Mesenchymal Stromal Cell-derived Extracellular Vesicles Promote Myeloid-biased Multipotent Hematopoietic Progenitor Expansion via Toll-Like Receptor Engagement

Abstract

Edited by Dennis VoelkerMesenchymal stromal cells (MSCs) present in the bone marrow microenvironment secrete cytokines and angiogenic factors that support the maintenance and regenerative expansion of hematopoietic stem and progenitor cells (HSPCs). Here, we tested the hypothesis that extracellular vesicles (EVs) released by MSCs contribute to the paracrine crosstalk that shapes hematopoietic function. We systematically characterized EV release by murine stromal cells and demonstrate that MSC-derived EVs prompt a loss of HSPC quiescence with concomitant expansion of murine myeloid progenitors. Our studies reveal that HSPC expansion by MSC EVs is mediated via the MyD88 adapter protein and is partially blocked by treatment with a TLR4 inhibitor. Imaging of fluorescence protein-tagged MSC EVs corroborated their cellular co-localization with TLR4 and endosomal Rab5 compartments in HSPCs. The dissection of downstream responses to TLR4 activation reveals that the mechanism by which MSC EVs impact HSPCs involves canonical NF-B signaling and downstream activation of Hif-1␣ and CCL2 target genes. Our aggregate data identify a previously unknown role for MSC-derived EVs in the regulation of hematopoiesis through innate immune mechanisms and illustrate the expansive cell-cell crosstalk in the bone marrow microenvironment.A small population of long-lived quiescent HSPCs 3 residing in the bone marrow sustains lifelong hematopoietic function and provides regenerative capacity through cycles of self-renewal and differentiation (1). Cell fate commitment yields a cascade of successively more restricted progenitor populations that give rise to circulating blood and mature immune cells (2). HSPC activation relies on cell-autonomous programs as well as extrinsic cues from the surrounding bone marrow microenvironment where mesenchymal stromal cells, osteoprogenitors, and endothelial cells act through the paracrine action of secreted cytokines and angiogenic factors (3-6).Recent studies indicate that HSPC self-renewal and emergence from quiescence are also regulated by type I and II interferons as well as Toll-like receptors (TLRs), signals associated with innate immunity (7-10), and known to contribute to regenerative HSPC responses (11)(12)(13)(14)(15). TLR signaling involves a number of transmembrane receptors and several critical adaptor molecules (16). Among them, the myeloid differentiation factor (MyD88) occupies a central role in transducing both surface and endosomal signals for most TLRs. Evidence supports TLR activation in the HSPC response to diverse stimuli, including radiation, bleeding, and infection (reviewed in Ref. 12). More recent reports, however, indicate that these mechanisms are also relevant for "tonic" homeostatic function and as well as developmental emergence of HSPCs (15,17,18). Indeed, many cytokines central to the inflammatory response are also critical to HSPC maintenance and differentiation (19 -21).Extracellular vesicles (EVs) are constitutively released from cells and are powerful paracrine regulator...

The Emerging Role of Rab5 in Membrane Receptor Trafficking and Signaling Pathways

“…e colocalization of TLR4 and Rab5 can be observed in bone marrow-derived macrophages and hematopoietic stem cells, and progenitor cells upon lipopolysaccharide stimulation [96]. Rab5 affects both TLR4 downstream NF-κB signaling and the downstream of target genes, such as Hif-1 and CCL2, ultimately promoting the amplification of bone marrowderived multifunctional hematopoietic stem cells [97].…”

Extracellular vesicles can act as a potent immunomodulators of human microglial cells

“…Another report showed that various types of body fluid EVs (exosomes) induced NFκB‐ and STAT3‐mediated secretion of inflammatory cytokines in TLR‐dependent fashion (Bretz et al, ). Furthermore, murine MSC‐derived EVs promoted myeloid‐biased multipotent haematopoietic progenitor expansion via TLR4 engagement (Goloviznina et al, ). Collectively, these observations indicate that TLR4/NFκB signalling pathway is a potential target of EVs.…”

“…We speculate that EVs may impair the assembly of TLR4 multireceptor complex and affect signal transduction from the plasma membrane. Although there is no experimental evidence about direct interaction between EVs and components of TLR4 multireceptor complex (TLR4 receptor, LPS binding protein, MD2, and CD14), recent report demonstrated quantitative colocalization of myristoylated tomato‐tagged EVs and TLR4 receptors (Goloviznina et al, ). EVs can also act as carriers for the endogenous TLR4 ligands.…”

Mesenchymal stromal cell‐derived extracellular vesicles as cell‐free biologics for the ex vivo expansion of hematopoietic stem cells

“…In a study, HSPC LSK cells showed prominent expansion of myeloid progenitor cells with MyD88‐dependent signaling when subjected to MSC‐EVs with the upregulation of nuclear factor‐κB (NF‐κB). This effect was abolished in the presence of a TLR4 inhibitor indicating TLR4 receptor involvement (Goloviznina et al, 2016).…”