RESUMO
Myocardial infarction (MI) induces the generation of proinflammatory Ly6Chigh monocytes in the spleen and the recruitment of these cells to the myocardium. CD4+ Foxp3+ CD25+ T-cells (Tregs) promote the healing process after myocardial infarction by engendering a pro-healing differentiation state in myocardial monocyte-derived macrophages. We aimed to study the effects of CD4+ T-cells on splenic myelopoiesis and monocyte differentiation. We instigated MI in mice and found that MI-induced splenic myelopoiesis is abrogated in CD4+ T-cell deficient animals. Conventional CD4+ T-cells promoted myelopoiesis in vitro by cell-cell-contact and paracrine mechanisms, including interferon-gamma (IFN-γ) signalling. Depletion of regulatory T-cells enhanced myelopoiesis in vivo, as evidenced by increases in progenitor cell numbers and proliferative activity in the spleen 5 days after MI. The frequency of CD4+ T-cells-producing factors that promote myelopoiesis increased within the spleen of Treg-depleted mice. Moreover, depletion of Tregs caused a proinflammatory bias in splenic Ly6Chigh monocytes, which showed predominantly upregulated expression of IFN-γ responsive genes after MI. Our results indicate that conventional CD4+ T-cells promote and Tregs attenuate splenic myelopoiesis and proinflammatory differentiation of monocytes.
Assuntos
Monócitos , Infarto do Miocárdio , Camundongos , Animais , Monócitos/metabolismo , Mielopoese , Baço/metabolismo , Infarto do Miocárdio/metabolismo , Linfócitos T Reguladores/metabolismo , Interferon gama/farmacologia , Camundongos Endogâmicos C57BLRESUMO
Though myocardial infarction (MI) in pigs is a well-established translational large animal model, it has not yet been widely used for immunotherapy studies, and a comprehensive description of the immune response to MI in this species is lacking. We induced MI in Landrace pigs by balloon occlusion of the left anterior descending artery over 90 min. Within 14 days, the necrotic myocardium was progressively replaced by scar tissue with involvement of myofibroblasts. We characterized the immune response in the heart ex vivo by (immuno)histology, flow cytometry, and RNA sequencing of myocardial tissue on days 3, 7, and 14 after MI. Besides a clear predominance of myeloid cells among heart-infiltrating leukocytes, we detected activated T cells and an increasing proportion of CD4+ Foxp3+ regulatory T cells (Treg), especially in the infarct core-findings that closely mirror what has been observed in mice and humans after MI. Transcriptome data indicated inflammatory activity that was persistent but markedly changing in character over time and linked to extracellular matrix biology. Analysis of lymphocytes in heart-draining lymph nodes revealed significantly higher proliferation rates of T helper cell subsets, including Treg on day 7 after MI, compared to sham controls. Elevated frequencies of myeloid progenitors in the spleen suggest that it might be a site of emergency myelopoiesis after MI in pigs, as previously shown in mice. We thus provide a first description of the immune response to MI in pigs, and our results can aid future research using the species for preclinical immunotherapy studies.
Assuntos
Modelos Animais de Doenças , Infarto do Miocárdio , Miocárdio , Linfócitos T Reguladores , Animais , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Linfócitos T Reguladores/imunologia , Miocárdio/patologia , Miocárdio/imunologia , Sus scrofa , Suínos , Ativação Linfocitária , Masculino , Transcriptoma , Feminino , Fatores de TempoRESUMO
Increased aerobic glycolysis is a metabolic hallmark of proinflammatory leukocytes including macrophages and T cells. To take up glucose from the environment and fuel glycolysis, activated leukocytes upregulate the glucose transporter GLUT1. The orally bioavailable selective GLUT1 inhibitor BAY-876 was developed primarily as an anti-tumor drug. Our study assessed its activity on activated macrophages and CD4+ T cells. BAY-876 significantly attenuated glucose uptake by cultured CD4+ T cells and macrophages by 41% and 15%, respectively. Extracellular flux analysis of activated CD4+ T cells in vitro showed that BAY-876 significantly decreases glycolytic proton flux rate and lactate production, effects that are accompanied by an increased oxidative phosphorylation-mediated ATP production rate, leaving intracellular ATP levels per cell unchanged. However, GLUT1 inhibition reduced CD4+ T cell proliferation without compromising cell viability and reduced IFN-γ secretion by 20%. Moreover, TNF secretion from macrophages was reduced by 27%. We conclude that GLUT1-specific inhibitors, like BAY-876, deserve further in vivo testing in a broad range of (auto-) inflammatory disease models.
Assuntos
Linfócitos T CD4-Positivos , Glucose , Transportador de Glucose Tipo 1/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Glucose/metabolismo , Glicólise , Macrófagos/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Both conventional and regulatory CD4+ T-cells rely on costimulatory signals mediated by cell surface receptors including CD28 for full activation. We showed previously that stimulation of CD4+ Foxp3+ regulatory T-cells by superagonistic anti-CD28 monoclonal antibodies (mAb) improves myocardial healing after experimental myocardial infarction (MI). However, the effect of ligand binding blocking anti-CD28 monoclonal antibodies has not yet been tested in this context. We hypothesize that ligand blocking anti-CD28 mAb treatment might favorably impact on healing after MI by limiting the activation of conventional CD4+ T-cells. Therefore, we studied the therapeutic effect of the recently characterized mAb E18 which blocks ligand binding to CD28 in a mouse permanent coronary ligation model. E18 or an irrelevant control mAb was applied once on day two after myocardial infarction to wildtype mice. Echocardiography was performed on day 7 after MI. E18 treatment improved the survival and reduced the incidence of left ventricular ruptures after experimental myocardial infarction. Accordingly, although we found no difference in infarct size, there was significantly less left ventricular dilation after E18 treatment in surviving animals as determined by echocardiography at day 7 after MI. In sham operated control mice neither antibody had an impact on body weight, survival, and echocardiographic parameters. Mechanistically, compared to control immunoglobulin, E18 treatment reduced the number of CD4+ T-cells and monocytes/macrophages within the infarct and periinfarct zone on day 5. This was accompanied by an upregulation of arginase which is a marker for alternatively differentiated macrophages. The data indicate that CD28-dependent costimulation of CD4+ T-cells impairs myocardial healing and anti-CD28 antibody treatment constitutes a potentially clinically translatable approach to improve the outcome early after MI.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD28/antagonistas & inibidores , Linfócitos T CD4-Positivos/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Animais , Anticorpos Monoclonais/uso terapêutico , Arginase/imunologia , Arginase/metabolismo , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Modelos Animais de Doenças , Ecocardiografia , Humanos , Ligantes , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Miocárdio/imunologia , Miocárdio/patologiaRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0227734.].
RESUMO
Dopamine is the predominant catecholamine in the brain and functions as a neurotransmitter. Dopamine is also a potent immune modulator. In this study, we have characterized the expression of dopamine receptors on murine microglia. We found that cultured primary microglia express dopamine D1, D2, D3, D4, and D5 receptors. We specifically focused on the D2 receptor (D2R), a major target of antipsychotic drugs. Whereas D2Rs were strongly expressed on striatal neurons in vivo, we did not detect any D2R expression on resident microglia in the healthy brains of wild-type mice or transgenic mice expressing the green fluorescent protein (GFP) under the control of the Drd2 promoter. However, cerebral ischemia induced the expression of D2R on Iba1-immunoreactive inflammatory cells in the infarct core and penumbra. Notably, D2R expression was confined to CD45(hi) cells, and GFP BM chimeras revealed that D2R was expressed on activated resident microglia as well as on peripherally derived macrophages in the ischemic brain. Importantly, the D2/3R agonist, pramipexole, enhanced the secretion of nitrite by cultured microglia in response to proinflammatory stimuli. Thus, dopamine may serve as a modulator of microglia function during neuroinflammation.
Assuntos
Microglia/metabolismo , Receptores de Dopamina D2/biossíntese , Receptores de Dopamina D2/genética , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismo , Animais , Benzotiazóis/farmacologia , Células da Medula Óssea , Células Cultivadas , Quimera , Agonistas de Dopamina/farmacologia , Proteínas de Fluorescência Verde , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Pramipexol , Regiões Promotoras Genéticas/genética , Receptores de Dopamina D2/efeitos dos fármacos , Receptores de Dopamina D3/efeitos dos fármacosRESUMO
Bone marrow-derived cells (BMDCs) are able to colonize the central nervous system (CNS) at sites of damage. This ability makes BMDCs an ideal cellular vehicle for transferring therapeutic genes/molecules to the CNS. However, conditioning is required for bone marrow-derived myeloid cells to engraft in the brain, which so far has been achieved by total body irradiation (TBI) and by chemotherapy (e.g. busulfan treatment). Unfortunately, both regimens massively disturb the host's hematopoietic compartment. Here, we established a conditioning protocol to target myeloid cells to sites of brain damage in mice using non-myeloablative focal head irradiation (HI). This treatment was associated with comparatively low inflammatory responses in the CNS despite cranial radiation doses which are identical to TBI, as revealed by gene expression analysis of cytokines/chemokines such as CCL2, CXCL10, TNF-α and CCL5. HI prior to bone marrow transplantation resulted in much lower levels of blood chimerism defined as the percentage of donor-derived cells in peripheral blood (< 5%) compared with TBI (> 95%) or busulfan treatment (> 50%). Nevertheless, HI effectively recruited myeloid cells to the area of motoneuron degeneration in the brainstem within 7 days after facial nerve axotomy. In contrast, no donor-derived cells were detected in the lesioned facial nucleus of busulfan-treated animals up to 2 weeks after transplantation. Our findings suggest that myeloid cells can be targeted to sites of brain damage even in the presence of very low levels of peripheral blood chimerism. We established a novel non-myeloablative conditioning protocol with minimal disturbance of the host's hematopoietic system for targeting BMDCs specifically to areas of pathology in the brain.