RESUMEN
Although tissue-resident memory T cells (TRM cells) have been shown to regulate host protection in infectious disorders, their function in inflammatory bowel disease (IBD) remains to be investigated. Here we characterized TRM cells in human IBD and in experimental models of intestinal inflammation. Pro-inflammatory TRM cells accumulated in the mucosa of patients with IBD, and the presence of CD4+CD69+CD103+ TRM cells was predictive of the development of flares. In vivo, functional impairment of TRM cells in mice with double knockout of the TRM-cell-associated transcription factors Hobit and Blimp-1 attenuated disease in several models of colitis, due to impaired cross-talk between the adaptive and innate immune system. Finally, depletion of TRM cells led to a suppression of colitis activity. Together, our data demonstrate a central role for TRM cells in the pathogenesis of chronic intestinal inflammation and suggest that these cells could be targets for future therapeutic approaches in IBD.
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Linfocitos T CD8-positivos/inmunología , Colitis/inmunología , Memoria Inmunológica/inmunología , Factor 1 de Unión al Dominio 1 de Regulación Positiva/inmunología , Factores de Transcripción/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Enfermedad Crónica , Colitis/genética , Colitis/metabolismo , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Memoria Inmunológica/genética , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Factor 1 de Unión al Dominio 1 de Regulación Positiva/deficiencia , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
In the version of this article initially published, a portion of the Acknowledgements section ("the Clinical Research Group CEDER of the German Research Council (DFG)") was incorrect. The correct statement is as follows: "...the Collaborative Research Center TRR241 of the German Research Council (DFG)...". The error has been corrected in the HTML and PDF version of the article.
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Checkpoints that limit stem cell self-renewal in response to DNA damage can contribute to cancer protection but may also promote tissue aging. Molecular components that control stem cell responses to DNA damage remain to be delineated. Using in vivo RNAi screens, we identified basic leucine zipper transcription factor, ATF-like (BATF) as a major component limiting self-renewal of hematopoietic stem cells (HSCs) in response to telomere dysfunction and γ-irradiation. DNA damage induces BATF in a G-CSF/STAT3-dependent manner resulting in lymphoid differentiation of HSCs. BATF deletion improves HSC self-renewal and function in response to γ-irradiation or telomere shortening but results in accumulation of DNA damage in HSCs. Analysis of bone marrow from patients with myelodysplastic syndrome supports the conclusion that DNA damage-dependent induction of BATF is conserved in human HSCs. Together, these results provide experimental evidence that a BATF-dependent differentiation checkpoint limits self-renewal of HSCs in response to DNA damage.
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Puntos de Control del Ciclo Celular , Diferenciación Celular , Senescencia Celular , Daño del ADN , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Organismos Libres de Patógenos Específicos , Acortamiento del TelómeroRESUMEN
T cells are considered to be critical drivers of intestinal inflammation in mice and people. The so called intra-epithelial lymphocyte (IEL) compartment largely consist of T cells. Interestingly, the specific regulation and contribution of IELs in the context of inflammatory bowel disease remains poorly understood, in part due to the lack of appropriate analysis tools. Powerful, label-free methods could ultimately provide access to this cell population and hence give valuable insight into IEL biology and even more to their disease-related functionalities. Raman spectroscopy has demonstrated over the last few years its potential for reliable cell characterization and differentiation, but its utility in regard to IEL exploration remains unknown. To address this question experimentally, we utilized a murine, T cell-driven experimental model system which is accepted to model human gut inflammation. Here, we repopulated the small intestinal IEL compartment (SI IELs) of Rag1-deficient mice endogenously lacking T cells by transferring naïve CD4+ T helper cells intraperitoneally. Using multivariate statistical analysis, high-throughput Raman spectroscopy managed to define a cell subpopulation ex vivo within the SI IEL pool of mice previously receiving T cells in vivo that displayed characteristic spectral features of lymphocytes. Raman data sets matched flow cytometry analyses with the latter identifying T cell receptor (TCR)αß+ CD4+ T cell population in SI IELs from T cell-transferred mice, but not from control mice, in an abundance comparable to the one detected by Raman spectroscopy. Hence, in this study, we provide experimental evidence for high-throughput Raman spectroscopy to be a novel, future tool to reliably identify and potentially further characterize the T cell pool of small intestinal IELs ex vivo.
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Receptores de Antígenos de Linfocitos T gamma-delta , Espectrometría Raman , Ratones , Humanos , Animales , Receptores de Antígenos de Linfocitos T gamma-delta/análisis , Linfocitos T , Intestino Delgado/química , Linfocitos/química , Receptores de Antígenos de Linfocitos T alfa-beta/análisis , Mucosa Intestinal/químicaRESUMEN
Intraepithelial lymphocytes (IEL) are widely distributed within the small intestinal epithelial cell (IEC) layer and represent one of the largest T cell pools of the body. While implicated in the pathogenesis of intestinal inflammation, detailed insight especially into the cellular cross-talk between IELs and IECs is largely missing in part due to lacking methodologies to monitor this interaction. To overcome this shortcoming, we employed and validated a murine IEL-IEC (organoids) ex vivo co-culture model system. Using livecell imaging we established a protocol to visualize and quantify the spatio-temporal migratory behavior of IELs within organoids over time. Applying this methodology, we found that IELs lacking CD103 (i.e., integrin alpha E, ITGAE) surface expression usually functioning as a retention receptor for IELs through binding to E-cadherin (CD324) expressing IECs displayed aberrant mobility and migration patterns. Specifically, CD103 deficiency affected the ability of IELs to migrate and reduced their speed during crawling within organoids. In summary, we report a new technology to monitor and quantitatively assess especially migratory characteristics of IELs communicating with IEC ex vivo. This approach is hence readily applicable to study the effects of targeted therapeutic interventions on IEL-IEC cross-talk.
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Antígenos CD/metabolismo , Movimiento Celular , Procesamiento de Imagen Asistido por Computador/métodos , Cadenas alfa de Integrinas/metabolismo , Mucosa Intestinal/metabolismo , Linfocitos Intraepiteliales/metabolismo , Organoides/metabolismo , Linfocitos T/fisiología , Animales , Técnicas de Cocultivo , Técnica del Anticuerpo Fluorescente , Mucosa Intestinal/citología , Linfocitos Intraepiteliales/citología , Ratones , Organoides/citología , Análisis Espacio-TemporalRESUMEN
Excessive inflammatory immune responses during infections with Plasmodium parasites are responsible for severe complications such as cerebral malaria (CM) that can be studied experimentally in mice. Dendritic cells (DCs) activate cytotoxic CD8+ T-cells and initiate immune responses against the parasites. Batf3-/- mice lack a DC subset, which efficiently induces strong CD8 T-cell responses by cross-presentation of exogenous antigens. Here we show that Batf3-/- mice infected with Plasmodium berghei ANKA (PbA) were protected from experimental CM (ECM), characterized by a stable blood-brain barrier (BBB) and significantly less infiltrated peripheral immune cells in the brain. Importantly, the absence of ECM in Batf3-/- mice correlated with attenuated responses of cytotoxic T-cells, as their parasite-specific lytic activity as well as the production of interferon gamma and granzyme B were significantly decreased. Remarkably, spleens of ECM-protected Batf3-/- mice had elevated levels of regulatory immune cells and interleukin 10. Thus, protection from ECM in PbA-infected Batf3-/- mice was associated with the absence of strong CD8+ T-cell activity and induction of immunoregulatory mediators and cells.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/deficiencia , Encéfalo/inmunología , Células Dendríticas/inmunología , Malaria Cerebral/prevención & control , Plasmodium berghei/patogenicidad , Proteínas Represoras/deficiencia , Linfocitos T Citotóxicos/inmunología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Barrera Hematoencefálica/inmunología , Barrera Hematoencefálica/parasitología , Encéfalo/metabolismo , Encéfalo/parasitología , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/parasitología , Modelos Animales de Enfermedad , Femenino , Granzimas/inmunología , Granzimas/metabolismo , Interacciones Huésped-Parásitos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-10/inmunología , Interleucina-10/metabolismo , Malaria Cerebral/inmunología , Malaria Cerebral/metabolismo , Malaria Cerebral/parasitología , Ratones Endogámicos C57BL , Ratones Noqueados , Plasmodium berghei/inmunología , Proteínas Represoras/genética , Bazo/inmunología , Bazo/metabolismo , Bazo/parasitología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/parasitologíaRESUMEN
CD8α(+) dendritic cells (DCs) prime cytotoxic T lymphocytes during viral infections and produce interleukin-12 in response to pathogens. Although the loss of CD8α(+) DCs in Batf3(-/-) mice increases their susceptibility to several pathogens, we observed that Batf3(-/-) mice exhibited enhanced resistance to the intracellular bacterium Listeria monocytogenes. In wild-type mice, Listeria organisms, initially located in the splenic marginal zone, migrated to the periarteriolar lymphoid sheath (PALS) where they grew exponentially and induced widespread lymphocyte apoptosis. In Batf3(-/-) mice, however, Listeria organisms remain trapped in the marginal zone, failed to traffic into the PALS, and were rapidly cleared by phagocytes. In addition, Batf3(-/-) mice, which lacked the normal population of hepatic CD103(+) peripheral DCs, also showed protection from liver infection. These results suggest that Batf3-dependent CD8α(+) and CD103(+) DCs provide initial cellular entry points within the reticuloendothelial system by which Listeria establishes productive infection.
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Células Dendríticas/metabolismo , Listeria monocytogenes/fisiología , Listeriosis/inmunología , Ganglios Linfáticos/metabolismo , Bazo/metabolismo , Animales , Antígenos CD/biosíntesis , Apoptosis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Antígenos CD8/biosíntesis , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Células Dendríticas/patología , Inmunidad Innata/genética , Cadenas alfa de Integrinas/biosíntesis , Listeria monocytogenes/patogenicidad , Listeriosis/microbiología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/microbiología , Ganglios Linfáticos/patología , Linfocitos/patología , Ratones , Ratones Endogámicos , Ratones Noqueados , Fagocitosis/genética , Proteínas Represoras/genética , Bazo/inmunología , Bazo/microbiología , Bazo/patología , VirulenciaRESUMEN
OBJECTIVE: Anti-tumour necrosis factor (TNF) antibodies are successfully used for treatment of Crohn's disease. Nevertheless, approximately 40% of patients display failure to anti-TNF therapy. Here, we characterised molecular mechanisms that are associated with endoscopic resistance to anti-TNF therapy. DESIGN: Mucosal and blood cells were isolated from patients with Crohn's disease prior and during anti-TNF therapy. Cytokine profiles, cell surface markers, signalling proteins and cell apoptosis were assessed by microarray, immunohistochemistry, qPCR, ELISA, whole organ cultures and FACS. RESULTS: Responders to anti-TNF therapy displayed a significantly higher expression of TNF receptor 2 (TNFR2) but not IL23R on T cells than non-responders prior to anti-TNF therapy. During anti-TNF therapy, there was a significant upregulation of mucosal IL-23p19, IL23R and IL-17A in anti-TNF non-responders but not in responders. Apoptosis-resistant TNFR2+IL23R+ T cells were significantly expanded in anti-TNF non-responders compared with responders, expressed the gut tropic integrins α4ß7, and exhibited increased expression of IFN-γ, T-bet, IL-17A and RORγt compared with TNFR2+IL23R- cells, indicating a mixed Th1/Th17-like phenotype. Intestinal TNFR2+IL23R+ T cells were activated by IL-23 derived from CD14+ macrophages, which were significantly more present in non-responders prior to anti-TNF treatment. Administration of IL-23 to anti-TNF-treated mucosal organ cultures led to the expansion of CD4+IL23R+TNFR2+ lymphocytes. Functional studies demonstrated that anti-TNF-induced apoptosis in mucosal T cells is abrogated by IL-23. CONCLUSIONS: Expansion of apoptosis-resistant intestinal TNFR2+IL23R+ T cells is associated with resistance to anti-TNF therapy in Crohn's disease. These findings identify IL-23 as a suitable molecular target in patients with Crohn's disease refractory to anti-TNF therapy.
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Enfermedad de Crohn/metabolismo , Resistencia a Medicamentos , Fármacos Gastrointestinales/uso terapéutico , Receptores de Interleucina/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Linfocitos T/fisiología , Adalimumab/uso terapéutico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/patología , Humanos , Infliximab/uso terapéutico , Interleucina-17/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND Confocal laser endomicroscopy (CLE) enables "in vivo" microscopic tissue diagnosis based on tissue reflectance or tissue fluorescence upon application of fluorescence agents. The aim of the present study was to evaluate CLE as a new diagnostic approach for differentiation between malignant versus non-malignant pleural effusions. MATERIAL AND METHODS In 100 patients with pleural effusions, thoracentesis was performed. Cresyl violet and acriflavine were used as contrast agents for probe-based CLE of effusions. CLE video sequences were assessed by 4 independent investigators (2 experienced in this technique, 2 with only basic knowledge). In addition, all CLE samples were evaluated by an expert pathologist (p). Results were compared with conventional cytology of effusions and histology of cell blocks. RESULTS CLE reliably permitted identification of malignant cells in pleural effusions. Sensitivity for detection of malignant effusions was 87% (p: 87%) and 81% (p: 72%) for acriflavine and cresyl violet, respectively. With regard to specificity, acriflavine and cresyl violet yielded a mean value of 99% (p: 100%) and 92% (p: 100%). CONCLUSIONS In this pilot study, CLE permitted simple and rapid detection of malignant pleural effusions. Larger prospective studies are warranted to corroborate our findings.
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Microscopía Confocal/métodos , Derrame Pleural Maligno/diagnóstico por imagen , Derrame Pleural/diagnóstico por imagen , Adulto , Anciano , Anciano de 80 o más Años , Medios de Contraste , Endoscopía/métodos , Femenino , Colorantes Fluorescentes , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
OBJECTIVES: IBDs have an increased risk for development of colorectal cancer (CRC). Here, we aimed at the characterisation of the functional role of Th17-associated transcription factors in sporadic and colitis-associated colon cancer in vivo. DESIGN: We used mice deficient or transgenic for the activating protein 1 family member basic leucine zipper transcription factor ATF-like (Batf) to evaluate the role of Th17 cells during sporadic and inflammation-induced colon carcinogenesis. We also studied the expression of Batf and RORγt in patients with IBD and CRC. RESULTS: Batf but not retinoic acid-related orphan receptor γt(RORγt) expression was significantly increased together with interleukin (IL) 23 expression in UC but not in Crohn's disease (CD) tissue samples. In CRC also Batf but not RORγt expression was increased and its expression correlated with the IL-23 and IL-23 receptor (IL-23R) expression. Finally, Batf but not RORγt was coexpressed with IL-17a, IL-23R and IL-6 within CRC-infiltrating CD4(+) T cells. Functional studies in mice revealed that Batf-dependent T cells are crucial regulators of sporadic and inflammation-induced CRC. Colitis-associated Batf(-/-) tumours lacked IL-17a(+)IL-23R(+)IL-6(+)CD4(+) T cells, hence displaying characteristics reminiscent of human CRC-infiltrating CD4(+) T cells. Strikingly, Batf(-/-) tumours contained low IL-23 but high IL-17a expression levels. Tumour formation and intratumoral IL-23 expression could be restored by administration of Hyper-IL-6 consisting of IL-6 and soluble IL-6 receptor. CONCLUSIONS: Batf-dependent IL-23R(+)IL-6(+)CD4(+) Th17 cells critically control IL-23 driven colitis-associated tumour formation and the progression of sporadic colon tumours. Batf-dependent IL-23R(+) T cells represent a potential future therapeutic target limiting CRC progression.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Colitis Ulcerosa/metabolismo , Neoplasias del Colon/metabolismo , Interleucina-23/metabolismo , Células Th17/metabolismo , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/deficiencia , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Transformación Celular Neoplásica , Colitis Ulcerosa/patología , Neoplasias del Colon/patología , Enfermedad de Crohn/metabolismo , Humanos , Interleucina-17/metabolismo , Interleucina-23/genética , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-6/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , ARN Mensajero/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-6/metabolismo , Transducción de SeñalAsunto(s)
Colitis Ulcerosa/diagnóstico , Colonoscopía/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Animales , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/genética , Colitis Ulcerosa/patología , Colon/diagnóstico por imagen , Colon/efectos de los fármacos , Colon/patología , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Fluoresceína/administración & dosificación , Humanos , Mucosa Intestinal/diagnóstico por imagen , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Ratones , Ratones NoqueadosRESUMEN
BACKGROUND: Inflammatory bowel diseases (IBDs) represent a group of chronic immune-mediated disorders that are influenced by a genetic predisposition and additional environmental triggers. Genome-wide association studies strongly implicate that a number of immune system-related genetic variations are critically contributing to the initiation and promotion of intestinal inflammation. Especially the identification of the strong association of a series of single nucleotide polymorphisms including interleukin (IL)-23R, CCR6, signal transducer and activator of transcription 3 (Stat3) and Stat4 with IBD susceptibility point at a critical involvement of T cells and especially of IL-17a-producing Th17 cells in the immune pathogenesis of IBD. In line with this hypothesis, a series of preclinical studies have unequivocally established that T cells are key drivers of immune-mediated colitis. Interestingly, especially Th17 cells were identified to be highly prevalent in inflamed IBD tissues, a finding that seems to be functionally relevant as genetic inactivation studies in the mouse resulted in almost complete suppression of colitis development. KEY MESSAGES: While targeting Th17 cell differentiation regulating transcription factors, as retinoic acid-related orphan receptor gamma t (RORγt) is effective in preventing murine colitis, one concern of drugs targeting RORγt in a clinical setting represents the large body of murine data unambiguously demonstrating that additional pathways within and outside the immune system are equally RORγt-dependent increasing the risk of undesirable side effects. The AP1 transcription factor Batf (B cell-activating transcription factor) appears to exclusively regulate pathways within lymphocytes. Importantly, Batf represents a central regulator of Th17 cell development and is strongly upregulated within IBD-affected tissues. Employing 2 acute colitis models, we demonstrate in this study that Batf-expressing T cells are critical drivers of T cell-mediated colitis while in contrast to Stat3 loss of Batf does not affect intestinal epithelial cell homeostasis ex vivo. CONCLUSIONS: Targeting Batf in IBD emerges as an attractive therapeutic approach disabling colitogenic T cell activities while sparing off-target effects in the intestinal epithelial cell compartment.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Colitis/genética , Enfermedades Inflamatorias del Intestino/genética , Células Th17/fisiología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/antagonistas & inhibidores , Diferenciación Celular/genética , Colitis/inducido químicamente , Colitis/metabolismo , Células Epiteliales/metabolismo , Humanos , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/metabolismo , Interleucina-17/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/patología , Ratones , Polimorfismo de Nucleótido Simple , Receptores CCR6/genética , Receptores de Interleucina/genética , Receptores de Ácido Retinoico/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT4/genética , Receptor de Ácido Retinoico gammaRESUMEN
The Citrobacter rodentium model mimics the pathogenesis of infectious colitis and requires sequential contributions from different immune cell populations, including innate lymphoid cells (ILCs) and CD4(+) lymphocytes. In this study, we addressed the role of STAT3 activation in CD4(+) cells during host defense in mice against C. rodentium. In mice with defective STAT3 in CD4(+) cells (Stat3(ΔCD4)), the course of infection was unchanged during the innate lymphoid cell-dependent early phase, but significantly altered during the lymphocyte-dependent later phase. Stat3(ΔCD4) mice exhibited intestinal epithelial barrier defects, including downregulation of antimicrobial peptides, increased systemic distribution of bacteria, and prolonged reduction in the overall burden of C. rodentium infection. Immunomonitoring of lamina propria cells revealed loss of virtually all IL-22-producing CD4(+) lymphocytes, suggesting that STAT3 activation was required for IL-22 production not only in Th17 cells, but also in Th22 cells. Notably, the defective host defense against C. rodentium in Stat3(∆CD4) mice could be fully restored by specific overexpression of IL-22 through a minicircle vector-based technology. Moreover, expression of a constitutive active STAT3 in CD4(+) cells shaped strong intestinal epithelial barrier function in vitro and in vivo through IL-22, and it promoted protection from enteropathogenic bacteria. Thus, our work indicates a critical role of STAT3 activation in Th17 and Th22 cells for control of the IL-22-mediated host defense, and strategies expanding STAT3-activated CD4(+) lymphocytes may be considered as future therapeutic options for improving intestinal barrier function in infectious colitis.
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Citrobacter rodentium/inmunología , Colitis/inmunología , Infecciones por Enterobacteriaceae/inmunología , Interleucinas/inmunología , Mucosa Intestinal/inmunología , Factor de Transcripción STAT3/inmunología , Células Th17/inmunología , Animales , Colitis/genética , Colitis/microbiología , Colitis/patología , Infecciones por Enterobacteriaceae/genética , Infecciones por Enterobacteriaceae/patología , Interleucinas/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Ratones , Ratones Transgénicos , Células Th17/patología , Interleucina-22RESUMEN
Tissue macrophages comprise a heterogeneous group of cell types differing in location, surface markers and function. Red pulp macrophages are a distinct splenic subset involved in removing senescent red blood cells. Transcription factors such as PU.1 (also known as Sfpi1) and C/EBPalpha (Cebpa) have general roles in myelomonocytic development, but the transcriptional basis for producing tissue macrophage subsets remains unknown. Here we show that Spi-C (encoded by Spic), a PU.1-related transcription factor, selectively controls the development of red pulp macrophages. Spi-C is highly expressed in red pulp macrophages, but not monocytes, dendritic cells or other tissue macrophages. Spic(-/-) mice have a cell-autonomous defect in the development of red pulp macrophages that is corrected by retroviral Spi-C expression in bone marrow cells, but have normal monocyte and other macrophage subsets. Red pulp macrophages highly express genes involved in capturing circulating haemoglobin and in iron regulation. Spic(-/-) mice show normal trapping of red blood cells in the spleen, but fail to phagocytose these red blood cells efficiently, and develop an iron overload localized selectively to splenic red pulp. Thus, Spi-C controls development of red pulp macrophages required for red blood cell recycling and iron homeostasis.
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Proteínas de Unión al ADN/metabolismo , Eritrocitos/metabolismo , Homeostasis , Hierro/metabolismo , Macrófagos/fisiología , Fagocitosis , Bazo/metabolismo , Animales , Recuento de Células , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Eritrocitos/citología , Regulación de la Expresión Génica , Macrófagos/citología , Ratones , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
Activator protein 1 (AP-1, also known as JUN) transcription factors are dimers of JUN, FOS, MAF and activating transcription factor (ATF) family proteins characterized by basic region and leucine zipper domains. Many AP-1 proteins contain defined transcriptional activation domains, but BATF and the closely related BATF3 (refs 2, 3) contain only a basic region and leucine zipper, and are considered to be inhibitors of AP-1 activity. Here we show that Batf is required for the differentiation of IL17-producing T helper (T(H)17) cells. T(H)17 cells comprise a CD4(+) T-cell subset that coordinates inflammatory responses in host defence but is pathogenic in autoimmunity. Batf(-/-) mice have normal T(H)1 and T(H)2 differentiation, but show a defect in T(H)17 differentiation, and are resistant to experimental autoimmune encephalomyelitis. Batf(-/-) T cells fail to induce known factors required for T(H)17 differentiation, such as RORgamma t (encoded by Rorc) and the cytokine IL21 (refs 14-17). Neither the addition of IL21 nor the overexpression of RORgamma t fully restores IL17 production in Batf(-/-) T cells. The Il17 promoter is BATF-responsive, and after T(H)17 differentiation, BATF binds conserved intergenic elements in the Il17a-Il17f locus and to the Il17, Il21 and Il22 (ref. 18) promoters. These results demonstrate that the AP-1 protein BATF has a critical role in T(H)17 differentiation.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Diferenciación Celular , Interleucina-17/metabolismo , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/deficiencia , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Encefalomielitis Autoinmune Experimental/genética , Femenino , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Interleucina-17/biosíntesis , Interleucina-17/genética , Interleucinas/genética , Interleucinas/metabolismo , Interleucinas/farmacología , Ganglios Linfáticos/metabolismo , Masculino , Ratones , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Regiones Promotoras Genéticas/genética , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Factor de Transcripción AP-1/deficiencia , Factor de Transcripción AP-1/genéticaRESUMEN
BACKGROUND: Mice without the basic leucine zipper transcription factor, ATF-like (BATF) gene (Batf(-/-)) lack TH17 and follicular helper T cells, which demonstrates that Batf is a transcription factor important for T- and B-cell differentiation. OBJECTIVE: In this study we examined whether BATF expression would influence allergic asthma. METHODS: In a cohort of preschool control children and children with asthma, we analyzed BATF mRNA expression using real-time PCR in PBMCs. In a murine model of allergic asthma, we analyzed differences in this allergic disease between wild-type, Batf transgenic, and Batf(-/-) mice. RESULTS: In the absence of corticosteroid treatment, children with recurrent asthma have a significant increase in BATF mRNA expression in their PBMCs. Batf(-/-) mice display a significant reduction in the pathophysiologic responses seen in asthmatic wild-type littermates. Moreover, we discovered a decrease in IL-3 production and IL-3-dependent mast cell development in Batf(-/-) mice. By contrast, IFN-γ was induced in lung CD4(+) and CD8(+) T cells. Intranasal delivery of anti-IFN-γ antibodies induced airway hyperresponsiveness and inflammation in wild-type but not in Batf(-/-) mice. Transgenic overexpression of Batf under the control of the CD2 promoter/enhancer augmented lung inflammation and IgE levels in the setting of experimental asthma. CONCLUSION: BATF is increased in non-steroid-treated asthmatic children. Targeting BATF expression resulted in amelioration of the pathophysiologic responses seen in children with allergic asthma, and BATF has emerged as a novel target for antiasthma interventions.
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Asma/inmunología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Interferón gamma/metabolismo , Mastocitos/inmunología , Animales , Anticuerpos Bloqueadores/administración & dosificación , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Niño , Preescolar , Estudios de Cohortes , Humanos , Inmunoglobulina E/sangre , Interferón gamma/inmunología , Interleucina-3/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , ARN Mensajero/análisis , Transgenes/genética , Regulación hacia ArribaRESUMEN
Confocal laser endomicroscopy is a novel endoscopic technique that may allow imaging of living cells in lung tissue in vivo. We assessed the potential of this technique for the detection of histology during screening bronchoscopy for lung cancer. 32 patients with suspected malignancies underwent bronchoscopy with endomicroscopy using acriflavine hydrochloride. Standardised areas and localised lesions were analysed by in vivo confocal imaging during bronchoscopy and biopsies were taken. Confocal images were graded and correlated prospectively with conventional histology from biopsies. Acriflavine hydrochloride yielded high-quality confocal images and strongly labelled airway epithelial cells. No side-effects were noted. 75,522 confocal images from 56 different locations were compared prospectively with histological data from biopsy specimens. Endomicroscopy allowed subsurface imaging with detailed analysis of cellular and subcellular structures. Neoplastic changes could be predicted with high accuracy (sensitivity 96.0%, specificity 87.1%, accuracy 91.0%). Confocal laser endomicroscopy with acriflavine is a novel diagnostic tool for the analysis of living cells during bronchoscopy and permits virtual histology of neoplastic changes in the airways with high accuracy. This technique may enable the rapid diagnosis of neoplasia during ongoing endoscopy in patients with suspected lung cancer.
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Broncoscopía/instrumentación , Rayos Láser , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patología , Microscopía Confocal/instrumentación , Acriflavina/farmacología , Anciano , Broncoscopía/métodos , Medios de Contraste/farmacología , Femenino , Colorantes Fluorescentes/farmacología , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Microscopía Confocal/métodos , Persona de Mediana Edad , Proyectos Piloto , Valor Predictivo de las Pruebas , Estudios Prospectivos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The analysis of cell types involved in cross-priming of particulate Ag is essential to understand and improve immunotherapies using microparticles. In this study, we show that murine splenic dendritic cells (DCs) as well as macrophages (MΦs) are able to efficiently endocytose poly(D,L-lactate-co-glycolate) acid (PLGA) microspheres (MS) and to cross-present encapsulated Ags in the context of MHC class I molecules in vitro. A comparison of purified CD8(+) and CD8(-) DCs indicated that both DC subtypes are able to present OVA-derived epitopes on MHC class I and II in vitro. To determine the contribution of DCs and MΦs to cross-priming of PLGA MS in vivo, DCs were depleted in transgenic CD11c-DTR mice, and MΦs were depleted by clodronate liposomes in wild-type mice before immunizing mice with OVA-encapsulated MS. Our results show that the depletion of DCs or MΦs alone only led to minor differences in the OVA-specific immune responses. However, simultaneous depletion of DCs and MΦs caused a strong reduction of primed effector cells, indicating a redundancy of both cell populations for the priming of PLGA MS-encapsulated Ag. Finally, we analyzed PLGA MS trafficking to draining lymph nodes after s.c. injection. It was evident that fluorescent particles accumulated within draining lymph nodes over time. Further analysis of PLGA MS-positive lymphatic cells revealed that mainly CD8(-) DCs and MΦs contained MS. Moreover, immune responses in BATF3 knockout mice lacking CD8(+) DCs were normal. The results presented in this work strongly suggest that in vivo cross-priming of PLGA MS-encapsulated Ag is performed by CD8(-) DCs and MΦs.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos CD8/inmunología , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Macrófagos/inmunología , Animales , Antígenos CD8/biosíntesis , Separación Celular , Células Dendríticas/metabolismo , Citometría de Flujo , Ácido Láctico/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microesferas , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido PoliglicólicoRESUMEN
Lymphoid organs are characterized by a complex network of phenotypically distinct dendritic cells (DC) with potentially unique roles in pathogen recognition and immunostimulation. Classical DC (cDC) include two major subsets distinguished in the mouse by the expression of CD8alpha. Here we describe a subset of CD8alpha(+) DC in lymphoid organs of naïve mice characterized by expression of the CX(3)CR1 chemokine receptor. CX(3)CR1(+) CD8alpha(+) DC lack hallmarks of classical CD8alpha(+) DC, including IL-12 secretion, the capacity to cross-present antigen, and their developmental dependence on the transcriptional factor BatF3. Gene-expression profiling showed that CX(3)CR1(+) CD8alpha(+) DC resemble CD8alpha(-) cDC. The microarray analysis further revealed a unique plasmacytoid DC (PDC) gene signature of CX(3)CR1(+) CD8alpha(+) DC. A PDC relationship of the cells is supported further by the fact that they harbor characteristic D-J Ig gene rearrangements and that development of CX(3)CR1(+) CD8alpha(+) DC requires E2-2, the critical transcriptional regulator of PDC. Thus, CX(3)CR1(+) CD8alpha(+) DC represent a unique DC subset, related to but distinct from PDC. Collectively, the expression-profiling data of this study refine the resolution of previous DC definitions, sharpen the border of classical CD8alpha(+) and CD8alpha(-) DC, and should assist the identification of human counterparts of murine DC subsets.