TLR Activation By MSC Derived Extracellular Vesicles Drives Myeloid Commitment

Hematopoietic stem and progenitor cells (HSPC) are responsive to paracrine signaling in the bone marrow microenvironment. We recently showed that leukemia derived extracellular vesicles (EVs) directly regulate HSPC in the leukemic niche and confer a net loss of progenitor function in part through direct action of a highly abundant miRNA on the hematopoietic transcription factor c-Myb. These observations motivated us to explore the potential regulation of HSPC by EVs present during steady state hematopoiesis in the unperturbed bone marrow. Mesenchymal stromal cells (MSC) comprise a diverse set of cells secreting supportive factors central to the maintenance of HSPC. We systematically characterized EVs released by murine MSCs as a candidate population and demonstrated that EVs themselves carry cytokines, predominantly interleukin (IL) -1, -2, -4,-6 and -17A, as well as G-CSF and Interferon g. As a functional corollary, we found that MSC EVs led to preferential granulocyte and monocyte differentiation in co-culture with C-kit enriched HSPCs, when compared to vesicle free condition (termed S100). These observations were further supported by the expansion of KSL (c-kit⁺,Sca-1⁺,lin^-) and MPP2-4 (CD48⁺, CD150^+/-) progenitor populations. To determine the in vivo relevance of these findings we performed a series of competitive transplantation experiments, that demonstrated that MSC-EV exposed HSPCs outperformed HSPCs cultured with EV depleted media, with striking advantage in early, but a significant deficit in later (week 16) myeloid recovery, compared to the S100 condition. These observations were broadly consistent with recent evidence for HSPC regulation by toll like receptors (TLRs) and we confirmed that the observed deficit in progenitor phenotypes was partially reversed in MSC-EV co-cultures with HSPCs from animals with genetic disruption of TLR4 or MyD88. EV-TLR engagement further induced a NF-kB response with downstream transcriptional activation of canonical transcriptional targets (STAT1, IRF1, -9) and significantly upregulated the release of IL-3, -6, IFNg, and G-CSF in exposed WT HSPC, compared with S100. Finally, we performed deconvolution microscopy studies of co-cultured HSPC and demonstrated the Pearson's spatial correlation between fluorescently tagged MSC-EV and TLR4 as well as the early and late endosomal marker proteins, Rab5 and Rab7, respectively. We conclude that MSC-derived EV trafficking of TLR contributes to the homeostatic regulation of HSPCs in the bone marrow microenvironment as a mechanism of tonic regulation of hematopoiesis.