RESUMEN
Regulatory T cells (Tregs) are key immune regulators that have shown promise in enhancing cardiac repair post-MI, although the mechanisms remain elusive. Here, we show that rapidly increasing Treg number in the circulation post-MI via systemic administration of exogenous Tregs improves cardiac function in male mice, by limiting cardiomyocyte death and reducing fibrosis. Mechanistically, exogenous Tregs quickly home to the infarcted heart and adopt an injury-specific transcriptome that mediates repair by modulating monocytes/macrophages. Specially, Tregs lead to a reduction in pro-inflammatory Ly6CHi CCR2+ monocytes/macrophages accompanied by a rapid shift of macrophages towards a pro-repair phenotype. Additionally, exogenous Treg-derived factors, including nidogen-1 and IL-10, along with a decrease in cardiac CD8+ T cell number, mediate the reduction of the pro-inflammatory monocyte/macrophage subset in the heart. Supporting the pivotal role of IL-10, exogenous Tregs knocked out for IL-10 lose their pro-repair capabilities. Together, this study highlights the beneficial use of a Treg-based therapeutic approach for cardiac repair with important mechanistic insights that could facilitate the development of novel immunotherapies for MI.
Asunto(s)
Interleucina-10 , Macrófagos , Ratones Endogámicos C57BL , Infarto del Miocardio , Linfocitos T Reguladores , Animales , Infarto del Miocardio/inmunología , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Linfocitos T Reguladores/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Interleucina-10/metabolismo , Interleucina-10/genética , Fenotipo , Miocardio/patología , Miocardio/inmunología , Miocardio/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/inmunología , Fibrosis , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Ratones NoqueadosRESUMEN
Mesenchymal stromal cells (MSCs) have significant therapeutic potential due to their ability to differentiate into musculoskeletal lineages suitable for tissue-engineering, as well as the immunomodulatory and pro-regenerative effects of the paracrine factors that these cells secrete. Cues from the extracellular environment, including physical stimuli such as substrate stiffness, are strong drivers of MSC differentiation, but their effects upon MSC paracrine activity are not well understood. This study, therefore sought to determine the impact of substrate stiffness on the paracrine activity of MSCs, analysing both effects on MSC fate and their effect on T-cell and macrophage activity and angiogenesis. The data show that conditioned medium (CM) from MSCs cultured on 0.2 kPa (soft) and 100 kPa (stiff) polyacrylamide hydrogels have differing effects on MSC proliferation and differentiation, with stiff CM promoting proliferation whilst soft CM promoted differentiation. There were also differences in the effects upon macrophage phagocytosis and angiogenesis, with the most beneficial effects from soft CM. Analysis of the media composition identified differences in the levels of proteins including IL-6, OPG, and TIMP-2. Using recombinant proteins and blocking antibodies, we confirmed a role for OPG in modulating MSC proliferation with a complex combination of factors involved in the regulation of MSC differentiation. Together the data confirm that the physical microenvironment has an important influence on the MSC secretome and that this can alter the differentiation and regenerative potential of the cells. These findings can be used to tailor the culture environment for manufacturing potent MSCs for specific clinical applications or to inform the design of biomaterials that enable the retention of MSC activity after delivery into the body. STATEMENT OF SIGNIFICANCE: ⢠MSCs cultured on 100 kPa matrices produce a secretome that boosts MSC proliferation ⢠MSCs cultured on 0.2 kPa matrices produce a secretome that promotes MSC osteogenesis and adipogenesis, as well as angiogenesis and macrophage phagocytosis ⢠IL-6 secretion is elevated in MSCs on 0.2 kPa substrates ⢠OPG, TIMP-2, MCP-1, and sTNFR1 secretion are elevated in MSCs on 100 kPa substrates.
Asunto(s)
Trasplante de Células Madre Mesenquimatosas , Inhibidor Tisular de Metaloproteinasa-2 , Inhibidor Tisular de Metaloproteinasa-2/farmacología , Interleucina-6 , Diferenciación Celular , Ingeniería de Tejidos , Medios de Cultivo Condicionados/farmacologíaRESUMEN
Chronic wounds are a major clinical problem where wound closure is prevented by pathologic factors, including immune dysregulation. To design efficient immunotherapies, an understanding of the key molecular pathways by which immunity impairs wound healing is needed. Interleukin-1 (IL-1) plays a central role in regulating the immune response to tissue injury through IL-1 receptor (IL-1R1). Generating a knockout mouse model, we demonstrate that the IL-1-IL-1R1 axis delays wound closure in diabetic conditions. We used a protein engineering approach to deliver IL-1 receptor antagonist (IL-1Ra) in a localised and sustained manner through binding extracellular matrix components. We demonstrate that matrix-binding IL-1Ra improves wound healing in diabetic mice by re-establishing a pro-healing microenvironment characterised by lower levels of pro-inflammatory cells, cytokines and senescent fibroblasts, and higher levels of anti-inflammatory cytokines and growth factors. Engineered IL-1Ra has translational potential for chronic wounds and other inflammatory conditions where IL-1R1 signalling should be dampened.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Proteína Antagonista del Receptor de Interleucina 1/genética , Cicatrización de Heridas/fisiología , Animales , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Masculino , Ratones , Ratones NoqueadosRESUMEN
Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.
Asunto(s)
Macrófagos/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/lesiones , Mioblastos/citología , Nicotinamida Fosforribosiltransferasa/metabolismo , Nicho de Células Madre , Pez Cebra/metabolismo , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Macrófagos/citología , Masculino , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Mioblastos/metabolismo , Nicotinamida Fosforribosiltransferasa/genética , Factor de Transcripción PAX7/metabolismo , RNA-Seq , Receptores CCR5/genética , Receptores CCR5/metabolismo , Regeneración/fisiología , Análisis de la Célula Individual , Pez Cebra/inmunologíaRESUMEN
Perivascular mesenchymal stem/stromal cells can be isolated from the human endometrium using the surface marker SUSD2 and are being investigated for use in tissue repair. Mesenchymal stem/stromal cells from other tissues modulate T cell responses via mechanisms including interleukin-10, prostaglandin E2, TGF-ß1 and regulatory T cells. Animal studies demonstrate that endometrial mesenchymal stem/stromal cells can also modify immune responses to implanted mesh, but the mechanism/s they employ have not been explored. We examined the immunomodulatory properties of human endometrial mesenchymal stem/stromal cells on lymphocyte proliferation using mouse splenocyte cultures. Endometrial mesenchymal stem/stromal cells inhibited mitogen-induced lymphocyte proliferation in vitro in a dose-dependent manner. Inhibition of lymphocyte proliferation was not affected by blocking the mouse interleukin-10 receptor or inhibiting prostaglandin production. Endometrial mesenchymal stem/stromal cells continued to restrain lymphocyte proliferation in the presence of an inhibitor of TGF-ß receptors, despite a reduction in regulatory T cells. Thus, the in vitro inhibition of mitogen-induced lymphocyte proliferation by endometrial mesenchymal stem/stromal cells occurs by a mechanism distinct from the interleukin-10, prostaglandin E2, TGF-ß1 and regulatory T cell-mediated mechanisms employed by MSC from other tissues. eMSCs were shown to produce interleukin-17A and Dickkopf-1 which may contribute to their immunomodulatory properties. In contrast to MSC from other sources, systemic administration of endometrial mesenchymal stem/stromal cells did not inhibit swelling in a T cell-mediated model of skin inflammation. We conclude that, while endometrial mesenchymal stem/stromal cells can modify immune responses, their immunomodulatory repertoire may not be sufficient to restrain some T cell-mediated inflammatory events.
Asunto(s)
Proliferación Celular , Endometrio/citología , Células Madre Mesenquimatosas/fisiología , Linfocitos T/fisiología , Animales , Comunicación Celular/fisiología , Células Cultivadas , Endometrio/inmunología , Endometrio/metabolismo , Femenino , Humanos , Glicoproteínas de Membrana/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/fisiologíaRESUMEN
Extracellular vesicles (EVs)-based therapeutics are based on the premise that EVs shed by stem cells exert similar therapeutic effects and these have been proposed as an alternative to cell therapies. EV-mediated delivery is an effective and efficient system of cell-to-cell communication which can confer therapeutic benefits to their target cells. EVs have been shown to promote tissue repair and regeneration in various animal models such as, wound healing, cardiac ischemia, diabetes, lung fibrosis, kidney injury, and many others. Given the unique attributes of EVs, considerable thought must be given to the preservation, formulation and cold chain strategies in order to effectively translate exciting preclinical observations to clinical and commercial success. This review summarizes current understanding around EV preservation, challenges in maintaining EV quality, and also bioengineering advances aimed at enhancing the long-term stability of EVs.
RESUMEN
Idiopathic pulmonary fibrosis (IPF) is characterized by chronic inflammation, severe scarring, and stem cell senescence. Stem cell-based therapies modulate inflammatory and fibrogenic pathways by release of soluble factors. Stem cell-derived extracellular vesicles should be explored as a potential therapy for IPF. Human amnion epithelial cell-derived exosomes (hAEC Exo) were isolated and compared against human lung fibroblasts exosomes. hAEC Exo were assessed as a potential therapy for lung fibrosis. Exosomes were isolated and evaluated for their protein and miRNA cargo. Direct effects of hAEC Exo on immune cell function, including macrophage polarization, phagocytosis, neutrophil myeloperoxidase activity and T cell proliferation and uptake, were measured. Their impact on immune response, histological outcomes, and bronchioalveolar stem cell (BASC) response was assessed in vivo following bleomycin challenge in young and aged mice. hAEC Exo carry protein cargo enriched for MAPK signaling pathways, apoptotic and developmental biology pathways and miRNA enriched for PI3K-Akt, Ras, Hippo, TGFß, and focal adhesion pathways. hAEC Exo polarized and increased macrophage phagocytosis, reduced neutrophil myeloperoxidases, and suppressed T cell proliferation directly. Intranasal instillation of 10 µg hAEC Exo 1 day following bleomycin challenge reduced lung inflammation, while treatment at day 7 improved tissue-to-airspace ratio and reduced fibrosis. Administration of hAEC Exo coincided with the proliferation of BASC. These effects were reproducible in bleomycin-challenged aged mice. The paracrine effects of hAECs can be largely attributed to their exosomes and exploitation of hAEC Exo as a therapy for IPF should be explored further. Stem Cells Translational Medicine 2018;7:180-196.
Asunto(s)
Amnios/citología , Células Epiteliales/citología , Exosomas/fisiología , Lesión Pulmonar/patología , Pulmón/patología , Animales , Proliferación Celular/fisiología , Células Cultivadas , Femenino , Fibroblastos/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Neutrófilos/fisiología , Neumonía/patología , Embarazo , Fibrosis Pulmonar/patología , Transducción de Señal/fisiología , Linfocitos T/fisiologíaRESUMEN
Human amnion epithelial cells (hAECs) have been shown to possess potent immunomodulatory properties across a number of disease models. Recently, we reported that hAECs influence macrophage polarization and activity, and that this step was dependent on regulatory T cells. In this study, we aimed to assess the effects of hAEC-derived proresolution lipoxin-A4 (LXA4) on T-cell, macrophage, and neutrophil phenotype and function during the acute phase of bleomycin-induced lung injury. Using C57Bl6 mice, we administered 4 million hAECs intraperitoneally 24 hours after bleomycin challenge. Outcomes were measured at days 3, 5, and 7. hAEC administration resulted in significant changes to T-cell, macrophage, dendritic cell, and monocyte/macrophage infiltration and phenotypes. Endogenous levels of lipoxygenases, LXA4, and the lipoxin receptor FPR2 were elevated in hAEC-treated animals. Furthermore, we showed that the effects of hAECs on macrophage phagocytic activity and T-cell suppression are LXA4 dependent, whereas the inhibition of neutrophil-derived myleoperoxidase by hAECs is independent of LXA4. This study provides the first evidence that lipid-based mediators contribute to the immunomodulatory effects of hAECs and further supports the growing body of evidence that LXA4 is proresolutionary in lung injury. This discovery of LXA4-dependent communication between hAECs, macrophages, T cells, and neutrophils is important to the understanding of hAEC biodynamics and would be expected to inform future clinical applications. Stem Cells Translational Medicine 2017;6:1085-1095.
Asunto(s)
Amnios/citología , Células Epiteliales/citología , Lipoxinas/metabolismo , Pulmón/citología , Animales , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Citocinas/metabolismo , Humanos , Inflamación/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Peroxidasa , Fagocitosis/fisiologíaRESUMEN
INTRODUCTION: The immunomodulatory properties of human amnion epithelial cells (hAECs) have been previously described in several disease models. We previously reported on the ability of hAECs to influence macrophage phenotype and chemotaxis. In this study, we aim to elucidate the contribution of regulatory T cells (Tregs) to macrophage polarisation and downstream effects on inflammation and fibrosis in a bleomycin model of lung injury. METHODS: Either CD45(+)/FoxP3(+) Tregs or CD45(+)/FoxP3 (-) non-Tregs were adoptively transferred into Rag1 (-/-) mice immediately prior to bleomycin challenge. Four million hAECs were administered 24 hours later. Outcomes were measured 7 or 14 days later. RESULTS: Mitigation of lung inflammation and fibrosis was observed only in animals that received both hAECs and Tregs. hAEC treatment also induced the maturation of non-Tregs into FoxP3-expressing Tregs. This event was found to be transforming growth factor-beta (TGFß)-dependent. Furthermore, polarisation of macrophages from M1 to M2 occurred only in animals that received hAECs and Tregs. CONCLUSIONS: This study provides the first evidence that Tregs are required for hAEC-mediated macrophage polarisation and consequential mitigation of bleomycin-induced lung injury. Uncovering the interactions between hAECs, macrophages, and T-cell subsets is central to understanding the mechanisms by which hAECs elicit lung repair.
Asunto(s)
Amnios/citología , Bleomicina/toxicidad , Lesión Pulmonar/etiología , Linfocitos T Reguladores/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Células Epiteliales/citología , Células Epiteliales/metabolismo , Fibrosis , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Inflamación , Antígenos Comunes de Leucocito/metabolismo , Pulmón/citología , Macrófagos/citología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/efectos de los fármacos , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.
Asunto(s)
Pletismografía Total/métodos , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Fenómenos Fisiológicos RespiratoriosRESUMEN
Human amnion epithelial cells (hAECs) have been shown to modulate inflammation and restore normal lung structure and respiratory function following bleomycin challenge in immune-competent mice. These effects are exerted despite a lack of significant engraftment of hAECs, suggesting that immunomodulatory effect mechanisms are at play. In this study, using the bleomycin model of injury, we explored the interactions between hAECs and macrophages. We administered 4 million hAECs intraperitoneally to C57Bl6 mice 24 h following a bleomycin challenge. Using FACS analysis and qPCR, we showed that hAEC administration significantly reduced macrophage infiltration into the lungs and that the majority of the pulmonary macrophages were of the M2 phenotype. Using bone marrow-derived macrophages, we then showed that hAEC-conditioned media could alter macrophage polarization, migration, and phagocytosis, without affecting macrophage survival or proliferation in vitro. This study provides the first evidence that hAECs directly influence macrophage behavior in a proreparative manner and suggests that hAECs are able to mediate these effects independently of other immune cell types.
Asunto(s)
Amnios/citología , Células Epiteliales/inmunología , Células Epiteliales/trasplante , Lesión Pulmonar/inmunología , Lesión Pulmonar/cirugía , Pulmón/patología , Macrófagos/patología , Animales , Bleomicina , Células Cultivadas , Humanos , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/patología , Inflamación/cirugía , Pulmón/efectos de los fármacos , Pulmón/inmunología , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/patología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BLRESUMEN
Since current treatments for both acute and chronic lung diseases are less than ideal, there has been recent interest in the use of cell-based therapies for inflammatory lung disease. Specifically, human amnion epithelial cells (hAECs) have been shown to reduce bleomycin-induced lung injury and prevent subsequent loss of respiratory function, primarily through modulation of the host immune response. The precise mechanisms of this effect remain unclear. We aimed to investigate the potential of hAECs to mitigate bleomycin-induced lung injury in surfactant protein C deficient (Sftpc(−/−)) mice, which are highly susceptible to pulmonary injury as a result of impairment of macrophage function. Primary hAECs were administered to wild-type (Sftpc(+/+)) and Sftpc(−/−) mice 24 h after exposure to bleomycin. Compared to Sftpc(+/+) mice receiving bleomycin alone, Sftpc(+/+) mice administered hAECs 24 h after bleomycin exposure had decreased expression of proinflammatory genes, decreased macrophage and neutrophil infiltration, fibrosis, collagen content, and α-smooth muscle actin as well as a significant improvement in lung function. Compared to Sftpc(−/−) mice given bleomycin alone, Sftpc(−/−) mice administered hAECs 24 h after bleomycin did not have a decrease in inflammatory gene expression or a reduction in macrophage pulmonary infiltration. Subsequently, Sftpc(−/−) mice did not show any decrease in pulmonary fibrosis or improvement of lung function after hAEC administration. The ability of hAECs to mitigate bleomycin-induced lung injury is abolished in Sftpc(−/−) mice, suggesting that hAECs require normal host macrophage function to exert their reparative effects.