Phagocytic Capacity Of Microglia Research Articles

HMGB1 mediates synaptic loss and cognitive impairment in an animal model of sepsis-associated encephalopathy

BackgroundMicroglial activation-mediated neuroinflammation is one of the essential pathogenic mechanisms of sepsis-associated encephalopathy (SAE). Mounting evidence suggests that high mobility group box-1 protein (HMGB1) plays a pivotal role in neuroinflammation and SAE, yet the mechanism by which HMGB1 induces cognitive impairment in SAE remains unclear. Therefore, this study aimed to investigate the mechanism of HMGB1 underlying cognitive impairment in SAE.MethodsAn SAE model was established by cecal ligation and puncture (CLP); animals in the sham group underwent cecum exposure alone without ligation and perforation. Mice in the inflachromene (ICM) group were continuously injected with ICM intraperitoneally at a daily dose of 10 mg/kg for 9 days starting 1 h before the CLP operation. The open field, novel object recognition, and Y maze tests were performed on days 14–18 after surgery to assess locomotor activity and cognitive function. HMGB1 secretion, the state of microglia, and neuronal activity were measured by immunofluorescence. Golgi staining was performed to detect changes in neuronal morphology and dendritic spine density. In vitro electrophysiology was performed to detect changes in long-term potentiation (LTP) in the CA1 of the hippocampus. In vivo electrophysiology was performed to detect the changes in neural oscillation of the hippocampus.ResultsCLP-induced cognitive impairment was accompanied by increased HMGB1 secretion and microglial activation. The phagocytic capacity of microglia was enhanced, resulting in aberrant pruning of excitatory synapses in the hippocampus. The loss of excitatory synapses reduced neuronal activity, impaired LTP, and decreased theta oscillation in the hippocampus. Inhibiting HMGB1 secretion by ICM treatment reversed these changes.ConclusionsHMGB1 induces microglial activation, aberrant synaptic pruning, and neuron dysfunction in an animal model of SAE, leading to cognitive impairment. These results suggest that HMGB1 might be a target for SAE treatment.

The effect of genetic background and immunophenotype on microglial response to AD pathology

AbstractBackgroundA major hurdle in developing effective treatments for Alzheimer’s Disease (AD) is the fact that pathological hallmarks, such as amyloid beta (Aβ) plaques, develop decades before the first clinical symptoms emerge. Moreover, the risk of developing AD is strongly influenced by genetic background. In addition, the role of microglia in the disease pathway is undeniable, albeit dual and complex. We therefore aim to determine the influence of patient genetic background and immunophenotype on the microglial response to AD pathology.MethodSubject material was sequenced and underwent deep immunophenotyping. Based on their scores patient iPSCs were divided in a high risk and low risk group of developing AD. This microglial response is determined by (i) their phagocytic capacity and (ii) their (change in) morphology. iPSC cells were differentiated into microglia (Mancuso et al., 2019), followed by RT‐qPCR to verify their identity. When microglia are incubated with pHrodo beads, these are phagocytosed and transported to the acidic lysosome, yielding a pH dependent increase in fluorescence, measured with an Operetta confocal microscope. Microglial morphology was recorded in an Incucyte and tracked and scored (e.g. amoeboid, rod‐like, size, motility …) automatically by AI based software. The change in morphology was assessed after addition of stressors such as LPS or Aβ1‐42.ResultPhagocytotic capacity of a reference microglia strain, measured by pHrodo intensity, reached a maximum level approximately 2 hours after addition of the pHrodo beads. When cytochalasin D, an Actin polymerization inhibitor, was added to the medium, the phagocytosis process is inhibited and baseline pHrodo intensity levels are observed. In addition, the addition of LPS seemed to change the morphology of microglia to a more elongated rod‐like shape with less motility.ConclusionThe phagocytic capacity of microglia could be dependent on the genetic background and immunophenotype of the iPSC strain. Similarly, the effect on morphology (e.g. more rod‐like shape) as an answer to stressors (e.g. LPS) could be dependent on the microglia strain. The differential microglial response based on genetic background and immunophenotype might yield insight into the further segmentation of different types of Alzheimer’s Disease and personalized therapeutic approaches.

Amyloid‐β plus cholesterol treatment impairs the ability of microglia to efflux excess cholesterol

AbstractBackgroundMicroglia, specialized phagocytic cells of the brain, play a critical role in the clearance of amyloid beta (Aβ), which is involved in the pathogenesis of Alzheimer’s disease. The ability of microglia to remove Aβ is influenced by their cellular cholesterol clearance capacity. It is not known to what extent Aβ impairs microglial cholesterol efflux capacity, especially in the context of excess cholesterol.MethodHMC3 microglia were treated with Aβ‐oligomer (AβO, 2µM), cholesterol loading (20µg), or AβO + cholesterol in combination, and with or without cholesterol acceptor, high‐density lipoprotein (HDL), at 0.1 mg/mL. Pooled HDL from 18 individuals in a 2‐hour postprandial state and a 36‐hour fasted state were compared. The cellular content of unesterified and esterified cholesterol was measured.ResultsThe addition of cholesterol, AβO, and cholesterol+AβO increased cellular cholesterol content, which was attenuated by the addition of HDL. Importantly, cells treated with cholesterol plus AβO had the highest overall cholesterol content (27.80+/‐2.85µM), and both fasted (20.67+/‐3.04µM, p≥0.05) and postprandial HDL (21.95+/‐3.84µM, p<0.05) were not able to reduce the cellular cholesterol content as well as in the untreated (fasted: 16.17+/‐2.28µM, p<0.05; postprandial: 18.55+/‐2.01µM, p>0.05), cholesterol only (fasted: 17.72+/‐2.17µM, p<0.05; postprandial: 18.14+/‐3.19µM, p<0.05), or AβO only condition (fasted: 16.80+/‐1.40µM, p<0.05; postprandial: 18.42+/‐2.05µM, p>0.05). Fasted HDL more efficiently removed cellular cholesterol in the untreated and in AβO+cholesterol treated cells. Intriguingly, cellular esterified cholesterol content was significantly lowered with fasted HDL but not postprandial HDL in both AβO alone (fasted: 5.73+/‐2.27 µM, p<0.05; postprandial: 10.32+/‐2.70µM, p>0.05) and AβO + cholesterol (fasted: 2.87+/‐2.13µM, p<0.05; postprandial: 9.03+/‐1.74µM, p>0.05).ConclusionThese findings suggest that microglia exposed to a double‐hit of AβO and excess cholesterol experience an impaired ability to efflux excess cholesterol. Additionally, HDL from fasted individuals displayed an enhanced capacity to efflux cholesterol, especially in microglia dually encumbered by AβO + cholesterol. Further studies are needed to examine the impact of this diminished cholesterol efflux capacity and cholesterol accumulation on the phagocytic capacity of microglia.

The endoplasmic reticulum Ca2+ -ATPase SERCA2b is upregulated in activated microglia and its inhibition causes opposite effects on migration and phagocytosis.

Activation of microglia is an early immune response to damage in the brain. Although a key role for Ca2+ as trigger of microglial activation has been considered, little is known about the molecular scenario for regulating Ca2+ homeostasis in these cells. Taking into account the importance of the endoplasmic reticulum as a cellular Ca2+ store, the sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA2b) is an interesting target to modulate intracellular Ca2+ dynamics. We found upregulation of SERCA2b in activated microglia of human brain with Alzheimer's disease and we further studied the participation of SERCA2b in microglial functions by using the BV2 murine microglial cell line and primary microglia isolated from mouse brain. To trigger microglia activation, we used the bacterial lipopolysaccharide (LPS), which is known to induce an increase of cytosolic Ca2+ . Our results showed an upregulated expression of SERCA2b in LPS-induced activated microglia likely associated to an attempt to restore the increased cytosolic Ca2+ concentration. We analyzed SERCA2b contribution in microglial migration by using the specific SERCA inhibitor thapsigargin in scratch assays. Microglial migration was strongly stimulated with thapsigargin, even more than with LPS-induction, but delayed in time. However, phagocytic capacity of microglia was blocked in the presence of the SERCA inhibitor, indicating the importance of a tight control of cytosolic Ca2+ in these processes. All together, these results provide for the first time compelling evidence for SERCA2b as a major player regulating microglial functions, affecting migration and phagocytosis in an opposite manner.

Propranolol diminished severity of rat EAE by enhancing immunoregulatory/protective properties of spinal cord microglia

Sympathetic dysfunction is suggested to contribute to development of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) alike. Considering important role of microglia in development/resolution of neuroinflammation, contribution of noradrenaline, the key sympathetic end-point mediator, in modulation of microglial phenotypic and functional properties in rat EAE model was examined. The study showed that noradrenaline acting in neurocrine and autocrine/paracrine way might influence microglia during EAE. Propranolol treatment over the effector phase moderated EAE course. This was associated with the increased proportion of microglia expressing CX3CR1, the key molecule in their immunomodulatory/neuroprotective action, and upregulation of CX3CR1 downstream Nrf2 gene. This correlated with the increased heme oxygenase-1 (HO-1) expression and phagocytic capacity of microglia, and their phenotypic changes mirrored in increased proportion of CD163- and CD83-expressing cells. The enhanced HO-1 expression was linked with the decreased proportion of microglial cells expressing IL-1β and IL-23, and possibly IL-6, followed by increased proportion of IL-10–expressing microglia, and downregulated MCP-1/CCL2 expression. Consistently, spinal cord infiltration with blood-borne myeloid and CD4+ T cells, as well as CD4+ T-cell reactivation/proliferation and differentiation into highly pathogenic IL-17+ cells co-producing IFN-γ and GM-CSF were decreased in propranolol-treated rats compared with saline-injected controls. The in vitro investigations of the effects of noradrenaline on microglia showed that noradrenaline through β-adrenoceptor may influence Nrf2 expression also via CX3CR1-independent route. The study suggests β-adrenoceptor–mediated neuroinflammation-promoting role of noradrenaline in EAE via modulation of microglial Nrf2 expression, and thereby forms the basis for further translational pharmacological research to improve multiple sclerosis therapy.

Age does not influence the disease course in a mouse model of Streptococcus pneumoniae serotype 3 meningitis

In order to elucidate the causes for the increased mortality of aged patients with bacterial central nervous system (CNS) infections, we compared the course of Streptococcus pneumoniae (S. pneumoniae) meningitis in aged and young mice. Aged (21.2 ± 3.1 months, n = 40) and young (3.2 ± 0.9 months, n = 42) C57BL/6N and B6/SJL mice were infected by intracerebral injection of 50–70 CFU S. pneumoniae serotype 3 and monitored for 15 days. Aged and young mice did not differ concerning mortality (35% versus 38%), weight loss, development of clinical symptoms, bacterial concentrations in cerebellum and spleen as well as the number of leukocytes infiltrating the CNS. In contrast to results from our geriatric mouse model of Escherichia coli (E. coli) meningitis, where aged mice showed a higher mortality and an impaired elimination of bacteria, we did not find any differences between aged and young mice after intracerebral infection with S. pneumoniae serotype 3. This indicates that the increased susceptibility of aged mice to bacterial CNS infections is pathogen-specific: It appears less prominent in infections caused by hardly phagocytable pathogens with thick capsules like S. pneumoniae serotype 3, where the age-related decline of the phagocytic capacity of microglia and macrophages has a minor influence on the disease course.

Absence of CX3CR1 impairs the internalization of Tau by microglia

BackgroundExtracellular Tau is toxic for neighboring cells, and it contributes to the progression of AD. The CX3CL1/CX3CR1 axis is an important neuron/microglia communication mechanism.MethodsWe studied Tau clearance by microglia both in vitro (microglia primary cultures treated with Cy5-Tau, affinity chromatography to study the binding of Tau to CX3CR1, and Tau-CX3CL1 competition assays) and in vivo (stereotaxic injection of Cy5-Tau into WT and CX3CR1−/− mice). The expression of CX3CR1, CX3CL1 and the microglial phagocytic phenotype were studied in brain tissue samples from AD patients.ResultsTau binding to CX3CR1 triggers the internalization of the former by microglia, whereas S396 Tau phosphorylation decreases the binding affinity of this protein to CX3CR1. Of note, the progressive increase in the levels of phosho-Tau occurred in parallel with an increase in CX3CR1. In addition, our studies suggest that the phagocytic capacity of microglia in brain tissue samples from AD patients is decreased. Furthermore, the CX3CR1/CX3CL1 axis may be impaired in late stages of the disease.ConclusionsOur data suggest that the CX3CR1/CX3CL1 axis plays a key role in the phagocytosis of Tau by microglia in vitro and in vivo and that it is affected as AD progresses. Taken together, our results reveal CX3CR1 as a novel target for the clearance of extracellular Tau.

Severe chronic cerebral hypoperfusion induces microglial dysfunction leading to memory loss in APPswe/PS1 mice.

Cerebral vasculature plays a key role in controlling brain homeostasis. Cerebral vasculature dysfunction, associated to irregularities in cerebral blood perfusion, has been proposed to directly contribute to Alzheimer's disease (AD) pathogenesis. More precisely, chronic cerebral hypoperfusion, which impairs brain homeostasis, was demonstrated to take place even before cognitive decline. However, the mechanisms underlying the implication of chronic cerebral hypoperfusion in AD pathogenesis remain elusive. Therefore, this study aims at investigating the role of severe chronic cerebral hypoperfusion (SCCH) in AD pathogenesis. For this purpose, SCCH was induced in young APPswe/PS1 in order to evaluate the progression of AD-like pathology in these mice. We observed that SCCH accelerated the cognitive decline of young APPswe/PS1 mice, which was associated with an increased amyloid plaque number in brain parenchyma. In addition, SCCH reduced the activity of extracellular signal-regulated kinases 1/2 (ERK1/2), which has been shown to play an important role in the adaptive responses of neurons. Importantly, SCCH impaired the function of microglial cells, which are implicated in amyloid-β (Aβ) elimination. In vitro approaches underlined the ability of a low-glucose microenvironment to decrease the general activity and phagocytic capacity of microglia. By using a new model of SCCH, our study unravels new insights into the implication of severe chronic cerebral hypoperfusion in AD pathogenesis, mainly by altering microglial cell activity and consequently Aβ clearance.

Bone Marrow-Derived Mesenchymal Stem Cells Maintain the Resting Phenotype of Microglia and Inhibit Microglial Activation

Many studies have shown that microglia in the activated state may be neurotoxic. It has been proven that uncontrolled or over-activated microglia play an important role in many neurodegenerative disorders. Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown in many animal models to have a therapeutic effect on neural damage. Such a therapeutic effect is attributed to the fact that BMSCs have the ability to differentiate into neurons and to produce trophic factors, but there is little information available in the literature concerning whether BMSCs play a therapeutic role by affecting microglial activity. In this study, we triggered an inflammatory response situation in vitro by stimulating microglia with the bacterial endotoxin lipopolysaccharide (LPS), and then culturing these microglia with BMSC-conditioned medium (BMSC-CM). We found that BMSC-CM significantly inhibited proliferation and secretion of pro-inflammatory factors by activated microglia. Furthermore, we found that the phagocytic capacity of microglia was also inhibited by BMSC-CM. Finally, we investigated whether the induction of apoptosis and the production of nitric oxide (NO) were involved in the inhibition of microglial activation. We found that BMSC-CM significantly induced apoptosis of microglia, while no apoptosis was apparent in the LPS-stimulated microglia. Our study also provides evidence that NO participates in the inhibitory effect of BMSCs. Our experimental results provide evidence that BMSCs have the ability to maintain the resting phenotype of microglia or to control microglial activation through their production of several factors, indicating that BMSCs could be a promising therapeutic tool for treatment of diseases associated with microglial activation.

Functional Impairment of Microglia Coincides with Beta-Amyloid Deposition in Mice with Alzheimer-Like Pathology

Microglial cells closely interact with senile plaques in Alzheimer’s disease and acquire the morphological appearance of an activated phenotype. The significance of this microglial phenotype and the impact of microglia for disease progression have remained controversial. To uncover and characterize putative changes in the functionality of microglia during Alzheimer’s disease, we directly assessed microglial behavior in two mouse models of Alzheimer’s disease. Using in vivo two-photon microscopy and acute brain slice preparations, we found that important microglial functions - directed process motility and phagocytic activity - were strongly impaired in mice with Alzheimer’s disease-like pathology compared to age-matched non-transgenic animals. Notably, impairment of microglial function temporally and spatially correlated with Aβ plaque deposition, and phagocytic capacity of microglia could be restored by interventionally decreasing amyloid burden by Aβ vaccination. These data suggest that major microglial functions progressively decline in Alzheimer’s disease with the appearance of Aβ plaques, and that this functional impairment is reversible by lowering Aβ burden, e.g. by means of Aβ vaccination.

Fibrillar prion peptide (106-126) and scrapie prion protein hamper phagocytosis in microglia.

The inflammatory response in prion diseases is dominated by microglial activation. As macrophages of the central nervous system, the phagocytic capacity of microglia is well recognized, and it is possible that microglia are involved in the removal and processing of amyloid fibrils, thus preventing their harmful effect. We have analyzed the effects of a synthetic peptide of the human prion protein, PrP(106-126), which can form fibrils, and the pathogenic form of prion protein, PrPsc, on phagocytosis in microglia isolated from neonatal rat brain cultures. To some extent, fibrillar PrP(106-126) is internalized and processed. However, both synthetic prion peptide PrP(106-126) in a fibrillar form and pathogenic prion protein PrPsc severely hamper the phagocytic activity as measured by the uptake of beads by microglia. At a concentration that does not induce microglial death, PrP(106-126) reduced the number of beads internalized and altered their cytoplasmic distribution. This effect was not due to decreased binding of beads to the cell surface, nor restricted to specific classes of receptors. Although the PrP(106-126) did not prevent F-actin and Rac1 accumulation at sites of particle engulfment, it appeared to interfere with a later step of the internalization process.