RESUMO
Eosinophilia is a hallmark characteristic of T helper type 2 (TH2) cell-associated diseases and is critically regulated by the central eosinophil growth factor interleukin 5 (IL-5). Here we demonstrate that IL-5 activity in eosinophils was regulated by paired immunoglobulin-like receptors PIR-A and PIR-B. Upon self-recognition of ß2-microglobulin (ß2M) molecules, PIR-B served as a permissive checkpoint for IL-5-induced development of eosinophils by suppressing the proapoptotic activities of PIR-A, which were mediated by the Grb2-Erk-Bim pathway. PIR-B-deficient bone marrow eosinophils underwent compartmentalized apoptosis, resulting in decreased blood eosinophilia in naive mice and in mice challenged with IL-5. Subsequently, Pirb(-/-) mice displayed impaired aeroallergen-induced lung eosinophilia and induction of lung TH2 cell responses. Collectively, these data uncover an intrinsic, self-limiting pathway regulating IL-5-induced expansion of eosinophils, which has broad implications for eosinophil-associated diseases.
Assuntos
Diferenciação Celular/imunologia , Eosinófilos/imunologia , Interleucina-5/imunologia , Receptores Imunológicos/imunologia , Animais , Apoptose/genética , Apoptose/imunologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Asma/genética , Asma/imunologia , Asma/metabolismo , Proteína 11 Semelhante a Bcl-2 , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias/métodos , Eosinófilos/citologia , Eosinófilos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Citometria de Fluxo , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/imunologia , Proteína Adaptadora GRB2/metabolismo , Expressão Gênica/imunologia , Interleucina-5/farmacologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologiaRESUMO
The production of mature eosinophils (Eos) is a tightly orchestrated process with the aim to sustain normal Eos levels in tissues while also maintaining low numbers of these complex and sensitive cells in the blood. To identify regulators of homeostatic eosinophilopoiesis in mice, we took a global approach to identify genome-wide transcriptome and epigenome changes that occur during homeostasis at critical developmental stages, including Eos-lineage commitment and lineage maturation. Our analyses revealed a markedly greater number of transcriptome alterations associated with Eos maturation (1199 genes) than with Eos-lineage commitment (490 genes), highlighting the greater transcriptional investment necessary for differentiation. Eos-lineage-committed progenitors (EoPs) were noted to express high levels of granule proteins and contain granules with an ultrastructure distinct from that of mature resting Eos. Our analyses also delineated a 976-gene Eos-lineage transcriptome that included a repertoire of 56 transcription factors, many of which have never previously been associated with Eos. EoPs and Eos, but not granulocyte-monocyte progenitors or neutrophils, expressed Helios and Aiolos, members of the Ikaros family of transcription factors, which regulate gene expression via modulation of chromatin structure and DNA accessibility. Epigenetic studies revealed a distinct distribution of active chromatin marks between genes induced with lineage commitment and genes induced with cell maturation during Eos development. In addition, Aiolos and Helios binding sites were significantly enriched in genes expressed by EoPs and Eos with active chromatin, highlighting a potential novel role for Helios and Aiolos in regulating gene expression during Eos development.
Assuntos
Proteínas de Ligação a DNA/genética , Eosinófilos/citologia , Hematopoese/genética , Transativadores/genética , Fatores de Transcrição/genética , Transcriptoma/genética , Animais , Sítios de Ligação/genética , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Células Cultivadas , Cromatina/genética , Grânulos Citoplasmáticos/metabolismo , Eosinófilos/imunologia , Regulação da Expressão Gênica/genética , Células Precursoras de Granulócitos , Hematopoese/imunologia , Fator de Transcrição Ikaros , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Transcrição/biossínteseRESUMO
Eosinophils originate in the bone marrow from an eosinophil lineage-committed, IL-5Rα-positive, hematopoietic progenitor (eosinophil progenitor). Indeed, IL-5 is recognized as a critical regulator of eosinophilia and has effects on eosinophil progenitors, eosinophil precursors, and mature eosinophils. However, substantial levels of eosinophils remain after IL-5 neutralization or genetic deletion, suggesting that there are alternative pathways for promoting eosinophilia. In this study, we investigated the contributory role of IL-5 accessory cytokines on the final stages of eosinophil differentiation. IL-5 stimulation of low-density bone marrow cells resulted in expression of a panel of cytokines and cytokine receptors, including several ligand-receptor pairs. Notably, IL-4 and IL-4Rα were expressed by eosinophil precursors and mature eosinophils. Signaling through IL-4Rα promoted eosinophil maturation when IL-5 was present, but IL-4 stimulation in the absence of IL-5 resulted in impaired eosinophil survival, suggesting that IL-4 cooperates with IL-5 to promote eosinophil differentiation. In contrast, CCL3, an eosinophil precursor-produced chemokine that signals through CCR1, promotes terminal differentiation of CCR1-positive eosinophil precursors in the absence of IL-5, highlighting an autocrine loop capable of sustaining eosinophil differentiation. These findings suggest that brief exposure to IL-5 is sufficient to initiate a cytokine cooperative network that promotes eosinophil differentiation of low-density bone marrow cells independent of further IL-5 stimulation.
Assuntos
Diferenciação Celular/imunologia , Eosinófilos/efeitos dos fármacos , Subunidade alfa de Receptor de Interleucina-5/imunologia , Interleucina-5/farmacologia , Animais , Células da Medula Óssea/citologia , Linhagem da Célula/imunologia , Células Cultivadas , Quimiocina CCL3/biossíntese , Quimiocina CCL3/imunologia , Eosinofilia/imunologia , Eosinófilos/imunologia , Feminino , Interleucina-4/biossíntese , Interleucina-4/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Receptores CCR1/biossíntese , Receptores CCR1/imunologia , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/imunologiaRESUMO
Eosinophils are major effector cells in type 2 inflammatory responses and become activated in response to IL-4 and IL-33, yet the molecular mechanisms and cooperative interaction between these cytokines remain unclear. Our objective was to investigate the molecular mechanism and cooperation of IL-4 and IL-33 in eosinophil activation. Eosinophils derived from bone marrow or isolated from Il5-transgenic mice were activated in the presence of IL-4 or IL-33 for 1 or 4 h, and the transcriptome was analyzed by RNA sequencing. The candidate genes were validated by quantitative PCR and ELISA. We demonstrated that murine-cultured eosinophils respond to IL-4 and IL-33 by phosphorylation of STAT-6 and NF-κB, respectively. RNA sequence analysis of murine-cultured eosinophils indicated that IL-33 induced 519 genes, whereas IL-4 induced only 28 genes, including 19 IL-33-regulated genes. Interestingly, IL-33 induced eosinophil activation via two distinct mechanisms, IL-4 independent and IL-4 secretion/autostimulation dependent. Anti-IL-4 or anti-IL-4Rα Ab-treated cultured and mature eosinophils, as well as Il4- or Stat6-deficient cultured eosinophils, had attenuated protein secretion of a subset of IL-33-induced genes, including Retnla and Ccl17. Additionally, IL-33 induced the rapid release of preformed IL-4 protein from eosinophils by a NF-κB-dependent mechanism. However, the induction of most IL-33-regulated transcripts (e.g., Il6 and Il13) was IL-4 independent and blocked by NF-κB inhibition. In conclusion, we have identified a novel activation pathway in murine eosinophils that is induced by IL-33 and differentially dependent upon an IL-4 auto-amplification loop.
Assuntos
Células da Medula Óssea/imunologia , Eosinófilos/imunologia , Interleucina-4/imunologia , Interleucinas/imunologia , Animais , Anticorpos/imunologia , Sequência de Bases , Células Cultivadas , Inflamação/imunologia , Interleucina-33 , Interleucina-4/genética , Interleucina-4/metabolismo , Interleucinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Fosforilação , Receptores de Interleucina-4/imunologia , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Análise de Sequência de RNA , Transdução de Sinais/imunologiaRESUMO
Human intestinal organoids (HIOs) derived from pluripotent stem cells provide a valuable model for investigating human intestinal organogenesis and physiology, but they lack the immune components required to fully recapitulate the complexity of human intestinal biology and diseases. To address this issue and to begin to decipher human intestinal-immune crosstalk during development, we generated HIOs containing immune cells by transplanting HIOs under the kidney capsule of mice with a humanized immune system. We found that human immune cells temporally migrate to the mucosa and form cellular aggregates that resemble human intestinal lymphoid follicles. Moreover, after microbial exposure, epithelial microfold cells are increased in number, leading to immune cell activation determined by the secretion of IgA antibodies in the HIO lumen. This in vivo HIO system with human immune cells provides a framework for future studies on infection- or allergen-driven intestinal diseases.
Assuntos
Células-Tronco Pluripotentes , Transplantes , Humanos , Animais , Camundongos , Intestinos , Mucosa Intestinal , OrganoidesRESUMO
Most organs have tissue-resident immune cells. Human organoids lack these immune cells, which limits their utility in modeling many normal and disease processes. Here, we describe that pluripotent stem cell-derived human colonic organoids (HCOs) co-develop a diverse population of immune cells, including hemogenic endothelium (HE)-like cells and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of functional macrophages. HCO macrophages acquired a transcriptional signature resembling human fetal small and large intestine tissue-resident macrophages. HCO macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals and were able to phagocytose and mount a robust response to pathogenic bacteria. When transplanted into mice, HCO macrophages were maintained within the colonic organoid tissue, established a close association with the colonic epithelium, and were not displaced by the host bone-marrow-derived macrophages. These studies suggest that HE in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages.
Assuntos
Células-Tronco Pluripotentes , Humanos , Camundongos , Animais , Células-Tronco Hematopoéticas , Colo , Organoides , MacrófagosRESUMO
The cation-chloride cotransporter NKCC1 plays a fundamental role in the central and peripheral nervous systems by setting the value of intracellular chloride concentration. Following peripheral nerve injury, NKCC1 phosphorylation-induced chloride accumulation contributes to neurite regrowth of sensory neurons. However, the molecules and signaling pathways that regulate NKCC1 activity remain to be identified. Functional analysis of cotransporter activity revealed that inhibition of endogenously produced cytokine interleukin-6 (IL-6), with anti-mouse IL-6 antibody or in IL-6â»/â» mice, prevented chloride accumulation in a subset of axotomized neurons. Nerve injury upregulated the transcript and protein levels of IL-6 receptor in myelinated, TrkB-positive sensory neurons of murine lumbar dorsal root ganglia. Expression of phospho-NKCC1 was observed mainly in sensory neurons expressing IL-6 receptor and was absent from IL-6â»/â» dorsal root ganglia. The use of IL-6 receptor blocking-function antibody or soluble IL-6 receptor, together with pharmacological inhibition of Janus kinase, confirmed the role of neuronal IL-6 signaling in chloride accumulation and neurite growth of a subset of axotomized sensory neurons. Cell-specific expression of interleukin-6 receptor under pathophysiological conditions is therefore a cellular response by which IL-6 contributes to nerve regeneration through neuronal NKCC1 phosphorylation and chloride accumulation.
Assuntos
Cloretos/fisiologia , Interleucina-6/fisiologia , Células Receptoras Sensoriais/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Animais , Axotomia/métodos , Células Cultivadas , Cloretos/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Gânglios Espinais/metabolismo , Interleucina-6/genética , Janus Quinases/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/fisiologia , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Técnicas de Patch-Clamp , Fosforilação , Receptores de Interleucina-6/biossíntese , Receptores de Interleucina-6/fisiologia , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/fisiologia , Simportadores de Cloreto de Sódio-Potássio/fisiologia , Membro 2 da Família 12 de Carreador de Soluto , Regulação para CimaRESUMO
Pluripotent-stem-cell-derived human intestinal organoids (HIOs) model some aspects of intestinal development and disease, but current culture methods do not fully recapitulate the diverse cell types and complex organization of the human intestine and are reliant on 3D extracellular matrix or hydrogel systems, which limit experimental control and translational potential for regenerative medicine. We describe suspension culture as a simple, low-maintenance method for culturing HIOs and for promoting in vitro differentiation of an organized serosal mesothelial layer that is similar to primary human intestinal serosal mesothelium based on single-cell RNA sequencing and histological analysis. Functionally, HIO serosal mesothelium has the capacity to differentiate into smooth-muscle-like cells and exhibits fibrinolytic activity. An inhibitor screen identifies Hedgehog and WNT signaling as regulators of human serosal mesothelial differentiation. Collectively, suspension HIOs represent a three-dimensional model to study the human serosal mesothelium.
Assuntos
Epitélio/crescimento & desenvolvimento , Intestinos/crescimento & desenvolvimento , Organoides/crescimento & desenvolvimento , Membrana Serosa/crescimento & desenvolvimento , Técnicas de Cultura de Tecidos , Alginatos/farmacologia , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Colágeno/farmacologia , Combinação de Medicamentos , Epitélio/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Humanos , Intestinos/ultraestrutura , Laminina/farmacologia , Músculo Liso/citologia , Organoides/efeitos dos fármacos , Organoides/ultraestrutura , Proteoglicanas/farmacologia , Membrana Serosa/efeitos dos fármacos , Membrana Serosa/ultraestrutura , Transdução de Sinais/efeitos dos fármacos , Suspensões , Proteínas Wnt/metabolismoRESUMO
OBJECTIVES: Mesenchymal stromal cells (MSC) are characterised by their capacity to suppress immune reactions. This function was reported to be shared in vitro by fibroblasts but their role has been poorly investigated in vivo. This study explored whether fibroblasts isolated from skin may suppress the host immune response in a model of autoimmune disorder. METHODS AND RESULTS: It was first confirmed that skin fibroblasts lack the capacity to differentiate into osteoblasts or chondrocytes but possess the capacity to inhibit in vitro the proliferation of T lymphocytes. Fibroblasts also secrete modulatory molecules, in particular prostaglandin E2 and nitric oxide, similar to MSC. To assess their role in vivo, the collagen-induced arthritis model was used, and showed that similar to MSC the intravenous injection of fibroblasts efficiently suppress clinical signs of arthritis and delay disease onset. This effect was associated with reduced inflammation as reflected by biological parameters and increased levels of IL-5, IL-10 and IL-13 in the spleens of treated mice. To characterise the mechanism of immunosuppression further, phenotypic analyses were performed and could not detect any induction of CD4 CD25 Foxp3(+) regulatory T (Treg) cells. A population of CD4 IL-10(+) T cells was, however, detected that was slightly increased after fibroblast injection and significantly upregulated after MSC administration. CONCLUSIONS: This study gives the first evidence for an immunosuppressive role of fibroblasts in vivo, and strongly suggests that fibroblasts induce a T-helper type 2 immune profile, although the possibility that IL-10-secreting Treg cells may be generated cannot be excluded.
Assuntos
Artrite Experimental/prevenção & controle , Doenças Autoimunes/prevenção & controle , Fibroblastos/transplante , Terapia de Imunossupressão/métodos , Pele/imunologia , Animais , Artrite Experimental/imunologia , Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Fibroblastos/imunologia , Tolerância Imunológica , Imunofenotipagem , Interleucina-10/biossíntese , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Endogâmicos DBA , Baço/imunologiaRESUMO
OBJECTIVE: RNA interference (RNAi) is a powerful tool for sequence-specific gene silencing, and interest in its application in human diseases is growing. Given the success of recent strategies for administering gene therapy in rheumatoid arthritis using recombinant vectors such as adeno-associated virus type 5 (rAAV5) for optimized intraarticular gene transfer, we undertook the present study to determine the feasibility of using rAAV5-mediated RNAi-based therapy in arthritis. METHODS: We developed rAAV5 vectors expressing short hairpin small interfering RNA (shRNA) against tumor necrosis factor alpha (TNFalpha) under H1 promoter, and carrying the enhanced green fluorescent protein (eGFP) reporter gene under cytomegalovirus promoter (rAAV5-shTNF). TNFalpha gene silencing was validated in vitro with mouse macrophages. Mice with collagen-induced arthritis were injected in the ankle and knee joints, at disease onset, with either rAAV5-shTNF or control rAAV5-eGFP vectors (5 x 10(9) particles). Arthritis severity was assessed clinically and histologically, and immunologic response was examined. Local and systemic transgene expression was monitored using quantitative reverse transcriptase-polymerase chain reaction, immunohistochemical analysis, and enzyme-linked immunosorbent assay. RESULTS: After a single injection of rAAV5-shTNF into inflamed joints, local TNFalpha gene silencing provided rapid and long-term suppression of arthritis progression and reduced joint damage compared with that observed in control groups. Treatment with rAAV5-shTNF was associated with decreased proliferation and interferon-gamma production by antigen-stimulated T cells from draining lymph nodes, and the potency of this treatment was similar to that observed with other treatment strategies targeting TNFalpha at the protein level, either locally or systemically. CONCLUSION: Our data present the first proof-of-concept for the application of rAAV5-mediated RNAi-based gene therapy for local blockade of inflammation in experimental arthritis.
Assuntos
Artrite Experimental/terapia , Interferência de RNA/fisiologia , RNA Interferente Pequeno/administração & dosagem , Fator de Necrose Tumoral alfa/genética , Animais , Dependovirus , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Inativação Gênica/fisiologia , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Imuno-Histoquímica , Injeções Intra-Articulares , Camundongos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Expression of Ikaros family transcription factor IKZF3 (Aiolos) increases during murine eosinophil lineage commitment and maturation. Herein, we investigated Aiolos expression and function in mature human and murine eosinophils. Murine eosinophils deficient in Aiolos demonstrated gene expression changes in pathways associated with granulocyte-mediated immunity, chemotaxis, degranulation, ERK/MAPK signaling, and extracellular matrix organization; these genes had ATAC peaks within 1 kB of the TSS that were enriched for Aiolos-binding motifs. Global Aiolos deficiency reduced eosinophil frequency within peripheral tissues during homeostasis; a chimeric mouse model demonstrated dependence on intrinsic Aiolos expression by eosinophils. Aiolos deficiency reduced eosinophil CCR3 surface expression, intracellular ERK1/2 signaling, and CCL11-induced actin polymerization, emphasizing an impaired functional response. Aiolos-deficient eosinophils had reduced tissue accumulation in chemokine-, antigen-, and IL-13-driven inflammatory experimental models, all of which at least partially depend on CCR3 signaling. Human Aiolos expression was associated with active chromatin marks enriched for IKZF3, PU.1, and GATA-1-binding motifs within eosinophil-specific histone ChIP-seq peaks. Furthermore, treating the EOL-1 human eosinophilic cell line with lenalidomide yielded a dose-dependent decrease in Aiolos. These collective data indicate that eosinophil homing during homeostatic and inflammatory allergic states is Aiolos-dependent, identifying Aiolos as a potential therapeutic target for eosinophilic disease.
Assuntos
Quimiotaxia de Leucócito/genética , Quimiotaxia de Leucócito/imunologia , Eosinófilos/imunologia , Eosinófilos/metabolismo , Fator de Transcrição Ikaros/genética , Alérgenos/imunologia , Animais , Suscetibilidade a Doenças , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Granulócitos/imunologia , Granulócitos/metabolismo , Humanos , Fator de Transcrição Ikaros/metabolismo , Imunidade Inata , Imunofenotipagem , Contagem de Leucócitos , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Receptores CCR3/genética , Receptores CCR3/metabolismo , Transdução de SinaisRESUMO
Multipotent mesenchymal stromal cells (MSCs) have raised interest mainly because of cartilage/bone differentiation potential which is now partly eclipsed by their capacity to counteract inflammation and suppress host immune responses as well as to prevent fibrosis. MSCs have been identified within joint tissues including synovium, cartilage, subchondral bone, periosteum or adipose tissue. They are characterized by their phenotype and their ability to differentiate into three lineages, chondrocytes, osteoblasts and adipocytes. MSCs have also paracrine effects through the secretion of a number of cytokines and growth factors. This may explain the trophic effects that may be of therapeutic value for rheumatic diseases including OA and RA. On the other hand, MSCs have been associated with tumour growth. MSCs migrate to the tumour stroma, express chemokines involved in the attraction of carcinoma cells in metastasis. Indeed, the aim of this review is not only to focus on new potential therapeutic applications in osteo-articular diseases, but also to assess the potential risk of MSC-based cell therapy.
Assuntos
Artrite Reumatoide/patologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/patologia , Células-Tronco Multipotentes/patologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/terapia , Doenças Autoimunes/patologia , Doenças Autoimunes/terapia , Humanos , Tolerância Imunológica , Células-Tronco Multipotentes/imunologia , Células-Tronco Multipotentes/transplanteRESUMO
Prostate cancer metastasis to bone results in mixed osteolytic and osteoblastic lesions associated with high morbidity, and there is mounting evidence that the urokinase-type plasminogen system is causatively involved in the progression of prostate cancer. Adult mesenchymal stem cells (MSCs) are promising tools for cell-mediated gene therapy with the advantage of osteogenic potential, a critical issue in the case of osteolytic metastases. In this study, we evaluated the therapeutic use of engineered murine MSCs for in vivo delivery of the urokinase-type plasminogen antagonist amino-terminal fragment (hATF) to impair osteolytic prostate cancer cell progression in bone and to repair bone lesions. Bioluminescence imaging revealed that both primary MSCs and the MSC line C3H10T1/2 (C3) expressing hATF (MSC-hATF) significantly inhibited intratibial PC-3 Luciferase (Luc) growth following coinjection in SCID mice. Furthermore, microcomputed tomography imaging of vascular network clearly demonstrated a significant decrease in tumor-associated angiogenesis and a protection from tumor-induced osteolysis in MSC-hATF-treated mice. Importantly, the osteogenic potential of MSC-hATF cells was unaffected, and an area of new bone formation was evidenced in 60% of animals. Together, these data support the concept of MSC-based therapy of tumor osteolysis disease, indicating that MSCs may combine properties of vehicle for angiostatic agent with osteogenic potential. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Neoplasias Ósseas/terapia , Células-Tronco Mesenquimais/citologia , Fragmentos de Peptídeos/biossíntese , Neoplasias da Próstata/terapia , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Adenoviridae/genética , Animais , Neoplasias Ósseas/irrigação sanguínea , Neoplasias Ósseas/secundário , Comunicação Celular , Linhagem Celular Tumoral , Estudos de Viabilidade , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos SCID , Neovascularização Patológica/terapia , Osteogênese , Osteólise/patologia , Osteólise/terapia , Fragmentos de Peptídeos/genética , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/patologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Although cartilage defects are common features of osteoarthritis and rheumatoid arthritis, current treatments can rarely restore the full function of native cartilage. Recent studies have provided new perspectives for cartilage engineering using multipotent mesenchymal stromal cells (MSC). Moreover, MSC have been used as immunosuppressant agents in autoimmune diseases and have tested successfully in animal models of arthritis. However, the sequential events occurring during chondrogenesis must be fully understood before we can reproduce the complex molecular events that lead to MSC differentiation and long-term maintenance of cartilage characteristics in the context of chronic joint inflammation. This chapter focuses on the potential of MSC to repair cartilage, with an emphasis on the factors that are known to be required in inducing chondrogenesis and on their immunosuppressive potential.
Assuntos
Cartilagem/imunologia , Células-Tronco Mesenquimais/imunologia , Animais , Artrite Reumatoide/terapia , Células da Medula Óssea/fisiologia , Cartilagem/citologia , Diferenciação Celular/fisiologia , Condrogênese/imunologia , Modelos Animais de Doenças , Humanos , Terapia de Imunossupressão , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologiaRESUMO
Dynamic gene expression is a major mechanism that directs hematopoietic lineage commitment and differentiation. Recent advances have revealed an association between chromatin signatures and functional genetic sequences such that these chromatin signatures can be used to predict regulatory elements such as enhancers that may direct lineage differentiation. Our understanding of the genetic elements that regulate eosinophil development is very limited, likely due to the technical challenges in working with a rare complex cell. Herein, we describe protocols to sort mature eosinophils from the bone marrow of mice and to prepare chromatin that can be used for ChIP studies for genome-wide mapping of histone marks and transcription factors in mature eosinophils. Comprehensive epigenomic profiling during critical stages in eosinophil development will ultimately aid in defining the gene regulatory networks necessary to regulate eosinophil production.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Eosinófilos/imunologia , Eosinófilos/metabolismo , Estudo de Associação Genômica Ampla , Animais , Separação Celular/métodos , Imunoprecipitação da Cromatina , Epigênese Genética , Citometria de Fluxo , Regulação da Expressão Gênica , CamundongosRESUMO
Multipotent mesenchymal stromal cells or mesenchymal stem cells (MSC) are isolated mainly from bone marrow and adipose tissue but are identified in other tissues such as synovium, periosteum or placenta. They are characterised by their property to adhere to plastic, their phenotype and their ability to differentiate into three lineages (chondrocytes, osteoblasts and adipocytes). More recently, these cells were shown to escape immune recognition and inhibit immune responses. MSC may modulate the function of the major immune cell populations, including antigen-presenting cells, T cells, B cells and natural killer cells. The aim of this review is to focus on the molecular mechanisms, still poorly understood, which are responsible of the immunosuppressive effects mediated by the MSC. Finally, the data obtained from in vivo experimentation in various animal models as well as potential therapeutic applications will be presented.
Assuntos
Tolerância Imunológica , Células-Tronco Multipotentes/imunologia , Animais , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Sistema Imunitário/citologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Células-Tronco Multipotentes/transplante , Células Estromais/imunologia , Células Estromais/transplanteRESUMO
Eosinophils and their associated cytokines IL-4 and IL-5 are emerging as central orchestrators of the immune-metabolic axis. Herein, we demonstrate that cross-talk between the Ig-superfamily receptor CD300f and IL-5 is a key checkpoint that modifies the ability of eosinophils to regulate metabolic outcomes. Generation of Il5 Tg /Cd300f -/- mice revealed marked and distinct increases in eosinophil levels and their production of IL-4 in the white and brown adipose tissues. Consequently, Il5 Tg /Cd300f -/- mice had increased alternatively activated macrophage accumulation in the adipose tissue. Cd300f -/- mice displayed age-related accumulation of eosinophils and macrophages in the adipose tissue and decreased adipose tissue weight, which was associated with decreased diet-induced weight gain and insulin resistance. Notably, Il5 Tg /CD300f -/- were protected from diet-induced weight gain and glucose intolerance. These findings highlight the cross-talk between IL-5 receptor and CD300f as a novel pathway regulating adipose tissue eosinophils and offer new entry points for therapeutic intervention for obesity and its complications.
Assuntos
Tecido Adiposo/citologia , Eosinófilos/metabolismo , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Receptores Imunológicos/metabolismo , Animais , Células Endoteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Intolerância à Glucose/metabolismo , Intolerância à Glucose/patologia , Ligantes , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Interleucina-5/metabolismo , Aumento de PesoRESUMO
BACKGROUND: Based on their capacity to suppress immune responses, multipotent mesenchymal stromal cells (MSC) are intensively studied for various clinical applications. Although it has been shown in vitro that the immunomodulatory effect of MSCs mainly occurs through the secretion of soluble mediators, the mechanism is still not completely understood. The aim of the present study was to better understand the mechanisms underlying the suppressive effect of MSCs in vivo, using cells isolated from mice deficient in the production of inducible nitric oxide synthase (iNOS) or interleukin (IL)-6 in the murine model of collagen-induced arthritis. PRINCIPAL FINDINGS: In the present study, we show that primary murine MSCs from various strains of mice or isolated from mice deficient for iNOS or IL-6 exhibit different immunosuppressive potential. The immunomodulatory function of MSCs was mainly attributed to IL-6-dependent secretion of prostaglandin E2 (PGE2) with a minor role for NO. To address the role of these molecules in vivo, we used the collagen-induced arthritis as an experimental model of immune-mediated disorder. MSCs effectively inhibited collagen-induced inflammation during a narrow therapeutic window. In contrast to wild type MSCs, IL-6-deficient MSCs and to a lesser extent iNOS-deficient MSCs were not able to reduce the clinical signs of arthritis. Finally, we show that, independently of NO or IL-6 secretion or Treg cell induction, MSCs modulate the host response by inducing a switch to a Th2 immune response. SIGNIFICANCE: Our data indicate that mscs mediate their immunosuppressive effect via two modes of action: locally, they reduce inflammation through the secretion of anti-proliferative mediators, such as NO and mainly PGE2, and systemically they switch the host response from a Th1/Th17 towards a Th2 immune profile.
Assuntos
Artrite Experimental/metabolismo , Dinoprostona/metabolismo , Interleucina-6/metabolismo , Células-Tronco Mesenquimais/citologia , Animais , Artrite/metabolismo , Diferenciação Celular , Colágeno/metabolismo , Imunossupressores/química , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Óxido Nítrico Sintase Tipo II/metabolismo , Fenótipo , Células Th2/metabolismoRESUMO
Mesenchymal stem cells (MSCs) are multipotential nonhematopoietic progenitor cells that are isolated from many adult tissues, in particular from the bone marrow and adipose tissue. Along with their capacity for differentiating into cells of mesodermal lineage, such as adipocytes, osteoblasts and chondrocytes, these cells have also generated great interest for their ability to display immunomodulatory capacities. Indeed, a major breakthrough came with the finding that they are able to induce peripheral tolerance, suggesting they may be used as therapeutic tools in immune-mediated disorders. The present review aims at discussing the current knowledge on the targets and mechanisms of MSC-mediated immunosuppression as well as the potential use of MSCs as modulators of immune responses in a variety of diseases related to alloreactive immunity or autoimmunity.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Tolerância Periférica/imunologia , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/terapia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/terapia , Humanos , Terapia de Imunossupressão , Células-Tronco Mesenquimais/metabolismo , Camundongos , Neutrófilos/imunologia , Imunologia de Transplantes/imunologiaRESUMO
Cartilage engineering using mesenchymal stem cells (MSC) will require the use of a scaffold which will act as a support for cell adhesion keeping the cells in the cartilage defect. Optimally, a tissue engineered construct should allow sustained delivery of bioactive factors capable of inducing MSC differentiation into chondrocytes and should be easily injected inside the cartilage lesions to avoid surgical operations. We therefore developed pharmacologically active microcarriers (PAM) made of poly-lactic-co-glycolic acid (PLGA) produced using an oil-in-water (o/w) emulsion method. The microspheres were coated with a biomimetic surface of fibronectin (FN) and engineered to release TGF-beta3 as a chondrogenic differentiation factor. When human MSCs were incubated in vitro with TGF-beta3 releasing FN-coated PAMs in chondrogenic medium, they firmly adhered onto the surface of PAMs rapidly forming cell aggregates. After 3 weeks, strong up-regulation of cartilage-specific markers was observed both at the mRNA and protein level whereas osteogenic or adipogenic genes could not be detected. Importantly, implantation of MSC/TGF-beta3 releasing PAM complexes in SCID mice resulted in the formation of histologically resembling cartilage which stained positive for chondrocyte markers, collagen II and aggrecan. The present study demonstrated that functionalized PLGA-based microparticles can provide an appropriate environment for chondrogenic differentiation of MSCs and should contribute to injectable biomedical device development improving in vivo cartilage engineering.