Microglial Toll-like Receptor Research Articles

Spinal HMGB1 participates in the early stages of paclitaxel-induced neuropathic pain via microglial TLR4 and RAGE activation.

Chemotherapy-induced neuropathic pain (CINP) is one of the main adverse effects of chemotherapy treatment. At the spinal level, CINP modulation involves glial cells that upregulate Toll-like receptor 4 (TLR4) and signaling pathways, which can be activated by pro-inflammatory mediators as the high mobility group box-1 (HMGB1). To evaluate the spinal role of HMGB1 in the paclitaxel-induced neuropathic pain via receptor for advanced glycation end products (RAGE) and TLR4 activation expressed in glial cells. Male C57BL/6 Wild type and TLR4 deficient mice were used in the paclitaxel-induced neuropathic pain model. The nociceptive threshold was measured using the von Frey filament test. In addition, recombinant HMGB1 was intrathecally (i.t.) injected to confirm its nociceptive potential. To evaluate the spinal participation of RAGE, TLR4, NF-kB, microglia, astrocytes, and MAPK p38 in HMGB1-mediated nociceptive effect during neuropathic pain and recombinant HMGB1-induced nociception, the drugs FPS-ZM1, LPS-RS, PDTC, minocycline, fluorocitrate, and SML0543 were respectively administrated by i.t. rout. Microglia, astrocytes, glial cells, RAGE, and TLR4 protein expression were analyzed by Western blot. ELISA immunoassay was also used to assess HMGB1, IL-1β, and TNF-α spinal levels. The pharmacological experiments demonstrated that spinal RAGE, TLR4, microglia, astrocytes, as well as MAPK p38 and NF-kB signaling are involved with HMGB1-induced nociception and paclitaxel-induced neuropathic pain. Furthermore, HMGB1 spinal levels were increased during the early stages of neuropathic pain and associated with RAGE, TLR4 and microglial activation. RAGE and TLR4 blockade decreased spinal levels of pro-inflammatory cytokines during neuropathic pain. Taken together, our findings indicate that HMGB1 may be released during the early stages of paclitaxel-induced neuropathic pain. This molecule activates RAGE and TLR4 receptors in spinal microglia, upregulating pro-inflammatory cytokines that may contribute to neuropathic pain.

Microglial TLR4 Mediates White Matter Injury in a Combined Model of Diesel Exhaust Exposure and Cerebral Hypoperfusion.

Air pollution particulate matter exposure and chronic cerebral hypoperfusion (CCH) contribute to white matter toxicity through shared mechanisms of neuroinflammation, oxidative stress, and myelin breakdown. Prior studies showed that exposure of mice to joint particulate matter and CCH caused supra-additive injury to corpus callosum white matter. This study examines the role of TLR4 (toll-like receptor 4) signaling in mediating neurotoxicity and myelin damage observed in joint particulate matter and CCH exposures. Experiments utilized a novel murine model of inducible monocyte/microglia-specific TLR4 knockout (i-mTLR4-ko). Bilateral carotid artery stenosis (BCAS) was induced surgically to model CCH. TLR4-intact (control) and i-mTLR4-ko mice were exposed to 8 weeks of either aerosolized diesel exhaust particulate (DEP) or filtered air (FA) in 8 experimental groups: (1) control/FA (n=10), (2) control/DEP (n=10), (3) control/FA+BCAS (n=9), (4) control/DEP+BCAS (n=10), (5) i-mTLR4-ko/FA (n=9), (6) i-mTLR4-ko/DEP (n=8), (7) i-mTLR4-ko/FA+BCAS (n=8), and (8) i-mTLR4-ko/DEP+BCAS (n=10). Corpus callosum levels of 4-hydroxynonenal, 8-Oxo-2'-deoxyguanosine, Iba-1 (ionized calcium-binding adapter molecule 1), and dMBP (degraded myelin basic protein) were assayed via immunofluorescence to measure oxidative stress, neuroinflammation, and myelin breakdown, respectively. Compared with control/FA mice, control/DEP+BCAS mice exhibited increased dMBP (41%; P<0.01), Iba-1 (51%; P<0.0001), 4-hydroxynonenal (100%; P<0.0001), and 8-Oxo-2'-deoxyguanosine (65%; P<0.05). I-mTLR4 knockout attenuated responses to DEP/BCAS for all markers. i-mTLR4-ko markedly reduced neuroinflammation and oxidative stress and attenuated white matter degradation following DEP and CCH exposures. This suggests a potential role for targeting TLR4 signaling in individuals with vascular cognitive impairment, particularly those exposed to substantial ambient air pollution.

Microglial activation and toll-like receptor 4-Dependent regulation of angiotensin II type I receptor-mu-opioid receptor 1 heterodimerization and hypertension in fructose-fed rats

Our previous study reported that the heterodimer of Angiotensin II Type I Receptor (AT1R) and Mu-Opioid Receptor 1 (MOR1) involves Nitric Oxide (NO) reduction which leads to elevation of blood pressure. Secondly, we showed that Toll-like Receptor 4 (TLR4) may be involved in the heterodimerization of AT1R and MOR1 in the brainstem Nucleus Tractus Solitarii (NTS), which regulates systemic blood pressure and gastric nitric oxide through the insulin pathway. Here, we investigated the role of microglial activation and TLR4 in the heterodimerization of AT1R and MOR1. Hypertensive rats were established after four weeks of fructose consumption. SBP of rats was measured using non-invasive blood pressure method. PLA technique was utilized to determine protein-protein interaction in the nucleus tractus solitarii. Results showed that the level of MOR-1 and AT1R was induced significantly in the fructose group compared with control. PLA signal potentially showed that AT1R and MOR1 were formed in the nucleus tractus solitarii after fructose consumption. Meanwhile, the innate immune cell in the CNS microglia was observed in the nucleus tractus solitarii using biomarkers and was activated. TLR4 inhibitor CLI-095, was administered to animals to suppress the neuroinflammation and microglial activation. CLI-095 treatment reduced the heterodimer formation of AT1R and MOR1 and restored nitric oxide production in the nucleus tractus solitarii. These findings imply that TLR4-primed neuroinflammation involves formation of heterodimers AT1R and MOR1 in the nucleus tractus solitarii which leads to increase in systemic blood pressure.

Inhibition of Microglial GSK3β Activity Is Common to Different Kinds of Antidepressants: A Proposal for an In Vitro Screen to Detect Novel Antidepressant Principles

Depression is a major public health concern. Unfortunately, the present antidepressants often are insufficiently effective, whilst the discovery of more effective antidepressants has been extremely sluggish. The objective of this review was to combine the literature on depression with the pharmacology of antidepressant compounds, in order to formulate a conceivable pathophysiological process, allowing proposals how to accelerate the discovery process. Risk factors for depression initiate an infection-like inflammation in the brain that involves activation microglial Toll-like receptors and glycogen synthase kinase-3β (GSK3β). GSK3β activity alters the balance between two competing transcription factors, the pro-inflammatory/pro-oxidative transcription factor NFκB and the neuroprotective, anti-inflammatory and anti-oxidative transcription factor NRF2. The antidepressant activity of tricyclic antidepressants is assumed to involve activation of GS-coupled microglial receptors, raising intracellular cAMP levels and activation of protein kinase A (PKA). PKA and similar kinases inhibit the enzyme activity of GSK3β. Experimental antidepressant principles, including cannabinoid receptor-2 activation, opioid μ receptor agonists, 5HT2 agonists, valproate, ketamine and electrical stimulation of the Vagus nerve, all activate microglial pathways that result in GSK3β-inhibition. An in vitro screen for NRF2-activation in microglial cells with TLR-activated GSK3β activity, might therefore lead to the detection of totally novel antidepressant principles with, hopefully, an improved therapeutic efficacy.

PKr-2 induces neurodegeneration via upregulation of microglial TLR4 in the hippocampus of AD brain

We recently demonstrated that prothrombin kringle-2 (pKr-2) derived from blood-brain barrier (BBB) disruption could induce hippocampal neurodegeneration and object recognition impairment through neurotoxic inflammatory responses in the five familial Alzheimer's disease mutation (5XFAD) mice. In the present study, we aimed to determine whether pKr-2 induces microglial activation by stimulating toll-like receptor 4 (TLR4) upregulation and examine whether this response contributes to pKr-2-induced neuroinflammatory damage in the hippocampi of mice models. We observed that inflammatory responses induced by pKr-2 administration in the hippocampi of wild-type mice were significantly abrogated in TLR4-deficient mice (TLR4−/−), and caffeine supply or rivaroxaban treatment that inhibits the overexpression of hippocampal pKr-2 reduced TLR4 upregulation in 5XFAD mice, resulting in the inhibition of neuroinflammatory responses. Similar to the expression patterns of pKr-2, TLR4, and the TLR4 transcription factors, PU.1 and p-c-Jun, seen in the postmortem hippocampal tissues of Alzheimer's disease (AD) patients, our results additionally showed the influence of transcriptional regulation on TLR4 expression following pKr-2 expression in triggering the production of neurotoxic inflammatory mediators. Therefore, we conclude that pKr-2 may play a role in initiating upregulation of microglial TLR4, consequently inducing hippocampal neurodegeneration. Furthermore, the control of pKr-2-induced microglial TLR4 could be a useful therapeutic strategy against hippocampal neurodegeneration in AD.

Gender differences in Alzheimer's may be associated with TLR4-LYN expression in damage associated microglia and neuronal phagocytosis.

The role of Aβ plaques and neurofibrillary tangles in Alzheimer's disease (AD) pathogenesis have recently come into question due to failure of many pharmaceutical agents targeting these deposits and detection of these misfolded proteins in normal human brains. Therefore, we investigated correlations between microglial activation and toll like receptor 4 (TLR4) and Lck/Yes novel tyrosine (LYN) kinase signaling in an AD mouse model. In this study, we used 5-6-month-old 5XFAD and wild type (WT) male and female mice. Immunohistochemistry (IHC) and flow cytometry (FC) were performed on their brains. Cognitive performance was assessed with the Barnes-Maze. IHC showed more Ab aggregation in microglia of female 5XFAD mice compared to their male counterparts. Increased co-localization of microglial TLR4 and LYN was also observed in AD more than WT and females more than males. IHC also suggests microglial phagocytosis of neurons in AD mice, which is supported by FC data. Our FC data also support the involvement of disease associated microglia (DAMs) in this process based on cytokine secretion. Cognitive assessment by the Barnes maze showed 5XFAD females performed worse than males. In this study, we investigated the relationship between microglial TLR4 and LYN kinase in 5XFAD male and females. Our data reveals a correlation between microglial TLR4 and LYN co-localization and AD pathogenesis, more in females than males. Targeting microglial TLR4 and Lyn in DAMs may offer new therapeutic opportunities in the treatment of AD.

Triptolide Alleviates Neuropathic Pain by Inhibiting the Activation of Microglial Toll-Like Receptor 3.

Current data indicates the incidence of neuropathic pain after surgical nerve injury is as high as 50%, thus representing a major problem for patients and for the medical system. Triptolide, a traditional Chinese herb, has anti-inflammatory effects on various neurodegenerative and neuroinflammatory diseases. This agent also reduces peripheral nerve injury-induced neuropathic pain, although the mechanism underlying this effect is still unknown. The effects of triptolide on spinal nerve ligation (SNL) injury-induced neuropathic pain was studied in an animal model using behavioral, morphological and molecular biological methods. Repeated administration of intrathecal triptolide was found to alleviate SNL- or Poly(I:C) (toll-like receptor 3 agonist) injection-induced mechanical allodynia without any motor impairment. The mechanism by which triptolide reduces SNL- and Poly(I:C) injection-induced microglial activation appears to be via the inhibition of OX42 expression, which is a microglial-specific marker. Intrathecal triptolide also suppressed SNL- and Poly(I:C) injection-induced expression of spinal TRIF. TRIF transmits signals from activated TLR3 and is the downstream adaptor of TLR3 in microglia. In addition, intrathecal triptolide inhibited the expression of spinal pro-inflammatory IL-1 β following SNL or Poly(I:C) injection. Intrathecal triptolide can suppress the TLR3/TRIF/IL-1 β pathway in spinal microglia following SNL. This could be the underlying mechanism by which triptolide alleviate neuropathic pain induced by peripheral nerve injury.

κ-opioid receptor is involved in electroacupuncture analgesia via inhibition of spinal microglial Toll-like receptor 4 in neuropathic pain rats

To observe the effect of electroacupuncture (EA) on the expression of lumbar spinal κ-opioid receptor (KOR) and Toll-like receptor 4(TLR4) in microglia in neuropathic pain rats, so as to explore the role of cross-talk between KOR and TLK4 in EA-induced alleviation of chronic neuropathic pain. Wistar male rats were randomized into control, model, EA and EA plus KOR inhibitor (EA+inhibitor) groups (n=18 in each group). The neuropathic pain model was established in rats by ligature of the right sciatic nerve. EA was applied at bilateral "Zusanli"(ST36) and "Yanglingquan"(GB34) for 30 min, once daily for 5 days. JDTic dihydrochloride (a KOR inhibitor) was administrated by intraperitoneal injection before EA intervention. The difference value of paw withdrawal thermal latency (PWLD) of the bilateral hind-limbs was used as the thermal pain reaction level. At the end of experiments, the rat's lumbar spinal cord (L2-L4) was taken for detecting the expression of CD68 mRNA (a marker of the activated microglia) and Iba-1 (a marker for the activated and resting microglia) immunoactivity, and dynorphin content, and KOR mRNA and TLR4 protein (in immunomagnetic microbead method separated microglia) by using fluorescence quantitative PCR, immunofluorescence, radioimmunoassay and Western blot, separately. Compared with the control group, a strong thermal hyperalgesia was induced, the expression levels of Iba-1 and CD68 mRNA in the spinal cord, TLR4 protein of the spinal microglia were significantly increased(P<0.01) in the model group. The microglia were characterized by somatic hypertrophy and thickened branches in the model group. After EA intervention, the PWLD, the expression of Iba-1, CD68 mRNA and TLR4 protein of the microglia were significantly decreased(P<0.05), while the content of spinal dynorphin and the expression of KOR mRNA of the microglia increased in the EA group relative to the model group(P<0.05). The hypertrophic microglia shrinked slightly in the EA group. After injection of KOR inhibitor, the PWLD and expression levels of Iba-1, CD68 mRNA and TLR4 protein were significantly increased(P<0.05), and the expression of KOR mRNA was significantly decreased(P<0.05) in the EA+inhibitor group in comparison with the EA group. The analgesia effect of EA may partly mediated by spinal microglial KOR and the activation of KOR of microglia may be a target for inhibition of microglial TLR4-induced pro-inflammatory signaling.

TLR4 and AT1R mediate blood-brain barrier disruption, neuroinflammation, and autonomic dysfunction in spontaneously hypertensive rats

Angiotensin II (AngII) is implicated in neuroinflammation, blood-brain barrier (BBB) disruption, and autonomic dysfunction in hypertension. We have previously shown that exogenous AngII stimulates Toll-like receptor 4 (TLR4) via AngII type 1 receptor (AT1R), inducing activation of hypothalamic microglia ex vivo, and that AngII-AT1R signaling is necessary for the loss of BBB integrity in spontaneously hypertensive rats (SHRs). Herein, we hypothesized that microglial TLR4 and AT1R signaling interactions represent a crucial mechanistic link between AngII-mediated neuroinflammation and BBB disruption, thereby contributing to sympathoexcitation in SHRs. Male SHRs were treated with TAK-242 (TLR4 inhibitor; 2 weeks), Losartan (AT1R inhibitor; 4 weeks), or vehicle, and age-matched to control Wistar Kyoto rats (WKYs). TLR4 and AT1R inhibitions normalized increased TLR4, interleukin-6, and tumor necrosis factor-α protein densities in SHR cardioregulatory nuclei (hypothalamic paraventricular nucleus [PVN], rostral ventrolateral medulla [RVLM], and nucleus tractus solitarius [NTS]), and abolished enhanced microglial activation. PVN, RVLM, and NTS BBB permeability analyses revealed complete restoration after TAK-242 treatment, whereas SHRs presented with elevated dye leakage. Mean arterial pressure was normalized in Losartan-treated SHRs, and attenuated with TLR4 inhibition. In conscious assessments, TLR4 blockade rescued SHR baroreflex sensitivity to vasoactive drugs, and reduced the SHR pressor response to ganglionic blockade to normal levels. These data suggest that TLR4 activation plays a substantial role in mediating a feed-forward pro-hypertensive cycle involving BBB disruption, neuroinflammation, and autonomic dysfunction, and that TLR4-specific therapeutic interventions may represent viable alternatives in the treatment of hypertension.

Cannabinoid receptor 2 is necessary to induce toll-like receptor-mediated microglial activation.

The tight regulation of microglia activity is key for precise responses to potential threats, while uncontrolled and exacerbated microglial activity is neurotoxic. Microglial toll-like receptors (TLRs) are indispensable for sensing different types of assaults and triggering an innate immune response. Cannabinoid receptor 2 (CB2) signaling is a key pathway to control microglial homeostasis and activation, and its activation is connected to changes in microglial activity. We aimed to investigate how CB2 signaling impacts TLR-mediated microglial activation. Here, we demonstrate that deletion of CB2 causes a dampened transcriptional response to prototypic TLR ligands in microglia. Loss of CB2 results in distinct microglial gene expression profiles, morphology, and activation. We show that the CB2-mediated attenuation of TLR-induced microglial activation is mainly p38 MAPK-dependent. Taken together, we demonstrate that CB2 expression and signaling are necessary to fine-tune TLR-induced activation programs in microglia.

Temporal Contribution of Myeloid-Lineage TLR4 to the Transition to Chronic Pain: A Focus on Sex Differences.

Complex regional pain syndrome (CRPS) is a chronic pain disorder with a clear acute-to-chronic transition. Preclinical studies demonstrate that toll-like receptor 4 (TLR4), expressed by myeloid-lineage cells, astrocytes, and neurons, mediates a sex-dependent transition to chronic pain; however, evidence is lacking on which exact TLR4-expressing cells are responsible. We used complementary pharmacologic and transgenic approaches in mice to more specifically manipulate myeloid-lineage TLR4 and outline its contribution to the transition from acute-to-chronic CRPS based on three key variables: location (peripheral vs central), timing (prevention vs treatment), and sex (male vs female). We demonstrate that systemic TLR4 antagonism is more effective at improving chronic allodynia trajectory when administered at the time of injury (early) in the tibial fracture model of CRPS in both sexes. In order to clarify the contribution of myeloid-lineage cells peripherally (macrophages) or centrally (microglia), we rigorously characterize a novel spatiotemporal transgenic mouse line, Cx3CR1-CreERT2-eYFP;TLR4fl/fl (TLR4 cKO) to specifically knock out TLR4 only in microglia and no other myeloid-lineage cells. Using this transgenic mouse, we find that early TLR4 cKO results in profound improvement in chronic, but not acute, allodynia in males, with a significant but less robust effect in females. In contrast, late TLR4 cKO results in partial improvement in allodynia in both sexes, suggesting that downstream cellular or molecular TLR4-independent events may have already been triggered. Overall, we find that the contribution of TLR4 is time- and microglia-dependent in both sexes; however, females also rely on peripheral myeloid-lineage (or other TLR4 expressing) cells to trigger chronic pain.SIGNIFICANCE STATEMENT The contribution of myeloid cell TLR4 to sex-specific pain progression remains controversial. We used complementary pharmacologic and transgenic approaches to specifically manipulate TLR4 based on three key variables: location (peripheral vs central), timing (prevention vs treatment), and sex (male vs female). We discovered that microglial TLR4 contributes to early pain progression in males, and to a lesser extent in females. We further found that maintenance of chronic pain likely occurs through myeloid TLR4-independent mechanisms in both sexes. Together, we define a more nuanced contribution of this receptor to the acute-to-chronic pain transition in a mouse model of complex regional pain syndrome.

Modulating neuroinflammation in neurodegeneration-related dementia: can microglial toll-like receptors pull the plug?

Neurodegeneration-associated dementia disorders (NADDs), namely Alzheimer and Parkinson diseases, are developed by a significant portion of the elderly population globally. Extensive research has provided critical insights into the molecular basis of the pathological advancements of these diseases, but an efficient curative therapy seems elusive. A common attribute of NADDs is neuroinflammation due to a chronic inflammatory response within the central nervous system (CNS), which is primarily modulated by microglia. This response within the CNS is positively regulated by cytokines, chemokines, secondary messengers or cyclic nucleotides, and free radicals. Microglia mediated immune activation is regulated by a positive feedback loop in NADDs. The present review focuses on evaluating the crosstalk between inflammatory mediators and microglia, which aggravates both the clinical progression and extent of NADDs by forming a persistent chronic inflammatory milieu within the CNS. We also discuss the role of the human gut microbiota and its effect on NADDs as well as the suitability of targeting toll-like receptors for an immunotherapeutic intervention targeting the deflation of an inflamed milieu within the CNS.

Microglia-Astrocyte Cross Talk through IL-18/IL-18R Signaling Modulates Migraine-like Behavior in Experimental Models of Migraine

Interleukin-18 (IL-18) is an important regulator of innate and immune responses, and is involved in the pain process, including neuropathic and cancer pain. The current study demonstrated that inflammatory soup (IS) dural infusions elicited the activation of microglia and astrocytes. In comparison, IS dural infusions induced the upregulation of IL-18 and IL-18R in microglia and astrocytes, respectively. Blocking the IL-18 signaling pathway attenuated nociceptive behavior. In comparison, blocking IL-18 signaling also suppressed the activation of astrocytes and nuclear factor-kappa B (NF-κB). IL-18 dural infusions induced nociceptive behavior and glia activation. IL-18 is a product of the activation of microglial toll-like receptor 4 (TLR4), and it acted on IL-18R expressed in astrocytes. Subsequently, it stimulated the activation of nuclear factor-kappa B (NF-κB), leading to the activation of astrocytes. In conclusion, IL-18-mediated microglia/astrocyte interactions in the medullary dorsal horn likely contribute to the development of hyperpathia or allodynia induced by migraines.

Involvement of the Hsp70/TLR4/IL-6 and TNF-α pathways in delayed-onset muscle soreness.

Delayed-onset muscle soreness (DOMS) is a very common condition in athletes and individuals not accustomed to physical activity that occurs after moderate/high-intensity exercise sessions. The activation of microglial Toll-like receptor 4 (TLR4) in the spinal cord has been described to be important for the induction and maintenance of persistent pain. Based on that, we hypothesize that 70 kilodalton heat-shock protein (Hsp70), a mediator released by exercise, could activate microglial TLR4 in the spinal cord, releasing proinflammatory cytokines and contributing to the start of DOMS. In fact, we found that the knockout of TLR4, myeloid differentiation primary response 88 (MyD88), interleukin-6 (IL-6), or both tumor necrosis factor-α (TNF-α) receptor 1 and TNF-α receptor 2 in mice prevented the development of DOMS following acute aerobic exercise in contrast to the findings in male C57BL/6 wild-type mice. Furthermore, DOMS in exercised wild-type mice was also prevented after pre-treatment with microglia inhibitor, TLR4 antagonist, and anti-Hsp70 antibody. During exercise-induced DOMS, Hsp70 mRNA, TLR4 mRNA, and protein levels, as well as Iba-1 (a microglial marker), IL-6, and TNF-α protein levels, were increased in the muscle and/or spinal cord. Together, these findings suggest that Hsp70 released during exercise-induced DOMS activates the microglial TLR4/IL-6/TNF-α pathway in the spinal cord. Thus, the blockade of TLR4 activation may be a new strategy to prevent the development of DOMS before intense exercise.

Defining the Mechanism of Subarachnoid Hemorrhage-Induced Pyrexia.

Fever can affect the majority of patients with subarachnoid hemorrhage (SAH) and many times no identifiable source is found for the fever whether infectious or sterile, like deep vein thrombosis. We hypothesized that fever in SAH is mediated by a NON-cyclo-oxygenase-dependent mechanism, which we neologized as subarachnoid hemorrhage-induced pyrexia (SAHiP). This hypothesis was investigated using genetically modified mice, pharmacological manipulation, cerebrospinal fluid from SAH patients, and a large cohort of SAH patients. Mice with deletions of neuronal prostaglandin EP3 receptor, global toll-like receptor 4 (TLR4), myeloid TLR4, and microglial TLR4 were subjected to SAH after being implanted with thermometers. Pathways necessary for SAHiP were identified. In SAH patients, cerebrospinal fluid was examined by flow cytometry and correlated with SAHiP. From a large cohort of SAH patients, independent associations with SAHiP were determined using logistic regression analysis. In our mouse model of SAH, microglial TLR4 is necessary for SAHiP, but independent of the neuronal prostaglandin EP3 receptor, cyclo-oxygenase, and prostaglandins. Macrophages from the cerebrospinal fluid of SAH patients with SAHiP expressed more TLR4-co-receptor than SAH patients without SAHiP. In a large cohort of SAH patients, SAHiP was found to be independently, yet inversely, associated with acetaminophen administration. SAHiP is independent of the neuronal prostaglandin EP3 receptor, cyclo-oxygenase, and prostaglandins, but dependent on microglial/macrophage TLR4 with evidence from both SAH mouse models and SAH patients.

Microglia clear neuron-released \u03b1-synuclein via selective autophagy and prevent neurodegeneration

Microglia maintain brain homeostasis by removing neuron-derived components such as myelin and cell debris. The evidence linking microglia to neurodegenerative diseases is growing; however, the precise mechanisms remain poorly understood. Herein, we report a neuroprotective role for microglia in the clearance of neuron-released α-synuclein. Neuronal α-synuclein activates microglia, which in turn engulf α-synuclein into autophagosomes for degradation via selective autophagy (termed synucleinphagy). Synucleinphagy requires the presence of microglial Toll-like receptor 4 (TLR4), which induces transcriptional upregulation of p62/SQSTM1 through the NF-κB signaling pathway. Induction of p62, an autophagy receptor, is necessary for the formation of α-synuclein/ubiquitin-positive puncta that are degraded by autophagy. Finally, disruption of microglial autophagy in mice expressing human α-synuclein promotes the accumulation of misfolded α-synuclein and causes midbrain dopaminergic neuron degeneration. Our study thus identifies a neuroprotective function of microglia in the clearance of α-synuclein via TLR4-NF-κB-p62 mediated synucleinphagy.

The role of TLR4 and HO-1 in neuroinflammation after subarachnoid hemorrhage.

This review on the mechanisms of neuroinflammation following subarachnoid hemorrhage will focus mainly on toll‐like receptor 4 (TLR4), Heme Oxygenase‐1 (HO‐1), and the role of microglia and macrophages in this process. Vasospasm has long been the focus of research in SAH; however, clinical trials have shown that amelioration of vasospasm does not lead to an improved clinical outcome. This necessitates the need for novel avenues of research. Our work has demonstrated that microglial TLR4 and microglial HO‐1, not only affects cognitive dysfunction, but also circadian dysrhythmia in a mouse model of SAH. To attempt to translate these findings, we have also begun investigating macrophages in the cerebrospinal fluid of SAH patients. The goal of this review is to provide an update on the role of TLR4, HO‐1, and other signal transduction pathways in SAH‐induced neuroinflammation.

Tenascin C regulates multiple microglial functions involving TLR4 signaling and HDAC1

Tenascin C (Tnc) is an extracellular matrix glycoprotein, expressed in the CNS during development, as well as in the setting of inflammation, fibrosis and cancer, which operates as an activator of Toll-like receptor 4 (TLR4). Although TLR4 is highly expressed in microglia, the effect of Tnc on microglia has not been elucidated to date. Herein, we demonstrate that Tnc regulates microglial phagocytic activity at an early postnatal age (P4), and that this process is partially dependent on microglial TLR4 expression. We further show that Tnc regulates proinflammatory cytokine/chemokine production, chemotaxis and phagocytosis in primary microglia in a TLR4-dependent fashion. Moreover, Tnc induces histone-deacetylase 1 (HDAC1) expression in microglia, such that HDAC1 inhibition by MS-275 decreases Tnc-induced microglial IL-6 and TNF-α production. Finally, Tnc−/− cortical microglia have reduced HDAC1 expression levels at P4. Taken together, these findings establish Tnc as a regulator of microglia function during early postnatal development.

TLR4 participates in sympathetic hyperactivity Post-MI in the PVN by regulating NF-κB pathway and ROS production

Sympathetic nerve hyperactivity is a primary reason for fatal ventricular arrhythmias (VAs) following myocardial infarction (MI). Pro-inflammatory cytokines produced in the paraventricular nucleus (PVN) post-MI are associated with sympathetic overexcitation; however, the precise mechanism needs further investigation. Our aim was to explore the mechanism of toll-like receptor 4 (TLR4) and its downstream molecular pathway in mediating sympathetic activity post-MI within the PVN. A rat MI model was developed via left anterior descending coronary artery ligation. TLR4 was primarily localized in microglia and increased markedly within the PVN at 3 days in MI rats. Sympathoexcitation also increased, as indicated by high levels of renal sympathetic nerve activity (RSNA) and norepinephrine (NE) concentration. TLR4 knockdown via shRNA microinjection to the PVN resulted in decreased activation of Fos protein (+) neurons in the PVN and peripheral sympathetic nerve activity. TLR4 knockdown also exhibited a lower arrhythmia score following programmed electrical stimulation than those treated with MI surgery only, indicating that the knockdown of TLR4 decreased the incidence of malignant ventricular arrhythmias following MI. LPS-induced inflammatory response was analyzed to explore the underlying mechanism of TLR4 in sympathetic hyperactivity. High levels of NF-κB protein, the pro-inflammatory cytokines IL-1β and TNF-α, and ROS production were observed in the LPS group. PVN-targeted injection of the NF-κB inhibitor PDTC attenuated NF-κB expression and sympathetic activity. Taken together, the results suggested that knockdown of microglial TLR4 within the PVN decreased sympathetic hyperactivity and subsequent VAs post-MI. The downstream NF-κB pathway and ROS production participated in the process. Interventions targeting TLR4 signaling in the PVN may be a novel approach to ameliorate the incidence of VAs post-MI.

Imbalance of Microglial TLR4/TREM2 in LPS-Treated APP/PS1 Transgenic Mice: A Potential Link Between Alzheimer's Disease and Systemic Inflammation.

Clinically, superimposed systemic inflammation generally has significant deleterious effects on the Alzheimer's disease (AD) progression. However, the related molecular mechanisms remain poorly understood. Microglial toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells 2 (TREM2) are two key regulators of inflammation that may play an essential role in this complex pathophysiological process. In this study, intraperitoneal injection of lipopolysaccharide (LPS) into APP/PS1 transgenic AD model was used to mimic systemic inflammation in the development of AD. Initial results from the cortex showed that compared with wild-type mice, APP/PS1 mice exhibited elevated gene and protein expression levels of both TLR4 and TREM2 with different degree. Interestingly, after LPS treatment, TLR4 expression was persistently up-regulated, while TREM2 expression was significantly down-regulated in APP/PS1 mice, suggesting that the negative regulatory effect of TREM2 on inflammation might be suppressed by LPS-induced hyperactive TLR4. This imbalance of TLR4/TREM2 contributed to microglial over-activation, followed by increased neuronal apoptosis in the cortex of APP/PS1 mice; these changes did not alter the expression level of Aβ1-42. Similar alterations were observed in our in vitro experiment with β-amyloid1-42 (Aβ1-42)-treated N9 microglia. Further, Morris water maze (MWM) testing data indicated that LPS administration acutely aggravated cognitive impairment in APP/PS1 mice, suggesting that the addition of systemic inflammation can potentially accelerate the progression of AD. Collectively, we conclude that an imbalance of TLR4/TREM2 may be a potential link between AD and systemic inflammation. TREM2 can serve as a potential therapeutic target for treating systemic inflammation in AD progression.