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Adaptive response to inflammation contributes to sustained myelopoiesis and confers a competitive advantage in myelodysplastic syndrome HSCs

Abstract

Despite evidence of chronic inflammation in myelodysplastic syndrome (MDS) and cell-intrinsic dysregulation of Toll-like receptor (TLR) signaling in MDS hematopoietic stem and progenitor cells (HSPCs), the mechanisms responsible for the competitive advantage of MDS HSPCs in an inflammatory milieu over normal HSPCs remain poorly defined. Here, we found that chronic inflammation was a determinant for the competitive advantage of MDS HSPCs and for disease progression. The cell-intrinsic response of MDS HSPCs, which involves signaling through the noncanonical NF-κB pathway, protected these cells from chronic inflammation as compared to normal HSPCs. In response to inflammation, MDS HSPCs switched from canonical to noncanonical NF-κB signaling, a process that was dependent on TLR-TRAF6-mediated activation of A20. The competitive advantage of TLR-TRAF6-primed HSPCs could be restored by deletion of A20 or inhibition of the noncanonical NF-κB pathway. These findings uncover the mechanistic basis for the clonal dominance of MDS HSPCs and indicate that interfering with noncanonical NF-κB signaling could prevent MDS progression.

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Fig. 1: MDS HSPC are associated with inflammatory states.
Fig. 2: TLR-TRAF6-primed HSPCs outcompete WT HSPCs with LD-LPS.
Fig. 3: Overexpression of TRAF6 alters the response of hematopoietic cells to LD-LPS.
Fig. 4: LD-LPS stimulation of TLR-TRAF6-primed HSPC results in noncanonical NF-κB signaling.
Fig. 5: Noncanonical NF-κB activation correlates with A20 expression in TLR-TRAF6 primed HSPCs.
Fig. 6: A20 expression and noncanonical NF-κB activation occurs in TET2-deficient HSPC.
Fig. 7: Deletion of A20 prevents noncanonical NF-κB signaling and rescues the competitive advantage of TLR-TRAF6-primed HSPCs during LD-LPS.

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Data availability

The data that support the findings of this study are available from the corresponding author upon request. All gene expression data are available at GSE142560, GSE19429, GSE58831 and GSE88949. Source data for Figs. 2–7 and Extended Data Figs. 1, 3, 5 and 8 are presented with the paper.

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Acknowledgements

This work was supported in parts by the National Institutes of Health (grant nos. R35HL135787, R01DK102759, R01DK113639 to D.T.S.), Cancer Free Kids (D.T.S.), Cincinnati Children’s Hospital Research Foundation (D.T.S.), The Uehara Memorial Foundation (T.M.), The Waksman Foundation of Japan (T.M.), The Mochida Memorial Foundation for Medical and Pharmaceutical Research (T.M.), Japan Society for the Promotion of Science (T.M.) and Ohio State University Comprehensive Cancer Center (T.M.). T.M. is a Leukemia and Lymphoma Society Special Fellow. D.T.S. is a Leukemia and Lymphoma Society Scholar. We thank J. Bailey and V. Summey for assistance with transplantations (Comprehensive Mouse and Cancer Core at CCHMC), and M.-D. Filippi, D. Lucas, D. Reynaud and the Starczynowski laboratory for insightful suggestions and feedback.

Author information

Authors and Affiliations

Authors

Contributions

T.M. and D.T.S. contributed to study conception and design. T.M., C.S.W., K.C., K.H. and M.S. acquired, analyzed and interpreted data. Z.G. and G.G.-M. provided samples. A.M. and Y.Z. provided reagents. T.M. and D.T.S. wrote and revised the manuscript.

Corresponding author

Correspondence to Daniel T. Starczynowski.

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Competing interests

D.T.S. serves on the scientific advisory board at Kurome Therapeutics. All other authors declare no competing interests.

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Peer review information Ioana Visan was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Inflammatory and immune pathway activation in MDS cells.

a, Survival analysis of MDS patients based on TRAF6 expression (probe: 227264_at) in CD34+ cells (GSE58831). Patients were stratified based on TRAF6 mRNA expression (top 20%, n = 28; bottom 20%, n = 26). Log-rank (Mantel-Cox) test. b, Overview of experimental design to examine inflammatory states in MDS and human TLR-TRAF6 primed HSPC. c, Human miR-146a deficient (miR146a-/-) and control (WT) CD34+ BM cells generated from healthy CD34+ BM using CRISPR-Cas9 gene editing or Vav-TRAF6 and WT LSK BM cells were stimulated in vitro for 90 min with 1 µg/mL of LPS (or PBS) (n = 3 each per group) and then examined for differential gene expression by RNA-sequencing. The inflammatory state for each group was determined using the GSEA. NES, normalized enrichment score. d, Expression of miR-146a in miR-146a deficient (miR146a-/-) and control (sg-CTL) CD34+ BM cells gene edited using CRISPR-Cas9. Results are presented as mean ± s.e.m. for n = 3 technical replicate samples. e, Immunoblot analysis of TRAF6 and IRAK1, two miR-146a targets, in miR146a-/- and control (sg-CTL) CD34+ BM cells gene edited using CRISPR-Cas9. Shown is an immunoblot from a single biological replicate.

Extended Data Fig. 2 Evaluation of Vav-TRAF6-YFP and WT-YFP versus WT BM cell competition with LD-LPS.

a, Overview of experimental design to directly measure hematopoietic cell competition in the presence of low-dose chronic inflammation. Vav-TRAF6 CD45.2 BM cells (co-expressing a YFP transgene referred to as Vav-TRAF6-YFP) and WT CD45.2 BM cells were transplanted in equal proportions into lethally irradiated recipient mice. Two months after transplantation, chimeric mice were treated with LD-LPS (1 µg/g) or vehicle twice a week for 30 days and then examined for hematopoietic contribution of Vav-TRAF6-YFP and WT cells in the PB and BM. After the last LPS treatment, BM cells were serially transplanted into lethally irradiated recipient mice. b, Overview of experimental design to directly measure hematopoietic cell competition in the presence of low-dose chronic inflammation. Wild-type (WT) CD45.2 BM cells (co-expressing a YFP transgene) and WT CD45.2 BM cells were transplanted in equal proportions into lethally irradiated recipient mice. Two months after transplantation, chimeric mice were treated with LD-LPS (1 µg/g) or vehicle twice a week for 4 weeks and then examined for hematopoietic contribution of WT-YFP and WT cells in the BM. c, Representative flow cytometric profiles and gating strategy of YFP+ (WT-YFP) cells in the BM of chimeric mice after LPS or vehicle (PBS) treatment. d, The proportion of YFP+ (WT-YFP) cells in LSK populations 4 weeks after treatment with LPS or vehicle (PBS). Data represent the mean ± s.e.m., n = 6 mice per group.

Extended Data Fig. 3 Overexpression of TRAF6 alters the response of hematopoietic progenitor cells to LD-LPS.

a, Overview of experimental design to examine the long-term effects of low-dose chronic inflammation on hematopoiesis. Vav-TRAF6 (T6) CD45.2 BM cells or WT CD45.2 BM cells were isolated from mice treated with LD-LPS (1 µg/g) or vehicle twice a week for 30 days and then transplanted with a ratio of 10:1 of CD45.1 competitor BM cells into lethally irradiated recipient mice. Three months after transplantation, BM cells were serially transplanted into lethally irradiated recipient mice. b, The proportion of donor-derived CD45.2+ cells in MPP2 (Flk2-CD150+CD48+LSK), MPP3 (Flk2-CD150-CD48+LSK), and MPP4 (Flk2+CD150-CD48+LSK) after tertiary transplantation. Results are presented as mean ± s.e.m. for n = 3 mice per group. Statistical analysis was performed by a two-tailed Student’s t-test. *, P < 0.05.

Source data

Extended Data Fig. 4 TLR-TRAF6 primed HSPC exhibit expression of non-canonical NF-κB gene signatures.

a, Normalized enrichment scores (NES) and P value of gene signatures established from WT and Vav-TRAF6 LSK stimulated with LPS evaluated in constitutively active (ca) NIK expressing LSK (GSE88949). b, GSEA plots established from caNIK LSK were evaluated in miR-146a deficient (miR146a-/-) CD34+ BM cells gene edited using CRISPR-Cas9 and then stimulated with 1 µg/mL of LPS (or PBS) for 90 min. The gene expression profiles are relative to unstimulated miR-146a-/- CD34+ BM and control (sg-CTL) (+/− LPS). (c) Capillary immunoassay of CD34+ cells isolated from healthy controls or MDS BM visualized by chemiluminescence using ProteinSimple. Shown is an immunoassay from a single biological replicate.

Extended Data Fig. 5 TET2 deficiency in hematopoietic cells results in increased myeloid-biased hematopoiesis without affecting the proportions of HSC after LD-LPS.

a, Overview of experimental design to examine the effects of low-dose chronic inflammation on hematopoiesis. Tet2f/f VavCre CD45.2 BM cells (Tet2-/-) or Tet2f/f (WT) CD45.2 BM cells were isolated from mice treated with LD-LPS (1 µg/g) or vehicle twice a week for 4 weeks and then transplanted into lethally irradiated recipient mice (along with CD45.1 competitor BM cells). One month after transplantation, PB and BM cells were evaluated by flow cytometry. b, The proportion of donor-derived CD45.2+ myeloid (CD11b+) and lymphoid (B220+ and CD3+) cells in the PB (n = 6 mice per group). * P = 0.02. c, The proportion of donor-derived CD45.2+ LSK and LT-HSC in the BM of mice after treatment with LD-LPS. Results are presented as mean ± s.e.m., n = 6 for all groups; n = 3 for WT LPS treated group. * P = 0.03. Statistical analysis in b was performed by a two-tailed Student’s t-test. Statistical analysis in c was performed by a one-tailed Student’s t-test.

Source data

Extended Data Fig. 6 Generation of Traf6- and Tet2-deficient mice.

Genotyping analysis of Tet2-/- VavCre and Traf6-/- Tet2-/- VavCre mice.

Extended Data Fig. 7 Generation of Vav-TRAF6 and A20-deficient mice.

Genotyping analysis of Vav-TRAF6 RosaCreER and A20-/- Vav-TRAF6 RosaCreER mice. A20 floxed allele recombination is shown after Tamoxifen treatment.

Extended Data Fig. 8 A20 knockdown impairs MDSL and THP1 cell function.

a, Immunoblotting of MDSL cells expressing independent shRNAs targeting A20 (shA20) or non-targeting shRNA (shControl). Shown is an immunoblot from a single biological replicate. b, Colony forming potential of MDSL cells expressing shRNAs targeting A20 (shA20) or non-targeting shRNA (shControl) in methylcellulose. Results are presented as mean ± s.e.m., for n = 3 independent biological replicates. * P = 0.006, ** P = 0.0004. c, Immunoblotting of THP1 cells expressing an shRNA targeting A20 (shA20) or non-targeting shRNA (shControl). Shown is an immunoblot from a single biological replicate. d, Colony forming potential of THP1 cells expressing shRNAs targeting A20 or non-targeting shRNA (shControl) in methylcellulose. Results are presented as mean ± s.e.m., for n = 3 independent biological replicates. *, P = 0.0001. Statistical analysis in b,d was performed by a two-tailed Student’s t-test.

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Muto, T., Walker, C.S., Choi, K. et al. Adaptive response to inflammation contributes to sustained myelopoiesis and confers a competitive advantage in myelodysplastic syndrome HSCs. Nat Immunol 21, 535–545 (2020). https://doi.org/10.1038/s41590-020-0663-z

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