Proximity-Based Single Cell Analysis of the Bone Marrow Niche Identifies Interleukin-18 As a Quiescence Regulator of Early Hematopoietic Progenitors
Authors: Silberstein Lev , Kharchenko Peter , Kfoury Youmna , Mercier Francois , Osawa Masatake , Hoggatt Jonathan , Tate Tiffany , Lin Charles , Scadden David T. 
Published: October 2019

Abstract
Background. Niche-derived signals are essential for maintenance and expansion of stem/progenitor cells in the bone marrow. Recently published studies used elegant genetic tools to dissect predominant cellular sources of known HSPC regulators, such as CXCL12 and kit-ligand (Ding et al, Nature 2012, Greenbaum et al, Nature 2013). However, strategies enabling identification of novel niche-derived factors are lacking. Here, we describe an approach which utilizes spatial proximity between osteolineage cells (OLC) and HSPC in the post-transplant bone marrow niche as a guide to niche factor discovery and reveals the role of Interleukin-18 (IL18) as a quiescence regulator of early hematopoietic progenitors.
Results. We established a neonatal bone marrow (BM) transplantation model, in which adult BM-derived LKS CD34-Flk2- HSPCs fluorescently labeled with DiI were transplanted into irradiated newborn col2.3GFP recipients (in this mouse strain, the majority of OLCs are labeled with GFP). Forty-eight hours later, we obtained sections of femoral trabecular bone from transplanted mice and located rare OLCs harboring DiI-positive HSPCs in their immediate proximity. We harvested individual OLCs located within two cell diameters (proximal OLCs) and greater than five cell diameters (distal OLCs) from DiI-labeled HSPCs and performed comparative transcriptome analysis by single cell RNA-Seq. Remarkably, we found that proximal OLCs were distinct from their distal counterparts and showed a higher expression levels of known niche-associated molecules, as well as secreted factors not previously linked to regulatory niche function, including a pro-inflammatory cytokine IL18.
Quantification of primitive subsets in the BM of IL18 knock-out (IL18KO) mice showed no abnormalities. However, cell cycle studies revealed that while long-term HSCs (which did not express the IL18 receptor) were unaffected by IL18 deletion, early progenitors - short-term HSC, multi-potent progenitor and common lymphoid progenitor – were more actively cycling. Accordingly, following sublethal exposure to 5-fluorouracil (5FU), IL18KO animals displayed a 2-fold increase in frequency of lin-kit+Sca1+ (LKS) cells, lin-kit+ myeloid progenitors and CLPs, as compared wild-type (WT) controls (Figure 1). On the other hand, exogenous administration of recombinant IL18 to 5FU-treated WT animals was associated with 25% mortality, while no deaths were observed in the vehicle-treated group. Taken together, these data illustrate that IL18 constrains the ability of the progenitor pool to respond to a genotoxic stress.
Next, we tested if IL18 acts in a non cell-autonomous fashion. We transplanted progenitor-enriched bone marrow fraction (LKS cells) into IL18KO or WT hosts and observed an approximately 2-fold increase in both myeloid and lymphoid cells in peripheral blood of IL18KO animals. This finding was recapitulated in a reciprocal experiment, when LKS cells from IL18 receptor knock-out animals were transplanted into WT hosts, supporting the possibility that the effect of IL18 on progenitor proliferation is direct.
Finally, we investigated if the loss of IL18-mediated quiescence can be therapeutically exploited in the transplant setting. To this end, we assessed survival of lethally irradiated WT and IL18KO mice following transplantation with a limiting BM dose, i.e. predicted to confer survival to 50% or less of WT animals. Notably, we observed reduced 30-day post-transplant mortality (33% versus 72%, p value = 0.05) in IL18KO animals, which likely resulted from accelerated progenitor expansion in the absence of IL18 in the host microenvironment (Figure 2).
Conclusions. Our study demonstrates the capability of proximity-based single cell analysis of the post-transplant bone marrow microenvironment to identify a novel niche factor – IL 18. We show that IL18 specifically regulates quiescence of early hematopoietic progenitors, particularly under the conditions of stress hematopoiesis, such as exposure to 5FU or post-transplant hematopoietic expansion. The results presented here provide an insight into poorly understood molecular mechanisms of progenitor regulation by the niche. Moreover, improved post-transplant survival of animals in the absence of IL18 suggests that pharmacological inhibition of IL18 has the potential of improving clinical outcomes in transplant recipients.
Figure 2 Figure 2.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.