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1.
Cell ; 187(17): 4637-4655.e26, 2024 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-39043180

RESUMO

The medical burden of stroke extends beyond the brain injury itself and is largely determined by chronic comorbidities that develop secondarily. We hypothesized that these comorbidities might share a common immunological cause, yet chronic effects post-stroke on systemic immunity are underexplored. Here, we identify myeloid innate immune memory as a cause of remote organ dysfunction after stroke. Single-cell sequencing revealed persistent pro-inflammatory changes in monocytes/macrophages in multiple organs up to 3 months after brain injury, notably in the heart, leading to cardiac fibrosis and dysfunction in both mice and stroke patients. IL-1ß was identified as a key driver of epigenetic changes in innate immune memory. These changes could be transplanted to naive mice, inducing cardiac dysfunction. By neutralizing post-stroke IL-1ß or blocking pro-inflammatory monocyte trafficking with a CCR2/5 inhibitor, we prevented post-stroke cardiac dysfunction. Such immune-targeted therapies could potentially prevent various IL-1ß-mediated comorbidities, offering a framework for secondary prevention immunotherapy.


Assuntos
Lesões Encefálicas , Imunidade Inata , Memória Imunológica , Inflamação , Interleucina-1beta , Camundongos Endogâmicos C57BL , Monócitos , Animais , Camundongos , Interleucina-1beta/metabolismo , Lesões Encefálicas/imunologia , Humanos , Masculino , Monócitos/metabolismo , Monócitos/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/imunologia , Cardiopatias/imunologia , Feminino , Receptores CCR2/metabolismo , Fibrose , Epigênese Genética , Imunidade Treinada
2.
Immunity ; 54(4): 648-659.e8, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33667383

RESUMO

Loss of lymphocytes, particularly T cell apoptosis, is a central pathological event after severe tissue injury that is associated with increased susceptibility for life-threatening infections. The precise immunological mechanisms leading to T cell death after acute injury are largely unknown. Here, we identified a monocyte-T cell interaction driving bystander cell death of T cells in ischemic stroke and burn injury. Specifically, we found that stroke induced a FasL-expressing monocyte population, which led to extrinsic T cell apoptosis. This phenomenon was driven by AIM2 inflammasome-dependent interleukin-1ß (IL-1ß) secretion after sensing cell-free DNA. Pharmacological inhibition of this pathway improved T cell survival and reduced post-stroke bacterial infections. As such, this study describes inflammasome-dependent monocyte activation as a previously unstudied cause of T cell death after injury and challenges the current paradigms of post-injury lymphopenia.


Assuntos
Coinfecção/imunologia , Proteínas de Ligação a DNA/imunologia , Tolerância Imunológica/imunologia , Inflamassomos/imunologia , Transdução de Sinais/imunologia , Animais , Apoptose/imunologia , Infecções Bacterianas/imunologia , Queimaduras/imunologia , Queimaduras/microbiologia , Coinfecção/microbiologia , Humanos , Interleucina-1beta/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Acidente Vascular Cerebral/imunologia , Acidente Vascular Cerebral/microbiologia , Linfócitos T/imunologia
3.
Nature ; 633(8029): 433-441, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39112714

RESUMO

The risk of early recurrent events after stroke remains high despite currently established secondary prevention strategies1. Risk is particularly high in patients with atherosclerosis, with more than 10% of patients experiencing early recurrent events1,2. However, despite the enormous medical burden of this clinical phenomenon, the underlying mechanisms leading to increased vascular risk and recurrent stroke are largely unknown. Here, using a novel mouse model of stroke-induced recurrent ischaemia, we show that stroke leads to activation of the AIM2 inflammasome in vulnerable atherosclerotic plaques via an increase of circulating cell-free DNA. Enhanced plaque inflammation post-stroke results in plaque destabilization and atherothrombosis, finally leading to arterioarterial embolism and recurrent stroke within days after the index stroke. We confirm key steps of plaque destabilization also after experimental myocardial infarction and in carotid artery plaque samples from patients with acute stroke. Rapid neutrophil NETosis was identified as the main source of cell-free DNA after stroke and NET-DNA as the causative agent leading to AIM2 inflammasome activation. Neutralization of cell-free DNA by DNase treatment or inhibition of inflammasome activation reduced the rate of stroke recurrence after experimental stroke. Our findings present an explanation for the high recurrence rate after incident ischaemic events in patients with atherosclerosis. The detailed mechanisms uncovered here provide clinically uncharted therapeutic targets for which we show high efficacy to prevent recurrent events. Targeting DNA-mediated inflammasome activation after remote tissue injury represents a promising avenue for further clinical development in the prevention of early recurrent events.


Assuntos
Aterosclerose , Inflamassomos , Placa Aterosclerótica , Recidiva , Acidente Vascular Cerebral , Adulto , Animais , Feminino , Humanos , Masculino , Camundongos , Aterosclerose/sangue , Aterosclerose/complicações , Aterosclerose/metabolismo , Aterosclerose/patologia , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/metabolismo , Modelos Animais de Doenças , Proteínas de Ligação a DNA/metabolismo , Armadilhas Extracelulares/metabolismo , Inflamassomos/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Neutrófilos/metabolismo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Desoxirribonucleases/metabolismo
4.
EMBO J ; 41(4): e109108, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35019161

RESUMO

Haploinsufficiency of the progranulin (PGRN)-encoding gene (GRN) causes frontotemporal lobar degeneration (GRN-FTLD) and results in microglial hyperactivation, TREM2 activation, lysosomal dysfunction, and TDP-43 deposition. To understand the contribution of microglial hyperactivation to pathology, we used genetic and pharmacological approaches to suppress TREM2-dependent transition of microglia from a homeostatic to a disease-associated state. Trem2 deficiency in Grn KO mice reduced microglia hyperactivation. To explore antibody-mediated pharmacological modulation of TREM2-dependent microglial states, we identified antagonistic TREM2 antibodies. Treatment of macrophages from GRN-FTLD patients with these antibodies led to reduced TREM2 signaling due to its enhanced shedding. Furthermore, TREM2 antibody-treated PGRN-deficient microglia derived from human-induced pluripotent stem cells showed reduced microglial hyperactivation, TREM2 signaling, and phagocytic activity, but lysosomal dysfunction was not rescued. Similarly, lysosomal dysfunction, lipid dysregulation, and glucose hypometabolism of Grn KO mice were not rescued by TREM2 ablation. Synaptic loss and neurofilament light-chain (NfL) levels, a biomarker for neurodegeneration, were further elevated in the Grn/Trem2 KO cerebrospinal fluid (CSF). These findings suggest that TREM2-dependent microglia hyperactivation in models of GRN deficiency does not promote neurotoxicity, but rather neuroprotection.


Assuntos
Degeneração Lobar Frontotemporal/patologia , Glicoproteínas de Membrana/metabolismo , Microglia/fisiologia , Monócitos/metabolismo , Progranulinas/deficiência , Receptores Imunológicos/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Encéfalo/diagnóstico por imagem , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Feminino , Degeneração Lobar Frontotemporal/metabolismo , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Quinase Syk/metabolismo
5.
Brain ; 147(3): 1057-1074, 2024 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-38153327

RESUMO

Incomplete reperfusion of the microvasculature ('no-reflow') after ischaemic stroke damages salvageable brain tissue. Previous ex vivo studies suggest pericytes are vulnerable to ischaemia and may exacerbate no-reflow, but the viability of pericytes and their association with no-reflow remains under-explored in vivo. Using longitudinal in vivo two-photon single-cell imaging over 7 days, we showed that 87% of pericytes constrict during cerebral ischaemia and remain constricted post reperfusion, and 50% of the pericyte population are acutely damaged. Moreover, we revealed ischaemic pericytes to be fundamentally implicated in capillary no-reflow by limiting and arresting blood flow within the first 24 h post stroke. Despite sustaining acute membrane damage, we observed that over half of all cortical pericytes survived ischaemia and responded to vasoactive stimuli, upregulated unique transcriptomic profiles and replicated. Finally, we demonstrated the delayed recovery of capillary diameter by ischaemic pericytes after reperfusion predicted vessel reconstriction in the subacute phase of stroke. Cumulatively, these findings demonstrate that surviving cortical pericytes remain both viable and promising therapeutic targets to counteract no-reflow after ischaemic stroke.


Assuntos
Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Humanos , Pericitos/fisiologia , Infarto Cerebral
6.
Stroke ; 55(10): 2522-2527, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39315830

RESUMO

BACKGROUND: The discrepancy between experimental research and clinical trial outcomes is a persistent challenge in preclinical studies, particularly in stroke research. A possible factor contributing to this issue is the lack of standardization across experimental stroke models, leading to poor reproducibility in multicenter studies. This study addresses this gap by aiming to enhance reproducibility and the efficacy of multicenter studies through the harmonization of protocols and training of involved personnel. METHODS: We established a set of standard operating procedures for various stroke models and the Neuroscore. These standard operating procedures were implemented across multiple research centers, followed by specialized, in-person training for all participants. We measured the variability in infarct volume both before and after the implementation of these standardized protocols and training sessions. RESULTS: The standardization process led to a significant reduction in variability of infarct volume across different stroke models (40%-50% reduction), demonstrating the effectiveness of our harmonized protocols and training. Additionally, the implementation of the Neuroscore system across centers showed low variability and consistent results up to 28 days poststroke, underscoring its utility in chronic phase evaluations. CONCLUSIONS: The harmonization of protocols and surgeon training significantly reduced variability in experimental outcomes across different centers. This improvement can increase the comparability of data between research groups and enhance the statistical power of multicenter studies. Our findings also establish the Neuroscore as a reliable tool for long-term assessment in stroke research, paving the way for more consistent and impactful multicenter preclinical studies.


Assuntos
Acidente Vascular Cerebral , Acidente Vascular Cerebral/terapia , Reprodutibilidade dos Testes , Animais , Modelos Animais de Doenças , Masculino , Humanos , Estudos Multicêntricos como Assunto/métodos , Estudos Multicêntricos como Assunto/normas
7.
Int J Mol Sci ; 25(2)2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38279234

RESUMO

Stroke is the main cause for acquired disabilities. Pharmaceutical or mechanical removal of the thrombus is the cornerstone of stroke treatment but can only be administered to a subset of patients and within a narrow time window. Novel treatment options are therefore required. Here we induced stroke by permanent occlusion of the distal medial cerebral artery of wild-type mice and knockout mice for the lactate receptor hydroxycarboxylic acid receptor 1 (HCA1). At 24 h and 48 h after stroke induction, we injected L-lactate intraperitoneal. The resulting atrophy was measured in Nissl-stained brain sections, and capillary density and neurogenesis were measured after immunolabeling and confocal imaging. In wild-type mice, L-lactate treatment resulted in an HCA1-dependent reduction in the lesion volume accompanied by enhanced angiogenesis. In HCA1 knockout mice, on the other hand, there was no increase in angiogenesis and no reduction in lesion volume in response to L-lactate treatment. Nevertheless, the lesion volumes in HCA1 knockout mice-regardless of L-lactate treatment-were smaller than in control mice, indicating a multifactorial role of HCA1 in stroke. Our findings suggest that L-lactate administered 24 h and 48 h after stroke is protective in stroke. This represents a time window where no effective treatment options are currently available.


Assuntos
Ácido Láctico , Acidente Vascular Cerebral , Humanos , Camundongos , Animais , Ácido Láctico/farmacologia , Encéfalo/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Camundongos Knockout
8.
Stroke ; 54(7): 1920-1929, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37021568

RESUMO

Ischemic stroke profoundly influences the peripheral immune system, which responds quickly to brain ischemia and participates in the evolution of poststroke neuroinflammation, while a period of systemic immunosuppression ensues. Poststroke immunosuppression brings harmful consequences, including increased infection rates and escalated death. As the most abundant cell population in the fast-responding innate immune system, myeloid cells including neutrophils and monocytes play an indispensable role in systemic immunosuppression after stroke. The change in myeloid response after stroke can be regulated by circulating DAMPs (damage-associated molecular patterns) and neuromodulatory mechanisms, which contain sympathetic nervous system, hypothalamic-pituitary-adrenal, and parasympathetic nervous system. In this review, we summarize the emerging roles and newly identified mechanisms underlying myeloid cell response in poststroke immunosuppression. Deeper understanding of the above points may pave the way for future development of novel therapeutic strategies to treat poststroke immunosuppression.


Assuntos
Isquemia Encefálica , Acidente Vascular Cerebral , Humanos , Terapia de Imunossupressão , Células Mieloides , Sistema Imunitário
9.
J Neuroinflammation ; 20(1): 301, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-38102677

RESUMO

Ischemic stroke is a major global health issue and characterized by acute vascular dysfunction and subsequent neuroinflammation. However, the relationship between these processes remains elusive. In the current study, we investigated whether alleviating vascular dysfunction by restoring vascular nitric oxide (NO) reduces post-stroke inflammation. Mice were subjected to experimental stroke and received inhaled NO (iNO; 50 ppm) after reperfusion. iNO normalized vascular cyclic guanosine monophosphate (cGMP) levels, reduced the elevated expression of intercellular adhesion molecule-1 (ICAM-1), and returned leukocyte adhesion to baseline levels. Reduction of vascular pathology significantly reduced the inflammatory cytokines interleukin-1ß (Il-1ß), interleukin-6 (Il-6), and tumor necrosis factor-α (TNF-α), within the brain parenchyma. These findings suggest that vascular dysfunction is responsible for leukocyte adhesion and that these processes drive parenchymal inflammation. Reversing vascular dysfunction may therefore emerge as a novel approach to diminish neuroinflammation after ischemic stroke and possibly other ischemic disorders.


Assuntos
AVC Isquêmico , Acidente Vascular Cerebral , Camundongos , Animais , Óxido Nítrico , Doenças Neuroinflamatórias , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/tratamento farmacológico , Fator de Necrose Tumoral alfa/metabolismo , Inflamação/tratamento farmacológico , Inflamação/patologia , Molécula 1 de Adesão Intercelular/metabolismo
10.
J Neuroinflammation ; 19(1): 256, 2022 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-36224611

RESUMO

BACKGROUND: Previous studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple models of brain diseases. However, tools for the selective and efficient targeting of these receptors are lacking. The development of new P2X7-specific nanobodies (nbs) has enabled us to effectively block the P2X7 channel. METHODS: Temporary middle cerebral artery occlusion (tMCAO) in wild-type (wt) and P2X7 transgenic (tg) mice was used to model ischemic stroke. Adenosine triphosphate (ATP) release was assessed in transgenic ATP sensor mice. Stroke size was measured after P2X7-specific nbs were injected intravenously (iv) and intracerebroventricularly (icv) directly before tMCAO surgery. In vitro cultured microglia were used to investigate calcium influx, pore formation via 4,6-diamidino-2-phenylindole (DAPI) uptake, caspase 1 activation and interleukin (IL)-1ß release after incubation with the P2X7-specific nbs. RESULTS: Transgenic ATP sensor mice showed an increase in ATP release in the ischemic hemisphere compared to the contralateral hemisphere or the sham-treated mice up to 24 h after stroke. P2X7-overexpressing mice had a significantly greater stroke size 24 h after tMCAO surgery. In vitro experiments with primary microglial cells demonstrated that P2X7-specific nbs could inhibit ATP-triggered calcium influx and the formation of membrane pores, as measured by Fluo4 fluorescence or DAPI uptake. In microglia, we found lower caspase 1 activity and subsequently lower IL-1ß release after P2X7-specific nb treatment. The intravenous injection of P2X7-specific nbs compared to isotype controls before tMCAO surgery did not result in a smaller stroke size. As demonstrated by fluorescence-activated cell sorting (FACS), after stroke, iv injected nbs bound to brain-infiltrated macrophages but not to brain resident microglia, indicating insufficient crossing of the blood-brain barrier of the nbs. Therefore, we directly icv injected the P2X7-specific nbs or the isotype nbs. After icv injection of 30 µg of P2X7 specific nbs, P2X7 specific nbs bound sufficiently to microglia and reduced stroke size. CONCLUSION: Mechanistically, we can show that there is a substantial increase of ATP locally after stroke and that blockage of the ATP receptor P2X7 by icv injected P2X7-specific nbs can reduce ischemic tissue damage.


Assuntos
Receptores Purinérgicos P2 , Anticorpos de Domínio Único , Acidente Vascular Cerebral , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Caspase 1/metabolismo , Infarto da Artéria Cerebral Média/patologia , Interleucina-1beta/metabolismo , Camundongos , Microglia/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Anticorpos de Domínio Único/metabolismo , Acidente Vascular Cerebral/metabolismo
11.
Circ Res ; 127(6): 811-823, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32546048

RESUMO

RATIONALE: Arterial inflammation manifested as atherosclerosis is the leading cause of mortality worldwide. Genome-wide association studies have identified a prominent role of HDAC (histone deacetylase)-9 in atherosclerosis and its clinical complications including stroke and myocardial infarction. OBJECTIVE: To determine the mechanisms linking HDAC9 to these vascular pathologies and explore its therapeutic potential for atheroprotection. METHODS AND RESULTS: We studied the effects of Hdac9 on features of plaque vulnerability using bone marrow reconstitution experiments and pharmacological targeting with a small molecule inhibitor in hyperlipidemic mice. We further used 2-photon and intravital microscopy to study endothelial activation and leukocyte-endothelial interactions. We show that hematopoietic Hdac9 deficiency reduces lesional macrophage content while increasing fibrous cap thickness thus conferring plaque stability. We demonstrate that HDAC9 binds to IKK (inhibitory kappa B kinase)-α and ß, resulting in their deacetylation and subsequent activation, which drives inflammatory responses in both macrophages and endothelial cells. Pharmacological inhibition of HDAC9 with the class IIa HDAC inhibitor TMP195 attenuates lesion formation by reducing endothelial activation and leukocyte recruitment along with limiting proinflammatory responses in macrophages. Transcriptional profiling using RNA sequencing revealed that TMP195 downregulates key inflammatory pathways consistent with inhibitory effects on IKKß. TMP195 mitigates the progression of established lesions and inhibits the infiltration of inflammatory cells. Moreover, TMP195 diminishes features of plaque vulnerability and thereby enhances plaque stability in advanced lesions. Ex vivo treatment of monocytes from patients with established atherosclerosis reduced the production of inflammatory cytokines including IL (interleukin)-1ß and IL-6. CONCLUSIONS: Our findings identify HDAC9 as a regulator of atherosclerotic plaque stability and IKK activation thus providing a mechanistic explanation for the prominence of HDAC9 as a vascular risk locus in genome-wide association studies. Its therapeutic inhibition may provide a potent lever to alleviate vascular inflammation. Graphical Abstract: A graphical abstract is available for this article.


Assuntos
Artérias/enzimologia , Aterosclerose/enzimologia , Histona Desacetilases/metabolismo , Quinase I-kappa B/metabolismo , Placa Aterosclerótica , Proteínas Repressoras/metabolismo , Acetilação , Idoso , Idoso de 80 Anos ou mais , Animais , Artérias/efeitos dos fármacos , Artérias/patologia , Aterosclerose/tratamento farmacológico , Aterosclerose/genética , Aterosclerose/patologia , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Ativação Enzimática , Feminino , Fibrose , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Humanos , Quinase I-kappa B/genética , Mediadores da Inflamação/metabolismo , Migração e Rolagem de Leucócitos , Macrófagos/enzimologia , Macrófagos/patologia , Masculino , Camundongos Knockout para ApoE , Pessoa de Meia-Idade , Monócitos/enzimologia , Monócitos/patologia , Ligação Proteica , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transdução de Sinais
12.
J Neurosci ; 40(5): 1162-1173, 2020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-31889008

RESUMO

Recovery after stroke is a multicellular process encompassing neurons, resident immune cells, and brain-invading cells. Stroke alters the gut microbiome, which in turn has considerable impact on stroke outcome. However, the mechanisms underlying gut-brain interaction and implications for long-term recovery are largely elusive. Here, we tested the hypothesis that short-chain fatty acids (SCFAs), key bioactive microbial metabolites, are the missing link along the gut-brain axis and might be able to modulate recovery after experimental stroke. SCFA supplementation in the drinking water of male mice significantly improved recovery of affected limb motor function. Using in vivo wide-field calcium imaging, we observed that SCFAs induced altered contralesional cortex connectivity. This was associated with SCFA-dependent changes in spine and synapse densities. RNA sequencing of the forebrain cortex indicated a potential involvement of microglial cells in contributing to the structural and functional remodeling. Further analyses confirmed a substantial impact of SCFAs on microglial activation, which depended on the recruitment of T cells to the infarcted brain. Our findings identified that microbiota-derived SCFAs modulate poststroke recovery via effects on systemic and brain resident immune cells.SIGNIFICANCE STATEMENT Previous studies have shown a bidirectional communication along the gut-brain axis after stroke. Stroke alters the gut microbiota composition, and in turn, microbiota dysbiosis has a substantial impact on stroke outcome by modulating the immune response. However, until now, the mediators derived from the gut microbiome affecting the gut-immune-brain axis and the molecular mechanisms involved in this process were unknown. Here, we demonstrate that short-chain fatty acids, fermentation products of the gut microbiome, are potent and proregenerative modulators of poststroke neuronal plasticity at various structural levels. We identified that this effect was mediated via circulating lymphocytes on microglial activation. These results identify short-chain fatty acids as a missing link along the gut-brain axis and as a potential therapeutic to improve recovery after stroke.


Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Ácidos Graxos Voláteis/administração & dosagem , Acidente Vascular Cerebral/imunologia , Animais , Encéfalo/metabolismo , Feminino , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/imunologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Acidente Vascular Cerebral/metabolismo , Transcriptoma/efeitos dos fármacos
13.
Stroke ; 52(12): e837-e841, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34807742

RESUMO

Immune cell infiltration to the injured brain is a key component of the neuroinflammatory response after ischemic stroke. In contrast to the large amount of proinflammatory immune cells, regulatory T cells, are an important subgroup of T cells that are involved in maintaining immune homeostasis and suppress an overshooting immune reaction after stroke. Numerous previous reports have consistently demonstrated the beneficial role of this immunosuppressive immune cell population during the acute phase after experimental stroke by limiting inflammatory lesion progression. Two recent studies expanded now this concept and demonstrate that regulatory T cells-mediated effects also promote chronic recovery after stroke by promoting a proregenerative tissue environment. These recent findings suggest that boosting regulatory T cells could be beneficial beyond modulating the immediate neuroinflammatory response and improve chronic functional recovery.


Assuntos
AVC Isquêmico/imunologia , Recuperação de Função Fisiológica/imunologia , Linfócitos T Reguladores/imunologia , Animais , Humanos
14.
EMBO J ; 36(5): 583-603, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28007893

RESUMO

Alzheimer's disease (AD) is characterized by deposition of amyloid plaques, neurofibrillary tangles, and neuroinflammation. In order to study microglial contribution to amyloid plaque phagocytosis, we developed a novel ex vivo model by co-culturing organotypic brain slices from up to 20-month-old, amyloid-bearing AD mouse model (APPPS1) and young, neonatal wild-type (WT) mice. Surprisingly, co-culturing resulted in proliferation, recruitment, and clustering of old microglial cells around amyloid plaques and clearance of the plaque halo. Depletion of either old or young microglial cells prevented amyloid plaque clearance, indicating a synergistic effect of both populations. Exposing old microglial cells to conditioned media of young microglia or addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) was sufficient to induce microglial proliferation and reduce amyloid plaque size. Our data suggest that microglial dysfunction in AD may be reversible and their phagocytic ability can be modulated to limit amyloid accumulation. This novel ex vivo model provides a valuable system for identification, screening, and testing of compounds aimed to therapeutically reinforce microglial phagocytosis.


Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Modelos Animais de Doenças , Microglia/metabolismo , Placa Amiloide/metabolismo , Animais , Proliferação de Células , Técnicas de Cocultura , Meios de Cultivo Condicionados , Camundongos , Microglia/fisiologia , Técnicas de Cultura de Órgãos
15.
Brain Behav Immun ; 91: 668-672, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33197540

RESUMO

Stroke causes severe and long-lasting symptoms in patients. Besides focal deficits such as speech impairment and limb weakness, stroke also results in neuropsychiatric symptoms, including fatigue, anxiety, and depression, which are debilitating and often impair post-stroke rehabilitation. However, in experimental stroke research, the study of neuropsychiatric symptoms and their therapeutic targeting has so far been largely neglected, which can be mainly attributed to the lack of appropriate tools to investigate such deficits in mice. Here, we report that neuropsychiatric symptoms can be differentiated from focal deficits and specifically modulated independent of treating the primary lesion. In order to achieve this, we developed a novel behavior analysis tool by assessing test performance of various tests, combining outcome parameters to cover functional domains of focal and neuropsychiatric symptoms, and finally weighted results into a time point-specific score. This weighted score enabled us to clearly differentiate focal deficits and neuropsychiatric symptoms and detect these until the chronic phase after stroke. Using this analysis tool, we detected that neutralizing systemic cytokines (TNF-α, IL-1ß and IL-6) specifically ameliorated neuropsychiatric symptoms but did not affect focal deficits or lesion volume. Hence, most conventional studies analyzing only focal deficits and lesion volume as primary outcome measures would have missed these significant and translationally relevant therapeutic effects. We anticipate that these findings will encourage more detailed analyses of neuropsychiatric symptoms particularly for anti-inflammatory therapies in stroke and that the presented weighted composite score will facilitate this development.


Assuntos
Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Animais , Isquemia Encefálica/complicações , Citocinas , Humanos , Comportamento de Doença , Camundongos , Acidente Vascular Cerebral/complicações
16.
Neuroimage ; 199: 570-584, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31181333

RESUMO

The organization of brain areas in functionally connected networks, their dynamic changes, and perturbations in disease states are subject of extensive investigations. Research on functional networks in humans predominantly uses functional magnetic resonance imaging (fMRI). However, adopting fMRI and other functional imaging methods to mice, the most widely used model to study brain physiology and disease, poses major technical challenges and faces important limitations. Hence, there is great demand for alternative imaging modalities for network characterization. Here, we present a refined protocol for in vivo widefield calcium imaging of both cerebral hemispheres in mice expressing a calcium sensor in excitatory neurons. We implemented a stringent protocol for minimizing anesthesia and excluding movement artifacts which both imposed problems in previous approaches. We further adopted a method for unbiased identification of functional cortical areas using independent component analysis (ICA) on resting-state imaging data. Biological relevance of identified components was confirmed using stimulus-dependent cortical activation. To explore this novel approach in a model of focal brain injury, we induced photothrombotic lesions of the motor cortex, determined changes in inter- and intrahemispheric connectivity at multiple time points up to 56 days post-stroke and correlated them with behavioral deficits. We observed a severe loss in interhemispheric connectivity after stroke, which was partially restored in the chronic phase and associated with corresponding behavioral motor deficits. Taken together, we present an improved widefield calcium imaging tool accounting for anesthesia and movement artifacts, adopting an advanced analysis pipeline based on human fMRI algorithms and with superior sensitivity to recovery mechanisms in mouse models compared to behavioral tests. This tool will enable new studies on interhemispheric connectivity in murine models with comparability to human imaging studies for a wide spectrum of neuroscience applications in health and disease.


Assuntos
Cálcio , Córtex Cerebral/fisiologia , Conectoma/métodos , Rede Nervosa/fisiologia , Neuroimagem/métodos , Imagem Óptica/métodos , Prosencéfalo/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Animais , Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/fisiopatologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Córtex Motor/lesões , Córtex Motor/fisiopatologia , Rede Nervosa/diagnóstico por imagem , Rede Nervosa/fisiopatologia , Prosencéfalo/diagnóstico por imagem , Prosencéfalo/fisiopatologia , Acidente Vascular Cerebral/diagnóstico por imagem
17.
Circ Res ; 121(8): 970-980, 2017 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-28724745

RESUMO

RATIONALE: Currently, there are no blood-based biomarkers with clinical utility for acute ischemic stroke (IS). MicroRNAs show promise as disease markers because of their cell type-specific expression patterns and stability in peripheral blood. OBJECTIVE: To identify circulating microRNAs associated with acute IS, determine their temporal course up to 90 days post-stroke, and explore their utility as an early diagnostic marker. METHODS AND RESULTS: We used RNA sequencing to study expression changes of circulating microRNAs in a discovery sample of 20 patients with IS and 20 matched healthy control subjects. We further applied quantitative real-time polymerase chain reaction in independent samples for validation (40 patients with IS and 40 matched controls), replication (200 patients with IS, 100 healthy control subjects), and in 72 patients with transient ischemic attacks. Sampling of patient plasma was done immediately upon hospital arrival. We identified, validated, and replicated 3 differentially expressed microRNAs, which were upregulated in patients with IS compared with both healthy control subjects (miR-125a-5p [1.8-fold; P=1.5×10-6], miR-125b-5p [2.5-fold; P=5.6×10-6], and miR-143-3p [4.8-fold; P=7.8×10-9]) and patients with transient ischemic attack (miR-125a-5p: P=0.003; miR-125b-5p: P=0.003; miR-143-3p: P=0.005). Longitudinal analysis of expression levels up to 90 days after stroke revealed a normalization to control levels for miR-125b-5p and miR-143-3p starting at day 2 while miR-125a-5p remained elevated. Levels of all 3 microRNAs depended on platelet numbers in a platelet spike-in experiment but were unaffected by chemical hypoxia in Neuro2a cells and in experimental stroke models. In a random forest classification, miR-125a-5p, miR-125b-5p, and miR-143-3p differentiated between healthy control subjects and patients with IS with an area under the curve of 0.90 (sensitivity: 85.6%; specificity: 76.3%), which was superior to multimodal cranial computed tomography obtained for routine diagnostics (sensitivity: 72.5%) and previously reported biomarkers of acute IS (neuron-specific enolase: area under the curve=0.69; interleukin 6: area under the curve=0.82). CONCLUSIONS: A set of circulating microRNAs (miR-125a-5p, miR-125b-5p, and miR-143-3p) associates with acute IS and might have clinical utility as an early diagnostic marker.


Assuntos
Isquemia Encefálica/sangue , MicroRNAs/sangue , Análise de Sequência de RNA , Acidente Vascular Cerebral/sangue , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Isquemia Encefálica/diagnóstico por imagem , Isquemia Encefálica/genética , Estudos de Casos e Controles , Diagnóstico Precoce , Feminino , Marcadores Genéticos , Humanos , Interleucina-6/sangue , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Fosfopiruvato Hidratase/sangue , Valor Preditivo dos Testes , Prognóstico , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/genética , Fatores de Tempo , Tomografia Computadorizada por Raios X
18.
Nucleic Acids Res ; 45(18): 10595-10613, 2017 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-28977635

RESUMO

The integrity of genome is a prerequisite for healthy life. Indeed, defects in DNA repair have been associated with several human diseases, including tissue-fibrosis, neurodegeneration and cancer. Despite decades of extensive research, the spatio-mechanical processes of double-strand break (DSB)-repair, especially the auxiliary factor(s) that can stimulate accurate and timely repair, have remained elusive. Here, we report an ATM-kinase dependent, unforeseen function of the nuclear isoform of the Receptor for Advanced Glycation End-products (nRAGE) in DSB-repair. RAGE is phosphorylated at Serine376 and Serine389 by the ATM kinase and is recruited to the site of DNA-DSBs via an early DNA damage response. nRAGE preferentially co-localized with the MRE11 nuclease subunit of the MRN complex and orchestrates its nucleolytic activity to the ATR kinase signaling. This promotes efficient RPA2S4-S8 and CHK1S345 phosphorylation and thereby prevents cellular senescence, IPF and carcinoma formation. Accordingly, loss of RAGE causatively linked to perpetual DSBs signaling, cellular senescence and fibrosis. Importantly, in a mouse model of idiopathic pulmonary fibrosis (RAGE-/-), reconstitution of RAGE efficiently restored DSB-repair and reversed pathological anomalies. Collectively, this study identifies nRAGE as a master regulator of DSB-repair, the absence of which orchestrates persistent DSB signaling to senescence, tissue-fibrosis and oncogenesis.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reparo do DNA , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Animais , Núcleo Celular/enzimologia , Núcleo Celular/metabolismo , Senescência Celular , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Homeostase , Pulmão/fisiopatologia , Proteína Homóloga a MRE11 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibrose Pulmonar/genética , Fibrose Pulmonar/fisiopatologia , Receptor para Produtos Finais de Glicação Avançada/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais
19.
J Neurosci ; 36(28): 7428-40, 2016 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-27413153

RESUMO

UNLABELLED: Acute brain ischemia induces a local neuroinflammatory reaction and alters peripheral immune homeostasis at the same time. Recent evidence has suggested a key role of the gut microbiota in autoimmune diseases by modulating immune homeostasis. Therefore, we investigated the mechanistic link among acute brain ischemia, microbiota alterations, and the immune response after brain injury. Using two distinct models of acute middle cerebral artery occlusion, we show by next-generation sequencing that large stroke lesions cause gut microbiota dysbiosis, which in turn affects stroke outcome via immune-mediated mechanisms. Reduced species diversity and bacterial overgrowth of bacteroidetes were identified as hallmarks of poststroke dysbiosis, which was associated with intestinal barrier dysfunction and reduced intestinal motility as determined by in vivo intestinal bolus tracking. Recolonizing germ-free mice with dysbiotic poststroke microbiota exacerbates lesion volume and functional deficits after experimental stroke compared with the recolonization with a normal control microbiota. In addition, recolonization of mice with a dysbiotic microbiome induces a proinflammatory T-cell polarization in the intestinal immune compartment and in the ischemic brain. Using in vivo cell-tracking studies, we demonstrate the migration of intestinal lymphocytes to the ischemic brain. Therapeutic transplantation of fecal microbiota normalizes brain lesion-induced dysbiosis and improves stroke outcome. These results support a novel mechanism in which the gut microbiome is a target of stroke-induced systemic alterations and an effector with substantial impact on stroke outcome. SIGNIFICANCE STATEMENT: We have identified a bidirectional communication along the brain-gut microbiota-immune axis and show that the gut microbiota is a central regulator of immune homeostasis. Acute brain lesions induced dysbiosis of the microbiome and, in turn, changes in the gut microbiota affected neuroinflammatory and functional outcome after brain injury. The microbiota impact on immunity and stroke outcome was transmissible by microbiota transplantation. Our findings support an emerging concept in which the gut microbiota is a key regulator in priming the neuroinflammatory response to brain injury. These findings highlight the key role of microbiota as a potential therapeutic target to protect brain function after injury.


Assuntos
Disbiose/etiologia , Encefalite/complicações , Encefalite/etiologia , Microbiota/fisiologia , Acidente Vascular Cerebral/complicações , Animais , Infarto Encefálico/etiologia , Complexo CD3/metabolismo , Modelos Animais de Doenças , Disbiose/imunologia , Disbiose/microbiologia , Fezes/microbiologia , Feminino , Gastroenteropatias/etiologia , Motilidade Gastrointestinal/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Íleus/imunologia , Íleus/microbiologia , Íleus/patologia , Infarto da Artéria Cerebral Média/complicações , Leucócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microbiota/imunologia , Acidente Vascular Cerebral/etiologia , Estruturas Linfoides Terciárias/patologia
20.
J Neurosci ; 36(38): 9962-75, 2016 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-27656033

RESUMO

UNLABELLED: After traumatic brain injury (TBI), neurons surviving the initial insult can undergo chronic (secondary) degeneration via poorly understood mechanisms, resulting in long-term cognitive impairment. Although a neuroinflammatory response is promptly activated after TBI, it is unknown whether it has a significant role in chronic phases of TBI (>1 year after injury). Using a closed-head injury model of TBI in mice, we showed by MRI scans that TBI caused substantial degeneration at the lesion site within a few weeks and these did not expand significantly thereafter. However, chronic alterations in neurons were observed, with reduced dendritic spine density lasting >1 year after injury. In parallel, we found a long-lasting inflammatory response throughout the entire brain. Deletion of one allele of CX3CR1, a chemokine receptor, limited infiltration of peripheral immune cells and largely prevented the chronic degeneration of the injured brain and provided a better functional recovery in female, but not male, mice. Therefore, targeting persistent neuroinflammation presents a new therapeutic option to reduce chronic neurodegeneration. SIGNIFICANCE STATEMENT: Traumatic brain injury (TBI) often causes chronic neurological problems including epilepsy, neuropsychiatric disorders, and dementia through unknown mechanisms. Our study demonstrates that inflammatory cells invading the brain lead to secondary brain damage. Sex-specific amelioration of chronic neuroinflammation rescues the brain degeneration and results in improved motor functions. Therefore, this study pinpoints an effective therapeutic approach to preventing secondary complications after TBI.


Assuntos
Lesões Encefálicas Traumáticas/complicações , Inflamação/etiologia , Degeneração Neural , Recuperação de Função Fisiológica/fisiologia , Animais , Encéfalo/patologia , Receptor 1 de Quimiocina CX3C , Proteínas de Ligação ao Cálcio/metabolismo , Doença Crônica , Espinhas Dendríticas/imunologia , Espinhas Dendríticas/patologia , Espinhas Dendríticas/ultraestrutura , Modelos Animais de Doenças , Comportamento Exploratório/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Atividade Motora , Degeneração Neural/diagnóstico por imagem , Degeneração Neural/etiologia , Degeneração Neural/patologia , Neurônios/metabolismo , Neurônios/patologia , Desempenho Psicomotor/fisiologia , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Fatores de Tempo
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