RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is characterized by increasing fibrosis, which can enhance tumor progression and spread. Here, we undertook an unbiased temporal assessment of the matrisome of the highly metastatic KPC (Pdx1-Cre, LSL-KrasG12D/+, LSL-Trp53R172H/+) and poorly metastatic KPflC (Pdx1-Cre, LSL-KrasG12D/+, Trp53fl/+) genetically engineered mouse models of pancreatic cancer using mass spectrometry proteomics. Our assessment at early-, mid-, and late-stage disease reveals an increased abundance of nidogen-2 (NID2) in the KPC model compared to KPflC, with further validation showing that NID2 is primarily expressed by cancer-associated fibroblasts (CAFs). Using biomechanical assessments, second harmonic generation imaging, and birefringence analysis, we show that NID2 reduction by CRISPR interference (CRISPRi) in CAFs reduces stiffness and matrix remodeling in three-dimensional models, leading to impaired cancer cell invasion. Intravital imaging revealed improved vascular patency in live NID2-depleted tumors, with enhanced response to gemcitabine/Abraxane. In orthotopic models, NID2 CRISPRi tumors had less liver metastasis and increased survival, highlighting NID2 as a potential PDAC cotarget.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Proteómica , Animales , Humanos , Ratones , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/genética , Moléculas de Adhesión Celular , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Modelos Animales de Enfermedad , Fibrosis , Gemcitabina , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Proteómica/métodosAsunto(s)
Glucuronidasa , Vigilancia Inmunológica , Células Asesinas Naturales , Invasividad Neoplásica , Humanos , Células Asesinas Naturales/inmunología , Animales , Glucuronidasa/metabolismo , Glucuronidasa/genética , Glucuronidasa/inmunología , Ratones , Neoplasias/inmunología , Neoplasias/patología , Neoplasias/enzimología , Neoplasias/genéticaRESUMEN
Medulloblastoma is the most common malignant pediatric brain tumor and there is an urgent need for molecularly targeted and subgroup-specific therapies. The stem cell factor SOX9, has been proposed as a potential therapeutic target for the treatment of Sonic Hedgehog medulloblastoma (SHH-MB) subgroup tumors, given its role as a downstream target of Hedgehog signaling and in functionally promoting SHH-MB metastasis and treatment resistance. However, the functional requirement for SOX9 in the genesis of medulloblastoma remains to be determined. Here we report a previously undocumented level of SOX9 expression exclusively in proliferating granule cell precursors (GCP) of the postnatal mouse cerebellum, which function as the medulloblastoma-initiating cells of SHH-MBs. Wild-type GCPs express comparatively lower levels of SOX9 than neural stem cells and mature astroglia and SOX9low GCP-like tumor cells constitute the bulk of both infant (Math1Cre:Ptch1lox/lox ) and adult (Ptch1LacZ/+ ) SHH-MB mouse models. Human medulloblastoma single-cell RNA data analyses reveal three distinct SOX9 populations present in SHH-MB and noticeably absent in other medulloblastoma subgroups: SOX9 + MATH1 + (GCP), SOX9 + GFAP + (astrocytes) and SOX9 + MATH1 + GFAP + (potential tumor-derived astrocytes). To functionally address whether SOX9 is required as a downstream effector of Hedgehog signaling in medulloblastoma tumor cells, we ablated Sox9 using a Math1Cre model system. Surprisingly, targeted ablation of Sox9 in GCPs (Math1Cre:Sox9lox/lox ) revealed no overt phenotype and loss of Sox9 in SHH-MB (Math1Cre:Ptch1lox/lox;Sox9lox/lox ) does not affect tumor formation. IMPLICATIONS: Despite preclinical data indicating SOX9 plays a key role in SHH-MB biology, our data argue against SOX9 as a viable therapeutic target.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Factor de Transcripción SOX9/metabolismo , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Meduloblastoma/fisiopatología , Ratones , Transducción de SeñalRESUMEN
Multiphoton fluorescence lifetime microscopy has revolutionized studies of pathophysiological and xenobiotic dynamics, enabling the spatial and temporal quantification of these processes in intact organs in vivo. We have previously used multiphoton fluorescence lifetime microscopy to characterise the morphology and amplitude weighted mean fluorescence lifetime of the endogenous fluorescent metabolic cofactor nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) of mouse livers in vivo following induction of various disease states. Here, we extend the characterisation of liver disease models by using nonlinear regression to estimate the unbound, bound fluorescence lifetimes for NAD(P)H, flavin adenine dinucleotide (FAD), along with metabolic ratios and examine the impact of using multiple segmentation methods. We found that NAD(P)H amplitude ratio, and fluorescence lifetime redox ratio can be used as discriminators of diseased liver from normal liver. The redox ratio provided a sensitive measure of the changes in hepatic fibrosis and biliary fibrosis. Hepatocellular carcinoma was associated with an increase in spatial heterogeneity and redox ratio coupled with a decrease in mean fluorescence lifetime. We conclude that multiphoton fluorescence lifetime microscopy parameters and metabolic ratios provided insights into the in vivo redox state of diseased compared to normal liver that were not apparent from a global, mean fluorescence lifetime measurement alone.
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Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Microscopía de Fluorescencia por Excitación Multifotónica , Animales , Tetracloruro de Carbono , Modelos Animales de Enfermedad , Cirrosis Hepática/inducido químicamente , Ratones , Ratones Noqueados , Oxidación-ReducciónRESUMEN
Intravital imaging of the immune system is a powerful technique for studying biology of the immune response in the spinal cord using a variety of disease models ranging from traumatic injury to autoimmune disorders. Here, we will discuss specific technical aspects as well as many intriguing biological phenomena that have been revealed with the use of intravital imaging for investigation of the immune system in the spinal cord. We will discuss surgical techniques for exposing and stabilizing the spine that are critical for obtaining images, visualizing immune and CNS cells with genetically expressed fluorescent proteins, fluorescent labeling techniques and briefly discuss some of the challenges of image analysis.
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Microscopía Intravital/métodos , Neuroimagen/métodos , Médula Espinal , Animales , HumanosRESUMEN
Extracellular adenosine is a key immunosuppressive metabolite that restricts activation of cytotoxic lymphocytes and impairs antitumor immune responses. Here, we show that engagement of A2A adenosine receptor (A2AR) acts as a checkpoint that limits the maturation of natural killer (NK) cells. Both global and NK-cell-specific conditional deletion of A2AR enhanced proportions of terminally mature NK cells at homeostasis, following reconstitution, and in the tumor microenvironment. Notably, A2AR-deficient, terminally mature NK cells retained proliferative capacity and exhibited heightened reconstitution in competitive transfer assays. Moreover, targeting A2AR specifically on NK cells also improved tumor control and delayed tumor initiation. Taken together, our results establish A2AR-mediated adenosine signaling as an intrinsic negative regulator of NK-cell maturation and antitumor immune responses. On the basis of these findings, we propose that administering A2AR antagonists concurrently with NK cell-based therapies may heighten therapeutic benefits by augmenting NK cell-mediated antitumor immunity.Significance: Ablating adenosine signaling is found to promote natural killer cell maturation and antitumor immunity and reduce tumor growth. Cancer Res; 78(4); 1003-16. ©2017 AACR.
Asunto(s)
Células Asesinas Naturales/patología , Melanoma Experimental/metabolismo , Receptor de Adenosina A2A/metabolismo , Animales , Línea Celular Tumoral , Xenoinjertos , Humanos , Células Asesinas Naturales/inmunología , Masculino , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptor de Adenosina A2A/deficiencia , Receptor de Adenosina A2A/inmunología , Transducción de Señal/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
The emerging role for CD73 in driving cancer growth and metastasis has presented opportunities to develop anti-CD73 monoclonal antibodies (mAbs) in the treatment of human cancers. Blockade of CD73 by antagonistic CD73 mAbs ameliorates tumor growth and metastasis via the inhibition of enzymatic and non-enzymatic CD73 pathways. In this study, we investigated whether Fc-receptor cross-linking represented a non-redundant mechanism by which anti-CD73 mAbs exert potent suppression of solid tumors and metastases. We engineered four anti-CD73 mAbs, each different in their ability to modulate CD73 enzymatic function and bind Fc receptors. mAbs recognizing a similar epitope of CD73 (CD73-04, TY/23 and 2C5) displayed the greatest antitumor activity. Importantly, we observed that the optimal control of metastasis by anti-CD73 mAbs involved primarily Fc receptor engagement, while suppression of solid tumors required both, enzyme inhibition and activation of Fc receptors. Engagement of Fc-receptors was also essential for optimal anti-metastatic effect in combination with either A2AR inhibitor or anti-PD-1 mAb treatment. The control of experimental metastases relied on the activation of host NK cells and IFNγ, while NK cells, CD8+ T cells and IFNγ were needed for effective antitumor effect in the spontaneous metastases model. These observations advance our understanding of the enzymatic and non-enzymatic functions of anti-CD73 mAbs in solid tumors and metastases. Altogether, these findings will greatly assist in the design of anti-CD73 mAbs to be used as either single agents or in combination with other immunotherapeutic molecules or targeted therapies.
RESUMEN
Increasing evidence exists for the role of immunosuppressive adenosine in promoting tumor growth and spread in a number of cancer types, resulting in poor clinical outcomes. In this study, we assessed whether the CD73-adenosinergic pathway is active in melanoma patients and whether adenosine restricts the efficacy of clinically approved targeted therapies for commonly mutated BRAFV600E melanoma. In AJCC stage III melanoma patients, CD73 expression (the enzyme that generates adenosine) correlated significantly with patients presenting nodal metastatic melanoma, suggesting that targeting this pathway may be effective in advanced stage disease. In addition, dabrafenib and trametinib treatment of CD73+ BRAFV600E-mutant melanomas caused profound CD73 downregulation in tumor cells. Inhibition of BRAF and MEK in combination with the A2A adenosine receptor provided significant protection against tumor initiation and metastasis formation in mice. Our results suggest that targeting adenosine may enhance therapeutic responses for melanoma patients receiving targeted or immune-based therapies. Cancer Res; 77(17); 4684-96. ©2017 AACR.
Asunto(s)
Adenosina/metabolismo , Neoplasias Pulmonares/prevención & control , Melanoma/prevención & control , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Receptor de Adenosina A2A/química , Neoplasias Cutáneas/prevención & control , 5'-Nucleotidasa/metabolismo , Animales , Quimioterapia Combinada , Proteínas Ligadas a GPI/metabolismo , Humanos , Imidazoles/farmacología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Melanoma/genética , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Terapia Molecular Dirigida , Mutación/genética , Oximas/farmacología , Piridonas/farmacología , Pirimidinonas/farmacología , Receptor de Adenosina A2A/fisiología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/secundarioRESUMEN
NK cells are highly efficient at preventing cancer metastasis but are infrequently found in the core of primary tumors. Here, have we demonstrated that freshly isolated mouse and human NK cells express low levels of the endo-ß-D-glucuronidase heparanase that increase upon NK cell activation. Heparanase deficiency did not affect development, differentiation, or tissue localization of NK cells under steady-state conditions. However, mice lacking heparanase specifically in NK cells (Hpsefl/fl NKp46-iCre mice) were highly tumor prone when challenged with the carcinogen methylcholanthrene (MCA). Hpsefl/fl NKp46-iCre mice were also more susceptible to tumor growth than were their littermate controls when challenged with the established mouse lymphoma cell line RMA-S-RAE-1ß, which overexpresses the NK cell group 2D (NKG2D) ligand RAE-1ß, or when inoculated with metastatic melanoma, prostate carcinoma, or mammary carcinoma cell lines. NK cell invasion of primary tumors and recruitment to the site of metastasis were strictly dependent on the presence of heparanase. Cytokine and immune checkpoint blockade immunotherapy for metastases was compromised when NK cells lacked heparanase. Our data suggest that heparanase plays a critical role in NK cell invasion into tumors and thereby tumor progression and metastases. This should be considered when systemically treating cancer patients with heparanase inhibitors, since the potential adverse effect on NK cell infiltration might limit the antitumor activity of the inhibitors.
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Liasa de Heparina/inmunología , Vigilancia Inmunológica , Células Asesinas Naturales/inmunología , Neoplasias Experimentales/inmunología , Animales , Línea Celular Tumoral , Citocinas/genética , Citocinas/inmunología , Femenino , Liasa de Heparina/genética , Humanos , Células Asesinas Naturales/patología , Masculino , Ratones , Ratones Noqueados , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Invasividad Neoplásica/genética , Invasividad Neoplásica/inmunología , Metástasis de la Neoplasia , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/inmunología , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/inmunologíaRESUMEN
Preclinical studies targeting the adenosinergic pathway have gained much attention for their clinical potential in overcoming tumor-induced immunosuppression. Here, we have identified that co-blockade of the ectonucleotidase that generates adenosine CD73 and the A2A adenosine receptor (A2AR) that mediates adenosine signaling in leuokocytes, by using compound gene-targeted mice or therapeutics that target these molecules, limits tumor initiation, growth, and metastasis. This tumor control requires effector lymphocytes and interferon-γ, while antibodies targeting CD73 promote an optimal therapeutic response in vivo when engaging activating Fc receptors. In a two-way mixed leukocyte reaction using a fully human anti-CD73, we demonstrated that Fc receptor binding augmented the production of proinflammatory cytokines.
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5'-Nucleotidasa/antagonistas & inhibidores , Antagonistas del Receptor de Adenosina A2/farmacología , Tolerancia Inmunológica/inmunología , Neoplasias/inmunología , Neoplasias/terapia , 5'-Nucleotidasa/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Femenino , Humanos , Masculino , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/terapia , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/patología , Receptor de Adenosina A2A/inmunología , Transducción de SeñalRESUMEN
Cancers often evade immune surveillance by adopting peripheral tissue- tolerance mechanisms, such as the expression of programmed cell death ligand 1 (PD-L1), the inhibition of which results in potent antitumor immunity. Here, we show that cyclin-dependent kinase 5 (Cdk5), a serine-threonine kinase that is highly active in postmitotic neurons and in many cancers, allows medulloblastoma (MB) to evade immune elimination. Interferon-γ (IFN-γ)-induced PD-L1 up-regulation on MB requires Cdk5, and disruption of Cdk5 expression in a mouse model of MB results in potent CD4(+) T cell-mediated tumor rejection. Loss of Cdk5 results in persistent expression of the PD-L1 transcriptional repressors, the interferon regulatory factors IRF2 and IRF2BP2, which likely leads to reduced PD-L1 expression on tumors. Our finding highlights a central role for Cdk5 in immune checkpoint regulation by tumor cells.
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Antígeno B7-H1/genética , Neoplasias Cerebelosas/inmunología , Quinasa 5 Dependiente de la Ciclina/fisiología , Regulación Neoplásica de la Expresión Génica , Meduloblastoma/inmunología , Neoplasias Experimentales/inmunología , Escape del Tumor/genética , Animales , Linfocitos T CD4-Positivos/inmunología , Línea Celular Tumoral , Neoplasias Cerebelosas/genética , Quinasa 5 Dependiente de la Ciclina/genética , Humanos , Vigilancia Inmunológica , Factor 2 Regulador del Interferón/genética , Factor 2 Regulador del Interferón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Neoplasias Experimentales/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The presence of colony stimulating factor-1 (CSF1)/CSF1 receptor (CSF1R)-driven tumor-infiltrating macrophages and myeloid-derived suppressor cells (MDSCs) is shown to promote targeted therapy resistance. In this study, we demonstrate the superior effect of a combination of CSF1R inhibitor, PLX3397 and BRAF inhibitor, PLX4720, in suppressing primary and metastatic mouse BRAFV600E melanoma. Using flow cytometry to assess SM1WT1 melanoma-infiltrating leukocytes immediately post therapy, we found that PLX3397 reduced the recruitment of CD11b+ Gr1lo and CD11b+ Gr1int M2-like macrophages, but this was accompanied by an accumulation of CD11b+ Gr1hi cells. PDL1 expression on remaining myeloid cells potentially dampened the antitumor efficacy of PLX3397 and PLX4720 in combination, since PD1/PDL1 axis blockade improved outcome. We also reveal a role for PLX3397 in reducing tumor-infiltrating lymphocytes, and interestingly, this feature was rescued by the co-administration of PLX4720. Our findings, from three different mouse models of BRAF-mutated melanoma, support clinical approaches that co-target BRAF oncogene and CSF1R.
RESUMEN
In inflamed lymph nodes, Ag-specific CD4(+) and CD8(+) T cells encounter Ag-bearing dendritic cells and, together, this complex enhances the release of CCL3 and CCL4, which facilitate additional interaction with naive CD8(+) T cells. Although blocking CCL3 and CCL4 has no effect on primary CD8(+) T cell responses, it dramatically impairs the development of memory CD8(+) T cells upon Ag rechallenge. Despite the absence of detectable surface CCR5 expression on circulating native CD8(+) T cells, these data imply that naive CD8(+) T cells are capable of expressing surface CCR5 prior to cognate Ag-induced TCR signaling in inflamed lymph nodes; however, the molecular mechanisms have not been characterized to date. In this study, we show that CCR5, the receptor for CCL3 and CCL4, can be transiently upregulated on a subset of naive CD8(+) T cells and that this upregulation is dependent on direct contact with the high endothelial venule in inflamed lymph node. Binding of CD62L and CD11a on T cells to their ligands CD34 and CD54 on the high endothelial venule can be enhanced during inflammation. This enhanced binding and subsequent signaling promote the translocation of CCR5 molecules from intracellular vesicles to the surface of the CD8(+) T cell. The upregulation of CCR5 on the surface of the CD8(+) T cells increases the number of contacts with Ag-bearing dendritic cells, which ultimately results in increased CD8(+) T cell response to Ag rechallenge.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Ganglios Linfáticos/inmunología , Receptores CCR5/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Presentación de Antígeno , Antígenos CD34/inmunología , Antígenos CD34/metabolismo , Antígeno CD11a/inmunología , Antígeno CD11a/metabolismo , Células Dendríticas/inmunología , Inflamación , Molécula 1 de Adhesión Intercelular/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Selectina L/inmunología , Selectina L/metabolismo , Ganglios Linfáticos/citología , Ganglios Linfáticos/patología , Activación de Linfocitos , Ratones , Receptores CCR5/genética , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
Peripheral immune cells are critical to the pathogenesis of neurodegenerative diseases including multiple sclerosis (MS) (Hendriks et al., 2005; Kasper and Shoemaker, 2010). However, the precise sequence of tissue events during the early asymptomatic induction phase of experimental autoimmune encephalomyelitis (EAE) pathogenesis remains poorly defined. Due to the spatial-temporal constrains of traditional methods used to study this disease, most studies had been performed in the spine during peak clinical disease; thus the debate continues as to whether tissue changes such as vessel disruption represent a cause or a byproduct of EAE pathophysiology in the cortex. Here, we provide dynamic, high-resolution information on the evolving structural and cellular processes within the gray matter of the mouse cortex during the first 12 asymptomatic days of EAE induction. We observed that transient focal vessel disruptions precede microglia activation, followed by infiltration of and directed interaction between circulating dendritic cells and T cells. Histamine antagonist minimizes but not completely ameliorates blood vessel leaks. Histamine H1 receptor blockade prevents early microglia function, resulting in subsequent reduction in immune cell accumulation, disease incidence and clinical severity.
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Corteza Cerebral/patología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Espacio Subaracnoideo/patología , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/patología , Barrera Hematoencefálica/patología , Encefalomielitis Autoinmune Experimental/inducido químicamente , Sustancia Gris/patología , Antagonistas de los Receptores Histamínicos H1/farmacología , Hidroxizina/farmacología , Activación de Macrófagos/efectos de los fármacos , Ratones , Ratones Transgénicos , Toxina del Pertussis , Linfocitos T/inmunologíaRESUMEN
Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.
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Axones/patología , Comunicación Celular/fisiología , Microscopía Intravital/métodos , Macrófagos/patología , Microglía/patología , Traumatismos de la Médula Espinal/patología , Animales , Modelos Animales de Enfermedad , Inflamación/patología , RatonesRESUMEN
Resident microglia and blood-borne macrophages have both been implicated to play a dominant role in mediating the neuroinflammatory response affecting implanted intracortical microelectrodes. However, the distinction between each cell type has not been demonstrated due to a lack of discriminating cellular markers. Understanding the subtle differences of each cell population in mediating neuroinflammation can aid in determining the appropriate therapeutic approaches to improve microelectrode performance. Therefore, the goal of this study is to characterize the role of infiltrating blood-derived cells, specifically macrophages, in mediating neuroinflammation following intracortical microelectrode implantation. Interestingly, we found no correlation between microglia and neuron populations at the microelectrode-tissue interface. On the other hand, blood-borne macrophages consistently dominated the infiltrating cell population following microelectrode implantation. Most importantly, we found a correlation between increased populations of blood-derived cells (including the total macrophage population) and neuron loss at the microelectrode-tissue interface. Specifically, the total macrophage population was greatest at two and sixteen weeks post implantation, at the same time points when we observed the lowest densities of neuronal survival in closest proximity to the implant. Together, our results suggest a dominant role of infiltrating macrophages, and not resident microglia, in mediating neurodegeneration following microelectrode implantation.
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Electrodos Implantados , Inflamación/patología , Macrófagos/citología , Microglía/patología , Animales , Astrocitos/citología , Barrera Hematoencefálica , Movimiento Celular , Proteínas Fluorescentes Verdes/metabolismo , Inmunoglobulina G/química , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Microelectrodos , Neuronas/metabolismo , Prótesis e ImplantesRESUMEN
After traumatic spinal cord injury, functional deficits increase as axons die back from the center of the lesion and the glial scar forms. Axonal dieback occurs in two phases: an initial axon intrinsic stage that occurs over the first several hours and a secondary phase which takes place over the first few weeks after injury. Here, we examine the secondary phase, which is marked by infiltration of macrophages. Using powerful time-lapse multi-photon imaging, we captured images of interactions between Cx3cr1(+/GFP) macrophages and microglia and Thy-1(YFP) axons in a mouse dorsal column crush spinal cord injury model. Over the first few weeks after injury, axonal retraction bulbs within the lesion are static except when axonal fragments are lost by a blebbing mechanism in response to physical contact followed by phagocytosis by mobile Cx3Cr1(+/GFP) cells. Utilizing a radiation chimera model to distinguish marrow-derived cells from radio-resistant CNS-resident microglia, we determined that the vast majority of accumulated cells in the lesion are derived from the blood and only these are associated with axonal damage. Interestingly, CNS-resident Cx3Cr1(+/GFP) microglia did not increasingly accumulate nor participate in neuronal destruction in the lesion during this time period. Additionally, we found that the blood-derived cells consisted mainly of singly labeled Ccr2(+/RFP) macrophages, singly labeled Cx3Cr1(+/GFP) macrophages and a small population of double-labeled cells. Since all axon destructive events were seen in contact with a Cx3Cr1(+/GFP) cell, we infer that the CCR2 single positive subset is likely not robustly involved in axonal dieback. Finally, in our model, deletion of CCR2, a chemokine receptor, did not alter the position of axons after dieback. Understanding the in vivo cellular interactions involved in secondary axonal injury may lead to clinical treatment candidates involving modulation of destructive infiltrating blood monocytes.
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Axones/patología , Macrófagos/patología , Microglía/patología , Traumatismos de la Médula Espinal/patología , Animales , Trasplante de Médula Ósea , Receptor 1 de Quimiocinas CX3C , Comunicación Celular/inmunología , Femenino , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/inmunología , Microscopía/métodos , Monocitos/inmunología , Monocitos/patología , Compresión Nerviosa , Fagocitosis/inmunología , Receptores CCR2/genética , Receptores de Quimiocina/genética , Nervio Ciático/patología , Traumatismos de la Médula Espinal/inmunología , Quimera por TrasplanteRESUMEN
Fluorescent imaging coupled with high-resolution femto-second pulsed infrared lasers allows for interrogation of cellular interactions deeper in living tissues than ever imagined. Intra-vital imaging of the central nervous system (CNS) has provided insights into neuronal development, synaptic transmission, and even immune interactions. In this review we will discuss the two most common intravital approaches for studying the cerebral cortex in the live mouse brain for pre-clinical studies, the thinned skull and cranial window techniques, and focus on the advantages and drawbacks of each approach. In addition, we will discuss the use of neuronal physiologic parameters as determinants of successful surgical and imaging preparation.
RESUMEN
Within the central nervous system (CNS), antigen-presenting cells (APCs) play a critical role in orchestrating inflammatory responses where they present CNS-derived antigens to immune cells that are recruited from the circulation to the cerebrospinal fluid, parenchyma, and perivascular space. Available data indicate that APCs do so indirectly from outside of CNS vessels without direct access to luminal contents. Here, we applied high-resolution, dynamic intravital two-photon laser scanning microscopy to directly visualize extravascular CX3CR1+ APC behavior deep within undisrupted CNS tissues in two distinct anatomical sites under three different inflammatory stimuli. Surprisingly, we observed that CNS-resident APCs dynamically extend their cellular processes across an intact vessel wall into the vascular lumen with preservation of vessel integrity. While only a small number of APCs displayed intravascular extensions in intact, noninflamed vessels in the brain and the spinal cord, the frequency of projections increased over days in an experimental autoimmune encephalomyelitis model, whereas the number of projections remained stable compared to baseline days after tissue injury such as CNS tumor infiltration and aseptic spinal cord trauma. Our observation of this unique behavior by parenchyma CX3CR1+ cells in the CNS argues for further exploration into their functional role in antigen sampling and immune cell recruitment.
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Sistema Nervioso Central/inmunología , Células Dendríticas/inmunología , Receptores de Quimiocina/análisis , Animales , Receptor 1 de Quimiocinas CX3C , Modelos Animales de Enfermedad , Encefalomielitis/inmunología , Encefalomielitis/patología , Ratones , Microscopía ConfocalRESUMEN
Human mesenchymal stem cells (hMSCs) have gained intense research interest due to their immune-modulatory, tissue differentiating, and homing properties to sites of inflammation. Despite evidence demonstrating the biodistribution of infused hMSCs in target organs using static fluorescence imaging or whole-body imaging techniques, surprisingly little is known about how hMSCs behave dynamically within host tissues on a single-cell level in vivo. Here, we infused fluorescently labeled clinical-grade hMSCs into immune-competent mice in which neutrophils and monocytes express a second fluorescent marker under the lysozyme M (LysM) promoter. Using intravital two-photon microscopy (TPM), we were able for the first time to capture dynamic interactions between hMSCs and LysM(+) granulocytes in the calvarium bone marrow of recipient mice during systemic LPS challenge in real time. Interestingly, many of the infused hMSCs remained intact despite repeated cellular contacts with host neutrophils. However, we were able to observe the destruction and subsequent phagocytosis of some hMSCs by surrounding granulocytes. Thus, our imaging platform provides opportunities to gain insight into the biology and therapeutic mechanisms of hMSCs in vivo at a single-cell level within live hosts.