Trem2 promotes anti-inflammatory responses in microglia and is suppressed under pro-inflammatory conditions

In the paper

“In addition, the soluble TREM2 protein level in the conditioned medium in response to LPS was decreased for the microglia from the WT littermates (Fig. 3D), and the level of TREM2 protein in the medium conditioned by cells from the homozygous Trem2 R47H KI mice was also decreased, supporting the transcriptional findings.”

In the paper

“Similar results were seen in microglia from the homozygous Trem2 R47H KI mice with no substantial change to Tnf expression but a decrease in Il1b expression (Supplementary Material, Fig. S7).”

In the paper

“The other anti-inflammatory marker Tgfb1 was significantly decreased in homozygous Trem2 R47H microglia independent of IL-4-treatment, and the fold-change in Tgfb1 up-regulation in response to treatment was similar for WT and homozygous Trem2 R47H cells (Fig. 5B).”

In the paper

“Arg1 expression was undetectable in our non-stimulated microglia, but reached levels well above detection threshold at 24 h after IL-4 treatment, and by 48 h expression levels had further doubled.”

In the paper

“Consistent with the transcriptional results, the protein level of TNF-alpha released from the microglial cells showed that LPS strongly up-regulated TNF-alpha, but there was no significant change caused by Trem2 knockdown (Supplementary Material, Fig. S6).”

In the paper

“The Tgfb1 expression also showed an increase with IL-4 stimulation, however, in contrast to Arg1 the effect of Trem2 knockdown and IL-4 showed no interaction.”

In the paper

“Interestingly, the expression of all of these genes was significantly lower in cells with acute Trem2 knockdown, and was not dependent on IL-4 stimulation (no significant interaction of knockdown versus IL-4).”

In the paper

“Upon IL-4 treatment, Arg1 expression showed a time-dependent up-regulation, however, this up-regulation was considerably decreased in the Trem2 knockdown group (two-way ANOVA: interaction between main effects of Trem2 knockdown and IL-4 treatment time, P = 0.05; Sidak’s multiple comparisons: significant difference between the Trem2 knockdown group and the control at 48 h post IL-4 treatment, P < 0”

In the paper

“Our findings were similar in the microglia from the Trem2 R47H KI mice, with Arg1 showing a strong induction by IL-4 treatment and the down-regulation of Trem2 in the microglia from Trem2 R47H KI mice significantly preventing this change in a gene dose-dependent manner (Fig. 5B, one-way ANOVA: P = 0.002, Sidak’s multiple comparisons: P = 0.08 for WT versus heterozygous, and P = 0.01 for WT versus”

In the paper

“This change was not primarily due to the total microglia number change because the proportion of CD68 positive microglia also showed a substantial decrease (two-way ANOVA: main effect of genotype P < 0.001; Fig. 1B and C).”

In the paper

“RNA-seq analysis showed that IL-4 induced the expression of a program of genes including Arg1 and Ap1b1 in microglia, which showed an attenuated response to IL-4 when Trem2 expression was decreased.”

In the paper

“IL-4 caused robust phosphorylation of STAT6, with phosphorylated STAT6 not detectable in the absence of IL-4 but clearly measurable 48 h after IL-4 treatment.”

In the paper

“While there was an overlap of genotype and IL-4 affecting common genes (such as Arg1), we also saw that Trem2 and IL-4 had independent effects. Thus, the strong drive of Trem2 R47H KI genotype and IL-4 meant there was no module that was significant for both genotype and IL-4 treatment.”

In the paper

“both manipulations displayed strong effects on gene expression.”

In the paper

“Genes showing increased expression in the Trem2 R47H KI microglia included App (amyloid precursor protein), Hspa1b (heat-shock 70 kDa protein 1B) and Ccnd2 (cyclin D2) (408 genes with increased expression, DESeq2 FDR < 0.05; Fig. 6A; Supplementary Material, Table S2).”

In the paper

“Igf1 expression was also decreased when Trem2 expression was knocked-down or if the Trem2 R47H variant was carried by the primary microglial cultures.”

In the paper

“the total AKT protein level was not significantly affected by Trem2 knockdown, although there was a slight increase in AKT levels in response to IL-4 treatment.”

In the paper

“we saw a decrease in the density of microglia in vivo in the hippocampus, and that the remaining microglia had dramatically reduced CD68 protein levels.”

In the paper

“Our findings, in line with previous studies (59,68–71), show that Igf1 expression is inhibited by pro-inflammatory stimuli and enhanced in anti-inflammatory environments.”

What the sources say

• Not checked yetcites [59] Butovsky, 2018, Microglial signatures and their role in health and disease, Nat. Rev. Neurosci. source
• Not checked yetcites [68] Arkins, 1995, Interferon-gamma inhibits macrophage insulin-like growth factor-I synthesis at the transcriptional level, Mol. Endocrinol.
• Not checked yetcites [69] Suh, 2013, Insulin-like growth factor 1 and 2 (IGF1, IGF2) expression in human microglia: differential regulation by inflammatory mediators, J. Neuroinflammation source
• Not checked yetcites [70] Wynes, 2003, Induction of macrophage insulin-like growth factor-I expression by the Th2 cytokines IL-4 and IL-13, J. Immunol. source
• Not checked yetcites [71] Barrett, 2015, Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp. Cell Res. source

In the paper

“In hippocampal homogenates from young adult Trem2 R47H mice, we did not see differences in expression of the genes we studied, including Stat6 and Igf1, despite a slight but significant reduction in the number of microglia.”

In the paper

“In the presence of LPS, decrease in expression of Trem2 began as early as 3 h after LPS application and reached more than an 85% decrease by 24 h.”

In the paper

“In contrast to IL-4 stimulation, which had little effect on Trem2 expression, LPS largely suppressed Trem2.”

In the paper

“These findings suggest that separate mechanisms regulate basal levels of some genes like Tgfb1 (which may be Trem2-dependent at least in part), compared with mechanisms that regulate levels following induction (which can be Trem2-independent).”

In the paper

“It is important to note that in the hippocampal tissue, the gene expression in microglia is greatly diluted by the much greater contribution of other cell types and so small changes in gene expression will be much more difficult to detect than in enriched microglia.”

In the paper

“This suggests that pro-inflammatory conditions inhibit microglial Trem2 expression, which might result in a state of temporary or chronic ‘TREM2-deficiency’ in microglia.”

In the paper

“TREM2 promotes an anti-inflammatory response via two mechanisms: (i) an IL-4 independent mechanism supporting the expression of common genes, such as Igf1, Cd68 and C1qa, which are induced by IL-4 in control cells and suppressed by Trem2 reduction, but there is lack of interaction between the effects of Trem2 and IL-4 on these genes, and so here TREM2 and IL-4 likely act via independent parallel”

In the paper

“(ii) an IL-4-dependent mechanism where the gene expression response to IL-4 depends on TREM2, genes such as Arg1, where there is a significant interaction between the effects of Trem2 and IL-4.”

In the paper

“It has been reported to be widely expressed in various cell types derived from the myeloid lineage, such as bone osteoclasts (12,13), and lung and peritoneal macrophages (14).”

What the sources say

• Not checked yetcites [12] Humphrey, 2006, TREM2, a DAP12-associated receptor, regulates osteoclast differentiation and function, J. Bone Miner. Res. source
• Not checked yetcites [13] Paloneva, 2003, DAP12/TREM2 deficiency results in impaired osteoclast differentiation and osteoporotic features, J. Exp. Med. source
• Not checked yetcites [14] Turnbull, 2006, Cutting edge: TREM-2 attenuates macrophage activation, J. Immunol. source

In the paper

“The same study also showed that Trem2 knockdown via a lentiviral strategy increased pro-inflammatory gene expression and decreased phagocytosis of labeled apoptotic neuronal membranes in primary microglia stressed with co-culture of apoptosis-induced primary neurons.”

What the sources say

• Not checked yetcites [17] Takahashi, 2005, Clearance of apoptotic neurons without inflammation by microglial triggering receptor expressed on myeloid cells-2, J. Exp. Med. source

In the paper

“LPS-induced pro-inflammatory stimulation suppressed Trem2 expression, thus preventing TREM2’s anti-inflammatory drive.”

In the paper

“TREM2 was also found to suppress LPS-induced pro-inflammatory markers up-regulated in primary alveolar macrophages in vitro (28), and the BV-2 microglial cell line (29).”

What the sources say

• Not checked yetcites [28] Gao, 2013, Silencing of triggering receptor expressed on myeloid cells-2 enhances the inflammatory responses of alveolar macrophages to lipopolysaccharide, Mol. Med. Rep. source
• Not checked yetcites [29] Zhong, 2015, DAP12 stabilizes the C-terminal fragment of the triggering receptor expressed on myeloid cells-2 (TREM2) and protects against LPS-induced pro-inflammatory response, J. Biol. Chem. source

In the paper

“Interestingly, the effects of IL-4 stimulation on gene expression mimic the changes seen in association with plaque development in transgenic mice (44), where Trem2 levels are also strongly increased, whereas the pro-inflammatory effects of LPS have the opposite effect.”

What the sources say

• Not checked yetcites [44] Salih, 2019, Genetic variability in response to amyloid beta deposition influences Alzheimer's disease risk, Brain Commun. source

In the paper

“Evidence from in vitro studies has accumulated to support the role of TREM2 in restricting inflammation and promoting phagocytosis.”

In the paper

“TREM2 expression was first observed in macrophages and dendritic cells, but not granulocytes or monocytes (9–11).”

What the sources say

• Not checked yetcites [9] Bouchon, 2000, Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes, J. Immunol. source
• Not checked yetcites [10] Bouchon, 2001, A DAP12-mediated pathway regulates expression of CC chemokine receptor 7 and maturation of human dendritic cells, J. Exp. Med. source
• Not checked yetcites [11] Daws, 2001, Cloning and characterization of a novel mouse myeloid DAP12-associated receptor family, Eur. J. Immunol. source

In the paper

“In addition to the R47H variant, further TREM2 variants have been revealed to be risk factors of AD or other CNS diseases, such as R62H (8,20), H157Y (25), Q33X, T66M and Y38C (26,27).”

What the sources say

• Not checked yetcites [8] Sims, 2017, Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease, Nat. Genet. source
• Not checked yetcites [20] Jin, 2014, Coding variants in TREM2 increase risk for Alzheimer's disease, Hum. Mol. Genet. source
• Not checked yetcites [25] Jiang, 2016, A rare coding variant in TREM2 increases risk for Alzheimer's disease in Han Chinese, Neurobiol. Aging source
• Not checked yetcites [26] Guerreiro, 2013, Novel compound heterozygous mutation in TREM2 found in a Turkish frontotemporal dementia-like family, Neurobiol. Aging source
• Not checked yetcites [27] Guerreiro, 2013, Using exome sequencing to reveal mutations in TREM2 presenting as a frontotemporal dementia-like syndrome without bone involvement, JAMA Neurol. source

In the paper

“It was first reported in a study using primary microglia transduced with flag epitope tagged TREM2 that TREM2 stimulated microglial chemotaxis, and phagocytosis of fluorescent beads (17).”

What the sources say

• Not checked yetcites [17] Takahashi, 2005, Clearance of apoptotic neurons without inflammation by microglial triggering receptor expressed on myeloid cells-2, J. Exp. Med. source

In the paper

“However, other cellular functions of TREM2, particularly anti-inflammatory or alternative activation, and the molecular mechanisms mediating the actions of TREM2, are not well understood.”

In the paper

“The expression level of the Stat6 gene was also decreased with reduced Trem2 expression (Fig. 9C), and so confirms the decreased levels of the translated protein.”

In the paper

“the genes down-regulated in cells carrying the Trem2 R47H KI alleles were associated with microglial activation, suggesting down-regulation of genes with reduced Trem2 expression may dampen some states of microglial activation (Supplementary Material, Fig. S12).”

In the paper

“Recently, it was further shown that apolipoproteins, lipids and Aβ itself acted as ligands of TREM2 to facilitate microglial phagocytosis and clearance of Aβ and apoptotic neurons (32–35).”

What the sources say

• Not checked yetcites [32] Atagi, 2015, Apolipoprotein E is a ligand for triggering receptor expressed on myeloid cells 2 (TREM2), J. Biol. Chem. source
• Not checked yetcites [33] Yeh, 2016, TREM2 binds to apolipoproteins, including APOE and CLU/APOJ, and thereby facilitates uptake of amyloid-Beta by microglia, Neuron source
• Not checked yetcites [34] Zhao, 2018, TREM2 is a receptor for beta-amyloid that mediates microglial function, Neuron source
• Not checked yetcites [35] Nugent, 2020, TREM2 regulates microglial cholesterol metabolism upon chronic phagocytic challenge, Neuron source

In the paper

“These results suggest that TREM2 deficiency leads to a smaller STAT6 pool in microglia, resulting in decreased amount of phosphorylated STAT6 upon IL-4 stimulation.”

In the paper

“In general, these studies suggest that Trem2 deficiency exacerbates microglial pro-inflammatory activation in response to various stimuli, and accompanies impaired phagocytosis.”

In the paper

“previous studies have shown that phosphorylation of STAT6 is a key step downstream in the IL-4 cascade that leads to Arg1 expression (49–51).”

What the sources say

• Not checked yetcites [49] Luzina, 2012, Regulation of inflammation by interleukin-4: a review of "alternatives", J. Leukoc. Biol. source
• Not checked yetcites [50] Nelms, 1999, The IL-4 receptor: signaling mechanisms and biologic functions, Annu. Rev. Immunol. source
• Not checked yetcites [51] Gordon, 2010, Alternative activation of macrophages: mechanism and functions, Immunity source

In the paper

“Trem2 knockdown also decreased the total levels of phosphorylated STAT6 but this could be explained by a decrease in the overall levels of STAT6 rather than an effect on phosphorylation per se.”

In the paper

“Pharmacological blockade of CSF1 receptors has been shown to impair microglial survival and can completely remove microglia from the mouse brain (53–55).”

What the sources say

• Not checked yetcites [53] Elmore, 2014, Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain, Neuron source
• Not checked yetcites [54] Dagher, 2015, Colony-stimulating factor 1 receptor inhibition prevents microglial plaque association and improves cognition in 3xTg-AD mice, J. Neuroinflammation source
• Not checked yetcites [55] Olmos-Alonso, 2016, Pharmacological targeting of CSF1R inhibits microglial proliferation and prevents the progression of Alzheimer's-like pathology, Brain source

In the paper

“While this may not affect microglial survival as the IGF1 receptor is found largely on neurons, it could contribute to neurodegeneration, being an important factor in neuronal survival and lifespan across many species (67).”

What the sources say

• Not checked yetcites [67] Broughton, 2009, Insulin/IGF-like signalling, the central nervous system and aging, Biochem. J. source

In the paper

“Interestingly no difference in Csf1r expression was seen in the remaining microglia in the brain tissue from the young adult Trem2 R47H KI mice, possibly suggesting different populations of microglia, with loss of those more vulnerable to the effects of Trem2 knockdown than others, at least in whole tissue.”

In the paper

“Thus, the Igf1 expression decrease caused by Trem2 deficiency may suggest a microglial phenotype shift towards pro-inflammatory activation and away from non-inflammatory, and a decrease of the pro-survival role of IGF1 on neurons.”

In the paper

“Hence, we can largely consider these mice as a model of Trem2 down-regulation, validated by our siRNA data in primary microglia.”

In the paper

“considering that PI3K-AKT signaling is downstream of TREM2”

What the sources say

• Not checked yetcites [40] Zheng, 2017, TREM2 promotes microglial survival by activating Wnt/beta-catenin pathway, J. Neurosci. source
• Not checked yetcites [52] Paradowska-Gorycka, 2013, Structure, expression pattern and biological activity of molecular complex TREM-2/DAP12, Hum. Immunol. source

In the paper

“Igf1 is abundantly expressed by microglia during postnatal development, aging or following brain injuries and plays a vital role in promoting neuronal survival (59,63–66).”

What the sources say

• Not checked yetcites [59] Butovsky, 2018, Microglial signatures and their role in health and disease, Nat. Rev. Neurosci. source
• Not checked yetcites [63] Ueno, 2013, Layer V cortical neurons require microglial support for survival during postnatal development, Nat. Neurosci. source
• Not checked yetcites [64] Fernandez, 2012, The many faces of insulin-like peptide signalling in the brain, Nat. Rev. Neurosci. source
• Not checked yetcites [65] Kohman, 2012, Wheel running attenuates microglia proliferation and increases expression of a proneurogenic phenotype in the hippocampus of aged mice, Brain Behav. Immun. source
• Not checked yetcites [66] Thored, 2009, Long-term accumulation of microglia with proneurogenic phenotype concomitant with persistent neurogenesis in adult subventricular zone after stroke, Glia source

In the paper

“Differences in gene expression profiles of different microglial populations have been recently described using single cell RNA-seq showing some microglial populations are selectively dependent on Trem2 (58–62).”

What the sources say

• Not checked yetcites [58] Keren-Shaul, 2017, A unique microglia type associated with restricting development of Alzheimer's disease, Cell source
• Not checked yetcites [59] Butovsky, 2018, Microglial signatures and their role in health and disease, Nat. Rev. Neurosci. source
• Not checked yetcites [60] Sala Frigerio, 2019, The major risk factors for Alzheimer's disease: age, sex, and genes modulate the microglia response to Abeta plaques, Cell Rep. source
• Not checked yetcites [61] Zhou, 2020, Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and TREM2-independent cellular responses in Alzheimer's disease, Nat. Med. source
• Not checked yetcites [62] Hammond, 2019, Single-cell RNA sequencing of microglia throughout the mouse lifespan and in the injured brain reveals complex cell-state changes, Immunity source

In the paper

“IL-33 ameliorated AD-associated phenotypes by promoting anti-inflammatory functions, which included induction of Arg1 (80).”

What the sources say

• Not checked yetcites [80] Fu, 2016, IL-33 ameliorates Alzheimer's disease-like pathology and cognitive decline, Proc. Natl. Acad. Sci. USA source

In the paper

“ARG1 in myeloid cells mainly functions as an enzyme to hydrolyze arginine to produce ornithine as part of the urea cycle, which both promotes tissue repair through generation of polyamines and collagens, and also reduces nitric oxide production from inducible nitric oxide synthase through decreasing intracellular arginine availability (76–78).”

What the sources say

• Not checked yetcites [76] Yang, 2014, Functions of arginase isoforms in macrophage inflammatory responses: impact on cardiovascular diseases and metabolic disorders, Front. Immunol. source
• Not checked yetcites [77] Mills, 2000, M-1/M-2 macrophages and the Th1/Th2 paradigm, J. Immunol. source
• Not checked yetcites [78] Modolell, 1995, Reciprocal regulation of the nitric oxide synthase/arginase balance in mouse bone marrow-derived macrophages by TH1 and TH2 cytokines, Eur. J. Immunol. source

In the paper

“Dusp4 null mice show impaired T cell activation (83).”

What the sources say

• Not checked yetcites [83] Barbour, 2016, MAP kinase phosphatase 2 deficient mice develop attenuated experimental autoimmune encephalomyelitis through regulating dendritic cells and T cells, Sci. Rep. source

In the paper

“work with a microglial cell line suggests that Aβ binding to scavenger receptors induces oxidative stress, which then may impair AP1B1-dependent internalization of the Aβ (82).”

What the sources say

• Not checked yetcites [82] Manzano-Leon, 2006, Beta-adaptin: key molecule for microglial scavenger receptor function under oxidative stress, Biochem. Bioph. Res. Co. source

In the paper

“ARG1-positive microglia have been shown to modify Aβ accumulation (79)”

What the sources say

• Not checked yetcites [79] Cherry, 2015, Arginase 1+ microglia reduce Abeta plaque deposition during IL-1beta-dependent neuroinflammation, J. Neuroinflammation source

In the paper

“Lack of gene expression changes in vivo may be due to a supportive environment from healthy neurons and astrocytes, providing regulating input to the microglia in young mice.”

In the paper

“Anti-inflammatory processes are associated with tissue repair, such as those mediated by IL-4, working through microglia and macrophages, and have been shown to provide benefits in models representing the early stages of neurodegenerative conditions, such as AD and amyotrophic lateral sclerosis, and other disorders related to cell death (45,72,73).”

What the sources say

• Not checked yetcites [45] Rossi, 2018, Interleukin 4 modulates microglia homeostasis and attenuates the early slowly progressive phase of amyotrophic lateral sclerosis, Cell Death Dis. source
• Not checked yetcites [72] Bosurgi, 2017, Macrophage function in tissue repair and remodeling requires IL-4 or IL-13 with apoptotic cells, Science source
• Not checked yetcites [73] Kiyota, 2010, CNS expression of anti-inflammatory cytokine interleukin-4 attenuates Alzheimer's disease-like pathogenesis in APP+PS1 bigenic mice, FASEB J. source

In the paper

“ITGAX-positive microglia are a source of Igf1 expression (59).”

What the sources say

• Not checked yetcites [59] Butovsky, 2018, Microglial signatures and their role in health and disease, Nat. Rev. Neurosci. source

In the paper

“AP1B1 is part of the clathrin-associated adaptor protein complex AP-1 and is involved in endocytosis and intracellular trafficking (81).”

What the sources say

• Not checked yetcites [81] Li, 2019, Developmental heterogeneity of microglia and brain myeloid cells revealed by deep single-cell RNA sequencing, Neuron source

In the paper

“Our findings in cultured primary microglia suggest an attenuated anti-inflammatory phenotype with reduced expression of Trem2 (Fig. 10), impacting cellular processes involving vesicle function, indicating that genes dependent on both Trem2 and IL-4 affect cytokine release or the response to extracellular cytokines.”

In the paper

“Itgax/CD11C is an integrin or cell surface antigen, and is up-regulated in a number of recent RNA-seq studies in response to amyloid plaques (58,59,61,84).”

What the sources say

• Not checked yetcites [58] Keren-Shaul, 2017, A unique microglia type associated with restricting development of Alzheimer's disease, Cell source
• Not checked yetcites [59] Butovsky, 2018, Microglial signatures and their role in health and disease, Nat. Rev. Neurosci. source
• Not checked yetcites [61] Zhou, 2020, Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and TREM2-independent cellular responses in Alzheimer's disease, Nat. Med. source
• Not checked yetcites [84] Krasemann, 2017, The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases, Immunity source

In the paper

“As the IL-4-induced up-regulation of a number of genes including Arg1 is critically dependent on phosphorylation of STAT6 (75)”

What the sources say

• Not checked yetcites [75] Gray, 2005, Induction of arginase I transcription by IL-4 requires a composite DNA response element for STAT6 and C/EBPbeta, Gene source

In the paper

“This finding is consistent with previous studies showing that LPS/IFNγ stimulation down-regulated Trem2 expression via TLR4 receptors in cell lines and primary cultures of microglia/macrophages and also in mouse brains (14,16,42,88,89).”

What the sources say

• Not checked yetcites [14] Turnbull, 2006, Cutting edge: TREM-2 attenuates macrophage activation, J. Immunol. source
• Not checked yetcites [16] Schmid, 2002, Heterogeneous expression of the triggering receptor expressed on myeloid cells-2 on adult murine microglia, J. Neurochem. source
• Not checked yetcites [42] Owens, 2017, Divergent Neuroinflammatory regulation of microglial TREM expression and involvement of NF-kappaB, Front. Cell. Neurosci.
• Not checked yetcites [88] Zheng, 2016, Opposing roles of the triggering receptor expressed on myeloid cells 2 and triggering receptor expressed on myeloid cells-like transcript 2 in microglia activation, Neurobiol. Aging source
• Not checked yetcites [89] Zhou, 2019, Imbalance of microglial TLR4/TREM2 in LPS-treated APP/PS1 transgenic mice: a potential Link between Alzheimer's disease and systemic inflammation, Neurochem. Res. source

In the paper

“The effects seen in the primary cultured microglia probably reflect a different mean activation state from microglia in the brain environment, although there remains a significant overlap with microglia expression profiles in vivo that are dependent on Trem2 levels and IL-4 simulation.”

In the paper

“in microglia originating from Trem2 R47H KI mice with a similar level of Trem2 down-regulation, there was about a 39% decrease in the activation of Il1b compared with WT mice possibly suggesting that the Trem2 R47H mutation underlies this effect.”

In the paper

“There are, however, other possible explanations for this difference, as the down-regulation of Trem2 in these mice is present throughout life and so these microglia may have a different state when isolated than those in which Trem2 is knocked down acutely.”

In the paper

“This might be one of the major mechanisms underlying various neurodegenerative diseases with advanced age by suppression of WT TREM2 in many sporadic cases, considering that TREM2 variants associated with loss-of-function are extremely rare in frequency in humans (2).”

What the sources say

• Not checked yetcites [2] Guerreiro, 2013, TREM2 variants in Alzheimer's disease, N. Engl. J. Med. source

In the paper

“Recent important studies have also shown that impaired/absent Trem2 function attenuates the microglial response to amyloid pathology by regulating microglial number around plaques, regulating microglial activity, and restricting neuritic damage (37,61,84–87,95–99).”

What the sources say

• Not checked yetcites [37] Cheng-Hathaway, 2018, The Trem2 R47H variant confers loss-of-function-like phenotypes in Alzheimer's disease, Mol. Neurodegener. source
• Not checked yetcites [61] Zhou, 2020, Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and TREM2-independent cellular responses in Alzheimer's disease, Nat. Med. source
• Not checked yetcites [84] Krasemann, 2017, The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases, Immunity source
• Not checked yetcites [85] Song, 2018, Humanized TREM2 mice reveal microglia-intrinsic and -extrinsic effects of R47H polymorphism, J. Exp. Med. source
• Not checked yetcites [86] Griciuc, 2019, TREM2 acts downstream of CD33 in modulating microglial pathology in Alzheimer's disease, Neuron source
• Not checked yetcites [87] Linnartz-Gerlach, 2019, TREM2 triggers microglial density and age-related neuronal loss, Glia source
• Not checked yetcites [95] Leyns, 2019, TREM2 function impedes tau seeding in neuritic plaques, Nat. Neurosci. source
• Not checked yetcites [96] Prokop, 2019, Impact of TREM2 risk variants on brain region-specific immune activation and plaque microenvironment in Alzheimer's disease patient brain samples, Acta Neuropathol. source
• Not checked yetcites [97] Sayed, 2018, Differential effects of partial and complete loss of TREM2 on microglial injury response and tauopathy, Proc. Natl. Acad. Sci. USA source
• Not checked yetcites [98] Meilandt, 2020, Trem2 deletion reduces late-stage amyloid plaque accumulation, elevates the Abeta42:Abeta40 ratio, and exacerbates axonal dystrophy and dendritic spine loss in the PS2APP Alzheimer's mouse model, J. Neurosci. source

In the paper

“This might reflect a mechanism for microglia to perform specific functional requirements under pro-inflammatory conditions.”

In the paper

“accumulated pro-inflammation and oxidative stress in the brain, which is often observed in aged brains and aged microglia (90–94), could cause sustained suppression of Trem2 expression and thus an impairment of TREM2-dependent microglial functions.”

What the sources say

• Not checked yetcites [90] Prolla, 2002, DNA microarray analysis of the aging brain, Chem. Senses source
• Not checked yetcites [91] Godbout, 2005, Exaggerated neuroinflammation and sickness behavior in aged mice following activation of the peripheral innate immune system, FASEB J. source
• Not checked yetcites [92] Lee, 2000, Gene-expression profile of the ageing brain in mice, Nat. Genet. source
• Not checked yetcites [93] Verbitsky, 2004, Altered hippocampal transcript profile accompanies an age-related spatial memory deficit in mice, Learn. Mem. source
• Not checked yetcites [94] Sierra, 2007, Microglia derived from aging mice exhibit an altered inflammatory profile, Glia source

In the paper

“Taken together, pro-inflammatory mediators, either locally produced, or infiltrating into the CNS from the periphery following blood–brain barrier breakdown, could induce down-regulation of microglial Trem2 expression and subsequently induce a microglial state with low TREM2-signaling (Fig. 10).”

In the paper

“In contrast to populations of microglia polarized with purified LPS or IL-4 stimuli in vitro, microglia in vivo probably never receive such simple stimuli to govern their activation state or at least not across the whole population.”

In the paper

“Cells pre-incubated with 10 μm cytochalasin-D for 30 min prior to the phagocytosis assay were used as a negative control.”

In the paper

“All RT-qPCR reactions were checked for a single peak with the melt-curve analysis reflecting a single PCR product.”

In the paper

“Unstained cells, and cells treated with 50 nM staurosporine for 24 h were used as negative and positive controls respectively to allow correct gating.”

In the paper

“Expression levels of the housekeeping genes Arf1, H3f3b and Rps28 were shown to be stable between the different experimental samples when higher molecular weight RNA was measured (Supplementary Material, Figs S9 and S10).”

In the paper

“To account for the differences in the cell number between different wells of microglia, cell lysates were collected in parallel with the supernatants from the same wells and then tested for levels of a housekeeping protein beta-actin (Ab8227, Abcam, RRID: AB_2305186) by western blot, which was used for normalization of TNF-alpha levels.”

In the paper

“Primers were designed to span at least two exons and tested for specificity against the entire mouse transcriptome using Primer-BLAST (NCBI) and supplied by Eurofins MWG Operon.”

In the paper

“Experimenter was blind to the genotype of the samples during quantification and analysis.”

In the paper

“Typical RNA Integrity values (RIN) around 9.0 from 10.0 were obtained regardless of siRNA, genotype of mice or treatment type.”