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Cited by 151Neuroinflammation and Neurodegeneration MechanismsPrion Diseases and Protein MisfoldingMicroRNA in disease regulation

MicroRNA 146a (miR-146a) Is Over-Expressed during Prion Disease and Modulates the Innate Immune Response and the Microglial Activation State

Open access
Saba, Reuben, Gushue, Shantel, Huzarewich, Rhiannon L. C. H., Manguiat, Kathy, Medina, Sarah, Robertson, Catherine, Booth, Stephanie A.·2012·PLoS ONE
DOI 10.1371/journal.pone.0030832PMC PMC3281888
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Abstract

Increasing evidence supports the involvement of microRNAs (miRNAs) in inflammatory and immune processes in prion neuropathogenesis. MiRNAs are small, non-coding RNA molecules which are emerging as key regulators of numerous cellular processes. We established miR-146a over-expression in prion-infected mouse brain tissues concurrent with the onset of prion deposition and appearance of activated microglia. Expression profiling of a variety of central nervous system derived cell-lines revealed that miR-146a is preferentially expressed in cells of microglial lineage. Prominent up-regulation of miR-146a was evident in the microglial cell lines BV-2 following TLR2 or TLR4 activation and also EOC 13.31 via TLR2 that reached a maximum 24-48 hours post-stimulation, concomitant with the return to basal levels of transcription of induced cytokines. Gain- and loss-of-function studies with miR-146a revealed a substantial deregulation of inflammatory response pathways in response to TLR2 stimulation. Significant transcriptional alterations in response to miR-146a perturbation included downstream mediators of the pro-inflammatory transcription factor, nuclear factor-kappa B (NF-κB) and the JAK-STAT signaling pathway. Microarray analysis also predicts a role for miR-146a regulation of morphological changes in microglial activation states as well as phagocytic mediators of the oxidative burst such as CYBA and NOS3. Based on our results, we propose a role for miR-146a as a potent modulator of microglial function by regulating the activation state during prion induced neurodegeneration.

AI snapshot

Written from the paper’s full text.

TL;DR

miR-146a is over-expressed in prion-infected mouse brain and is enriched in microglial cells, where TLR2/TLR4 stimulation induces it with delayed kinetics relative to cytokines. Gain- and loss-of-function studies indicate miR-146a broadly dampens microglial inflammatory and phagocytosis-related gene programs, suggesting it modulates the microglial activation state during prion disease.

Key findings

  • miR-146a was consistently up-regulated in prion-infected mouse brain: 3.3-fold in 22A whole brain, 8.3-fold in RML hippocampus, 16.3-fold in RML cerebellum, and 10.0-fold in BSE cerebellum versus age-matched mock-infected controls (Table 1).
  • miR-146a expression was enriched in microglial cell lines and induced by TLR2 or TLR4 agonists in BV-2 and EOC 13.31 cells, reaching a maximum at 24–48 hours and remaining elevated at 72 hours, while IL-6, CSF2/GM-CSF, and TNF transcripts peaked before 24 hours and returned to basal levels by 48 hours.
  • In EOC 13.31 cells, miR-146a over-expression altered 1,668 genes by ≥1.5-fold (FDR ≤2%) in resting cells; after TLR2/LPS stimulation, 977 genes were deregulated at 24 hours (778 down, 199 up), with enrichment in NF-κB, JNK, and MAPK signaling.
  • miR-146a over-expression significantly reduced IL-6 and GM-CSF protein after TLR2 stimulation (p<0.001), while anti-miR knock-down caused a small but significant increase in IL-6; predicted targets included IL1B, SMAD3, NOS2, QK1, and BCL2L1.

Methods

  • CD-1 mice were inoculated intracerebrally with 20 µl of 1% RML scrapie brain homogenate; at clinical symptoms, 6 mice per group were sacrificed and hippocampus or cerebellar tissue was used for RNA extraction, with equal numbers of age-matched mock-infected controls.
  • Murine microglial cell lines BV-2 and EOC 13.31 were stimulated with LPS or heat-killed Listeria monocytogenes (HKLM), with anti-TLR2 neutralization; miR-146a was over-expressed with 30 nM pre-miR mimic or knocked down with 50 nM anti-miR, followed by qRT-PCR (normalized to U6, 2−ΔΔCt) and IL-6/GM-CSF ELISA.
  • miRNA profiling used an in-house microarray; mRNA expression used Agilent whole mouse genome 4×44K arrays, analyzed with Feature Extraction, SAM (p=0.01), Ingenuity Pathway Analysis, and TargetScan 5.1; raw data deposited under GEO accession GSE17759.

Limitations

  • The paper states that in situ hybridization for miR-146a was inconclusive, that miR-146a may be expressed at low levels in other cell types including neurons, and that attempts to induce miR-146a in cultured microglia with recombinant PrP or PrP106–126 were unsuccessful.
  • The paper states that in vitro culture lacks multiple cell types, cell-to-cell signaling, normal cellular functions, and the long disease time course, and that TLR4 transcripts were below detection in their prion model; it also notes that MyD88 knockout data imply TLR2 signaling may not be essential in disease and that their model may not fully mimic disease processes.
  • Evident from the text: findings rely on mouse models and microglial cell lines rather than human tissue; direct miR-146a targets are largely inferred from microarray intersections and bioinformatic prediction, with limited validation; some in vivo miR-146a increases were not statistically significant (22A p=9.4×10−2; BSE p=7.6×10−2), and IL-10 up-regulation was not detectable by ELISA.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1371/journal.pone.0030832

Cites

57 of 57 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

6 of 55 cited sources checked · 1 supported · 0 flagged · accuracy not yet measured

  • Findingcitation

    The microglial cell-line enriched cluster contained 20 miRNAs including miR-146a and other inflammation-related miRNAs such as miR-155, miR-221 and miR-147.

    “The microglial cell-line enriched cluster contained 20 miRNAs including miR-146a and a number of other miRNAs reportedly involved in inflammation, such as miR-155, miR-221 [23] and miR-147, a microRNA that is induced upon Toll-like receptor (TLR) stimulation and regulates murine macrophage inflammatory responses [24].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [23] G Hu, 2010, miR-221 suppresses ICAM-1 translation and regulates interferon-gamma-induced ICAM-1 expression in human cholangiocytes., Am J Physiol Gastrointest Liver Physiol source
    • cites [24] G Liu, 2009, miR-147, a microRNA that is induced upon toll-like receptor stimulation, regulates murine macrophage inflammatory responses., Proc Natl Acad Sci U S A source
  • Premisecitation

    TRAF6, IRAK1, and IRAK2 are regulated primarily at the protein level.

    “These targets have been shown to be regulated primarily at the protein level”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [16] KD Taganov, 2006, NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses., Proc Natl Acad Sci U S A source

      The Results (page unavailable) show miR-146-dependent repression of IRAK1 and TRAF6 reporters, and the Discussion describes possible down-regulation of their protein levels. The copy does not establish that regulation is primarily at the protein level, and it does not address IRAK2. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [37] J Hou, 2009, MicroRNA-146a feedback inhibits RIG-I-dependent type I IFN production in macrophages by targeting TRAF6, IRAK1, and IRAK2., J Immunol source

      In “miR-146a targets TRAF6, IRAK1, and IRAK2” (page unavailable), the paper reports changes in TRAF6 and IRAK1 protein expression after miR-146a manipulation. It reports that IRAK2 changes at both the protein and mRNA levels. The copy therefore supports protein-level regulation, but not the claim that regulation of all three targets is primarily at the protein level. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Findingown data

    In our model, the scavenger receptors SCARB1, SCARB2 and AGER were not up-regulated during disease, contrary to previous reports.

    “Contrary to previous reports, we did not find the scavenger receptors SCARB1, SCARB2 and AGER to be up-regulated during disease [39].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [39] MM Hughes, 2010, Microglia in the degenerating brain are capable of phagocytosis of beads and of apoptotic cells, but do not efficiently remove PrPSc, even upon LPS stimulation., Glia source

      The Results section (‘Upregulation of Phagocytosis-Related Genes in ME7 Versus NBH Animals’; page unavailable) reports increased RAGE (AGER) expression during disease, supporting that part of the cited contrast. It reports CD36, SRA2 and CD68 as the other up-regulated scavenger receptors, but does not report up-regulation of SCARB1 or SCARB2. The citing authors’ own failure to find up-regulation is not a finding attributed to this source. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Findingcitation

    miR-146a is over-expressed in brain tissue of prion-infected mice compared with age-matched controls.

    “We established miR-146a over-expression in prion-infected mouse brain tissues concurrent with the onset of prion deposition and appearance of activated microglia.”
    • Also stated in: § Introduction · plain
      “Recent work in our own laboratory showed the amount of miR-146a in the brain of scrapie infected mice to be significantly increased in comparison with age-matched control mice [17].”
    • Also stated in: § MiR-146a is consistently up-regulated in the brain's of prion infected mice · boosted
      “Quantitative real time-PCR (qRT-PCR) analysis using TaqMan® miRNA probes, specific for mouse miR-146a, revealed that this increase in expression is consistently found in mice clinically infected with a number of scrapie strains (22A, RML, and BSE) in comparison to age matched, mock-infected mice (Table 1).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [17] R Saba, 2008, A miRNA signature of prion induced neurodegeneration., PLoS One source
  • Findingown data

    miR-146a is up-regulated in EOC 13.31 microglial cells via TLR2.

    “Treatment with an ultra-pure TLR2 agonist, HKLM, resulted in an induction of miR-146a in both cell lines (Figure 2D).”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 31
  • May combine two claims. 42
  • The quote only partly states this claim. 4

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductcitation

    The neuronal cluster included miR-124, a miRNA especially abundant in neurons, affirming specificity of methodology.

    “The neuronal cluster included miR-124, a miRNA that is especially abundant in neurons affirming the specificity of the methodology [20]–[22].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [20] M Lagos-Quintana, 2002, Identification of tissue-specific microRNAs from mouse., Curr Biol source
    • cites [21] LF Sempere, 2004, Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation., Genome Biol source

      In “Diverse miRNA expression patterns in mammalian organs” (page not supplied), the paper places miR-124a and miR-124b in a brain-expressed cluster. In “A subset of brain-expressed miRNAs is induced in retinoic acid-treated EC cells” (page not supplied), it reports their induction during neuronal differentiation, but says it did not examine expression in glial cells. It therefore supports the cluster finding, not the stronger assertion that miR-124 is especially abundant in neurons specifically. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [22] AM Krichevsky, 2006, Specific microRNAs modulate embryonic stem cell-derived neurogenesis., Stem Cells source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    miR-146a can regulate NF-κB itself, as well as key genes downstream of NF-κB.

    “Previous studies have determined that miR-146a can regulate NF-κB itself, as well as key genes downstream of NF-κB [16].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [16] KD Taganov, 2006, NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses., Proc Natl Acad Sci U S A source

      The Results show that miR-146a represses reporters bearing the IRAK1 and TRAF6 3′ UTRs, and the Discussion proposes that this could reduce NF-κB pathway activity through negative feedback. But the paper describes IRAK1 and TRAF6 as mediators of NF-κB activation, not genes downstream of NF-κB; it also does not show direct regulation of NF-κB itself. No page numbers are available in our copy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingnone given

    BV-2 cells divided at least once during a 24 h period, whereas EOC 13.31 cells doubled only every 48 h.

    “While the BV-2 cell line divided very rapidly, at least once during a 24 hour period, EOC 13.31 cells doubled only every 48 hours.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Fc receptors FCGR1 and FCGR3, complement receptors ITGAM and ITGAX, and TREM2 were over-expressed in scrapie-infected mouse brain.

    “Other receptors including Fc (FCGR1 and FCGR3) and complement receptors (ITGAM and ITGAX), as well as TREM2 a myeloid specific receptor triggered by amyloid to promote active phagocytosis, were also found to be over-expressed.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    mRNAs of other TLRs, including TLR4, were barely detectable in the study, so it could not be determined whether their expression increased in infected animals.

    “However, the mRNAs of other TLRs, including TLR4, were barely detectable in our study and so we could not determine whether there was an increase in expression of these receptors in the infected animals.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Treatment with anti-TLR2 antibody prior to crude LPS prevented induction of miR-146a in EOC 13.31 cells but not in BV-2 cells.

    “This treatment, in turn, prevented the induction of miR-146a in EOC 13.31 cells stimulated via the crude LPS preparation, but not similarly treated BV-2 cells (Figure 2C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In BV-2 and EOC 13.31 microglial cells, TLR agonist treatment significantly induced miR-146a expression, which increased over time to a maximum at 48 h and remained elevated at 72 h.

    “Agonist treatment uniformly resulted in the significant induction of miR-146a expression that increased over time to a maximum at 48 hours post-stimulation, but was still elevated even at 72 hours post stimulation (Figure 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In EOC 13.31 cells stimulated with LPS, transcripts for IL-6, CSF2, and TNF peaked well before 24 h and returned to basal levels by 48 h.

    “All three were found to peak well before 24 hours post-stimulation and had all returned to basal levels by 48 hours (Figure 3).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    In situ hybridization to detect miR-146a expression proved inconclusive.

    “In situ hybridization to detect miR-146a expression proved inconclusive in confirming this (data not shown).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Other up-regulated genes were involved in cell cycle progression, cell death, cellular movement, and morphology.

    “Other groups of up-regulated genes included genes involved in cell cycle progression, cell death, cellular movement and morphology.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    SMAD3 was identified as a potential miR-146a target in both resting and LPS-stimulated cells and has not been previously described as a target.

    “The transcription regulator SMAD3 was identified as a potential target of miR-146a in both resting cells and those stimulated with LPS and has not been described to be a target of miR-146a.”
    Quote in the paperNot a substantial claim
    • The authors state this as an assumption: Most translational repression is accompanied by miRNA-mediated mRNA degradation.
    • Rests on an assumption: Direct targets of miR-146a important in immune activation are likely to be those found at the intersection of genes up-regulated following knock-down and down-regulated after over-expression, provided they exhibit altered expression detectable at the mRNA level.

  • May combine two claims.

    Findingown data

    Following miR-146a over-expression and LPS, 22 genes (3 down, 19 up) were deregulated at 8 h and 977 (778 down, 199 up) at 24 h.

    “A significant number of genes were de-regulated under these conditions, 22 (3 down-regulated, 19 up-regulated) at 8 hours post transfection and 977 (778 down-regulated, 199 up-regulated) at 24 hours.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    miR-146a expression follows alternate kinetics compared to the rapid and transient induction of inflammatory mediators such as IL-6 and TNF; it is induced at a slower rate, peaks at 24 hours, and is maintained at 48 and 72 hours post-stimulation, when cytokine expression levels have returned to baseline.

    “Our first finding was that in contrast to the rapid and transient induction of transcription of inflammatory mediators such as cytokines IL-6 and TNF, miR-146a expression follows alternate kinetics. MiR-146a was induced at a slower rate, reaching a peak after 24 hours that is maintained even at 48 and 72 hours post-stimulation; time-points when cytokine expression levels have returned to base-line”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Up-regulated genes after miR-146a knock-down and LPS included infection process genes NR3C1, PTPRC, SFRS1, SFRS5, SMAD3, C3, and DDX58.

    “Examination of the ontologies enriched in this set of up-regulated genes showed genes known to play a role in infection processes, including NR3C1, PTPRC, SFRS1, SFRS5, SMAD3, C3 and DDX58.”
    Quote in the paperNot a substantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    PAX6, MDM2, JAK2, SMAS3, CASP7, CASP8, and BCL2L1 were increased following miR-146a knock-down and LPS.

    “A number of important transcriptional regulators PAX6, MDM2, JAK2 and SMAS3 were increased, as were some caspases, CASP7 and CASP8, and the apoptotic regulator BCL2L1.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Ribosomal proteins and mitochondrial cytochrome C oxidase subunits were up-regulated following miR-146a knock-down (IPA p = 1.13×10−54 for ribosomal proteins).

    “Ribosomal proteins were strikingly up-regulated resulting in a significance score in IPA of p = 1.13×10−54, similarly numerous mitochondrial cytochrome C oxidase subunits involved in oxidative phosphorylation were also up-regulated.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    IL-10 gene was up-regulated but expressed at very low levels and not detectable by ELISA.

    “One up-regulated cytokine coding gene was IL-10, a negative regulator of cytokine production during the immune response; however, this gene was expressed at very low levels based on the spot intensity observed by microarray and we were unable to detect IL-10 expression by ELISA.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Following miR-146a anti-miR transfection and LPS stimulation, 691 annotated genes were up-regulated at 8 h and 354 at 24 h.

    “Indeed, we did find a significant number of up-regulated genes at 8 hours (691 annotated genes), with a smaller number (354 annotated genes) up-regulated at 24 hours post stimulation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In our study, IRAK1 mRNA levels were unchanged, but TRAF6 gene expression was slightly but significantly down-regulated in EOC 13.31 cells transfected with a miR-146a mimic.

    “The mRNA levels of IRAK1 were similarly found to be unchanged in our study, although we measured a small, but significant, down-regulation of TRAF6 gene expression in EOC 13.31 cells transfected with a miR-146a mimic.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    miR-146a over-expression down-regulated NOS1, NOS2, NOS3, NOX4, CYBA, CYBB, and SOD3, while up-regulating SOD1.

    “These included the genes NOS1, NOS2 and NOS3, nitric oxide synthases that are inducible by TLR stimulation and NOX4, a gene involved in H2O2 production. Significantly, the genes CYBA and CYBB proposed to be primary components of the microbicidal oxidase system of phagocytes, were also down-regulated, as was SOD3. SOD1, however, was up-regulated in our work.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IL-1B is down-regulated following miR-146a over-expression and up-regulated after knock-down.

    “In particular, IL-1B, a key mediator of the inflammatory response, is both down-regulated following miR-146a over-expression and up-regulated after knock-down.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    miR-146a is expressed at low levels in laser capture microdissected neurons isolated from mouse hippocampus.

    “In subsequent studies using TaqMan® qRT-PCR assays we have found that miR-146a is expressed at low levels in laser capture microdissected (LCM) neurons isolated from mouse hippocampus (data not shown)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    IL-1B was down-regulated by miR-146a over-expression and up-regulated following knock-down.

    “This includes the down-regulation of the cytokine IL-1B by miR-146a over-expression and up-regulation following knock-down.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    Knock-down of miR-146a in EOC 13.31 cells dysregulated substantially fewer genes, mostly up-regulated.

    “Substantially fewer genes were dysregulated following transfection of miR-146a anti-miR and these were for the most part up-regulated.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    miR-142-3p, miR-34a, and miR-223 were enriched in microglial cell lines in this study, and were also up-regulated in multiple sclerosis glial cells and scrapie infection.

    “Additionally, a number of miRNAs found to be up-regulated in a recent study profiling glial cells in multiple sclerosis brain lesions, as well as our previous study to determine deregulated miRNAs in scrapie infection, were also identified to be enriched in microglial cell lines in this study [18], [38]. These were miR-142-3p, miR-34a, and miR-223.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [18] VG Tusher, 2001, Significance analysis of microarrays applied to the ionizing radiation response., Proc Natl Acad Sci U S A source

      Not checked in the free run.

    • cites [38] A Junker, 2009, MicroRNA profiling of multiple sclerosis lesions identifies modulators of the regulatory protein CD47., Brain source

  • May combine two claims.

    Findingown data

    miR-146a is over-expressed in the brains of mice during scrapie infection and is most likely highly enriched in cells of microglial origin based on miRNA expression profiling of various brain-derived cell lines.

    “Firstly, we confirmed that miR-146a is over-expressed in the brain's of mice during scrapie infection and that this expression is, most likely, highly enriched in cells of microglial origin based on miRNA expression profiling of various brain-derived cell lines.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IL-1B expression is decreased following transfection of miR-146a mimic and increased following knock-down with an anti-miR in both resting and TLR-stimulated cells.

    “Interestingly, IL-1B appeared to be tightly regulated by miR-146a; its expression is decreased following transfection of the miR-146a mimic and increased following knock-down with an anti-miR in both resting and TLR-stimulated cells.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Transfection of 30 nM miR-146a mimic over-expressed miR-146a in EOC 13.31 cells.

    “Over-expression was achieved by transfecting 30 nM of miR-146a mimic (Figure 4A).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    miR-147 is induced upon TLR stimulation and regulates murine macrophage inflammatory responses.

    “The microglial cell-line enriched cluster contained 20 miRNAs including miR-146a and a number of other miRNAs reportedly involved in inflammation, such as miR-155, miR-221 [23] and miR-147, a microRNA that is induced upon Toll-like receptor (TLR) stimulation and regulates murine macrophage inflammatory responses [24].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [24] G Liu, 2009, miR-147, a microRNA that is induced upon toll-like receptor stimulation, regulates murine macrophage inflammatory responses., Proc Natl Acad Sci U S A source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    miR-146a is reported to be up-regulated in LPS stimulated human macrophages.

    “MiR-146a is reported to be up-regulated in LPS stimulated human macrophages, however, the microglial surface receptor that is engaged leading to activation during prion pathogenesis is unknown [16].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] KD Taganov, 2006, NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses., Proc Natl Acad Sci U S A source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    CFH has been reported to be a direct target of miR-146a and potentially important in inflammation in Alzheimer's disease.

    “CFH has also been reported to be a direct target of miR-146a and potentially important in inflammation in Alzheimer's disease [32], [33].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [32] AI Pogue, 2009, Characterization of an NF-kappaB-regulated, miRNA-146a-mediated down-regulation of complement factor H (CFH) in metal-sulfate-stressed human brain cells., J Inorg Biochem source
    • cites [33] YY Li, 2011, Increased expression of miRNA-146a in alzheimer&apos;s disease transgenic mouse models., Neurosci Lett source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All procedures involving live animals were approved by the Canadian Science Centre for Human and Animal Health - Animal Care Committee.

    “All procedures involving live animals were approved by the Canadian Science Centre for Human and Animal Health - Animal Care Committee (CSCHAH-ACC).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Taganov et al. (2006) showed that TLR2 stimulation by peptidoglycan and Pam3CSK4 produced up-regulation of miR-146a in human monocytes, in contrast to TLR3, TLR7 and TLR9 agonists.

    “Taganov et al (2006) previously showed that TLR2 stimulation by exposure to peptidoglycan and its synthetic analog, Pam3CSK4, produced an up-regulation of miR-146a in human monocytes in contrast to TLR3, TLR7 and TLR9 agonists.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Taganov et al (2006) source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    BV-2 cells form spineous processes and express inwardly rectifying, but not outwardly rectifying K+ channels.

    “Additionally, like microglial cells, they form spineous processes and express inwardly rectifying, but not outwardly rectifying K+ channels [25].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25] A Henn, 2009, The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation., ALTEX source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In macrophages, miR-146a expression can dampen the immediate innate immune response mediated by TLR2 and TLR4.

    “This has been previously described in macrophages where miR-146a expression was found to be capable of dampening the immediate innate immune response mediated by TLR2 and TLR4 [16], [30], [31].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [16] KD Taganov, 2006, NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses., Proc Natl Acad Sci U S A source

      Not checked in the free run.

    • cites [30] MM Perry, 2008, Rapid changes in microRNA-146a expression negatively regulate the IL-1beta-induced inflammatory response in human lung alveolar epithelial cells., J Immunol source
    • cites [31] MA Nahid, 2009, miR-146a is critical for endotoxin-induced tolerance: IMPLICATION IN INNATE IMMUNITY., J Biol Chem source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    One pathological feature of prion disease is activation of microglia and accumulation of astrocytes, detectable prior to clinical symptoms and other signs of neurodegeneration.

    “As is the case in other neurodegenerative conditions, such as Alzheimer's disease, one of the pathological features of disease is the activation of the brain's resident immune cells, the microglia, and the accumulation of astrocytes. In many instances, this feature can be detected prior to clinical symptoms and other signs of neurodegeneration, such as spongiosis and neuronal cell death become app”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [2] A Giese, 1998, Role of microglia in neuronal cell death in prion disease., Brain Pathol source
    • cites [3] LW Shepard, 2001, Constitutive activation of NF-kappa B and secretion of interleukin-8 induced by the G protein-coupled receptor of kaposi&apos;s sarcoma-associated herpesvirus involve G alpha(13) and RhoA., J Biol Chem source

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    In prion disease, microglia appear to express low levels of pro-inflammatory cytokines during chronic neurodegeneration but may be in a "primed" state.

    “In prion disease, microglia appear to express low levels of pro-inflammatory cytokines during chronic neurodegeneration but may be in a “primed” state [8].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [8] C Cunningham, 2009, Systemic inflammation induces acute behavioral and cognitive changes and accelerates neurodegenerative disease., Biol Psychiatry source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    U6 snRNA was expressed at high levels with negligible variance under the experimental conditions used.

    “MiRNA expression was normalized to the U6 snRNA level which was expressed at high levels with negligible variance under the experimental conditions used in our study.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Un-methylated CpG DNA, an agonist of TLR9 signaling, prolongs scrapie incubation period in mice.

    “Un-methylated CpG DNA, which is an agonist of TLR9 signaling, has been shown to prolong the scrapie incubation period in mice, suggesting that innate immune activation can interfere with prion disease progression [9], [10].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [9] S Sethi, 2002, Postexposure prophylaxis against prion disease with a stimulator of innate immunity., Lancet source
    • cites [10] DS Spinner, 2007, CpG oligodeoxynucleotide-enhanced humoral immune response and production of antibodies to prion protein PrPSc in mice immunized with 139A scrapie-associated fibrils., J Leukoc Biol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Pathogenic changes in prion disease appear independent of MyD88, as ablation of this molecule did not lead to changes in incubation periods and neuropathological profiles of scrapie-infected mice.

    “Pathogenic changes in prion disease appear to be independent of MyD88, a signaling intermediate for several TLR family members, as ablation of this molecule did not lead to changes in the incubation periods, and neuropathological profiles of scrapie-infected mice [12].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] M Prinz, 2003, Prion pathogenesis in the absence of toll-like receptor signalling., EMBO Rep source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The 3′UTRs of IRAK1 and TRAF6 are responsive to interaction with miR-146a, but regulation is at the level of translation; mRNA levels remained unchanged.

    “The 3′UTRs of these genes are responsive to interaction with miR-146a, however, regulation was shown to be at the level of translation; mRNA levels remained unchanged.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] KD Taganov, 2006, NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses., Proc Natl Acad Sci U S A source

      Not checked in the free run.

  • The quote only partly states this claim.

    Interpretationown data

    NF-κB, JNK, and MAPK signaling pathways are implicated in the gene expression changes.

    “Signaling pathways involving NF-κB, JNK and MAPK are implicated in Figure 6B.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Injection of amyloid into the hippocampus provokes increased TLR2 expression.

    “Furthermore, an injection of amyloid into the hippocampus provokes increased TLR2 expression [41].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [41] KL Richard, 2008, Toll-like receptor 2 acts as a natural innate immune receptor to clear amyloid beta 1–42 and delay the cognitive decline in a mouse model of alzheimer&apos;s disease., J Neurosci source

  • May combine two claims.

    Interpretationcitation

    TLR2-signaling is not essential to the disease process and our model may not mimic processes that occur during disease.

    “This data implies that TLR2-signaling is not essential to the disease process and that our model may not mimic processes that occur during disease.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: MyD88 is the main adaptor protein implicated in TLR signaling and is considered essential for the induction of inflammatory cytokines triggered by most TLRs, including TLR2.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    IL-10 has been found to be up-regulated in human patients with CJD.

    “Conversely, we found a small number of genes to increase in expression upon transfection of a miR-146a mimic. One of these was IL-10, an anti-inflammatory cytokine that can down-regulate the expression of cytokines, MHC class II molecules, and co-stimulatory molecules on macrophages, and has been found to be up-regulated in human patients with CJD [50].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [50] K Stoeck, 2006, Pro- and anti-inflammatory cytokines in the CSF of patients with creutzfeldt-jakob disease., J Neuroimmunol source

  • May combine two claims.

    Interpretationreasoning

    The considerable increase in expression of TLR2 observed at clinical stages of prion disease could be indicative of accumulation of microglia not in an activation state optimal for phagocytosis, and could contribute to defective clearing of debris and prion accumulation during later stages of disease.

    “In terms of prion disease this suggests the considerable increase in expression of TLR2 observed at clinical stages of prion disease could be indicative of the accumulation of microglia that are not in an activation state that is optimal for phagocytosis. This could therefore contribute to defective clearing of debris and prion accumulation during later stages of disease.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: A reduction in TLR2 expression is linked with an increase in phagocytosis, implying an inverse relation between TLR2 expression and phagocytic activity.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    TRAF6, IRAK1, and IRAK2 have been identified and validated as putative targets of miR-146a in macrophages.

    “Previously, TRAF6, IRAK1 and IRAK2 have been identified and validated as putative targets of miR-146a in macrophages [16], [37].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [16] KD Taganov, 2006, NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses., Proc Natl Acad Sci U S A source

      Not checked in the free run.

    • cites [37] J Hou, 2009, MicroRNA-146a feedback inhibits RIG-I-dependent type I IFN production in macrophages by targeting TRAF6, IRAK1, and IRAK2., J Immunol source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    miR-146a is transcribed by NF-κB and may operate as part of a feedback inhibitory regulatory loop to modulate inflammation by this transcription factor.

    “MiR-146a is also transcribed by NF-κB and therefore may operate as part of a feedback inhibitory regulatory-loop to modulate inflammation by this transcription factor [46].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [46] K Pichler, 2008, MicroRNA miR-146a and further oncogenesis-related cellular microRNAs are dysregulated in HTLV-1-transformed T lymphocytes., Retrovirology source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Cells lacking TLR4, TLR2 and CD14 could not initiate a Src-Vav-Rac signaling cascade required to stimulate phagocytosis of amyloid beta deposits.

    “Cells lacking these receptors could not initiate a Src-Vav-Rac signaling cascade required to stimulate phagocytosis of amyloid beta deposits [45].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [45] GE Landreth, 2009, Toll-like receptors in alzheimer&apos;s disease., Curr Top Microbiol Immunol

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    TLR2 and TLR7 have been shown to display profoundly increased expression levels in APP transgenic mice that accumulate amyloid deposits in their brain.

    “Interestingly TLR2, and TLR7 which was also up-regulated in our prion model, has also been shown to display profoundly increased expression levels in APP transgenic mice that accumulate amyloid deposits in their brain [40].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] M Letiembre, 2009, Screening of innate immune receptors in neurodegenerative diseases: A similar pattern., Neurobiol Aging source

  • May combine two claims.

    Premisecitation

    MyD88 knock-out mice were readily infectable with scrapie and did not show any significant alteration in the time-course or pathology of the disease.

    “Nevertheless, the study determined by Prinz et al. (2003) showed that MyD88 knock-out mice were readily infectable with scrapie and did not show any significant alteration in the time-course or pathology of the disease [12].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [12] M Prinz, 2003, Prion pathogenesis in the absence of toll-like receptor signalling., EMBO Rep source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    TLR 2, 4, and 9 signaling modulates phagocytosis and clearance of neurotoxic amyloid deposition in neurodegenerative diseases, perhaps by MyD88 independent signaling pathways.

    “It has also been reported by others that TLR 2, 4, and 9 signaling modulates phacogytosis and clearance of neurotoxic amyloid deposition in neurodegenerative diseases, perhaps by MyD88 independent signaling pathways [43], [55].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [43] P Iribarren, 2005, CpG-containing oligodeoxynucleotide promotes microglial cell uptake of amyloid beta 1–42 peptide by up-regulating the expression of the G-protein- coupled receptor mFPR2., FASEB J source
    • cites [55] K Tahara, 2006, Role of toll-like receptor signalling in abeta uptake and clearance., Brain source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The microarray data had very high reproducibility.

    “confirming very high reproducibility of the microarray data.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    CFH has been shown to be targeted by miR-146a and down-regulated in Alzheimer's disease and in Herpes simplex virus type 1 infection.

    “Interestingly, this gene has been shown to be targeted by miR-146a and down-regulated in Alzheimer's disease as well as in other diseases such as Herpes simplex virus type1 infection [47], [48].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [47] WJ Lukiw, 2008, An NF-kappaB-sensitive micro RNA-146a-mediated inflammatory circuit in alzheimer disease and in stressed human brain cells., J Biol Chem source
    • cites [48] JM Hill, 2009, HSV-1 infection of human brain cells induces miRNA-146a and alzheimer-type inflammatory signaling., Neuroreport source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationown data

    NOS2, QK1, and BCL2L1 are highly related based on IPA interactions, potentially a co-ordinately regulated gene set.

    “They are also highly related based on potential interactions identified using the IPA tool, potentially a co-ordinately regulated gene set.”
    Quote in the paperNot a substantial claim
    • The authors state this as an assumption: Most translational repression is accompanied by miRNA-mediated mRNA degradation.
    • Rests on an assumption: Direct targets of miR-146a important in immune activation are likely to be those found at the intersection of genes up-regulated following knock-down and down-regulated after over-expression, provided they exhibit altered expression detectable at the mRNA level.

  • May combine two claims.

    Interpretationown data

    NOS2, QK1, and BCL2L1 are predicted as strong candidates for miR-146a targeting and have not been previously described.

    “In addition, NOS2, QK1 and BCL2L1 are also predicted from this study as being strong candidates for targeting by miR-146a that have not previously been described.”
    Quote in the paperNot a substantial claim
    • The authors state this as an assumption: Most translational repression is accompanied by miRNA-mediated mRNA degradation.
    • Rests on an assumption: Direct targets of miR-146a important in immune activation are likely to be those found at the intersection of genes up-regulated following knock-down and down-regulated after over-expression, provided they exhibit altered expression detectable at the mRNA level.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    IL-1B has been reported to be negatively regulated by miR-146a.

    “The gene IL-1B has previously been reported to be negatively regulated by miR-146a [30].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [30] MM Perry, 2008, Rapid changes in microRNA-146a expression negatively regulate the IL-1beta-induced inflammatory response in human lung alveolar epithelial cells., J Immunol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The response of microglia to fibrillar Aβ is reliant upon the expression of TLR4, TLR2 and the co-receptor CD14 to activate intracellular signaling.

    “Landreth and Reed-Geaghan (2009) have also shown that the response of microglia to fibrillar Aβ is reliant upon the expression of TLR4, TLR2 and the co-receptor CD14 to activate intracellular signaling [45].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [45] GE Landreth, 2009, Toll-like receptors in alzheimer&apos;s disease., Curr Top Microbiol Immunol

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    SMAD3 knock-out mice exhibit alterations in many aspects of glial function, including altered scar formation and immune response to injury, with fewer neutrophils, macrophages/microglia, NG2-positive cells and GFAP-positive cells around lesions.

    “SMAD3 knock-out mice exhibit alterations in many aspects of glial function, including altered scar formation and immune response to injury [51]. Fewer neutrophils, macrophages/microglia, NG2-positive cells and GFAP-positive cells were reported around lesions in Smad3 null mice brains.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [51] Y Wang, 2007, Smad3 null mice display more rapid wound closure and reduced scar formation after a stab wound to the cerebral cortex., Exp Neurol source

  • May combine two claims.

    Interpretationown data

    Genes involved in cytokine and other aspects of the inflammatory response are regulated by miR-146a expression, and genes involved in morphological changes, chemotaxis and oxidative burst may also be targets for direct or indirect miR-146a regulation.

    “Not only are genes involved in cytokine and other aspects of the inflammatory response regulated by its expression, but we found in this study that genes involved in morphological changes that accompany activation, chemotaxis and oxidative burst-related genes may also be targets for either direct or indirect miR-146a regulation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Some evidence suggests that this response is independent of phagocytic clearance pathways that appear to be MyD88 dependent.

    “Some evidence also exists suggesting that this response is independent of phagocytic clearance pathways that appear to be MyD88 dependent [53].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [53] P Henneke, 2002, Cellular activation, phagocytosis, and bactericidal activity against group B streptococcus involve parallel myeloid differentiation factor 88-dependent and independent signaling pathways., J Immunol source

  • May combine two claims.

    Interpretationreasoning

    The discrepancy between in vitro and whole brain transcriptional responses is likely multi-factorial, with contributing factors including lack of multiple cell types and cell-to-cell signaling in vitro, lack of normal cellular functions under in vitro conditions, and the time required for disease progression.

    “This discrepancy is very likely multi-factorial and contributing factors include, but are certainly not limited to, lack of multiple cell types and associated cell-to-cell signaling in in vitro culture, lack of normal cellular functions under in vitro conditions, and also the time required for disease progression, which is often months to years in whole organism/animal.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Caspase-8 has non-apoptotic functions and is required for TLR signaling and regulation of NF-κB function.

    “Caspase-8 is known to have non-apoptotic functions and has been shown to be required for TLR signaling and in the regulation of NF-κB function [36].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] B Lemmers, 2007, Essential role for caspase-8 in toll-like receptors and NFkappaB signaling., J Biol Chem source

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Transfected anti-miRs or mimics were stably detected over a 24-48 h period post-transfection.

    “Transfected anti-miRs or mimics were stably detected over a 24–48 hour period post transfection; therefore cells were transfected and allowed to recover overnight, thus reducing the likelihood of differential expression induced by the transfection process, prior to stimulation.”
    Quote in the paperSubstantial claim
64 of 64

  • The paper relies on this without support.

    PrPSc induces inflammatory responses by the brain's glial cells (astrocytes and microglia) by activating innate immunity pathways.

    “PrPSc induces inflammatory responses by the brain's glial cells (astrocytes and microglia) by activating innate immunity pathways.”

  • The paper relies on this without support.

    The microglial surface receptor that is engaged leading to activation during prion pathogenesis is unknown.

    “MiR-146a is reported to be up-regulated in LPS stimulated human macrophages, however, the microglial surface receptor that is engaged leading to activation during prion pathogenesis is unknown [16].”

  • The paper relies on this without support.

    The specific stimuli and signaling pathways that lead to modulations of microglial functions, and the mechanisms by which microglial activation influences chronic neurodegeneration, are as yet unknown.

    “The specific stimuli and signaling pathways that lead to these modulations of functions, and the mechanisms by which microglial activation influences chronic neurodegeneration, are as yet unknown.”

  • The paper relies on this without support.

    Whether activation of the innate immune response is beneficial or harmful in prion-like pathologies is still unknown.

    “Such an inflammatory milieu has been recognized in the prion infected brain for some time; however, whether the activation of this innate immune response is beneficial or harmful in prion-like pathologies is still unknown.”

  • The authors state this as an assumption.

    Most translational repression is accompanied by miRNA-mediated mRNA degradation.

    “It has been previously reported that most translational repression is accompanied by miRNA-mediated mRNA degradation, we therefore used this assumption to predict potential direct targets of miR-146a in this system [34], [35].”

  • The paper relies on this without support.

    Direct targets of miR-146a important in immune activation are likely to be those found at the intersection of genes up-regulated following knock-down and down-regulated after over-expression, provided they exhibit altered expression detectable at the mRNA level.

    “Direct targets of miR-146a important in immune activation are likely to be those found at the intersection of genes that are up-regulated following knock-down and down-regulated after over-expression, provided that they exhibit altered expression that is detectable at the mRNA level.”

  • The paper relies on this without support.

    Microglial cells respond to prion deposition by converting from a resting to an activated phenotype, but synthesize low levels of pro-inflammatory cytokines.

    “Microglial cells, the brain's resident immune effector cells, respond to prion deposition by the conversion of a resting phenotype into one that is ‘activated’. However, although they take on an activated phenotype they synthesize fairly low levels of pro-inflammatory cytokines”

  • The paper relies on this without support.

    Phenotypic changes in microglia are one of the earliest apparent alterations in tissue pathology detectable during prion disease.

    “However, it is clear that phenotypic changes in microglia are one of the earliest apparent alterations in tissue pathology detectable during disease.”

  • The paper relies on this without support.

    The in vivo transcriptional response likely represents the host's response to the process of neurodegeneration as much, if not more than, the response specifically to prions.

    “It is often speculated that the in vivo transcriptional response likely represents the host's response to the process of neurodegeneration as much, if not more than, the response specifically to prions, which may account for our inability to detect a robust miR-146a induction in the cultured microglia.”

  • The paper relies on this without support.

    Effector molecules downstream of multiple cell surface receptors, including TLR2 and TLR4 signals, are similar.

    “However, effector molecules downstream of multiple cell surface receptors, including TLR2 (and TLR4) signals, are similar”

  • The paper relies on this without support.

    A reduction in TLR2 expression is linked with an increase in phagocytosis, implying an inverse relation between TLR2 expression and phagocytic activity.

    “Studies also link a reduction in TLR2 expression with an increase in phagocytosis, implying an inverse relation between TLR2 expression and phagocytic activity.”

  • The paper relies on this without support.

    MyD88 is the main adaptor protein implicated in TLR signaling and is considered essential for the induction of inflammatory cytokines triggered by most TLRs, including TLR2.

    “MyD88 is the main adaptor protein implicated in TLR signaling and is considered essential for the induction of inflammatory cytokines triggered by most TLRs, including TLR2.”
12 of 12

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • MiR-146a is elevated in reactive astrocytes in AD and is generally considered a compensatory anti-inflammatory regulator.

    “MiR-146a is also elevated in reactive astrocytes in AD and is generally considered a compensatory anti-inflammatory regulator.”

    From Cell-specific MicroRNA networks orchestrate the pathogenesis of Alzheimer’s disease (2026) · cites this paper as Saba et al., 2012

    The Discussion describes miR-146a as a possible inhibitory feedback regulator of inflammation, and the Results report reduced inflammatory cytokine production when it is overexpressed in stimulated microglia. The paper does not establish that miR-146a is elevated in reactive astrocytes in Alzheimer’s disease; its cell-line profiling instead places miR-146a in a microglia-enriched cluster. Page numbers were not available in our copy. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • MiR-146a serves as a classical NF-κB-induced negative-feedback regulator; elevated miR-146a in microglia acts to inhibit IRAK1 and TRAF6.

    “MiR-146a serves as a classical NF-κB–induced negative-feedback regulator. Elevated miR-146a in microglia acts to inhibit IRAK1 and TRAF6 (Fan et al., 2020; Lukiw et al., 2008; Saba et al., 2012; Saba et al., 2014).”

    From Cell-specific MicroRNA networks orchestrate the pathogenesis of Alzheimer’s disease (2026) · cites this paper as Saba et al., 2012

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 4B is cited for pathways it does not depict

    Minor

    The discussion directs readers to Figure 4B for alterations in NF-κB and JAK-STAT pathway mediators, but Figure 4B is described as a measurement of miR-146a knock-down over time.

  • An 8-hour stimulated result is dated from transfection rather than stimulation

    Minor

    The paper says LPS stimulation occurred 24 hours after mimic transfection, then describes a stimulated result at 8 hours post transfection. A result eight hours after transfection would precede the stated LPS stimulation.

  • TLR1 is called significantly up-regulated despite its reported p-value

    Minor

    The text names TLR1 as significantly up-regulated, but Table 2 reports p = 0.081 for TLR1. That exceeds the conventional 0.05 threshold, as well as the paper's stated microarray differential-expression threshold of 0.01.

  • The named up-regulated Fc receptor differs from Table 2

    Minor

    The results name FCGR3 as an over-expressed Fc receptor, whereas Table 2 lists FCGR1 and FCGR2, not FCGR3.

4 of 4

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Authors
Saba, R., Gushue, S., Huzarewich, R. L. C. H., Manguiat, K., Medina, S., Robertson, C., & Booth, S. A.
Affiliations
Public Health Agency of Canada (CA)
Corresponding author
Booth, Stephanie A.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-02-17
Volume
7
Issue
2
Pages
e30832
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0030832
PMC
PMC3281888
OpenAlex
W2084713640
Semantic Scholar
12a9d810cdceb96df038db57ecb7ad335db78d45
Cited by
151
References
57
Citations by year
2026: 4 · 2025: 5 · 2024: 4 · 2023: 4 · 2022: 10 · 2021: 10 · 2020: 13 · 2019: 9 · 2018: 13 · 2017: 15 · 2016: 11 · 2015: 16 · 2014: 14 · 2013: 16 · 2012: 7
FWCI
8.04
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Public Health Agency of Canada
  • Public Health Agency
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Prion Diseases and Protein Misfolding
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
Keywords
miR-146a, prion disease, microglial activation, innate immune response, TLR2, TLR4, NF-κB signaling, JAK/STAT signaling, neuroinflammation, respiratory burst
MeSH terms
Animals, Brain, Cell Line, Cell Movement, Immunity, Innate, Kinetics, Lipopolysaccharides, Oxidative Phosphorylation, Prions, Time Factors, Transcription, Genetic, Protein Biosynthesis, Signal Transduction, Up-Regulation, Cluster Analysis, Cytokines, Prion Diseases, Microglia, Inflammation Mediators, Gene Expression Profiling, MicroRNAs, Toll-Like Receptor 2, Toll-Like Receptor 4, Mice

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-06.

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