RESUMEN
Behçet disease (BD) and relapsing polychondritis (RP) are chronic multisystem disorders characterized by recurrent flare-ups of tissue inflammation. Major clinical manifestations of BD are oral aphthae, genital aphthous ulcers, skin lesions, arthritis, and uveitis. Patients with BD may develop rare but serious neural, intestinal, and vascular complications, with high relapse rates. Meanwhile, RP is characterized by the inflammation of the cartilaginous tissues of the ears, nose, peripheral joints, and tracheobronchial tree. Additionally, it affects the proteoglycan-rich structures in the eyes, inner ear, heart, blood vessels, and kidneys. The mouth and genital ulcers with inflamed cartilage (MAGIC) syndrome is a common characteristic of BD and RP. The immunopathology of these two diseases may be closely related. It is established that the genetic predisposition to BD is related to the human leukocyte antigen (HLA)-B51 gene. Skin histopathology demonstrates the overactivation of innate immunity, such as neutrophilic dermatitis/panniculitis, in patients with BD. Monocytes and neutrophils frequently infiltrate cartilaginous tissues of patients with RP. Somatic mutations in UBA1, which encodes a ubiquitylation-related enzyme, cause vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic syndrome (VEXAS) with severe systemic inflammation and activation of myeloid cells. VEXAS prompts auricular and/or nasal chondritis, with neutrophilic infiltration around the cartilage in 52-60% of patients. Thus, innate immune cells may play an important role in the initiation of inflammatory processes underlying both diseases. This review summarizes the recent advances in our understanding of the innate cell-mediated immunopathology of BD and RP, with a focus on the common and distinct features of these mechanisms.
RESUMEN
Homozygous Dab1 yotari mutant mice, Dab1yot (yot/yot) mice, have an autosomal recessive mutation of Dab1 and show reeler-like phenotype including histological abnormality of the cerebellum, hippocampus, and cerebral cortex. We here show abnormal hippocampal development of yot/yot mice where granule cells and pyramidal cells fail to form orderly rows but are dispersed diffusely in vague multiplicative layers. Possibly due to the positioning failure of granule cells and pyramidal cells and insufficient synaptogenesis, axons of the granule cells did not extend purposefully to connect with neighboring regions in yot/yot mice. We found that both hippocampal granule cells and pyramidal cells of yot/yot mice expressed proteins reactive with the anti-Dab1 antibody. We found that Y198- phosphorylated Dab1 of yot/yot mice was greatly decreased. Accordingly the downstream molecule, Akt was hardly phosphorylated. Especially, synapse formation was defective and the distribution of neurons was scattered in hippocampus of yot/yot mice. Some of neural cell adhesion molecules and hippocampus associated transcription factors of the neurons were expressed aberrantly, suggesting that the Reelin-Dab1 signaling pathway seemed to be importantly involved in not only neural migration as having been shown previously but also neural maturation and/or synaptogenesis of the mice. It is interesting to clarify whether the defective neural maturation is a direct consequence of the dysfunctional Dab1, or alternatively secondarily due to the Reelin-Dab1 intracellular signaling pathways.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/fisiología , Proteínas de la Matriz Extracelular/fisiología , Hipocampo/anomalías , Ratones Mutantes/anomalías , Proteínas del Tejido Nervioso/fisiología , Serina Endopeptidasas/fisiología , Transducción de Señal/fisiología , Animales , Moléculas de Adhesión Celular Neuronal/deficiencia , Movimiento Celular , Activación Enzimática , Proteínas de la Matriz Extracelular/deficiencia , Genes Recesivos , Hipocampo/embriología , Hipocampo/metabolismo , Hipocampo/patología , Homocigoto , Ratones , Ratones Mutantes/genética , Ratones Mutantes/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Moléculas de Adhesión de Célula Nerviosa/biosíntesis , Moléculas de Adhesión de Célula Nerviosa/genética , Fenotipo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Reelina , Serina Endopeptidasas/deficiencia , Sinapsis/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Studies have shown that mutant calreticulin (CALR) constitutively activates the thrombopoietin (TPO) receptor MPL and thus plays a causal role in the development of myeloproliferative neoplasms (MPNs). To further elucidate the molecular mechanism by which mutant CALR promotes MPN development, we studied the subcellular localization of mutant CALR and its importance for the oncogenic properties of mutant CALR. Here, mutant CALR accumulated in the Golgi apparatus, and its entrance into the secretion pathway and capacity to interact with N-glycan were required for its oncogenic capacity via the constitutive activation of MPL. Mutant CALR-dependent MPL activation was resistant to blockade of intracellular protein trafficking, suggesting that MPL is activated before reaching the cell surface. However, removal of MPL from the cell surface with trypsin shut down downstream activation, implying that the surface localization of MPL is required for mutant CALR-dependent activation. Furthermore, we found that mutant CALR and MPL interact on the cell surface. Based on these findings, we propose a model in which mutant CALR induces MPL activation on the cell surface to promote MPN development.
Asunto(s)
Calreticulina/genética , Mutación/genética , Receptores de Trombopoyetina/genética , Vías Secretoras/genética , Línea Celular , Transformación Celular Neoplásica/genética , Humanos , Trastornos Mieloproliferativos/genética , Transducción de Señal/genética , Tripsina/genéticaRESUMEN
Studies have previously shown that mutant calreticulin (CALR), found in a subset of patients with myeloproliferative neoplasms (MPNs), interacts with and subsequently promotes the activation of the thrombopoietin receptor (MPL). However, the molecular mechanism behind the activity of mutant CALR remains unknown. Here we show that mutant, but not wild-type, CALR interacts to form a homomultimeric complex. This intermolecular interaction among mutant CALR proteins depends on their carboxyl-terminal domain, which is generated by a unique frameshift mutation found in patients with MPN. With a competition assay, we demonstrated that the formation of mutant CALR homomultimers is required for the binding and activation of MPL. Since association with MPL is required for the oncogenicity of mutant CALR, we propose a model in which the constitutive activation of the MPL downstream pathway by mutant CALR multimers induces the development of MPN. This study provides a potential novel therapeutic strategy against mutant CALR-dependent tumorigenesis via targeting the intermolecular interaction among mutant CALR proteins.
Asunto(s)
Calreticulina/química , Transformación Celular Neoplásica/patología , Leucemia Eritroblástica Aguda/patología , Proteínas Mutantes/química , Mutación , Receptores de Trombopoyetina/metabolismo , Calreticulina/genética , Calreticulina/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Humanos , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformación Proteica , Multimerización de Proteína , Trombopoyetina/genética , Trombopoyetina/metabolismo , Células Tumorales CultivadasRESUMEN
Somatic mutations in the calreticulin (CALR) gene have been found in most patients with JAK2- and MPL-unmutated Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). It has recently been shown that mutant CALR constitutively activates the thrombopoietin receptor MPL and, thus, plays a causal role in the development of MPNs. However, the roles of mutant CALR in human haematopoietic cell differentiation remain predominantly elusive. To examine the impact of the 5-base insertion mutant CALR gene (Ins5) on haematopoietic cell differentiation, we generated induced pluripotent stem cells from an essential thrombocythaemia (ET) patient harbouring a CALR-Ins5 mutation and from a healthy individual (WT). Megakaryopoiesis was more prominent in Ins5-haematopoietic progenitor cells (Ins5-HPCs) than in WT-HPCs, implying that the system recapitulates megakaryocytosis observed in the bone marrow of CALR-mutant ET patients. Ins5-HPCs exhibited elevated expression levels of GATA1 and GATA2, suggesting a premature commitment to megakaryocytic differentiation in progenitor cells. We also demonstrated that 3-hydroxy anagrelide markedly perturbed megakaryopoiesis, but not erythropoiesis. Collectively, we established an in vitro model system that recapitulates megakaryopoiesis caused by mutant CALR. This system can be used to validate therapeutic compounds for MPN patients harbouring CALR mutations and in detailed studies on mutant CALR in human haematological cell differentiation.
Asunto(s)
Calreticulina/metabolismo , Diferenciación Celular , Células Madre Hematopoyéticas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Megacariocitos/metabolismo , Mutación , Mielopoyesis , Calreticulina/genética , Femenino , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo , Células Madre Hematopoyéticas/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Masculino , Megacariocitos/citologíaRESUMEN
Recurrent somatic mutations of calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains elusive. Here, we found that the expression of mutant but not wild-type CALR induces the thrombopoietin (TPO)-independent growth of UT-7/TPO cells. We demonstrated that c-MPL, the TPO receptor, is required for this cytokine-independent growth of UT-7/TPO cells. Mutant CALR preferentially associates with c-MPL that is bound to Janus kinase 2 (JAK2) over the wild-type protein. Furthermore, we demonstrated that the mutant-specific carboxyl terminus portion of CALR interferes with the P-domain of CALR to allow the N-domain to interact with c-MPL, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induces the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked by JAK2 inhibitor treatment. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in induced pluripotent stem cell-derived hematopoietic stem cells harboring the CALR mutation. These findings imply that mutant CALR activates the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes myeloproliferative neoplasm development by activating c-MPL and its downstream pathway.
Asunto(s)
Calreticulina/metabolismo , Neoplasias Hematológicas/metabolismo , Trastornos Mieloproliferativos/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Trombopoyetina/metabolismo , Calreticulina/genética , Línea Celular Tumoral , Células HEK293 , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/mortalidad , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Janus Quinasa 2/metabolismo , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Proteínas de Neoplasias/genética , Fosforilación , Estructura Terciaria de Proteína , Receptores de Trombopoyetina/genética , Trombopoyesis/genética , Trombopoyetina/metabolismoRESUMEN
Recent studies have revealed negligible immunogenicity of induced pluripotent stem (iPS) cells in syngeneic mice and in autologous monkeys. Therefore, human iPS cells would not elicit immune responses in the autologous setting. However, given that human leukocyte antigen (HLA)-matched allogeneic iPS cells would likely be used for medical applications, a more faithful model system is needed to reflect HLA-matched allogeneic settings. Here we examined whether iPS cells induce immune responses in the swine leukocyte antigen (SLA)-matched setting. iPS cells were generated from the SLA-defined C1 strain of Clawn miniature swine, which were confirmed to develop teratomas in mice, and transplanted into the testes (nâ=â4) and ovary (nâ=â1) of C1 pigs. No teratomas were found in pigs on 47 to 125 days after transplantation. A Mixed lymphocyte reaction revealed that T-cell responses to the transplanted MHC-matched (C1) iPS cells were significantly lower compared to allogeneic cells. The humoral immune responses were also attenuated in the C1-to-C1 setting. More importantly, even MHC-matched iPS cells were susceptible to innate immunity, NK cells and serum complement. iPS cells lacked the expression of SLA class I and sialic acids. The in vitro cytotoxic assay showed that C1 iPS cells were targeted by NK cells and serum complement of C1. In vivo, the C1 iPS cells developed larger teratomas in NK-deficient NOG (T-B-NK-) mice (nâ=â10) than in NK-competent NOD/SCID (T-B-NK+) mice (nâ=â8) (p<0.01). In addition, C1 iPS cell failed to form teratomas after incubation with the porcine complement-active serum. Taken together, MHC-matched iPS cells can attenuate cellular and humoral immune responses, but still susceptible to innate immunity in pigs.
Asunto(s)
Células Madre Pluripotentes Inducidas/trasplante , Células Asesinas Naturales/inmunología , Complejo Mayor de Histocompatibilidad , Porcinos Enanos/inmunología , Porcinos/inmunología , Teratoma/etiología , Animales , Células Cultivadas , Femenino , Antígenos de Histocompatibilidad Clase I , Antígenos de Histocompatibilidad Clase II , Inmunidad Humoral , Inmunidad Innata , Células Madre Pluripotentes Inducidas/inmunología , Masculino , Ratones , Ratones SCID , Ovario/inmunología , Testículo/inmunología , Trasplante AutólogoRESUMEN
BACKGROUND: The development and validation of stem cell therapies using induced pluripotent stem (iPS) cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naïve type characteristics, it is an indispensable challenge to develop naïve type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells. METHODOLOGY/SIGNIFICANT PRINCIPAL FINDINGS: In this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM) from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4-8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM) oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3%) was similar to that after the injection of ICMs into morulae (24/29, 82.8%). We also found that 4-8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%). After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively. CONCLUSION/SIGNIFICANCE: Our findings indicate that the aggregation method using parthenogenetic morulae or 4-8 cell-stage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated cells, such as iPS cells, based on their ability to form chimeras.
Asunto(s)
Blastocisto/fisiología , Quimera/embriología , Células Madre Pluripotentes Inducidas/fisiología , Mórula/fisiología , Partenogénesis/genética , Animales , Blastocisto/citología , Diferenciación Celular , Células Cultivadas , Quimera/crecimiento & desarrollo , Femenino , Fertilización In Vitro , Feto , Humanos , Células Madre Pluripotentes Inducidas/citología , Masculino , Mórula/citología , Oocitos/fisiología , Técnicas Reproductivas Asistidas , PorcinosRESUMEN
In pluripotent stem cells (PSCs), there are 2 types: naive and primed. Only the naive type has the capacity for producing chimeric offspring. Mouse PSCs are naive, but human PSCs are in the primed state. Previously reported porcine PSCs appear in the primed state. In this study, putative naive porcine-induced pluripotent stem cells (iPSCs) were generated. Porcine embryonic fibroblasts were transduced with retroviral vectors expressing Yamanaka's 4 genes. Emergent colonies were propagated in the presence of porcine leukemia inhibitory factor (pLIF) and forskolin. The cells expressed pluripotency markers and formed embryoid bodies, which gave rise to cell types from all 3 embryonic germ layers. The naive state of the cells was demonstrated by pLIF dependency, 2 active X chromosomes (when female), absent MHC class I expression, and characteristic gene expression profiles. The porcine iPSCs contributed to the in vitro embryonic development (11/24, 45.8%) as assessed by fluorescent markers. They also contributed to the in utero fetal development (11/71, 15.5% at day 23; 1/13, 7.7% at day 65). This is the first demonstration of macroscopic fluorescent chimeras derived from naive-like porcine PSCs, although adult chimeras remain to be produced.