Comparative Inflammasome Analysis of Bone Marrow Stroma in Aging and Myelofibrosis

Background

In the development of Myelofibrosis (MF), disease which is often associated with aging, deregulation of stem cell activity by somatic mutations occurs in the setting of deregulation of the bone marrow microenvironment. While somatic mutations in stem and progenitor cells have been shown to promote pathological myeloproliferation, functional involvement of stromal cells in MF pathogenesis is not well defined. Chronic inflammation has long been regarded as a driving force for MF development, which is intensified by continuous release of pro-inflammatory cytokines and chemokines. We have recently reported that inflammatory cytokine signaling in the aging bone marrow niche via aberrant RNA editing and splicing may predispose patients to leukemia stem cell generation. However, little is known about the functional effects of stromal cell driven inflammasome signaling in MF pathogenesis. Here we investigate the impact of the inflammasome on aging of the BM niche and how changes in the activity of BM niche cells contribute to MF pathogenesis.

Methods

Stromal monolayers were established from CD34-negative bone marrow cells obtained from young (<3 y/o; y-NBM), aged (> 60 y/o; a-NBM) healthy donors and myelofibrosis (MF) patient samples. Conditioned media from those stromal cultures was collected after 7 d post confluence and was analyzed for secreted factors by Human multi-analyte (MAP) Luminex-based analysis (Myriad). Some stromal cultures were used for RNA transcriptome analysis. The other stromal cultures were utilized for the co-culture experiments with CD34⁺ cells (hematopoietic stem cells, HSC) selected from y-NBM, a-NBM and cord blood (CB), which were subsequently subjected to survival and self-renewal assays.

Results

MF and a-NBM derived stroma severely impaired survival and self-renewal of CB (n=5) and y-NBM (n=5) HSC in co-culture models. In similar experiments HSC from a-NBM (n=6) demonstrated significantly higher survival and self-renewal capacity when co-cultured with y-NBM stroma (n=3) compared to a-NBM stroma (n=3). Conditioned media (CM) from both a-NBM and MF stroma by itself also greatly reduced HSC survival and self-renewal. Notably, co-culture conditions resulted in a greater reduction in survival and self-renewal capacity than CM alone, suggesting that cell-cell contact or unstable secreted factors exacerbate the effects.

Focused analysis of 45 inflammation-associated secreted factors in 3 y-NBM and 4 a-NBM stromal CM samples revealed no substantial alterations in inflammatory factors However, a-NBM stroma demonstrated a significant reduction in production of a variety of HSC-regulatory factors (BDNF, IL-17, IL-12p40, SCF, ICAM-1,VEGF, Eotaxin-1, RANTES). Moreover, RNA transcriptome analysis of 3 y-NBM, 4 a-NBM and 3 MF stromal monolayers revealed significant variations in differentially expressed genes (DEG) between y-NBM and a-NBM. Specifically, aged stroma was typified by upregulated expression of inflammation and immune response genes and of genes associated with the lysosome (ELANE, CLU, DEFA1, LBP). Downregulation of collagen genes and energy metabolism genes, particularly mitochondrial genes, as well as alterations in the expression of apoptosis, cell cycle and cellular senescence biomarkers (IL1B, IDS, TPST2, SERPINB2) was observed.

Comparisons in gene expression between a-NBM and MF stroma revealed upregulated inflammatory response(PLIN2, IL1B,PDK4,) and TGF-β signaling pathways(PTGDS,) and downregulated ability to support HSC (RARRES2, FGF7, FGF23, and IGF2) Notably, some transcripts (RUNX3, BCL2) were identified within the MF stromal niche, while they were absent in a-NBM stroma or HSC from a-NBM and MF samples.

Conclusions

These data indicate that MF and aged stroma exhibit a severely compromised ability to maintain normal hematopoiesis. Molecular inflammasome signatures of aging and MF reflect a combination of degenerative processes and transcriptional responses. Targeting this pathological interplay between the MF niche and MF stem cells could represent a novel avenue for the treatment of MF.