Chronic infection drives Dnmt3a-loss-of-function clonal hematopoiesis via IFNγ signaling

In the paper

“inflammation has been shown to be a driver of Tet2-mutant CH.”

What the sources say

• ?Not checked — no full text of the sourcecites Abegunde et al., 2018 source
• Supported

  The Results (page unavailable in our copy) report that LPS-induced inflammation increased the ratio of Tet2-KO to wild-type cells in a mouse model of clonal hematopoiesis, while anti-inflammatory treatment repressed Tet2-KO cell expansion. This directly supports inflammation as a driver of Tet2-mutant clonal hematopoiesis in the studied model. (LLM, conf=0.93)

  cites Cai et al., 2018 source
• Disputed

  The paper reports the opposite causal direction: TET2 deficiency increases inflammatory cytokine production, while clonal expansion of TET2-deficient cells contributes to atherosclerosis. In the indexed passage labeled Abstract (page unavailable), NLRP3 inhibition reduced atherosclerosis but “did not affect the expansion of TET2-deficient cells”; it does not show inflammation driving that expansion. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  cites Fuster et al., 2017 sourceExperimental — accuracy not yet measured

In the paper

“Both NR4A1 and SOCS3 have been noted to play a role in supporting HSC homeostasis by dampening inflammatory responses (Freire and Conneely, 2018; Kim et al., 2017).”

What the sources say

• Supported

  The Discussion (page not supplied) says NR4A receptors help maintain HSC quiescence and suppress inflammatory responses, including NF-κB-driven signaling. In this jointly cited sentence, Freire and Conneely support the NR4A1 portion; the SOCS3 portion is not attributed specifically to this paper. (LLM, conf=0.90)

  cites Freire and Conneely, 2018 source
• Disputed

  The Results and discussion section, “Level of Socs3 expression dictates HSC homeostasis in the BM” (page unavailable), reports that Socs3 overexpression impairs HSC homeostasis—the opposite of the role attributed to SOCS3 here. The Concluding remarks instead link preserved homeostasis to repression of Socs3 and discuss Tpo/Jak2 signaling, not dampening inflammatory responses. This paper does not address NR4A1. (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  cites Kim et al., 2017 sourceExperimental — accuracy not yet measured

In the paper

“Strikingly, loss of the histone demethylase Kdm6b leads to opposite effects on Fos and Jun”

What the sources say

• Disputed

  The Results section (page not supplied) says that both Fos and Jun were overexpressed in Kdm6b-deficient hematopoietic stem cells, at baseline and more strongly under stress. It therefore reports effects in the same direction, not opposite effects. (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  cites Mallaney et al., 2019 sourceExperimental — accuracy not yet measured

In the paper

“DNMT3A, the gene most commonly mutated in CH”

What the sources say

• Partly supported

  The Results (Error-corrected NGS; page unavailable) report that 64% of exonic clonal variants occurred in DNMT3A and TET2 combined. The Discussion says DNMT3A mutations are frequent in clonal haematopoiesis, but the paper does not establish that DNMT3A alone is the most commonly mutated gene. (LLM, conf=0.84)

  cites Young et al., 2016 sourceExperimental — accuracy not yet measured

In the paper

“mutations in DNMT3A, the gene most commonly mutated in CH, are nearly universal in individuals by the age of 50 (Young et al., 2016)”

What the sources say

• Partly supported

  The Abstract (page unavailable) reports clonal haematopoiesis in 95% of the healthy 50–60-year-olds studied, with mutations frequently in DNMT3A or TET2. The Results (Error-corrected NGS; page unavailable) show DNMT3A is a frequent mutation site, but the paper does not say DNMT3A mutations themselves are nearly universal, or establish that they are present by age 50. (LLM, conf=0.90)

  cites Young et al., 2016 sourceExperimental — accuracy not yet measured

In the paper

“Dnmt3a loss of function mutations, which comprise the majority of human CH-associated mutations (Russler-Germain et al., 2014)”

What the sources say

• Partly supported

  The Discussion describes DNMT3A mutations that impair its function, and the Introduction reports DNMT3A mutation frequencies in acute myeloid leukemia (AML). Neither passage establishes that DNMT3A loss-of-function mutations comprise the majority of mutations associated with human clonal hematopoiesis; the reported population is AML, not clonal hematopoiesis. (LLM, conf=0.83)

  cites Russler-Germain et al., 2014 sourceExperimental — accuracy not yet measured

In the paper

“prior studies showed that Dnmt3a−/− HSCs demonstrate defective differentiation and enhanced self-renewal (Challen et al., 2011; Jeong et al., 2018)”

What the sources say

• Supported

  The Results section, “Dnmt3a loss impairs long-term HSC differentiation in vivo,” reports that Dnmt3a-null HSCs’ differentiation capacity declined sharply during serial transplantation while their self-renewal potential increased and remained sustained. This supports both parts of the attributed claim. (LLM, conf=0.94)

  cites Challen et al., 2011 source
• Supported

  In Results, the paper reports that Dnmt3aKO HSCs failed to generate substantial peripheral-blood progeny after the third transplant, yet repopulated bone marrow through 12 transplant rounds; it says this self-renewal far exceeds that of normal HSCs. A separate Results section reports severely compromised myeloid differentiation in late-passage Dnmt3aKO HSCs. Page numbers were not provided in our copy. (LLM, conf=0.94)

  cites Jeong et al., 2018 source

In the paper

“Nr4a1 has been reported to be reduced in Dnmt3a-mutant hematopoietic progenitors”

What the sources say

• Supported

  The Results section ('Nr4a1 is downregulated in Dnmt3aKO DN2 thymic progenitors') reports 2.3-fold lower Nr4a1 expression in Dnmt3aKO DN2 cells and also reports downregulation in Dnmt3aKO early thymic progenitors. These are the hematopoietic progenitors supporting the citing sentence. (LLM, conf=0.91)

  cites Kramer et al., 2017 source

In the paper

“although increased red cell distribution width (RDW) has been reported (Jaiswal et al., 2014).”

What the sources say

• Supported

  The Results section, “Blood-Cell Indexes of Persons with Somatic Mutations,” reports significantly higher red-cell distribution width in participants with detectable somatic mutations than in those without mutations (13.8% vs. 13.4% among normocytic subjects). This supports the claim that increased RDW has been reported in humans with clonal hematopoiesis. (LLM, conf=0.94)

  cites Jaiswal et al., 2014 source

In the paper

“leading to suppression of apoptosis (Kramer et al., 2017).”

What the sources say

• Partly supported

  The Discussion says Dnmt3aKO-derived DN2 thymic progenitors have reduced apoptosis partially due to Nr4a1 downregulation. The claim omits “partially” and extends this finding from DN2 thymic progenitors to Dnmt3a-mutant hematopoietic progenitors generally. (LLM, conf=0.86)

  cites Kramer et al., 2017 sourceExperimental — accuracy not yet measured

In the paper

“While hypermethylation in the setting of a Dnmt3a deletion seems counterintuitive, it has also been reported in other studies”

What the sources say

• Supported

  The Results section (‘Dnmt3a Controls DNA Methylation at HSC Regulatory Elements’) reports increased DNA methylation in bivalent and inactive canyons of Dnmt3a-knockout HSCs. This supports the claim that hypermethylation has been observed following Dnmt3a deletion, even though the paper also reports overall hypomethylation. (LLM, conf=0.93)

  cites Jeong et al., 2018 source

In the paper

“suggesting an interrelated regulatory mechanism (Mallaney et al., 2019).”

What the sources say

• Partly supported

  The Results and Discussion report that Kdm6b loss dysregulates Fos and Jun in hematopoietic stem cells, and that reducing their expression improves engraftment. They do not establish an interrelated regulatory mechanism involving Dnmt3a loss; searches for Dnmt3a and the proposed mechanism found no supporting passage. The Discussion also says the mechanism linking Kdm6b and AP-1 remains unclear. Page numbers were not available in our copy. (LLM, conf=0.86)

  cites Mallaney et al., 2019 sourceExperimental — accuracy not yet measured

In the paper

“Aging itself is associated with increased inflammation (Piber et al., 2019)”

What the sources say

• Supported

  The Abstract states that systemic inflammation increases with age. The Discussion also reports higher levels of specific inflammatory markers with increasing age among adults over 60, while noting that other markers were not significantly associated with age. (LLM, conf=0.94)

  cites Piber et al., 2019 source

In the paper

“and may be related to compensatory effects of alternative methyltransferases (Jeong et al., 2018).”

What the sources say

• Partly supported

  The Results section, “Dnmt3a Controls DNA Methylation at HSC Regulatory Elements” (page unavailable), reports hypermethylated regions in Dnmt3a-knockout HSCs, including increased methylation in bivalent and inactive canyons. It does not attribute that hypermethylation to compensatory activity by alternative methyltransferases, so the proposed explanation is unsupported by our copy. (LLM, conf=0.84)

  cites Jeong et al., 2018 sourceExperimental — accuracy not yet measured

In the paper

“Immune signaling pathways are known to contribute to DNMT3A-associated hematologic conditions including aplastic anemia, myeloproliferative neoplasms, myelodysplastic syndrome, and acute myelogenous leukemia (Azrakhsh et al., 2019; Jacquelin et al., 2018; Yoshizato et al., 2015).”

What the sources say

• ?Not checked — no full text of the sourcecites Azrakhsh et al., 2019 source
• ?Not checked — no full text of the sourcecites Jacquelin et al., 2018 source
• Partly supported

  The Discussion (page unavailable) links DNMT3A-mutated clones in aplastic anemia to progression toward MDS/AML, and the paper describes aplastic anemia as immune-mediated. It does not establish that immune signaling pathways contribute to DNMT3A-associated disease across the listed conditions or address that mechanism in myeloproliferative neoplasms. The Discussion instead says it is unlikely that DNMT3A-mutated clones initiate a pathogenic immune response. (LLM, conf=0.86)

  cites Yoshizato et al., 2015 sourceExperimental — accuracy not yet measured

In the paper

“Whereas the percentage of Dnmt3a−/− CD45.2 peripheral blood mononuclear cells (PBMCs) expanded to an insignificant degree in this time frame for naïve mice, the percentage increased significantly for infected mosaic mice at both 8- and 12-months post infection (Figure 2B–G).”

In the paper

“the total number of differentially expressed genes was lower in the Dnmt3a−/− background than WT (Figure 6D), with 155 genes upregulated in WT but not Dnmt3a−/− HSCs.”

In the paper

“whereas HSCs were depleted upon chronic repeated infection of WT mice, the number of HSCs remained relatively intact in Batf2 KO mice, similar to the protection observed in chronically infected Dnmt3a−/− mice (Figure 3F).”

In the paper

“As shown in Figure 5C, Dnmt3a−/− clonal expansion occurred as expected upon M. avium infection, but no clonal expansion was evident among mosaics with the DKO minor population or in mosaic mice with a minor population of Ifngr1−/− HSCs.”

In the paper

“Whereas infection significantly altered gene expression in WT HSCs, there were fewer differences in the Dnmt3a−/− background (Figure 6A, 6B, Supplemental Tables 2–3).”

In the paper

“At low concentrations (1 ng/mL), IFNγ stimulated increased WT colony formation, whereas this increase was diminished for Dnmt3a−/− HSCs (Figure 4B, S4C).”

In the paper

“PIPC treatment slightly augmented the serial replating capacity of Dnmt3a-mutant WBM (compare maroon to orange), extending the potential for replating from 9 to 12 passages.”

In the paper

“Upon IFNγ stimulation, control CD34+ cells differentiated at a high rate, as indicated by a loss of CD34+ CD38− HSCs and a significant increase in CD38+ progenitors and CD66b+ neutrophils. In contrast, DNMT3A−/− human CD34+ cells did not differentiate normally, showing little change in either their HSC or neutrophil numbers after three days in culture with IFNγ (Figure 4F).”

In the paper

“At high concentrations (100 ng/mL), IFNγ resulted in a loss of total WT colonies, whereas Dnmt3a−/− HSCs were relatively resistant to these toxic effects (Figure 4A, S4B).”

In the paper

“Furthermore, daily injection of IL1β induced a clear trend toward expansion, whereas no trend was observed in the setting of daily LPS, TNFa, or polyinosinic-polycytidylic acid (pIpC) treatment (Figure 5E).”

In the paper

“Collectively, these experiments indicate that sustained IFNγ signaling is necessary and sufficient to induce infection-associated Dnmt3a−/− HSC expansion.”

In the paper

“we did not see expansion among more downstream populations such as granulocyte-monocyte progenitors, megakaryocyte progenitors, or B and T cells, macrophage/monocytes, or granulocytes (Figures S1E–L), suggesting a differentiation block in the Dnmt3a-mutant population at the level of the MPP.”

In the paper

“Collectively, these studies indicate that the phenotypically defined Dnmt3a−/− HSCs that expand upon infection remain functional in long-term reconstitution studies, are more resistant to loss by secondary stress-induced apoptosis and differentiation, and retain their self-renewal capacity better than WT HSCs when stressed by chronic infection with M. avium.”

In the paper

“Collectively, these experiments show that Dnmt3a−/− HSCs have improved survival at high IFNγ concentrations and reduced differentiation at low IFNγ concentrations, both of which may contribute to a survival advantage.”

In the paper

“Quality control was conducted using FastQC (Andrews, 2010) and CGmapTools (Guo et al., 2018).”

What the sources say

• ?Not matched

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

  cites Andrews, 2010
• ?Not checked — no full text of the sourcecites Guo et al., 2018 source

In the paper

“there have been recent reports of human epidemiologic data correlating CH with severe infections (Bolton et al., 2020a)”

What the sources say

• ?Not matched

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

  cites Bolton et al., 2020a

In the paper

“Our controlled biological experiments provide proof of concept that an infection can promote Dnmt3a-loss of function CH through increased resistance to stress-induced apoptosis and a defect in differentiation, driven by impaired expression of an IFNγ-dependent transcriptional network (Figure S7).”

In the paper

“Differentially methylated regions (DMRs) were identified with CGmapTools (Guo et al., 2018) using all CG sites with a minimum of 4x and maximum of 100x coverage and Benjamini & Hochberg correction for multiple testing.”

What the sources say

• ?Not checked — no full text of the sourcecites Guo et al., 2018 source

In the paper

“We find that inflammatory IFNγ signaling is both necessary and sufficient for Dnmt3a-loss of function CH.”

In the paper

“To check the efficiency of deletion at the end of the experiment, CD45.2+ HSCs from mock-infected and infected mosaic mice were sorted into StemSpan media (Stem Cell Technologies), incubated for 2 weeks, and then the colonies were genotyped.”

In the paper

“Each experimental replicate was conducted with cells from a different biological sample, representing a different individual.”

In the paper

“Our findings lend further support to the emerging theme that inflammation is a major driver of both aging and age-associated CH”

In the paper

“Gene deletion was confirmed by PCR and realtime using the following primers”

In the paper

“The phenotypes reported in this study largely focus on KO mice”