RESUMEN
The proinflammatory microRNA-155 (miR-155) is highly expressed in the serum and CNS lesions of patients with multiple sclerosis (MS). Global knockout (KO) of miR-155 in mice confers resistance to a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), by reducing the encephalogenic potential of CNS-infiltrating Th17 T cells. However, cell-intrinsic roles for miR-155 during EAE have not been formally determined. In this study, we use single-cell RNA sequencing and cell-specific conditional miR-155 KOs to determine the importance of miR-155 expression in distinct immune cell populations. Time-course single-cell sequencing revealed reductions in T cells, macrophages, and dendritic cells (DCs) in global miR-155 KO mice compared with wild-type controls at day 21 after EAE induction. Deletion of miR-155 in T cells, driven by CD4 Cre, significantly reduced disease severity similar to global miR-155 KOs. CD11c Cre-mediated deletion of miR-155 in DCs also resulted in a modest yet significant reduction in the development of EAE, with both T cell- and DC-specific KOs showing a reduction in Th17 T cell infiltration into the CNS. Although miR-155 is highly expressed in infiltrating macrophages during EAE, deletion of miR-155 using LysM Cre did not impact disease severity. Taken together, these data show that although miR-155 is highly expressed in most infiltrating immune cells, miR-155 has distinct roles and requirements depending on the cell type, and we have demonstrated this using the gold standard conditional KO approach. This provides insights into which functionally relevant cell types should be targeted by the next generation of miRNA therapeutics.
Asunto(s)
Encefalomielitis Autoinmune Experimental , MicroARNs , Esclerosis Múltiple , Animales , Ratones , Enfermedades Neuroinflamatorias , Células Th17/metabolismo , Encéfalo/patología , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The importance of individual target genes for miRNA activity has been difficult to establish. In this issue of Immunity, Lu et al. (2015) disrupt the miR-155 binding site in the SOCS1 3' UTR in the mouse germline and show that this axis is important for T and NK cell function.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Células Asesinas Naturales/inmunología , MicroARNs/genética , Proteínas Supresoras de la Señalización de Citocinas/genética , Linfocitos T Reguladores/inmunología , AnimalesRESUMEN
Chronic inflammation is a contributing factor to most life-shortening human diseases. However, the molecular and cellular mechanisms that sustain chronic inflammatory responses remain poorly understood, making it difficult to treat this deleterious condition. Using a mouse model of age-dependent inflammation that results from a deficiency in miR-146a, we demonstrate that miR-155 contributed to the progressive inflammatory disease that emerged as Mir146a(-/-) mice grew older. Upon analyzing lymphocytes from inflamed versus healthy middle-aged mice, we found elevated numbers of T follicular helper (Tfh) cells, germinal center (GC) B cells, and autoantibodies, all occurring in a miR-155-dependent manner. Further, Cd4-cre Mir155(fl/fl) mice were generated and demonstrated that miR-155 functions in T cells, in addition to its established role in B cells, to promote humoral immunity in a variety of contexts. Taken together, our study discovers that miR-146a and miR-155 counterregulate Tfh cell development that drives aberrant GC reactions during chronic inflammation.
Asunto(s)
Centro Germinal/inmunología , Inflamación/inmunología , MicroARNs/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Traslado Adoptivo , Animales , Autoanticuerpos/biosíntesis , Autoanticuerpos/inmunología , Linfocitos B/inmunología , Antígenos CD4/biosíntesis , Diferenciación Celular/inmunología , Modelos Animales de Enfermedad , Antígeno 2 Relacionado con Fos/genética , Centro Germinal/citología , Inmunidad Humoral , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
Impaired recovery of aged muscle following a disuse event is an unresolved issue facing the older adult population. Although investigations in young animals have suggested that rapid regrowth of skeletal muscle following a disuse event entails a coordinated involvement of skeletal muscle macrophages, this phenomenon has not yet been thoroughly tested as an explanation for impaired muscle recovery in aging. To examine this hypothesis, young (4-5 mo) and old (24-26 mo) male mice were examined as controls following 2 wk of hindlimb unloading (HU) and following 4 (RL4) and 7 (RL7) days of reloading after HU. Muscles were harvested to assess muscle weight, myofiber-specifc cross-sectional area, and skeletal muscle macrophages via immunofluorescence. Flow cytometry was used on gastrocnemius and soleus muscle (at RL4) single-cell suspensions to immunophenotype skeletal muscle macrophages. Our data demonstrated impaired muscle regrowth in aged compared with young mice following disuse, which was characterized by divergent muscle macrophage polarization patterns and muscle-specifc macrophage abundance. During reloading, young mice exhibited the classical increase in M1-like (MHC II+CD206-) macrophages that preceeded the increase in percentage of M2-like macrophages (MHC II-CD206+); however, old mice did not demonstrate this pattern. Also, at RL4, the soleus demonstrated reduced macrophage abundance with aging. Together, these data suggest that dysregulated macrophage phenotype patterns in aged muscle during recovery from disuse may be related to impaired muscle growth. Further investigation is needed to determine whether the dysregulated macrophage response in the old during regrowth from disuse is related to a reduced ability to recruit or activate specific immune cells.
Asunto(s)
Envejecimiento/fisiología , Polaridad Celular/fisiología , Suspensión Trasera/fisiología , Macrófagos/fisiología , Músculo Esquelético/patología , Atrofia Muscular/rehabilitación , Animales , Activación de Macrófagos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/citología , Músculo Esquelético/inmunología , Atrofia Muscular/patología , Condicionamiento Físico Animal/fisiologíaRESUMEN
FLT3-ITD+ acute myeloid leukemia (AML) accounts for â¼25% of all AML cases and is a subtype that carries a poor prognosis. microRNA-155 (miR-155) is specifically overexpressed in FLT3-ITD+ AML compared with FLT3 wild-type (FLT3-WT) AML and is critical for the growth of FLT3-ITD+ AML cells in vitro. However, miR-155's role in regulating FLT3-ITD-mediated disease in vivo remains unclear. In this study, we used a genetic mouse model to determine whether miR-155 influences the development of FLT3-ITD-induced myeloproliferative disease. Results indicate that miR-155 promotes FLT3-ITD-induced myeloid expansion in the bone marrow, spleen, and peripheral blood. Mechanistically, miR-155 increases proliferation of the hematopoietic stem and progenitor cell compartments by reducing the growth-inhibitory effects of the interferon (IFN) response, and this involves targeting of Cebpb. Consistent with our observations in mice, primary FLT3-ITD+ AML clinical samples have significantly higher miR-155 levels and a lower IFN response compared with FLT3-WT AML samples. Further, inhibition of miR-155 in FLT3-ITD+ AML cell lines using CRISPR/Cas9, or primary FLT3-ITD+ AML samples using locked nucleic acid antisense inhibitors, results in an elevated IFN response and reduces colony formation. Altogether, our data reveal that miR-155 collaborates with FLT3-ITD to promote myeloid cell expansion in vivo and that this involves a multitarget mechanism that includes repression of IFN signaling.
Asunto(s)
Interferones/biosíntesis , MicroARNs/genética , Trastornos Mieloproliferativos/etiología , Tirosina Quinasa 3 Similar a fms/genética , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Leucemia Mieloide Aguda/etiología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , MicroARNs/antagonistas & inhibidores , Mutación , Células Progenitoras Mieloides/inmunología , Células Progenitoras Mieloides/patología , Mielopoyesis/genética , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/inmunología , Ensayo de Tumor de Célula MadreRESUMEN
Extracellular vesicles, including exosomes, have recently been implicated as novel mediators of immune cell communication in mammals. However, roles for endogenously produced exosomes in regulating immune cell functions in vivo are just beginning to be identified. In this article, we demonstrate that Rab27a and Rab27b double-knockout (Rab27DKO) mice that are deficient in exosome secretion have a chronic, low-grade inflammatory phenotype characterized by elevated inflammatory cytokines and myeloproliferation. Upon further investigation, we found that some of these phenotypes could be complemented by wild-type (WT) hematopoietic cells or administration of exosomes produced by GM-CSF-expanded bone marrow cells. In addition, chronically inflamed Rab27DKO mice had a blunted response to bacterial LPS, resembling endotoxin tolerance. This defect was rescued by bone marrow exosomes from WT, but not miR-155-/-, cells, suggesting that uptake of miR-155-containing exosomes is important for a proper LPS response. Further, we found that SHIP1 and IRAK-M, direct targets of miR-155 that are known negative regulators of the LPS response, were elevated in Rab27DKO mice and decreased after treatment with WT, but not miR-155-/-, exosomes. Together, our study finds that Rab27-dependent exosome production contributes to homeostasis within the hematopoietic system and appropriate responsiveness to inflammatory stimuli.
Asunto(s)
Exosomas/metabolismo , Inflamación/inmunología , MicroARNs/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas rab27 de Unión a GTP/metabolismo , Enfermedad Aguda , Animales , Proliferación Celular , Células Cultivadas , Enfermedad Crónica , Citocinas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Tolerancia Inmunológica , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/patología , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTP/genéticaRESUMEN
MicroRNA-155 (miR-155) regulates antitumor immune responses. However, its specific functions within distinct immune cell types have not been delineated in conditional KO mouse models. In this study, we investigated the role of miR-155 specifically within T cells during the immune response to syngeneic tumors. We found that miR-155 expression within T cells is required to limit syngeneic tumor growth and promote IFNγ production by T cells within the tumor microenvironment. Consequently, we found that miR-155 expression by T cells is necessary for proper tumor-associated macrophage expression of IFNγ-inducible genes. We also found that immune checkpoint-blocking (ICB) antibodies against programmed cell death protein 1/programmed death ligand 1 (PD-1/PD-L1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) restored antitumor immunity in miR-155 T cell-conditional KO mice. We noted that these ICB antibodies rescued the levels of IFNγ-expressing T cells, expression of multiple activation and effector genes expressed by tumor-infiltrating CD8+ and CD4+ T cells, and tumor-associated macrophage activation. Moreover, the ICB approach partially restored expression of several derepressed miR-155 targets in tumor-infiltrating, miR-155-deficient CD8+ T cells, suggesting that miR-155 and ICB regulate overlapping pathways to promote antitumor immunity. Taken together, our findings highlight the multifaceted role of miR-155 in T cells, in which it promotes antitumor immunity. These results suggest that the augmentation of miR-155 expression could be used to improve anticancer immunotherapies.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Antígeno CTLA-4/antagonistas & inhibidores , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Melanoma/tratamiento farmacológico , MicroARNs/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Animales , Anticuerpos Bloqueadores/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Antígeno B7-H1/metabolismo , Antígeno CTLA-4/metabolismo , Línea Celular Tumoral , Cruzamientos Genéticos , Vigilancia Inmunológica/efectos de los fármacos , Interferón gamma/antagonistas & inhibidores , Interferón gamma/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Melanoma/inmunología , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , MicroARNs/genética , Trasplante de Neoplasias , Receptor de Muerte Celular Programada 1/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patología , Carga Tumoral/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacosRESUMEN
Th17 cells are central to the pathogenesis of autoimmune disease, and recently specific noncoding microRNAs have been shown to regulate their development. However, it remains unclear whether microRNAs are also involved in modulating Th17 cell effector functions. Consequently, we examined the role of miR-155 in differentiated Th17 cells during their induction of experimental autoimmune encephalomyelitis. Using adoptive transfer experiments, we found that highly purified, myelin oligodendrocyte glycoprotein Ag-specific Th17 cells lacking miR-155 were defective in their capacity to cause experimental autoimmune encephalomyelitis. Gene expression profiling of purified miR-155(-/-)IL-17F(+) Th17 cells identified a subset of effector genes that are dependent on miR-155 for their proper expression through a mechanism involving repression of the transcription factor Ets1. Among the genes reduced in the absence of miR-155 was IL-23R, resulting in miR-155(-/-) Th17 cells being hyporesponsive to IL-23. Taken together, our study demonstrates a critical role for miR-155 in Th17 cells as they unleash autoimmune inflammation and finds that this occurs through a signaling network involving miR-155, Ets1, and the clinically relevant IL-23-IL-23R pathway.
Asunto(s)
Encefalomielitis Autoinmune Experimental/genética , Regulación de la Expresión Génica/inmunología , MicroARNs/genética , Células Th17/inmunología , Traslado Adoptivo , Animales , Encefalomielitis Autoinmune Experimental/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Immunoblotting , Ratones , Ratones Noqueados , MicroARNs/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/inmunología , TranscriptomaRESUMEN
Tumor associated macrophage responses are regulated by distinct metabolic states that affect their function. However, the ability of specific signals in the local tumor microenvironment to program macrophage metabolism remains under investigation. Here, we identify NAMPT, the rate limiting enzyme in NAD salvage synthesis, as a target of STAT1 during cellular activation by interferon gamma, an important driver of macrophage polarization and antitumor responses. We demonstrate that STAT1 occupies a conserved element within the first intron of Nampt, termed Nampt-Regulatory Element-1 (NRE1). Through disruption of NRE1 or pharmacological inhibition, a subset of M1 genes is sensitive to NAMPT activity through its impact on glycolytic processes. scRNAseq is used to profile in vivo responses by NRE1-deficient, tumor-associated leukocytes in melanoma tumors through the creation of a unique mouse strain. Reduced Nampt and inflammatory gene expression are present in specific myeloid and APC populations; moreover, targeted ablation of NRE1 in macrophage lineages results in greater tumor burden. Finally, elevated NAMPT expression correlates with IFNγ responses and melanoma patient survival. This study identifies IFN and STAT1-inducible Nampt as an important factor that shapes the metabolic program and function of tumor associated macrophages.
Asunto(s)
Citocinas/genética , Melanoma/genética , Nicotinamida Fosforribosiltransferasa/genética , Factor de Transcripción STAT1/metabolismo , Neoplasias Cutáneas/genética , Macrófagos Asociados a Tumores/inmunología , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Elementos de Facilitación Genéticos , Femenino , Regulación Neoplásica de la Expresión Génica/inmunología , Células HEK293 , Humanos , Interferón gamma/metabolismo , Estimación de Kaplan-Meier , Masculino , Melanoma/inmunología , Melanoma/mortalidad , Melanoma/patología , Ratones , Ratones Noqueados , Nicotinamida Fosforribosiltransferasa/metabolismo , Células RAW 264.7 , RNA-Seq , Receptores de Interferón/genética , Receptores de Interferón/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Células THP-1 , Macrófagos Asociados a Tumores/metabolismo , Regulación hacia Arriba , Efecto Warburg en Oncología , Receptor de Interferón gammaRESUMEN
miR-155 has recently emerged as an important promoter of antitumor immunity through its functions in T lymphocytes. However, the impact of T cell-expressed miR-155 on immune cell dynamics in solid tumors remains unclear. In the present study, we used single-cell RNA sequencing to define the CD45+ immune cell populations at different time points within B16F10 murine melanoma tumors growing in either wild-type or miR-155 T cell conditional knockout (TCKO) mice. miR-155 was required for optimal T cell activation and reinforced the T cell response at the expense of infiltrating myeloid cells. Further, myeloid cells from tumors growing in TCKO mice were defined by an increase in wound healing genes and a decreased IFN-γ-response gene signature. Finally, we found that miR-155 expression predicted a favorable outcome in human melanoma patients and was associated with a strong immune signature. Moreover, gene expression analysis of The Cancer Genome Atlas (TCGA) data revealed that miR-155 expression also correlates with an immune-enriched subtype in 29 other human solid tumors. Together, our study provides an unprecedented analysis of the cell types and gene expression signatures of immune cells within experimental melanoma tumors and elucidates the role of miR-155 in coordinating antitumor immune responses in mammalian tumors.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/inmunología , Melanoma Experimental/genética , MicroARNs/metabolismo , Neoplasias/genética , Animales , Línea Celular Tumoral/trasplante , Conjuntos de Datos como Asunto , Perfilación de la Expresión Génica , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Estimación de Kaplan-Meier , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Noqueados , MicroARNs/genética , Neoplasias/inmunología , Neoplasias/mortalidad , Neoplasias/patología , Pronóstico , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Linfocitos T/inmunología , Linfocitos T/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
MicroRNA (miRNA) regulation of gene expression is becoming an increasingly recognized mechanism by which host immune responses are governed following microbial infection. miRNAs are short, non-coding RNAs that repress translation of target genes, and have been implicated in a number of activities that modulate host immune responses, including the regulation of immune cell proliferation, survival, expansion, differentiation, migration, polarization, and effector function. This review highlights several examples in which mammalian-encoded miR-155 influences immune responses following viral infection of the CNS.
Asunto(s)
Encefalitis , MicroARNs/genética , Mielitis , Virosis/complicaciones , Animales , Encefalitis/etiología , Encefalitis/inmunología , Encefalitis/virología , Regulación Viral de la Expresión Génica/inmunología , Humanos , MicroARNs/metabolismo , Mielitis/etiología , Mielitis/inmunología , Mielitis/virologíaRESUMEN
An increased understanding of antitumor immunity is necessary for improving cell-based immunotherapies against human cancers. Here, we investigated the roles of two immune system-expressed microRNAs (miRNAs), miR-155 and miR-146a, in the regulation of antitumor immune responses. Our results indicate that miR-155 promotes and miR-146a inhibits interferon γ (IFNγ) responses by T cells and reduces solid tumor growth in vivo. Using a double-knockout (DKO) mouse strain deficient in both miR-155 and miR-146a, we have also identified an epistatic relationship between these two miRNAs. DKO mice had defective T cell responses and tumor growth phenotypes similar to miR-155(-/-) mice. Further analysis of the T cell compartment revealed that miR-155 modulates IFNγ expression through a mechanism involving repression of Ship1. Our work reveals critical roles for miRNAs in the reciprocal regulation of CD4(+) and CD8(+) T cell-mediated antitumor immunity and demonstrates the dominant nature of miR-155 during its promotion of immune responses.