RESUMEN
Fat-associated lymphoid clusters (FALCs) are a type of lymphoid tissue associated with visceral fat. Here we found that the distribution of FALCs was heterogeneous, with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 cells in the peritoneal cavity through high expression of the chemokine CXCL13, and they supported B cell proliferation and germinal center differentiation during peritoneal immunological challenges. FALC formation was induced by inflammation, which triggered the recruitment of myeloid cells that expressed tumor-necrosis factor (TNF) necessary for signaling via the TNF receptors in stromal cells. Natural killer T cells (NKT cells) restricted by the antigen-presenting molecule CD1d were likewise required for the inducible formation of FALCs. Thus, FALCs supported and coordinated the activation of innate B cells and T cells during serosal immune responses.
Asunto(s)
Inflamación/inmunología , Grasa Intraabdominal/inmunología , Linfocitos/inmunología , Tejido Linfoide/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Quimiocina CXCL13/genética , Quimiocina CXCL13/inmunología , Quimiocina CXCL13/metabolismo , Citometría de Flujo , Expresión Génica/inmunología , Inflamación/genética , Inflamación/metabolismo , Grasa Intraabdominal/metabolismo , Linfocitos/metabolismo , Tejido Linfoide/citología , Tejido Linfoide/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Células Mieloides/inmunología , Células Mieloides/metabolismo , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/inmunología , Células del Estroma/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The alternative (noncanonical) nuclear factor-κB (NF-κB) signaling pathway predominantly regulates the function of the p52/RelB heterodimer. Germline Nfkb2 deficiency in mice leads to loss of p100/p52 protein and offers protection against a variety of gastrointestinal conditions, including azoxymethane/dextran sulfate sodium (DSS)-induced colitis-associated cancer and lipopolysaccharide (LPS)-induced small intestinal epithelial apoptosis. However, the common underlying protective mechanisms have not yet been fully elucidated. We applied high-throughput RNA-Seq and proteomic analyses to characterize the transcriptional and protein signatures of the small intestinal mucosa of naïve adult Nfkb2-/- mice. Those data were validated by immunohistochemistry and quantitative ELISA using both small intestinal tissue lysates and serum. We identified a B-lymphocyte defect as a major transcriptional signature in the small intestinal mucosa and immunoglobulin A as the most downregulated protein by proteomic analysis in Nfkb2-/- mice. Small intestinal immunoglobulins were dramatically dysregulated, with undetectable levels of immunoglobulin A and greatly increased amounts of immunoglobulin M being detected. The numbers of IgA-producing, cluster of differentiation (CD)138-positive plasma cells were also reduced in the lamina propria of the small intestinal villi of Nfkb2-/- mice. This phenotype was even more striking in the small intestinal mucosa of RelB-/- mice, although these mice were equally sensitive to LPS-induced intestinal apoptosis as their RelB+/+ wild-type counterparts. NF-κB2/p52 deficiency confers resistance to LPS-induced small intestinal apoptosis and also appears to regulate the plasma cell population and immunoglobulin levels within the gut.NEW & NOTEWORTHY Novel transcriptomic analysis of murine proximal intestinal mucosa revealed an unexpected B cell signature in Nfkb2-/- mice. In-depth analysis revealed a defect in the CD38+ B cell population and a gut-specific dysregulation of immunoglobulin levels.
Asunto(s)
Subunidad p52 de NF-kappa B , Células Plasmáticas , Animales , Inmunoglobulina A/metabolismo , Inmunoglobulinas/metabolismo , Mucosa Intestinal/metabolismo , Lipopolisacáridos/farmacología , Ratones , FN-kappa B/metabolismo , Subunidad p52 de NF-kappa B/genética , Subunidad p52 de NF-kappa B/metabolismo , Células Plasmáticas/metabolismo , ProteómicaRESUMEN
Lymph node development during embryogenesis involves lymphotoxin-ß receptor engagement and subsequent differentiation of a poorly defined population of mesenchymal cells into lymphoid tissue organizer cells. Here, we showed that embryonic mesenchymal cells with characteristics of adipocyte precursors present in the microenvironment of lymph nodes gave rise to lymph node organizer cells. Signaling through the lymphotoxin-ß receptor controlled the fate of adipocyte precursor cells by blocking adipogenesis and instead promoting lymphoid tissue stromal cell differentiation. This effect involved activation of the NF-κB2-RelB signaling pathway and inhibition of the expression of the key adipogenic factors Pparγ and Cebpα. In vivo organogenesis assays show that embryonic and adult adipocyte precursor cells can migrate into newborn lymph nodes and differentiate into a variety of lymph node stromal cells. Thus, we propose that adipose tissues act as a source of lymphoid stroma for lymph nodes and other lymphoid structures associated with fat.
Asunto(s)
Adipocitos/inmunología , Ganglios Linfáticos/inmunología , Transducción de Señal , Adipocitos/citología , Animales , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Receptor beta de Linfotoxina/inmunología , Ratones , Subunidad p52 de NF-kappa B/inmunología , Subunidad p52 de NF-kappa B/metabolismo , Fenotipo , Células del Estroma/inmunología , Factor de Transcripción ReIB/inmunología , Factor de Transcripción ReIB/metabolismoRESUMEN
The thymic medulla provides a specialized microenvironment for the negative selection of T cells, with the presence of autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) during the embryonic-neonatal period being both necessary and sufficient to establish long-lasting tolerance. Here we showed that emergence of the first cohorts of Aire(+) mTECs at this key developmental stage, prior to αß T cell repertoire selection, was jointly directed by Rankl(+) lymphoid tissue inducer cells and invariant Vγ5(+) dendritic epidermal T cell (DETC) progenitors that are the first thymocytes to express the products of gene rearrangement. In turn, generation of Aire(+) mTECs then fostered Skint-1-dependent, but Aire-independent, DETC progenitor maturation and the emergence of an invariant DETC repertoire. Hence, our data attributed a functional importance to the temporal development of Vγ5(+) γδ T cells during thymus medulla formation for αß T cell tolerance induction and demonstrated a Rank-mediated reciprocal link between DETC and Aire(+) mTEC maturation.
Asunto(s)
Células Precursoras de Linfocitos T/citología , Células Precursoras de Linfocitos T/inmunología , Receptor Activador del Factor Nuclear kappa-B/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Factores de Transcripción/inmunología , Animales , Diferenciación Celular/inmunología , Microambiente Celular , Células Epiteliales/inmunología , Femenino , Feto/citología , Feto/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Embarazo , Transducción de Señal/inmunología , Timo/citología , Timo/inmunología , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Proteína AIRERESUMEN
Lymphangiogenesis associated with tertiary lymphoid structure (TLS) has been reported in numerous studies. However, the kinetics and dynamic changes occurring to the lymphatic vascular network during TLS development have not been studied. Using a viral-induced, resolving model of TLS formation in the salivary glands of adult mice we demonstrate that the expansion of the lymphatic vascular network is tightly regulated. Lymphatic vessel expansion occurs in two distinct phases. The first wave of expansion is dependent on IL-7. The second phase, responsible for leukocyte exit from the glands, is regulated by lymphotoxin (LT)ßR signaling. These findings, while highlighting the tight regulation of the lymphatic response to inflammation, suggest that targeting the LTα1ß2/LTßR pathway in TLS-associated pathologies might impair a natural proresolving mechanism for lymphocyte exit from the tissues and account for the failure of therapeutic strategies that target these molecules in diseases such as rheumatoid arthritis.
Asunto(s)
Interleucina-7/metabolismo , Linfangiogénesis , Vasos Linfáticos/inmunología , Heterotrímero de Linfotoxina alfa1 y beta2/inmunología , Heterotrímero de Linfotoxina alfa1 y beta2/metabolismo , Estructuras Linfoides Terciarias/inmunología , Animales , Regulación de la Expresión Génica , Inflamación , Interleucina-7/genética , Interleucina-7/inmunología , Vasos Linfáticos/metabolismo , Heterotrímero de Linfotoxina alfa1 y beta2/genética , Ratones , Glándulas Salivales/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Estructuras Linfoides Terciarias/patologíaRESUMEN
The importance of CLEC-2, a natural ligand/receptor for Gp38/Podoplanin, in the formation of the lymphatic vasculature has recently been demonstrated. As the development and maintenance of lymph nodes (LNs) is dependent on the formation of the lymphatic vasculature and the differentiation of Gp38/Podoplanin(+) stromal cells, we investigated the role of CLEC-2 in lymphoneogenesis and LN homeostasis. Using constitutive Clec1b(-/-) mice, we showed that while CLEC-2 was not necessary for initiation of the LN anlage, it was required at late stages of development. Constitutive deletion of CLEC-2 induced a profound defect in lymphatic endothelial cell proliferation, resulting in lack of LNs at birth. In contrast, conditional deletion of CLEC-2 in the megakaryocyte/platelet lineage in Clec1b(fl/fl)PF4-Cre mice led to the development of blood-filled LNs and fibrosis, in absence of a proliferative defect of the lymphatic endothelial compartment. This phenotype was also observed in chimeric mice reconstituted with Clec1b(fl/fl)PF4-Cre bone marrow, indicating that CLEC-2 expression in platelets was required for LN integrity. We demonstrated that LNs of Clec1b(fl/fl)PF4-Cre mice are able to sustain primary immune responses but show a defect in immune cell recirculation after repeated immunizations, thus suggesting CLEC-2 as target in chronic immune response.
Asunto(s)
Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ganglios Linfáticos/crecimiento & desarrollo , Animales , Plaquetas/metabolismo , Proliferación Celular , Células Cultivadas , Endotelio Linfático/citología , Endotelio Linfático/metabolismo , Eliminación de Gen , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Linfangiogénesis , Megacariocitos/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
NF-κB signalling is an important factor in the development of inflammation-associated cancers. Mouse models of Helicobacter-induced gastric cancer and colitis-associated colorectal cancer have demonstrated that classical NF-κB signalling is an important regulator of these processes. In the stomach, it has also been demonstrated that signalling involving specific NF-κB proteins, including NF-κB1/p50, NF-κB2/p52, and c-Rel, differentially regulate the development of gastric pre-neoplasia. To investigate the effect of NF-κB subunit loss on colitis-associated carcinogenesis, we administered azoxymethane followed by pulsed dextran sodium sulphate to C57BL/6, Nfkb1(-/-), Nfkb2(-/-), and c-Rel(-/-) mice. Animals lacking the c-Rel subunit were more susceptible to colitis-associated cancer than wild-type mice, developing 3.5 times more colonic polyps per animal than wild-type mice. Nfkb2(-/-) mice were resistant to colitis-associated cancer, developing fewer polyps per colon than wild-type mice (median 1 compared to 4). To investigate the mechanisms underlying these trends, azoxymethane and dextran sodium sulphate were administered separately to mice of each genotype. Nfkb2(-/-) mice developed fewer clinical signs of colitis and exhibited less severe colitis and an attenuated cytokine response compared with all other groups following DSS administration. Azoxymethane administration did not fully suppress colonic epithelial mitosis in c-Rel(-/-) mice and less colonic epithelial apoptosis was also observed in this genotype compared to wild-type counterparts. These observations demonstrate different functions of specific NF-κB subunits in this model of colitis-associated carcinogenesis. NF-κB2/p52 is necessary for the development of colitis, whilst c-Rel-mediated signalling regulates colonic epithelial cell turnover following DNA damage.
Asunto(s)
Adenoma/metabolismo , Colitis/complicaciones , Neoplasias del Colon/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Subunidad p52 de NF-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Adenoma/inducido químicamente , Adenoma/etiología , Animales , Azoximetano/toxicidad , Transformación Celular Neoplásica/metabolismo , Colitis/inducido químicamente , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/etiología , Citocinas/metabolismo , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Células Epiteliales/metabolismo , Femenino , Inflamación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de SeñalRESUMEN
In this study we describe a previously unreported function for NFκB2, an NFκB family transcription factor, in antiviral immunity. NFκB2 is induced in response to poly(I:C), a mimic of viral dsRNA. Poly(I:C), acting via TLR3, induces p52-dependent transactivation of a reporter gene in a manner that requires the kinase activity of IκB kinase ε (IKKε) and the transactivating potential of RelA/p65. We identify a novel NFκB2 binding site in the promoter of the transcription factor Sp1 that is required for Sp1 gene transcription activated by poly(I:C). We show that Sp1 is required for IL-15 induction by both poly(I:C) and respiratory syncytial virus, a response that also requires NFκB2 and IKKε. Our study identifies NFκB2 as a target for IKKε in antiviral immunity and describes, for the first time, a role for NFκB2 in the regulation of gene expression in response to viral infection.
Asunto(s)
Quinasa I-kappa B/inmunología , Interleucina-15/metabolismo , Subunidad p52 de NF-kappa B/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/inmunología , Factor de Transcripción Sp1/inmunología , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Células HEK293 , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Inductores de Interferón/farmacología , Interleucina-15/genética , Ratones , Ratones Noqueados , Subunidad p52 de NF-kappa B/genética , Subunidad p52 de NF-kappa B/metabolismo , Poli I-C/farmacología , Infecciones por Virus Sincitial Respiratorio/genética , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/patología , Virus Sincitiales Respiratorios/genética , Virus Sincitiales Respiratorios/metabolismo , Elementos de Respuesta/genética , Elementos de Respuesta/inmunología , Factor de Transcripción Sp1/biosíntesis , Factor de Transcripción Sp1/genética , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 3/metabolismo , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/inmunología , Factor de Transcripción ReIA/metabolismoRESUMEN
Cardiac remodeling and hypertrophy are the pathological consequences of cardiovascular disease and are correlated with its associated mortality. Activity of the transcription factor NF-κB is increased in the diseased heart; however, our present understanding of how the individual subunits contribute to cardiovascular disease is limited. We assign a new role for the c-Rel subunit as a stimulator of cardiac hypertrophy and fibrosis. We discovered that c-Rel-deficient mice have smaller hearts at birth, as well as during adulthood, and are protected from developing cardiac hypertrophy and fibrosis after chronic angiotensin infusion. Results of both gene expression and cross-linked chromatin immunoprecipitation assay analyses identified transcriptional activators of hypertrophy, myocyte enhancer family, Gata4, and Tbx proteins as Rel gene targets. We suggest that the p50 subunit could limit the prohypertrophic actions of c-Rel in the normal heart, because p50 overexpression in H9c2 cells repressed c-Rel levels and the absence of cardiac p50 was associated with increases in both c-Rel levels and cardiac hypertrophy. We report for the first time that c-Rel is highly expressed and confined to the nuclei of diseased adult human hearts but is restricted to the cytoplasm of normal cardiac tissues. We conclude that c-Rel-dependent signaling is critical for both cardiac remodeling and hypertrophy. Targeting its activities could offer a novel therapeutic strategy to limit the effects of cardiac disease.
Asunto(s)
Cardiomegalia/etiología , Miocardio/patología , FN-kappa B/fisiología , Proteínas Proto-Oncogénicas c-rel/fisiología , Angiotensinas/farmacología , Animales , Presión Sanguínea/fisiología , Cardiomegalia/metabolismo , Cardiomegalia/patología , Fibrosis , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Subunidad p50 de NF-kappa B/metabolismo , Subunidad p50 de NF-kappa B/fisiología , Proteínas Proto-Oncogénicas c-rel/deficiencia , Proteínas Proto-Oncogénicas c-rel/genética , Transducción de Señal/fisiología , Remodelación Ventricular/fisiologíaRESUMEN
OBJECTIVE: Tissue glucocorticoid (GC) levels are regulated by the GC-activating enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1). This enzyme is expressed in cells and tissues arising from mesenchymal stromal cells. Proinflammatory cytokines dramatically increase expression of 11ß-HSD1 in stromal cells, an effect that has been implicated in inflammatory arthritis, osteoporosis, obesity, and myopathy. Additionally, GCs act synergistically with proinflammatory cytokines to further increase enzyme expression. The present study was undertaken to investigate the mechanisms underlying this regulation. METHODS: Gene reporter analysis, rapid amplification of complementary DNA ends (RACE), chemical inhibition experiments, and genetic disruption of intracellular signaling pathways in mouse embryonic fibroblasts (MEFs) were used to define the molecular mechanisms underlying the regulation of 11ß-HSD1 expression. RESULTS: Gene reporter, RACE, and chemical inhibitor studies demonstrated that the increase in 11ß-HSD1 expression with tumor necrosis factor α (TNFα)/interleukin-1ß (IL-1ß) occurred via the proximal HSD11B1 gene promoter and depended on NF-κB signaling. These findings were confirmed using MEFs with targeted disruption of NF-κB signaling, in which RelA (p65) deletion prevented TNFα/IL-1ß induction of 11ß-HSD1. GC treatment did not prevent TNFα-induced NF-κB nuclear translocation. The synergistic enhancement of TNFα-induced 11ß-HSD1 expression with GCs was reproduced by specific inhibitors of p38 MAPK. Inhibitor and gene deletion studies indicated that the effects of GCs on p38 MAPK activity occurred primarily through induction of dual-specificity phosphatase 1 expression. CONCLUSION: The mechanism by which stromal cell expression of 11ß-HSD1 is regulated is novel and distinct from that in other tissues. These findings open new opportunities for development of therapeutic interventions aimed at inhibiting or stimulating local GC levels in cells of mesenchymal stromal lineage during inflammation.
Asunto(s)
Artritis Reumatoide/metabolismo , Glucocorticoides/metabolismo , Células Madre Mesenquimatosas/metabolismo , Osteoartritis/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Animales , Artritis Reumatoide/patología , Células Cultivadas , Citocinas/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Inflamación/metabolismo , Inflamación/patología , Interleucina-1beta/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/patología , Ratones , FN-kappa B/metabolismo , Osteoartritis/patología , Membrana Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
NF-κB1-dependent signaling directs the development of CD4(+) Th2 cells during allergic airway inflammation and protective responses to helminth infection. Here, we show that IL-4 and IL-13 production is NF-κB1-dependent in mouse OVA-specific CD4(+) (OTII) T cells responding to alum-precipitated OVA (alumOVA) immunization. More surprisingly, we found that NF-κB1 deficiency in OTII cells also selectively impairs their CXCR5 induction by alumOVA without affecting upregulation of BCL6, IL-21, OX40 and CXCR4 mRNA and PD-1 protein. This results in functional impairment of follicular helper T cells. Thus, fewer germinal center B cells develop in LN responses to alumOVA in T-cell-deficient mice reconstituted with NF-κB1(-/-) OTII cells as opposed to NF-κB1(+/+) OTII cells, while plasma cell numbers are comparable. Unlike CXCR5 induction in CD4(+) T cells, NF-κB1-deficient recirculating follicular B cells are shown to express normal levels of CXCR5. The selective effects of NF-κB1-deficiency on Th2 and follicular helper T cell induction do not appear to be due to altered expression of the Th2-associated transcription factors - GATA-3, c-Maf and Ikaros. Altogether, these results suggest that NF-κB1 regulates the expression of CXCR5 on CD4(+) T cells primed in vivo, and thus selectively controls the T-cell-dependent germinal center component of B-cell response to alumOVA.
Asunto(s)
Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/metabolismo , FN-kappa B/metabolismo , Receptores CXCR5/metabolismo , Células Th2/metabolismo , Traslado Adoptivo , Compuestos de Alumbre/administración & dosificación , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Células Cultivadas , Centro Germinal/patología , Inmunización , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , FN-kappa B/genética , FN-kappa B/inmunología , Ovalbúmina/inmunología , Receptores CXCR5/genética , Receptores CXCR5/inmunología , Células Th2/inmunología , Células Th2/patología , Regulación hacia Arriba/genéticaRESUMEN
The development of secondary lymphoid organs, such as lymph nodes (LNs), in the embryo results from the reciprocal action between lymphoid tissue inducer (LTi) cells and stromal cells. However, the initial events inducing LN anlagen formation before the LTi stromal cells cross-talk interactions take place are not fully elucidated. In this study, we show that the inguinal LN anlagen in mouse embryos developed from mesenchymal cells surrounding the lymph sacs, spherical structures of endothelial cells that bud from veins. Using inguinal and mesenteric LNs (mLNs), we provide evidence supporting a two-step maturation model for stromal cells: first, ICAM-1(-)VCAM-1(-) mesenchymal precursor cells become ICAM-1(int)VCAM-1(int) cells, in a process independent of LTi cells and lymphotoxin beta receptor (LTbetaR) signaling. The second step involves the maturation of ICAM-1(int)VCAM-1(int) cells to ICAM-1(high)VCAM-1(high) mucosal addressin cell adhesion molecule-1(+) organizer cells and depends on both LTi cells and LTbetaR. Addition of alphaLTbetaR agonist to LN organ cultures was sufficient to induce ICAM-1(int)VCAM-1(int) cells to mature. In LtbetaR(-/-) embryos, both inguinal and mLN stromal cells showed a block at the ICAM-1(int)VCAM-1(int) stage, and, contrary to inguinal LNs, mLNs persist longer and contained LTi cells, which correlated with the sustained gene expression of Il-7, Cxcl13, and, to a lesser degree, Ccl21. Taken together, these results highlight the importance of the signals and cellular interactions that induce the maturation of stromal cells and ultimately lead to the formation of lymphoid tissues.
Asunto(s)
Diferenciación Celular/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/embriología , Animales , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Diferenciación Celular/genética , Endotelio Linfático/citología , Endotelio Linfático/embriología , Endotelio Linfático/metabolismo , Inmunofenotipificación , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de Órganos , Células del Estroma/citología , Células del Estroma/inmunología , Células del Estroma/metabolismo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
The thymic medulla represents a key site for the induction of T cell tolerance. In particular, autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) provide a spectrum of tissue-restricted Ags that, through both direct presentation and cross-presentation by dendritic cells, purge the developing T cell repertoire of autoimmune specificities. Despite this role, the mechanisms of Aire(+) mTEC development remain unclear, particularly those stages that occur post-Aire expression and represent mTEC terminal differentiation. In this study, in mouse thymus, we analyze late-stage mTEC development in relation to the timing and requirements for Aire and involucrin expression, the latter a marker of terminally differentiated epithelium including Hassall's corpuscles. We show that Aire expression and terminal differentiation within the mTEC lineage are temporally separable events that are controlled by distinct mechanisms. We find that whereas mature thymocytes are not essential for Aire(+) mTEC development, use of an inducible ZAP70 transgenic mouse line--in which positive selection can be temporally controlled--demonstrates that the emergence of involucrin(+) mTECs critically depends upon the presence of mature single positive thymocytes. Finally, although initial formation of Aire(+) mTECs depends upon RANK signaling, continued mTEC development to the involucrin(+) stage maps to activation of the LTα-LTßR axis by mature thymocytes. Collectively, our results reveal further complexity in the mechanisms regulating thymus medulla development and highlight the role of distinct TNFRs in initial and terminal differentiation stages in mTECs.
Asunto(s)
Diferenciación Celular/inmunología , Células Epiteliales/citología , Linfotoxina-alfa/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Timo/citología , Animales , Separación Celular , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Linfotoxina-alfa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Precursores de Proteínas/inmunología , Precursores de Proteínas/metabolismo , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Autotolerancia/inmunología , Linfocitos T/metabolismo , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismo , Proteína AIRERESUMEN
UNLABELLED: In this study, we determined the role of the nuclear factor-kappaB (NF-kappaB) subunit c-Rel in liver injury and regeneration. In response to toxic injury of the liver, c-Rel null (c-rel(-/-)) mice displayed a defect in the neutrophilic inflammatory response, associated with impaired induction of RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted; also known as CCL5). The subsequent fibrogenic/wound-healing response to both chronic carbon tetrachloride and bile duct ligation induced injury was also impaired and this was associated with deficiencies in the expression of fibrogenic genes, collagen I and alpha-smooth muscle actin, by hepatic stellate cells. We additionally report that c-Rel is required for the normal proliferative regeneration of hepatocytes in response to toxic injury and partial hepatectomy. Absence of c-Rel was associated with blunted and delayed induction of forkhead box M1 (FoxM1) and its downstream targets cyclin B1 and Cdc25C. Furthermore, isolated c-rel(-/-) hepatocytes expressed reduced levels of FoxM1 and a reduced rate of basal and epidermal growth factor-induced DNA synthesis. Chromatin immunoprecipitation revealed that c-Rel binding to the FoxM1 promoter is induced in the regenerating liver. CONCLUSION: c-Rel has multiple functions in the control of liver homeostasis and regeneration and is a transcriptional regulator of FoxM1 and compensatory hepatocyte proliferation.
Asunto(s)
Hepatitis/etiología , Hepatocitos/citología , FN-kappa B/fisiología , Cicatrización de Heridas/fisiología , Animales , Proliferación Celular , Regeneración Hepática , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Alternative pathway NF-κB signalling regulates susceptibility towards developing inflammatory bowel disease (IBD), colitis-associated cancer and sepsis-associated intestinal epithelial cell apoptosis and shedding. However, the cell populations responsible for the perturbed alternative pathway NF-κB signalling in intestinal mucosal pathology remain unclear. In order to investigate the contribution of the epithelial compartment, we have tested whether NF-κB2 regulated transcription in intestinal epithelial cells controls the intestinal epithelial response to cytokines that are known to disrupt intestinal barrier permeability. Enteroids were generated from the proximal, middle and distal regions of small intestine (SI) from C57BL/6J wild-type mice and displayed region-specific morphology that was maintained during sub-culture. Enteroids treated with 100 ng/mL TNF were compared with corresponding regions of SI from C57BL/6J mice treated systemically with 0.33 mg/kg TNF for 1.5 h. TNF-induced apoptosis in all regions of the intestine in vitro and in vivo but resulted in Paneth cell degranulation only in proximal tissue-derived SI and enteroids. TNF also resulted in increased enteroid sphericity (quantified as circularity from two-dimensional bright field images). This response was dose and time-dependent and correlated with active caspase-3 immunopositivity. Proximal tissue-derived enteroids generated from Nfκb2-/- mice showed a significantly blunted circularity response following the addition of TNF, IFNγ, lipopolysaccharide (LPS) activated C57BL/6J-derived bone marrow-derived dendritic cells (BMDC) and secreted factors from LPS-activated BMDCs. However, Nfκb1-/- mouse-derived enteroids showed no significant changes in response to these stimuli. In conclusion, the selection of SI region is important when designing enteroid studies as region-specific identity and response to stimuli such as TNF are maintained in culture. Intestinal epithelial cells are at least partially responsible for regulating their own fate by modulating NF-κB2 signalling in response to stimuli known to be involved in multiple intestinal and systemic diseases. Future studies are warranted to investigate the therapeutic potential of intestinal epithelial NF-κB2 inhibition.
Asunto(s)
Células de la Médula Ósea/citología , Células Dendríticas/metabolismo , Enterocitos/metabolismo , Subunidad p52 de NF-kappa B/metabolismo , Organoides/metabolismo , Transducción de Señal , Animales , Apoptosis/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Degranulación de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Células Dendríticas/efectos de los fármacos , Enterocitos/citología , Enterocitos/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Interferón gamma/farmacología , Intestino Delgado/metabolismo , Lipopolisacáridos/farmacología , Ratones Endogámicos C57BL , Células de Paneth/efectos de los fármacos , Células de Paneth/metabolismo , Reproducibilidad de los Resultados , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The oncogenic Epstein-Barr virus (EBV)-encoded latent infection membrane protein 1 (LMP1) constitutively activates the 'canonical' NF-kappaB pathway that involves the phosphorylation and degradation of IkappaBalpha downstream of the IkappaB kinases (IKKs). In this study, we show that LMP1 also promotes the proteasome-mediated proteolysis of p100 NF-kappaB2 resulting in the generation of active p52, which translocates to the nucleus in complex with the p65 and RelB NF-kappaB subunits. LMP1-induced NF-kappaB transactivation is reduced in nf-kb2(-/-) mouse embryo fibroblasts, suggesting that p100 processing contributes to LMP1-mediated NF-kappaB transcriptional effects. This pathway is likely to operate in vivo, as the expression of LMP1 in primary EBV-positive Hodgkin's lymphoma and nasopharyngeal carcinoma biopsies correlates with the nuclear accumulation of p52. Interestingly, while the ability of LMP1 to activate the canonical NF-kappaB pathway is impaired in cells lacking IKKgamma/NEMO, the regulatory subunit of the IKK complex, p100 processing remains unaffected. As a result, nuclear translocation of p52, but not p65, occurs in the absence of IKKgamma. These data point to the existence of a novel signalling pathway that regulates NF-kappaB in LMP1-expressing cells, and may thereby play a role in both oncogenic transformation and the establishment of persistent EBV infection.
Asunto(s)
FN-kappa B/metabolismo , Transducción de Señal , Proteínas de la Matriz Viral/metabolismo , Transporte Activo de Núcleo Celular , Animales , Línea Celular Tumoral , Ensayo de Cambio de Movilidad Electroforética , Humanos , Quinasa I-kappa B , Ratones , Subunidad p52 de NF-kappa B , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Transcripción ReIA , Activación Transcripcional , Transfección , Proteínas de la Matriz Viral/genéticaRESUMEN
The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (Bénézech et al.). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP(+) embryonic adipose tissue and make a LN/ EYFP(+) fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP(+) fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.
Asunto(s)
Tejido Adiposo/citología , Quimera/fisiología , Ganglios Linfáticos/citología , Células del Estroma/citología , Tejido Adiposo/embriología , Animales , Animales Recién Nacidos , Quimera/embriología , Transferencia de Embrión/métodos , Embrión de Mamíferos/citología , Femenino , Ganglios Linfáticos/embriología , Masculino , Ratones , EmbarazoRESUMEN
A key role of the thymic medulla is to negatively select autoreactive CD4(+) and CD8(+) thymocytes, a process important for T cell tolerance induction. However, the involvement of the thymic medulla in other aspects of αß T cell development, including the generation of Foxp3(+) natural regulatory T cells (nTreg cells) and the continued maturation of positively selected conventional αß T cells, is unclear. We show that newly generated conventional CD69(+)Qa2(-) CD4 single-positive thymocytes mature to the late CD69(-)Qa2(+) stage in the absence of RelB-dependent medullary thymic epithelial cells (mTECs). Furthermore, an increasing ability to continue maturation extrathymically is observed within the CD69(+)CCR7(-/lo)CCR9(+) subset of conventional SP4 thymocytes, providing evidence for an independence from medullary support by the earliest stages after positive selection. In contrast, Foxp3(+) nTreg cell development is medullary dependent, with mTECs fostering the generation of Foxp3(-)CD25(+) nTreg cell precursors at the CD69(+)CCR7(+)CCR9(-) stage. Our results demonstrate a differential requirement for the thymic medulla in relation to CD4 conventional and Foxp3(+) thymocyte lineages, in which an intact mTEC compartment is a prerequisite for Foxp3(+) nTreg cell development through the generation of Foxp3(-)CD25(+) nTreg cell precursors.
Asunto(s)
Diferenciación Celular/fisiología , Factores de Transcripción Forkhead/inmunología , Linfocitos T Reguladores/inmunología , Timocitos/inmunología , Timo/inmunología , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/inmunología , Factores de Transcripción Forkhead/genética , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores CCR/genética , Receptores CCR/inmunología , Receptores CCR7/genética , Receptores CCR7/inmunología , Linfocitos T Reguladores/citología , Timocitos/citología , Timo/citología , Factor de Transcripción ReIB/genética , Factor de Transcripción ReIB/inmunologíaRESUMEN
The gut barrier, composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, prevents the entrance of harmful microorganisms, antigens and toxins from the gut lumen into the blood. Small intestinal homeostasis is normally maintained by the rate of shedding of senescent enterocytes from the villus tip exactly matching the rate of generation of new cells in the crypt. However, in various localized and systemic inflammatory conditions, intestinal homeostasis can be disturbed as a result of increased IEC shedding. Such pathological IEC shedding can cause transient gaps to develop in the epithelial barrier and result in increased intestinal permeability. Although pathological IEC shedding has been implicated in the pathogenesis of conditions such as inflammatory bowel disease, our understanding of the underlying mechanisms remains limited. We have therefore developed a murine model to study this phenomenon, because IEC shedding in this species is morphologically analogous to humans. IEC shedding was induced by systemic lipopolysaccharide (LPS) administration in wild-type C57BL/6 mice, and in mice deficient in TNF-receptor 1 (Tnfr1(-/-)), Tnfr2 (Tnfr2(-/-)), nuclear factor kappa B1 (Nfκb1(-/-)) or Nfĸb2 (Nfĸb2(-/-)). Apoptosis and cell shedding was quantified using immunohistochemistry for active caspase-3, and gut-to-circulation permeability was assessed by measuring plasma fluorescence following fluorescein-isothiocyanate-dextran gavage. LPS, at doses ≥0.125 mg/kg body weight, induced rapid villus IEC apoptosis, with peak cell shedding occurring at 1.5 hours after treatment. This coincided with significant villus shortening, fluid exudation into the gut lumen and diarrhea. A significant increase in gut-to-circulation permeability was observed at 5 hours. TNFR1 was essential for LPS-induced IEC apoptosis and shedding, and the fate of the IECs was also dependent on NFκB, with signaling via NFκB1 favoring cell survival and via NFκB2 favoring apoptosis. This model will enable investigation of the importance and regulation of pathological IEC apoptosis and cell shedding in various diseases.
Asunto(s)
Apoptosis/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Lipopolisacáridos/farmacología , Modelos Animales , Animales , Caspasa 3/metabolismo , Activación Enzimática , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , Intestino Delgado/enzimología , Intestino Delgado/patología , Lipopolisacáridos/administración & dosificación , Ratones , FN-kappa B/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Secondary lymphoid tissues such as lymph nodes are essential for the interactions between antigen presenting cells and lymphocytes that result in adaptive immune responses that protect the host against invading pathogens. The specialized architecture of these organs facilitates the cognate interactions between antigen-loaded dendritic cells and lymphocytes expressing their specific receptor as well as B-T cell interactions that are at the core of long lasting adaptive immune responses. Lymph nodes develop during embryogenesis as a result of a series of cross-talk interactions between a hematopoietically derived cell lineage called lymphoid tissue inducer cells and stromal cells of mesenchymal origin to form the anlagen of these organs. This review will present an overview of the different signaling pathways and maturation steps that mesenchymal cells undergo during the process of lymph node formation such as cell specification, priming, and maturation to become lymphoid tissue stromal organizer cells.