BackgroundNeuroinflammation is implicated in the development and progression of many neurodegenerative diseases. Conditions that lead to a peripheral immune response are often associated with inflammation in the central nervous system (CNS), suggesting a communication between the peripheral immune system and the neuroimmune system. The underlying mechanism of this relationship remains largely unknown; however, experimental studies have demonstrated that exposure to infectious stimuli, such as lipopolysaccharide (LPS) or high-fat diet (HFD) feeding, result in profound peripheral- and neuro-inflammation.MethodsUsing the model of endotoxemia with LPS, we studied the role of serum-derived exosomes in mediating neuroinflammation. We purified circulating exosomes from the sera of LPS-challenged mice, which were then intravenously injected into normal adult mice.ResultsWe found that the recipient mice that received serum-derived exosomes from LPS-challenged mice exhibited elevated microglial activation. Moreover, we observed astrogliosis, increased systemic pro-inflammatory cytokine production, and elevated CNS expression of pro-inflammatory cytokine mRNA and the inflammation-associated microRNA (miR-155) in these recipient mice. Gene expression analysis confirmed that many inflammatory microRNAs were significantly upregulated in the purified exosomes under LPS-challenged conditions. We observed accumulated signaling within the microglia of mice that received tail-vein injections of fluorescently labeled exosomes though the percentage of those microglial cells was found low. Finally, purified LPS-stimulated exosomes from blood when infused directly into the cerebral ventricles provoked significant microgliosis and, to a lesser extent, astrogliosis.ConclusionsThe experimental results suggest that circulating exosomes may act as a neuroinflammatory mediator in systemic inflammation.Electronic supplementary materialThe online version of this article (10.1186/s12974-017-1038-8) contains supplementary material, which is available to authorized users.

Sepsis-associated encephalopathy (SAE) is an acutely progressing brain dysfunction induced by systemic inflammation. The mechanism of initiation of neuroinflammation during SAE, which ultimately leads to delirium and cognitive dysfunction, remains elusive. We aimed to study the molecular events of SAE to capture its onset and progression into the central nervous system (CNS), and further identify the cellular players involved in mediating acute inflammatory signaling. Gene expression profiling on the cerebral vessels isolated from the brains of the mice treated with peripheral lipopolysaccharide (LPS) revealed that the cerebral vasculature responds within minutes to acute systemic inflammation by upregulating the expression of immediate early response genes, followed by activation of the nuclear factor-κB pathway. To identify the earliest responding cell type, we used fluorescence-activated cell sorting (FACS) to sort the glial and vascular cells from the brains of the mice treated with LPS at different time points, and RNA-seq was performed on microglia and cerebral endothelial cells (CECs). Bioinformatic analysis followed by further validation in all the cell types revealed that panendothelitis. i.e., the activation of CECs is the earliest event in the CNS during the inception of acute neuroinflammation. Microglial activation occurs later than that of CECs, suggesting that CECs are the most likely initial source of proinflammatory mediators, which could further initiate glial cell activation. This is then followed by the activation of apoptotic signaling in the CECs, which is known to lead to the blood–brain barrier disruption and allow peripheral cytokines to leak into the CNS, exacerbate the gliosis, and result in the vicious neuroinflammatory cascade. Together, our results model the earliest sequential events during the advancement of systemic inflammation into the CNS and facilitate to understand the interplay between the vascular and glial cells in initiating and driving acute neuroinflammation during SAE.

Vascular cognitive impairment and dementia (VCID) is the second most common form of dementia after Alzheimer’s disease (AD). Currently, the mechanistic insights into the evolution and progression of VCID remain elusive. White matter change represents an invariant feature. Compelling clinical neuroimaging and pathological evidence suggest a link between white matter changes and neurodegeneration. Our prior study detected hypoperfused lesions in mice with partial deficiency of endothelial nitric oxide (eNOS) at very young age, precisely matching to those hypoperfused areas identified in preclinical AD patients. White matter tracts are particularly susceptible to the vascular damage induced by chronic hypoperfusion. Using immunohistochemistry, we detected severe demyelination in the middle-aged eNOS-deficient mice. The demyelinated areas were confined to cortical and subcortical areas including the corpus callosum and hippocampus. The intensity of demyelination correlated with behavioral deficits of gait and associative recognition memory performances. By Evans blue angiography, we detected blood–brain barrier (BBB) leakage as another early pathological change affecting frontal and parietal cortex in eNOS-deficient mice. Sodium nitrate fortified drinking water provided to young and middle-aged eNOS-deficient mice completely prevented non-perfusion, BBB leakage, and white matter pathology, indicating that impaired endothelium-derived NO signaling may have caused these pathological events. Furthermore, genome-wide transcriptomic analysis revealed altered gene clusters most related to mitochondrial respiratory pathways selectively in the white matter of young eNOS-deficient mice. Using eNOS-deficient mice, we identified BBB breakdown and hypoperfusion as the two earliest pathological events, resulting from insufficient vascular NO signaling. We speculate that the compromised BBB and mild chronic hypoperfusion trigger vascular damage, along with oxidative stress and astrogliosis, accounting for the white matter pathological changes in the eNOS-deficient mouse model. We conclude that eNOS-deficient mice represent an ideal spontaneous evolving model for studying the earliest events leading to white matter changes, which will be instrumental to future therapeutic testing of drug candidates and for targeting novel/specific vascular mechanisms contributing to VCID and AD.

Background: Vascular cognitive impairment and dementia (VCID) is the second most common form of dementia after Alzheimer’s disease (AD). Currently, the mechanistic insights into the evolution and progression of VCID are not fully understood. White matter change represents an invariant feature of both VCID and AD. Compelling clinical neuroimaging and pathological evidence suggest a link between white matter changes and neurodegeneration. Our prior study detected non-perfusion lesions in mice with partial deficiency of endothelial nitric oxide (eNOS) expression at a very young age. These lesions developed in multiple brain regions in an age-dependent manner, precisely matching to those hypoperfused areas identified in preclinical AD patients (i.e., temporoparietal and retrosplenial granular cortexes, and hippocampus). We therefore reasoned that eNOS- deficient mice could serve as a spontaneous model of chronic hypoperfusion.Methods/Results: White matter tracts are particularly susceptible to the vascular damage induced by chronic hypoperfusion. Using immunohistochemistry, we detected massive demyelination in the middle aged eNOS-deficient mice. The demyelinated areas were confined to cortical and subcortical areas including the corpus callosum and hippocampus, but did not involve the striatum. The intensity of demyelination correlated with behavioral gait deficits. By Evans blue angiography, we detected blood-brain barrier (BBB) leakage as another early pathological change affecting frontal and parietal cortex in eNOS-deficient mice, which occurs in as early as 3-4 months of age. Sodium nitrate fortified drinking water provided to young and middle aged eNOS-deficient mice completely prevented non-perfusion, BBB leakage, and white matter pathology, indicating that impaired endothelium-derived NO signaling may have caused these pathological events. Conclusions: Using eNOS-deficient mice, we identified BBB breakdown and non-perfusion as the two earliest pathological events, resulting from insufficient vascular NO signaling. We speculate that the compromised BBB and chronic hypoperfusion trigger vascular damage, along with oxidative stress and astrogliosis, accounting for the white matter pathological changes in the eNOS-deficient mouse model. We conclude that eNOS-deficient mice represent an ideal spontaneous evolving model for studying the earliest events leading to spontaneous white matter changes, which will be instrumental to future therapeutic testing of drug candidates and for targeting novel/specific vascular mechanisms contributing to VCID and AD.

Sepsis-associated encephalopathy (SAE) is an acutely progressing brain dysfunction induced by systemic inflammation. The mechanism of initiation of neuroinflammation during SAE, which ultimately leads to delirium and cognitive dysfunction, remains elusive. The goal of this project was to study the molecular events of SAE to capture its onset and progression into the central nervous system (CNS), and further identify the cellular players involved in mediating acute inflammatory signaling. Gene expression profiling on the cerebral vessels isolated from the brains of the mice treated with peripheral lipopolysaccharide (LPS) revealed that the cerebral vasculature responds within minutes to acute systemic inflammation by upregulating the expression of immediate early response genes, followed by activation of the NF-κB pathway. To identify the earliest responding cell type, fluorescence-activated cell sorting (FACS) was utilized to sort the immunolabelled glial and vascular cells from the brains of the mice treated with LPS at different time points and gene expression profiling was performed. Bioinformatic analysis of the sequencing data followed by further validation revealed that the cerebral endothelial cells (CECs) activation is the earliest event in the CNS and that they are the most likely source of proinflammatory mediators that could further initiate glial cell activation. This is further followed by the activation of apoptotic signaling in the CECs which is known to lead to blood brain barrier (BBB) disruption and allow the peripheral cytokines to leak into the CNS, exacerbate the gliosis and result in neuroinflammatory cascade. Together, these results model the sequential events during the advancement of systemic inflammation into the CNS, and facilitate better understanding of the interplay between the vascular and glial cells in initiating and driving acute neuroinflammation during SAE. Systemic inflammation does lead to neuroinflammation, thereby linking the peripheral inflammatory conditions to the CNS. However, the mechanisms through which systemic inflammation exerts its effect on the CNS are poorly understood. Exosomes are small (30 to 100 nanometers) membrane bound extracellular vesicles released by most of the mammalian cells. Exosomes play a vital role in cell to cell communication. This includes regulation of inflammatory responses by shuttling mRNAs, miRNAs and cytokines both locally and systemically to the neighboring as well as distant cells to further modulate their transcriptional and/or translational states and affect the functional phenotype of those cells which have taken up these exosomes. The role of circulating blood exosomes in mediating neuroinflammation during systemic inflammation was thus studied. Serum derived exosomes from LPS-challenged mice (SDEL) were freshly isolated from the sera of the mice which were earlier treated with LPS and used to study SDEL effects on neuroinflammation. Exosomes isolated from the sera of the mice injected with saline were used as control. In vitro studies showed that the SDEL upregulate pro-inflammatory cytokine gene expression in the cell lines of microglia (BV2), astrocytes (C8-D1A) and cerebral microvascular endothelial cells (Bend.3). To further study their effects in vivo, SDEL were then intravenously injected into normal adult mice. The recipient mice that received SDEL exhibited elevated microglial activation. Moreover, increased astrogliosis, and elevated CNS expression of pro-inflammatory cytokine mRNA were observed in SDEL recipient mice. Additionally, SDEL injected directly into the cerebral ventricles resulted in significant microgliosis as well as, astrogliosis. Together, these results demonstrate a novel role of peripheral circulating exosomes in causing neuroinflammation during systemic inflammatory conditions.