RESUMEN
Pianp (also known as Leda-1) is a type I transmembrane protein with preferential expression in the mammalian CNS. Its processing is characterized by proteolytic cleavage by a range of proteases including Adam10, Adam17, MMPs, and the γ-secretase complex. Pianp can interact with Pilrα and the GB1a subunit of the GABAB receptor (GBR) complex. A recent case description of a boy with global developmental delay and homozygous nonsense variant in PIANP supports the hypothesis that PIANP is involved in the control of behavioral traits in mammals. To investigate the physiological functions of Pianp, constitutive, global knockout mice were generated and comprehensively analyzed. Broad assessment did not indicate malformation or malfunction of internal organs. In the brain, however, decreased sizes and altered cellular compositions of the dentate gyrus as well as the cerebellum, including a lower number of cerebellar Purkinje cells, were identified. Functionally, loss of Pianp led to impaired presynaptic GBR-mediated inhibition of glutamate release and altered gene expression in the cortex, hippocampus, amygdala, and hypothalamus including downregulation of Erdr1, a gene linked to autism-like behavior. Behavioral phenotyping revealed that Pianp deficiency leads to context-dependent enhanced anxiety and spatial learning deficits, an altered stress response, severely impaired social interaction, and enhanced repetitive behavior, which all represent characteristic features of an autism spectrum disorder-like phenotype. Altogether, Pianp represents a novel candidate gene involved in autism-like behavior, cerebellar and hippocampal pathology, and GBR signaling.
Asunto(s)
Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Cerebelo/patología , Eliminación de Gen , Hipocampo/patología , Proteínas del Tejido Nervioso/deficiencia , Receptores de GABA-B/metabolismo , Animales , Trastorno Autístico/genética , Trastorno Autístico/metabolismo , Cerebelo/metabolismo , Modelos Animales de Enfermedad , Femenino , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Cofilin is an essential actin remodeling protein promoting depolymerization and severing of actin filaments. To address the relevance of cofilin for the development and function of T cells in vivo, we generated knock-in mice in which T-cell-specific nonfunctional (nf) cofilin was expressed instead of wild-type (WT) cofilin. Nf cofilin mice lacked peripheral αß T cells and showed a severe thymus atrophy. This was caused by an early developmental arrest of thymocytes at the double negative (DN) stage. Importantly, even though DN thymocytes expressed the TCRß chain intracellularly, they completely lacked TCRß surface expression. In contrast, nf cofilin mice possessed normal numbers of γδ T cells. Their functionality was confirmed in the γδ T-cell-driven, imiquimod (IMQ)-induced, psoriasis-like murine model. Overall, this study not only highlights the importance of cofilin for early αß T-cell development but also shows for the first time that an actin-binding protein is differentially involved in αß versus γδ T-cell development.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Timo/metabolismo , Factores Despolimerizantes de la Actina/química , Animales , Movimiento Celular , Técnicas de Sustitución del Gen , Humanos , Células Jurkat , Ratones , Mutación/genética , Prolina/metabolismo , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Timocitos/metabolismoRESUMEN
Chronic tubulointerstitial damage with tubular epithelial atrophy and interstitial fibrosis is the hallmark of chronic kidney disease (CKD) and a predictor for progression of CKD.Several experiments have now provided evidence that the Wnt signaling pathways are significantly contributing to atrophy and fibrosis; in contrast, it also has been shown that the Wnt system fosters regenerative processes in acute tubular injury.We now have demonstrated that Dickkopf 3 (DKK3) is an agonist for canonical Wnt signaling in CKD and fosters chronic fibrosing inflammation of the tubulointerstitial compartment. Genetic- and antibody-mediated inhibition of DKK3 leads to a pronounced improvement of tubular differentiation and a reduction in fibrosis.In addition, the secreted glycoprotein DKK3 can be used as a non-invasive urinary marker for the extent of CKD in man.
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Péptidos y Proteínas de Señalización Intercelular/metabolismo , Insuficiencia Renal Crónica/metabolismo , Vía de Señalización Wnt , Proteínas Adaptadoras Transductoras de Señales , Animales , Quimiocinas , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Insuficiencia Renal Crónica/genéticaRESUMEN
One third of patients with inflammatory bowel disease (IBD) inadequately respond to anti-TNF treatment and preclinical data suggest that matrix metalloproteinase-9 (MMP-9) is a novel therapeutic target. Here we show that IBD clinical and histopathological parameters found in wild type mice challenged with three different models of colitis, acute and chronic dextran sodium sulphate (DSS), and acute 2,4,6-trinitrobenzenesulfonic acid-induced colitis are not attenuated in MMP-9 knockout mice. We find similar colonic gene expression profiles in wild type and MMP-9 knockout mice in control and acute DSS conditions with the exception of eleven genes involved in antimicrobial response during colitis. Parameters of chronic colitis are similar in wild type and MMP-9 knockout mice. Pharmacological inhibition of MMP-9 with bio-active peptides does not improve DSS colitis. We suggest that MMP-9 upregulation is a consequence rather than a cause of intestinal inflammation and we question whether MMP-9 represents a disease target in IBD.
Asunto(s)
Colitis Ulcerosa/tratamiento farmacológico , Enfermedad de Crohn/tratamiento farmacológico , Fármacos Gastrointestinales/uso terapéutico , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz/uso terapéutico , Animales , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/patología , Colon/efectos de los fármacos , Colon/patología , Enfermedad de Crohn/inducido químicamente , Enfermedad de Crohn/patología , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Femenino , Fármacos Gastrointestinales/farmacología , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Resultado del Tratamiento , Ácido Trinitrobencenosulfónico/toxicidad , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
Microvascular endothelial cells (ECs) are increasingly recognized as organ-specific gatekeepers of their microenvironment. Microvascular ECs instruct neighboring cells in their organ-specific vascular niches through angiocrine factors, which include secreted growth factors (angiokines), extracellular matrix molecules, and transmembrane proteins. However, the molecular regulators that drive organ-specific microvascular transcriptional programs and thereby regulate angiodiversity are largely elusive. In contrast to other ECs, which form a continuous cell layer, liver sinusoidal ECs (LSECs) constitute discontinuous, permeable microvessels. Here, we have shown that the transcription factor GATA4 controls murine LSEC specification and function. LSEC-restricted deletion of Gata4 caused transformation of discontinuous liver sinusoids into continuous capillaries. Capillarization was characterized by ectopic basement membrane deposition, formation of a continuous EC layer, and increased expression of VE-cadherin. Correspondingly, ectopic expression of GATA4 in cultured continuous ECs mediated the downregulation of continuous EC-associated transcripts and upregulation of LSEC-associated genes. The switch from discontinuous LSECs to continuous ECs during embryogenesis caused liver hypoplasia, fibrosis, and impaired colonization by hematopoietic progenitor cells, resulting in anemia and embryonic lethality. Thus, GATA4 acts as master regulator of hepatic microvascular specification and acquisition of organ-specific vascular competence, which are indispensable for liver development. The data also establish an essential role of the hepatic microvasculature in embryonic hematopoiesis.
Asunto(s)
Diferenciación Celular/fisiología , Embrión de Mamíferos/enzimología , Células Endoteliales/metabolismo , Endotelio/embriología , Factor de Transcripción GATA4/metabolismo , Hematopoyesis/fisiología , Hígado/embriología , Animales , Capilares/embriología , Factor de Transcripción GATA4/genética , Hígado/irrigación sanguínea , Ratones , Ratones Transgénicos , Especificidad de Órganos/fisiologíaRESUMEN
Microvascular endothelial cells (ECs) display a high degree of phenotypic and functional heterogeneity among different organs. Organ-specific ECs control their tissue microenvironment by angiocrine factors in health and disease. Liver sinusoidal endothelial cells (LSECs) are uniquely differentiated to fulfill important organ-specific functions in development, under homeostatic conditions, and in regeneration and liver pathology. Recently, Bmp2 has been identified by us as an organ-specific angiokine derived from LSECs. To study angiocrine Bmp2 signaling in the liver, we conditionally deleted Bmp2 in LSECs using EC subtype-specific Stab2-Cre mice. Genetic inactivation of hepatic angiocrine Bmp2 signaling in Stab2-Cre;Bmp2fl/fl (Bmp2LSECKO) mice caused massive iron overload in the liver and increased serum iron levels and iron deposition in several organs similar to classic hereditary hemochromatosis. Iron overload was mediated by decreased hepatic expression of hepcidin, a key regulator of iron homeostasis. Thus, angiocrine Bmp2 signaling within the hepatic vascular niche represents a constitutive pathway indispensable for iron homeostasis in vivo that is nonredundant with Bmp6. Notably, we demonstrate that organ-specific angiocrine signaling is essential not only for the homeostasis of the respective organ but also for the homeostasis of the whole organism.
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Proteína Morfogenética Ósea 2/genética , Células Endoteliales/metabolismo , Hemocromatosis/genética , Hepcidinas/genética , Homeostasis/genética , Hierro/metabolismo , Hígado/metabolismo , Animales , Proteína Morfogenética Ósea 2/deficiencia , Proteína Morfogenética Ósea 6/genética , Proteína Morfogenética Ósea 6/metabolismo , Capilares/metabolismo , Capilares/patología , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Células Endoteliales/patología , Femenino , Regulación de la Expresión Génica , Hemocromatosis/metabolismo , Hemocromatosis/patología , Hepatocitos/metabolismo , Hepatocitos/patología , Hepcidinas/metabolismo , Integrasas/genética , Integrasas/metabolismo , Hígado/irrigación sanguínea , Hígado/patología , Masculino , Ratones , Ratones Transgénicos , Comunicación Paracrina , Transducción de Señal , Transcripción GenéticaRESUMEN
Suppressor of cytokine signaling 1 (SOCS1) is a negative feedback inhibitor of cytoplasmic Janus kinase and signal transducer and activator of transcription (STAT) signaling. SOCS1 also contains a nuclear localization sequence (NLS), yet, the in vivo importance of nuclear translocation is unknown. We generated transgenic mice containing mutated Socs1ΔNLS that fails to translocate in the cell nucleus (MGLtg mice). Whereas mice fully deficient for SOCS1 die within the first 3 weeks due to excessive interferon signaling and multiorgan inflammation, mice expressing only non-nuclear Socs1ΔNLS (Socs1-/-MGLtg mice) were rescued from early lethality. Canonical interferon gamma signaling was still functional in Socs1-/-MGLtg mice as shown by unaltered tyrosine phosphorylation of STAT1 and whole genome expression analysis. However, a subset of NFκB inducible genes was dysregulated. Socs1-/-MGLtg mice spontaneously developed low-grade inflammation in the lung and had elevated Th2-type cytokines. Upon ovalbumin sensitization and challenge, airway eosinophilia was increased in Socs1-/-MGLtg mice. Decreased transepithelial electrical resistance in trachea epithelial cells from Socs1-/-MGLtg mice suggests disrupted epithelial cell barrier. The results indicate that nuclear SOCS1 is a regulator of local immunity in the lung and unravel a so far unrecognized function for SOCS1 in the cell nucleus.
RESUMEN
Renal tubular atrophy and interstitial fibrosis are common hallmarks of etiologically different progressive chronic kidney diseases (CKD) that eventually result in organ failure. Even though these pathological manifestations constitute a major public health problem, diagnostic tests, as well as therapeutic options, are currently limited. Members of the dickkopf (DKK) family, DKK1 and -2, have been associated with inhibition of Wnt signaling and organ fibrosis. Here, we identify DKK3 as a stress-induced, tubular epithelia-derived, secreted glycoprotein that mediates kidney fibrosis. Genetic as well as antibody-mediated abrogation of DKK3 led to reduced tubular atrophy and decreased interstitial matrix accumulation in two mouse models of renal fibrosis. This was facilitated by an amplified, antifibrogenic, inflammatory T cell response and diminished canonical Wnt/ß-catenin signaling in stressed tubular epithelial cells. Moreover, in humans, urinary DKK3 levels specifically correlated with the extent of tubular atrophy and interstitial fibrosis in different glomerular and tubulointerstitial diseases. In summary, our data suggest that DKK3 constitutes an immunosuppressive and a profibrotic epithelial protein that might serve as a potential therapeutic target and diagnostic marker in renal fibrosis.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Enfermedades Renales/patología , Riñón/patología , Insuficiencia Renal Crónica/patología , Proteínas Adaptadoras Transductoras de Señales , Animales , Atrofia , Fibrosis , Enfermedades Renales/metabolismo , Túbulos Renales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Nefritis Intersticial/metabolismo , Nefritis Intersticial/patología , Insuficiencia Renal Crónica/metabolismo , Vía de Señalización WntRESUMEN
Stabilin-1 is a multifunctional scavenger receptor expressed on alternatively-activated macrophages. Stabilin-1 mediates phagocytosis of "unwanted-self" components, intracellular sorting, and endocytic clearance of extracellular ligands including SPARC that modulates breast cancer growth. The expression of stabilin-1 was found on tumor-associated macrophages (TAM) in mouse and human cancers including melanoma, lymphoma, glioblastoma, and pancreatic insulinoma. Despite its tumor-promoting role in mouse models of melanoma and lymphoma the expression and functional role of stabilin-1 in breast cancer was unknown. Here, we demonstrate that stabilin-1 is expressed on TAM in human breast cancer, and its expression is most pronounced on stage I disease. Using stabilin-1 knockout (ko) mice we show that stabilin-1 facilitates growth of mouse TS/A mammary adenocarcinoma. Endocytosis assay on stabilin-1 ko TAM demonstrated impaired clearance of stabilin-1 ligands including SPARC that was capable of inducing cell death in TS/A cells. Affymetrix microarray analysis on purified TAM and reporter assays in stabilin-1 expressing cell lines demonstrated no influence of stabilin-1 expression on intracellular signalling. Our results suggest stabilin-1 mediated silent clearance of extracellular tumor growth-inhibiting factors (e.g. SPARC) as a mechanism of stabilin-1 induced tumor growth. Silent clearance function of stabilin-1 makes it an attractive candidate for delivery of immunomodulatory anti-cancer therapeutic drugs to TAM.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias de la Mama/metabolismo , Moléculas de Adhesión Celular Neuronal/biosíntesis , Neoplasias Mamarias Experimentales/metabolismo , Receptores Mensajeros de Linfocitos/biosíntesis , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular Neuronal/genética , Procesos de Crecimiento Celular/fisiología , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptores Mensajeros de Linfocitos/genética , TransfecciónRESUMEN
In adult mice, lymphopenia-induced proliferation (LIP) leads to T cell activation, memory differentiation, tissue destruction, and a loss of TCR diversity. Neonatal mice are lymphopenic within the first week of life. This enables some recent thymic emigrants to undergo LIP and convert into long-lived memory T cells. Surprisingly, however, most neonatal T cells do not undergo LIP. We therefore asked whether neonate-specific mechanisms prevent lymphopenia-driven T cell activation. In this study, we show that IL-7R-dependent innate lymphoid cells (ILCs) block LIP of CD8(+) T cells in neonatal but not adult mice. Importantly, CD8(+) T cell responses against a foreign Ag are not inhibited by neonatal ILCs. This ILC-based inhibition of LIP ensures the generation of a diverse naive T cell pool in lymphopenic neonates that is mandatory for the maintenance of T cell homeostasis and immunological self-tolerance later in life.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Tolerancia Inmunológica , Memoria Inmunológica , Activación de Linfocitos , Linfopenia/inmunología , Traslado Adoptivo , Animales , Animales Recién Nacidos , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular , Proliferación Celular , Homeostasis , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Receptores de Interleucina-7/inmunología , Transducción de SeñalRESUMEN
Understanding the molecular mechanisms regulating T cell reactivity is required for successful reprogramming of immune responses in medical conditions, characterized by dysfunctions of the immune system. Nck proteins are cytoplasmic adaptors mediating diverse cellular functions, including TCR signaling. By enhancing TCR signal strength, Nck proteins influence thymic selection and regulate the size and sensitivity of the peripheral T cell repertoire. Here, we investigated the contribution of Nck proteins to CD4(+) T cell differentiation and effector function using Nck.T(-/-) mice. Impaired GC formation and reduced Tfh were observed in Nck.T(-/-) mice after immunization with T cell-dependent antigens. Th2/Tfh-related cytokines, such as IL-4, IL-10, and IL-21, were decreased in Nck.T(-/-) mice T cells. Moreover, an increased susceptibility to cell death of Tfh cells in Nck.T(-/-) mice was associated with decreased levels of Akt phosphorylation. As a result of this dysregulation in Tfh cells of Nck.T(-/-) mice, we found impaired production and affinity maturation of antibodies against T cell-dependent antigens. Thus, Nck proteins not only participate in thymic selection and generation of the peripheral T cell repertoire but also are involved in the differentiation and effector functions of CD4(+) T cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Diferenciación Celular , Proteínas Oncogénicas/metabolismo , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Animales , Formación de Anticuerpos , Apoptosis , Citocinas/biosíntesis , Eliminación de Gen , Centro Germinal/citología , Humanos , Ratones , Proteínas Oncogénicas/deficiencia , Factores de Transcripción/metabolismoRESUMEN
Keratinocytes have a pivotal role in the regulation of immune responses, but the impact of antigen presentation by these cells is still poorly understood, particularly in a situation where the antigen will be presented only in adult life. Here, we generated a transgenic mouse model in which keratinocytes exclusively present a myelin basic protein (MBP) peptide covalently linked to the major histocompatibility complex class II ß-chain, solely under inflammatory conditions. In these mice, inflammation caused by epicutaneous contact sensitizer treatment resulted in keratinocyte-mediated expansion of MBP-specific CD4(+) T cells in the skin. Moreover, repeated contact sensitizer application preceding a systemic MBP immunization reduced the reactivity of the respective CD4(+) T cells and lowered the symptoms of the resulting experimental autoimmune encephalomyelitis. This downregulation was CD4(+) T-cell-mediated and dependent on the presence of the immune modulator Dickkopf-3. Thus, presentation of a neo self-antigen by keratinocytes in the inflamed, adult skin can modulate CD4(+) T-cell auto-aggression at a distal organ.
Asunto(s)
Autoantígenos/metabolismo , Linfocitos T CD4-Positivos/patología , Dermatitis/metabolismo , Dermatitis/patología , Queratinocitos/metabolismo , Queratinocitos/patología , Proteínas Adaptadoras Transductoras de Señales , Animales , Linfocitos T CD4-Positivos/metabolismo , Ciclopropanos/farmacología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/prevención & control , Genes MHC Clase II/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteína Básica de Mielina/genética , Oxazoles/farmacología , Piel/efectos de los fármacos , Piel/metabolismo , Piel/patologíaRESUMEN
Neuropathic pain is a major, intractable clinical problem and its pathophysiology is not well understood. Although recent gene expression profiling studies have enabled the identification of novel targets for pain therapy, classical study designs provide unclear results owing to the differential expression of hundreds of genes across sham and nerve-injured groups, which can be difficult to validate, particularly with respect to the specificity of pain modulation. To circumvent this, we used two outbred lines of rats, which are genetically similar except for being genetically segregated as a result of selective breeding for differences in neuropathic pain hypersensitivity. SerpinA3N, a serine protease inhibitor, was upregulated in the dorsal root ganglia (DRG) after nerve injury, which was further validated for its mouse homolog. Mice lacking SerpinA3N developed more neuropathic mechanical allodynia than wild-type (WT) mice, and exogenous delivery of SerpinA3N attenuated mechanical allodynia in WT mice. T lymphocytes infiltrate the DRG after nerve injury and release leukocyte elastase (LE), which was inhibited by SerpinA3N derived from DRG neurons. Genetic loss of LE or exogenous application of a LE inhibitor (Sivelastat) in WT mice attenuated neuropathic mechanical allodynia. Overall, we reveal a novel and clinically relevant role for a member of the serpin superfamily and a leukocyte elastase and crosstalk between neurons and T cells in the modulation of neuropathic pain.
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Proteínas de Fase Aguda/metabolismo , Inhibidores Enzimáticos/farmacología , Elastasa de Leucocito/antagonistas & inhibidores , Serpinas/metabolismo , Linfocitos T/citología , Animales , Separación Celular , Dependovirus/genética , Femenino , Ganglios Espinales/metabolismo , Hiperalgesia/fisiopatología , Hibridación in Situ , Masculino , Ratones , Ratones Transgénicos , Neuralgia , Neuronas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Dolor/fisiopatología , Fenotipo , Reacción en Cadena de la Polimerasa , Ratas , Regulación hacia ArribaRESUMEN
The adaptive immune system protects organisms from harmful environmental insults. In parallel, regulatory mechanisms control immune responses in order to assure preservation of organ integrity. Yet, molecules involved in the control of T-cell responses in peripheral tissues are poorly characterized. Here, we investigated the function of Dickkopf-3 in the modulation of local T-cell reactivity. Dkk3 is a secreted, mainly tissue-derived protein with highest expression in organs considered as immune-privileged such as the eye, embryo, placenta, and brain. While T-cell development and activation status in naïve Dkk3-deficient mice was comparable to littermate controls, we found that Dkk3 contributes to the immunosuppressive microenvironment that protects transplanted, class-I mismatched embryoid bodies from T-cell-mediated rejection. Moreover, genetic deletion or antibody-mediated neutralization of Dkk3 led to an exacerbated experimental autoimmune encephalomyelitis (EAE). This phenotype was accompanied by a change of T-cell polarization displayed by an increase of IFNγ-producing T cells within the central nervous system. In the wild-type situation, Dkk3 expression in the brain was up-regulated during the course of EAE in an IFNγ-dependent manner. In turn, Dkk3 decreased IFNγ activity and served as part of a negative feedback mechanism. Thus, our findings suggest that Dkk3 functions as a tissue-derived modulator of local CD4(+) and CD8(+) T-cell responses.
RESUMEN
The mechanisms responsible for the generation of a mature B1 and B2 cell compartment are still poorly understood. In this study, we demonstrated that absence of Dickkopf-3 (DKK3) led to changes in the composition of the B cell compartment, which were due to an altered development and maintenance program of B cells. Development of B2 cells was impaired at the pre- and immature B cell stage, resulting in decreased numbers of follicular B cells in adult DKK3-deficient mice. Furthermore, DKK3 limited B1 cell self-maintenance in the periphery, by decreasing the survival and proliferation behavior of B1 cells. DKK3 may act via the BCR signaling pathway, as Ca(2+) influx upon BCR stimulation was increased and SiglecG, a molecule shown to inhibit Calcium signaling, was downregulated in the absence of DKK3. DKK3-deficient mice exhibited altered Ab responses and an increased secretion of the cytokine IL-10. Additionally, DKK3 limited autoimmunity in a model of systemic lupus erythematosus. In summary, we identified DKK3 as a novel modulator interfering with B cell fate as well as the maintenance program of B cells, leading to changes in B cell immune responses.
Asunto(s)
Linfocitos B/inmunología , Proliferación Celular , Péptidos y Proteínas de Señalización Intercelular/inmunología , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Apoptosis/genética , Apoptosis/inmunología , Autoanticuerpos/inmunología , Autoanticuerpos/metabolismo , Linfocitos B/metabolismo , Calcio/inmunología , Calcio/metabolismo , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Células Cultivadas , Citocinas/inmunología , Citocinas/metabolismo , Citometría de Flujo , Expresión Génica/inmunología , Humanos , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Péptidos y Proteínas de Señalización Intercelular/genética , Lectinas/genética , Lectinas/inmunología , Lectinas/metabolismo , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Recuento de Linfocitos , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Ratones Noqueados , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico , Transducción de Señal/genéticaRESUMEN
Mesenchymal stem cells (MSCs) are known to limit immune responses in vivo by multiple soluble factors. Dickkopf-3 (DKK3), a secreted glycoprotein, has recently been identified as a novel immune modulator. Since DKK3 has been reported to be produced by MSCs, we investigated whether DKK3 contributes to the immune suppression of anti-tumor responses by MSCs. Whereas wild-type MSCs inhibited immune responses against two different transplantation tumors, DKK3-deficient MSCs did not affect the rejection process. Increased CD8(+) T cell and reduced M2-type macrophages infiltration was observed in tumors inoculated together with DKK3-deficient MSCs. Thus, DKK3 could alter the composition of the tumor stroma, thereby supporting the MSCs-mediated suppression of immune responses against these tumor transplants.
RESUMEN
L1 cell adhesion molecule (L1CAM) is overexpressed in many human cancers, confers bad prognosis and augments cell motility, invasion and metastasis. Results from xenograft mouse models suggested that L1CAM antibodies might be promising tools for cancer therapy. Here, we generated human L1CAM-transgenic mice to study therapeutic efficacy and putative side effects in a model system. We established three transgenic lines (M2, M3 and F4) expressing the human L1CAM transgene in brain, kidney and colon with decreasing intensity (M2, M3 > F4). The expression pattern was similar to that of L1CAM in humans. No interference of the transgene with the expression of endogenous L1CAM was observed. Immunohistochemical analysis revealed correct expression of the transgene in mouse cortex and collective duct of the kidney. Injection of (125)I-labeled L1CAM antibodies resulted in specific enrichment in the kidney but not in the brain. The injection of the therapeutic anti-human L1CAM mAb L1-9.3/2a into transgenic mice even at high doses did not cause behavioral changes or other side effects. Similar results were obtained using a mouse specific L1CAM mAb in normal mice. Tumor therapy experiments were performed using syngeneic mouse tumor cells (RET melanoma and Panc02 pancreatic adenocarcinoma) transduced with human L1CAM. MAb L1-9.3/2a efficiently and specifically attenuated local tumor growth in both model systems without apparent side effects. The therapeutic effect was dependent on immune effector mechanisms. Analysis of Panc02-huL1CAM tumors after therapy showed elevated levels of EGF and evidence of immune-induced epithelial-mesenchymal transition. The results suggest that our transgenic mice are valuable tools to study L1CAM-based antibody therapy.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal , Melanoma/terapia , Molécula L1 de Adhesión de Célula Nerviosa/antagonistas & inhibidores , Neoplasias Pancreáticas/terapia , Adenocarcinoma/genética , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Adenocarcinoma/terapia , Animales , Western Blotting , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Técnicas para Inmunoenzimas , Radioisótopos de Yodo/uso terapéutico , Melanoma/genética , Melanoma/inmunología , Melanoma/patología , Ratones , Ratones Transgénicos , Molécula L1 de Adhesión de Célula Nerviosa/inmunología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , ARN Mensajero/genética , Radioinmunoterapia , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation.
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Exosomas/metabolismo , Sistema Hematopoyético/metabolismo , Inflamación/metabolismo , Células de Purkinje/metabolismo , ARN Mensajero/metabolismo , Animales , Integrasas , Ratones Transgénicos , Recombinación GenéticaRESUMEN
The limited availability of experimental tumor models that faithfully mimic the progression of human tumors and their response to therapy remains a major bottleneck to the clinical translation and application of novel therapeutic principles. To address this challenge in hepatocellular carcinoma (HCC), one of the deadliest and most common cancers in the world, we developed and validated an inducible model of hepatocarcinogenesis in adult mice. Tumorigenesis was triggered by intravenous adenoviral delivery of Cre recombinase in transgenic mice expressing the hepatocyte-specific albumin promoter, a loxP-flanked stop cassette, and the SV40 large T-antigen (iAST). Cre recombinase-mediated excision of the stop cassette led to a transient viral hepatitis and resulted in multinodular tumorigenesis within 5 to 8 weeks. Tumor nodules with histologic characteristics of human HCC established a functional vasculature by cooption, remodeling, and angiogenic expansion of the preexisting sinusoidal liver vasculature with increasing signs of vascular immaturity during tumor progression. Treatment of mice with sorafenib rapidly resulted in the induction of vascular regression, inhibition of tumor growth, and enhanced overall survival. Vascular regression was characterized by loss of endothelial cells leaving behind avascular type IV collagen-positive empty sleeves with remaining pericytes. Sorafenib treatment led to transcriptional changes of Igf1, Id1, and cMet over time, which may reflect the emergence of potential escape mechanisms. Taken together, our results established the iAST model of inducible hepatocarcinogenesis as a robust and versatile preclinical model to study HCC progression and validate novel therapies.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Modelos Animales de Enfermedad , Neoplasias Hepáticas Experimentales/irrigación sanguínea , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Niacinamida/análogos & derivados , Compuestos de Fenilurea/farmacología , Animales , Humanos , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Niacinamida/farmacología , SorafenibRESUMEN
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a malignant tumour that is characterized by extensive vascular remodelling and responsiveness to treatment with the anti-angiogenic multikinase inhibitor sorafenib. The aim was to study endothelial remodelling in HCC. METHODS: The murine inducible albumin-SV40-large T-antigen model and two tissue microarrays (TMA) with 295 tumourous and 83 peri-tumourous samples of 296 patients with HCC were analysed for expression of liver sinusoidal endothelial cell (LSEC)-specific marker proteins, stabilin-1 and stabilin-2, LYVE-1 and CD32b. RESULTS: LSEC marker proteins were sequentially lost during HCC progression in the murine HCC model being absent from tumour nodules larger than 800 µm in diameter. Similarly, the TMA analysis of human HCCs revealed loss of all four marker proteins in the majority of tumourous tissue samples. Preservation of LYVE-1 expression showed a significant correlation with low grading (G1). In corresponding peri-tumourous liver tissue, loss of all marker proteins was seen in a minor proportion of cases (34%) while the majority of cases retained expression of at least one of the marker proteins. Loss of stabilin-2 expression in peri-tumourous liver tissue of patients with HCC was significantly less likely to occur (38%) than loss of the other marker proteins (63-95%) and it was associated with significantly longer tumour-specific (P = 0.0523) and overall (P = 0.0338) survival. Loss of stabilin-2 may enhance survival in HCC by preventing endothelial-tumour cell adhesive interactions and microvascular invasion. CONCLUSIONS: In summary, endothelial transdifferentiation is a major pathogenic event in HCC development indicating a switch from vessel co-option/intussusceptive angiogenesis to sprouting angiogenesis.