Interleukin-1/Toll-like Receptor Inhibition Can Restore the Disrupted Bone Marrow Microenvironment in Mouse Model of Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by bone marrow (BM) failure and associated with aging. We have previously shown that reversal of BM microenvironment (BMME) dysfunction in MDS mitigates MDS associated marrow failure and delays progression to acute leukemia. However, the exact mechanisms driving BMME dysfunction in MDS remain unknown. We recently reported that interleukin-1 (IL1) Receptor Type1 (IL1R1) signaling is a driver in myeloid bias via disruption of BMME in aging. In addition, we have found that IL1R1 signaling is involved in disease progression of AML. Therefore, to assess the role of IL1R1 signaling in MDS associated BMME dysfunction and marrow failure, we employed an age appropriate murine transplant model for MDS utilizing NUP98-HOXD13 (NHD13) transgenic mice.

Methods: BM cells (NHD13 transgenic or wild type (WT), 7 weeks) and competitor cells were transplanted into irradiated aged recipients (WT or IL1R1 KO, 60 weeks), and subsequently monitored for development of marrow failure. When marrow failure developed, mice were euthanized and peripheral blood, BM, BM extracellular fluid (BMEF), and collagenase-1 digested bone associated cells were analyzed including flow cytometry, colony forming units (CFU) assay, and cytokine analyses. Next, BM from NHD13 (8-10 weeks) and competitor cells were transplanted into lethally irradiated aged recipients (WT, 50-60 weeks). At onset of marrow failure, mice were treated with inhibitors of IL1/Toll-like receptor signaling (IL1R antagonist, MCC950, or IL1R-associated kinase 4 protein (IRAK4) inhibitor) for fourteen days, and then euthanized and analyzed as above. Finally, we evaluated cytokine profile in the BM serum from the patients with MDS and normal donors.

Results: Transplant of NHD13 BM cells into aged IL1R1 ^wt recipients (NHD13→IL1R1 ^wt) was not associated with a significant difference in survival rates or levels of NHD13 engraftment compared to NHD13 into IL1R1 ^ko recipients (NHD13→IL1R1 ^ko). IL1R1 ^wt developed macrocytic anemia compared to IL1R1 ^ko recipient (Hb 11.3±0.57 v.s 13.1±0.42 g/dL, n=12 and 9, p<0.05). In CFU-C assays, NHD13→IL1R1 ^wt and NHD13→IL1R1 ^ko demonstrated similar levels of CFU-activity whereas CFU-fibroblast (CFU-F) assays of IL1R1 ^wt recipients demonstrated lower numbers of large colonies (reported to contain highly proliferative mesenchymal stem cells (MSC)). Flow cytometry analysis of hematopoietic stem and progenitor cells (HSPC) population in IL1R1 ^wt recipients showed increased myeloid progenitors and decreased long-term HSC (LT-HSC) compared to IL1R1 ^ko recipients. Cytokine/chemokine profiles revealed that the inflammatory cytokines (IL-6 and TNFα) and macrophage activating cytokines (MCP-1 and M-CSF) were significantly decreased in NHD13→IL1R1 ^ko compared to NHD13→IL1R1 ^wt. Next, we treated MDS model mice with inhibitors. The pharmacological targeting of IL1R1 signaling in vivo was associated with decreased NHD13 cell burden and improvement of macrocytic anemia. CFU-C assays demonstrated some decreases in NHD13 CFU capacity post-treatment. Interestingly, non-NHD13 HSPCs assessed by CFU capacity increased in IRAK4 inhibitor treated mice. Consistent with this, flow cytometric analyses of HSPC pools demonstrated decreased NHD13 HSPCs (LT-HSC and granulocyte-monocyte progenitor cells) and increased non-NHD13 HSCs and myeloid progenitors compared to vehicle group. BMME cell populations showed that arteriolar endothelial cells and MSCs were also affected by drug treatment and both IL1R antagonist and MCC950 increased the number of large CFU-F colonies. Analysis of BMEF revealed the decreased IFNγ and IL-18 and upregulated M-CSF in MCC950 treated group. The cytokines of human BM serum revealed higher concentrations of soluble formed IL1R1, IL-18, CXCL1, and osteopontin in MDS compared to young or aged normal donors.

Conclusions: Collectively, our findings demonstrate that IL1R1 signaling alters the BMME and contributes to the disease phenotype of MDS and that the effects of targeting IL1R1 pharmacologically have differing effects based on the modality of inhibition as well as the cell population. IL1R1 signaling can be a promising target to alleviate the complexity of MDS via improving inflammatory status in BMME.