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Cited by 329Acute Myeloid Leukemia ResearchHematopoietic Stem Cell TransplantationBlood disorders and treatments

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

Open access
Hormaechea-Agulla, Daniel, Matatall, Katie A., Le, Duy T., Kain, Bailee, Long, Xiaochen, Kus, Pawel, Jaksik, Roman, Challen, Grant A. +2 more·2021·Cell Stem Cell
DOI 10.1016/j.stem.2021.03.002PMC PMC8349829
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Abstract

Age-related clonal hematopoiesis (CH) is a risk factor for malignancy, cardiovascular disease, and all-cause mortality. Somatic mutations in DNMT3A are drivers of CH, but decades may elapse between the acquisition of a mutation and CH, suggesting that environmental factors contribute to clonal expansion. We tested whether infection provides selective pressure favoring the expansion of Dnmt3a mutant hematopoietic stem cells (HSCs) in mouse chimeras. We created Dnmt3a-mosaic mice by transplanting Dnmt3a-/- and WT HSCs into WT mice and observed the substantial expansion of Dnmt3a-/- HSCs during chronic mycobacterial infection. Injection of recombinant IFNγ alone was sufficient to phenocopy CH by Dnmt3a-/- HSCs upon infection. Transcriptional and epigenetic profiling and functional studies indicate reduced differentiation associated with widespread methylation alterations, and reduced secondary stress-induced apoptosis accounts for Dnmt3a-/- clonal expansion during infection. DNMT3A mutant human HSCs similarly exhibit defective IFNγ-induced differentiation. We thus demonstrate that IFNγ signaling induced during chronic infection can drive DNMT3A-loss-of-function CH.

AI snapshot

Written from the paper’s full text.

TL;DR

Chronic *Mycobacterium avium* infection drives expansion of Dnmt3a-loss-of-function hematopoietic stem cell clones in mouse chimeras, and IFNγ signaling is necessary and sufficient for this clonal hematopoiesis. Mechanistically, Dnmt3a−/− HSCs show reduced differentiation and secondary stress-induced apoptosis, with epigenetic suppression of pro-differentiation genes.

Key findings

  • In Dnmt3a-mosaic mice, chronic *M. avium* infection expanded Dnmt3a−/− HSCs; by 12 months post infection, Dnmt3a−/− cells averaged 82.8% of total HSCs in infected mice versus 19.5% in uninfected controls.
  • IFNγ signaling was required and sufficient: Dnmt3a−/− Ifngr1−/− double-knockout HSCs did not expand, while daily recombinant IFNγ for one month induced Dnmt3a−/− clonal expansion; IL1β showed a trend, whereas LPS, TNFα, and pIpC did not.
  • Dnmt3a−/− HSCs had reduced differentiation and less secondary stress-induced apoptosis; RNAseq showed 155 genes upregulated in WT but not Dnmt3a−/− HSCs, including Fos/Jun/Batf2, and Batf2−/− HSCs partially phenocopied Dnmt3a−/− expansion.
  • Human DNMT3A−/− CD34+ HSPCs showed defective IFNγ-induced differentiation, and murine Dnmt3a−/− whole bone marrow serially replated for 8–9 rounds versus 2–3 rounds for WT; prior pIpC treatment extended Dnmt3a-mutant replating to 12 passages.

Methods

  • Mouse mosaic chimeras were created by transplanting CD45.2 Dnmt3a−/− or WT whole bone marrow with CD45.1 competitor marrow into lethally irradiated CD45.1 WT recipients; mice were infected IV with 2 × 10^6 CFU *M. avium*, repeatedly for chronic infection, and analyzed by flow cytometry, colony assays, and secondary transplants.
  • Functional assays included methylcellulose differentiation with IFNγ, serial replating, Annexin/Caspase-Glo apoptosis assays, cytokine bead arrays, and human CD34+ HSPC differentiation after CRISPR/Cas9 DNMT3A deletion.
  • RNAseq and whole-genome bisulfite sequencing were performed on sorted HSCs; differential expression used Limma-Voom with FDR < 0.05, and a differential equation model was used to predict competing HSC population dynamics.

Limitations

  • The paper states a lack of validating human data, noting the challenge of decades-long human longitudinal studies.
  • The paper states that Dnmt3a−/− mice become anemic with infection, which is not a common feature of human CH, and that the phenotypes largely focus on knockout mice, giving stronger effects than heterozygous human mutations and not modeling gain-of-function mutations.
  • Evident from the text: the 12-month peripheral blood analysis did not include WT:WT mosaics, and the post-transplant mosaic infection model may exaggerate competitive differences expected to take longer in non-transplant conditions.

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

Integrity signals

Registry

Crossref · 10.1016/j.stem.2021.03.002

Cites

64 of 64 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

25 of 35 cited sources checked · 10 supported · 3 flagged · accuracy not yet measured

  • Premisecitation

    The Mx1-Cre+ Dnmt3af/f mouse model approximates DNMT3A loss of function mutations commonly found in human clonal hematopoiesis.

    “we used a Mx1-Cre+ Dnmt3af/f mouse model that approximates DNMT3A loss of function mutations commonly found in human CH (Sandoval et al., 2019).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Sandoval et al., 2019 source

      The Discussion and murine embryonic stem-cell results report that some DNMT3A mutations identified in AML patients reduce or abolish methyltransferase activity. The paper does not establish that such mutations are common in human clonal hematopoiesis or assess whether the Mx1-Cre+ Dnmt3af/f mouse model approximates them (pages unavailable in our copy). (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Premisecitation

    DNMT3A mutations confer little phenotype on steady state hematopoiesis.

    “many have noted that DNMT3A mutations confer little phenotype on steady state hematopoiesis (Buscarlet et al., 2017)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Buscarlet et al., 2017 source
  • Premisecitation

    Many CH-associated mutations themselves contribute to increased inflammatory signaling, including mutations in TET2, FLT3, JAK2, STAT1, CBL, and RAS.

    “Since many CH-associated mutations themselves contribute to increased inflammatory signaling, including mutations in TET2, FLT3, JAK2, STAT1, CBL, and RAS (Azrakhsh et al., 2019; Cull et al., 2017; Jaiswal et al., 2017)”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Azrakhsh et al., 2019 source
    • cites Cull et al., 2017 source
    • cites Jaiswal et al., 2017 source

      The Results (page not provided) show increased inflammatory chemokines and cytokines in Tet2-deficient macrophages, supporting the TET2 portion. The Discussion explicitly says it is unclear whether JAK2 mutations increase coronary risk through inflammation, and the passages found do not establish inflammatory signaling for FLT3, STAT1, CBL, or RAS. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
  • Findingcitation

    Many alterations in genome methylation occurred in bivalent canyon sites that have been associated previously with developmentally important genes.

    “many alterations in genome methylation occurred in bivalent canyon sites that have been associated previously with developmentally important genes (Jeong et al., 2014).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Jeong et al., 2014 source
  • Premisecitation

    In serially transplanted Dnmt3a−/− HSCs compared with WT HSCs, these genes are also differentially suppressed in the Dnmt3a−/− cells.

    “In a recently published study examining gene expression differences between serially transplanted Dnmt3a−/− HSCs and WT HSCs, these genes are also differentially suppressed in the Dnmt3a−/− cells”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Jeong et al., 2018 source

      The Results section, “Dnmt3a Controls DNA Methylation at HSC Regulatory Elements” (page unavailable), reports RNA-seq comparisons of control and serially transplanted Dnmt3aKO HSCs and says genes in bivalent canyons showed repression with extended passage. The citing sentence does not identify what “these genes” refers to, so our copy cannot establish that the particular genes intended were suppressed. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 21
  • May combine two claims. 18

  • Classified as probably not a substantial claim.

    Premisecitation

    Inflammation has been shown to be a driver of Tet2-mutant clonal hematopoiesis.

    “inflammation has been shown to be a driver of Tet2-mutant CH.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Abegunde et al., 2018 source
    • cites Cai et al., 2018 source
    • cites Fuster et al., 2017 source

      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.

      Experimental — accuracy not yet measured

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

    Premisecitation

    NR4A1 and SOCS3 play a role in supporting HSC homeostasis by dampening inflammatory responses.

    “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).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Freire and Conneely, 2018 source
    • cites Kim et al., 2017 source

      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.

      Experimental — accuracy not yet measured

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

    Premisecitation

    Loss of the histone demethylase Kdm6b leads to opposite effects on Fos and Jun.

    “Strikingly, loss of the histone demethylase Kdm6b leads to opposite effects on Fos and Jun”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Mallaney et al., 2019 source

      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.

      Experimental — accuracy not yet measured

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

    Premisecitation

    DNMT3A is the gene most commonly mutated in clonal hematopoiesis.

    “DNMT3A, the gene most commonly mutated in CH”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Young et al., 2016 source

      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)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    DNMT3A mutations are nearly universal in individuals by the age of 50.

    “mutations in DNMT3A, the gene most commonly mutated in CH, are nearly universal in individuals by the age of 50 (Young et al., 2016)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Young et al., 2016 source

      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)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Dnmt3a loss of function mutations comprise the majority of human clonal hematopoiesis-associated mutations.

    “Dnmt3a loss of function mutations, which comprise the majority of human CH-associated mutations (Russler-Germain et al., 2014)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Russler-Germain et al., 2014 source

      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)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Dnmt3a−/− HSCs demonstrate defective differentiation and enhanced self-renewal.

    “prior studies showed that Dnmt3a−/− HSCs demonstrate defective differentiation and enhanced self-renewal (Challen et al., 2011; Jeong et al., 2018)”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Challen et al., 2011 source
    • cites Jeong et al., 2018 source

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

    Premisecitation

    Nr4a1 is reduced in Dnmt3a-mutant hematopoietic progenitors.

    “Nr4a1 has been reported to be reduced in Dnmt3a-mutant hematopoietic progenitors”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Kramer et al., 2017 source

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

    Premisecitation

    Increased red cell distribution width (RDW) has been reported in humans with CH.

    “although increased red cell distribution width (RDW) has been reported (Jaiswal et al., 2014).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Jaiswal et al., 2014 source

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

    Premisecitation

    Reduced Nr4a1 in Dnmt3a-mutant hematopoietic progenitors leads to suppression of apoptosis.

    “leading to suppression of apoptosis (Kramer et al., 2017).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Kramer et al., 2017 source

      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)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Hypermethylation in the setting of a Dnmt3a deletion has also been reported in other studies.

    “While hypermethylation in the setting of a Dnmt3a deletion seems counterintuitive, it has also been reported in other studies”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Jeong et al., 2018 source

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

    Interpretationcitation

    Loss of Kdm6b and Dnmt3a loss have an interrelated regulatory mechanism affecting Fos and Jun.

    “suggesting an interrelated regulatory mechanism (Mallaney et al., 2019).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Mallaney et al., 2019 source

      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)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Aging is associated with increased inflammation.

    “Aging itself is associated with increased inflammation (Piber et al., 2019)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Piber et al., 2019 source

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

    Interpretationcitation

    Hypermethylation in the setting of a Dnmt3a deletion may be related to compensatory effects of alternative methyltransferases.

    “and may be related to compensatory effects of alternative methyltransferases (Jeong et al., 2018).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Jeong et al., 2018 source

      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)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Immune signaling pathways contribute to DNMT3A-associated hematologic conditions including aplastic anemia, myeloproliferative neoplasms, myelodysplastic syndrome, and acute myelogenous leukemia.

    “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).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Azrakhsh et al., 2019 source
    • cites Jacquelin et al., 2018 source
    • cites Yoshizato et al., 2015 source

      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)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    In 12-month monitoring, the percentage of Dnmt3a−/− CD45.2 PBMCs expanded to an insignificant degree in naïve mice, but increased significantly for infected mosaic mice at both 8 and 12 months post infection.

    “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).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The total number of differentially expressed genes was lower in the Dnmt3a−/− background than WT, with 155 genes upregulated in WT but not Dnmt3a−/− HSCs.

    “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.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Batf2 KO mice, HSCs were depleted upon chronic repeated infection of WT mice, but the number of HSCs remained relatively intact in Batf2 KO mice, similar to the protection observed in chronically infected Dnmt3a−/− mice.

    “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).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    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.

    “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.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Infection significantly altered gene expression in WT HSCs, but there were fewer differences in the Dnmt3a−/− background.

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

  • May combine two claims.

    Findingown data

    At low IFNγ (1 ng/mL), IFNγ stimulated increased WT colony formation, whereas this increase was diminished for Dnmt3a−/− HSCs.

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

  • May combine two claims.

    Findingown data

    PIPC treatment slightly augmented the serial replating capacity of Dnmt3a-mutant WBM, extending the potential for replating from 9 to 12 passages.

    “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.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Upon IFNγ stimulation, control human CD34+ cells differentiated at a high rate, indicated by loss of CD34+ CD38− HSCs and significant increase in CD38+ progenitors and CD66b+ neutrophils; DNMT3A−/− human CD34+ cells did not differentiate normally, showing little change in HSC or neutrophil numbers after three days in culture with IFNγ.

    “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).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

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

    “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).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Daily injection of IL1β induced a clear trend toward expansion, whereas no trend was observed with daily LPS, TNFa, or polyinosinic-polycytidylic acid (pIpC) treatment.

    “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).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Sustained IFNγ signaling is necessary and sufficient to induce infection-associated Dnmt3a−/− HSC expansion.

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

  • May combine two claims.

    Interpretationown data

    There was no expansion among more downstream populations such as granulocyte-monocyte progenitors, megakaryocyte progenitors, or B and T cells, macrophage/monocytes, or granulocytes, suggesting a differentiation block in the Dnmt3a-mutant population at the level of the MPP.

    “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.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

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

    “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.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    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.

    “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.”
    Quote in the paperSubstantial claim

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

    Study design and conductcitation

    Quality control was conducted using FastQC and CGmapTools.

    “Quality control was conducted using FastQC (Andrews, 2010) and CGmapTools (Guo et al., 2018).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Andrews, 2010

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

    • cites Guo et al., 2018 source

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

    Premisecitation

    Human epidemiologic data correlate CH with severe infections.

    “there have been recent reports of human epidemiologic data correlating CH with severe infections (Bolton et al., 2020a)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Bolton et al., 2020a

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

  • May combine two claims.

    Interpretationown data

    Infection can promote Dnmt3a-loss of function clonal hematopoiesis through increased resistance to stress-induced apoptosis and a defect in differentiation, driven by impaired expression of an IFNγ-dependent transcriptional network.

    “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).”
    Quote in the paperSubstantial claim

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

    Study design and conductcitation

    Differentially methylated regions were identified with Benjamini & Hochberg correction for multiple testing.

    “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.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Guo et al., 2018 source

  • May combine two claims.

    Interpretationown data

    Inflammatory IFNγ signaling is both necessary and sufficient for Dnmt3a-loss of function clonal hematopoiesis.

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

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

    Study design and conductown data

    Deletion efficiency was checked at the end of the experiment by genotyping CD45.2+ HSCs from mock-infected and infected mosaic mice.

    “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.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Each experimental replicate in the human HSPC differentiation assay was conducted with cells from a different biological sample, representing a different individual.

    “Each experimental replicate was conducted with cells from a different biological sample, representing a different individual.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Inflammation is a major driver of both aging and age-associated CH.

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

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Gene deletion in human CD34+ cells was confirmed by PCR and real-time PCR.

    “Gene deletion was confirmed by PCR and realtime using the following primers”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    The phenotypes reported in this study largely focus on KO mice.

    “The phenotypes reported in this study largely focus on KO mice”
    Quote in the paperNot a substantial claim
39 of 39

  • The paper relies on this without support.

    Age and smoking are both associated with inflammation.

    “given that age and smoking are both associated with inflammation”

  • The paper relies on this without support.

    Specific environmental drivers of DNMT3A-mutant clonal hematopoiesis have not been determined.

    “specific environmental drivers of DNMT3A-mutant CH have not been determined.”

  • The paper relies on this without support.

    BATF2 is a critical IFNγ-induced transcription factor that drives myeloid differentiation of HSCs.

    “As a case in point, we previously identified BATF2 to be a critical IFNγ-induced transcription factor that drives myeloid differentiation of HSCs.”

  • The paper relies on this without support.

    Anemia is not a common feature of humans with CH.

    “but this is not a common feature of humans with CH although increased red cell distribution width (RDW) has been reported (Jaiswal et al., 2014).”
4 of 4

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?

  • No direct experiments have been performed to quantify fitness between mutant and wild-type hematopoietic stem cells in response to IFNγ.

    “Although the competitive advantage of Dnmt3a-mutant HSCs during inflammation has been implied (20,23), no direct experiments have been performed to quantify fitness between mutant and WT HSCs in response to IFNγ.”

    From <i>Txnip</i> Enhances Fitness of <i>Dnmt3a</i> -Mutant Hematopoietic Stem Cells via <i>p21</i> (2022) · cites this paper as [20]

    The Results section ('Dnmt3a loss of function blunts HSC response to infection'; page unavailable) reports that daily recombinant IFNγ injections induced expansion of Dnmt3a-mutant HSC clones. The Methods describe the corresponding mosaic-mouse experiment, in which mutant marrow competed against WT marrow. Thus, the paper reports a direct comparison under IFNγ treatment, contrary to the claim that no such experiment had been performed. (LLM, conf=0.90)

    Checked by a reasoning model — may be wrong.

    Experimental — accuracy not yet measured

  • IFN-γ signaling is necessary and sufficient for the fitness of Dnmt3a knockout hematopoiesis.

    “supporting that IFN-γ signaling is necessary and sufficient for the fitness of Dnmt3a knockout hematopoiesis.”

    From Clonal hematopoiesis and inflammation – the perpetual cycle (2023) · cites this paper as [49]

  • In competitive transplantation into recipient wild-type mice without additional stimuli, the advantage of Dnmt3a mutant cells is not significant and is only apparent in the context of chronic Mycobacterium avium infection.

    “However, in competitive transplantation into recipient wild-type mice without additional stimuli, the advantage of Dnmt3a mutant cells is not significant and is only apparent in the context of chronic Mycobacterium avium (M. avium) infection [49].”

    From Clonal hematopoiesis and inflammation – the perpetual cycle (2023) · cites this paper as [49]

  • The competitive advantage of Dnmt3a-mutant hematopoietic stem cells is exacerbated by hematopoietic stress such as serial transplantation and microbial infection.

    “The competitive advantage of Dnmt3a-mutant HSCs is exacerbated by hematopoietic stress such as serial transplantation (19) and microbial infection (20).”

    From <i>Txnip</i> Enhances Fitness of <i>Dnmt3a</i> -Mutant Hematopoietic Stem Cells via <i>p21</i> (2022) · cites this paper as [20]

  • There is emerging evidence that clonal HSCs can become inflammation-adapted and by altering the response to inflammatory challenges, mutations can thus confer a fitness advantage to HSCs.

    “There is emerging evidence that clonal HSCs can become inflammation-adapted47–49 and by altering the response to inflammatory challenges, mutations can thus confer a fitness advantage to HSCs.”

    From Single-cell multi-omics identifies chronic inflammation as a driver of TP53-mutant leukemic evolution (2023) · cites this paper as [48]

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.

  • Mathematical predictions assigned to different supplemental figures

    Minor

    The mathematical predictions are identified as Figures S3C-F in the Results but as Supplemental Figures 2C-F in the Code Availability statement.

  • Materials statement conflicts with generation of Batf2-KO mice

    Minor

    The Materials Availability statement says the study generated no new unique reagents, but the Methods state that the study generated Batf2-KO mice by CRISPR editing.

2 of 2

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Authors
Hormaechea-Agulla, D., Matatall, K. A., Le, D. T., Kain, B., Long, X., Kus, P., Jaksik, R., Challen, G. A., Kimmel, M., & King, K. Y.
Affiliations
  • Baylor College of Medicine (US)
  • Rice University (US)
  • Silesian University of Technology (PL)
  • Washington University in St. Louis (US)
Corresponding author
King, Katherine Y.
ORCID
Venue
Cell Stem Cell
Publisher
Elsevier BV
Published
2021-08
Volume
28
Issue
8
Pages
1428-1442.e6
ISSN
  • 1875-9777 (ISSN-L)
  • 1934-5909
DOI
10.1016/j.stem.2021.03.002
PMC
PMC8349829
OpenAlex
W3138449921
Cited by
329
References
65
Citations by year
2026: 43 · 2025: 66 · 2024: 63 · 2023: 68 · 2022: 64 · 2021: 25
FWCI
36.22
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell stem cell (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institutes of Health (R01HL134880, R01HL136333, T32AI053831, T32DK060445, T32HL092332)
  • National Institute of Allergy and Infectious Diseases (AI036211, P30CA125123, S10 OD020066)
  • National Center for Research Resources (S10RR024574)
  • Baylor College of Medicine (NCI-CA125123, NIDDK-DK56338, P30 CA125123)
Topics
  • Health Sciences › Medicine › Hematology › Acute Myeloid Leukemia Research
  • Health Sciences › Medicine › Hematology › Hematopoietic Stem Cell Transplantation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Blood disorders and treatments
Keywords
clonal hematopoiesis, DNMT3A, loss-of-function mutations, chronic infection, mycobacterial infection, interferon-gamma signaling, hematopoietic stem cells, clonal expansion, DNA methylation, epigenetic modifications, apoptosis, age-related diseases, somatic mutations, mouse chimeras
MeSH terms
Clonal Hematopoiesis, Animals, DNA (Cytosine-5-)-Methyltransferases, Hematopoiesis, Hematopoietic Stem Cells, Mutation, Mice

About these checks

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 2026-10-28.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-28.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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