RESUMEN
Human embryonic stem cells (hESCs) have an abbreviated G1 phase of the cell cycle that allows rapid proliferation and maintenance of pluripotency. Lengthening of G1 corresponds to loss of pluripotency during differentiation. However, precise mechanisms that link alterations in the cell cycle and early differentiation remain to be defined. We investigated initial stages of mesendodermal lineage commitment in hESCs, and observed a cell cycle pause. Transcriptome profiling identified several genes with known roles in regulation of the G2/M transition that were differentially expressed early during lineage commitment. WEE1 kinase, which blocks entry into mitosis by phosphorylating CDK1 at Y15, was the most highly expressed of these genes. Inhibition of CDK1 phosphorylation by a specific inhibitor of WEE1 restored cell cycle progression by preventing the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal, but not ectodermal, lineages. Functionally, WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. Our findings identify a novel G2 cell cycle pause that is required for endodermal differentiation and provide important new mechanistic insights into early events of lineage commitment. Stem Cells 2016;34:1765-1775.
Asunto(s)
Puntos de Control del Ciclo Celular , Diferenciación Celular , Linaje de la Célula , Células Madre Embrionarias/citología , Fase G2 , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/genética , Linaje de la Célula/genética , Análisis por Conglomerados , Células Madre Embrionarias/metabolismo , Endodermo/citología , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Mesodermo/citología , Modelos Biológicos , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Histamine (HA) is a key regulator of experimental allergic encephalomyelitis (EAE), the autoimmune model of multiple sclerosis. HA exerts its effects through four known G-protein-coupled receptors: H1, H2, H3, and H4 (histamine receptors; H(1-4)R). Using HR-deficient mice, our laboratory has demonstrated that H1R, H2R, H3R, and H4R play important roles in EAE pathogenesis, by regulating encephalitogenic T cell responses, cytokine production by APCs, blood-brain barrier permeability, and T regulatory cell activity, respectively. Histidine decarboxylase-deficient mice (HDCKO), which lack systemic HA, exhibit more severe EAE and increased Th1 effector cytokine production by splenocytes in response to myelin oligodendrocyte gp35-55. In an inverse approach, we tested the effect of depleting systemic canonical HA signaling on susceptibility to EAE by generating mice lacking all four known G-protein-coupled-HRs (H(1-4)RKO mice). In this article, we report that in contrast to HDCKO mice, H(1-4)RKO mice develop less severe EAE compared with wild-type animals. Furthermore, splenocytes from immunized H(1-4)RKO mice, compared with wild-type mice, produce a lower amount of Th1/Th17 effector cytokines. The opposing results seen between HDCKO and H1-4RKO mice suggest that HA may signal independently of H1-4R and support the existence of an alternative HAergic pathway in regulating EAE resistance. Understanding and exploiting this pathway has the potential to lead to new disease-modifying therapies in multiple sclerosis and other autoimmune and allergic diseases.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Histamina/metabolismo , Histidina Descarboxilasa/genética , Receptores Histamínicos/genética , Receptores Histamínicos/metabolismo , Animales , Células Presentadoras de Antígenos , Barrera Hematoencefálica/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular , Células Cultivadas , Citocinas/biosíntesis , Encefalomielitis Autoinmune Experimental/metabolismo , Histidina Descarboxilasa/deficiencia , Histidina Descarboxilasa/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esclerosis Múltiple/inmunología , Glicoproteína Mielina-Oligodendrócito/farmacología , Fragmentos de Péptidos/farmacología , Receptores Histamínicos/deficiencia , Transducción de SeñalRESUMEN
Experimental autoimmune orchitis (EAO), the principal model of non-infectious testicular inflammatory disease, can be induced in susceptible mouse strains by immunization with autologous testicular homogenate and appropriate adjuvants. As previously established, the genome of DBA/2J mice encodes genes that are capable of conferring dominant resistance to EAO, while the genome of BALB/cByJ mice does not and they are therefore susceptible to EAO. In a genome scan, we previously identified Orch3 as the major quantitative trait locus controlling dominant resistance to EAO and mapped it to chromosome 11. Here, by utilizing a forward genetic approach, we identified kinesin family member 1C (Kif1c) as a positional candidate for Orch3 and, using a transgenic approach, demonstrated that Kif1c is Orch3. Mechanistically, we showed that the resistant Kif1c(D2) allele leads to a reduced antigen-specific T cell proliferative response as a consequence of decreased MHC class II expression by antigen presenting cells, and that the L(578) â P(578) and S(1027) â P(1027) polymorphisms distinguishing the BALB/cByJ and DBA/2J alleles, respectively, can play a role in transcriptional regulation. These findings may provide mechanistic insight into how polymorphism in other kinesins such as KIF21B and KIF5A influence susceptibility and resistance to human autoimmune diseases.
Asunto(s)
Resistencia a la Enfermedad/genética , Genes Dominantes , Cinesinas/genética , Orquitis , Alelos , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Expresión Génica , Genes MHC Clase II , Predisposición Genética a la Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Orquitis/genética , Orquitis/inmunología , Sitios de Carácter Cuantitativo/genética , Testículo/inmunologíaRESUMEN
The uterotropic response of the uterus to 17ß-estradiol (E2) is genetically controlled, with marked variation observed depending on the mouse strain studied. Previous genetic studies from our laboratory using inbred mice that are high [C57BL/6J (B6)] or low [C3H/HeJ (C3H)] responders to E2 led to the identification of quantitative trait (QT) loci associated with phenotypic variation in uterine growth and leukocyte infiltration. The mechanisms underlying differential responsiveness to E2, and the genes involved, are unknown. Therefore, we used a microarray approach to show association of distinct E2-regulated transcriptional signatures with genetically controlled high and low responses to E2 and their segregation in (C57BL/6J×C3H/HeJ) F1 hybrids. Among the 6664 E2-regulated transcripts, analysis of cellular functions of those that were strain specific indicated C3H-selective enrichment of apoptosis, consistent with a 7-fold increase in the apoptosis indicator CASP3, and a 2.4-fold decrease in the apoptosis inhibitor Naip1 (Birc1a) in C3H vs. B6 following treatment with E2. In addition, several differentially expressed transcripts reside within our previously identified QT loci, including the ERα-tethering factor Runx1, demonstrated to enhance E2-mediated transcript regulation. The level of RUNX1 in uterine epithelial cells was shown to be 3.5-fold greater in B6 compared to C3H. Our novel insights into the mechanisms underlying the genetic control of tissue sensitivity to estrogen have great potential to advance understanding of individualized effects in physiological and disease states.
Asunto(s)
Caspasa 3/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Estradiol/farmacología , Receptor alfa de Estrógeno/genética , Proteína Inhibidora de la Apoptosis Neuronal/genética , Transcripción Genética/genética , Útero/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proliferación Celular/efectos de los fármacos , Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Células Epiteliales/metabolismo , Femenino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos , Peroxidasa/genética , Análisis por Matrices de Proteínas , Sitios de Carácter Cuantitativo/fisiología , Transcripción Genética/efectos de los fármacos , Transcriptoma , Útero/efectos de los fármacos , Útero/crecimiento & desarrolloRESUMEN
Histamine is a biogenic amine that mediates multiple physiological processes, including immunomodulatory effects in allergic and inflammatory reactions, and also plays a key regulatory role in experimental allergic encephalomyelitis, the autoimmune model of multiple sclerosis. The pleiotropic effects of histamine are mediated by four G protein-coupled receptors, as follows: Hrh1/H(1)R, Hrh2/H(2)R, Hrh3/H(3)R, and Hrh4/H(4)R. H(4)R expression is primarily restricted to hematopoietic cells, and its role in autoimmune inflammatory demyelinating disease of the CNS has not been studied. In this study, we show that, compared with wild-type mice, animals with a disrupted Hrh4 (H(4)RKO) develop more severe myelin oligodendrocyte glycoprotein (MOG)(35\x{2013}55)-induced experimental allergic encephalomyelitis. Mechanistically, we also show that H(4)R plays a role in determining the frequency of T regulatory (T(R)) cells in secondary lymphoid tissues, and regulates T(R) cell chemotaxis and suppressor activity. Moreover, the lack of H(4)R leads to an impairment of an anti-inflammatory response due to fewer T(R) cells in the CNS during the acute phase of the disease and an increase in the proportion of Th17 cells.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Receptores Acoplados a Proteínas G/fisiología , Receptores Histamínicos/fisiología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Animales , Barrera Hematoencefálica/inmunología , Recuento de Linfocito CD4 , Permeabilidad de la Membrana Celular/genética , Permeabilidad de la Membrana Celular/inmunología , Células Cultivadas , Encefalomielitis Autoinmune Experimental/genética , Glicoproteínas/administración & dosificación , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Glicoproteína Mielina-Oligodendrócito , Neuronas/inmunología , Neuronas/patología , Fragmentos de Péptidos/administración & dosificación , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Receptores Histamínicos/deficiencia , Receptores Histamínicos/genética , Receptores Histamínicos H4 , Índice de Severidad de la Enfermedad , Linfocitos T Reguladores/metabolismoRESUMEN
Vasectomy is a well accepted global contraceptive approach frequently associated with epididymal granuloma and sperm autoantibody formation. To understand the long-term sequelae of vasectomy, we investigated the early immune response in vasectomized mice. Vasectomy leads to rapid epithelial cell apoptosis and necrosis, persistent inflammation, and sperm granuloma formation in the epididymis. Vasectomized B6AF1 mice did not mount autoimmune response but instead developed sperm antigen-specific tolerance, documented as resistance to immunization-induced experimental autoimmune orchitis (EAO) but not experimental autoimmune encephalomyelitis. Strikingly, tolerance switches over to pathologic autoimmune state following concomitant CD4(+)CD25(+)Foxp3(+) regulatory T cell (Treg) depletion: unilaterally vasectomized mice produce dominant autoantibodies to an orchitogenic antigen (zonadhesin), and develop CD4 T-cell- and antibody-dependent bilateral autoimmune orchitis. Therefore, (i) Treg normally prevents spontaneous organ-specific autoimmunity induction by persistent endogenous danger signal, and (ii) autoantigenic stimulation with sterile autoinflammation can lead to tolerance. Finally, postvasectomy tolerance occurs in B6AF1, C57BL/6, and A/J strains. However, C57BL/6 mice resisted EAO after 60% Treg depletion, but developed EAO after 97% Treg reduction. Therefore, variance in intrinsic Treg function--a possible genetic trait--can influence the divergent tolerogenic versus autoimmune response to vasectomy.
Asunto(s)
Autoinmunidad/inmunología , Tolerancia Inmunológica/inmunología , Espermatozoides/inmunología , Linfocitos T Reguladores/inmunología , Vasectomía , Animales , Autoanticuerpos/inmunología , Western Blotting , Proliferación Celular , Electroforesis en Gel de Poliacrilamida , Masculino , Proteínas de la Membrana/inmunología , Ratones , Ratones Mutantes , Estadísticas no ParamétricasRESUMEN
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system in which histamine (HA) and its receptors have been implicated in disease pathogenesis. HA exerts its effects through four different G protein-coupled receptors designated H(1)-H(4). We previously examined the effects of traditional single HA receptor (HR) knockouts (KOs) in experimental allergic encephalomyelitis (EAE), the autoimmune model of MS. Our results revealed that H(1) R and H(2) R are propathogenic, while H(3) R and H(4) R are antipathogenic. This suggests that combinatorial targeting of HRs may be an effective disease-modifying therapy (DMT) in MS. To test this hypothesis, we generated H(1) H(2) RKO and H(3) H(4) RKO mice and studied them for susceptibility to EAE. Compared with wild-type (WT) mice, H(1) H(2) RKO mice developed a less severe clinical disease course, whereas the disease course of H(3) H(4) RKO mice was more severe. H(1) H(2) RKO mice also developed less neuropathology and disrupted blood brain barrier permeability compared with WT and H(3) H(4) RKO mice. Additionally, splenocytes from immunized H(1) H(2) RKO mice produced less interferon(IFN)-γ and interleukin(IL)-17. These findings support the concept that combined pharmacological targeting of HRs may be an appropriate ancillary DMT in MS and other immunopathologic diseases.
Asunto(s)
Encefalomielitis Autoinmune Experimental/etiología , Esclerosis Múltiple/etiología , Receptores Histamínicos/fisiología , Animales , Linfocitos T CD4-Positivos/inmunología , Polaridad Celular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Acoplados a Proteínas G/fisiología , Receptores Histamínicos H1/fisiología , Receptores Histamínicos H2/fisiología , Receptores Histamínicos H3/fisiología , Receptores Histamínicos H4RESUMEN
Although several transcription factors have been shown to be critical for the induction and maintenance of IL-17 expression by CD4 Th cells, less is known about the role of nontranscriptional mechanisms. Here we show that the p38 MAPK signaling pathway is essential for in vitro and in vivo IL-17 production by regulating IL-17 synthesis in CD4 T cells through the activation of the eukaryotic translation initiation factor 4E/MAPK-interacting kinase (eIF-4E/MNK) pathway. We also show that p38 MAPK activation is required for the development and progression of both chronic and relapsing-remitting forms of experimental allergic encephalomyelitis (EAE), the principal autoimmune model of multiple sclerosis. Furthermore, we show that regulation of p38 MAPK activity specifically in T cells is sufficient to modulate EAE severity. Thus, mechanisms other than the regulation of gene expression also contribute to Th17 cell effector functions and, potentially, to the pathogenesis of other Th17 cell-mediated diseases.
Asunto(s)
Autoinmunidad , Encefalomielitis Autoinmune Experimental/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Interleucina-17/biosíntesis , Activación de Linfocitos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células Th17/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Proliferación Celular , Separación Celular , Células Cultivadas , Enfermedad Crónica , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/inmunología , Femenino , Citometría de Flujo , Humanos , Interleucina-17/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Fosforilación/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Células Th17/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/inmunologíaRESUMEN
Day 3 thymectomy (D3Tx) results in a loss of peripheral tolerance mediated by natural regulatory T cells (nTregs) and development of autoimmune ovarian dysgenesis (AOD) and autoimmune dacryoadenitis (ADA) in A/J and (C57BL/6J × A/J) F(1) hybrids (B6A), but not in C57BL/6J (B6) mice. Previously, using quantitative trait locus (QTL) linkage analysis, we showed that D3Tx-AOD is controlled by five unlinked QTL (Aod1-Aod5) and H2. In this study, using D3Tx B6-Chr(A/J)/NaJ chromosome (Chr) substitution strains, we confirm that QTL on Chr16 (Aod1a/Aod1b), Chr3 (Aod2), Chr1 (Aod3), Chr2 (Aod4), Chr7 (Aod5), and Chr17 (H2) control D3Tx-AOD susceptibility. In addition, we also present data mapping QTL controlling D3Tx-ADA to Chr17 (Ada1/H2), Chr1 (Ada2), and Chr3 (Ada3). Importantly, B6-ChrX(A/J) mice were as resistant to D3Tx-AOD and D3Tx-ADA as B6 mice, thereby excluding Foxp3 as a susceptibility gene in these models. Moreover, we report quantitative differences in the frequency of nTregs in the lymph nodes (LNs), but not spleen or thymus, of AOD/ADA-resistant B6 and AOD/ADA-susceptible A/J, B6A, and B6-Chr17(A/J) mice. Similar results correlating with experimental allergic encephalomyelitis and orchitis susceptibility were seen with B10.S and SJL/J mice. Using H2-congenic mice, we show that the observed difference in frequency of LN nTregs is controlled by Ada1/H2. These data support the existence of an LN-specific, H2-controlled mechanism regulating the prevalence of nTregs in autoimmune disease susceptibility.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Antígenos H-2/fisiología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ooforitis/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Timectomía , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/cirugía , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Cromosomas/genética , Dacriocistitis/genética , Dacriocistitis/inmunología , Susceptibilidad a Enfermedades/inmunología , Femenino , Ligamiento Genético/inmunología , Ganglios Linfáticos/metabolismo , Recuento de Linfocitos , Ratones , Ratones Endogámicos A , Ratones Endogámicos C57BL , Ratones Transgénicos , Ooforitis/genética , Sitios de Carácter Cuantitativo/inmunologíaRESUMEN
OBJECTIVE: The major histocompatibility complex (MHC) is the primary genetic contributor to multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), but multiple additional interacting loci are required for genetic susceptibility. The identity of most of these non-MHC genes is unknown. In this report, we identify genes within evolutionarily conserved genetic pathways leading to MS and EAE. METHODS: To identify non-MHC binary and quantitative trait loci (BTL/QTL) important in the pathogenesis of EAE, we generated phenotype-selected congenic mice using EAE-resistant B10.S and EAE-susceptible SJL mice. We hypothesized that genes linked to EAE BTL/QTL and MS-GWAS can be identified if they belong to common evolutionarily conserved pathways, which can be identified with a bioinformatic approach using Ingenuity software. RESULTS: Many known BTL/QTL were retained and linked to susceptibility during phenotype selection, the most significant being a region on chromosome 17 distal to H2 (Eae5). We show in pathway analysis that T helper (T(H))-cell differentiation genes are critical for both diseases. Bioinformatic analyses predicted that Eae5 is important in CD4 T-effector and/or Foxp3(+) T-regulatory cells (Tregs), and we found that B10.S-Eae5(SJL) congenic mice have significantly greater numbers of lymph node CD4 and Tregs than B10.S mice. INTERPRETATION: These results support the polygenic model of MS/EAE, whereby MHC and multiple minor loci are required for full susceptibility, and confirm a critical genetic dependence on CD4 T(H)-cell differentiation and function in the pathogenesis of both diseases.
Asunto(s)
Susceptibilidad a Enfermedades , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Complejo Mayor de Histocompatibilidad/genética , Esclerosis Múltiple/genética , Linfocitos T Colaboradores-Inductores/patología , Animales , Linfocitos T CD4-Positivos/fisiología , Biología Computacional , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/etiología , Citometría de Flujo , Adyuvante de Freund/efectos adversos , Ligamiento Genético , Estudio de Asociación del Genoma Completo , Ratones , Ratones Congénicos , Fenotipo , Sitios de Carácter Cuantitativo/genética , Estadísticas no ParamétricasRESUMEN
T cell (TC) activation requires the coordinated signaling of the T cell receptor (TCR) and coreceptor molecules, allowing TCs to respond to lower degrees of TCR occupancy. Coreceptor molecules set the threshold for TC activation by controlling different regulatory signaling loops. The Cbl family members prevent undesired activation of T cells by regulating TCR signals. In this report, we show that TC prestimulation by the CD43 coreceptor molecule before TCR engagement inhibits TCR-dependent c-Cbl tyrosine phosphorylation, c-Cbl interaction with the adapter molecule Crk-L and promotes Cbl-b degradation in a PKCθ-dependent manner. Consequently, the prolonged tyrosine phosphorylation and delayed degradation of ZAP-70 and of the ζ chain lead to enhanced mitogen-activated protein kinase activation and robust TC response. These data indicates that CD43-mediated signals lower the threshold for TC activation by restricting the c-Cbl and Cbl-b inhibitory effects on TCR signaling. In addition to the strength and duration of intracellular signals, our data underscore temporality with which certain molecules are engaged as yet another mechanism to fine tune TC signal quality, and ultimately immune function.
Asunto(s)
Leucosialina/metabolismo , Activación de Linfocitos/fisiología , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Transducción de Señal/fisiología , Linfocitos T/fisiología , Animales , Proliferación Celular , Humanos , Immunoblotting , Inmunoprecipitación , Células Jurkat , Activación de Linfocitos/inmunología , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Linfocitos T/inmunologíaRESUMEN
Histamine receptor H1 (H1R) is a susceptibility gene in both experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune orchitis (EAO), 2 classical T cell-mediated models of organ-specific autoimmune disease. Here we showed that expression of H1R in naive CD4+ T cells was required for maximal IFN-gamma production but was dispensable for proliferation. Moreover, H1R signaling at the time of TCR ligation was required for activation of p38 MAPK, a known regulator of IFN-gamma expression. Importantly, selective reexpression of H1R in CD4+ T cells fully complemented both the IFN-gamma production and the EAE susceptibility of H1R-deficient mice. These data suggest that the presence of H1R in CD4+ T cells and its interaction with histamine regulates early TCR signals that lead to Th1 differentiation and autoimmune disease.
Asunto(s)
Interferón gamma/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Histamínicos H1/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Células Cultivadas , Encefalomielitis Autoinmune Experimental/inmunología , Activación Enzimática , Histamina/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Orquitis/inmunología , Receptores Histamínicos H1/genética , Transducción de Señal/fisiología , Células TH1/citología , Células TH1/inmunologíaRESUMEN
The postnatal maternal environment is known to increase susceptibility to a number of autoimmune diseases. Here we asked whether the postnatal maternal environment could influence autoimmune disease development to day 3 thymectomy (d3tx)-induced autoimmune ovarian disease (AOD) and experimental allergic encephalomyelitis (EAE) in cross-fostered A/J and B6 mice. A/J pups foster-nursed by B6 mothers exhibit an increase in autoimmune disease development while cross-fostering B6 pups on A/J mothers did not alter their susceptibility. The increase in AOD incidence seen in foster-nursed d3tx A/J mice correlated with a decrease in the total number of CD4(+) T cells in the lymph nodes of these animals. Analysis of the cellular composition in the milk revealed that B6 mice shed significantly more maternally derived lymphocytes into their milk compared to A/J mothers. These data suggest that there are maternally derived postnatal factors that influence the development of autoimmune disease in A/J mice.
Asunto(s)
Animales Recién Nacidos/inmunología , Animales Lactantes/inmunología , Enfermedades Autoinmunes/inmunología , Susceptibilidad a Enfermedades/inmunología , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Quimiocina CXCL1/metabolismo , Citocinas/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Inmunidad Materno-Adquirida/inmunología , Interleucina-13/metabolismo , Interleucina-9/metabolismo , Lactancia/inmunología , Lactancia/metabolismo , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Enfermedades del Ovario/inmunología , TimectomíaRESUMEN
Weibel-Palade bodies within endothelial cells are secretory granules known to release von Willebrand Factor (VWF), P-selectin, chemokines, and other stored molecules following histamine exposure. Mice with a disrupted VWF gene (VWFKO) have endothelial cells that are deficient in Weibel-Palade bodies. These mice were used to evaluate the role of VWF and/or Weibel-Palade bodies in Bordetella pertussis toxin-induced hypersensitivity to histamine, a subphenotype of experimental allergic encephalomyelitis, the principal autoimmune model of multiple sclerosis. No significant differences in susceptibility to histamine between wild-type and VWFKO mice were detected after 3 days; however, histamine sensitivity persisted significantly longer in VWFKO mice. Correspondingly, encephalomyelitis onset was earlier, disease was more severe, and blood brain barrier (BBB) permeability was significantly increased in VWFKO mice, as compared with wild-type mice. Moreover, inflammation was selectively increased in the brains, but not spinal cords, of VWFKO mice as compared with wild-type mice. Early increases in BBB permeability in VWFKO mice were not due to increased encephalitogenic T-cell activity since BBB permeability did not differ in adjuvant-treated VWFKO mice as compared with littermates immunized with encephalitogenic peptide plus adjuvant. Taken together, these data indicate that VWF and/or Weibel-Palade bodies negatively regulate BBB permeability changes and autoimmune inflammatory lesion formation within the brain elicited by peripheral inflammatory stimuli.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Encéfalo/patología , Permeabilidad Capilar/fisiología , Encefalomielitis Autoinmune Experimental/patología , Factor de von Willebrand/metabolismo , Adyuvantes Inmunológicos/metabolismo , Animales , Barrera Hematoencefálica/patología , Encéfalo/inmunología , Encéfalo/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Histamina/inmunología , Hipersensibilidad/inmunología , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cuerpos de Weibel-Palade/metabolismo , Factor de von Willebrand/genéticaRESUMEN
UNLABELLED: Apical membrane antigen 1 (AMA1) is a receptor protein on the surface of Toxoplasma gondii that plays a critical role in host cell invasion. The ligand to which T gondii AMA1 (TgAMA1) binds, TgRON2, is secreted into the host cell membrane by the parasite during the early stages of invasion. The TgAMA1-TgRON2 complex forms the core of the "moving junction," a ring-shaped zone of tight contact between the parasite and host cell membranes, through which the parasite pushes itself during invasion. Paradoxically, the parasite also expresses rhomboid proteases that constitutively cleave the TgAMA1 transmembrane domain. How can TgAMA1 function effectively in host cell binding if its extracellular domain is constantly shed from the parasite surface? We show here that when TgAMA1 binds the domain 3 (D3) peptide of TgRON2, its susceptibility to cleavage by rhomboid protease(s) is greatly reduced. This likely serves to maintain parasite-host cell binding at the moving junction, a hypothesis supported by data showing that parasites expressing a hypercleavable version of TgAMA1 invade less efficiently than wild-type parasites do. Treatment of parasites with the D3 peptide was also found to reduce phosphorylation of S527 on the cytoplasmic tail of TgAMA1, and parasites expressing a phosphomimetic S527D allele of TgAMA1 showed an invasion defect. Taken together, these data suggest that TgAMA1-TgRON2 interaction at the moving junction protects TgAMA1 molecules that are actively engaged in host cell penetration from rhomboid-mediated cleavage and generates an outside-in signal that leads to dephosphorylation of the TgAMA1 cytosolic tail. Both of these effects are required for maximally efficient host cell invasion. IMPORTANCE: Nearly one-third of the world's population is infected with the protozoan parasite Toxoplasma gondii, which causes life-threatening disease in neonates and immunocompromised individuals. T. gondii is a member of the phylum Apicomplexa, which includes many other parasites of veterinary and medical importance, such as those that cause coccidiosis, babesiosis, and malaria. Apicomplexan parasites grow within their hosts through repeated cycles of host cell invasion, parasite replication, and host cell lysis. Parasites that cannot invade host cells cannot survive or cause disease. AMA1 is a highly conserved protein on the surface of apicomplexan parasites that is known to be important for invasion, and the work presented here reveals new and unexpected insights into AMA1 function. A more complete understanding of the role of AMA1 in invasion may ultimately contribute to the development of new chemotherapeutics designed to disrupt AMA1 function and invasion-related signaling in this important group of human pathogens.
Asunto(s)
Antígenos de Protozoos/metabolismo , Péptido Hidrolasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteolisis , Proteínas Protozoarias/metabolismo , Toxoplasma/fisiología , Células Cultivadas , Fibroblastos/parasitología , Humanos , Fosforilación , Unión ProteicaRESUMEN
The CD43 coreceptor molecule has been shown to participate in lymphocyte adhesion and activation. Leukocyte homotypic aggregation results from a cascade of intracellular signals delivered to the cells upon engagement of different cell-surface molecules with their natural ligands. This phenomenon requires an active metabolism, reorganization of the cytoskeleton, and relocalization of cell-surface molecules. The aim of this study was to identify some of the key members of the signaling cascade leading to T lymphocyte homotypic aggregation following CD43 engagement. CD43-mediated homotypic aggregation of T lymphocytes required the participation of Src kinases, phospholipase C-gamma2, protein kinase C, phosphatidylinositol-3 kinase, as well as extracellular-regulated kinase 1/2 and p38. Data shown here suggest that these signaling molecules play a central role in regulating actin cytoskeleton remodeling after CD43 ligation. We also evaluated the ability of immunomodulatory drugs such as leflunomide to block the CD43-mediated homotypic aggregation. Leflunomide blocked the recruitment of targets of the Src family kinases as well as actin polymerization, diminishing the ability of T lymphocytes to aggregate in response to CD43-specific signals, suggesting that this drug might control the migration and recruitment of lymphoid cells to inflamed tissues.
Asunto(s)
Antígenos CD , Inmunosupresores/farmacología , Isoxazoles/farmacología , Sialoglicoproteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T/metabolismo , Actinas/metabolismo , Agregación Celular , Precursores Enzimáticos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Células Jurkat , Leflunamida , Leucosialina , Activación de Linfocitos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfolipasa C gamma , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Quinasa Syk , Fosfolipasas de Tipo C/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos , Familia-src Quinasas/metabolismoRESUMEN
Histamine H(3) receptor (Hrh3/H(3)R) is primarily expressed by neurons in the central nervous system (CNS) where it functions as a presynaptic inhibitory autoreceptor and heteroreceptor. Previously, we identified an H(3)R-mediated central component in susceptibility to experimental allergic encephalomyelitis (EAE), the principal autoimmune model of multiple sclerosis (MS), related to neurogenic control of blood brain barrier permeability and peripheral T cell effector responses. Furthermore, we identified Hrh3 as a positional candidate for the EAE susceptibility locus Eae8. Here, we characterize Hrh3 polymorphisms between EAE-susceptible and resistant SJL and B10.S mice, respectively, and show that Hrh3 isoform expression in the CNS is differentially regulated by acute peripheral inflammatory stimuli in an allele-specific fashion. Next, we show that Hrh3 is not expressed in any subpopulations of the immune compartment, and that secondary lymphoid tissue is anatomically poised to be regulated by central H(3)R signaling. Accordingly, using transcriptome analysis, we show that, inflammatory stimuli elicit unique transcriptional profiles in the lymph nodes of H(3)RKO mice compared to WT mice, which is indicative of negative regulation of peripheral immune responses by central H(3)R signaling. These results further support a functional link between the neurogenic control of T cell responses and susceptibility to CNS autoimmune disease coincident with acute and/or chronic peripheral inflammation. Pharmacological targeting of H(3)R may therefore be useful in preventing the development and formation of new lesions in MS, thereby limiting disease progression.
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Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Predisposición Genética a la Enfermedad/genética , Receptores Histamínicos H3/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Secuencia de Aminoácidos , Animales , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Regulación de la Expresión Génica , Hematopoyesis/genética , Hematopoyesis/inmunología , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Espacio Intracelular/metabolismo , Ganglios Linfáticos/inmunología , Masculino , Ratones , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estructura Terciaria de Proteína , Receptores Histamínicos H3/químicaRESUMEN
CD4+ CD25+ regulatory T cells (Tregs) strongly influence the early and late autoimmune responses to meiotic germ cell antigens (MGCA) and the gonadal immunopathology in vasectomized mice. This is supported by the published and recently acquired information presented here. Within 24h of unilateral vasectomy (uni-vx) the ipsilateral epididymis undergoes epithelial cell apoptosis followed by necrosis, severe inflammation, and granuloma formation. Unexpectedly, vasectomy alone induced MGCA-specific tolerance. In contrast, uni-vx plus simultaneous Treg depletion resulted in MGCA-specific autoimmune response and bilateral autoimmune orchitis. Both tolerance and autoimmunity were strictly linked to the early epididymal injury. We now discovered that testicular autoimmunity in uni-vx mice did not occur when Treg depletion was delayed by one week. Remarkably, this delayed Treg depletion also prevented tolerance induction. Therefore, tolerance depends on a rapid de novo Treg response to MGCA exposed after vasectomy. Moreover, tolerance was blunted in mice genetically deficient in PD-1 ligand, suggesting the involvement of induced Treg. We conclude that pre-existing natural Treg prevents post-vasectomy autoimmunity, whereas vasectomy-induced Treg maintains post-vasectomy tolerance. We further discovered that vasectomized mice were still resistant to autoimmune orchitis induction for at least 12-16 months; thus, tolerance is long-lasting. Although significant sperm autoantibodies of low titers became detectable in uni-vx mice at 7 months, the antibody titers fluctuated over time, suggesting a dynamic "balance" between the autoimmune and tolerance states. Finally, we observed severe epididymal fibrosis and hypo-spermatogenesis at 12 months after uni-vx: findings of highly critical clinical significance.
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Epidídimo/patología , Orquitis/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Vasectomía , Animales , Autoanticuerpos/sangre , Autoinmunidad/genética , Antígenos CD4/metabolismo , Fibrosis/etiología , Humanos , Tolerancia Inmunológica/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Depleción Linfocítica , Masculino , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Orquitis/etiología , Receptor de Muerte Celular Programada 1/genética , Vasectomía/efectos adversosRESUMEN
Intrauterine or intraperitoneal administration of lipopolysaccharide (LPS) into normal mice at midgestation induces preterm delivery (PTD) within 24 h through a mechanism dependent on Toll-like receptor signaling and expression of inflammatory cytokines. The exact participants in the cellular network involved in PTD are not known. Although the activities of innate immune cells are thought to be important, the extent to which this process depends on T and B cells has yet to be examined. Mice deficient in T and B cells due to genetic deficiency in the recombination activating gene 1 (Rag1(-/-)) were given LPS intraperitoneally on Day 15 of gestation and found to be susceptible to LPS-induced PTD. This was found to involve many of the inflammatory mediators reported as important in normal mice. Moreover, at a low dose (3 microg), pregnant Rag1(-/-) mice were found to be more susceptible to PTD than a cohort of normal mice on the same genetic background. This increased susceptibility was partially reversed by transfer, on Day 10 of gestation, of whole lymphocytes or purified CD4(+) T cells. Transfer of purified CD4(+) T cells to Rag1(-/-) mice resulted in a uterine draining node population of FOXP3(+) cells, suggesting that these cells may contribute to resistance to LPS-induced PTD. Overall, the data suggest that, although T and B lymphocytes are not critical positive regulators of LPS-induced PTD, CD4(+) T cells play a protective and regulatory role, and thus could be a target for preventive or therapeutic manipulation.
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Lipopolisacáridos/toxicidad , Nacimiento Prematuro/inmunología , Nacimiento Prematuro/prevención & control , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Linfocitos B/inmunología , Secuencia de Bases , ADN Complementario/genética , Femenino , Factores de Transcripción Forkhead/inmunología , Genes RAG-1 , Lipopolisacáridos/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Embarazo , Nacimiento Prematuro/inducido químicamente , Linfocitos T/inmunología , Linfocitos T Reguladores/trasplante , Receptor Toll-Like 4/inmunologíaRESUMEN
Structural polymorphisms (L263P, M313V, and S331P) in the third intracellular loop of the murine histamine receptor H(1) (H(1)R) are candidates for Bphs, a shared autoimmune disease locus in experimental allergic encephalomyelitis and experimental allergic orchitis. The P-V-P haplotype is associated with increased disease susceptibility (H(1)R(S)) whereas the L-M-S haplotype is associated with less severe disease (H(1)R(R)). In this study, we show that selective re-expression of the H(1)R(S) allele in T cells fully complements experimental allergic encephalomyelitis susceptibility and the production of disease-associated cytokines while selective re-expression of the H(1)R(R) allele does not. Mechanistically, we show that the two H(1)R alleles exhibit differential cell surface expression and altered intracellular trafficking, with the H(1)R(R) allele being retained within the endoplasmic reticulum. Moreover, we show that all three residues (L-M-S) comprising the H(1)R(R) haplotype are required for altered expression. These data are the first to demonstrate that structural polymorphisms influencing cell surface expression of a G protein-coupled receptor in T cells regulates immune functions and autoimmune disease susceptibility.