Coordination of Pro- and Anti-Inflammatory Signals Determine Human Hematopoietic Stem and Progenitor Cell Expansion
Authors: Chagraoui Jalila , Tomellini Elisa , Aubert Leo , Fares Iman , Spinella Jean-Francois , Girard Simon , Mayotte Nadine , Roux Philippe P , Sauvageau Guy 
Published: October 2019

Abstract
Recently, we have identified a small molecule, UM171 that enables the expansion of human HSC with both short- and long-term repopulating capacity. Ongoing clinical trials further confirm the beneficial effects of this molecule in patients.
Transcriptome analysis of cord blood-derived CD34+ cells and AML cell lines exposed to UM171 revealed unsuspected and strong inflammatory signatures pointing to simultaneous activation of pro-inflammatory (including NFKB and IFN signaling) and anti-inflammatory (including detoxification responses) programs. Moreover, characterisation of cell subsets expanded differentially in presence of UM171, showed a significant and preferential expansion of functional dendritic cell progenitors (CD34+CD86+CD45RA+) and mast cells (FCER1A+c-kit+).
In line with this result, immuno-suppressors such as glucocorticoids (dexamethasone) or cAMP elevating agents (IBMX, forskolin) suppressed UM171-mediated pro-inflammatory signaling. These drugs also compromise the ability of UM171 to expand HSCs with long-term repopulating activity and generate dendritic and mast cell progenitors ex vivo. Moreover, knockdown of glucocorticoid receptor (NR3C1) reverts the antagonistic effect of glucocorticoids on UM171 inflammatory properties.
Importantly, detrimental effects of glucocorticoid on HSC function was also observed in absence of UM171, thus indicating that inflammation is critical to HSC expansion, independently of the culture conditions.
We also found that UM171 induces the expression of the inflammatory mediator CD86 on HSC subsets and knockdown of this receptor leads to a loss of long-term HSCs and substantial depletion of lymphoid compartment.
Previously, we have shown that EPCR, a well known anti-inflammatory mediator, is induced by UM171 on CD34+ cord blood cells and defines a cell population with sustained short- and long-term repopulating activity (Fares et al., Blood 2017). We now show that disruption of EPCR function markedly skews UM171 effects towards pro-inflammatory response, thus exacerbating inflammation. Importantly, this uncontrolled inflammation impairs both immune cells generation and HSC activity suggesting that EPCR-driven anti-inflammatory signals provide a critical negative feedback constraining excessive inflammation in HSC and dendritic cell progenitors.
Altogether our results strongly suggest that UM171-mediated positive regulation of HSC function occurs through a coordinated and biphasic mechanism allowing a controlled and adequate pro- and anti-inflammatory status, thus creating a permissive environment for stem and immune cell survival and expansion. Most importantly, our data indicate that integration of pro- and anti-inflammatory signals is essential for human HSC self-renewal in the presence or in the absence of UM171, thus revealing a critical balance of pro- and anti-inflammatory activities in human HSC.

Disclosures
Sauvageau: ExCellThera: Employment, Equity Ownership.