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1.
Nat Immunol ; 19(12): 1319-1329, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30397348

RESUMEN

Many tumors evolve sophisticated strategies to evade the immune system, and these represent major obstacles for efficient antitumor immune responses. Here we explored a molecular mechanism of metabolic communication deployed by highly glycolytic tumors for immunoevasion. In contrast to colon adenocarcinomas, melanomas showed comparatively high glycolytic activity, which resulted in high acidification of the tumor microenvironment. This tumor acidosis induced Gprotein-coupled receptor-dependent expression of the transcriptional repressor ICER in tumor-associated macrophages that led to their functional polarization toward a non-inflammatory phenotype and promoted tumor growth. Collectively, our findings identify a molecular mechanism of metabolic communication between non-lymphoid tissue and the immune system that was exploited by high-glycolytic-rate tumors for evasion of the immune system.


Asunto(s)
Adenocarcinoma/inmunología , Macrófagos/inmunología , Melanoma/inmunología , Escape del Tumor/inmunología , Microambiente Tumoral/inmunología , Acidosis/inmunología , Adenocarcinoma/metabolismo , Animales , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Glucólisis/inmunología , Humanos , Melanoma/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos
2.
Immunity ; 54(11): 2531-2546.e5, 2021 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-34644537

RESUMEN

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.


Asunto(s)
Metabolismo Energético , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-33/metabolismo , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Biomarcadores , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Inflamación/etiología , Activación de Macrófagos/genética , Mitocondrias/genética , Mitocondrias/inmunología , Mitocondrias/metabolismo , Fagocitos , Transducción de Señal
3.
Nat Immunol ; 15(7): 676-86, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24908389

RESUMEN

The molecular checkpoints that drive inflammatory bowel diseases are incompletely understood. Here we found more T cells expressing the transcription factor PU.1 and interleukin 9 (IL-9) in patients with ulcerative colitis. In an animal model, citrine reporter mice had more IL-9-expressing mucosal T cells in experimental oxazolone-induced colitis. IL-9 deficiency suppressed acute and chronic colitis. Mice with PU.1 deficiency in T cells were protected from colitis, whereas treatment with antibody to IL-9 suppressed colitis. Functionally, IL-9 impaired intestinal barrier function and prevented mucosal wound healing in vivo. Thus, our findings suggest that the TH9 subset of helper T cells serves an important role in driving ulcerative colitis by regulating intestinal epithelial cells and that TH9 cells represent a likely target for the treatment of chronic intestinal inflammation.


Asunto(s)
Colitis/etiología , Mucosa Intestinal/inmunología , Proteínas Proto-Oncogénicas/fisiología , Receptores de Interleucina-9/fisiología , Transducción de Señal/fisiología , Subgrupos de Linfocitos T/fisiología , Linfocitos T Colaboradores-Inductores/inmunología , Transactivadores/fisiología , Animales , Claudina-2/genética , Colitis/inmunología , Colitis Ulcerosa/inmunología , Humanos , Interleucina-9/inmunología , Ratones , Ratones Endogámicos BALB C , Células Th2/inmunología , Cicatrización de Heridas
4.
Allergy ; 79(10): 2826-2839, 2024 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-39073174

RESUMEN

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.


Asunto(s)
Alérgenos , Betula , Corylus , Reacciones Cruzadas , Células Dendríticas , Tolerancia Inmunológica , Interleucina-10 , Polen , Linfocitos T Reguladores , Humanos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Interleucina-10/metabolismo , Reacciones Cruzadas/inmunología , Alérgenos/inmunología , Corylus/inmunología , Animales , Ratones , Betula/inmunología , Polen/inmunología , Modelos Animales de Enfermedad , Hipersensibilidad a la Nuez/inmunología , Rinitis Alérgica Estacional/inmunología , Rinitis Alérgica Estacional/terapia , Inmunomodulación
5.
Gut ; 72(11): 2081-2094, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37541770

RESUMEN

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.


Asunto(s)
Colitis , Enfermedades Inflamatorias del Intestino , Humanos , Linfocitos T Reguladores , Receptores de Interleucina-3/metabolismo , Interleucina-3/metabolismo , Inflamación/metabolismo , Colitis/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo
6.
Gut ; 72(2): 275-294, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-35241625

RESUMEN

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.


Asunto(s)
Citoesqueleto , Enfermedades Inflamatorias del Intestino , Animales , Humanos , Ratones , Células Epiteliales , Inflamación , Enfermedades Inflamatorias del Intestino/genética , Mucosa Intestinal/fisiología , Ratones Noqueados , Proteína de Unión al GTP rac1
7.
Int J Mol Sci ; 24(22)2023 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-38003220

RESUMEN

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.


Asunto(s)
Colitis , Enfermedades Inflamatorias del Intestino , Humanos , Animales , Flavonoides/farmacología , Flavonoides/uso terapéutico , Apigenina/farmacología , Apigenina/uso terapéutico , Células CACO-2 , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Citocinas , Inflamación/tratamiento farmacológico , Sulfato de Dextran , Modelos Animales de Enfermedad
8.
Int J Mol Sci ; 24(5)2023 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-36901885

RESUMEN

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.


Asunto(s)
Enfermedades Inflamatorias del Intestino , Nanopartículas , Humanos , Flavonoides/farmacología , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Colon , Polímeros/farmacología
9.
Gastroenterology ; 161(4): 1270-1287.e19, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34224738

RESUMEN

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.


Asunto(s)
Antiinflamatorios/farmacología , Colitis Ulcerosa/prevención & control , Colon/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Linfocitos Intraepiteliales/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Colitis Ulcerosa/enzimología , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Colon/enzimología , Colon/inmunología , Colon/patología , Ciclosporina/farmacología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Mucosa Intestinal/enzimología , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Linfocitos Intraepiteliales/enzimología , Linfocitos Intraepiteliales/inmunología , Linfocitos Intraepiteliales/patología , Ratones Noqueados , Terapia Molecular Dirigida , Fosforilación , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal
10.
J Allergy Clin Immunol ; 148(4): 1081-1087.e2, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34019913

RESUMEN

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.


Asunto(s)
Colitis/inmunología , Células T Asesinas Naturales/inmunología , Hipersensibilidad Respiratoria/inmunología , Rinitis Alérgica Estacional/inmunología , Alérgenos/inmunología , Animales , Betula/inmunología , Complejo CD3/inmunología , Antígeno CD56/inmunología , Colitis/patología , Colitis/fisiopatología , Colon/inmunología , Colon/patología , Femenino , Humanos , Inmunoglobulina E/sangre , Pulmón/inmunología , Pulmón/patología , Pulmón/fisiopatología , Masculino , Ratones Transgénicos , Poaceae/inmunología , Polen/inmunología , Hipersensibilidad Respiratoria/patología , Hipersensibilidad Respiratoria/fisiopatología , Rinitis Alérgica Estacional/patología , Rinitis Alérgica Estacional/fisiopatología
11.
J Allergy Clin Immunol ; 148(2): 506-522.e8, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-33617860

RESUMEN

BACKGROUND: Mas gene-related G protein-coupled receptors (MRGPRs) are a G protein-coupled receptor family responsive to various exogenous and endogenous agonists, playing a fundamental role in pain and itch sensation. The primate-specific family member MRGPRX2 and its murine orthologue MRGPRB2 are expressed by mast cells mediating IgE-independent signaling and pseudoallergic drug reactions. OBJECTIVES: Our aim was to increase knowledge about the function and regulation of MRGPRX2/MRGPRB2, which is of major importance in prevention of drug hypersensitivity reactions and drug-induced pruritus. METHODS: To identify novel MRGPR (ant)agonists, we screened a library of pharmacologically active compounds by utilizing a high-throughput calcium mobilization assay. The identified hit compounds were analyzed for their pseudoallergic and pruritogenic effects in mice and human. RESULTS: We found a class of commonly used drugs activating MRGPRX2 that, to a large extent, consists of antidepressants, antiallergic drugs, and antipsychotics. Three-dimensional pharmacophore modeling revealed structural similarities of the identified agonists, classifying them as cationic amphiphilic drugs. Mast cell activation was investigated by using the 3 representatively selected antidepressants clomipramine, paroxetine, and desipramine. Indeed, we were able to show a concentration-dependent activation and MRGPRX2-dependent degranulation of the human mast cell line LAD2 (Laboratory of Allergic Diseases-2). Furthermore, clomipramine, paroxetine, and desipramine were able to induce degranulation of human skin and murine peritoneal mast cells. These substances elicited dose-dependent scratching behavior following intradermal injection into C57BL/6 mice but less so in MRGPRB2-mutant mice, as well as wheal-and-flare reactions following intradermal injections in humans. CONCLUSION: Our results contribute to the characterization of structure-activity relationships and functionality of MRGPRX2 ligands and facilitate prediction of adverse reactions such as drug-induced pruritus to prevent severe drug hypersensitivity reactions.


Asunto(s)
Antidepresivos/efectos adversos , Conducta Animal/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Hipersensibilidad a las Drogas/inmunología , Mastocitos/inmunología , Proteínas del Tejido Nervioso/inmunología , Receptores Acoplados a Proteínas G/inmunología , Receptores de Neuropéptido/inmunología , Animales , Antidepresivos/farmacología , Línea Celular , Hipersensibilidad a las Drogas/patología , Humanos , Mastocitos/patología , Ratones , Proteínas del Tejido Nervioso/agonistas , Receptores Acoplados a Proteínas G/agonistas , Receptores de Neuropéptido/agonistas
12.
Int J Mol Sci ; 23(22)2022 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-36430411

RESUMEN

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.


Asunto(s)
Nanopartículas , Polímeros , Humanos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéutico , Sistemas de Liberación de Medicamentos , Anticuerpos , Lípidos
13.
J Immunol ; 203(2): 544-556, 2019 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-31152078

RESUMEN

The nucleotide-binding oligomerization domain (NOD)-like receptors belong to the family of pattern recognition receptors (PRRs). NOD-like receptors play a role in regulation of innate immune response by recognition of both pathogen-associated molecular patterns that are engulfed during phagocytic process and danger-associated molecular patterns that are mainly byproducts of cell stress mediated response. NOD-like family pyrin domain containing 6 (NLRP6) is one of the 14 pyrin domain-containing receptors. NLRP6 is highly expressed by epithelial and goblet cells to regulate epithelial renewal and mucus production in mice and humans, but its function in T cells is rather unknown. Increased caspase-1 activation and cell death were observed in mouse Nlrp6-deficient T cells following adoptive transfer into Rag2-deficient mice, indicating that Nlrp6 deficiency in CD4+ T cells led to decreased survival.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Supervivencia Celular/inmunología , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/inmunología , Traslado Adoptivo/métodos , Animales , Muerte Celular , Células Epiteliales/inmunología , Células Caliciformes/inmunología , Inmunidad Innata/inmunología , Ratones , Ratones Endogámicos C57BL
14.
Gastroenterology ; 156(3): 692-707.e7, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30315770

RESUMEN

BACKGROUND & AIMS: The CYLD lysine 63 deubiquitinase gene (CYLD) encodes tumor suppressor protein that is mutated in familial cylindromatosus, and variants have been associated with Crohn disease (CD). Splice forms of CYLD that lack exons 7 and 8 regulate transcription factors and functions of immune cells. We examined the expression of splice forms of CYLD in colon tissues from patients with CD and their effects in mice. METHODS: We performed immunohistochemical analyses of colon tissues from patients with untreated CD and patients without inflammatory bowel diseases (controls). We obtained mice that expressed splice forms of CYLD (sCYLD mice) without or with SMAD7 (sCYLD/SMAD7 mice) from transgenes and CYLD-knockout mice (with or without transgenic expression of SMAD7) and performed endoscopic analyses. Colitis was induced in Rag1-/- mice by transfer of CD4+ CD62L+ T cells from C57/Bl6 or transgenic mice. T cells were isolated from mice and analyzed by flow cytometry and quantitative real-time polymerase chain reaction and intestinal tissues were analyzed by histology and immunohistochemistry. CYLD forms were expressed in mouse embryonic fibroblasts, primary T cells, and HEK293T cells, which were analyzed by immunoblot, mobility shift, and immunoprecipitation assays. RESULTS: The colonic lamina propria from patients with CD was infiltrated by T cells and had higher levels of sCYLD (but not full-length CYLD) and SMAD7 than tissues from controls. Incubation of mouse embryonic fibroblasts and T cells with transforming growth factor ß increased their production of sCYLD and decreased full-length CYLD. Transgenic expression of sCYLD and SMAD7 in T cells prevented the differentiation of regulatory T cells and T-helper type 17 cells and increased the differentiation of T-helper type 1 cells. The same effects were observed in colon tissues from sCYLD/SMAD7 mice but not in those from CYLD-knockout SMAD7 mice. The sCYLD mice had significant increases in the numbers of T-helper type 1 cells and CD44high CD62Llow memory-effector CD4+ T cells in the spleen and mesenteric lymph nodes compared with wild-type mice; sCYLD/SMAD7 mice had even larger increases. The sCYLD/SMAD7 mice spontaneously developed severe colitis, with infiltration of the colon by dendritic cells, neutrophils, macrophages, and CD4+ T cells and increased levels of Ifng, Il6, Il12a, Il23a, and Tnf mRNAs. Co-transfer of regulatory T cells from wild-type, but not from sCYLD/SMAD7, mice prevented the induction of colitis in Rag1-/- mice by CD4+ T cells. We found increased levels of poly-ubiquitinated SMAD7 in sCYLD CD4+ T cells. CYLD formed a nuclear complex with SMAD3, whereas sCYLD recruited SMAD7 to the nucleus, which inhibited the expression of genes regulated by SMAD3 and SMAD4. We found that sCYLD mediated lysine 63-linked ubiquitination of SMAD7. The sCYLD-SMAD7 complex inhibited transforming growth factor ß signaling in CD4+ T cells. CONCLUSIONS: Levels of the spliced form of CYLD are increased in colon tissues from patients with CD. sCYLD mediates ubiquitination and nuclear translocation of SMAD7 and thereby decreases transforming growth factor ß signaling in T cells. This prevents immune regulatory mechanisms and leads to colitis in mice.


Asunto(s)
Enfermedad de Crohn/genética , Enfermedad de Crohn/patología , Cisteína Endopeptidasas/genética , Proteína smad7/genética , Ubiquitinación/genética , Animales , Biopsia con Aguja , Enzima Desubiquitinante CYLD , Modelos Animales de Enfermedad , Citometría de Flujo , Humanos , Inmunohistoquímica , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Distribución Aleatoria , Valores de Referencia , Transducción de Señal , Factor de Crecimiento Transformador beta1/genética
15.
J Allergy Clin Immunol ; 143(1): 201-212.e4, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-29574077

RESUMEN

BACKGROUND: Amylase-trypsin inhibitors (ATIs) in wheat and related cereals are potent activators of myeloid innate immune cells via engagement of TLR4. Furthermore, ATIs have been shown to serve as adjuvants in experimental intestinal inflammatory diseases. OBJECTIVE: The aim of this study was to analyze whether ATIs are also modifiers of allergic inflammation. METHODS: Therefore, CD4+ T cells from donors sensitized to grass or birch pollen were stimulated with autologous allergen-pulsed dendritic cells in the presence or absence of ATIs or the control storage protein zein from corn. To analyze allergen-induced gut and lung inflammation, immunodeficient mice were engrafted with PBMCs from these allergic donors plus the respective allergen, and fed with selected diets. Three weeks later, inflammation was induced by rectal or intranasal allergen challenge and monitored by mini endoscopy or airway hyperreactivity, respectively. RESULTS: Allergen-specific T-cell proliferation and cytokine production was significantly exacerbated by ATIs and not by zein. In vivo, allergen-specific human IgE level was strongly elevated in sera of mice receiving an ATI-containing diet compared with mice that were fed gluten-free and thus ATI-free diet. Importantly, allergen-induced IgE-dependent colitis and airway hyperreactivity were also enhanced in ATI-fed mice. Gut inflammation was further increased in mice receiving an additional ATI injection and even detectable in the absence of the aeroallergen, whereas zein had no such effect. Injection of anti-human TLR4 mAbs or the anti-human IgE mAb omalizumab completely abolished ATI-induced allergic inflammation. CONCLUSIONS: These results underline that wheat ATIs are important nutritional activators and adjuvants of allergy, which might be exploited for nutritional therapeutic strategies.


Asunto(s)
Asma/inmunología , Linfocitos T CD4-Positivos/inmunología , Inmunidad Innata/efectos de los fármacos , Proteínas de Plantas/farmacología , Triticum/química , Inhibidores de Tripsina/farmacología , Amilasas/antagonistas & inhibidores , Animales , Asma/dietoterapia , Asma/patología , Linfocitos T CD4-Positivos/patología , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Proteínas de Plantas/química , Células THP-1 , Inhibidores de Tripsina/química
17.
Proc Natl Acad Sci U S A ; 113(43): E6659-E6668, 2016 10 25.
Artículo en Inglés | MEDLINE | ID: mdl-27791035

RESUMEN

Aberrant immune activation mediated by T effector cell populations is pivotal in the onset of autoimmunity in type 1 diabetes (T1D). T follicular helper (TFH) cells are essential in the induction of high-affinity antibodies, and their precursor memory compartment circulates in the blood. The role of TFH precursors in the onset of islet autoimmunity and signaling pathways regulating their differentiation is incompletely understood. Here, we provide direct evidence that during onset of islet autoimmunity, the insulin-specific target T-cell population is enriched with a C-X-C chemokine receptor type 5 (CXCR5)+CD4+ TFH precursor phenotype. During onset of islet autoimmunity, the frequency of TFH precursors was controlled by high expression of microRNA92a (miRNA92a). miRNA92a-mediated TFH precursor induction was regulated by phosphatase and tension homolog (PTEN) - phosphoinositol-3-kinase (PI3K) signaling involving PTEN and forkhead box protein O1 (Foxo1), supporting autoantibody generation and triggering the onset of islet autoimmunity. Moreover, we identify Krueppel-like factor 2 (KLF2) as a target of miRNA92a in regulating human TFH precursor induction. Importantly, a miRNA92a antagomir completely blocked induction of human TFH precursors in vitro. More importantly, in vivo application of a miRNA92a antagomir to nonobese diabetic (NOD) mice with ongoing islet autoimmunity resulted in a significant reduction of TFH precursors in peripheral blood and pancreatic lymph nodes. Moreover, miRNA92a antagomir application reduced immune infiltration and activation in pancreata of NOD mice as well as humanized NOD Scid IL2 receptor gamma chain knockout (NSG) human leucocyte antigen (HLA)-DQ8 transgenic animals. We therefore propose that miRNA92a and the PTEN-PI3K-KLF2 signaling network could function as targets for innovative precision medicines to reduce T1D islet autoimmunity.


Asunto(s)
Autoinmunidad , Diabetes Mellitus Tipo 1/inmunología , Factores de Transcripción de Tipo Kruppel/inmunología , MicroARNs/inmunología , Fosfohidrolasa PTEN/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Adolescente , Animales , Antagomirs/genética , Antagomirs/inmunología , Autoanticuerpos/biosíntesis , Niño , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Femenino , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/inmunología , Regulación de la Expresión Génica , Humanos , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/patología , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/inmunología , Cultivo Primario de Células , Receptores CXCR5/genética , Receptores CXCR5/inmunología , Transducción de Señal , Linfocitos T Colaboradores-Inductores/patología
18.
Gastroenterology ; 152(1): 176-192.e5, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27639807

RESUMEN

BACKGROUND & AIMS: GATA3 is a transcription factor that regulates T-cell production of cytokines. We investigated the role of GATA3 in development of colitis in mice. METHODS: We performed quantitative polymerase chain reaction and immunofluorescence analyses of colon tissues from patients with Crohn's disease (n = 61) or ulcerative colitis (UC, n = 74) or from patients without inflammatory bowel diseases (n = 22), to measure levels of GATA3. Colitis was induced by administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid to control mice, mice with T-cell-specific deletion of GATA3, and mice with deletion of tumor necrosis factor receptor (TNFR) 1 and TNFR2 (TNFR double knockouts); some mice were given a GATA3-specific DNAzyme (hgd40) or a control DNAzyme via intrarectal administration, or systemic injections of an antibody to TNF before or during sensitization and challenge phase of colitis induction. Colon tissues were collected and immunofluorescence and histochemical analyses were performed. Lamina propria mononuclear cells and T cells were isolated and analyzed by flow cytometry or cytokine assays. Colonic distribution of labeled DNAzyme and inflammation were monitored by in vivo imaging (endoscopy) of mice. RESULTS: Levels of GATA3 messenger RNA were higher in colon tissues from patients with UC, but not ileal Crohn's disease, than control tissues; levels of GATA3 correlated with levels of inflammatory cytokines (interleukin [IL] 9, IL17A, IL6, IL5, IL4, IL13, and TNF). We observed increased expression of GATA3 by lamina propria T cells from mice with colitis compared with controls. Mice with T-cell-specific deletion of GATA3 did not develop colitis and their colonic tissues did not produce inflammatory cytokines (IL6, IL9, or IL13). The DNAzyme hgd40 inhibited expression of GATA3 messenger RNA by unstimulated and stimulated T cells, and distributed throughout the inflamed colons of mice with colitis. Colon tissues from mice given hgd40 had reduced expression of GATA3 messenger RNA, compared with mice given a control DNAzyme. Mice given hgd40 did not develop colitis after administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid; lamina propria cells from these mice expressed lower levels of IL6, IL9, and IL13 than cells from mice given the control DNAzyme. Mini-endoscopic images revealed that hgd40 and anti-TNF reduced colon inflammation over 3 days; hgd40 reduced colitis in TNFR double-knockout mice. CONCLUSIONS: Levels of GATA3 are increased in patients with UC and correlate with production of inflammatory cytokines in mice and humans. A DNAzyme that prevents expression of GATA3 reduces colitis in mice, independently of TNF, and reduces levels of cytokines in the colon. This DNAzyme might be developed for treatment of patients with UC.


Asunto(s)
Colitis/metabolismo , Colitis/prevención & control , ADN Catalítico/administración & dosificación , Factor de Transcripción GATA3/antagonistas & inhibidores , Factor de Transcripción GATA3/metabolismo , ARN Mensajero/análisis , Administración Rectal , Adolescente , Adulto , Anciano , Animales , Estudios de Casos y Controles , Niño , Colitis/inducido químicamente , Colitis Ulcerosa/genética , Colitis Ulcerosa/metabolismo , Colon/química , Colon/metabolismo , Enfermedad de Crohn/genética , Enfermedad de Crohn/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Factor de Transcripción GATA3/genética , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Oxazolona , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Linfocitos T/metabolismo , Ácido Trinitrobencenosulfónico , Adulto Joven
19.
Gastroenterology ; 153(1): 123-138.e8, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28342759

RESUMEN

BACKGROUND & AIMS: We investigated the roles of interleukin 28A (also called IL28A or interferon λ2) in intestinal epithelial cell (IEC) activation, studying its effects in mouse models of inflammatory bowel diseases (IBD) and intestinal mucosal healing. METHODS: Colitis was induced in C57BL/6JCrl mice (controls), mice with IEC-specific disruption of Stat1 (Stat1IEC-KO), mice with disruption of the interferon λ receptor 1 gene (Il28ra-/-), and mice with disruption of the interferon regulatory factor 3 gene (Irf3-/-), with or without disruption of Irf7 (Irf7-/-). We used high-resolution mini-endoscopy and in vivo imaging methods to assess colitis progression. We used 3-dimensional small intestine and colon organoids, along with RNA-Seq and gene ontology methods, to characterize the effects of IL28 on primary IECs. We studied the effects of IL28 on the human intestinal cancer cell line Caco-2 in a wound-healing assay, and in mice colon wounds. Colonic biopsies and resected tissue from patients with IBD (n = 62) and patients without colon inflammation (controls, n = 23) were analyzed by quantitative polymerase chain rection to measure expression of IL28A, IL28RA, and other related cytokines; biopsy samples were also analyzed by immunofluorescence to identify sources of IL28 production. IECs were isolated from patient tissues and incubated with IL28; signal transducer and activator of transcription 1 (STAT1) phosphorylation was measured by immunoblots and confocal imaging. RESULTS: Lamina propria cells in colon tissues of patients with IBD, and mice with colitis, had increased expression of IL28 compared with controls; levels of IL28R were increased in the colonic epithelium of patients with IBD and mice with colitis. Administration of IL28 induced phosphorylation of STAT1 in primary human and mouse IECs, increasing with dose. Il28ra-/-, Irf3-/-, Irf3-/-Irf7-/-, as well as Stat1IEC-KO mice, developed more severe colitis after administration of dextran sulfate sodium than control mice, with reduced epithelial restitution. Il28ra-/- and Stat1IEC-KO mice also developed more severe colitis in response to oxazolone than control mice. We found IL28 to induce phosphorylation (activation) of STAT1 in epithelial cells, leading to their proliferation in organoid culture. Administration of IL28 to mice with induced colonic wounds promoted mucosal healing. CONCLUSIONS: IL28 controls proliferation of IECs in mice with colitis and accelerates mucosal healing by activating STAT1. IL28 might be developed as a therapeutic agent for patients with IBD.


Asunto(s)
Colitis/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Interleucinas/metabolismo , Factor de Transcripción STAT1/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Células CACO-2 , Proliferación Celular , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Células Dendríticas/metabolismo , Sulfato de Dextran , Modelos Animales de Enfermedad , Células Epiteliales , Femenino , Expresión Génica , Humanos , Enfermedades Inflamatorias del Intestino/genética , Factor 3 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/genética , Interleucinas/genética , Interleucinas/farmacología , Mucosa Intestinal/metabolismo , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Organoides , Fosforilación , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Receptores de Interferón/genética , Factor de Transcripción STAT1/genética , Transducción de Señal , Cicatrización de Heridas , Adulto Joven
20.
Nature ; 477(7364): 335-9, 2011 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-21921917

RESUMEN

Dysfunction of the intestinal epithelium is believed to result in the excessive translocation of commensal bacteria into the bowel wall that drives chronic mucosal inflammation in Crohn's disease, an incurable inflammatory bowel disease in humans characterized by inflammation of the terminal ileum. In healthy individuals, the intestinal epithelium maintains a physical barrier, established by the tight contact of cells. Moreover, specialized epithelial cells such as Paneth cells and goblet cells provide innate immune defence functions by secreting mucus and antimicrobial peptides, which hamper access and survival of bacteria adjacent to the epithelium. Epithelial cell death is a hallmark of intestinal inflammation and has been discussed as a possible pathogenic mechanism driving Crohn's disease in humans. However, the regulation of epithelial cell death and its role in intestinal homeostasis remain poorly understood. Here we demonstrate a critical role for caspase-8 in regulating necroptosis of intestinal epithelial cells (IECs) and terminal ileitis. Mice with a conditional deletion of caspase-8 in the intestinal epithelium (Casp8(ΔIEC)) spontaneously developed inflammatory lesions in the terminal ileum and were highly susceptible to colitis. Casp8(ΔIEC) mice lacked Paneth cells and showed reduced numbers of goblet cells, indicating dysregulated antimicrobial immune cell functions of the intestinal epithelium. Casp8(ΔIEC) mice showed increased cell death in the Paneth cell area of small intestinal crypts. Epithelial cell death was induced by tumour necrosis factor (TNF)-α, was associated with increased expression of receptor-interacting protein 3 (Rip3; also known as Ripk3) and could be inhibited on blockade of necroptosis. Lastly, we identified high levels of RIP3 in human Paneth cells and increased necroptosis in the terminal ileum of patients with Crohn's disease, suggesting a potential role of necroptosis in the pathogenesis of this disease. Together, our data demonstrate a critical function of caspase-8 in regulating intestinal homeostasis and in protecting IECs from TNF-α-induced necroptotic cell death.


Asunto(s)
Apoptosis , Caspasa 8/metabolismo , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/patología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Caspasa 8/genética , Colitis/enzimología , Colitis/inmunología , Colitis/metabolismo , Colitis/patología , Enfermedad de Crohn/enzimología , Enfermedad de Crohn/inmunología , Eliminación de Gen , Células Caliciformes/patología , Humanos , Técnicas In Vitro , Ratones , Necrosis , Células de Paneth/enzimología , Células de Paneth/inmunología , Células de Paneth/metabolismo , Células de Paneth/patología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo
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