohistochemistry. IL-9 citrine reporter mice, IL-9 knockout mice, and PU.1 and GATA3 CD4-Cre conditional knockout mice were studied in the AOM/DSS model. DNA minicircles or hyper-IL-6 were used for overexpression of cytokines in vivo. Effects of IL-6 and IL-9 were determined in organoid and T cell cultures. Claudin2/3 expression was studied by western blotting and bacterial translocation by FISH. RESULTS: We uncovered a significant expansion of IL-9- and PU.1-expressing mucosal Th9 cells in CRC patients, with particularly high levels in patients with colitis-associated neoplasias. PU.1+âTh9 cells accumulated in experimental colorectal neoplasias. Deficiency of IL-9 or inactivation of PU.1 in T cells led to impaired tumour growth in vivo, suggesting a protumoral role of Th9 cells. In contrast, GATA3 inactivation did not affect Th9-mediated tumour growth. Mechanistically, IL-9 controls claudin2/3 expression and T cell-derived IL-6 production in colorectal tumours. IL-6 abrogated the anti-proliferative effects of IL-9 in epithelial organoids in vivo. IL-9-producing Th9 cells expand in CRC and control IL-6 production by T cells. CONCLUSIONS: IL-9 is a crucial regulator of tumour growth in colitis-associated neoplasias and emerges as potential target for therapy.
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Colite , Neoplasias Colorretais , Camundongos , Animais , Interleucina-9/metabolismo , Interleucina-6/metabolismo , Linfócitos T Auxiliares-Indutores/patologia , Colite/patologia , Células Epiteliais/metabolismo , Citocinas/metabolismo , Neoplasias Colorretais/patologia , Camundongos Knockout , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Clinical challenges in inflammatory bowel diseases require microscopic in vivo evaluation of inflammation. Here, label-free imaging holds great potential, and recently, our group demonstrated the advantage of using in vivo multiphoton endomicroscopy for longitudinal animal studies. This article extends our previous work by in-depth analysis of label-free tissue features in common colitis models quantified by the multiphoton colitis score (MCS). METHODS: Fresh mucosal tissues were evaluated from acute and chronic dextran sulfate sodium (DSS), TNBS, oxazolone, and transfer colitis. Label-free imaging was performed by using second harmonic generation and natural autofluorescence. Morphological changes in mucosal crypts, collagen fibers, and cellularity in the stroma were analyzed and graded. RESULTS: Our approach discriminated between healthy (mean MCSâ =â 2.5) and inflamed tissue (mean MCSâ >â 5) in all models, and the MCS was validated by hematoxylin and eosin scoring of the same samples (85.2% agreement). Moreover, specific characteristics of each phenotype were identified. While TNBS, oxazolone, and transfer colitis showed high cellularity in stroma, epithelial damage seemed specific for chronic, acute DSS and transfer colitis. Crypt deformations were mostly observed in acute DSS. CONCLUSIONS: Quantification of label-free imaging is promising for in vivo endoscopy. In the future, this could be valuable for monitoring of inflammatory pathways in murine models, which is highly relevant for the development of new inflammatory bowel disease therapeutics.
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Colite , Doenças Inflamatórias Intestinais , Camundongos , Animais , Sulfato de Dextrana , Oxazolona , Modelos Animais de Doenças , InflamaçãoRESUMO
INTRODUCTION: Inflammatory bowel disease (IBD) is the inflammatory condition of the gastrointestinal tract particularly affecting the colon and the ileum. IBD patients can have a very poor quality of life because of the limited therapeutic efficacy and accompanied adverse effects. AREAS COVERED: The potential ways to employ nanoparticles to deliver drugs to a certain site of inflammation are discussed. The focus was set on the microenvironment in the gut as well as the mucosa, epithelial layer and the microbiota. Moreover, experimental animal colitis models were nanoparticles were used as a potential treatment are presented. Lastly, challenges for the potential clinical use in humans are discussed. EXPERT OPINION: Although there still remain many open questions e.g. regarding the toxicity, the metabolism or the pharmacokinetics of nanoparticles further research on this topic could overcome these challenges. For example, instead of synthetically engineered particles, biodegradable components could be used. Since there have been a lot pf promising results in the recent years, we are sure that in the future nanoparticles will be developed in a way to ensure safe and targeted delivery of drugs to the site of inflammation.
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Colite , Doenças Inflamatórias Intestinais , Nanopartículas , Animais , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Qualidade de VidaRESUMO
Inflammatory bowel disease (IBD) is a comprehensive term for chronic or relapsing inflammatory diseases occurring in the intestinal tract, generally including Crohn's disease (CD) and ulcerative colitis (UC). Presently, the pathogenesis of IBD is unknown, yet multiple factors have been reported to be related with the development of IBD. Flavonoids are phytochemicals with biological activity, which are ubiquitously distributed in edible plants, such as fruits and vegetables. Recent studies have demonstrated impressively that flavonoids have anti-IBD effects through multiple mechanisms. These include anti-inflammatory and antioxidant actions; the preservation of the epithelial barrier integrity, the intestinal immunomodulatory property, and the shaping microbiota composition and function. In addition, a few studies have shown the impact of flavonoids on enterohormones release; nonetheless, there is hardly any work showing the link between flavonoids, enterohormones release and IBD. So far, the interaction between flavonoids, enterohormones and IBD is elucidated for the first time in this review. Furthermore, the inference can be drawn that flavonoids may protect against IBD through modulating enterohormones, such as glucagon-like peptide 1 (GLP-1), GLP-2, dipeptidyl peptidase-4 inhibitors (DPP-4 inhibitors), ghrelin and cholecystokinin (CCK). In conclusion, this manuscript explores a possible mechanism of flavonoids protecting against IBD.
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BACKGROUND: Platelets play an important role in the pathogenesis of inflammatory and proliferative vascular changes. The aim of this study was to investigate whether human platelets are able to induce transplant arteriosclerosis in a humanized C57/Bl6-Rag2-/-γc-/- mouse xenograft model. METHODS: Nonactivated and in vitro-activated human platelets were analyzed and phenotyped for surface markers by flow cytometry. Side branches of human mammary arteries were implanted into the infrarenal aorta of recipients, followed by daily application of human platelets and histological analyzed on day 30 after transplantation. RESULTS: Human platelets collected by apheresis had low levels of platelet activation markers. However, after in vitro activation, expression was markedly increased. Sixty minutes after injection in recipient mice, nonactivated human platelets become significantly activated. Increased adhesion of platelets to the vascular endothelium was detected by in vivo fluorescence microscopy. After intravenous injection of nonactivated or activated platelets, human xenografts showed pronounced intimal proliferation. Immunohistological analysis showed that the group treated with activated human platelets exhibited significantly increased intragraft protein expression of intracellular adhesion molecule-1 and platelet-derived growth factor receptor beta and smooth muscle cell migration into the neointima. CONCLUSIONS: These data demonstrate that an isolated daily application of both in vivo- and in vitro-activated human platelets results in the development of transplant arteriosclerosis in a humanized mouse transplantation model.
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Arteriosclerose , Plaquetas , Animais , Aorta Abdominal/patologia , Arteriosclerose/etiologia , Arteriosclerose/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , NeointimaRESUMO
Alternatively activated macrophages (AAMs) contribute to the resolution of inflammation and tissue repair. However, molecular pathways that govern their differentiation have remained incompletely understood. Here, we show that uncoupling protein-2-mediated mitochondrial reprogramming and the transcription factor GATA3 specifically controlled the differentiation of pro-resolving AAMs in response to the alarmin IL-33. In macrophages, IL-33 sequentially triggered early expression of pro-inflammatory genes and subsequent differentiation into AAMs. Global analysis of underlying signaling events revealed that IL-33 induced a rapid metabolic rewiring of macrophages that involved uncoupling of the respiratory chain and increased production of the metabolite itaconate, which subsequently triggered a GATA3-mediated AAM polarization. Conditional deletion of GATA3 in mononuclear phagocytes accordingly abrogated IL-33-induced differentiation of AAMs and tissue repair upon muscle injury. Our data thus identify an IL-4-independent and GATA3-dependent pathway in mononuclear phagocytes that results from mitochondrial rewiring and controls macrophage plasticity and the resolution of inflammation.
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Metabolismo Energético , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-33/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Biomarcadores , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Inflamação/etiologia , Ativação de Macrófagos/genética , Mitocôndrias/genética , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Fagócitos , Transdução de SinaisRESUMO
BACKGROUND & AIMS: The molecular checkpoints driving T cell activation and cytokine responses in ulcerative colitis (UC) are incompletely understood. Here, we studied the Tec kinase ITK in UC. METHODS: We analyzed patients with inflammatory bowel disease (n = 223) and evaluated ITK activity as well as the functional effects of cyclosporine-A (CsA). In addition, 3 independent murine colitis models were used to investigate the functional role of ITK. Finally, the activity of ITK was blocked via pharmacological inhibitors and genetically engineered mice. Readout parameters were mini-endoscopy, histopathology, mucosal T cell apoptosis, and cytokine production. RESULTS: We found an expansion of pITK-expressing mucosal CD4+ T cells in UC rather than Crohn's disease that correlated with disease severity. CsA suppressed activation of ITK in cultured CD4+ T cells and calcineurin-containing microclusters adjacent to the T cell receptor signaling complex. Functionally, the capacity of CsA to suppress activity of experimental colitis was critically dependent on ITK. Genetic inactivation of Itk via gene targeting or induction of allele-sensitive Itk mutants prevented experimental colitis in 3 colitis models, and treatment with pharmacological ITK blockers suppressed established colitis. In addition, ITK controlled apoptosis and activation of mucosal Th2 and Th17 lymphocytes via NFATc2 signaling pathways. CONCLUSIONS: ITK activation was detected in UC and could be down-regulated in cultured T cells by CsA administration. Selective targeting of ITK emerges as an attractive approach for treatment of chronic intestinal inflammation and potentially UC by driving resolution of mucosal inflammation.
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Anti-Inflamatórios/farmacologia , Colite Ulcerativa/prevenção & controle , Colo/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Linfócitos Intraepiteliais/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Colite Ulcerativa/enzimologia , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Colo/enzimologia , Colo/imunologia , Colo/patologia , Ciclosporina/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Linfócitos Intraepiteliais/enzimologia , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/patologia , Camundongos Knockout , Terapia de Alvo Molecular , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: CD56-expressing natural killer (NK) cells as well as invariant NK T (iNKT) cells have been shown to either promote or inhibit allergic immune responses. OBJECTIVE: The aim of the present study was to investigate the impact of these cells in a recently developed humanized mouse model of allergen-induced IgE-dependent gut and lung inflammation. METHODS: Nonobese diabetic-severe combined immunodeficiency γ-chain knockout mice were injected intraperitoneally with human PBMCs or CD56-depleted (CD56neg) PBMCs from highly sensitized donors with birch or grass pollen allergy together with the respective allergen or with NaCl as a control. Three weeks later, the mice were challenged with the allergen rectally and gut inflammation was monitored by video miniendoscopy and by histology. Furthermore, airway inflammation was measured after an additional intranasal allergen challenge. RESULTS: Allergen-specific human IgE in mouse sera, detectable only after coinjection of the respective allergen, was reduced in mice being injected with CD56neg PBMCs compared with in mice receiving nondepleted PBMCs. Consequently, allergen-induced IgE-dependent colitis, airway hyperreactivity, and mucus-producing goblet cells were significantly inhibited in these mice. Interestingly, reconstitution of CD56neg PBMCs with nondepleted CD56+ cells and with CD56+CD3+ iNKT cells restored gut as well as lung inflammation, whereas addition of CD3-depleted CD56+ cells did not. CONCLUSION: These results demonstrate that allergen-specific gut and lung inflammation in PBMC-engrafted humanized mice is promoted by CD56+CD3+ iNKT cells, which opens new possibilities of therapeutic intervention in allergic diseases.