Adult Microglia Research Articles

Serpina3n in neonatal microglia mediates its protective role for damaged adult microglia by alleviating extracellular matrix remodeling-induced tunneling nanotubes degradation in a cell model of traumatic brain injury

Traumatic brain injury (TBI) induces significant neuroinflammation, primarily driven by microglia. Neonatal microglia (NMG) may have therapeutic potential by modulating the inflammatory response of damaged adult microglia (AMG). This study investigates the influence of NMG on AMG function through extracellular matrix (ECM) remodeling and the formation of tunneling nanotubes (TnTs), with a focus on the role of Serpina3n. We established an in vitro TBI model using a 3D Transwell system, co-culturing damaged AMG with NMG. Viral vector transfection was employed to manipulate Serpina3n expression in NMG. Quantitative real-time PCR, Western blotting, and ELISA were utilized to assess inflammatory markers, ECM remodeling proteins, and TnTs-related proteins. Co-culturing with NMG significantly inhibited M1 polarization of AMG and reduced the release of pro-inflammatory cytokines while promoting M2 polarization and increasing the production of anti-inflammatory cytokines. NMG expressed higher levels of Serpina3n, which played a crucial role in reducing Granzyme B, matrix metalloproteinase (MMP) 2 and MMP9 expression, thereby mitigating ECM remodeling. Inhibition of Serpina3n in NMG increased pro-inflammatory markers and decreased TnTs formation proteins, whereas overexpression of M−sec in AMG counteracted these effects. This highlights the importance of TnTs in maintaining microglial function and promoting an anti-inflammatory environment. In conclusion, NMG improve the function of damaged AMG by modulating ECM remodeling and promoting TnTs formation through the action of Serpina3n.

Serotonin-2B receptor (5-HT2BR) expression and binding in the brain of APPswe/PS1dE9 transgenic mice and in Alzheimer’s disease brain tissue

Despite well-documented dysregulation in central serotonergic signaling in Alzheimer’s disease (AD), knowledge about the potential involvement of the serotonin-2B receptor (5-HT2BR) subtype remains sparse. Here, we assessed the levels of 5-HT2BRs in brain tissue from APPswe/PS1dE9 transgenic (TG) mice, AD patients, and adult microglial cells. 5-HT2BR mRNA was measured by RT-qPCR in ageing TG and wild-type (WT) mice, in samples from the middle frontal gyrus of female, AD and control subjects, and in microglia from the cerebral cortex of WT mice. The density of 5-HT2BRs was measured by autoradiography using [3H]RS 127445. Both mouse and human brains had low levels of 5-HT2BR mRNA. In whole-brain mouse samples, mRNA expression was significantly lower in TG mice compared to WT at > 18 months of age. In the Aβ-plaque-burdened neocortex and hippocampus of old TG mice, however, levels of 5-HT2BR mRNA were two-fold higher over control, with similar elevations observed in the Aβ-plaque-burdened frontal cortex of human AD patients. 5-HT2BR mRNA expression varied widely in adult microglia and was higher compared to other cortical cell subtypes. In mice, specific [3H]RS-127445 binding in the cortex was first detected after 3 months of age. The density of 5-HT2BRs was low and overall reduced in TG, compared to WT mice. Binding was detectable but too low to be reliably quantified in the human cortex. Our results document Aβ-associated increases in 5-HT2BR mRNA expression and suggest reduced receptor binding in the context of AD. Studies investigating the functional involvement of microglial 5-HT2BRs in AD are considered relevant.

Acute postnatal inflammation alters adult microglial responses to LPS that are sex-, region- and timing of postnatal inflammation-dependent

BackgroundAdverse events in early life can have impact lasting into adulthood. We investigated the long-term effects of systemic inflammation during postnatal development on adult microglial responses to lipopolysaccharide (LPS) in two CNS regions (cortex, cervical spinal cord) in male and female rats.MethodsInflammation was induced in Sprague-Dawley rats by LPS (1 mg/kg) administered intraperitoneally during postnatal development at P7, P12 or P18. As adults (12 weeks of age), the rats received a second LPS dose (1 mg/kg). Control rats received saline. Microglia were isolated 3 h post-LPS followed by gene expression analysis via qRT-PCR for pro-inflammatory (IL-6, iNOS, Ptgs2, C/EBPb, CD14, CXCL10), anti-inflammatory (CD68, Arg-1), and homeostatic genes (P2Y12, Tmemm119). CSF-1 and CX3CL1 mRNAs were analyzed in microglia-free homogenates.ResultsBasal gene expression in adult microglia was largely unaffected by postnatal inflammation. Adult cortical microglial pro-inflammatory gene responses to LPS were either unchanged or attenuated in rats exposed to LPS during postnatal development. Ptgs2, C/EBPb, CXCL10 and Arg-1 were the most affected genes, with expression significantly downregulated vs. rats without postnatal LPS. Spinal microglia were affected most by LPS at P18, with mixed and sometimes opposing effects on proinflammatory genes in males vs. females. Overall, male cortical vs. spinal microglia were more affected by postnatal LPS. Females were affected in both cortex and spinal cord, but the effect was dependent on timing of postnatal LPS. Overall, inflammatory challenge at P18 had greater effect on adult microglia vs. challenge at P12 or P7.ConclusionsLong-lasting effects of postnatal inflammation on adult microglia depend on postnatal timing, CNS region and sex.

A Custom Panel for Profiling Microglia Gene Expression.

Despite being immune cells of the central nervous system (CNS), microglia contribute to CNS development, maturation, and homeostasis, and microglia dysfunction has been implicated in several neurological disorders. Recent advancements in single-cell studies have uncovered unique microglia-specific gene expression. However, there is a need for a simple yet elegant multiplexed approach to quantifying microglia gene expression. To address this, we have designed a NanoString nCounter technology-based murine microglia-specific custom codeset comprising 178 genes. We analyzed RNA extracted from ex vivo adult mouse microglia, primary mouse microglia, the BV2 microglia cell line, and mouse bone marrow monocytes using our custom panel. Our findings reveal a pattern where homeostatic genes exhibit heightened expression in adult microglia, followed by primary cells, and are absent in BV2 cells, while reactive markers are elevated in primary microglia and BV2 cells. Analysis of publicly available data sets for the genes present in the panel revealed that the panel could reliably reflect the changes in microglia gene expression in response to various factors. These findings highlight that the microglia panel used offers a swift and cost-effective means to assess microglial cells and can be used to study them in varying contexts, ranging from normal homeostasis to disease models.

Frequent low-impact exposure to THC during adolescence causes persistent sexually dimorphic alterations in the response to viral infection in mice

Adolescent exposure to Δ9-tetrahydrocannabinol (THC) has enduring effects on energy metabolism and immune function. Prior work showed that daily administration of a low-impact dose of THC (5 mg/kg, intraperitoneal) during adolescence alters transcription in adult microglia and disrupts their response to bacterial endotoxin or social stress. To explore the lasting impact of adolescent THC exposure on the brain’s reaction to viral infection, we administered THC (5 mg/kg, intraperitoneal) in male and female mice once daily on postnatal day (PND) 30–43. When the mice reached adulthood (PND 70), we challenged them with the viral mimic, polyinosinic acid:polycytidylic acid [Poly(I:C)], and assessed sickness behavior (motor activity, body temperature) and whole brain gene transcription. Poly(I:C) caused an elevation in body temperature which was lessened by prior THC exposure in female but not male mice. Adolescent THC exposure did not affect the locomotor response to Poly(I:C) in either sex. Transcriptomic analyses showed that Poly(I:C) produced a substantial upregulation of immune-related genes in the brain, which was decreased by THC in females. Additionally, the viral mimic caused a male-selective downregulation in transcription of genes involved in neurodevelopment and synaptic transmission, which was abrogated by adolescent THC treatment. The results indicate that Poly(I:C) produces complex transcriptional alterations in the mouse brain, which are sexually dimorphic and differentially affected by early-life THC exposure. In particular, adolescent THC dampens the brain’s antiviral response to Poly(I:C) in female mice and prevents the transcriptional downregulation of neuron-related genes caused by the viral mimic in male mice.

Ikzf1 as a novel regulator of microglial homeostasis in inflammation and neurodegeneration

In the last two decades, microglia have emerged as key contributors to disease progression in many neurological disorders, not only by exerting their classical immunological functions but also as extremely dynamic cells with the ability to modulate synaptic and neural activity. This dynamic behavior, together with their heterogeneous roles and response to diverse perturbations in the brain parenchyma has raised the idea that microglia activation is more diverse than anticipated and that understanding the molecular mechanisms underlying microglial states is essential to unravel their role in health and disease from development to aging. The Ikzf1 (a.k.a. Ikaros) gene plays crucial roles in modulating the function and maturation of circulating monocytes and lymphocytes, but whether it regulates microglial functions and states is unknown. Using genetic tools, here we describe that Ikzf1 is specifically expressed in the adult microglia in brain regions such as cortex and hippocampus. By characterizing the Ikzf1 deficient mice, we observed that these mice displayed spatial learning deficits, impaired hippocampal CA3-CA1 long-term potentiation, and decreased spine density in pyramidal neurons of the CA1, which correlates with an increased expression of synaptic markers within microglia. Additionally, these Ikzf1 deficient microglia exhibited a severe abnormal morphology in the hippocampus, which is accompanied by astrogliosis, an aberrant composition of the inflammasome, and an altered expression of disease-associated microglia molecules. Interestingly, the lack of Ikzf1 induced changes on histone 3 acetylation and methylation levels in the hippocampus. Since the lack of Ikzf1 in mice appears to induce the internalization of synaptic markers within microglia, and severe gliosis we then analyzed hippocampal Ikzf1 levels in several models of neurological disorders. Ikzf1 levels were increased in the hippocampus of these neurological models, as well as in postmortem hippocampal samples from Alzheimer’s disease patients. Finally, over-expressing Ikzf1 in cultured microglia made these cells hyporeactive upon treatment with lipopolysaccharide, and less phagocytic compared to control microglia. Altogether, these results suggest that altered Ikzf1 levels in the adult hippocampus are sufficient to induce synaptic plasticity and memory deficits via altering microglial state and function.

Homotypic Neonatal and Adult Inflammation Primes Adult Microglia Without Impairing Adult Breathing

A population of adults are predisposed to breathing deficits during subsequent illness and disease, yet it is unknown what factor(s) contribute to this adult vulnerability. Since neonatal inflammation primes adult microglia in non‐respiratory control regions and augments peripheral immune responses, we hypothesized that neonatal and adult homotypic inflammation (same inflammatory stimulus at both ages) primes male and female microglia in respiratory control regions, contributing to impairments in adult breathing during subsequent inflammatory challenges. Using lipopolysaccharide (LPS) to induce homotypic inflammation in neonates (1mg/kg LPS, P4) and adults (24µg/kg LPS, i.p.), medullary microglia were primed in adult males (neonatal LPS + adult LPS: 25±4% microglia, n=9; neonatal LPS + adult saline: 18±2% microglia, n=9; neonatal saline + adult saline: 10±1% microglia, n=9; p<0.01) and adult females (neonatal LPS + adult LPS: 25±3% microglia, n=9; neonatal LPS + adult saline: 14±2% microglia, n=6; neonatal saline + adult saline: 10±2% microglia, n=8; p<0.0001). However, spinal cord microglia were only primed in adult males (neonatal LPS + adult LPS: 13±2% microglia, n=9; neonatal LPS + adult saline: 9±2% microglia, n=9; neonatal saline + adult saline: 10±2% microglia, n=9; p<0.01) and not adult females (neonatal LPS + adult LPS: 13±2% microglia, n=9; neonatal LPS + adult saline: 9±1% microglia, n=6; neonatal saline + adult saline: 9±1% microglia, n=8; p<0.01). Thus, neonatal and adult homotypic inflammation region‐ and sex‐dependently prime microglia in respiratory control regions. To test whether primed microglia in respiratory control regions contribute to deficits in adult breathing, breathing was assessed by plethysmography in adults at two time points: during the peak inflammatory response (3hrs) and peak microglial migration and proliferation (24hrs). Neither adult eupneic breathing nor chemoreflexes (hypercapnic ventilatory response, HCVR, and hypoxic ventilatory response, HVR) were primed in males or females by neonatal and adult homotypic inflammation at either time point. Interestingly, adult female chemoreflexes were time‐dependently increased by neonatal and adult homotypic inflammation. In adult females, neonatal and adult homotypic inflammation increased the peak HCVR at 3hrs (neonatal LPS + adult LPS: 298±112% ∆VE from baseline, n=6) compared to 24hrs (neonatal LPS + adult LPS: 169±41% ∆VE from baseline, n=6, p=0.04), and increased the peak HVR at 3hrs (neonatal LPS + adult LPS: 159±37% ∆VE from baseline) compared to 24hrs (neonatal LPS + adult LPS: 62±23% ∆VE from baseline, n=6, p=0.02). Thus, adult female chemoreflexes were time‐dependently impacted by the combination of neonatal and adult homotypic inflammation, suggesting time‐specific effects of adult inflammation. Despite no significant impact of primed microglia on baseline breathing and chemoreflexes, primed respiratory control microglia have the potential to significantly impair respiratory control during more severe inflammatory challenges associated with illness and disease. Further, sex differences in primed respiratory control microglia suggest sex‐specific vulnerability to adult ventilatory control disorders.

Distinct phases of adult microglia proliferation: a Myc-mediated early phase and a Tnfaip3-mediated late phase

Microgliosis is a hallmark of many neurological diseases, including Alzheimer’s disease, stroke, seizure, traumatic brain and spinal cord injuries, and peripheral and optic nerve injuries. Recent studies have shown that the newly self-renewed microglia have specific neurological functions. However, the mechanism of adult microglia proliferation remains largely unclear. Here, with single-cell RNA sequencing, flow cytometry, and immunohistochemistry, we demonstrate that the sciatic nerve injury induced two distinct phases of microglia proliferation in mouse spinal cord, each with different gene expression profiles. We demonstrate that the transcription factor Myc was transiently upregulated in spinal cord microglia after nerve injury to mediate an early phase microglia proliferation. On the other hand, we reveal that the tumor-necrosis factor alpha-induced protein 3 (Tnfaip3) was downregulated to mediate the Myc-independent late-phase microglia proliferation. We show that cyclin dependent kinase 1, a kinase with important function in the M phase of the cell cycle, was involved only in the early phase. We reveal that although the early phase was neither necessary nor sufficient for the late phase proliferation, the late-phase suppressed the early phase microglia proliferation in the spinal cord. Finally, we demonstrate that the termination of spinal cord microglia proliferation required both Myc and Tnfaip3 to resume their baseline expression. Thus, we have delineated an interactive signaling network in the proliferation of differentiated microglia.

Cx3Cr1-Cre induction leads to microglial activation and IFN-1 signaling caused by DNA damage in early postnatal brain.

Cx3cr1CreER-driven Cre recombinase (Cre) is a widely used genetic tool for enabling gene manipulation in microglia and macrophages. However, an in-depth analysis of the possible detrimental effects of Cre activity in microglia, surprisingly, remains missing. Here, we demonstrate an age-dependent sensitivity of microglia to Cx3cr1-Cre toxicity, wherein Cre induction, specifically in early postnatal microglia, is detrimental to microglial development, proliferation, and function. Tamoxifen (TAM)-induced Cre activity leads to microglial activation, type 1 interferon (IFN-1) signaling, and increased phagocytosis, causing aberrant synaptic pruning during the early postnatal period and anxious behavior at later age. The detrimental effects of Cre induction are caused by DNA-damage-induced toxicity in microglia and are limited to the early postnatal period, showing no detrimental effects in adult microglia. Thus, our study reveals an age-dependent vulnerability of microglia to Cre activity, thereby highlighting age dependency of Cre action, which could be especially applicable in the broader context of environment-responsive cell types.

A new method for obtaining bankable and expandable adult-like microglia in mice

BackgroundThe emerging role of microglia in neurological disorders requires a novel method for obtaining massive amounts of adult microglia. We aim to develop a new method for obtaining bankable and expandable adult-like microglia in mice.MethodsThe head neuroepithelial layer (NEL) that composed of microglial progenitor and neuroepithelial cells at mouse E13.5 was dissected and then cultured or banked. Microglia (MG) isolated from the cultured NEL by magnetic-activated cell sorting system were obtained and named NEL-MG.ResultsThe NEL included microglia progenitors that proliferate and ramify over time with neuroepithelial cells as feeder. In functional analysis, NEL-MG exhibited microglial functions, such as phagocytosis (microbeads, amyloid β, synaptosome), migration, and inflammatory response following lipopolysaccharide (LPS) stimulation. NEL was passage cultured and the NEL-MG exhibited a higher expression of microglia signature genes than the neonatal microglia, a widely used in vitro surrogate. Banking or long-term passage culture of NEL did not affect NEL-MG characteristics. Transcriptome analysis revealed that NEL-MG exhibited better conservation of microglia signature genes with a closer fidelity to freshly isolated adult microglia than neonatal microglia. NEL-MG could be re-expandable when they were plated again on neuroepithelial cells.ConclusionsThis new method effectively contributes to obtaining sufficient matured form of microglia (adult-like microglia), even when only a small number of experimental animals are available, leading to a broad application in the field of neuroscience.

Innate immune responses after stimulation with Toll-like receptor agonists in ex vivo microglial cultures and an in vivo model using mice with reduced microglia

BackgroundPast experiments studying innate immunity in the central nervous system (CNS) utilized microglia obtained from neonatal mouse brain, which differ developmentally from adult microglia. These differences might impact our current understanding of the role of microglia in CNS development, function, and disease.MethodsCytokine protein secretion was compared in ex vivo P3 and adult microglial cultures after exposure to agonists for three different toll-like receptors (TLR4, lipopolysaccharide [LPS]; TLR7, imiquimod [IMQ]; and TLR9, CpG Oligodeoxynucleotide [CpG-ODN] 1585). In addition, changes in inflammatory gene expression in ex vivo adult microglia in response to the TLR agonists was assessed. Furthermore, in vivo experiments evaluated changes in gene expression associated with inflammation and TLR signaling in brains of mice with or without treatment with PLX5622 to reduce microglia.ResultsEx vivo adult and P3 microglia increased cytokine secretion when exposed to TLR4 agonist LPS and to TLR7 agonist IMQ. However, adult microglia decreased expression of numerous genes after exposure to TLR 9 agonist CpG-ODN 1585. In contrast, in vivo studies indicated a core group of inflammatory and TLR signaling genes increased when each of the TLR agonists was introduced into the CNS. Reducing microglia in the brain led to decreased expression of various inflammatory and TLR signaling genes. Mice with reduced microglia showed extreme impairment in upregulation of genes after exposure to TLR7 agonist IMQ.ConclusionsCultured adult microglia were more reactive than P3 microglia to LPS or IMQ exposure. In vivo results indicated microglial influences on neuroinflammation were agonist specific, with responses to TLR7 agonist IMQ more dysregulated in mice with reduced microglia. Thus, TLR7-mediated innate immune responses in the CNS appeared more dependent on the presence of microglia. Furthermore, partial responses to TLR4 and TLR9 agonists in mice with reduced microglia suggested other cell types in the CNS can compensate for their absence.

Acute Neonatal Inflammation Differentially Activates Adult Microglia In Respiratory Neural Control Regions

Adult respiratory motor plasticity is abolished by neonatal inflammation, but is restored with an adult anti-inflammatory treatment, suggesting lasting inflammatory signaling underlies deficiencies in adult respiratory motor plasticity. While neonatal inflammation impairs adult hippocampal plasticity by increasing adult microglial number (resident immune cells of the brain) and inflammatory gene expression, it is unclear if adult microglia in respiratory control regions are similarly affected by neonatal inflammation. Thus, we hypothesized that acute neonatal inflammation would increase the number of and inflammatory transcriptomic profiles in adult microglia in respiratory control regions (medulla and cervical spinal cord). Using flow cytometry, a single neonatal inflammatory challenge (1 mg/kg LPS, i.p., postnatal day 4) significantly increased adult medullary microglial frequency in males (neonatal LPS: 25 ± 3% microglia, n=11; neonatal saline: 12 ± 2% microglia, n=11, p<0.0001), although the effect was not statistically significant in females (neonatal LPS: 21 ± 4% microglia, n=10; neonatal saline: 12 ± 3% microglia, n=11, p=0.0626). Interestingly, these differences were not observed in spinal microglia in either sex (male neonatal LPS: 13 ± 5% microglia, n=12; male neonatal saline: 11 ± 3% microglia, n=10, p=0.9; female neonatal LPS: 16 ± 6% microglia, n=12; female neonatal saline: 13 ± 5% microglia, n=12, p=0.9), suggesting a neonatal LPS challenge region-specifically increases adult microglia. Similar regional differences were also observed in preliminary transcriptomic data from adult females challenged acutely as neonates; more microglial genes were differentially increased in isolated medullary microglia (271 genes, n=3) than in spinal microglia (23 genes, n=6). In addition, preliminary data suggest potential sex differences in adult microglial inflammatory gene responses in respiratory control regions following an acute neonatal LPS challenge. More inflammatory genes were increased in adult female microglia (18 genes, n=9) than in male microglia (7 genes, n=7). In conclusion, neonatal inflammation region-dependently increased medullary microglial frequency in adult males, while medullary microglia have increased inflammatory transcriptomic profiles in females. These microglial changes may contribute to the mechanisms underlying abolition of adult respiratory motor plasticity following an early life neonatal inflammatory challenge.

Dopamine induces functional extracellular traps in microglia

SummaryDopamine (DA) plays many roles in the brain, especially in movement, motivation, and reinforcement of behavior; however, its role in regulating innate immunity is not clear. Here, we show that DA can induce DNA-based extracellular traps in primary, adult, human microglia and BV2 microglia cell line. These DNA-based extracellular traps are formed independent of reactive oxygen species, actin polymerization, and cell death. These traps are functional and capture fluorescein (FITC)-tagged Escherichia coli even when reactive oxygen species production or actin polymerization is inhibited. We show that microglial extracellular traps are present in Glioblastoma multiforme. This is crucial because Glioblastoma multiforme cells are known to secrete DA. Our findings demonstrate that DA plays a significant role in sterile neuro-inflammation by inducing microglia extracellular traps.

Organotypic Brain Slice Culture Microglia Exhibit Molecular Similarity to Acutely-Isolated Adult Microglia and Provide a Platform to Study Neuroinflammation.

Microglia are central nervous system (CNS) resident immune cells that have been implicated in neuroinflammatory pathogenesis of a variety of neurological conditions. Their manifold context-dependent contributions to neuroinflammation are only beginning to be elucidated, which can be attributed in part to the challenges of studying microglia in vivo and the lack of tractable in vitro systems to study microglia function. Organotypic brain slice cultures offer a tissue-relevant context that enables the study of CNS resident cells and the analysis of brain slice microglial phenotypes has provided important insights, in particular into neuroprotective functions. Here we use RNA sequencing, direct digital quantification of gene expression with nCounter® technology and targeted analysis of individual microglial signature genes, to characterize brain slice microglia relative to acutely-isolated counterparts and 2-dimensional (2D) primary microglia cultures, a widely used in vitro surrogate. Analysis using single cell and population-based methods found brain slice microglia exhibited better preservation of canonical microglia markers and overall gene expression with stronger fidelity to acutely-isolated adult microglia, relative to in vitro cells. We characterized the dynamic phenotypic changes of brain slice microglia over time, after plating in culture. Mechanical damage associated with slice preparation prompted an initial period of inflammation, which resolved over time. Based on flow cytometry and gene expression profiling we identified the 2-week timepoint as optimal for investigation of microglia responses to exogenously-applied stimuli as exemplified by treatment-induced neuroinflammatory changes observed in microglia following LPS, TNF and GM-CSF addition to the culture medium. Altogether these findings indicate that brain slice cultures provide an experimental system superior to in vitro culture of microglia as a surrogate to investigate microglia functions, and the impact of soluble factors and cellular context on their physiology.

A MAC2-positive progenitor-like microglial population is resistant to CSF1R inhibition in adult mouse brain.

Microglia are the resident myeloid cells in the central nervous system (CNS). The majority of microglia rely on CSF1R signaling for survival. However, a small subset of microglia in mouse brains can survive without CSF1R signaling and reestablish the microglial homeostatic population after CSF1R signaling returns. Using single-cell transcriptomic analysis, we characterized the heterogeneous microglial populations under CSF1R inhibition, including microglia with reduced homeostatic markers and elevated markers of inflammatory chemokines and proliferation. Importantly, MAC2/Lgals3 was upregulated under CSF1R inhibition, and shared striking similarities with microglial progenitors in the yolk sac and immature microglia in early embryos. Lineage-tracing studies revealed that these MAC2+ cells were of microglial origin. MAC2+ microglia were also present in non-treated adult mouse brains and exhibited immature transcriptomic signatures indistinguishable from those that survived CSF1R inhibition, supporting the notion that MAC2+ progenitor-like cells are present among adult microglia.

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent Aβ fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Microglia Dysfunction Caused by the Loss of Rhoa Disrupts Neuronal Physiology and Leads to Neurodegeneration

Nervous tissue homeostasis requires the regulation of microglia activity. Using conditional gene targeting in mice, we demonstrate that genetic ablation of the small GTPase Rhoa in adult microglia is sufficient to trigger spontaneous microglia activation, producing a neurological phenotype (including synapse and neuron loss, impairment of long-term potentiation [LTP], formation of β-amyloid plaques, and memory deficits). Mechanistically, loss of Rhoa in microglia triggers Src activation and Src-mediated tumor necrosis factor (TNF) production, leading to excitotoxic glutamate secretion. Inhibiting Src in microglia Rhoa-deficient mice attenuates microglia dysregulation and the ensuing neurological phenotype. We also find that the Rhoa/Src signaling pathway is disrupted in microglia of the APP/PS1 mouse model of Alzheimer disease and that low doses of Aβ oligomers trigger microglia neurotoxic polarization through the disruption of Rhoa-to-Src signaling. Overall, our results indicate that disturbing Rho GTPase signaling in microglia can directly cause neurodegeneration.

Neonatal Inflammation Sex‐dependently Impairs Adult, Spinal Microglia

Neonatal inflammation is common and disrupts adult respiratory control, yet the mechanism(s) underlying the impairments are not well understood. Since microglia are key mediators of central inflammation and are important in the lasting effects of neonatal inflammation in non‐respiratory brain regions, we hypothesized lasting dysfunction in microglia underlie impairments in adult respiratory control. Specifically, we hypothesized neonatal inflammation would increase the number of and inflammatory gene expression in adult microglia. Since adult respiratory motor plasticity in the cervical spinal cord is impaired after neonatal inflammation, we analyzed immunomagnetically isolated microglia from the ventral cervical spinal cord. Interestingly, the number of microglia measured by flow cytometry calibrated with counting beads from adults after neonatal inflammation (LPS; 1 mg/kg, i.p.; postnatal day 4) were unchanged in males (LPS: 14,488 ± 4000, n=4; saline: 12,784 ± 4000, n=4, p=0.97) or females (LPS: 14,611 ± 4500, n=4; saline: 9,062 ± 4500, n=5, p=0.79). Additionally, the number of microglia was not significantly different between males and females (p&gt;0.05). Despite no changes in microglia number, we hypothesized microglia function was impaired by neonatal inflammation. Preliminary transcriptomic analysis suggests neonatal inflammation in adult females (n=2) up‐regulated 7 genes and down‐regulated 1 gene, and in adult males (n=3) down‐regulated 1 gene. Gene ontology revealed these differentially expressed genes are associated with inflammatory signaling. While neonatal inflammation has no impact on the number of microglia, it sex‐dependently increases inflammatory‐related gene expression in females with surprisingly few changes in adult males. Thus, adult, female microglia likely contribute to the lasting impairments in respiratory control following neonatal inflammation. Further, the lasting impairments in adult males are likely mediated by a sex‐specific distinct mechanism. Understanding the sex‐specific inflammatory mechanisms impairing respiratory control is crucial to developing treatment strategies to promote breathing during ventilatory control disorders.Support or Funding InformationSupported by NIH HL141249 (AGH)

Repression of phagocytosis by human CD33 is not conserved with mouse CD33

CD33 is an immunomodulatory receptor linked to Alzheimer’s disease (AD) susceptibility via regulation of phagocytosis in microglia. Divergent features between human CD33 (hCD33) and murine CD33 (mCD33) include a unique transmembrane lysine in mCD33 and cytoplasmic tyrosine in hCD33. The functional consequences of these differences in restraining phagocytosis remains poorly understood. Using a new αmCD33 monoclonal antibody, we show that mCD33 is expressed at high levels on neutrophils and low levels on microglia. Notably, cell surface expression of mCD33 is entirely dependent on Dap12 due to an interaction with the transmembrane lysine in mCD33. In RAW264.7 cultured macrophages, BV-2 cultured microglia, primary neonatal and adult microglia, uptake of cargo — including aggregated Aβ1–42 — is not altered upon genetic ablation of mCD33. Alternatively, deletion of hCD33 in monocytic cell lines increased cargo uptake. Moreover, transgenic mice expressing hCD33 in the microglial cell lineage showed repressed cargo uptake in primary microglia. Therefore, mCD33 and hCD33 have divergent roles in regulating phagocytosis, highlighting the importance of studying hCD33 in AD susceptibility.

Loss of NF-\u03baB p50 function synergistically augments microglial priming in the middle-aged brain

BackgroundWhile NF-κB p50 function is impaired in central nervous system disease, aging in non-CNS tissues, and response to reactive oxygen species, the role of NF-κB p50 in aging-associated microglial pro-inflammatory priming is poorly understood.MethodsMale NF-κB p50+/+ and NF-κB p50−/− mice at three different ages (1.5–3.0 month old, 8.0–11.0 month old, and 16.0–18.0 month old) were treated with LPS (5 mg/kg, IP) to trigger peripheral inflammation, where circulating cytokines, neuroinflammation, microglia morphology, and NF-κB p50/p65 function in brain tissue were determined 3 h later.ResultsPeripheral LPS injection in 9-month-old C57BL/6 mice resulted in lower NF-κB p50 DNA binding of nuclear extracts from the whole brain, when compared to 3-week-old C57BL/6 mice, revealing differences in LPS-induced NF-κB p50 activity in the brain across the mouse lifespan. To examine the consequences of loss NF-κB p50 function with aging, NF-κB p50+/+ and NF-κB p50−/− mice of three different age groups (1.5–3.0 month old, 8.0–11.0 month old, and 16.0–18.0 month old) were injected with LPS (5 mg/kg, IP). NF-κB p50−/− mice showed markedly elevated circulating, midbrain, and microglial TNFα when compared to NF-κB p50+/+ mice at all ages. Notably, the 16.0–18.0-month-old (middle aged) NF-κB p50−/− mice exhibited synergistically augmented LPS-induced serum and midbrain TNFα when compared to the younger (1.5–3.0 month old, young adult) NF-κB p50−/− mice. The 16.0–18.0-month-old LPS-treated NF-κB p50−/− mice also had the highest midbrain IL-1β expression, largest number of microglia with changes in morphology, and greatest elevation of pro-inflammatory factors in isolated adult microglia. Interestingly, aging NF-κB p50−/− mice exhibited decreased brain NF-κB p65 expression and activity.ConclusionsThese findings support that loss of NF-κB p50 function and aging in middle-aged mice may interact to excessively augment peripheral/microglial pro-inflammatory responses and point to a novel neuroinflammation signaling mechanism independent the NF-κB p50/p65 transcription factor in this process.