Abstract
Somatic mutations in tet methylcytosine dioxygenase 2 (TET2), which encodes an epigenetic modifier enzyme, drive the development of haematopoietic malignancies1,2,3,4,5,6,7. In both humans and mice, TET2 deficiency leads to increased self-renewal of haematopoietic stem cells with a net developmental bias towards the myeloid lineage1,4,8,9. However, pre-leukaemic myeloproliferation (PMP) occurs in only a fraction of Tet2β/β mice8,9 and humans with TET2 mutations1,3,5,6,7, suggesting that extrinsic non-cell-autonomous factors are required for disease onset. Here we show that bacterial translocation and increased interleukin-6 production, resulting from dysfunction of the small-intestinal barrier, are critical for the development of PMP in mice that lack Tet2 expression in haematopoietic cells. Furthermore, in symptom-free Tet2β/β mice, PMP can be induced by disrupting intestinal barrier integrity, or in response to systemic bacterial stimuli such as the toll-like receptor 2 agonist. PMP was reversed by antibiotic treatment and failed to develop in germ-free Tet2β/β mice, which illustrates the importance of microbial signals in the development of this condition. Our findings demonstrate the requirement for microbial-dependent inflammation in the development of PMP and provide a mechanistic basis for the variation in PMP penetrance observed in Tet2β/β mice. This study will prompt new lines of investigation that may profoundly affect the prevention and management of haematopoietic malignancies.
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Acknowledgements
We thank the University of Chicago DNA Sequencing Core facility for assistance with sequencing; the Calcul QuΓ©bec and Compute Canada for providing access to the supercomputer Briaree from the University of Montreal; the Histology Core facility at the University of Chicago Human Tissue Resource Center for assistance with histology; S. Hwang (University of Chicago) for providing LysM-Cre mice; and V. Abadie (UniversitΓ© de MontrΓ©al) for discussions and critical reading of the manuscript. This work was supported by grants from the Cancer Center Support Grant P30CA014599 to B.J.; Digestive Diseases Research Core Center P30DK42086 at the University of Chicago to R.H., E.B.C., C.R.W. and B.J.; P01DK072201, R01DK110352 and 5R01CA161373 to S.A.L. and G.C.F.; F32 DK105728-01A1 to J.F.P.; CCFA Research Fellowship Award (ID: 480735) to M.M.; FWF Austrian Science Fund (P30324-B21) and Christian Doppler Society (I-CARE) to G.B.; and by a CIHR grant MOP (#20003029), a Canada Research Chair to E.F.V. and a Canada Research Chair to L.B.B. A.P. was supported by a fellowship from FRQS.
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Nature thanks S. Grivennikov, K. Shannon, Y. Xiong and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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Authors and Affiliations
Contributions
M.M., R.H. and B.J. conceived and designed the research, guided the interpretation of the results and wrote the manuscript. M.M. and R.H. performed the majority of the experiments and data analysis. A.P. and L.B.B. performed RNA sequencing experiments and data analysis. L.C. helped with various experiments. Z.M.E. and D.L.R. performed bacterial cultures and species identification. T.M. performed cell-sorting experiments. J.F.P. performed histology experiments. J.D.E. and M.W.M. helped with intestinal permeability assays. H.J.G. helped with germ-free mouse experiments. N.T. performed cytokine measurements. R.B. performed RT-PCR experiments. M.B. performed 16S PCR experiments. Y.W., Y.L. and C.R.W. performed ZO-1 immunofluorescence experiments and analysis. B.D.M. helped with flow cytometry experiments. V.D., S.M.K. and M.A.Z. helped with intestinal tissue sample collection. G.C.F. performed TUNEL and caspase 3 experiments and analysis. A.M.E. performed 16S analysis. S.A.L., G.B., E.B.C., A.M.E., C.R.W., L.B., L.A.G., E.F.V. and L.B.B. provided critical intellectual input and technical support. T.M., L.A.G., L.B.B. and B.J. critically edited the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Tet2β/β mice display variability in PMP penetrance that correlates with bacteraemia.
a, Penetrance of PMP assessed by splenomegaly, splenic LSK cell expansion and CD11b+Gr1+ myeloid cell expansion in spleen and peripheral blood. bβe, Correlation between frequency of peripheral-blood CD11b+Gr1+ myeloid cells (peripheral-blood biomarker) and numbers of CD11b+Gr1+ myeloid cells in spleen (b) and peripheral blood (c), numbers of LSK cells (d) and splenomegaly (e), in Tet2+/+ and Tet2β/β mice. In bβe, dotted vertical lines indicate level of 16% for peripheral-blood CD11b+Gr1+ myeloid cells (level determined by the distribution in wild-type littermates). The peripheral-blood biomarker (frequency of CD11b+Gr1+ myeloid cells) was used to categorize Tet2β/β mice either as symptom free (β€16% CD11b+Gr1+ myeloid cells) or as mice with PMP (>16% CD11b+Gr1+ myeloid cells). Spearman correlation test excluding Tet2+/+ mice. Red lines indicate regression line calculated for Tet2β/β data points (nβ=β10 (blue), 23 mice (red). fβi, Symptom-free, Tet2β/β mice with PMP and littermate controls were used. f, Representative dot blots are shown of LSK cells in the spleen, CD11b+Gr1+ myeloid cells in the spleen and peripheral blood, and lymphocytes in peripheral blood. Data are representative of five independent experiments with similar results. g, Spleen weight (top; nβ=β11 (blue), 9 (red, PMP) or 5 mice (red, symptom free)) and numbers of CD11b+Gr1+ myeloid cells in the spleen (bottom; nβ=β10 (blue), 12 (red, PMP) or 7 mice (red, symptom free)). h, Numbers per ml of peripheral blood of live CD45+ gated white blood cells (WBC) (top left), lymphocytes (top right), CD11b+ monocytes (bottom left) and CD11b+Gr1+ myeloid cells (bottom right) (nβ=β15 (blue), 15 (red, PMP) or 32 mice (red, symptom free)). i, In vitro HSC self-renewal colony-forming assay of haematopoietic progenitors of the bone marrow (nβ=β3 mice). MeanβΒ±βs.e.m. j, k, Correlation between 16S gene copies in the peripheral blood and spleen weight (nβ=β16 mice) (j) or numbers of CD11b+ monocytes (k top left), percentage of lymphocytes (k top right), numbers of lymphocytes (k bottom left), and numbers of leukocytes (WBC) (k bottom right) in the peripheral blood (nβ=β40 mice). Pearson correlation test. l, Bacterial colonies from Fig.Β 1e, f identified by 16S sequencing; blue rectangles indicate presence of bacteria. In g, h, boxes represent median values and interquartile ranges; whiskers represent minimum and maximum values. One-way ANOVA, Sidakβs post hoc test. Data are representative of at least three independent experiments. *Pβ<β0.05, **Pβ<β0.01, ***Pβ<β0.001, ****Pβ<β0.0001.
Extended Data Fig. 2 Tet2β/β mice display no anatomical changes in the jejunum.
a, Representative images of haematoxylin and eosin-stained sections of the jejunum. b, c, Immunofluorescence analyses of sections of the jejunum of Tet2+/+ and Tet2β/β mice. b, No difference was observed between the groups in the number of apoptotic cells using the TUNEL assay (red). Nuclei of intestinal cells were visualized with DAPI (blue). c, No difference was observed in the number of apoptotic (cleaved caspase 3-positive (CC3), red) epithelial cells (pan-keratin-positive, (PanK), green) between the groups. In b, c, Jejunum sections of diphtheria-toxin-treated VDTR mice were used as a positive control. Data are representative of at least two independent experiments.
Extended Data Fig. 3 Disruption of barrier integrity drives PMP.
a, FITCβdextran concentration in blood plasma correlates with numbers of LSK cells (left, nβ=β12 mice), frequency of CD11b+Gr1+ myeloid cells in the spleen (middle left, nβ=β10 mice), peripheral blood (middle right, nβ=β15 mice) and frequency of lymphocytes (right, nβ=β15 mice). Pearson correlation test. b, Schematic of DSS treatment of symptom-free Tet2f/fVavcre mice that are over 20 weeks old, and littermate controls. c, Numbers of CD11b+ monocytes (left), lymphocytes (middle) and leukocytes (WBC) (right) before, during and after DSS treatment (nβ=β5 (blue) or 8 (orange) mice). MeanβΒ±βs.e.m. d, Percentage of CD11b+Gr1+ myeloid cells (left) and numbers of GMP (right) at end point analysis (nβ=β5 (Tet2f/fcreβ, without DSS), 4 (Tet2f/fVavcre, without DSS), 6 (Tet2f/fcreβ, with DSS) or 8 (Tet2f/fVavcre, with DSS) mice). Centre is median, KruskalβWallis, Dunnβs post hoc test. *Pβ<β0.05, **Pβ<β0.01. Data are representative of at least two independent experiments.
Extended Data Fig. 4 Tight junction ZO-1 is markedly reduced in the jejunum but not in the colon of Tet2β/β mice.
a, RNA sequencing of whole intestinal tissue of duodenum, jejunum, ileum and colon of three Tet2β/β and three Tet2+/+ mice. Venn diagram illustrating the number of differentially expressed genes (|log2(fold change)|β>β0.5 and false discovery rateβ<β0.1); see Supplementary Table in Tet2β/β mice compared to littermate controls. b, RNA sequencing of the jejunum of seven Tet2β/β and seven Tet2+/+ mice (the full list of differentially expressed genes is shown in Supplementary Table. Heat map of a selection of antimicrobial peptides and tight junction genes. c, Gene expression of tight junction gene (ZO-1, Tjp1) in the jejunum (left; nβ=β12 (Tet2+/+) or 9 (Tet2β/β) mice) and colon (right; nβ=β9 mice in both cases). Centre is mean, two-tailed unpaired t-test. d, ZO-1 immunofluorescence shows reduced tight junction staining of epithelial cells in the jejunum (left) but no difference in colonic epithelial cells (right) of Tet2β/β mice. Representative images are shown from Tet2+/+ (nβ=β3 mice) and Tet2β/β mice (nβ=β7 mice). Scale bars, 100βΞΌm. Green, ZO-1; blue, DAPI. **Pβ<β0.01. Data are representative of at least two independent experiments.
Extended Data Fig. 5 Absence of Tet2 expression in haematopoietic cells leads to barrier dysfunction and PMP.
a, Intestinal permeability measurement by blood plasma FITCβdextran concentrations (nβ=β16 (Tet2f/fcreβ), 6 (Tet2f/fVavcre), 12 (Tet2f/fVillincre) or 12 (Tet2f/fLysMcre) mice). b, c, Gene expression of antimicrobial peptides Retlnb (b left), Ang4 (b right) and (c) tight junction genes ZO-1 (Tjp1) (left), occludin (Ocln) (middle), and desmoplakin (Dsp) (right) in the jejunum (nβ=β13 (Tet2f/fcreβ), 7 (Tet2f/fVavcre), 9 (Tet2f/fVillincre) or 10 (Tet2f/fLysMcre) mice). d, Representative dot blots (left) and numbers of LSK cells (right) in the spleen, CD11b+Gr1+ myeloid cells in the spleen (nβ=β6 (Tet2f/fcreβ), 12 (Tet2f/fVavcre), 10 (Tet2f/fVillincre) or 10 (Tet2f/fLysMcre) mice) and peripheral blood, and lymphocytes in the peripheral blood (nβ=β10 (Tet2f/fcreβ), 8 (Tet2f/fVavcre), 10 (Tet2f/fVillincre) or 10 (Tet2f/fLysMcre) mice). e, White blood cell (WBC) count (top) and cell number per ml of blood of CD11b+ monocytes (bottom) (nβ=β10 (Tet2f/fcreβ), 8 (Tet2f/fVavcre), 10 (Tet2f/fVillincre) or 10(Tet2f/fLysMcre) mice). f, Spleen weight is shown (nβ=β10 (Tet2f/fcreβ), 6 (Tet2f/fVavcre), 10 (Tet2f/fVillincre) or 10 (Tet2f/fLysMcre) mice). In aβf, centre is mean, one-way ANOVA, Sidakβs post hoc test. *Pβ<β0.05, **Pβ<β0.01, ***Pβ<β0.001, ****Pβ<β0.0001. Data are representative of three independent experiments.
Extended Data Fig. 6 Microbial signals are sufficient for PMP in Tet2β/β mice.
a, TLR2 activation measured using a HEK-TLR2 reporter assay (nβ=β5 biological replicates). b, Schematic of Pam3CSK4 in vivo treatment of symptom-free Tet2f/fVavcre mice that are over 20 weeks old, and littermate controls. c, Numbers of CD11b+ monocytes prior (day 0), during treatment (day 2) and at end point analysis (day 14) (nβ=β6 mice). MeanβΒ±βs.e.m. d, Percentage of CD11b+Gr1+ myeloid cells (left, nβ=β5 (Tet2f/fcreβ, no Pam3CSK4), 6 (Tet2f/fcreβ, with Pam3CSK4), 7 (Tet2f/fVavcre, no Pam3CSK4) or 6 (Tet2f/fVavcre, with Pam3CSK4) mice) and numbers of GMP cells (right, nβ=β6 (Tet2f/fcreβ, no Pam3CSK4), 6 (Tet2f/fcreβ, with Pam3CSK4), 7 (Tet2f/fVavcre, no Pam3CSK4) or 6 (Tet2f/fVavcre, with Pam3CSK4) mice). e, Intestinal permeability measurement by blood plasma FITCβdextran concentrations at end point analysis (nβ=β6 mice in all cases). a, cβe, Centre is mean. a, d, One-way ANOVA, Sidakβs post hoc test, *Pβ<β0.05, **Pβ<β0.01, ***Pβ<β0.001, ****Pβ<β0.0001. Data are representative of at least two independent experiments.
Extended Data Fig. 7 Microbial community structures are similar between Tet2β/β mice and littermate controls.
a, b, Non-metric multidimensional scaling of samples based on their oligotype profiles (with BrayβCurtis distance). a, Microbial community structures within the same body siteβthat is, jejunum (left, nβ=β14 (Tet2β/β (KO)) or 9 mice (Tet2+/+(WT))), colon (middle, nβ=β7 (KO) or 5 mice (WT)) and faeces (right, nβ=β6 (KO) or 5 mice (WT))βdo not differ significantly across genotypes based on envfit test with 999 permutations (Pβ>β0.05, two-tailed). b, Microbial community structures within the same genotypeβWT (top, nβ=β5 (colonic sample (COL) or faecal sample (FEC)) or 9 mice (jejunal sample (JEJ)) and KO (bottom, nβ=β6 (FEC), 7 (COL) or 14 mice (JEJ))βsignificantly differ across body sites based on the same test (Pβ<β0.001). c, d, Heat-map displays of oligotypes across samples. Clustering dendrograms are computed with BrayβCurtis distance and average linkage algorithm using the oligotype profiles. The intensity of colours indicates the per cent abundance of a given oligotype (rows) in a given sample (columns). c, Comparison of genotypes for samples collected from a single body site. d, Comparison of genotypes for samples collected across body sites. e, Co-housing does not affect the development of PMP in Tet2f/fVavcre mice. Representative cage configurations of 3βlitters from Tet2f/fVavcre mice with PMP that are over 20 weeks old (red), symptom-free Tet2f/fVavcre mice (grey), and littermate controls (blue). CON, luminal content; SCR, scraping samples.
Extended Data Fig. 8 Microbial signals are required for PMP in Tet2β/β mice.
a, b, Germ-free, SPF-housed Tet2β/β mice that are over 40 weeks old, and germ-free wild-type controls, analysed for percentage of lymphocytes (a left), numbers of lymphocytes (a middle left), numbers of CD11b+ monocytes (a middle right) and numbers of leukocytes (WBC) (a right) (nβ=β6 (Tet2+/+, GF), 7 (Tet2β/β, GF) or 7 (Tet2β/β, SPF) mice), and percentage of CD11b+Gr1+ myeloid cells (b left) and numbers of GMP cells (b right), (nβ=β7 (Tet2+/+, GF), 4 (Tet2β/β, GF) or 5 (Tet2β/β, SPF) mice). c, Mice treated with antibiotics (ABX) before onset of PMP (see schematic in Fig. Β 3c. Numbers of CD11b+Gr1+ myeloid cells (left; nβ=β14 (Tet2+/+, no ABX), 11 (Tet2β/β, no ABX), 7 (Tet2+/+, with ABX) or 7 (Tet2β/β, with ABX) mice) and LSK cells (right; nβ=β12 (Tet2+/+, no ABX), 8 (Tet2β/β, no ABX), 7 (Tet2+/+, with ABX) or 7 (Tet2β/β, with ABX) mice). d, Tet2β/β mice monitored for the number of CD11b+ monocytes (left, nβ=β7 mice) and 16S gene copies in the faeces (right, nβ=β6 mice) before, during and after antibiotics treatment. MeanβΒ±βs.e.m., repeated measures one-way ANOVA, Sidakβs post hoc test. In aβc, centre is mean, one-way ANOVA, Sidakβs post hoc test, *Pβ<β0.05, **Pβ<β0.01, ***Pβ<β0.001, ****Pβ<β0.0001. Data are representative of at least two independent experiments.
Extended Data Fig. 9 Antibiotic treatment reverses PMP in Tet2β/β mice.
aβj, Tet2β/β mice with PMP that are over 20 weeks old, and littermate controls, were treated with and without antibiotics (ABX) for four weeks. a, Numbers of peripheral-blood CD11b+ monocytes (nβ=β13 mice). Lines connect values obtained from the same mouse sampled before (β) and after (+) ABX treatment. b, c, Representative dot blots and percentage of CD11b+Gr1+ myeloid cells (b) (nβ=β20 (Tet2+/+, no ABX), 21 (Tet2β/β, no ABX), 6 (Tet2+/+, with ABX) or 14 (Tet2β/β, with ABX) mice) and LSK cells in the spleen (c) (nβ=β11(Tet2+/+, no ABX), 12 (Tet2β/β, no ABX), 6 (Tet2+/+, with ABX) or 12 (Tet2β/β, with ABX) mice). d, Representative dot blots and numbers of bone marrow-derived LSK cells (nβ=β11(Tet2+/+, no ABX), 13 (Tet2β/β, no ABX), 6 (Tet2+/+, with ABX) or 12 (Tet2β/β, with ABX) mice). e, f, Representative dot blots and numbers of splenic (e) (nβ=β6 (Tet2+/+, no ABX), 12 (Tet2β/β, no ABX), 5 (Tet2+/+, with ABX) or 12 (Tet2β/β, with ABX) mice) and bone marrow-derived (f) LSK gated CD34+Flt3β (Flt3 is also known as CD135) short-term (ST)-HSCs and CD34βFlt3β long-term (LT)-HSCs (nβ=β6 (Tet2+/+, no ABX), 13 (Tet2β/β, no ABX), 5 (Tet2+/+, with ABX) or 12 (Tet2β/β, with ABX) mice). g, h, Representative dot blots and numbers of splenic (g) (nβ=β5(Tet2+/+, no ABX), 10 (Tet2β/β, no ABX), 5 (Tet2+/+, with ABX) or 10 (Tet2β/β, with ABX) mice) and bone marrow-derived (h) LSK gated CD150+ CD48β cells (nβ=β5 (Tet2+/+, no ABX), 11 (Tet2β/β, no ABX), 5 (Tet2+/+, with ABX) or 10 (Tet2β/β, with ABX) mice). i, j, Representative dot blots and numbers of splenicΒ (i) (nβ=β6 (Tet2+/+, no ABX), 12 (Tet2β/β, no ABX), 5 (Tet2+/+, with ABX) or 12 (Tet2β/β, with ABX) mice) and bone marrow-derived (j) c-Kit+Sca-1β (LK gated) GMP, common myeloid progenitor (CMP) and megakaryocyteβerythroid progenitor (MEP) cells (nβ=β6 (Tet2+/+, no ABX), 13 (Tet2β/β, no ABX), 5 (Tet2+/+, with ABX) or 12 (Tet2β/β, with ABX) mice). In bβj, centre is mean, one-way ANOVA, Sidakβs post hoc test. Data are representative of at least three independent experiments. *Pβ<β0.05, **Pβ<β0.01, ***Pβ<β0.001, ****Pβ<β0.0001.
Extended Data Fig. 10 Bacteria-induced IL-6 is required for PMP in Tet2β/β mice.
a, Gene expression of Il6 in the spleen of SPF-housed Tet2β/β mice, littermates and germ-free Tet2β/β mice (nβ=β7 (Tet2+/+, SPF), 10 (Tet2β/β, SPF) or 4 (Tet2β/β, GF) mice). b, IL-6 cytokine levels in blood plasma of Tet2β/β mice with PMP treated with antibiotics (ABX) for four weeks (nβ=β5 mice). Lines connect values obtained from the same mouse sampled before and after antibiotics treatment. Two-tailed paired t-test. c, IL-6 cytokine levels in blood plasma of DSS-treated symptom-free Tet2f/fVavcre mice that are over 20 weeks old, and littermate controls (see schematic in Extended Data Fig.Β 3b (nβ=β6 (Tet2+/+) or 9 (Tet2β/β) mice). d, IL-6 cytokine levels in blood plasma of Tet2f/fVavcre mice that are over 20Β weeks old, and littermate controls, treated with the TLR1/2 agonist Pam3CSK4 (see schematic in Extended Data Fig. Β 6b (nβ=β6 (Tet2+/+) or 8 (Tet2β/β) mice). e, Correlation between IL-6 cytokine levels in blood plasma of Tet2β/β mice and numbers of peripheral-blood CD11b+ monocytes (nβ=β49 mice). Pearson correlation test. f, g, In vitro HSC self-renewal colony-forming assay of haematopoietic progenitors of the spleen from Tet2β/β mice (red lines) and littermate controls (blue lines) in the presence of anti-IL-6 antibody or isotype control (ISO) after the first replating (nβ=β3 mice). g, Representative images of colonies after the 5th replating. Scale bars, 100βΞΌm. h, Representative histogram (left) and quantification of (right) mean fluorescence intensity (MFI) of IL-6RΞ±+c-Kit+Sca-1β (LK gated) CD34+FcΞ³RIII/II+ GMPs from the bone marrow, (nβ=β5 (Tet2+/+, SPF), 6 (Tet2β/β, SPF), 4 (Tet2+/+, GF) or 3 (Tet2β/β, GF) mice). i, Representative flow cytometry plot of Stat3 phosphorylation (pY705) response after 30-min stimulation with 10βng mlβ1 IL-6 in splenic c-Kit+Sca-1β (LK gated) CD34+ and CD34β myeloid progenitors (MP). j, Numbers of CD11b+ myeloid cells (nβ=β7 (Tet2+/+) or 6 (Tet2β/β) mice). k, l, CD11b+F4/80+ macrophages and CD11b+Gr1+ myeloid cells of the spleen, from Tet2β/β mice and littermate controls, were FACS sorted. Gene expression of Il6 in macrophages (k) and CD11b+Gr1+ cells (l) (nβ=β7 (Tet2+/+) or 6 (Tet2β/β) mice). Centre is median. m, Representative histogram (left) and quantification of (right) MFI of IL-6RΞ±+CD11b+Gr1+ myeloid cells in the spleen (nβ=β5 (Tet2+/+, SPF), 6 (Tet2β/β, SPF), 4 (Tet2+/+, GF) or 4 (Tet2β/β, GF) mice). n, Representative histogram (left) and quantification of (right) MFI of IL-6RΞ±+c-Kit+Sca-1β (LK gated) CD34+FcΞ³RIII/II+ GMPs from the spleen of Tet2f/fLysMcre mice and littermate controls (nβ=β5 mice). Centre is mean. o, p, anti-IL-6 antibody (+) or ISO treatment (β) of Tet2β/β mice with PMP that are over 20 weeks old, and littermate controls (see schematic in Fig. Β 4g. o, Percentage of CD11b+Gr1+ myeloid cells (left; nβ=β10 (Tet2+/+, with anti-IL-6), 6 (Tet2β/β, with anti-IL-6) or 7 (Tet2β/β, without anti-IL-6) mice) and numbers of GMPs (right; nβ=β11 (Tet2+/+, with anti-IL-6), 6 (Tet2β/β, with anti-IL-6) or 8 (Tet2β/β, without anti-IL-6) mice) in the spleen. p, Intestinal permeability was assessed by blood plasma FITCβdextran concentrations (nβ=β6 mice in all cases). q, IL-6 in supernatants from intestinal explants: jejunum (left; nβ=β6 (Tet2+/+) or 5 (Tet2β/β) mice) and colon (right; nβ=β6 mice in both cases). Centre is mean. a, h, m, p, Centre is mean, one-way ANOVA, Sidakβs post hoc test. c, d, j, Centre is median, two-tailed MannβWhitney U-test. o, Centre is median, KruskalβWallis, Dunnβs post hoc test. Data are representative of at least three independent experiments; *Pβ<β0.05, **Pβ<β0.01, ***Pβ<β0.001. r, Model showing that extrinsic (microbial-induced inflammatory) and intrinsic (IL-6RΞ± expression) signals are required for PMP in Tet2β/β mice. In detail, small-intestinal barrier dysfunction (reduced ZO-1 and upregulation of defence response genes), which occurs spontaneously or upon intestinal damage, leads to bacterial translocation and to high levels of IL-6. Bacterial translocation can be bypassed when Tet2-deficient mice receive systemic microbial signals. Microbial-induced IL-6 is sensed by Tet2β/β myeloid progenitor (MP) cells that overexpress IL-6RΞ± and are highly sensitive to IL-6 (Stat3 (pY705)). Subsequently, MPs expand upon IL-6 signals and preferentially differentiate into mature myeloid cells with IL-6-producing capacities. This cycle results in the development of PMP. Treatment with antibiotics or neutralizing anti-IL-6 antibody can revert PMP, indicating that microbial inflammatory signals are required for PMP in the context of Tet2 deficiency. However, whether bacteria-induced inflammatory signals also create a permissive environment that induces the acquisition of cooperative oncogenic mutations that lead to the development of leukaemia remains to be determined. The mechanisms through which Tet2 deficiency in haematopoietic cells leads to a microbiota-dependent impairment of gut barrier function remains to be addressed.
Supplementary information
Supplementary Figure 1
Flow cytometry gating strategy
Supplementary Table 1
RNA-seq mapping statistics
Supplementary Table 2
A list of protein-coding genes ranked by statistical evidence of differential expression in Tet2β/β mice (Duodenum, Jejunum, Ileum, Colon)
Supplementary Table 3
A list of protein-coding genes ranked by statistical evidence of differential expression in Tet2β/β mice jejunum (7 samples)
Supplementary Table 4
16S rRNA Matrix counts
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Meisel, M., Hinterleitner, R., Pacis, A. et al. Microbial signals drive pre-leukaemic myeloproliferation in a Tet2-deficient host. Nature 557, 580β584 (2018). https://doi.org/10.1038/s41586-018-0125-z
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DOI: https://doi.org/10.1038/s41586-018-0125-z
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