RESUMO
B cells and their progeny are the sources of highly expressed antibodies. Their high protein expression capabilities together with their abundance, easy accessibility via peripheral blood, and amenability to simple adoptive transfers have made them an attractive target for gene editing approaches to express recombinant antibodies or other therapeutic proteins. The gene editing of mouse and human primary B cells is efficient, and mouse models for in vivo studies have shown promise, but feasibility and scalability for larger animal models have so far not been demonstrated. We, therefore, developed a protocol to edit rhesus macaque primary B cells in vitro to enable such studies. We report conditions for in vitro culture and gene-editing of primary rhesus macaque B cells from peripheral blood mononuclear cells or splenocytes using CRISPR/Cas9. To achieve the targeted integration of large (<4.5 kb) cassettes, a fast and efficient protocol was included for preparing recombinant adeno-associated virus serotype 6 as a homology-directed repair template using a tetracycline-enabled self-silencing adenoviral helper vector. These protocols enable the study of prospective B cell therapeutics in rhesus macaques.
Assuntos
Edição de Genes , Leucócitos Mononucleares , Animais , Humanos , Edição de Genes/métodos , Macaca mulatta/genética , Estudos Prospectivos , Linfócitos B , Sistemas CRISPR-CasRESUMO
Mesenchymal stem cells (MSCs) have emerged as a promising treatment for inflammatory diseases. The immunomodulatory effect of MSCs takes place both by direct cell-to-cell contact and by means of soluble factors that leads to an increased accumulation of regulatory immune cells at the sites of inflammation. Similar efficacy of MSCs has been described regardless of the route of administration used, the inflammation conditions and the major histocompatibility complex context. These observations raise the question of whether the migration of the MSCs to the inflamed tissues is a pre-requisite to achieve their beneficial effect. To address this, we examined the biodistribution and the efficacy of intraperitoneal luciferase-expressing human expanded adipose-derived stem cells (Luci-eASCs) in a mouse model of colitis. Luci-eASC-infused mice were stratified according to their response to the Luci-eASC treatment. According to the stratification criteria, there was a tendency to increase the bioluminescence signal in the intestine at the expense of a decrease in the bioluminescence signal in the liver in the "responder" mice. These data thus suggest that the accumulation of the eASCs to the inflamed tissues is beneficial for achieving an optimal modulation of inflammation.
Assuntos
Tecido Adiposo/citologia , Colite/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Tecido Adiposo/metabolismo , Animais , Comunicação Celular , Diferenciação Celular , Movimento Celular , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Modelos Animais de Doenças , Genes Reporter , Humanos , Injeções Intraperitoneais , Mucosa Intestinal/metabolismo , Intestinos/patologia , Fígado/metabolismo , Fígado/patologia , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Baço/metabolismo , Baço/patologia , Ácido TrinitrobenzenossulfônicoRESUMO
Mesenchymal stem cells (MSCs) have a large potential in cell therapy for treatment of inflammatory and autoimmune diseases, thanks to their immunomodulatory properties. The encouraging results in animal models have initiated the translation of MSC therapy to clinical trials. In cell therapy protocols with MSCs, administered intravenously, several studies have shown that a small proportion of infused MSCs can traffic to the draining lymph nodes (LNs). This is accompanied with an increase of different types of regulatory immune cells in the LNs, suggesting the importance of migration of MSCs to the LNs in order to contribute to immunomodulatory response. Intranodal (IN), also referred as intralymphatic, injection of cells, like dendritic cells, is being proposed in the clinic for the treatment of cancer and allergy, showing that this route of administration is clinically safe and efficient. In this study, we investigated, for the first time, the biodistribution and the efficacy of Luciferase+ adipose-derived MSCs (Luci-eASCs), infused through the inguinal LNs (iLNs), in normal mice and in inflamed mice with colitis. Most of the Luci-eASCs remain in the iLNs and in the adipose tissue surrounding the inguinal LNs. A small proportion of Luci-eASCs can migrate to other locations within the lymphatic system and to other tissues and organs, having a preferential migration toward the intestine in colitic mice. Our results show that the infused Luci-eASCs protected 58% of the mice against induced colitis. Importantly, a correlation between the response to eASC treatment and a higher accumulation of eASCs in popliteal, parathymic, parathyroid, and mesenteric LNs were found. Altogether, these results suggest that IN administration of eASCs is feasible and may represent an effective strategy for cell therapy protocols with human adipose-derived MSCs in the clinic for the treatment of immune-mediated disorders.
RESUMO
Emerging evidence indicates that the metalloproteinase Adamts-1 plays a significant role in the pathophysiology of vessel remodeling, but little is known about the signaling pathways that control Adamts-1 expression. We show that vascular endothelial growth factor (VEGF), angiotensin-II, interleukin-1ß, and tumor necrosis factor α, stimuli implicated in pathological vascular remodeling, increase Adamts-1 expression in endothelial and vascular smooth muscle cells. Analysis of the intracellular signaling pathways implicated in this process revealed that VEGF and angiotensin-II upregulate Adamts-1 expression via activation of differential signaling pathways that ultimately promote functional binding of the NFAT or C/EBPß transcription factors, respectively, to the Adamts-1 promoter. Infusion of mice with angiotensin-II triggered phosphorylation and nuclear translocation of C/EBPß proteins in aortic cells concomitantly with an increase in the expression of Adamts-1, further underscoring the importance of C/EBPß signaling in angiotensin-II-induced upregulation of Adamts-1. Similarly, VEGF promoted NFAT activation and subsequent Adamts-1 induction in aortic wall in a calcineurin-dependent manner. Our results demonstrate that Adamts-1 upregulation by inducers of pathological vascular remodeling is mediated by specific signal transduction pathways involving NFAT or C/EBPß transcription factors. Targeting of these pathways may prove useful in the treatment of vascular disease.
Assuntos
Proteínas ADAM/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Fatores de Transcrição NFATC/metabolismo , Remodelação Vascular , Proteínas ADAM/genética , Proteína ADAMTS1 , Animais , Aorta/enzimologia , Sequência de Bases , Calcineurina/metabolismo , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Camundongos Knockout , Dados de Sequência Molecular , Transdução de Sinais , Ativação Transcricional , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Osteoclasts are bone-resorbing cells that are important for maintenance of bone remodeling and mineral homeostasis. Regulation of osteoclast differentiation and activity is important for the pathogenesis and treatment of diseases associated with bone loss. Here, we demonstrate that retinoid X receptors (RXRs) are key elements of the transcriptional program of differentiating osteoclasts. Loss of RXR function in hematopoietic cells resulted in formation of giant, nonresorbing osteoclasts and increased bone mass in male mice and protected female mice from bone loss following ovariectomy, which induces osteoporosis in WT females. The increase in bone mass associated with RXR deficiency was due to lack of expression of the RXR-dependent transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (MAFB) in osteoclast progenitors. Evaluation of osteoclast progenitor cells revealed that RXR homodimers directly target and bind to the Mafb promoter, and this interaction is required for proper osteoclast proliferation, differentiation, and activity. Pharmacological activation of RXRs inhibited osteoclast differentiation due to the formation of RXR/liver X receptor (LXR) heterodimers, which induced expression of sterol regulatory element binding protein-1c (SREBP-1c), resulting in indirect MAFB upregulation. Our study reveals that RXR signaling mediates bone homeostasis and suggests that RXRs have potential as targets for the treatment of bone pathologies such as osteoporosis.
Assuntos
Remodelação Óssea/fisiologia , Diferenciação Celular/fisiologia , Receptores Nucleares Órfãos/metabolismo , Osteoclastos/metabolismo , Multimerização Proteica/fisiologia , Receptores X de Retinoides/metabolismo , Animais , Feminino , Receptores X do Fígado , Fator de Transcrição MafB/biossíntese , Fator de Transcrição MafB/genética , Masculino , Camundongos , Camundongos Knockout , Receptores Nucleares Órfãos/genética , Osteoclastos/citologia , Osteoporose/genética , Osteoporose/metabolismo , Receptores X de Retinoides/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Transcrição Gênica/fisiologia , Regulação para Cima/fisiologiaRESUMO
OBJECTIVE: Vascular endothelial growth factor (VEGF) has been identified as a crucial regulator of physiological and pathological angiogenesis. Among the intracellular signaling pathways triggered by VEGF, activation of the calcineurin/nuclear factor of activated T cells (NFAT) signaling axis has emerged as a critical mediator of angiogenic processes. We and others previously reported a novel role for the plasma membrane calcium ATPase (PMCA) as an endogenous inhibitor of the calcineurin/NFAT pathway, via interaction with calcineurin, in cardiomyocytes and breast cancer cells. However, the functional significance of the PMCA/calcineurin interaction in endothelial pathophysiology has not been addressed thus far. APPROACH AND RESULTS: Using in vitro and in vivo assays, we here demonstrate that the interaction between PMCA4 and calcineurin in VEGF-stimulated endothelial cells leads to downregulation of the calcineurin/NFAT pathway and to a significant reduction in the subsequent expression of the NFAT-dependent, VEGF-activated, proangiogenic genes RCAN1.4 and Cox-2. PMCA4-dependent inhibition of calcineurin signaling translates into a reduction in endothelial cell motility and blood vessel formation that ultimately impairs in vivo angiogenesis by VEGF. CONCLUSIONS: Given the importance of the calcineurin/NFAT pathway in the regulation of pathological angiogenesis, targeted modulation of PMCA4 functionality might open novel therapeutic avenues to promote or attenuate new vessel formation in diseases that occur with angiogenesis.
Assuntos
Indutores da Angiogênese/farmacologia , Calcineurina/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Células Endoteliais/efeitos dos fármacos , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica/efeitos dos fármacos , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Proteínas de Ligação ao Cálcio , ATPases Transportadoras de Cálcio/deficiência , ATPases Transportadoras de Cálcio/genética , Movimento Celular , Proliferação de Células , Ciclo-Oxigenase 2/metabolismo , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Células Endoteliais/enzimologia , Células HEK293 , Membro Posterior , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isquemia/enzimologia , Isquemia/fisiopatologia , Camundongos , Camundongos Knockout , Proteínas Musculares/metabolismo , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
Macrophages contribute to tissue homeostasis and influence inflammatory responses by modulating their phenotype in response to the local environment. Understanding the molecular mechanisms governing this plasticity would open new avenues for the treatment for inflammatory disorders. We show that deletion of calcineurin (CN) or its inhibition with LxVP peptide in macrophages induces an anti-inflammatory population that confers resistance to arthritis and contact hypersensitivity. Transfer of CN-targeted macrophages or direct injection of LxVP-encoding lentivirus has anti-inflammatory effects in these models. Specific CN targeting in macrophages induces p38 MAPK activity by downregulating MKP-1 expression. However, pharmacological CN inhibition with cyclosporin A (CsA) or FK506 did not reproduce these effects and failed to induce p38 activity. The CN-inhibitory peptide VIVIT also failed to reproduce the effects of LxVP. p38 inhibition prevented the anti-inflammatory phenotype of CN-targeted macrophages, and mice with defective p38-activation were resistant to the anti-inflammatory effect of LxVP. Our results identify a key role for CN and p38 in the modulation of macrophage phenotype and suggest an alternative treatment for inflammation based on redirecting macrophages toward an anti-inflammatory status.
Assuntos
Calcineurina/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , Macrófagos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Calcineurina/genética , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Fosfatase 1 de Especificidade Dupla/genética , Citometria de Fluxo , Humanos , Immunoblotting , Imuno-Histoquímica , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Osteoclastos/citologia , Osteoclastos/metabolismo , Fagocitose/genética , Fagocitose/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Atherosclerosis is a complex inflammatory disease involving extensive vascular vessel remodelling and migration of vascular cells. As RCAN1 is implicated in cell migration, we investigated its contribution to atherosclerosis. We show RCAN1 induction in atherosclerotic human and mouse tissues. Rcan1 was expressed in lesional macrophages, endothelial cells and vascular smooth muscle cells and was induced by treatment of these cells with oxidized LDLs (oxLDLs). Rcan1 regulates CD36 expression and its genetic inactivation reduced atherosclerosis extension and severity in Apoe(-/-) mice. This effect was mechanistically linked to diminished oxLDL uptake, resistance to oxLDL-mediated inhibition of macrophage migration and increased lesional IL-10 and mannose receptor expression. Moreover, Apoe(-/-) Rcan1(-/-) macrophages expressed higher-than-Apoe(-/-) levels of anti-inflammatory markers. We previously showed that Rcan1 mediates aneurysm development and that its expression is not required in haematopoietic cells for this process. However, transplantation of Apoe(-/-) Rcan1(-/-) bone-marrow (BM) cells into Apoe(-/-) recipients confers atherosclerosis resistance. Our data define a major role for haematopoietic Rcan1 in atherosclerosis and suggest that therapies aimed at inhibiting RCAN1 expression or function might significantly reduce atherosclerosis burden.
Assuntos
Aterosclerose/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Musculares/metabolismo , Aneurisma/metabolismo , Aneurisma/patologia , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Antígenos CD36/genética , Antígenos CD36/metabolismo , Proteínas de Ligação ao Cálcio , Movimento Celular/efeitos dos fármacos , Progressão da Doença , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Espumosas/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipoproteínas LDL/toxicidade , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , FenótipoRESUMO
Dendritic cells (DCs), monocytes, and macrophages are closely related phagocytes that share many phenotypic features and, in some cases, a common developmental origin. Although the requirement for DCs in initiating adaptive immune responses is well appreciated, the role of monocytes and macrophages remains largely undefined, in part because of the lack of genetic tools enabling their specific depletion. Here, we describe a two-gene approach that requires overlapping expression of LysM and Csf1r to define and deplete monocytes and macrophages. The role of monocytes and macrophages in immunity to pathogens was tested by their selective depletion during infection with Citrobacter rodentium. Although neither cell type was required to initiate immunity, monocytes and macrophages contributed to the adaptive immune response by secreting IL-12, which induced Th1 polarization and IFN-γ secretion. Thus, whereas DCs are indispensable for priming naive CD4(+) T cells, monocytes and macrophages participate in intestinal immunity by producing mediators that direct T cell polarization.
Assuntos
Citrobacter rodentium/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Macrófagos/imunologia , Monócitos/imunologia , Subpopulações de Linfócitos T/imunologia , Imunidade Adaptativa , Animais , Movimento Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Infecções por Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/imunologia , Ordem dos Genes , Interleucina-12/biossíntese , Interleucina-12/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Monócitos/metabolismo , Muramidase/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
The nuclear factor of activated T cells (NFAT) family of transcription factors plays important roles in many biologic processes, including the development and function of the immune and vascular systems. Cells usually express more than one NFAT member, raising the question of whether NFATs play overlapping roles or if each member has selective functions. Using mRNA knock-down, we show that NFATc3 is specifically required for IL2 and cyclooxygenase-2 (COX2) gene expression in transformed and primary T cells and for T-cell proliferation. We also show that NFATc3 regulates COX2 in endothelial cells, where it is required for COX2, dependent migration and angiogenesis in vivo. These results indicate that individual NFAT members mediate specific functions through the differential regulation of the transcription of target genes. These effects, observed on short-term suppression by mRNA knock-down, are likely to have been masked by compensatory effects in gene-knockout studies.
Assuntos
Ciclo-Oxigenase 2/genética , Interleucina-2/genética , Ativação Linfocitária/fisiologia , Fatores de Transcrição NFATC , Neovascularização Fisiológica/fisiologia , Linfócitos T/fisiologia , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Ciclo-Oxigenase 2/imunologia , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Interleucina-2/imunologia , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/imunologia , Fatores de Transcrição NFATC/metabolismo , Regiões Promotoras Genéticas/fisiologia , Transcrição Gênica/fisiologiaRESUMO
OBJECTIVE: Inducible expression of cyclooxygenase-2 (COX-2) and terminal prostaglandin synthases (tPGS) has been mainly analyzed in tumor, stromal, and inflammatory cells, and little is known about the regulation of prostanoid biosynthesis by endothelial cells. Here we characterize the profile of prostanoids produced by activated HUVECs and analyze the expression and activities of tPGS. METHODS AND RESULTS: Enzyme immunoassays indicated increased endothelial prostanoid production after proangiogenic stimulation, but without parallel upregulation of tPGS. Endothelial prostanoid production instead depended on the induction of COX-2 and was abolished by COX-2 silencing or pharmacological inhibition. COX-2 is functionally coupled to prostacyclin and thromboxane synthases in HUVECs, but these cells show no detectable PGE(2) synthase (PGES) activity. Endothelial PGE(2) production is partly mediated by nonenzymatic decomposition of COX-2-derived PGH(2), but endothelial-produced PGH(2) can also be metabolized enzymatically by microsomal PGES-1 in cocultured tumor cells. CONCLUSIONS: Our findings identify a novel transcellular metabolism of PGE(2) between the endothelial and tumor compartments. Given the role of PGE(2) as a mediator of COX-2 proangiogenic effects, transcellular metabolism of endothelial-derived PGH(2) is a potential target for treatment of pathological angiogenesis.