PLD4 Is Involved in Phagocytosis of Microglia: Expression and Localization Changes of PLD4 Are Correlated with Activation State of Microglia

Phospholipase D4 (PLD4) is a recently identified protein that is mainly expressed in the ionized calcium binding adapter molecule 1 (Iba1)-positive microglia in the early postnatal mouse cerebellar white matter. Unlike PLD1 and PLD2, PLD4 exhibits no enzymatic activity for conversion of phosphatidylcholine into choline and phosphatidic acid, and its function is completely unknown. In the present study, we examined the distribution of PLD4 in mouse cerebellar white matter during development and under pathological conditions. Immunohistochemical analysis revealed that PLD4 expression was associated with microglial activation under such two different circumstances. A primary cultured microglia and microglial cell line (MG6) showed that PLD4 was mainly present in the nucleus, except the nucleolus, and expression of PLD4 was upregulated by lipopolysaccharide (LPS) stimulation. In the analysis of phagocytosis of LPS-stimulated microglia, PLD4 was co-localized with phagosomes that contained BioParticles. Inhibition of PLD4 expression using PLD4 specific small interfering RNA (siRNA) in MG6 cells significantly reduced the ratio of phagocytotic cell numbers. These results suggest that the increased PLD4 in the activation process is involved in phagocytosis of activated microglia in the developmental stages and pathological conditions of white matter.     

Phospholipase D family member 4, a transmembrane glycoprotein with no phospholipase D activity, expression in spleen and early postnatal microglia

Background

Phospholipase D (PLD) catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x)₄-Asp (HKD) motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4.

Methodology/Principal Findings

PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity.

Conclusions/Significance

Results showed that PLD4 is a non-PLD, HKD motif-carrying, transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus. The spatiotemporally restricted expression patterns suggested that PLD4 might play a role in common function(s) among microglia during early postnatal brain development and splenic marginal zone cells.     

Accumulating microglia phagocytose injured neurons in hippocampal slice cultures: involvement of p38 MAP kinase

In this study, microglial migration and phagocytosis were examined in mouse organotypic hippocampal slice cultures, which were treated with N-methyl-D-aspartate (NMDA) to selectively injure neuronal cells. Microglial cells were visualized by the expression of enhanced green fluorescent protein. Daily observation revealed microglial accumulation in the pyramidal cell layer, which peaked 5 to 6 days after NMDA treatment. Time-lapse imaging showed that microglia migrated to the pyramidal cell layer from adjacent and/or remote areas. There was no difference in the number of proliferating microglia between control and NMDA-treated slices in both the pyramidal cell layer and stratum radiatum, suggesting that microglial accumulation in the injured areas is mainly due to microglial migration, not to proliferation. Time-lapse imaging also showed that the injured neurons, which were visualized by propidium iodide (PI), disappeared just after being surrounded by microglia. Daily observation revealed that the intensity of PI fluorescence gradually attenuated, and this attenuation was suppressed by pretreatment with clodronate, a microglia toxin. These findings suggest that accumulating microglia phagocytosed injured neurons, and that PI fluorescence could be a useful indicator for microglial phagocytosis. Using this advantage to examine microglial phagocytosis in living slice cultures, we investigated the involvements of mitogen-activated protein (MAP) kinases in microglial accumulation and phagocytosis. p38 MAP kinase inhibitor SB203580, but not MAP kinase/extracellular signal-regulated kinase inhibitor PD98059 or c-Jun N-terminal kinase inhibitor SP600125, suppressed the attenuation of PI fluorescence. On the other hand, microglial accumulation in the injured areas was not inhibited by any of these inhibitors. These data suggest that p38 MAP kinase plays an important role in microglial phagocytosis of injured neurons.     

Accelerated phagocytosis of amyloid-beta by mouse and human microglia overexpressing the macrophage colony-stimulating factor receptor

Microglia surrounding A beta plaques in Alzheimer's disease and in the APPV717F transgenic mouse model of Alzheimer's disease have enhanced immunoreactivity for the macrophage colony-stimulating factor receptor (M-CSFR), encoded by the proto-oncogene c-fms. Increased expression of M-CSFR on cultured microglia results in proliferation and release of pro-inflammatory cytokines and expression of inducible nitric-oxide synthase. We transfected mouse BV-2 and human SV-A3 microglia to overexpress M-CSFR and examined microglial phagocytosis of fluorescein-conjugated A beta. Flow cytometry and laser confocal microscopy showed accelerated phagocytosis of A beta in mouse and human microglia because of M-CSFR overexpression that was time- and concentration-dependent. In contrast, microglial uptake of 1-microm diameter polystyrene microspheres was not enhanced by M-CSFR overexpression. Microglial uptake of A beta was blocked by cytochalasin D, which inhibits phagocytosis. M-CSFR overexpression increased the mRNA for macrophage scavenger receptor A, and fucoidan blocking of macrophage scavenger receptors inhibited uptake of A beta. M-CSFR antibody blocking experiments demonstrated that increased A beta uptake depended on the interaction of the M-CSFR with its ligand. These results suggest that overexpression of M-CSFR in APPV717F mice may prime microglia for phagocytosis of A beta after immunization.     

Annexin A1: a central player in the anti-inflammatory and neuroprotective role of microglia

The brain microenvironment is continuously monitored by microglia with the detection of apoptotic cells or pathogens being rapidly followed by their phagocytosis to prevent inflammatory responses. The protein annexin A1 (ANXA1) is key to the phagocytosis of apoptotic leukocytes during peripheral inflammatory resolution, but the pathophysiological significance of its expression in the CNS that is restricted almost exclusively to microglia is unclear. In this study, we test the hypothesis that ANXA1 is important in the microglial clearance of apoptotic neurons in both noninflammatory and inflammatory conditions. We have identified ANXA1 to be sparingly expressed in microglia of normally aged human brains and to be more strongly expressed in Alzheimer's disease. Using an in vitro model comprising microglial and neuronal cell lines, as well as primary microglia from wild-type and ANXA1 null mice, we have identified two distinct roles for microglial ANXA1: 1) controlling the noninflammatory phagocytosis of apoptotic neurons and 2) promoting resolution of inflammatory microglial activation. In particular, we showed that microglial-derived ANXA1 targets apoptotic neurons, serving as both an "eat me" signal and a bridge between phosphatidylserine on the dying cell and formyl peptide receptor 2 on the phagocytosing microglia. Moreover, inflammatory activation of microglia impairs their ability to discriminate between apoptotic and nonapoptotic cells, an ability restored by exogenous ANXA1. We thus show that ANXA1 is fundamental for brain homeostasis, and we suggest that ANXA1 and its peptidomimetics can be novel therapeutic targets in neuroinflammation.     

USP18 lack in microglia causes destructive interferonopathy of the mouse brain

Microglia are tissue macrophages of the central nervous system (CNS) that control tissue homeostasis. Microglia dysregulation is thought to be causal for a group of neuropsychiatric, neurodegenerative and neuroinflammatory diseases, called “microgliopathies”. However, how the intracellular stimulation machinery in microglia is controlled is poorly understood. Here, we identified the ubiquitin‐specific protease (Usp) 18 in white matter microglia that essentially contributes to microglial quiescence. We further found that microglial Usp18 negatively regulates the activation of Stat1 and concomitant induction of interferon‐induced genes, thereby terminating IFN signaling. The Usp18‐mediated control was independent from its catalytic activity but instead required the interaction with Ifnar2. Additionally, the absence of Ifnar1 restored microglial activation, indicating a tonic IFN signal which needs to be negatively controlled by Usp18 under non‐diseased conditions. These results identify Usp18 as a critical negative regulator of microglia activation and demonstrate a protective role of Usp18 for microglia function by regulating the Ifnar pathway. The findings establish Usp18 as a new molecule preventing destructive microgliopathy.     

Carnosic acid, a pro-electrophilic compound, inhibits LPS-induced activation of microglia

In the previous studies, we reported that carnosic acid (CA) protects cortical neurons by activating the Keap1/Nrf2 pathway, which activation is initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the "electrophilic"quinone-type CA. Here, we found that the pro-electrophilic CA inhibited the in vitro lipopolysaccharide (LPS)-induced activation of cells of the mouse microglial cell line MG6. LPS induced the expression of IL-1β and IL-6, typical inflammatory cytokines released from microglial cells. CA inhibited the NO production associated with a decrease in the level of inducible NO synthase. Neither CA nor LPS affected cell survival at the concentrations used here. These actions of CA seemed to be mediated by induction of phase 2 genes (gclc, gclm, nqo1 and xct). We propose that an inducer of phase 2 genes may be a critical regulator of microglial activation. Thus, CA is a unique pro-electrophilic compound that provides both a protective effect on neurons and an anti-inflammatory one on microglia through induction of phase 2 genes.     

Involvement of phosphatidylinositol 3-kinase-mediated up-regulation of I kappa B alpha in anti-inflammatory effect of gemfibrozil in microglia

The present study underlines the importance of PI3K in mediating the anti-inflammatory effect of gemfibrozil, a prescribed lipid-lowering drug for humans, in mouse microglia. Gemfibrozil inhibited LPS-induced expression of inducible NO synthase (iNOS) and proinflammatory cytokines in mouse BV-2 microglial cells and primary microglia. By overexpressing wild-type and dominant-negative constructs of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) in microglial cells and isolating primary microglia from PPAR-alpha-/- mice, we have demonstrated that gemfibrozil inhibits the activation of microglia independent of PPAR-alpha. Interestingly, gemfibrozil induced the activation of p85alpha-associated PI3K (p110beta but not p110alpha) and inhibition of that PI3K by either chemical inhibitors or dominant-negative mutants abrogated the inhibitory effect of gemfibrozil. Conversely, overexpression of the constitutively active mutant of p110 enhanced the inhibitory effect of gemfibrozil on LPS-induced expression of proinflammatory molecules. Similarly, gemfibrozil also inhibited fibrillar amyloid beta (Abeta)-, prion peptide (PrP)-, dsRNA (poly IC)-, HIV-1 Tat-, and 1-methyl-4-phenylpyridinium (MPP+)-, but not IFN-gamma-, induced microglial expression of iNOS. Inhibition of PI3K also abolished the inhibitory effect of gemfibrozil on Abeta-, PrP-, poly IC-, Tat-, and MPP+-induced microglial expression of iNOS. Involvement of NF-kappaB activation in LPS-, Abeta-, PrP-, poly IC-, Tat-, and MPP+-, but not IFN-gamma-, induced microglial expression of iNOS and stimulation of IkappaBalpha expression and inhibition of NF-kappaB activation by gemfibrozil via the PI3K pathway suggests that gemfibrozil inhibits the activation of NF-kappaB and the expression of proinflammatory molecules in microglia via PI3K-mediated up-regulation of IkappaBalpha.     

Fibrillar amyloid-beta peptides activate microglia via TLR2: implications for Alzheimer's disease

Microglial activation is an important pathological component in brains of patients with Alzheimer's disease (AD), and fibrillar amyloid-beta (Abeta) peptides play an important role in microglial activation in AD. However, mechanisms by which Abeta peptides induce the activation of microglia are poorly understood. The present study underlines the importance of TLR2 in mediating Abeta peptide-induced activation of microglia. Fibrillar Abeta1-42 peptides induced the expression of inducible NO synthase, proinflammatory cytokines (TNF-alpha, IL-1beta, and IL-6), and integrin markers (CD11b, CD11c, and CD68) in mouse primary microglia and BV-2 microglial cells. However, either antisense knockdown of TLR2 or functional blocking Abs against TLR2 suppressed Abeta1-42-induced expression of proinflammatory molecules and integrin markers in microglia. Abeta1-42 peptides were also unable to induce the expression of proinflammatory molecules and increase the expression of CD11b in microglia isolated from TLR2(-/-) mice. Finally, the inability of Abeta1-42 peptides to induce the expression of inducible NO synthase and to stimulate the expression of CD11b in vivo in the cortex of TLR2(-/-) mice highlights the importance of TLR2 in Abeta-induced microglial activation. In addition, ligation of TLR2 alone was also sufficient to induce microglial activation. Consistent to the importance of MyD88 in mediating the function of various TLRs, antisense knockdown of MyD88 also inhibited Abeta1-42 peptide-induced expression of proinflammatory molecules. Taken together, these studies delineate a novel role of TLR2 signaling pathway in mediating fibrillar Abeta peptide-induced activation of microglia.     

Role of antiproliferative B cell translocation gene-1 as an apoptotic sensitizer in activation-induced cell death of brain microglia

Mouse brain microglial cells undergo apoptosis on exposure to inflammatory stimuli, which is considered as an autoregulatory mechanism to control their own activation. Here, we present evidence that an antiproliferative B cell translocation gene 1 (BTG1) constitutes a novel apoptotic pathway of LPS/IFN-gamma-activated microglia. The expression of BTG1 was synergistically enhanced by LPS and IFN-gamma in BV-2 mouse microglial cells as well as in primary microglia cultures. Levels of BTG1 expression inversely correlated with a proliferative capacity of the microglial cells. Tetracycline-based conditional expression of BTG1 not only suppressed microglial proliferation but also increased the sensitivity of microglial cells to NO-induced apoptosis, suggesting a novel mechanism of cooperation between LPS and IFN-gamma in the induction of microglial apoptosis. An increase in BTG1 expression, however, did not affect microglial production of NO, TNF-alpha, or IL-1beta, indicating that the antiproliferative BTG1 is important in the activation-induced apoptosis of microglia, but not in the activation itself. The synergistic action of LPS and IFN-gamma in the microglial BTG1 induction and apoptosis was dependent on the Janus kinase/STAT1 pathway, but not IFN-regulatory factor-1, as demonstrated by a pharmacological inhibitor of Janus kinase (AG490), STAT1 dominant negative mutant, and IFN-regulatory factor-1-deficient mice. Taken together, antiproliferative BTG1 may participate in the activation-induced cell death of microglia by lowering the threshold for apoptosis; BTG1 increases the sensitivity of microglia to apoptogenic action of autocrine cytotoxic mediator, NO. Our results point out an important link between the proliferative state of microglia and their sensitivity to apoptogenic agents.     

Young microglia restore amyloid plaque clearance of aged microglia

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

Phagocytosis is regulated by nitric oxide in murine microglia.

Nitric oxide (NO) is produced by inducible nitric oxide synthase (iNOS) in activated microglia and has been shown to participate in host defense mechanisms. However, the role of NO produced by constitutive nitric oxide synthase (cNOS) in microglia is poorly understood. In this report, NO was found to regulate phagocytosis in murine BV-2 microglial cells as quantified by flow cytometry. Addition of NO-generating compounds caused impaired phagocytosis as compared to untreated microglia. The addition of nitric oxide synthase (NOS) inhibitors to microglial cells resulted in potentiation of phagocytosis, suggesting that constitutive NO was participating in the regulation of phagocytosis. The inverse correlation between NO production and phagocytosis was also observed when Alzheimer's beta-amyloid peptide was added. With beta-amyloid treatment, constitutive NO production decreased while phagocytosis increased. Cell extracts prepared from untreated microglia were found to contain both neuronal and endothelial NOS isoforms, but not the inducible form. The correlation of spontaneous NO production with attenuated phagocytosis suggests that constitutive NOS enzymes participate in microglial regulation.     

Phosphoinositide 3-kinase-gamma expression is upregulated in brain microglia and contributes to ischemia-induced microglial activation in acute experimental stroke

Microglia, the resident microphages of the CNS, are rapidly activated after ischemic stroke. Inhibition of microglial activation may protect the brain by attenuating blood-brain barrier damage and neuronal apoptosis after ischemic stroke. However, the mechanisms by which microglia is activated following cerebral ischemia is not well defined. In this study, we investigated the expression of PI3Kγ in normal and ischemic brains and found that PI3Kγ mRNA and protein are constitutively expressed in normal brain microvessels, but significantly upregulated in postischemic brain primarily in activated microglia following cerebral ischemia. In vitro, the expression of PI3Kγ mRNA and protein was verified in mouse brain endothelial and microglial cell lines. Importantly, absence of PI3Kγ blocked the early microglia activation (at 4 h) and subsequent expansion (at 24-72 h) in PI3Kγ knockout mice. The results suggest that PI3Kγ is an ischemia-responsive gene in brain microglia and contributes to ischemia-induced microglial activation and expansion.     

Minocycline selectively inhibits M1 polarization of microglia

Minocycline is commonly used to inhibit microglial activation. It is widely accepted that activated microglia exert dual functions, that is, pro-inflammatory (M1) and anti-inflammatory (M2) functions. The in vivo status of activated microglia is probably on a continuum between these two extreme states. However, the mechanisms regulating microglial polarity remain elusive. Here, we addressed this question focusing on minocycline. We used SOD1^G93A mice as a model, which exhibit the motor neuron-specific neurodegenerative disease, amyotrophic lateral sclerosis. Administration of minocycline attenuated the induction of the expression of M1 microglia markers during the progressive phase, whereas it did not affect the transient enhancement of expression of M2 microglia markers during the early pathogenesis phase. This selective inhibitory effect was confirmed using primary cultured microglia stimulated by lipopolysaccharide (LPS) or interleukin (IL)-4, which induced M1 or M2 polarization, respectively. Furthermore, minocycline inhibited the upregulation of NF-κB in the LPS-stimulated primary cultured microglia and in the spinal cord of SOD1^G93A mice. On the other hand, IL-4 did not induce upregulation of NF-κB. This study indicates that minocycline selectively inhibits the microglia polarization to a proinflammatory state, and provides a basis for understanding pathogeneses of many diseases accompanied by microglial activation.     

Reactive oxygen species up-regulate CD11b in microglia via nitric oxide: Implications for neurodegenerative diseases

Microglial activation is considered as a hallmark of several neurodegenerative disorders. During microglial activation, the expression of CD11b, the beta-integrin marker of microglia, is increased. However, the molecular mechanism behind increased microglial CD11b expression is poorly understood. The present study was undertaken to explore the role of reactive oxygen species (ROS) in the expression of CD11b in microglial cells. Bacterial lipopolysaccharide (LPS) stimulated the expression of CD11b in mouse BV-2 microglial cells and primary microglia, the effect that was blocked by antioxidants such as N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC). Furthermore, comicroinjection of either NAC or PDTC with LPS was also able to suppress LPS-stimulated expression of CD11b in striatum in vivo. Similarly, other neurotoxic molecules, such as interleukin-1beta (IL-1beta), IL-12 p40(2), fibrillar amyloid-beta (Abeta) peptides, HIV-1 gp120, and double-stranded RNA (poly(IC)), also stimulated the expression of CD11b in microglia through the involvement of ROS. Complete inhibition of LPS-stimulated expression of CD11b by catalase, induction of CD11b expression by H2O2 alone, and inhibition of superoxide-stimulated CD11b expression by catalase suggest that H2O2, but not superoxide, is in fact involved in the expression of CD11b. Interestingly, we also demonstrate that ROS stimulated the expression of CD11b after the induction of nitric oxide (NO) production and failed to stimulate CD11b when NO production was inhibited by either 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) or L-N6-(1-iminoethyl)-L-lysine (L-NIL). Taken together, these studies suggest that the up-regulation of CD11b in microglia is redox sensitive and that ROS up-regulates CD11b via NO.     

Dectin-1 mediates in vitro phagocytosis of Candida albicans yeast cells by retinal microglia

We have investigated the expression of TLR2 and Dectin-1 in retinal microglia and their involvement in Candida albicans phagocytosis using a cytometric approach. The expression of both receptors has been demonstrated in CD11b(+) retinal cells. Phagocytosis of pHrodo-labelled C. albicans yeasts by microglial CD11b(+) cells of C57BL/6 mice was inhibited both by the Dectin-1 antagonist laminarin and anti-Dectin-1 antibodies, whereas phagocytosis of yeasts by retinal microglia of TLR2 KO mice was unaffected. These data indicate that phagocytosis of C. albicans yeasts by retinal microglia is mediated by Dectin-1, whereas TLR2 does not play a significant role in this process.     

Prion protein expression differences in microglia and astroglia influence scrapie-induced neurodegeneration in the retina and brain of transgenic mice

Activated microglia and astroglia are known to be involved in a variety of neurodegenerative diseases, including prion diseases. In the present experiments, we studied activation of astroglia and microglia after intraocular scrapie infection in transgenic mice expressing prion protein (PrP) in multiple cell types (tg7 mice) or in neurons only (tgNSE mice). In this model, scrapie infection and protease-resistant PrP deposition occurs in the retinas of both strains of mice, but retinal degeneration is observed only in tg7 mice. Our results showed that the retinas of tg7 and tgNSE mice both had astroglial activation with increased chemokine expression during the course of infection. However, only tg7 retinas exhibited strong microglial activation compared to tgNSE retinas, which showed little microglial activation by biochemical or morphological criteria. Therefore, microglial PrP expression might be required for scrapie-induced retinal microglial activation and damage. Furthermore, microglial activation preceded retinal neurodegeneration in tg7 mice, suggesting that activated microglia might contribute to the degenerative process, rather than being a response to the damage. Surprisingly, brain differed from retina in that an altered profile of microglial activation markers was upregulated, and the profiles in the two mouse strains were indistinguishable. Microglial activation in the brain was associated with severe brain vacuolation and neurodegeneration, leading to death. Thus, retinal and brain microglia appeared to differ in their requirements for activation, suggesting that different activation pathways occur in the two tissues.     

Optogenetic activation of spinal microglia triggers chronic pain in mice

Spinal microglia are highly responsive to peripheral nerve injury and are known to be a key player in pain. However, there has not been direct evidence showing that selective microglial activation in vivo is sufficient to induce chronic pain. Here, we used optogenetic approaches in microglia to address this question employing CX3CR1^creER/+: R26^LSL-ReaChR/+ transgenic mice, in which red-activated channelrhodopsin (ReaChR) is inducibly and specifically expressed in microglia. We found that activation of ReaChR by red light in spinal microglia evoked reliable inward currents and membrane depolarization. In vivo optogenetic activation of microglial ReaChR in the spinal cord triggered chronic pain hypersensitivity in both male and female mice. In addition, activation of microglial ReaChR up-regulated neuronal c-Fos expression and enhanced C-fiber responses. Mechanistically, ReaChR activation led to a reactive microglial phenotype with increased interleukin (IL)-1β production, which is likely mediated by inflammasome activation and calcium elevation. IL-1 receptor antagonist (IL-1ra) was able to reverse the pain hypersensitivity and neuronal hyperactivity induced by microglial ReaChR activation. Therefore, our work demonstrates that optogenetic activation of spinal microglia is sufficient to trigger chronic pain phenotypes by increasing neuronal activity via IL-1 signaling.

This study uses red light activation of channelrhodopsin in spinal microglia to trigger chronic pain hypersensitivity in awake mice, revealing that optogenetic activation of microglia increases IL-1β production via inflammasome activation and calcium elevation, leading to neuronal hyperactivity and chronic pain.