Triggering Receptor Expressed On Myeloid Cells 2 Research Articles

INPP5D regulates the amyloid pathology in Alzheimer’s disease.

AbstractBackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline, robust microgliosis, neuroinflammation, and neuronal loss. Genome‐wide association studies highlighted a prominent role for microglia in late‐onset AD (LOAD). Specifically, inositol polyphosphate‐5‐phosphatase (INPP5D) is selectively expressed in brain microglia and one of its common intronic variants (rs35349669; OR=1.08, 95%CI=1.06‐1.11) has been reported to be associated with increased risk of LOAD. INPP5D is linked to the triggering receptor expressed on myeloid cells 2 (TREM2) signaling, but little is known about the function of INPP5D in microglia and how INPP5D regulates TREM2‐related AD pathogenesis. Therefore, we aim to understand the role of INPP5D in microglia and AD pathology.MethodWe performed differential gene expression analysis to investigate INPP5D expression in LOAD and its association with plaque density using transcriptomic (RNA‐Seq) data from the Accelerating Medicines Partnership for Alzheimer’s Disease (AMP‐AD) cohort. We also performed quantitative real‐time PCR, immunoblotting, and immunofluorescence assays to assess INPP5D expression and microglial markers in the 5xFAD amyloid mouse model with INPP5D deficiency. Using the INPP5D knockout animal crossed with the 5xFAD, we perform the behavioral assays and IHC staining to reveal how the INPP5D regulates the amyloid pathology.Result INPP5D gene expression was upregulated in LOAD and positively correlated with the amyloid plaque density. Inpp5d expression increased along with the disease progression in the 5xFAD mice, and is selectively expressed in plaque‐associated microglia. Also, Inpp5d inhibition mitigated the plaque burdens in the 5xFAD mice and further protected against behavioral deficits induced by amyloid pathology.ConclusionOur findings show that INPP5D expression increases throughout the AD progression and is predominantly in the plaque‐associated microglia. Importantly, inhibited INPP5D expression reduces amyloid pathology, highlighting INPP5D as a potential therapeutic target.

INPP5D as a potential therapeutic target against Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline, robust microgliosis, neuroinflammation, and neuronal loss. Genome-wide association studies recently highlighted a prominent role for microglia in late-onset AD (LOAD). Specifically, inositol polyphosphate-5-phosphatase (INPP5D) is selectively expressed in brain microglia and one of its common intronic variants (rs35349669; OR=1.08, 95%CI=1.06-1.11) has been reported to be associated with increased risk of LOAD. INPP5D is linked to the triggering receptor expressed on myeloid cells 2 (TREM2) signaling, but little is known about the function of INPP5D in microglia and how INPP5D regulates TREM2-related AD pathogenesis. Therefore, we aim to understand the role of INPP5D in microglia and AD pathology. We performed differential gene expression analysis to investigate INPP5D expression in LOAD and its association with plaque density using transcriptomic (RNA-Seq) data from the Accelerating Medicines Partnership for Alzheimer's Disease (AMP-AD) cohort. We also performed quantitative real-time PCR, immunoblotting, and immunofluorescence assays to assess INPP5D expression and microglial markers in the 5xFAD amyloid mouse model with INPP5D deficiency. Treatment of fibril amyloid-beta (fAβ) on immortalized human microglial cell line (IMhu) was assessed to investigate the effect of INPP5D under amyloid pathology. INPP5D gene expression was upregulated in LOAD and positively correlated with amyloid plaque density. Inpp5d expression increased as the disease progressed in the 5xFAD mice, and selectively in plaque-associated microglia. Also, the protein expression of INPP5D was induced by fAβ treatment in IMhu cells. Inpp5d deficiency mitigated the plaque burdens and neuropathology in the 5xFAD mice and further protected against behavioral deficits induced by amyloid pathology. Our findings show that INPP5D expression increases throughout the AD progression and is predominantly in plaque-associated microglia. Importantly, inhibited INPP5D expression reduces amyloid pathology, highlighting INPP5D as a potential therapeutic target.

Older adults with Alzheimer’s disease exhibit an altered relationship between plasma sTREM2 and inflammatory markers

AbstractBackgroundAlzheimer’s disease (AD) is the 6th leading cause of death in the US, and currently lacks a blood‐based biomarker for early detection. Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune regulator shown to be protective against AD; a soluble form (sTREM2) circulates in peripheral blood and is a potential indicator of disease progression. Genetic variants of TREM2 are risk factors for AD and other neurodegenerative diseases. Multiple studies also have reported an altered peripheral immune response in AD. However, little is known about the relationship between plasma sTREM2 and the altered peripheral immune response in AD. Our objective was to examine the relationship between sTREM2 and inflammatory markers in a spectrum of older adults including cognitively normal (CN) controls, amnestic mild cognitive impairment (MCI) and AD. The hypothesis of this exploratory study was that TREM2‐related inflammatory activity is a stage specific (MCI or AD dementia) biomarker of AD.MethodStages of AD progression were characterized based on symptoms, designated in our study as CN controls, MCI, and AD. Alternatively, CSF amyloid beta, phosphorylated tau and total tau were measured, as described by the NIA‐AA Research Framework “ATN continuum”. Our study aims were to (1) define the relationship between sTREM2‐related inflammatory activity and AD clinical diagnosis, and (2) characterize sTREM2‐related inflammatory activity within the ATN continuum. Plasma sTREM2 and 38 cytokines were measured using the Luminex platform.ResultWe investigated this hypothesis in an exploratory cohort of CN, MCI, and AD subjects. We observed differential plasma sTREM2 and cytokines levels, correlations, and patterns across both AD groups defined by symptoms and the ATN continuum. This suggests the existence of alteration in normal peripheral sTREM2‐related inflammatory activity along AD and ATN stages.ConclusionThese data provide important support to the hypothesis that TREM2‐related inflammatory activity is a stage specific biomarker of AD progression. Our results provides the groundwork for future functional investigation and therapeutic strategy research.

Development and analytical performance testing of a novel sTREM2 ELISA method to support drug development

AbstractBackgroundThe triggering receptor expressed on myeloid cells 2 (TREM2) is a cell surface receptor of microglia cells in the central nerve system and is associated with neuroinflammation. sTREM2 can be used as pharmacodynamic biomarker for inflammation or microglial activation in therapeutic development programs. There is a need for well characterized assays that are suitable for measurement of neuroinflammatory biomarkers in pre‐clinical and clinical drug development. This abstract describes the characterization of a new robust sTREM2 prototype immunoassay based on monoclonal recombinant rabbit antibodies(mAbs).MethodA colorimetric sandwich ELISA was developed using capture mAb B02 (code ADx601) and detector mAb C10 (code ADx602) reactive towards the sTREM2 extracellular ectodomain (aa 19‐172). The analytical assay performance will be reported: the repeatability and reproducibility was determined using a neat CSF based QC panel (n=3; 6 test runs; duplicate testing). Accuracy was assessed by testing parallelism and calibrator point back calculated concentrations. Passing Bablok regression analysis was used to demonstrate consistency of sample testing results (43 samples; duplicate testing; 3 test runs). Finally clinical routine CSF samples were analysed (19 patients diagnosed with AD and 20 controls based upon biomarker profile).ResultA prototype sTREM2 ELISA assay was used to generate the assay characterization data: The %CV for repeatability respectively reproducibility were <12.2% and 15.4%. Accuracy: a slope value of 0.992 was obtained after Deming regression between the theoretical and experimentally obtained levels of 8 samples diluted over the measuring range (25‐3500 pg/mL) while the accuracy of the calibrator points was between 0 and 12.6% difference in back calculated concentration over 6 runs. Testing of the lot consistency (Figure 1) resulted in a slope and intercept value of respectively 1.07 (95%CI: 0.90‐1.17) and ‐80.9 (95%CI: ‐294.70‐154.30).Testing of clinical samples showed an increased sTREM2 concentration in AD versus controls (p<0.01).ConclusionA novel CSF prototype ELISA for sTREM2 was developed and characterized including lot consistency, precision, accuracy and others. The generated data indicate a robust sTREM2 immunoassay for use in drug development programs. The next step is the transfer to a manufacturing environment to make the assay available as a commercial test kit (RUO).

Transcriptomic Profiling Reveals a Role for TREM-1 Activation in Enterovirus D68 Infection-Induced Proinflammatory Responses.

Increasing cases related to the pathogenicity of Enterovirus D68 (EV-D68) have made it a growing worldwide public health concern, especially due to increased severe respiratory illness and acute flaccid myelitis (AFM) in children. There are currently no vaccines or medicines to prevent or treat EV-D68 infections. Herein, we performed genome-wide transcriptional profiling of EV-D68-infected human rhabdomyosarcoma (RD) cells to investigate host-pathogen interplay. RNA sequencing and subsequent experiments revealed that EV-D68 infection induced a profound transcriptional dysregulation of host genes, causing significantly elevated inflammatory responses and altered antiviral immune responses. In particular, triggering receptor expressed on myeloid cells 1 (TREM-1) is involved in highly activated TREM-1 signaling processes, acting as an important mediator in EV-D68 infection, and it is related to upregulation of interleukin 8 (IL-8), IL-6, IL-12p70, IL-1β, and tumor necrosis factor alpha (TNF-α). Further results demonstrated that NF-κB p65 was essential for EV-D68-induced TREM-1 upregulation. Moreover, inhibition of the TREM1 signaling pathway by the specific inhibitor LP17 dampened activation of the p38 mitogen-activated protein kinase (MAPK) signaling cascade, suggesting that TREM-1 mainly transmits activation signals to phosphorylate p38 MAPK. Interestingly, treatment with LP17 to inhibit TREM-1 inhibited viral replication and infection. These findings imply the pathogenic mechanisms of EV-D68 and provide critical insight into therapeutic intervention in enterovirus diseases.

Protective effect of quercetin against the metabolic dysfunction of glucose and lipids and its associated learning and memory impairments in NAFLD rats

BackgroundQuercetin (QUE) is a flavonol reported with anti-inflammatory and antioxidant activities, and previous results from the group of this study have demonstrated its neuroprotective effect against lipopolysaccharide-induced neuropsychiatric injuries. However, little is known about its potential effect on neuropsychiatric injuries induced or accompanied by metabolic dysfunction of glucose and lipids.MethodsA nonalcoholic fatty liver disease (NAFLD) rat model was induced via a high-fat diet (HFD), and glucolipid parameters and liver function were measured. Behavioral performance was observed via the open field test (OFT) and the Morris water maze (MWM). The plasma levels of triggering receptor expressed on myeloid cells-1 (TREM1) and TREM2 were measured via enzyme-linked immunosorbent assay (ELISA). The protein expression levels of Synapsin-1 (Syn-1), Synaptatogmin-1 (Syt-1), TREM1 and TREM2 in the hippocampus were detected using western blotting. Morphological changes in the liver and hippocampus were detected by HE and Oil red or silver staining.ResultsCompared with the control rats, HFD-induced NAFLD model rats presented significant metabolic dysfunction, hepatocyte steatosis, and impaired learning and memory ability, as indicated by the increased plasma concentrations of total cholesterol (TC) and triglyceride (TG), the impaired glucose tolerance, the accumulated fat droplets and balloon-like changes in the liver, and the increased escaping latency but decreased duration in the target quadrant in the Morris water maze. All these changes were reversed in QUE-treated rats. Moreover, apart from improving the morphological injuries in the hippocampus, treatment with QUE could increase the decreased plasma concentration and hippocampal protein expression of TREM1 in NAFLD rats and increase the decreased expression of Syn-1 and Syt-1 in the hippocampus.ConclusionsThese results suggested the therapeutic potential of QUE against NAFLD-associated impairment of learning and memory, and the mechanism might involve regulating the metabolic dysfunction of glucose and lipids and balancing the protein expression of synaptic plasticity markers and TREM1/2 in the hippocampus.

Age Matching Is Essential for the Study of Cerebrospinal Fluid sTREM2 Levels and Alzheimer's Disease Risk: A Meta-Analysis.

Background: Both the genetic and pathological studies link Alzheimer’s disease (AD) to the triggering receptor expressed on myeloid cells 2 (TREM2). A large number of studies have explored the value of cerebrospinal fluid (CSF) soluble TREM2 (sTREM2) levels as a biomarker for the diagnosis and prediction of AD; however, the findings are inconsistent. We aimed to review the studies that investigated the association of CSF sTREM2 levels and AD risk, and to provide the recommendations for future research.Methods and Results: A systematic literature search was performed using the MEDLINE, EMBASE, and Web of Science (all databases) databases. The meta-analysis for the association between the CSF sTREM2 levels and AD risk included 15 studies (17 comparisons) with a total of 1,153 cases and 1,626 controls. The total results showed that the higher CSF sTREM2 levels and AD risk were associated [standardized mean difference (SMD) = 0.428, 95% CI (0.213, 0.643), I2 = 81.1%]. However, the analysis of the subgroup of “age difference ≤ 2 years” indicated that sTREM2 was not associated with AD [SMD = 0.090, 95% CI (−0.092, 0.272), I2 = 27.4%] and had a significantly lower heterogeneity. Combining the results of the “age difference of 5–10 years” [SMD = 0.497, 95% CI (0.139, 0.855), I2 = 82.5%] and “age difference > 10 years” [SMD = 0.747, 95% CI (0.472, 1.023), I2 = 50.0%] subgroups showed that the difference in CSF sTREM2 between the AD and control groups was positively correlated with the age difference. A meta-regression analysis showed that the age difference can explain 33.4% of the between-study variance. By conducting further subgroup analyses of the five age-matched studies (495 cases and 364 controls) according to the measurement method, and whether inclusion criteria containing the requirement for pathological evidence of AD, no changes were observed in the corresponding pooled SMD or in the significance of the results. The meta-analysis result of “age difference ≤ 2 years” group was robust in the sensitivity analysis.Conclusion: The available high-quality evidence does not yet support an association between the CSF sTREM2 levels and AD risk. Age matching between the patients with AD and cognitively unimpaired controls was a major influencing factor in the results.

Soluble TREM-1, as a new ligand for the membrane receptor Robo2, promotes hepatic stellate cells activation and liver fibrosis.

Triggering receptor expressed on myeloid cells‐1 (TREM‐1) exists in two forms: a transmembrane form and a soluble form (sTREM‐1). The levels of sTREM‐1 are elevated in supernatants of activated HSCs. However, the role of sTREM‐1 in HSC activation and liver fibrosis remains undefined. Previous studies have primarily focused on the transmembrane form of TREM‐1; we innovatively observed the function of sTREM‐1 as a ligand in liver fibrosis and screened its receptor. Here, recombinant sTREM‐1 was used as a stimulator which induced HSC activation and further aggravated liver fibrosis. Then, screening for sTREM‐1 interacting membrane receptors was performed using pull‐down assay followed by mass spectrometry, and the membrane receptor roundabout guidance receptor 2 (Robo2) was identified as a candidate receptor for sTREM‐1. The interaction between sTREM‐1 and Robo2 was verified by pull‐down and immunofluorescence. The role of Robo2 on sTREM‐1‐induced HSC activation and its downstream signal pathways was assessed by knockdown of Robo2 in LX‐2 cells. Furthermore, HSC‐specific knockdown of Robo2 was achieved in a mouse model of liver fibrosis by using a recombinant adeno‐associated virus (AAV) vector to confirm the role of the receptor, and we proved that Robo2 knockdown inhibited the activation of HSC and liver fibrosis, which also led to the inactivation of Smad2/3 and PI3K/Akt pathways in sTREM‐1‐induced HSC activation and liver fibrosis. In conclusion, sTREM‐1 acts as a new ligand of Robo2; the binding of sTREM‐1 to Robo2 initiates the activation of the downstream Smad2/3 and PI3K/Akt signalling pathways, thereby promoting HSC activation and liver fibrosis.

Higher Expression of TREM-2 on M-MDSC Cell Surface Independently Predicts Worse Survival in Adults with Newly-Diagnosed and Treatment-Naïve Non-Hodgkin Lymphoma

IntroductionMyeloid-derived suppressive cells (MDSCs) represent a collection of myeloid-lineage immature cells owning immunosuppressive ability to facilitate tumor growth and can be phenotyped into two major groups: monocytic MDSC (M-MDSC) and granulocytic MDSC (G-MDSC). Earlier studies demonstrated the association between M-MDSC and prognosis in lymphoma patients. Triggering Receptors Expressed on Myeloid cells 2 (TREM-2) is a transmembrane glycoprotein and well-known to polarize macrophage to M2-like phenotype presenting anti-inflammation properties. Given that both the macrophage and M-MDSC belong to monocytic lineage, we wondered if TREM-2 was also expressed on M-MDSC cell surface and tried to elucidate the clinical impact of surface TREM-2 on M-MDSC derived from patients with newly-diagnosed and treatment-naïve non-Hodgkin lymphoma (NHL).MethodsThis was a prospective observational study conducted in a tertiary medical center (Taipei Veterans General Hospital) in Taiwan. Adult patients with newly-diagnosed and treatment-naïve NHL were eligible for enrollment from May 15, 2019 to February 28, 2021. Patients with HIV infection or other concurrent malignancies were excluded. The Mann-Whitney U test was used for quantitative data, the Kaplan-Meier estimate and log-rank test for survival data, and Cox regression models for hazard ratios (HRs).Human M-MDSC (defined as CD14 + CD11b + CD33 + HLA-DR - CD15 - cells, Figure A) was obtained from freshly isolated PBMC before initiating any treatment, including steroid. To increase inter-experiment reproducibility, the peripheral blood (PB) from the principal investigator (Hao-yuan Wang) was utilized as an internal control (Healthy Control), and every patient's blood sample was paired to the Healthy Control at the same performance of flow cytometric analysis to calculate the normalized MFI change of TREM-2 as the following: (Patient's TREM2 MFI - Healthy Control's TREM2 MFI) / (Healthy Control's TREM2 MFI) x100 %ResultsOne hundred and one adults with newly-diagnosed and treatment-naïve NHL were prospectively enrolled, with median age of 70 years, DLBCL as the most common pathological subtype (64.4%), 8.9% of patients as the T-cell lymphoma, 18.8% of patients being indolent subtypes, 52.5% of patients having stage IV, 36.6% of patients showing high-risk IPI score, and the median percentage of M-MDSC among peripheral-blood CD45 + cells indicating 0.45%.After a medium follow-up of 14.5 months, we confirmed that patients with higher percentage (>0.82%) of M-MDSC among peripheral-blood CD45 + cells had significantly worse overall survival (non-reach v.s. non-reach, P =0.011) when comparing to those with lower percentage (≦0.82%) of M-MDSC in PB.With regard to the expression of TREM-2 on M-MDSC cell surface, M-MDSC from patients with aggressive lymphoma showed significantly higher normalized MFI change of surface TREM-2 when comparing to M-MDSC from patients with indolent lymphoma (Figure B); the normalized MFI change of surface TREM-2 on M-MDSC derived from patients with higher IPI scores is also significantly higher than those from patients with lower IPI scores (Figure C). Furthermore, patients whose M-MDSCs showed higher normalized MFI change of TREM-2 had significantly worse progression-free and overall survival than those with lower normalized MFI change of surface TREM-2 (Figure D & E).To validate the clinical significance of TREM-2 on M-MDSC cell surface in adults with newly-diagnosed and treatment-naïve NHL, seven factors (age > 70 years, male gender, T-cell subtype, aggressive subtype, high-risk IPI score, percentage of M-MDSC among CD45 + cells in PB >0.82%, and normalized change of surface TREM-2 on M-MDSC >0%) were initially verified by univariate analysis and subsequently put into multivariate analysis if qualified, as shown by Table 1. At last, age (>70 years) and normalized change of TREM-2 on M-MDSC cell surface (>0%) were independent predictors of poor PFS and OS.Conclusion:For adults with newly-diagnosed and treatment-naïve NHL, patients with higher percentage of M-MDSC in PB is associated with worse prognosis; more importantly, high expression of TREM-2 on M-MDSC cell surface is an independent predictor for worse progression and overall survival. The functional role of TREM-2 on M-MDSC warranted further investigation for validating its possibility as an immunotherapeutic novel target. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

TREM-1 amplifies trophoblastic inflammation via activating NF-κB pathway during preeclampsia.

Excessive activation of maternal systemic inflammation is one of the underlying causes of pathology during the disease course of preeclampsia (PE). The triggering receptor expressed on myeloid cells-1 (TREM-1) participates in the development and persistence of inflammation. We hypothesized that dysregulated TREM-1 may be involved in the pathogenesis of PE by promoting the secretion of trophoblastic pro-inflammatory cytokines that augment inflammation. The localization of TREM-1 in placenta and the extravillous trophoblast cell line (TEV-1) was determined by immunohistochemical staining. The expression level of TREM-1 and pro-inflammatory cytokines in placentas were compared between normal pregnancies and PE. We used lipopolysaccharide (LPS) to simulate trophoblastic inflammation. TEV-1cells were transfected with TREM-1 plasmid and si-TREM-1 respectively, and then were incubated with LPS. The expression levels of pro-inflammatory cytokines and key molecules featured in nuclear transcription factor-kappaB (NF-κB) pathway were detected. Transwell assays were used to detect the effects of TREM-1 on cell migration and invasion. TREM-1 was localized on both villous trophoblasts (VTs) and extravillous trophoblasts (EVTs). TREM-1 and pro-inflammatory cytokines were up-regulated in preeclamptic placenta. Overexpression of TREM-1 promoted the activation of NF-κB pathway and the release of pro-inflammatory factors induced by LPS, and enhanced migration and invasion of TEV-1cells. Inhibition of TREM-1 significantly attenuated LPS-induced effects and suppressed migration and invasion. This study suggested that TREM-1 was up-regulated in PE, and may promote the production of downstream inflammatory factors by activating NF-κB pathway in trophoblastic cells, thus exerting pro-inflammatory effects in the pathogenesis of PE.

Differential Stimulation of Pluripotent Stem Cell-Derived Human Microglia Leads to Exosomal Proteomic Changes Affecting Neurons.

Microglial exosomes are an emerging communication pathway, implicated in fulfilling homeostatic microglial functions and transmitting neurodegenerative signals. Gene variants of triggering receptor expressed on myeloid cells-2 (TREM2) are associated with an increased risk of developing dementia. We investigated the influence of the TREM2 Alzheimer’s disease risk variant, R47Hhet, on the microglial exosomal proteome consisting of 3019 proteins secreted from human iPS-derived microglia (iPS-Mg). Exosomal protein content changed according to how the iPS-Mg were stimulated. Thus lipopolysaccharide (LPS) induced microglial exosomes to contain more inflammatory signals, whilst stimulation with the TREM2 ligand phosphatidylserine (PS+) increased metabolic signals within the microglial exosomes. We tested the effect of these exosomes on neurons and found that the exosomal protein changes were functionally relevant and influenced downstream functions in both neurons and microglia. Exosomes from R47Hhet iPS-Mg contained disease-associated microglial (DAM) signature proteins and were less able to promote the outgrowth of neuronal processes and increase mitochondrial metabolism in neurons compared with exosomes from the common TREM2 variant iPS-Mg. Taken together, these data highlight the importance of microglial exosomes in fulfilling microglial functions. Additionally, variations in the exosomal proteome influenced by the R47Hhet TREM2 variant may underlie the increased risk of Alzheimer’s disease associated with this variant.

Sphingosine-1-phosphate, a novel TREM2 ligand, promotes microglial phagocytosis to protect against ischemic brain injury

The mechanism of sphingosine-1-phosphate (S1P)-mediated phagocytosis remains unknown. Here, we found that S1P or FTY720 (an analog of S1P) promoted microglial phagocytosis in stroke independent of S1PRs. First, we used computer simulation of molecular docking to predict that S1P might be a ligand for triggering receptor expressed on myeloid cells 2 (TREM2). Next, microscale thermophoresis (MST), surface plasmon resonance (SPR) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) were performed to reveal that S1P was a novel TREM2 ligand. Then, we confirmed the pro-phagocytosis of S1P targeting in Trem2-Dap12 transfected CHO cells and TREM2 knockdown microglia. Point mutation analysis showed that D104 was the critical binding residue. Trem2−/− mice were used to demonstrate the role of S1P-induced phagocytosis targeting on TREM2 in protecting against ischemic brain injury. Finally, further studies revealed that apolipoprotein E (APOE) loaded with S1P was released by microglia and bound to apoptotic neurons via LDL receptor related protein 1B (LRP1B) and thereby induced microglia to phagocytose apoptotic neurons. Overall, the present work reveals for the first time that S1P acts as a novel endogenous ligand of TREM2 to effectively promote microglial phagocytosis. Our findings provide a new lead compound for developing immunomodulator targeting on TREM2.

TREM2 modulates differential deposition of modified and non-modified A\u03b2 species in extracellular plaques and intraneuronal deposits

Progressive accumulation of Amyloid-β (Aβ) deposits in the brain is a characteristic neuropathological hallmark of Alzheimer’s disease (AD). During disease progression, extracellular Aβ plaques undergo specific changes in their composition by the sequential deposition of different modified Aβ species. Microglia are implicated in the restriction of amyloid deposits and play a major role in internalization and degradation of Aβ. Recent studies showed that rare variants of the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) are associated with an increased risk for AD. Post-translational modifications of Aβ could modulate the interaction with TREM2, and the uptake by microglia. Here, we demonstrate that genetic deletion of TREM2 or expression of a disease associated TREM2 variant in mice lead to differential accumulation of modified and non-modified Aβ species in extracellular plaques and intraneuronal deposits. Human brains with rare TREM2 AD risk variants also showed altered deposition of modified Aβ species in the different brain lesions as compared to cases with the common variant of TREM2. These findings indicate that TREM2 plays a critical role in the development and the composition of Aβ deposits, not only in extracellular plaques, but also intraneuronally, that both could contribute to the pathogenesis of AD.

Spinal Neuronal GRK2 Contributes to Preventive Effect by Electroacupuncture on Cisplatin-Induced Peripheral Neuropathy in Mice.

BACKGROUND:The main symptoms of chemotherapy-induced peripheral neuropathy (CIPN) include pain and numbness. Neuronal G protein–coupled receptor kinase 2 (GRK2) plays an important role in various pain models. Cisplatin treatment can induce the activation of proinflammatory microglia in spinal cord. The purpose of this study was to investigate the role of spinal neuronal GRK2 in cisplatin-induced CIPN and in the prevention of CIPN by electroacupuncture (EA).METHODS:The pain and sensory deficit behaviors of mice were examined by von Frey test and adhesive removal test. The expression of neuronal GRK2 in the spinal cord is regulated by intraspinal injection of adeno-associated virus (AAV) containing neuron-specific promoters. The protein levels of GRK2, triggering receptor expressed on myeloid cells 2 (TREM2), and DNAX-activating protein of 12 kDa (DAP12) in spinal dorsal horn were detected by Western blot, the density of intraepidermal nerve fibers (IENFs) was detected by immunofluorescence, and microglia activation were evaluated by real-time polymerase chain reaction (PCR).RESULTS:In this study, cisplatin treatment led to the decrease of GRK2 expression in the dorsal horn of spinal cord. Overexpression of neuronal GRK2 in spinal cord by intraspinal injection of an AAV vector expressing GRK2 with human synapsin (hSyn) promotor significantly inhibited the loss of IENFs and alleviated the mechanical pain and sensory deficits induced by cisplatin. Real-time PCR analysis showed that the overexpression of neuronal GRK2 significantly inhibited the messenger RNA (mRNA) upregulation of proinflammatory cytokine interleukin (IL)-1β, IL-6, inducible nitric oxide synthase (iNOS), and M1 microglia marker cluster of differentiation (CD)16 induced by cisplatin. Furthermore, the TREM2 and DAP12, which has been demonstrated to play a role in microglia activation and in the development of CIPN, were also downregulated by overexpression of neuronal GRK2 in this study. Interestingly, preventive treatment with EA completely mimics the effect of overexpression of neuronal GRK2 in the spinal cord in this mouse model of cisplatin-induced CIPN. EA increased GRK2 level in spinal dorsal horn after cisplatin treatment. Intraspinal injection of AAV vector specifically downregulated neuronal GRK2, completely reversed the regulatory effect of EA on CIPN and microglia activation. All these indicated that the neuronal GRK2 mediated microglial activation contributed to the process of CIPN.CONCLUSIONS:Neuronal GRK2 in the spinal cord contributed to the preventive effect of EA on CIPN. The neuronal GRK2 may be a potential target for CIPN intervention.

TREM1 Regulates Neuroinflammatory Injury by Modulate Proinflammatory Subtype Transition of Microglia and Formation of Neutrophil Extracellular Traps via Interaction With SYK in Experimental Subarachnoid Hemorrhage.

Neuroinflammation is a key process in the pathogenesis of subarachnoid hemorrhage (SAH) and contributes to poor outcome in patients. The purpose of this study is to explore the effect of triggering receptor expressed on myeloid cells 1 (TREM1) in the SAH, as well as its potential mechanism. In our study, plasma levels of soluble TREM1 was increased significantly after SAH and correlated to SAH severity and serum C-reactiveprotein. TREM1 inhibitory peptide LP17 alleviated the neurological deficits, attenuated brain water content, and reduced neuronal damage after SAH. Meanwhile, TREM1 inhibitory peptide decreased neuroinflammation (evidenced by the decreased levels of markers including IL-6, IL-1β, TNF-α) by attenuating proinflammatory subtype transition of microglia (evidenced by the decreased levels of markers including CD68, CD16, CD86) and decreasing the formation of neutrophil extracellular traps (evidenced by the decreased levels of markers including CitH3, MPO, and NE). Further mechanistic study identified that TREM1 can activate downstream proinflammatory pathways through interacting with spleen tyrosine kinase (SYK). In conclusion, inhibition of TREM1 alleviates neuroinflammation by attenuating proinflammatory subtype transition of microglia and decreasing the formation of neutrophil extracellular traps through interacting with SYK after SAH. TREM1 may be a a promising therapeutic target for SAH.

Keratinocytes and Activation of TREM-1 Pathway in Cutaneous Leishmaniasis Lesions

Triggering Receptor Expressed on Myeloid Cells 1 (TREM-1) amplifies the immune response, operating synergistically with Toll-Like Receptors (TLRs) in the production of inflammatory mediators. TREM-1 signaling depends on the adapter protein DAP12, which results in the activation of NFkB, the expression of inflammatory genes, and the release of antimicrobial peptides, such as Beta-defensin 2. We evaluated the activation of the TREM-1 signaling pathways in Cutaneous Leishmaniasis (CL) caused by Leishmania braziliensis and linage human keratinocytes exposed to these parasites since the host immune response against Leishmania plays a critical role in promoting parasite killing but also participates in inflammation and tissue damage. We analyzed publicly available transcriptome data from the lesions of CL patients. In the CL biopsies, we found increased expression of the molecules involved in the TREM-1 pathway. We then validated these findings with RT-qPCR and immunohistochemistry in newly obtained biopsies. Surprisingly, we found a strong labeling of TREM-1 in keratinocytes, prompting the hypothesis that increased TREM-1 activation may be the result of tissue damage. However, increased TREM-1 expression was only seen in human lineage keratinocytes following parasite stimulation. Moreover, no up-regulation of TREM-1 expression was observed in the skin lesions caused by other non-infectious inflammatory diseases. Together, these findings indicate that L. braziliensis (Lb) induces the expression of the TREM-1 receptor in tissue keratinocytes regardless of tissue damage, suggesting that non-immune skin cells may play a role in the inflammatory response of CL.

Identification and Validation of TREM2 in Intracranial Aneurysms.

Background: Intracranial aneurysm (IA) is a cerebrovascular disease that seriously endangers human heath and life. However, the pathogenesis of IA has not been clarified. Objective: In this study, we explored the role of the triggering receptor expressed on myeloid cells-2 (TREM2) gene to explore a novel mechanism underlying IA. Methods: First, we verified the role of the candidate gene, TREM2 in a modified mouse model of IA. Second, we verified elevated expression of TREM2 using the Gene Expression Omnibus (GEO) database (GSE54083 and GSE75436) and developed protein interaction (PPI) network analysis using the top one hundred DEGs from GSE75436 dataset. Finally, we predicted a likely mechanism by which TREM2 is involved in the pathology of IA using single-gene Gene Set Enrichment Analysis (GSEA). Results: The expression of TREM2 and inflammatory factors was significantly increased in the modified mouse IA model, and showed a positive correlation. Elevated expression of TREM2 was also found in IA patients tissues from the GSE54083 and GSE75436 data sets. PPI network analyses suggested that the DEGs were involved in a variety of inflammatory processes. The GSEA results suggest that TREM2 may participate in IA progression by regulating macrophage function. Conclusion: TREM2 is highly expressed in both human and mouse IA tissues, and may participate in IA progression by regulating macrophage function and inflammatory factor expression. The molecular mechanism of TREM2 involvement in the IA process can be further studied using our modified mouse IA model.

Discovery of Active Ingredients Targeted TREM2 by SPR Biosensor-UPLC/MS Recognition System, and Investigating the Mechanism of Anti-Neuroinflammatory Activity on the Lignin-Amides from Datura metel Seeds.

As a new target protein for Alzheimer’s disease (AD), the triggering receptor expressed on myeloid Cells 2 (TREM2) was expressed on the surface of microglia, which was shown to regulate neuroinflammation, be associated with a variety of neuropathologic, and regarded as a potential indicator for monitoring AD. In this study, a novel recognition system based on surface plasmon resonance (SPR) for the TREM2 target spot was established coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-MS), in order to screen the active ingredients targeting TREM2 from Datura metel seeds. The results showed that four lignan-amides were discovered as candidate compounds by SPR biosensor-UPLC/MS recognition analysis. According to the guidance of the active ingredients discovered by the system, the lignin-amides from Datura metel seeds (LDS) were preliminarily identified as containing 27 lignan-amides, which were enriched compositions by the HP-20 of Datura metel seeds. Meanwhile, the anti-inflammatory activity of LDS was evaluated in BV2 microglia induced by LPS. Our experimental results demonstrated that LDS could reduce NO release in LPS-treated BV2 microglia cells and significantly reduce the expression of the proteins of inducible Nitric Oxide Synthase (iNOS), cyclooxygenase 2 (COX-2), microtubule-associated protein tau (Tau), and ionized calcium-binding adapter molecule 1 (IBA-1). Accordingly, LDS might increase the expression of TREM2/DNAX-activating protein of 12 kDa (DAP12) and suppress the Toll-like receptor SX4 (TLR4) pathway and Recombinant NLR Family, Pyrin Domain Containing Protein 3 (NLRP3)/cysteinyl aspartate specific proteinase-1 (Caspase-1) inflammasome expression by LDS in LPS-induced BV2 microglial cells. Then, the inhibitory release of inflammatory factors Interleukin 1 beta (IL-1β), Interleukin 6 (IL-6), and Tumor necrosis factor-alpha (TNFα) inflammatory cytokines were detected to inhibit neuroinflammatory responses. The present results propose that LDS has potential as an anti-neuroinflammatory agent against microglia-mediated neuroinflammatory disorders.

Soluble Triggering Receptor Expressed on Myeloid Cells 2 From Cerebrospinal Fluid in Sleep Disorders Related to Parkinson's Disease.

Background: Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial receptor exclusively expressed in the central nervous system (CNS). It contributes to abnormal protein aggregation in neurodegenerative disorders, but its role in Parkinson’s disease (PD) is still unclear.Methods: In this case-control study, we measured the concentration of the soluble fragment of TREM2 (sTREM2) in PD patients, evaluated their sleep conditions by the PD sleep scale (PDSS), and analyzed the relationship between sTREM2 and PD symptoms.Results: We recruited 80 sporadic PD patients and 65 healthy controls without disease-related variants in TREM2. The concentration of sTREM2 in the CSF was significantly higher in PD patients than in healthy controls (p < 0.01). In the PD group, the concentration of sTREM2 had a positive correlation with α-syn in the CSF (Pearson r = 0.248, p = 0.027). Receiver operating characteristic curve (ROC) analyses showed that sTREM2 in the CSF had a significant diagnostic value for PD (AUC, 0.791; 95% CI, 0.711–0.871, p < 0.05). The subgroup analysis showed that PD patients with sleep disorders had a significantly higher concentration of sTREM2 in their CSF (p < 0.01). The concentration of sTREM2 in the CSF had a negative correlation with the PDSS score in PD patients (Pearson r = −0.555, p < 0.01). The ROC analyses showed that sTREM2 in the CSF had a significant diagnostic value for sleep disorders in PD (AUC, 0.733; 95% CI, 0.619–0.846, p < 0.05).Conclusion: Our findings suggest that CSF sTREM2 may be a potential biomarker for PD and it could help predict sleep disorders in PD patients, but multicenter prospective studies with more participants are still needed to confirm its diagnostic value in future.

Reduced TREM2 activation in microglia of patients with Alzheimer's disease.

Loss‐of‐function variants of triggering receptor expressed on myeloid cells 2 (TREM2) increase the risk of developing Alzheimer's disease (AD). The mechanism through which TREM2 contributes to the disease (TREM2 activation vs inactivation) is largely unknown. Here, we analyzed changes in a gene set downstream of TREM2 to determine whether TREM2 signaling is modified by AD progression. We generated an anti‐human TREM2 agonistic antibody and defined TREM2 activation in terms of the downstream expression changes induced by this antibody in microglia developed from human induced pluripotent stem cells (iPSC). Differentially expressed genes (DEGs) following TREM2 activation were compared with the gene set extracted from microglial single nuclear RNA sequencing data of patients with AD, using gene set enrichment analysis. We isolated an anti‐TREM2‐specific agonistic antibody, Hyb87, from anti‐human TREM2 antibodies generated using binding and agonism assays, which helped us identify 300 upregulated and 251 downregulated DEGs. Pathway enrichment analysis suggested that TREM2 activation may be associated with Th2‐related pathways. TREM2 activation was lower in AD microglia than in microglia from healthy subjects or patients with mild cognitive impairment. TREM2 activation also showed a significant negative correlation with disease progression. Pathway enrichment analysis of DEGs controlled by TREM2 activity indicated that TREM2 activation in AD may lead to anti‐apoptotic signaling, immune response, and cytoskeletal changes in the microglia. We showed that TREM2 activation decreases with AD progression, in support of a protective role of TREM2 activation in AD. In addition, the agonistic anti‐TREM2 antibody can be used to identify TREM2 activation state in AD microglia.