TET2 Regulates the Neuroinflammatory Response in Microglia

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

In the brain, under sterile inflammatory conditions, there are increasing reports showing activation of the Type I IFN response in ischemia, spinal cord injury, and in two different AD mouse models as well as in AD patients.

“However, in the brain, and under sterile inflammatory conditions, there are increasing numbers of reports showing activation of the Type I IFN response in ischemia (McDonough et al., 2017), spinal cord injury (Impellizzeri et al., 2015) and in two different AD mouse models, the 5xFAD (Landel et al., 2014) and APP/PS1 (Taylor et al., 2014), as well as in AD patients (Taylor et al., 2014).”

• cites McDonough et al., 2017 sourceSupported
• cites Impellizzeri et al., 2015 sourceNot checked — no full text of the source
• cites Landel et al., 2014 sourceDisputed

  The Landel citation is attached to the 5XFAD model, not the other conditions or populations in the sentence. In Results and discussion, “Altered expression of interferon gamma-induced genes” and “Potential role of IRF8 in the expression of interferon gamma-regulated genes” (pages unavailable), the paper identifies the increased gene expression in 5XFAD brain as an IFN-γ response, rather than the claimed Type I IFN response. (LLM, conf=0.85)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

TET2 is an important player in the resolution of inflammation by repressing IL-6 expression through recruitment of HDAC2, a histone deacetylase, into the Il-6 promoter.

“The latter study showed that TET2 is an important player in the resolution of inflammation by repressing IL-6 expression through recruitment of HDAC2, a histone deacetylase, into the Il-6 promoter.”

• cites Zhang et al., 2015 sourceSupported

May combine two claims.

The upstream region of Tet2 is an enhancer element that becomes active upon TLR-4 activation.

“providing support for the upstream region being an enhancer element that becomes active upon TLR-4 activation (Creyghton et al., 2010).”

• cites Creyghton et al., 2010 sourcePartly supported

  The Results (p. 5) describe enhancers poised to become active after an inflammatory stimulus, and the paper identifies H3K27ac as a marker distinguishing active from poised enhancers (p. 2). Our copy does not identify the upstream Tet2 region as an enhancer or show that it becomes active upon TLR-4 activation. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

TET2 regulates the inflammatory response in dendritic cells and bone marrow-derived macrophages independently of its dioxygenase activity.

“TET2 also regulates the inflammatory response in dendritic cells and bone marrow-derived macrophages independently of its dioxygenase activity (Zhang et al., 2015).”

• cites Zhang et al., 2015 sourceSupported

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

TET2 has been shown to act via both 5mC oxidation and catalytic-independent mechanisms, such as recruitment of epigenetic modifiers.

“TET2 has been shown to act via both 5mC oxidation and catalytic-independent mechanisms, such as recruitment of epigenetic modifiers (Ichiyama et al., 2015), (Zhang et al., 2015).”

• cites Ichiyama et al., 2015 sourcePartly supported

  The Introduction describes TET-mediated oxidation of 5mC, and the Results report reduced p300 recruitment in Tet2-deficient Th1 and Th17 cells (page numbers unavailable in our copy). These findings support a link between Tet2 and an epigenetic modifier, but the paper does not establish that the p300 effect is independent of Tet2’s catalytic activity. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites Zhang et al., 2015 sourceSupported

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

The age-dependent increase in microglial IL-1β levels is seemingly driven by the age-dependent loss of microglial SIRT1, a NAD-dependent deacetylase that can regulate the activity of DNA methyltransferase 1 (DNMT1).

“which is seemingly driven by the age-dependent loss of microglial SIRT1, a NADdependent deacetylase that can regulate the activity of DNA methyltransferase 1 (DNMT1) (Cho et al., 2015).”

• cites Cho et al., 2015 sourceSupported

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

TET2 and TET3 are responsible for the development and proliferation of CD4+CD8+ double-positive thymocytes into invariant natural killer T cells (iNKT cells).

“For instance, TET2 and TET3 are responsible for the development and proliferation of CD4+CD8+ double-positive thymocytes into invariant natural killer T cells (iNKT cells) (Tsagaratou et al., 2016).”

• cites Tsagaratou et al., 2016 sourcePartly supported

  The Abstract reports that deleting Tet2 and Tet3 in mouse CD4+CD8+ double-positive thymocytes dysregulated iNKT-cell development and proliferation. It does not say that the double-positive thymocytes proliferate into iNKT cells; rather, TET proteins support proper iNKT development while suppressing aberrant iNKT-cell proliferation. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

In T helper cells, TET2 mediates demethylation of putative regulatory elements in genes associated with T cell differentiation, regulating the Th1 and Th17 cytokine expression in vitro.

“In T helper cells, TET2 mediates demethylation of putative regulatory elements in genes associated with T cell differentiation, regulating the Th1 and Th17 cytokine expression in vitro (Ichiyama et al., 2015).”

• cites Ichiyama et al., 2015 sourceSupported

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

The contribution of epigenetic modifications to neurodegenerative diseases such as PD and AD has been addressed in a number of studies.

“Indeed, the contribution of epigenetic modifications to neurodegenerative diseases such as PD (Wüllner et al., 2016) (Park et al., 2015) and AD (Watson et al., 2016), (Phipps et al., 2016) has been addressed in a number of studies.”

• cites Wüllner et al., 2016 sourceSupported
• cites Park et al., 2015Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Watson et al., 2016 sourceSupported

Classified as probably not a substantial claim.

In pro-inflammatory (M1) macrophages, aerobic glycolysis is a consequence of glucose uptake and the conversion of pyruvate into lactate.

“In pro-inflammatory (M1) macrophages, aerobic glycolysis is a consequence of glucose uptake and the conversion of pyruvate into lactate (Galván-Peña and O'Neill, 2014).”

• cites Galván-Peña and O'Neill, 2014 sourceSupported

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

Treatment with LPS in macrophages, dendritic and microglia cells provokes a metabolic shift from oxidative phosphorylation (OXPHOS) towards aerobic glycolysis, a process required to quickly supply high energy demands of the inflammatory response.

“Treatment with LPS in macrophages, dendritic and microglia cells provokes a metabolic shift from oxidative phosphorylation (OXPHOS) towards aerobic glycolysis, a process required to quickly supply high energy demands of the inflammatory response (Galván-Peña and O'Neill, 2014),(Ruiz-García et al., 2011), (Ganeshan and Chawla, 2014),(Orihuela et al., 2016).”

• cites Galván-Peña and O'Neill, 2014 sourcePartly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites Ruiz-García et al., 2011 sourcePartly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites Ganeshan and Chawla, 2014 sourcePartly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured

May combine two claims.

The in vitro finding that TET2 is necessary for a full proinflammatory response contrasts with findings in peripheral macrophages after intraperitoneal injection in vivo.

“These results contrast with findings in peripheral macrophages after intraperitoneal injection in vivo (Zhang et al., 2015).”

• cites Zhang et al., 2015 sourcePartly supported

  The in vivo endotoxin-shock passage (section labeled Abstract; page unavailable) reports greater IL-6 production and inflammation in myeloid Tet2-deficient mice after systemic LPS challenge, supporting the stated contrast. Extended Data Figure 1 describes peritoneal macrophages isolated from deficient mice and then stimulated with LPS, but our copy does not establish an in vivo finding specifically in peripheral macrophages after intraperitoneal injection. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

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

NO from NOS-2 inhibits mitochondria.

“potentially mediated by the well-known inhibition of mitochondria by NO from NOS-2 (BalPrice et al., 2002) (Kelly and O'Neill, 2015) (Doulias et al., 2013).”

• cites BalPrice et al., 2002Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Kelly and O'Neill, 2015Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Doulias et al., 2013 sourceCould not verify

  The Discussion (page unavailable) reports that eNOS-derived nitric oxide activates a mitochondrial fatty-acid oxidation enzyme; it does not address whether NO from NOS-2 inhibits mitochondria. Searches for NOS-2, inducible nitric oxide synthase, and mitochondrial respiration inhibition found no passage making that claim. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

May combine two claims.

Inhibition of hexokinase activity by 2-deoxy-D-glucose (2-DG) inhibited the inflammatory response measured as NOS-2 expression in BV2 at 6h without inducing cell death.

“inhibition of hexokinase activity by using 2deoxy-D-glucose (2-DG) inhibited the inflammatory response measured as NOS-2 expression in BV2 at 6h without inducing cell death (Vilalta and Brown, 2014), (Tannahill et al., 2013) (Figure S5F-G).”

• cites Vilalta and Brown, 2014 sourcePartly supported

  The Methods (Flow cytometry; page unavailable) confirm that BV-2 viability was assessed after 6 hours of deoxyglucose treatment. The Results (Microglia are killed by deoxyglucose via ATP depletion and phagocytosis; page unavailable) report ATP depletion before cell death, but do not report reduced NOS-2 expression or establish the claimed absence of cell death at 6 hours. They instead report a small increase in TNFα release from primary microglia at 3 hours. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites Tannahill et al., 2013 sourcePartly supported

  The passage labelled Abstract reports that 2DG inhibits induced IL-1β transcription in bone marrow-derived macrophages, supporting the general point that 2DG can suppress an inflammatory response. It does not report NOS-2 expression in BV2 cells at 6 hours or establish that the treatment did not cause cell death. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

Hk3 and Pfkfb3 play an important role during the aerobic glycolysis process.

“both playing an important role during the aerobic glycolysis process (Galván-Peña and O'Neill, 2014)(Ruiz-García et al., 2011).”

• cites Galván-Peña and O'Neill, 2014 sourcePartly supported

  The “Glycolytic M1 Versus Oxidative M2 Macrophages” section says that induction of a gene it calls PFKB3 accompanies a switch that increases glycolytic flux in activated macrophages, supporting the Pfkfb3 part of the claim. Our copy does not mention Hk3 or establish its role in aerobic glycolysis. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites Ruiz-García et al., 2011 sourcePartly supported

  The Discussion (page not provided) reports that increased PFKFB3 expression and activity raise glycolytic flux in activated macrophages. The paper also describes macrophage glycolysis in highly oxygenated environments, but the passages do not address HK3; the claim about both genes is therefore only partly supported. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

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

TET2 is required for the repression of IL-6 upon LPS treatment in peripheral macrophages to ensure termination of inflammation.

“It was previously shown that TET2 is required for the repression of IL-6 upon LPS treatment in peripheral macrophages to ensure termination of inflammation (Zhang et al., 2015).”

• cites Zhang et al., 2015 sourceSupported

Classified as probably not a substantial claim.

Microglia play a fundamental role in the demise of the neuronal population in several neurodegenerative diseases.

“Since microglia play a fundamental role in the demise of the neuronal population in several neurodegenerative diseases, we tested whether Tet2 expression could also be affected by different pathological protein aggregates seen in a number of neurodegenerative diseases typically associated to neuroinflammation (Ugalde et al., 2016).”

• cites Ugalde et al., 2016 sourceCould not verify

  The “Neurotoxicity of misfolded protein” section describes neuronal damage and loss across proteinopathies, and an Author Manuscript passage mentions microgliosis alongside disease in a mouse model. Neither says that microglia play a fundamental role in causing neuronal loss. Searches for microglia and neuroinflammation found no passage establishing that attributed role; page numbers were unavailable in our copy. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

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

In a severe neurodegeneration model (CK-p25 mouse model) for AD, 'late-stage' activated microglia is characterized by the expression of many Type I and Type II Interferon response genes.

“In fact, using single-cell RNA sequencing of microglia at different stages in a severe neurodegeneration model (CK-p25 mouse model) for AD (Mathys et al., 2017), the authors described that 'late-stage' activated microglia is characterized by the expression of many Type I and Type II Interferon response genes.”

• cites Mathys et al., 2017 sourceSupported

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

The 5xFAD model recapitulates in a few months the main features of AD.

“This model recapitulates in a few months the main features of AD (Oakley et al., 2006).”

• cites Oakley et al., 2006 sourceSupported

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

The fibrillar and oligomeric Aβ-amyloid-induced neuroinflammatory response has been linked to TLR4.

“Similarly, the fibrillar and oligomeric A β -amyloid-induced neuroinflammatory response has been linked to TLR4 as well (Reed-Geaghan et al., 2009; Wang et al., 2018).”

• cites Reed-Geaghan et al., 2009 sourcePartly supported

  The Discussion links TLR4 to microglial activation and inflammatory signaling induced by fibrillar Aβ. The paper studies fibrillar Aβ, not oligomeric Aβ, so it does not support that part of the jointly cited claim. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Wang et al., 2018 sourceNot checked — no full text of the source

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

Traditionally, Type I IFN response was considered solely for defense against viral and bacterial infections.

“Traditionally, Type I IFN response was considered solely for defense against viral and bacterial infections (Stifter and Feng, 2015)(Kovarik et al., 2016).”

• cites Stifter and Feng, 2015 sourceNot checked — no full text of the source
• cites Kovarik et al., 2016 sourcePartly supported

  The ‘Type I IFN Induction by Bacteria’ section says type I interferons were originally described as interfering with viral replication, and the review discusses their roles in bacterial defense. It does not support the claim that they were traditionally considered solely a defense against both viruses and bacteria: it also describes responses to fungi and parasites, and notes immunosuppressive activities. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

The action of TET2 is highly context-specific, with fundamental differences in the regulation of innate immune responses between peripheral immune cells (and specifically monocyte-derived macrophages) and microglia.

“This effect differs from the result reported here and highlights fundamental differences in the regulation of the innate immune responses between peripheral immune cells (and specifically monocyte-derived macrophages) and microglia (Burm et al., 2015) (London et al., 2013; Zarruk et al., 2018), suggesting that the action of TET2 is highly context-specific.”

• cites Burm et al., 2015 sourceSupported
• cites London et al., 2013 sourceSupported
• cites Zarruk et al., 2018 sourceNot checked — no full text of the source

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

TET2 plays important roles during the inflammatory response in different peripheral immune cells.

“Recent reports have shown that TET2 plays important roles during the inflammatory response in different peripheral immune cells (Ichiyama et al., 2015)(Tsagaratou et al., 2016)(Zhang et al., 2015)(Cull et al., 2017).”

• cites Ichiyama et al., 2015 sourceSupported
• cites Tsagaratou et al., 2016 sourcePartly supported

  The Results (iNKT cell expansion; no page supplied) report expansion of iNKT cells in peripheral lymphoid organs after Tet2–Tet3 deletion. The Discussion (no page supplied) also says TET2 modulates cytokine production in CD4+ helper T cells. These passages support an immune-regulatory role, but this study primarily tests combined Tet2 and Tet3 loss and does not establish TET2 alone as regulating inflammatory responses across different peripheral immune cells. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites Zhang et al., 2015 sourceSupported

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

α-synuclein aggregates induce microglial activation in a TLR-4 dependent manner.

“For instance, α -synuclein aggregates induce microglial activation (Boza-Serrano et al., 2014) in a TLR-4 dependent manner (Fellner et al., 2013).”

• cites Boza-Serrano et al., 2014Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Fellner et al., 2013 sourceSupported

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

TET2 specifically represses Il-6 transcription at late phase in LPS-treated macrophages to terminate the inflammatory response.

“Notably, TET2 has been shown to specifically repress Il-6 transcription at late phase in LPS-treated macrophages to terminate the inflammatory response (Zhang et al., 2015).”

• cites Zhang et al., 2015 sourceSupported

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

These aggregated forms play leading roles in driving main pathogenic events in these neurodegenerative diseases with the active involvement of highly activated microglia, more recently referred to as disease-associated microglia.

“In fact, these aggregated forms play leading roles in driving main pathogenic events in these neurodegenerative diseases with the active involvement of highly activated microglia, more recently referred to as disease-associated microglia (Krasemann et al., 2017) (Keren-Shaul et al., 2017) (Mathys et al., 2017).”

• cites Krasemann et al., 2017 sourcePartly supported

  The Results identify a disease-associated microglial phenotype in models of AD, ALS and MS, and associate it with neuritic Aβ plaques in AD. The Discussion suggests that microglia, amyloid plaques and dystrophic neurites may contribute to AD progression. It does not establish that aggregated forms drive the main pathogenic events across these neurodegenerative diseases; the claimed causal role is broader and stronger than the paper's findings. Page numbers are unavailable in our copy. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites Keren-Shaul et al., 2017 sourcePartly supported

  The Results identify disease-associated microglia (DAM) in an Alzheimer’s mouse model and find them near amyloid-β plaques. The Discussion also links DAM to clearance of aggregated proteins in neurodegenerative disease. It does not establish the broader claim that aggregated forms drive the main pathogenic events across these diseases, and it describes DAM as potentially protective rather than as drivers of pathology. The supplied copy gives section labels but no page numbers. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites Mathys et al., 2017 sourcePartly supported

  The Introduction describes microglia responding to Aβ plaques, and the Discussion (‘Two Distinct Reactive Microglia Phenotypes in Neurodegeneration’; page unavailable) reports that the authors’ late-response microglia resemble disease-associated microglia. The paper does not establish that aggregated forms drive the main pathogenic events in these diseases, and it says whether the DAM-like response is protective, neutral, or harmful remains unknown. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

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

Hexokinase 3 catalyzes the first committed step of glycolysis, while PFKFB3 catalyzes both the synthesis and degradation of fructose-2,6-bisphosphate (F2,6BP), a regulatory molecule that controls glycolysis in eukaryotes.

“Hexokinase 3 catalyzes the first committed step of glycolysis (Nishizawa et al., 2014), while PFKFB3 catalyzes both the synthesis and degradation of fructose-2,6-bisphosphate (F2,6BP), a regulatory molecule that controls glycolysis in eukaryotes.”

• cites Nishizawa et al., 2014 sourcePartly supported

  The Results and Discussion identify Hk3 as one of the hexokinases that catalyze the first, irreversible reaction in glycolysis, converting glucose to glucose-6-phosphate. The passage does not call that reaction the first committed step, so it supports the enzyme and reaction but not that qualifier. The PFKFB3 clause follows the citation and is not attributed to this source. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

May combine two claims.

Microglial TET2 is upregulated in vivo in two well-defined models of microglia polarization: a neuroinflammatory mouse model induced by repeated intraperitoneal injections of LPS, and AD, including human patients and transgenic mice (5xFAD).

“We also found that microglial TET2 is upregulated in vivo in two-well defined models of microglia polarization; in a neuroinflammatory mouse model induced by repeated intraperitoneal injections of LPS (Krasemann et al., 2017; Bodea et al., 2014), and in AD, including human patients and transgenic mice (5xFAD).”

• cites Krasemann et al., 2017 sourcePartly supported

  Split into 2 atoms — 0 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites Bodea et al., 2014 sourceSupported

May combine two claims.

BV2 cells are enriched for H3K27ac at the Tet2 promoter and at the putative regulatory region upstream of Tet2.

“BV2 cells are enriched for H3K27ac at the Tet2 promoter and at the putative regulatory region upstream of Tet2 (Figure 2B).”

May combine two claims.

The levels of H3K27ac increased in the upstream region (E1 and E2) after 1-hour treatment with KLA, and this was concomitant with the recruitment of p65 to both the promoter and upstream regions upon KLA treatment.

“Notably, the levels of H3K27ac increased in the upstream region (E1 and E2) after 1-hour treatment with KLA (a TLR-4 agonist), and this was concomitant with the recruitment of p65 to both the promoter and upstream regions upon KLA treatment (Figure 2C).”

• AssumptionThe authors state this as an assumption: TLR-4-induced regulatory events in macrophages are a model for those in microglia.

May combine two claims.

RNA-seq data from postnatal primary microglia cell culture experiments showed that Tet2 expression was increased both at low and high levels of LPS treatment, as well as after treatment with fibronectin.

“We further validated our observations by analyzing RNA sequencing (RNA-seq) data from postnatal primary microglia cell culture experiments (Janova et al., 2016), which showed that Tet2 expression was increased both at low and high levels of LPS treatment, as well as after treatment with fibronectin, which also promotes inflammation (Figure S1A).”

• cites Janova et al., 2016 sourceNot checked — no full text of the source

May combine two claims.

Neither LPS treatment nor knockdown of TET2 resulted in a significant change in global 5mC levels.

“Neither LPS treatment nor knockdown of TET2 resulted in a significant change in global 5mC levels (Figure S4A)”

May combine two claims.

LPS treatment did not change global 5hmC levels, despite the increase in TET2 expression.

“Interestingly, LPS treatment did not change global 5hmC levels, despite the increase in TET2 expression.”

May combine two claims.

TET2 is an NF-κB-responsive gene that regulates both early transcription (after only a few hours) of genes affecting several pathways (including control of the immune response and cell cycle) and the late inflammatory response.

“We found that TET2 is an NF- B-responsive gene that regulates both early transcription (after only a few hours) of genes affecting several pathways (including control of the immune response and cell cycle) and the late inflammatory response.”

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

The expression pattern in BV2 cells after LPS treatment is very similar to previously published RNA-seq data from LPS-treated primary microglia cells.

“We first confirmed that the expression pattern in our BV2 cells after LPS treatment is very similar to previously published RNA-seq data from LPS-treated primary microglia cells in (Janova et al., 2016) (Figure S3A).”

• cites Janova et al., 2016 sourceNot checked — no full text of the source

May combine two claims.

In response to LPS, Tet2 flox/flox Cx3cr1 WT/WT mice showed very low presence of homeostatic microglia and very robust increases of activated microglial cells.

“In response to LPS, Tet2 flox/flox Cx3cr1 WT/WT mice showed very low presence of homeostatic microglia, with very robust increases of activated microglial cells (Figure 6C).”

May combine two claims.

This led to decreased Tet2 expression in Tet2 flox/flox Cx3cr1 Cre/WT in basal conditions and complete abrogation of Tet2 upregulation upon treatment with 100 ng/ml of LPS for 24 hours.

“Importantly, this led to decreased Tet2 expression in Tet2 flox/flox Cx3cr1 Cre/WT in basal conditions and complete abrogation of Tet2 upregulation upon treatment with 100 ng/ml of LPS for 24 hours (Figure S5C).”

May combine two claims.

In BV2 cells, while there was no difference at 3h after LPS treatment, the expression of Il-1β, Il-6 and Nos-2 was reduced at 6 and 24h post-LPS treatment upon TET2 depletion.

“We observed in BV2 cells that, while there was no difference at 3h after LPS treatment, the expression of Il-1 β , Il-6 and Nos-2 was reduced at 6 and 24h post-LPS treatment upon TET2 depletion (Figure 5A-5C).”

May combine two claims.

LPS-treated siControl cells consume glucose from the media, but LPS-treated siTET2 cells show a substantial reduction in glucose uptake after 3h LPS treatment.

“We analyzed the extra-cellular glucose levels after 3h LPS treatment and found that siControl LPS treated cells consume glucose from the media, but that LPS-treated siTET2 cells show a substantial reduction in the glucose uptake (Figure 5J).”

May combine two claims.

TET2 knockdown reduced the basal and maximal oxygen consumption of the cells after 3h LPS treatment, but after 24h LPS treatment, the cellular oxygen consumption levels increase.

“Indeed, TET2 knockdown reduced the basal and maximal oxygen consumption of the cells after 3h LPS treatment (Figure 5I and Figure S5E), but after 24h LPS treatment, the cellular oxygen consumption levels increase (Figure 5I).”

May combine two claims.

TET2 binding increases substantially at target genes upon LPS treatment, whereas a control locus (Oct4) shows no alterations.

“Our data show that, indeed, TET2 binding increases substantially at target genes upon LPS treatment, whereas a control locus ( Oct4 ) shows no alterations (Figure 4G).”

May combine two claims.

In one AD patient (patient 1), there is significant upregulation of TET2 in plaque-associated microglia, while no statistical significance was observed in the other two patients.

“Our analysis showed that in one patient (patient 1), there is significant upregulation of TET2 in plaque-associated microglia, while no statistical significance was observed in the other two patients (Figure 7D).”

May combine two claims.

In one of the patients, microglial TET2 expression is highly expressed in plaque-associated microglia, while microglia localized away from plaques showed little or no induction of TET2 expression.

“Importantly, similar to the result obtained in the 5xFAD mouse model, in one of the patients microglial TET2 expression is highly expressed in the plaque-associated microglia (DAM microglia), while microglia localized away from the plaques (homeostatic) showed little or no induction of TET2 expression (Figure 7A-D).”

May combine two claims.

TET2 played central roles in driving both the classical proinflammatory response and metabolic reprogramming during TLR-dependent microglia activation.

“Notably, TET2 played central roles in driving both the classical proinflammatory response and metabolic reprogramming that take place during the TLR-dependent microglia activation.”

May combine two claims.

TET2 regulates primarily genes involved in the innate immune response and more specifically genes related to the TLR induced-Type I IFN response.

“We found that TET2 regulates primarily genes involved in the innate immune response and more specifically genes related to the TLR induced-Type I IFN response (e.g., Stat1, Stat3, Irf1, Irf7 ) (Noppert et al., 2007)(Luu et al., 2014).”

• cites Noppert et al., 2007 sourceNot checked — no full text of the source
• cites Luu et al., 2014 sourceNot checked — no full text of the source

May combine two claims.

The Iba-1+/TET2+ ratio is relatively high in patient 1, while patients 2 and 3 present a lower ratio.

“The Iba-1+/TET2+ ratio is relatively high in patient number 1, but on the other hand, patients number 2 and 3 present a lower ratio (Figure 7D).”

May combine two claims.

Using the same in vivo model used by Zhang and colleagues, TET2 is also upregulated in microglia but TET2 is necessary for a full proinflammatory response.

“Using the same in vivo model used by Zhang and colleagues, we observe that TET2 is also upregulated in microglia but TET2 is necessary for a full proinflammatory response.”

May combine two claims.

GBP2 and GBP3 appear in our RNA-seq analysis as genes under TET2 control upon LPS treatment, which could potentially affect the inflammatory response.

“GBP2 and GBP3 appear in our RNA-seq analysis as genes under TET2 control upon LPS treatment, which could potentially affect the inflammatory response.”

May combine two claims.

TET2 drives early expression of multiple genes associated to the immune system including the Type I IFN response upon TLR stimulation, an effect largely independent of its oxidative activity.

“We provide strong evidence that TET2 drives early expression of multiple genes associated to the immune system including the Type I IFN response upon TLR stimulation, an effect that was largely independent from its oxidative activity.”

May combine two claims.

TET2 regulates the early (3 h) transcriptional response to LPS but without an immediate effect on traditional markers for the inflammatory response.

“Our RNA-seq data show that TET2 regulates the early (3h) transcriptional response to LPS but without an immediate effect on traditional markers for the inflammatory response.”

May combine two claims.

All patients present some Iba-1 positive cells expressing TET2, but the numbers vary greatly within the three patients.

“While all the patients present some Iba-1 positive cells expressing TET2, the numbers vary greatly within the three patients.”

May combine two claims.

Direct inhibition of glycolysis using 2-D-Deoxyglucose prevented LPS induction of NOS-2, suggesting that TET2 regulation of glycolysis may mediate the LPS induced inflammatory response.

“Direct inhibition of glycolysis (using 2-D- Deoxyglucose to inhibit hexokinase activity) prevented LPS induction of NOS-2, suggesting that the TET2 regulation of glycolysis may mediate the LPS induced inflammatory response.”

May combine two claims.

TET2 depletion leads to reduced glucose consumption and lactate production, an effect that precedes the reduction in classic inflammatory markers.

“We found that TET2 depletion leads to reduced glucose consumption and lactate production, an effect that precedes the reduction in classic inflammatory markers.”

May combine two claims.

Using an antibody against TET2 validated in knockout mice, analysis of temporal cortex of human AD and hippocampus of 5xFAD mice demonstrated unequivocal upregulation of TET2 in disease plaque-related microglia but not in homeostatic microglia.

“This is interesting because our analysis using an antibody against TET2 (validated in Knockout mice) in temporal cortex of human AD and hippocampus of 5xFAD mice demonstrated the unequivocal upregulation of TET2 in disease plaque-related microglia but not in homeostatic microglia.”

May combine two claims.

p65 has a potential role in Tet2 expression through binding to an upstream enhancer element, increasing its activity, reflected by higher H3K27ac levels in LPS-treated cells.

“These results suggest a potential role of p65 in Tet2 expression through binding to an upstream enhancer element, increasing its activity, which is reflected by the higher H3K27ac levels in LPS-treated cells.”

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

DNA methylation can be removed by the action of Ten-eleven Translocation (TET) enzymes, which are dioxygenases that catalyze the oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and other oxidative derivatives.

“Importantly, DNA methylation can be removed by the action of Ten-eleven Translocation (TET) enzymes, which are dioxygenases that catalyze the oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and other oxidative derivatives (Branco et al., 2012).”

• cites Branco et al., 2012 sourceNot checked — no full text of the source

May combine two claims.

TET2 not only plays a role in the regulation of the inflammatory response, but also in other aspects of microglia physiology, such as cell cycle regulation.

“Altogether, our results suggest that TET2 not only plays a role in the regulation of the inflammatory response, but also in other aspects of microglia physiology, such as cell cycle regulation.”

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

A role for TET2 during the resolution of inflammation has also been described in bone-derived macrophages.

“A role for TET2 during the resolution of inflammation has also been described in bone-derived macrophages (Cull et al., 2017).”

• cites Cull et al., 2017 sourceNot checked — no full text of the source

May combine two claims.

TET2 plays a role in the LPS-induced inflammatory response, in particular the response to interferon β (Type I IFN response).

“Our RNA-seq data suggested that TET2 plays a role in the LPS-induced inflammatory response, in particular the response to interferon β (or Type I IFN response) (Figure 3E).”

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

TLR-4 stimulation induces a rapid and robust transcriptional response which involves genes that regulate metabolic reprogramming.

“TLR-4 stimulation induces a rapid and robust transcriptional response which involves genes that regulate metabolic reprogramming (Medzhitov and Horng, 2009).”

• cites Medzhitov and Horng, 2009 sourceNot checked — no full text of the source

May combine two claims.

TET2 acts directly on these genes but its effect upon LPS is not predominantly mediated through its catalytic activity.

“These results suggest that TET2 acts directly on these genes but that its effect upon LPS is not predominantly mediated through its catalytic activity.”

May combine two claims.

The 18-month-old 5xFAD mouse model mirrors the main features of AD through five mutations linked to familial forms of AD and develops extracellular amyloid plaques typically associated with clusters of highly reactive microglia.

“We used the 18 month-old 5xFAD mouse model, which mirrors the main features of AD through five mutations linked to familial forms of AD, and develops extracellular amyloid plaques typically associated with clusters of highly reactive microglia.”

May combine two claims.

IFIT2 is one of the TET2-regulated genes and has been shown to play a role in the amplification of the secretion of TNFα and IL-6 in vivo in a LPS-induced endotoxin shock model.

“Of note, one of the TET2-regulated genes that forms part of this response is the IFN-induced protein with tetratricopeptide repeats 2 (IFIT2), which has been shown to play a role in the amplification of the secretion of TNFα and IL-6 in vivo in a LPS-induced endotoxin shock model (Siegfried et al., 2013).”

• cites Siegfried et al., 2013 sourceNot checked — no full text of the source

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

There is growing acknowledgement of the role that the Type I IFN response plays over the TLR-induced classical inflammatory response.

“There is growing acknowledgement of the role that the Type I IFN response plays over the TLRinduced classical inflammatory response (Luu et al., 2014)(Taylor et al., 2014).”

• cites Luu et al., 2014 sourceNot checked — no full text of the source
• cites Taylor et al., 2014 sourceNot checked — no full text of the source

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

Data in Figure 7B are based on three independent experiments.

“Data shown in B is mean ± s.e.m of 3 independent experiments.”

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

Data in Figure 5H, 5I and 5J are based on 3 independent experiments.

“Data shown in H, I and J are mean ± s.d of 3 independent experiments (H, I and J).”

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

In Figure 6D and 6E, the number of independent experiments is three for Tet2 flox/flox Cx3cr1 WT/WT + LPS and seven for Tet2 flox/flox Cx3cr1 Cre/WT + LPS.

“In panels D and E the number of independent experiments is equal to three for Tet2 flox/flox Cx3cr1 WT/WT + LPS and seven for Tet2 flox/flox Cx3cr1 Cre/WT + LPS.”

May combine two claims.

TET2 could drive the proinflammatory activation of microglia and induction of metabolic reprogramming upon inflammatory stimulus.

“Our results strongly support the idea that TET2 could drive the proinflammatory activation of microglia and induction of metabolic reprogramming upon inflammatory stimulus.”

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

Results from an independent biological replicate of data in Figure 4H are shown in Figure S4H.

“Results from an independent biological replicate of data in H are shown in Figure S4H.”

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

Data in Figure 5E are based on seven independent experiments.

“Data shown in E are mean ± s.d of seven independent experiments.”

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

Data in Figure 5A and 5C are based on five and three independent experiments, respectively.

“Data shown are mean ± s.d. of five (A) and three (C) independent experiments.”

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

Data in Figure 5D are based on nine (3h), six (6h) and eight (24h) independent experiments.

“Data shown in D are mean ± s.e.m of nine (3h), six (6h) and eight (24h) independent experiments.”

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

Increased levels of TET2 protein present in microglia cells might result from a post-translational mechanism, for instance a decrease in the rate of protein degradation.

“This suggests that increased levels of TET2 protein present in microglia cells might result from a post-translational mechanism (for instance a decrease in the rate of protein degradation which allows an increase of protein content).”

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

Data in Figure 5F and 5G are based on 9 and 3 independent experiments, respectively.

“Data shown in F and G are mean ± s.e.m of 9 (F) and 3 (G) independent experiments.”

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

Results in Figure 6B and 6C correspond to three independent experiments.

“The results correspond to three independent experiments in panels B and C.”

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

Data in Figure 4 panels A-H are based on two technical replicates.

“Data shown in A-H represent the mean ± s.d of two technical replicates.”

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

In Figure 6G, all treatments are three independent experiments except Tet2 flox/flox Cx3cr1 Cre/WT + LPS, which is four independent experiments.

“In panel G all treatments are 3 independent experiments except Tet2 flox/flox Cx3cr1 Cre/WT + LPS which is four independent experiments.”

Classified as probably not a substantial claim.

An active role of TET2 in the switch from homeostatic to disease-associated microglia is anticipated.

“Keeping in mind the chronic nature of inflammation in neurodegenerative diseases, an active role of TET2 in the switch from homeostatic to disease-associated microglia is anticipated.”