RESUMO
Navigation of fast migrating cells such as amoeba Dictyostelium and immune cells are tightly associated with their morphologies that range from steady polarized forms that support high directionality to those more complex and variable when making frequent turns. Model simulations are essential for quantitative understanding of these features and their origins, however systematic comparisons with real data are underdeveloped. Here, by employing deep-learning-based feature extraction combined with phase-field modeling framework, we show that a low dimensional feature space for 2D migrating cell morphologies obtained from the shape stereotype of keratocytes, Dictyostelium and neutrophils can be fully mapped by an interlinked signaling network of cell-polarization and protrusion dynamics. Our analysis links the data-driven shape analysis to the underlying causalities by identifying key parameters critical for migratory morphologies both normal and aberrant under genetic and pharmacological perturbations. The results underscore the importance of deciphering self-organizing states and their interplay when characterizing morphological phenotypes.
Assuntos
Movimento Celular/fisiologia , Aprendizado Profundo , Modelos Biológicos , Animais , Polaridade Celular/fisiologia , Forma Celular/fisiologia , Extensões da Superfície Celular/fisiologia , Células Cultivadas , Ciclídeos , Biologia Computacional , Simulação por Computador , Dictyostelium/citologia , Dictyostelium/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Células HL-60 , HumanosRESUMO
BACKGROUND: Lymphocytes circulate between peripheral lymphoid tissues via blood and lymphatic systems, and chemokine-induced migration is important in trafficking lymphocytes to distant sites. The small GTPase Rap1 is important in mediating lymphocyte motility, and Rap1-GEFs are involved in chemokine-mediated Rap1 activation. Here, we describe the roles and mechanisms of Rap1-GEFs in lymphocyte trafficking. RESULTS: In this study, we show that RA-GEF-1 and 2 (also known as Rapgef2 and 6) are key guanine nucleotide exchange factors (GEF) for Rap1 in lymphocyte trafficking. Mice harboring T cell-specific knockouts of Rapgef2/6 demonstrate defective homing and egress of T cells. Sphingosine-1-phosphate (S1P) as well as chemokines activates Rap1 in a RA-GEF-1/2-dependent manner, and their deficiency in T cells impairs Mst1 phosphorylation, cell polarization, and chemotaxis toward S1P gradient. On the other hand, B cell-specific knockouts of Rapgef2/6 impair chemokine-dependent retention of B cells in the bone marrow and passively facilitate egress. Phospholipase D2-dependent production of phosphatidic acid by these chemotactic factors determines spatial distribution of Rap1-GTP subsequent to membrane localization of RA-GEFs and induces the development of front membrane. On the other hand, basal de-phosphorylation of RA-GEFs is necessary for chemotactic factor-dependent increase in GEF activity for Rap1. CONCLUSIONS: We demonstrate here that subcellular distribution and activation of RA-GEFs are key factors for a directional movement of lymphocytes and that phosphatidic acid is critical for membrane translocation of RA-GEFs with chemokine stimulation.
Assuntos
Movimento Celular , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Linfócitos/fisiologia , Ácidos Fosfatídicos/metabolismo , Animais , Linhagem Celular , Feminino , Humanos , Masculino , Camundongos , FosforilaçãoRESUMO
Integrin activation by Rap1-GTP is pivotal for lymphocyte trafficking. In this study, we show the phosphatidic acid (PA)-dependent membrane distribution of RA-GEF-1 and -2 (also known as Rapgef2 and 6), which are guanine nucleotide exchange factors for Rap1, plays important roles in lymphocyte migration. RA-GEF-1 associates with PA through 919-967 aa within CDC25 homology domain, and the deletion of this region of RA-GEF-1 inhibits chemokine-dependent migration. Chemokine stimulation induces temporal production of PA on the plasma membrane, which is not necessary for Rap1 activation, but the translocation of RA-GEFs. Thus, chemokine-dependent generation of PA is critical for lymphocyte migration through membrane localization of RA-GEFs.
Assuntos
Movimento Celular , Quimiocinas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ácidos Fosfatídicos/metabolismo , Animais , Linhagem Celular , Células HEK293 , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , CamundongosRESUMO
RAPL (an alternative spliced form of Rassf5) is a critical Ras-related protein1 (Rap1) effector that regulates lymphocyte adhesion. Here, we have shown that in addition to this previously described function, RAPL also negatively controls lymphocyte proliferation and prevents autoimmunity and lymphoma. RAPL-deficient mice experienced age-related lupus-like glomerulonephritis and developed B cell lymphomas. RAPL-deficient lymphocytes showed hyperproliferation by enhanced S phase entry after antigen receptor ligation. Compared to wild-type cells, RAPL-deficient naive lymphocytes had a 2- to 3-fold increase in Cdk2 kinase activity with a cytoplasmic mislocalization of the cyclin-dependent kinase inhibitor p27(kip1). RAPL was found to suppress the phosphorylation of p27(kip1) on serine 10 (S10) and promoted p27(kip1) nuclear translocation. An S10A mutation in p27(kip1) corrected its cytoplasmic accumulation, reduced hyperproliferation in RAPL-deficient lymphocytes, and suppressed glomerulonephritis and development of B cell lymphoma. Thus, RAPL serves as a checkpoint for S phase entry to prevent lymphoproliferative disorders through the spatial regulation of p27(kip1).
Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Linfoma de Células B/genética , Transtornos Linfoproliferativos/genética , Proteínas rap1 de Ligação ao GTP/genética , Animais , Autoimunidade/genética , Adesão Celular/genética , Adesão Celular/imunologia , Proliferação de Células , Células Cultivadas , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/imunologia , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/imunologia , Nefrite Lúpica/genética , Nefrite Lúpica/imunologia , Linfócitos/imunologia , Camundongos , Camundongos Knockout , Mutação/genética , Fosforilação/genética , Transporte Proteico/genética , Transporte Proteico/imunologia , Proteínas rap1 de Ligação ao GTP/imunologia , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) causes both a neoplastic disease and inflammatory diseases, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 basic leucine zipper factor (HBZ) gene is encoded in the minus strand of the proviral DNA and is constitutively expressed in infected cells and ATL cells. HBZ increases the number of regulatory T (Treg) cells by inducing the Foxp3 gene transcription. Recent studies have revealed that some CD4âºFoxp3⺠T cells are not terminally differentiated but have a plasticity to convert to other T-cell subsets. Induced Treg (iTreg) cells tend to lose Foxp3 expression, and may acquire an effector phenotype accompanied by the production of inflammatory cytokines, such as interferon-γ (IFN-γ). In this study, we analyzed a pathogenic mechanism of chronic inflammation related with HTLV-1 infection via focusing on HBZ and Foxp3. Infiltration of lymphocytes was observed in the skin, lung and intestine of HBZ-Tg mice. As mechanisms, adhesion and migration of HBZ-expressing CD4⺠T cells were enhanced in these mice. Foxp3â»CD4⺠T cells produced higher amounts of IFN-γ compared to those from non-Tg mice. Expression of Helios was reduced in Treg cells from HBZ-Tg mice and HAM/TSP patients, indicating that iTreg cells are predominant. Consistent with this finding, the conserved non-coding sequence 2 region of the Foxp3 gene was hypermethylated in Treg cells of HBZ-Tg mice, which is a characteristic of iTreg cells. Furthermore, Treg cells in the spleen of HBZ-transgenic mice tended to lose Foxp3 expression and produced an excessive amount of IFN-γ, while Foxp3 expression was stable in natural Treg cells of the thymus. HBZ enhances the generation of iTreg cells, which likely convert to Foxp3â»T cells producing IFN-γ. The HBZ-mediated proinflammatory phenotype of CD4⺠T cells is implicated in the pathogenesis of HTLV-1-associated inflammation.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Infecções por HTLV-I/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Linfócitos T Reguladores/imunologia , Proteínas Virais/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Adesão Celular , Movimento Celular , Células Cultivadas , Metilação de DNA , Fatores de Transcrição Forkhead/genética , Infecções por HTLV-I/patologia , Infecções por HTLV-I/fisiopatologia , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Leucócitos Mononucleares/virologia , Ativação Linfocitária , Masculino , Camundongos Transgênicos , Paraparesia Espástica Tropical/etiologia , Proteínas Recombinantes/metabolismo , Proteínas dos Retroviridae , Baço/imunologia , Baço/metabolismo , Baço/patologia , Baço/virologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Linfócitos T Reguladores/virologia , Timo/imunologia , Timo/metabolismo , Timo/patologia , Timo/virologia , Proteínas Virais/genéticaRESUMO
The regulation of lymphocyte adhesion and migration plays crucial roles in lymphocyte trafficking during immunosurveillance. However, our understanding of the intracellular signalling that regulates these processes is still limited. Here, we show that the Ste20-like kinase Mst1 plays crucial roles in lymphocyte trafficking in vivo. Mst1(-/-) lymphocytes exhibited an impairment of firm adhesion to high endothelial venules, resulting in an inefficient homing capacity. In vitro lymphocyte adhesion cascade assays under physiological shear flow revealed that the stopping time of Mst1(-/-) lymphocytes on endothelium was markedly reduced, whereas their L-selectin-dependent rolling/tethering and transition to LFA-1-mediated arrest were not affected. Mst1(-/-) lymphocytes were also defective in the stabilization of adhesion through alpha4 integrins. Consequently, Mst1(-/-) mice had hypotrophic peripheral lymphoid tissues and reduced marginal zone B cells and dendritic cells in the spleen, and defective emigration of single positive thymocytes. Furthermore, Mst1(-/-) lymphocytes had impaired motility over lymph node-derived stromal cells and within lymph nodes. Thus, our data indicate that Mst1 is a key enzyme involved in lymphocyte entry and interstitial migration.
Assuntos
Movimento Celular/fisiologia , Fator de Crescimento de Hepatócito/fisiologia , Linfócitos/citologia , Linfócitos/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Adesão Celular/fisiologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Quimiocina CCL21/farmacologia , Citometria de Fluxo , Imunofluorescência , Fator de Crescimento de Hepatócito/genética , Immunoblotting , Técnicas In Vitro , Linfonodos/citologia , Tecido Linfoide/citologia , Tecido Linfoide/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/genética , Baço/citologia , Baço/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
Background and Aims: To examine the roles of microRNAs in the development of colitis, we conducted the RNA-sequencing studies using RNA derived from normal and colitogenic CD4+ T cells. Colitogenic CD4+ T cells demonstrated the increased expression of miR-150. We focused on the involvement of miR-150 in the colitis. Methods: We crossed miR-150 knockout mice and T-cell-specific Rap1KO mice, which is colitis model mice and spontaneously develop the colitis with tubular adenomas in microbiota-dependent manner. Results: MiR-150 silencing completely inhibited the expansion of pathogenic Th17 cells and the development of colitis. Conclusion: MiR-150 is a potential therapeutic target of inflammatory bowel diseases.
RESUMO
The small GTPase Rap1 and its effector RAPL regulate lymphocyte adhesion and motility. However, their precise regulatory roles in the adhesion cascade preceding entry into lymph nodes and during interstitial migration are unclear. Here, we show that Rap1 is indispensably required for the chemokine-triggered initial arrest step of rolling lymphocytes through LFA-1, whereas RAPL is not involved in rapid arrest. RAPL and talin play a critical role in stabilizing lymphocyte arrest to the endothelium of blood vessels under flow or to the high endothelial venules of peripheral lymph nodes in vivo. Further, mutagenesis and peptide studies suggest that release of a trans-acting restraint from the beta2 cytoplasmic region of LFA-1 is critical for Rap1-dependent initial arrest. Rap1 or RAPL deficiency severely impaired lymphocyte motility over lymph node stromal cells in vitro, and RAPL deficiency impaired high-velocity directional movement within lymph nodes. These findings reveal the several critical steps of Rap1, which are RAPL-dependent and -independent, in lymphocyte trafficking.
Assuntos
Adesão Celular/imunologia , Movimento Celular/imunologia , Linfócitos/citologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular , Humanos , Molécula 1 de Adesão Intercelular/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Migração e Rolagem de Leucócitos/fisiologia , Linfonodos/citologia , Linfonodos/fisiologia , Antígeno-1 Associado à Função Linfocitária/química , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/metabolismo , Linfócitos/metabolismo , Camundongos , Mutagênese , Estrutura Terciária de Proteína , Interferência de RNA , Transdução de Sinais/imunologia , Talina/genética , Talina/metabolismo , Transfecção , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
T-cell-specific Rap1 deletion causes spontaneous colitis in mice. In the present study, we revealed that Rap1 deficiency in T cells impaired the preceding induction of intestinal RORγt+ Treg cells. In the large intestinal lamina propria (LILP) of T-cell-specific Rap1-knockout mice (Rap1KO mice), Th17 cells were found to increase in a microbiota-dependent manner, and the inhibition of IL-17A production prevented the development of colitis. In the LILP of Rap1KO mice, RORγt+ Treg cells were scarcely induced by 4 weeks of age. The expression of CTLA-4 on Rap1-deficient Treg cells was reduced and the expression of CD80 and CD86 on dendritic cells was consequently elevated in Rap1KO mice. When cultured under each polarizing condition, Rap1-deficient naïve CD4+ T cells did not show biased differentiation into Th17 cells; their differentiation into Treg cells as well as Th1 and Th2 cells was lesser than that of wild-type cells. Rap1-deficient naïve CD4+ T cells were found to exhibit the defective nuclear translocation of NFAT and formation of actin foci in response to TCR engagement. These data suggest that Rap1 amplifies the TCR signaling required for Treg-mediated control of intestinal colitogenic Th17 responses.
Assuntos
Colite , Células Th17 , Proteínas rap1 de Ligação ao GTP , Animais , Diferenciação Celular , Colite/metabolismo , Colite/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Células Th17/metabolismo , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
Integrin regulation by Rap1 is indispensable for lymphocyte recirculation. In mice having B-cell-specific Rap1a/b double knockouts (DKO), the number of B cells in lymph nodes decreased to approximately 4% of that of control mice, and B cells were present in the spleen and blood. Upon the immunization with NP-CGG, DKO mice demonstrated the defective GC formation in the spleen, and the reduced NP-specific antibody production. In vitro, Rap1 deficiency impaired the movement of activated B cells along the gradients of chemoattractants known to be critical for their localization in the follicles. Furthermore, B-1a cells were almost completely absent in the peritoneal cavity, spleen and blood of adult DKO mice, and the number of B-cell progenitor/precursor (B-p) were reduced in neonatal and fetal livers. However, DKO B-ps normally proliferated, and differentiated into IgM+ cells in the presence of IL-7. CXCL12-dependent migration of B-ps on the VCAM-1 was severely impaired by Rap1 deficiency. Immunostaining study of fetal livers revealed defects in the co-localization of DKO B-ps and IL-7-producing stromal cells. This study proposes that the profound effects of Rap1-deficiency on humoral responses and B-1a cell generation may be due to or in part caused by impairments of the chemoattractant-dependent positioning and the contact with stromal cells.
Assuntos
Linfócitos B/metabolismo , Quimiotaxia de Leucócito , Centro Germinativo/metabolismo , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Quimiocina CXCL12/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Centro Germinativo/citologia , Centro Germinativo/efeitos dos fármacos , Centro Germinativo/imunologia , Imunidade Humoral , Imunização , Molécula 1 de Adesão Intercelular/metabolismo , Fígado/imunologia , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Precursoras de Linfócitos B/imunologia , Células Precursoras de Linfócitos B/metabolismo , Baço/imunologia , Baço/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , gama-Globulinas/farmacologia , Proteínas rap de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
Mesenchymal stromal cells are crucial components of secondary lymphoid organs (SLOs). Organogenesis of SLOs involves specialized stromal cells, designated lymphoid tissue organizer (LTo) in the embryonic anlagen; in the adult, several distinct stromal lineages construct elaborate tissue architecture and regulate lymphocyte compartmentalization. The relationship between the LTo and adult stromal cells, however, remains unclear, as does the precise number of stromal cell types that constitute mature SLOs are unclear. From mouse lymph nodes, we established a VCAM-1(+)ICAM-1(+)MAdCAM-1(+) reticular cell line that can produce CXCL13 upon LTbetaR stimulation and support primary B cell adhesion and migration in vitro. A similar stromal population sharing many characteristics with the LTo, designated marginal reticular cells (MRCs), was found in the outer follicular region immediately underneath the subcapsular sinus of lymph nodes. Moreover, MRCs were commonly observed at particular sites in various SLOs even in Rag2(-/-) mice, but were not found in ectopic lymphoid tissues, suggesting that MRCs are a developmentally determined element. These findings lead to a comprehensive view of the stromal composition and architecture of SLOs.
Assuntos
Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Mesoderma/citologia , Mesoderma/imunologia , Envelhecimento/imunologia , Animais , Animais Recém-Nascidos , Linfócitos B/citologia , Linfócitos B/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Quimiocina CXCL13/biossíntese , Fibroblastos/imunologia , Fibroblastos/metabolismo , Humanos , Tecido Linfoide/embriologia , Tecido Linfoide/metabolismo , Receptor beta de Linfotoxina/fisiologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais/imunologia , Células Estromais/citologia , Células Estromais/imunologia , Células Estromais/metabolismo , Quinase Induzida por NF-kappaBRESUMO
The Rap1 small GTPase has been implicated in regulation of integrin-mediated leukocyte adhesion downstream of various chemokines and cytokines in many aspects of inflammatory and immune responses. However, the mechanism for Rap1 regulation in the adhesion signaling remains unclear. RA-GEF-2 is a member of the multiple-member family of guanine nucleotide exchange factors (GEFs) for Rap1 and characterized by the possession of a Ras/Rap1-associating domain, interacting with M-Ras-GTP as an effector, in addition to the GEF catalytic domain. Here, we show that RA-GEF-2 is specifically responsible for the activation of Rap1 that mediates tumor necrosis factor-alpha (TNF-alpha)-triggered integrin activation. In BAF3 hematopoietic cells, activated M-Ras potently induced lymphocyte function-associated antigen 1 (LFA-1)-mediated cell aggregation. This activation was totally abrogated by knockdown of RA-GEF-2 or Rap1. TNF-alpha treatment activated LFA-1 in a manner dependent on M-Ras, RA-GEF-2, and Rap1 and induced activation of M-Ras and Rap1 in the plasma membrane, which was accompanied by recruitment of RA-GEF-2. Finally, we demonstrated that M-Ras and RA-GEF-2 were indeed involved in TNF-alpha-stimulated and Rap1-mediated LFA-1 activation in splenocytes by using mice deficient in RA-GEF-2. These findings proved a crucial role of the cross-talk between two Ras-family GTPases M-Ras and Rap1, mediated by RA-GEF-2, in adhesion signaling.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Integrinas/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Baço/citologia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Marcação de Genes , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Modelos Biológicos , Baço/efeitos dos fármacosAssuntos
Movimento Celular/fisiologia , Linfócitos/citologia , Proteínas de Ligação a Telômeros/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Humanos , Transporte Proteico , Complexo Shelterina , Proteínas de Ligação a Telômeros/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
HIV reactivation from latency is induced by cytokines but also by cell contact with other cells. To better understand this, J1.1 cells, a latent HIV-1-infected Jurkat derivative, were cocultured with its parental Jurkat. J1.1 cells became p17MA-positive and produced a high level of HIV p24CA antigen, only when they were cocultured with stimulated Jurkat with cell-to-cell contact. In contrast, very little p24CA was produced when they were cocultured without cell contact. Similar results were obtained when latent ACH-2 and its parental A3.01 cells were cocultured. Confocal microscopy revealed that not only HIV-1 p17MA and gp120Env but also LFA-1, CD81, CD59, and TCR CD3 accumulated at the cell contact site, suggesting formation of the virological synapse-like structure. LFA-1-ICAM-1 interaction was involved in the cell-to-cell contact. When J1.1 was cocultured with TCR-deficient Jurkat, the p17MA-positive rate was significantly lower, although the cell-to-cell contact was not impaired. Quantitative proteomics identified 54 membrane molecules, one of which was MHC class I, that accumulated at the cell contact site. Reactivation from latency was also influenced by the presence of stromal cells. Our study indicated that latent HIV-1 in J1.1/ACH-2 cells was efficiently reactivated by cell-to-cell contact with stimulated parental cells, accompanying the virological synapse-like structure.
Assuntos
Comunicação Celular , Infecções por HIV/virologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Ativação Viral , Latência Viral , Técnicas de Cocultura , Imunofluorescência , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Células Jurkat , Ativação Linfocitária/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Ligação Proteica , Células Estromais , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Integrin activation is associated with conformational regulation. In this study, we developed a system to evaluate conformational changes in α4ß7 integrin. We first inserted the PA tag into the plexin-semaphorin-integrin (PSI) domain of ß7 chain, which reacted with an anti-PA tag antibody (NZ-1) in an Mn2+-dependent manner. The small GTPase Rap1 deficiency, as well as chemokine stimulation and the introduction of the active form of Rap1, Rap1V12, enhanced the binding of NZ-1 to the PA-tagged mutant integrin, and increased the binding affinity to mucosal addressing cell adhesion molecule-1 (MAdCAM-1). Furthermore, we generated two kinds of hybridomas producing monoclonal antibodies (mAbs) that recognized Mn2+-dependent epitopes of ß7. Both epitopes were exposed to bind to mAbs on the cells by the introduction of Rap1V12. Although one epitope in the PSI domain of ß7 was exposed on Rap1-deficienct cells, the other epitope in the hybrid domain of ß7 was not. These data indicate that the conversion of Rap1-GDP to GTP exerts two distinct effects stepwise on the conformation of α4ß7. The induction of colitis by Rap1-deficient CD4+ effector/memory T cells suggests that the removal of constraining effect by Rap1-GDP on α4ß7 is sufficient for homing of these pathogenic T cells into colon lamina propria (LP).
Assuntos
Anticorpos Monoclonais/imunologia , Integrinas/metabolismo , Proteínas rap1 de Ligação ao GTP/fisiologia , Animais , Linhagem Celular , Humanos , Integrinas/química , Integrinas/imunologia , Células Jurkat , Glicoproteínas de Membrana/química , Camundongos , Peptídeos/imunologia , Conformação Proteica , Domínios Proteicos , Ratos , Relação Estrutura-AtividadeRESUMO
MicroRNAs (miRNAs), one of small non-coding RNAs, regulate many cell functions through their post-transcriptionally downregulation of target genes. Accumulated studies have revealed that miRNAs are involved in hematopoiesis. In the present study, we investigated effects of miR-669m overexpression on hematopoiesis in mouse in vivo, and found that erythroid differentiation was inhibited by the overexpression. Our bioinformatic analyses showed that candidate targets of miR-669m which are involved in the erythropoiesis inhibition are A-kinase anchoring protein 7 (Akap7) and X-linked Kx blood group (Xk) genes. These two genes were predicted as targets of miR-669m by two different in silico methods and were upregulated in late erythroblasts in a public RNA-seq data, which was confirmed with qPCR. Further, miR-669m suppressed luciferase reporters for 3' untranslated regions of Akap7 and Xk genes, which supports these genes are direct targets of miR-669m. Physiologically, miR-669m was not expressed in the erythroblast. In conclusion, using miR-669m, we found Akap7 and Xk, which may be involved in erythroid differentiation, implying that manipulating these genes could be a therapeutic way for diseases associated with erythropoiesis dysfunction.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Diferenciação Celular , Eritroblastos/citologia , Eritropoese , MicroRNAs/genética , Proteínas de Ancoragem à Quinase A/genética , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Eritroblastos/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
In classical Hodgkin lymphoma (cHL)-characterized by the presence of Hodgkin and Reed-Sternberg (HRS) cells-tumor-associated macrophages (TAMs) play a pivotal role in tumor formation. However, the significance of direct contact between HRS cells and TAMs has not been elucidated. HRS cells and TAMs are known to express PD-L1, which leads to PD-1+ CD4+ T cell exhaustion in cHL. Here, we found that PD-L1/L2 expression was elevated in monocytes co-cultured with HRS cells within 1 h, but not in monocytes cultured with supernatants of HRS cells. Immunofluorescence analysis of PD-L1/L2 revealed that their upregulation resulted in membrane transfer called "trogocytosis" from HRS cells to monocytes. PD-L1/L2 upregulation was not observed in monocytes co-cultured with PD-L1/L2-deficient HRS cells, validating the hypothesis that there is a direct transfer of PD-L1/L2 from HRS cells to monocytes. In the patients, both ligands (PD-L1/L2) were upregulated in TAMs in contact with HRS cells, but not in TAMs distant from HRS cells, suggesting that trogocytosis occurs in cHL patients. Taken together, trogocytosis may be one of the mechanisms that induces rapid upregulation of PD-L1/L2 in monocytes to evade antitumor immunity through the suppression of T cells as mediated by MHC antigen presentation.
Assuntos
Antígeno B7-H1/metabolismo , Doença de Hodgkin/metabolismo , Monócitos/metabolismo , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Doença de Hodgkin/imunologia , Doença de Hodgkin/patologia , Humanos , Complexo Principal de Histocompatibilidade/imunologia , Microambiente Tumoral/imunologiaRESUMO
Chemokines arrest circulating lymphocytes within the vasculature through the rapid up-regulation of leukocyte integrin adhesive activity, promoting subsequent lymphocyte transmigration. However, the key regulatory molecules regulating this process have remained elusive. Here, we demonstrate that Rap1 plays a pivotal role in chemokine-induced integrin activation and migration. Rap1 was activated by secondary lymphoid tissue chemokine (SLC; CCL21) and stromal-derived factor 1 (CXCL4) treatment in lymphocytes within seconds. Inhibition of Rap1 by Spa1, a Rap1-specific GTPase-activating protein, abrogated chemokine-stimulated lymphocyte rapid adhesion to endothelial cells under flow via intercellular adhesion molecule 1. Expression of a dominant active Rap1V12 in lymphocytes stimulated shear-resistant adhesion, robust cell migration on immobilized intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, and transendothelial migration under flow. We also demonstrated that Rap1V12 expression in lymphocytes induced a polarized morphology, accompanied by the redistribution of CXCR4 and CD44 to the leading edge and uropod, respectively. Spa1 effectively suppressed this polarization after SLC treatment. This unique characteristic of Rap1 may control chemokine-induced lymphocyte extravasation.
Assuntos
Polaridade Celular/fisiologia , Quimiocinas/metabolismo , Quimiotaxia de Leucócito/fisiologia , Endotélio Vascular/metabolismo , Proteínas Ativadoras de GTPase , Integrinas/metabolismo , Linfócitos/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL21 , Quimiocina CXCL12 , Quimiocinas CC/metabolismo , Quimiocinas CC/farmacologia , Quimiocinas CXC/metabolismo , Quimiocinas CXC/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Hemodinâmica/fisiologia , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Nucleares/metabolismo , Proteínas Nucleares/farmacologia , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Proteínas rap1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
INTRODUCTION: Collagen peptides have been widely used as a food supplement. After ingestion of collagen peptides, oligopeptides containing hydroxyproline (Hyp), which are known to have some physiological activities, are detected in peripheral blood. However, the effects of collagen-peptide administration on immune response are unclear. In the present study, we tested the effects of collagen-peptide ingestion on allergic response and the effects of collagen-derived oligopeptides on CD4+ T-cell differentiation. METHODS: BALB/c mice fed a collagen-peptide diet were immunized with ovalbumin (OVA), and their serum IgE and IgG levels, active cutaneous anaphylaxis, and cytokine secretion by splenocytes were examined. Naive CD4+ T cells were stimulated with anti-CD3 and anti-CD28 in the presence of collagen-derived oligopeptides, and the expression of IFN-γ, IL-4, and Foxp3 was analyzed. RESULTS: In an active anaphylaxis model, oral administration of collagen peptides suppressed serum OVA-specific immunoglobulin E (IgE) production and diminished anaphylaxis responses. In this model, the ingestion of collagen peptides skewed the pattern of cytokine production by splenocytes toward T-helper (Th) type 1 and regulatory T (Treg) cells. In vitro T-helper cell differentiation assays showed that Hyp-containing oligopeptides promoted Th1 differentiation by upregulating IFN-γ-induced signal transducer and activator of transcription 1 (STAT1) signaling. These oligopeptides also promoted the development of Foxp3+ Treg cells in response to antigen stimulation in the presence of TGF-ß. CONCLUSIONS: Collagen-peptide ingestion suppresses allergic responses by skewing the balance of CD4+ T cells toward Th1 and Treg cells and seems to be a promising agent for preventing allergies and inflammatory diseases.
Assuntos
Anafilaxia/prevenção & controle , Colágeno/administração & dosagem , Suplementos Nutricionais , Linfócitos T Reguladores/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Administração Oral , Anafilaxia/sangue , Anafilaxia/dietoterapia , Anafilaxia/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Colágeno/imunologia , Modelos Animais de Doenças , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Peptídeos/administração & dosagem , Peptídeos/imunologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologiaRESUMO
Activation of T cells by antigen requires adhesive interactions with antigen-presenting cells (APC) in which leukocyte function-associated antigen 1 (LFA-1) and intercellular adhesion molecules (ICAMs) are important. However, it is not well understood what signaling molecules regulate this process and how the modulation of adhesive events influences T-cell activation. Here we show that Rap1 is activated in T cells in an antigen-dependent manner and accumulated at the contact site of T-cell and antigen-loaded APC. Inhibition of Rap1 activation by a dominant-negative Rap1 or SPA-1, a Rap1 GTPase-activating protein, abrogates LFA-1-ICAM-1-mediated adhesive interactions with antigen-pulsed APC and the subsequent T-cell-receptor triggering and interleukin-2 production. Conversely, augmented antigen-dependent Rap1 activation by the expression of wild-type Rap1 enhances these responses but culminates in apoptosis by Fas and FasL. Thus, Rap1 functions as a key regulator of T-cell and APC interactions and modulates T-cell responses from productive activation to activation-induced cell death by regulating the strength of adhesive interactions. Moreover, constitutive Rap1 activation rendered T cells unresponsive with accumulation of p27(Kip1). Our study indicates that the activation state of Rap1 has a decisive effect on the T-cell response to antigen.