Primary Murine Microglia Research Articles

Interleukin-6 and interferon-alpha differentially regulate microglia function.

Previous reports have shown that IL-6 and IFN-⍺ induce distinct transcriptomic and morphological changes in microglia. Here, we demonstrate that IL-6 increases tissue surveillance, migration and phagocytosis in primary murine microglia, whereas IFN-⍺ inhibits these functions. Our results provide a crucial link between transcriptome and function. It holds the potential to serve as the foundation for future studies aimed at identifying therapeutic targets for cytokine-mediated neuroinflammatory diseases.

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons.

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adoptingreactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

LRRK2 kinase activity restricts NRF2-dependent mitochondrial protection in microglia.

Mounting evidence supports a critical role for central nervous system (CNS) glial cells in neuroinflammation and neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's Disease (PD), Multiple Sclerosis (MS), as well as neurovascular ischemic stroke. Previously, we found that loss of the PD-associated gene leucine-rich repeat kinase 2 (Lrrk2) in macrophages, peripheral innate immune cells, induced mitochondrial stress and elevated basal expression of type I interferon (IFN) stimulated genes (ISGs) due to chronic mitochondrial DNA engagement with the cGAS/STING DNA sensing pathway. Here, we report that loss of LRRK2 results in a paradoxical response in microglial cells, a CNS-specific macrophage population. In primary murine microglia and microglial cell lines, loss of Lrrk2 reduces tonic IFN signaling leading to a reduction in ISG expression. Consistent with reduced type I IFN, mitochondria from Lrrk2 KO microglia are protected from stress and have elevated metabolism. These protective phenotypes involve upregulation of NRF2, an important transcription factor in the response to oxidative stress and are restricted by LRRK2 kinase activity. Collectively, these findings illustrate a dichotomous role for LRRK2 within different immune cell populations and give insight into the fundamental differences between immune regulation in the CNS and the periphery.

Naringenin-loaded poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-based devices have an anti-inflammatory activity on microglia

Retinal degenerative diseases, such as retinitis pigmentosa, represent a significant fraction of the burden of blindness. This kind of diseases involves the activation of microglial cells which causes neuroinflammation. Flavonoids, such as naringenin (Nar), are able to modulate the pro-inflammatory behavior of the microglia cells but their low bioavailability often hinders this activity. In this context, the aim of this research activity was the development of implantable drug delivery systems through Nar encapsulation into a polymeric matrix, as a valuable way to increase its bioavailability. For this purpose, a blend between microbial poly(3-hydroxybutyrate-co-3-hydroxyvaerate) (PHBV) and poly(d,l-lactide-co-glycolide (PLGA) was processed by means of two different fabrication techniques, i.e., melt-spinning and wet-spinning, to fabricate rods designed for ocular implantation. The two developed kinds of rod had different morphology and thermal properties, as demonstrated by scanning electron microscopy, thermogravimetric analysis, and differential scanning calorimetry. In addition, an increase in Nar concentration in the starting polymeric mixture (1–5 wt% respect to device weight) determined a higher drug encapsulation efficiency, with a resulting drug loading increasing from 0.5 to 3.5 % and from 0.4 to 4.4 % for melt- and wet-spun rods, respectively. A significant effect of processing technique and drug loading on the resulting release kinetics was also observed, with the devices loaded with a higher percentage of Nar reaching a 100 % cumulative drug release after 38 and 73 days in the case of wet- and melt-spun rods, respectively. In addition, the wet-spun rods showed a first order drug release kinetics while the melt-spun rods a zero order one. The bioactivity of the wet-spun rods was tested on lipopolysaccharide-activated BV2 as well as primary murine microglia cells, thus demonstrating the ability of the released flavonoid to favor cell maturation from a pro-inflammatory to an anti-inflammatory phenotype.

The gut microbe‐derived methylamine TMAO protects against neuroinflammation through restraint of pro‐inflammatory glial behaviour

AbstractBackgroundWhile the ultimate cause(s) for Alzheimer’s disease (AD) are unknown, factors that can modify the risk of its development are clearer. Consumption of a seafood‐rich diet has repeatedly been shown to protect against AD. What is less clear however, is why. Much attention has focussed on omega‐3 fatty acids given their anti‐inflammatory effects in vitro, but translation to human studies have been disappointing. Omega‐3 fatty acids are not the only bioactive components in seafood however. In particular, we have recently shown the dietary methylamine trimethylamine N‐oxide (TMAO), produced by gut microbial metabolism of seafood‐enriched choline, to enhance blood‐brain barrier integrity, prevents glial activation and protect cognitive function following peripheral inflammatory challenge.MethodsWe investigated whether protective effects of TMAO treatment could be extended to neuroinflammatory challenge. Male C57Bl/6 (n = 6) were pre‐treated for 4 weeks with or without TMAO through the drinking water (0.5g/l) prior to unilateral i.c.v. injection of 4 mg/kg streptozotocin (STZ) or saline vehicle. Two weeks post‐lesioning (continuing TMAO treatment as appropriate) animals were killed and assessment was made of hippocampal inflammation. In parallel, the molecular mechanism of action of TMAO was investigated in vitro using immortalised and primary murine microglia, challenged with oligomeric β‐amyloid (100nM).ResultsAnimals treated with STZ showed significant increases in both microglial and astrocytic density in both the CA1 and dentate gyrus regions of the hippocampus, two weeks post‐lesioning, effects that were almost completely attenuated by TMAO pre‐treatment. Study of oxidative and inflammatory markers is ongoing. In vitro analyses of immortalised and primary microglia showed TMAO pre‐treatment (20 µM, 24 h) to prevent lipopolysaccharide (10 ng/ml) or oligomeric β‐amyloid (100nM)‐induced secretion of TNFα, nitric oxide and reactive oxygen species.ConclusionTogether, these data further highlight the importance of TMAO in the gut‐brain axis, and emphasise gut microbial metabolism as a conduit for dietary effects upon the brain. Given that TMAO is ultimately derived from dietary choline, these changes may help explain the well‐known beneficial effects of consumption of a seafood‐rich diet upon dementia risk.

NLRP3-directed antisense oligonucleotides reduce microglial immunoactivities in vitro.

Alzheimer's disease (AD) is associated with the cerebral deposition of Amyloid-β (Aβ) peptide, which leads to NLRP3 inflammasome activation and subsequent release of interleukin-1β (IL-1β) and interleukin-18 (IL-18). NLRP3 reduction has been found to increase microglial clearance, protect from synapse loss, and suppress both the changes to synaptic plasticity and spatial memory dysfunction observed in murine AD models. Here, we test whether NLRP3-directed antisense oligonucleotides (ASOs) can be harnessed as immune modulators in primary murine microglia and human THP-1 cells. NLRP3 mRNA degradation was achieved at 72 h of ASO treatment in primary murine microglia. Consequently, NLRP3-directed ASOs significantly reduced the levels of cleaved caspase-1 and mature IL-1β when microglia were either activated by LPS and nigericin or LPS and Aβ. In human THP-1 cells NLRP3-targeted ASOs also significantly reduced the LPS plus nigericin- or LPS plus Aβ-induced release of mature IL-1β. Together, NLRP3-directed ASOs can suppress NLRP3 inflammasome activity and subsequent release of IL-1β in primary murine microglia and THP-1 cells. ASOs may represent a new and alternative approach to modulate NLRP3 inflammasome activation in neurodegenerative diseases, in addition to attempts to inhibit the complex pharmacologically.

Identification of oxytocin expression in human and murine microglia

BackgroundOxytocin is a neuropeptide synthesized in the hypothalamus. In addition to its role in parturition and lactation, oxytocin mediates social behavior and pair bonding. The possibility of using oxytocin to modify behavior in neurodevelopmental disorders, such as autism spectrum disorder, is of clinical interest. Microglia are tissue-resident macrophages with roles in neurogenesis, synapse pruning, and immunological mediation of brain homeostasis. Recently, oxytocin was found to attenuate microglial secretion of proinflammatory cytokines, but the source of this oxytocin was not established. This prompted us to investigate whether microglia themselves were the source. MethodsWe examined oxytocin expression in human and murine brain tissue in both sexes using immunohistochemistry. Oxytocin mRNA expression and secretion were examined in isolated murine microglia from wild type and oxytocin-knockout mice. Also, secretion of oxytocin and cytokines was measured in cultured microglia (MG6) stimulated with lipopolysaccharide (LPS). ResultsWe identified oxytocin expression in microglia of human brain tissue, cultured microglia (MG6), and primary murine microglia. Furthermore, LPS stimulation increased oxytocin mRNA expression in primary murine microglia and MG6 cells, and oxytocin secretion as well. A positive correlation between oxytocin and IL-1β, IL-10 secretion emerged, respectively. ConclusionThis may be the first demonstration of oxytocin expression in microglia. Functionally, oxytocin might regulate inflammatory cytokine release from microglia in a paracrine/autocrine manner.

Cysteine Peptidase Cathepsin X as a Therapeutic Target for Simultaneous TLR3/4-mediated Microglia Activation

Microglia are resident macrophages in the central nervous system that are involved in immune responses driven by Toll-like receptors (TLRs). Microglia-mediated inflammation can lead to central nervous system disorders, and more than one TLR might be involved in these pathological processes. The cysteine peptidase cathepsin X has been recognized as a pathogenic factor for inflammation-induced neurodegeneration. Here, we hypothesized that simultaneous TLR3 and TLR4 activation induces synergized microglia responses and that these phenotype changes affect cathepsin X expression and activity. Murine microglia BV2 cells and primary murine microglia were exposed to the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)) and the TLR4 ligand lipopolysaccharide (LPS), individually and simultaneously. TLR3 and TLR4 co-activation resulted in increased inflammatory responses compared to individual TLR activation, where poly(I:C) and LPS induced distinct patterns of proinflammatory factors together with different patterns of cathepsin X expression and activity. TLR co-activation decreased intracellular cathepsin X activity and increased cathepsin X localization at the plasma membrane with concomitant increased extracellular cathepsin X protein levels and activity. Inhibition of cathepsin X in BV2 cells by AMS36, cathepsin X inhibitor, significantly reduced the poly(I:C)- and LPS-induced production of proinflammatory cytokines as well as apoptosis. Additionally, inhibiting the TLR3 and TLR4 common signaling pathway, PI3K, with LY294002 reduced the inflammatory responses of the poly(I:C)- and LPS-activated microglia and recovered cathepsin X activity. We here provide evidence that microglial cathepsin X strengthens microglia activation and leads to subsequent inflammation-induced neurodegeneration. As such, cathepsin X represents a therapeutic target for treating neurodegenerative diseases related to excess inflammation.

Predictive high-throughput screening of PEGylated lipids in oligonucleotide-loaded lipid nanoparticles for neuronal gene silencing.

Lipid nanoparticles (LNPs) are gaining traction in the field of nucleic acid delivery following the success of two mRNA vaccines against COVID-19. As one of the constituent lipids on LNP surfaces, PEGylated lipids (PEG-lipids) play an important role in defining LNP physicochemical properties and biological interactions. Previous studies indicate that LNP performance is modulated by tuning PEG-lipid parameters including PEG size and architecture, carbon tail type and length, as well as the PEG-lipid molar ratio in LNPs. Owing to these numerous degrees of freedom, a high-throughput approach is necessary to fully understand LNP behavioral trends over a broad range of PEG-lipid variables. To this end, we report a low-volume, automated, high-throughput screening (HTS) workflow for the preparation, characterization, and in vitro assessment of LNPs loaded with a therapeutic antisense oligonucleotide (ASO). A library of 54 ASO-LNP formulations with distinct PEG-lipid compositions was prepared using a liquid handling robot and assessed for their physiochemical properties as well as gene silencing efficacy in murine cortical neurons. Our results show that the molar ratio of anionic PEG-lipid in LNPs regulates particle size and PEG-lipid carbon tail length controls ASO-LNP gene silencing activity. ASO-LNPs formulated using PEG-lipids with optimal carbon tail lengths achieved up to 5-fold lower mRNA expression in neurons as compared to naked ASO. Representative ASO-LNP formulations were further characterized using dose–response curves and small-angle X-ray scattering to understand structure–activity relationships. Identified hits were also tested for efficacy in primary murine microglia and were scaled-up using a microfluidic formulation technique, demonstrating a smooth translation of ASO-LNP properties and in vitro efficacy. The reported HTS workflow can be used to screen additional multivariate parameters of LNPs with significant time and material savings, therefore guiding the selection and scale-up of optimal formulations for nucleic acid delivery to a variety of cellular targets.

A novel phosphoproteomic landscape evoked in response to type I interferon in the brain and in glial cells

BackgroundType I interferons (IFN-I) are key responders to central nervous system infection and injury and are also increased in common neurodegenerative diseases. Their effects are primarily mediated via transcriptional regulation of several hundred interferon-regulated genes. In addition, IFN-I activate several kinases including members of the MAPK and PI3K families. Yet, how changes to the global protein phosphoproteome contribute to the cellular response to IFN-I is unknown.MethodsThe cerebral phosphoproteome of mice with brain-targeted chronic production of the IFN-I, IFN-α, was obtained. Changes in phosphorylation were analyzed by ontology and pathway analysis and kinase enrichment predictions. These were verified by phenotypic analysis, immunohistochemistry and immunoblots. In addition, primary murine microglia and astrocytes, the brain's primary IFN-I-responding cells, were acutely treated with IFN-α and the global phosphoproteome was similarly analyzed.ResultsWe identified widespread protein phosphorylation as a novel mechanism by which IFN-I mediate their effects. In our mouse model for IFN-I-induced neurodegeneration, protein phosphorylation, rather than the proteome, aligned with the clinical hallmarks and pathological outcome, including impaired development, motor dysfunction and seizures. In vitro experiments revealed extensive and rapid IFN-I-induced protein phosphorylation in microglia and astrocytes. Response to acute IFN-I stimulation was independent of gene expression and mediated by a small number of kinase families. The changes in the phosphoproteome affected a diverse range of cellular processes and functional analysis suggested that this response induced an immediate reactive state and prepared cells for subsequent transcriptional responses.ConclusionsOur studies reveal a hitherto unappreciated role for changes in the protein phosphorylation landscape in cellular responses to IFN-I and thus provide insights for novel diagnostic and therapeutic strategies for neurological diseases caused by IFN-I.

Modulation of inflammatory responses by fractalkine signaling in microglia.

Reactive microglia are suggested to be involved in neurological disorders, and the mechanisms underlying microglial activity may provide insights into therapeutic strategies for neurological diseases. Microglia produce immunological responses to various stimuli, which include fractalkine (FKN or CX3CL1). CX3CR1, a FKN receptor, is present in microglial cells, and when FKN is applied before lipopolysaccharide (LPS) administration, LPS-induced inflammatory responses are inhibited, suggesting that the activation of the FKN signal is beneficial. Considering the practical administration for treatment, we investigated the influence of FKN on immunoreactive microglia using murine primary microglia and BV-2, a microglial cell line. The administration of LPS leads to nitric oxide (NO) production. NO was reduced when FKN was administered 4 h after LPS administration without a change in inducible nitric oxide synthase expression. In contrast, morphological changes, migratory activity, and proliferation were not altered by delayed FKN treatment. LPS decreases the CX3CR1 mRNA concentration, and the overexpression of CX3CR1 restores the FKN-mediated decrease in NO. CX3CR1 overexpression decreased the NO production that is mediated by LPS even without the application of FKN. ATP and ethanol also reduced CX3CR1 mRNA concentrations. In conclusion, the delayed FKN administration modified the LPS-induced microglial activation. The FKN signals were attenuated by a reduction in CX3CR1 by some inflammatory stimuli, and this modulated the inflammatory response of microglial cells, at least partially.

A novel long intergenic non-coding RNA, Nostrill, regulates iNOS gene transcription and neurotoxicity in microglia

BackgroundMicroglia are resident immunocompetent and phagocytic cells in the CNS. Pro-inflammatory microglia, stimulated by microbial signals such as bacterial lipopolysaccharide (LPS), viral RNAs, or inflammatory cytokines, are neurotoxic and associated with pathogenesis of several neurodegenerative diseases. Long non-coding RNAs (lncRNA) are emerging as important tissue-specific regulatory molecules directing cell differentiation and functional states and may help direct proinflammatory responses of microglia. Characterization of lncRNAs upregulated in proinflammatory microglia, such as NR_126553 or 2500002B13Rik, now termed Nostrill (iNOS Transcriptional Regulatory Intergenic LncRNA Locus) increases our understanding of molecular mechanisms in CNS innate immunity.MethodsMicroglial gene expression array analyses and qRT-PCR were used to identify a novel long intergenic non-coding RNA, Nostrill, upregulated in LPS-stimulated microglial cell lines, LPS-stimulated primary microglia, and LPS-injected mouse cortical tissue. Silencing and overexpression studies, RNA immunoprecipitation, chromatin immunoprecipitation, chromatin isolation by RNA purification assays, and qRT-PCR were used to study the function of this long non-coding RNA in microglia. In vitro assays were used to examine the effects of silencing the novel long non-coding RNA in LPS-stimulated microglia on neurotoxicity.ResultsWe report here characterization of intergenic lncRNA, NR_126553, or 2500002B13Rik now termed Nostrill (iNOS Transcriptional Regulatory Intergenic LncRNA Locus). Nostrill is induced by LPS stimulation in BV2 cells, primary murine microglia, and in cortical tissue of LPS-injected mice. Induction of Nostrill is NF-κB dependent and silencing of Nostrill decreased inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in BV2 and primary microglial cells. Overexpression of Nostrill increased iNOS expression and NO production. RNA immunoprecipitation assays demonstrated that Nostrill is physically associated with NF-κB subunit p65 following LPS stimulation. Silencing of Nostrill significantly reduced NF-κB p65 and RNA polymerase II recruitment to the iNOS promoter and decreased H3K4me3 activating histone modifications at iNOS gene loci. In vitro studies demonstrated that silencing of Nostrill in microglia reduced LPS-stimulated microglial neurotoxicity.ConclusionsOur data indicate a new regulatory role of the NF-κB-induced Nostrill and suggest that Nostrill acts as a co-activator of transcription of iNOS resulting in the production of nitric oxide by microglia through modulation of epigenetic chromatin remodeling. Nostrill may be a target for reducing the neurotoxicity associated with iNOS-mediated inflammatory processes in microglia during neurodegeneration.

P2RX7 inhibitor suppresses exosome secretion and disease phenotype in P301S tau transgenic mice

BackgroundNeuronal accumulation of misfolded microtubule-associated protein tau is a hallmark of neuropathology in Alzheimer’s disease, frontotemporal dementia, and other tauopathies, and has been a therapeutic target. Microglia can spread tau pathology by secreting tau-containing exosomes, although the specific molecular target is yet to be identified for the therapeutic intervention. P2X purinoceptor 7 (P2RX7) is an ATP-gated cation channel, enriched in microglia and triggers exosome secretion. The purpose of the study is to examine the therapeutic effect of an orally applicable, CNS-penetrant P2RX7 specific inhibitor on the early disease stage of a tauopathy mouse model.MethodsThree-months-old P301S tau mice were treated with P2RX7-specific inhibitor GSK1482160 or vehicle for 30 days, followed by behavioral, biochemical and immunohistochemical assessment. GSK1482160 was also tested for exosome secretion from primary cultured murine astrocytes, neurons and microglia in vitro.ResultsOral administration of GSK1482160 significantly reduced accumulation of MC1+ and Alz50+ misfolded tau in hippocampal regions, which was accompanied with reduced accumulation of Tsg101, an exosome marker, in hippocampal neurons. Proximity ligation assay demonstrated complex formation of Alz50+ tau and Tsg101 in hippocampal neurons, which was reduced by GSK1482160. On the other hand, GSK1482160 had no effect on microglial ramification or CD68 expression, which was significantly enhanced in P301S mice, or pro/anti-inflammatory cytokine gene expression. Strikingly, GSK1482160-treated P301S mice show significantly improved working and contextual memory as determined by Y-maze and fear conditioning tests. GSK1482160 also significantly increased accumulation of Tsg101 and CD81 in microglia in vivo, suggesting its suppression of P2RX7-induced exosome secretion from microglia. This effect was confirmed in vitro, as ATP-induced secretion of tau-containing exosome was significantly suppressed by GSK1482160 treatment from primary murine microglia, but not from neurons or astrocytes.DiscussionThe oral administration of P2RX7 inhibition mitigates disease phenotypes in P301S mice, likely by suppressing release of microglial exosomes. P2RX7 could be a novel therapeutic target for the early stage tauopathy development.

A Novel Interaction of Translocator Protein 18\xa0kDa (TSPO) with NADPH Oxidase in Microglia

In the brain neuropil, translocator protein 18 kDa (TSPO) is a stress response protein that is upregulated in microglia and astrocytes in diverse central nervous system pathologies. TSPO is widely used as a biomarker of neuroinflammation in preclinical and clinical neuroimaging studies. However, there is a paucity of knowledge on the function(s) of TSPO in glial cells. In this study, we explored a putative interaction between TSPO and NADPH oxidase 2 (NOX2) in microglia. We found that TSPO associates with gp91phox and p22phox, the principal subunits of NOX2 in primary murine microglia. The association of TSPO with gp91phox and p22phox was observed using co-immunoprecipitation, confocal immunofluorescence imaging, and proximity ligation assay. We found that besides gp91phox and p22phox, voltage-dependent anion channel (VDAC) also co-immunoprecipitated with TSPO consistent with previous reports. When we compared lipopolysaccharide (LPS) stimulated microglia to vehicle control, we found that a lower amount of gp91phox and p22phox protein co-immunoprecipitated with TSPO suggesting a disruption of the TSPO-NOX2 subunits association. TSPO immuno-gold electron microscopy confirmed that TSPO is present in the outer mitochondrial membrane but it is also found in the endoplasmic reticulum (ER), mitochondria-associated ER membrane (MAM), and in the plasma membrane. TSPO localization at the MAM may represent a subcellular site where TSPO interacts with gp91phox and p22phox since the MAM is a point of communication between outer mitochondria membrane proteins (TSPO) and ER proteins (gp91phox and p22phox) where they mature and form the cytochrome b558 (Cytb558) heterodimer. We also found that an acute burst of reactive oxygen species (ROS) increased TSPO levels on the surface of microglia and this effect was abrogated by a ROS scavenger. These results suggest that ROS production may alter the subcellular distribution of TSPO. Collectively, our findings suggest that in microglia, TSPO is associated with the major NOX2 subunits gp91phox and p22phox. We hypothesize that this interaction may regulate Cytb558 formation and modulate NOX2 levels, ROS production, and redox homeostasis in microglia.

Modulation of microglial activity by salt load and SGK1

Microglial cells are derived from myelogenous cells and their chronic activation elicits brain inflammation, which influences neurological activity. Comprehensive understanding of the regulation of microglial activation could therefore contribute to overcoming neuropsychiatric disorders. Recently, the importance of serum- and glucocorticoid-inducible kinases (SGKs) has been explored in immune cells such as T cells, dendritic cells and macrophages. We have already shown that SGK1 and SGK3 are expressed in microglial cells and associated with the regulation of lipopolysaccharide (LPS)-induced inflammatory molecules. Here we investigate whether salt load influences expression of SGK1 and inflammatory responses in murine primary microglia and an immortalized microglial cell line, BV-2. Additional amounts of NaCl were administrated and immunoblotting was carried out, and SGK1 was induced in dose- and time-dependent manners. Next, the dynamics of inflammatory mediators iNOS and TNFα were investigated by administration of LPS. iNOS mRNA was induced by LPS application and enhanced by NaCl preload. In support of these results, nitric oxide was produced by LPS and accelerated by NaCl preload. In contrast, however, NaCl preload reduced the release of TNFα, suggesting the modulation of immune responses by salt load. The effects of salt load on both cases were attenuated in SGK1-deleted cells. Taken together, these results indicate that salt load modulates inflammatory responses and that SGK1 assists salt load-induced inflammatory responses.

Phenotypic Differences in Primary Murine Microglia Treated with NOD1, NOD2, and NOD1/2 Agonists.

The purpose of the study was studying the influence of different NOD agonists on the morphological phenotype of primary murine microglia and to examine their influence on characteristic cytokines. Primary CD11b-positive cells were isolated from the brain of neonatal mice. The microglial phenotype of the cells was examined by ionized calcium-binding adapter molecule (Iba)1 staining. After14 days in culture, these cells were stimulated by iE-DAP, L18-MDP, or M-TriDAP as NOD1, NOD2, and NOD1/2 agonists, respectively. The cellular morphology was recorded and compared to the phenotype of cells cultured in medium alone or after LPS stimulation. The cells developed a specific phenotype only after treatment with the NOD2 agonist L18-MDP. These cells were characterized by straight extensions carrying tiny spikes and had a high ramification index. This was in sharp contrast to all other treatments, which always resulted in an amoeboid phenotype typically shown by activated microglia in vivo and by cultured microglia in vitro. The staining intensity of IL-6 and TNF-α did not reveal any clear difference independent of the NOD agonist treatment. In contrast, an increased staining intensity was observed for IL-10 after L18-MDP treatment. The NOD2 agonist L18-MDP induced a morphologically distinct phenotype characterized by microspike-decorated dendritiform extensions and a high degree of ramification in primary murine microglia. Increased ramification index and elevated staining intensity of anti-inflammatory IL-10 as hallmarks suggest that a M2-like phenotype of microglia was induced.

Molecular Insight Into the IRE1α-Mediated Type I Interferon Response Induced by Proteasome Impairment in Myeloid Cells of the Brain.

Proteostasis is critical for cells to maintain the balance between protein synthesis, quality control, and degradation. This is particularly important for myeloid cells of the central nervous system as their immunological function relies on proper intracellular protein turnover by the ubiquitin-proteasome system. Accordingly, disruption of proteasome activity due to, e.g., loss-of-function mutations within genes encoding proteasome subunits, results in systemic autoinflammation. On the molecular level, pharmacological inhibition of proteasome results in endoplasmic reticulum (ER) stress-activated unfolded protein response (UPR) as well as an induction of type I interferons (IFN). Nevertheless, our understanding as to whether and to which extent UPR signaling regulates type I IFN response is limited. To address this issue, we have tested the effects of proteasome dysfunction upon treatment with proteasome inhibitors in primary murine microglia and microglia-like cell line BV-2. Our data show that proteasome impairment by bortezomib is a stimulus that activates all three intracellular ER-stress transducers activation transcription factor 6, protein kinase R-like endoplasmic reticulum kinase and inositol-requiring protein 1 alpha (IRE1α), causing a full activation of the UPR. We further demonstrate that impaired proteasome activity in microglia cells triggers an induction of IFNβ1 in an IRE1-dependent manner. An inhibition of the IRE1 endoribonuclease activity significantly attenuates TANK-binding kinase 1-mediated activation of type I IFN. Moreover, interfering with TANK-binding kinase 1 activity also compromised the expression of C/EBP homologous protein 10, thereby emphasizing a multilayered interplay between UPR and type IFN response pathway. Interestingly, the induced protein kinase R-like endoplasmic reticulum kinase-activation transcription factor 4-C/EBP homologous protein 10 and IRE1-X-box-binding protein 1 axes caused a significant upregulation of proinflammatory cytokine interleukin 6 expression that exacerbates STAT1/STAT3 signaling in cells with dysfunctional proteasomes. Altogether, these findings indicate that proteasome impairment disrupts ER homeostasis and triggers a complex interchange between ER-stress sensors and type I IFN signaling, thus inducing in myeloid cells a state of chronic inflammation.

Dual PPAR\u03b1/\u03b3 agonist aleglitazar confers stroke protection in a model of mild focal brain ischemia in mice

Peroxisome proliferator-activated receptors (PPARs) control the expression of genes involved in glucose homeostasis, lipid metabolism, inflammation, and cell differentiation. Here, we analyzed the effects of aleglitazar, a dual PPARα and PPARγ agonist with balanced affinity for either subtype, on subacute stroke outcome. Healthy young adult mice were subjected to transient 30 min middle cerebral artery occlusion (MCAo)/reperfusion. Daily treatment with aleglitazar was begun on the day of MCAo and continued until sacrifice. Blood glucose measurements and lipid profile did not differ between mice receiving aleglitazar and mice receiving vehicle after MCAo. Aleglitazar reduced the size of the ischemic lesion as assessed using NeuN immunohistochemistry on day 7. Sensorimotor performance on the rotarod was impaired during the first week after MCAo, an effect that was significantly attenuated by treatment with aleglitazar. Smaller lesion volume in mice treated with aleglitazar was accompanied by a decrease in mRNA transcription of IL-1β, Vcam-1, and Icam-1, suggesting that reduced proinflammatory signaling and reduced vascular inflammation in the ischemic hemisphere contribute to the beneficial effects of aleglitazar during the first week after stroke. Further experiments in primary murine microglia confirmed that aleglitazar reduces key aspects of microglia activation including NO production, release of proinflammatory cytokines, migration, and phagocytosis. In aggregate, a brief course of PPARα/γ agonist aleglitazar initiated post-event affords stroke protection and functional recovery in a model of mild brain ischemia. Our data underscores the theme of delayed injury processes such as neuroinflammation as promising therapeutic targets in stroke.Key messagesPPARα/γ agonist aleglitazar improves stroke outcome after transient brain ischemia.Aleglitazar attenuates inflammatory responses in post-ischemic brain.Aleglitazar reduces microglia migration, phagocytosis, and release of cytokines.Beneficial effects of aleglitazar independent of glucose regulation.Aleglitazar provides extended window of opportunity for stroke treatment.

Diverse Protein Profiles in CNS Myeloid Cells and CNS Tissue From Lipopolysaccharide- and Vehicle-Injected APPSWE/PS1ΔE9 Transgenic Mice Implicate Cathepsin Z in Alzheimer's Disease.

Neuroinflammation, characterized by chronic activation of the myeloid-derived microglia, is a hallmark of Alzheimer’s disease (AD). Systemic inflammation, typically resulting from infection, has been linked to the progression of AD due to exacerbation of the chronic microglial reaction. However, the mechanism and the consequences of this exacerbation are largely unknown. Here, we mimicked systemic inflammation in AD with weekly intraperitoneal (i.p.) injections of APPSWE/PS1ΔE9 transgenic mice with E. coli lipopolysaccharide (LPS) from 9 to 12 months of age, corresponding to the period with the steepest increase in amyloid pathology. We found that the repeated LPS injections ameliorated amyloid pathology in the neocortex while increasing the neuroinflammatory reaction. To elucidate mechanisms, we analyzed the proteome of the hippocampus from the same mice as well as in unique samples of CNS myeloid cells. The repeated LPS injections stimulated protein pathways of the complement system, retinoid receptor activation and oxidative stress. CNS myeloid cells from transgenic mice showed enrichment in pathways of amyloid-beta clearance and elevated levels of the lysosomal protease cathepsin Z, as well as amyloid precursor protein, apolipoprotein E and clusterin. These proteins were found elevated in the proteome of both LPS and vehicle injected transgenics, and co-localized to CD11b+ microglia in transgenic mice and in primary murine microglia. Additionally, cathepsin Z, amyloid precursor protein, and apolipoprotein E appeared associated with amyloid plaques in neocortex of AD cases. Interestingly, cathepsin Z was expressed in microglial-like cells and co-localized to CD68+ microglial lysosomes in AD cases, and it was expressed in perivascular cells in AD and control cases. Taken together, our results implicate systemic LPS administration in ameliorating amyloid pathology in early-to-mid stage disease in the APPSWE/PS1ΔE9 mouse and attract attention to the potential disease involvement of cathepsin Z expressed in CNS myeloid cells in AD.

Alleviation of Microglial Activation Induced by p38 MAPK/MK2/PGE2 Axis by Capsaicin: Potential Involvement of other than TRPV1 Mechanism/s

Exaggerated inflammatory responses in microglia represent one of the major risk factors for various central nervous system’s (CNS) associated pathologies. Release of excessive inflammatory mediators such as prostaglandins and cytokines are the hallmark of hyper-activated microglia. Here we have investigated the hitherto unknown effects of capsaicin (cap) - a transient receptor potential vanilloid 1 (TRPV1) agonist- in murine primary microglia, organotypic hippocampal slice cultures (OHSCs) and human primary monocytes. Results demonstrate that cap (0.1–25 µM) significantly (p < 0.05) inhibited the release of prostaglandin E2 (PGE2), 8-iso-PGF2α, and differentially regulated the levels of cytokines (TNF-α, IL-6 & IL-1β). Pharmacological blockade (via capsazepine & SB366791) and genetic deficiency of TRPV1 (TRPV1−/−) did not prevent cap-mediated suppression of PGE2 in activated microglia and OHSCs. Inhibition of PGE2 was partially dependent on the reduced levels of PGE2 synthesising enzymes, COX-2 and mPGES-1. To evaluate potential molecular targets, we discovered that cap significantly suppressed the activation of p38 MAPK and MAPKAPK2 (MK2). Altogether, we demonstrate that cap alleviates excessive inflammatory events by targeting the PGE2 pathway in in vitro and ex vivo immune cell models. These findings have broad relevance in understanding and paving new avenues for ongoing TRPV1 based drug therapies in neuroinflammatory-associated diseases.