Protection of Transplanted Hematopoietic Stem Cells from Inflammatory Recipient Marrow Environment through Specific TNF-α Signal Blockade

Introduction

Hematopoietic stem cell (HSC) transplantation (HSCT) for hematological malignancy generally requires aggressive chemotherapy and irradiation, which exceed dose-limiting toxicity. These treatments are necessary to eradicate malignant cells and to create a niche for graft HSCs. However, as the intense preconditioning may also induce inflammation in recipient bone marrow (BM), transplanted HSCs thus should be inevitably exposed to this devastating environment. Whilst triggered inflammation within BM is hypothesized to deteriorate transplanted HSCs, how this BM environmental change affects graft HSCs remains largely unknown. We therefore sought to clarify how the pre-conditioned BM environment might affect donor HSCs, focusing especially on inflammatory effects and on developing protective measures against them.

Methods & Results

Our experiments revealed that total body irradiation (TBI) could induce local inflammation peaking around 2-3 days within marrow environment. In vivo exposure of HSCs to irradiated BM environment 2-3 days after TBI exhibited negative effects on HSC function.

Then, we tested how the TBI-conditioned BM environment could affect donor HSCs. We first conducted comprehensive gene expression analysis on BM-resident stromal cells considered to constitute the HSC niche. We found that expression of 3 major inflammatory cytokines, IFN-γ, IL-1β, and TNF-α was induced after TBI treatments, thus we compared the effects of these cytokines by an in vitro HSC colony forming assay. Only TNF-α inhibited colony formation of HSCs in a dose dependent manner.

We then sought to elucidate mechanisms by which TNF-α impaired HSCs' reconstitution abilities. Based on previous reports, TNF-α is a major stimulus of reactive oxygen species (ROS) through activating Nicotinamide Adenine Dinucleotidemono Phosphate Hydride (NADPH) oxidase. We therefore determined whether TNF-α stimulation produced excessive levels of ROS in highly purified murine HSPCs by culturing them with or without TNF-α for up to 48 hours and staining them with dichlorodihydrofluorescein (DCF) to quantify the accumulation of ROS. The addition of TNF-α was found to induce ROS production in HSPCs in a dose- and time-dependent manner. We next examined if overproduction of TNF-α-mediated ROS impaired reconstitution ability of HSCs; we compared reconstitution abilities of HSCs between groups, one exhibiting high levels of ROS and the other with low/medium levels of ROS. Transplantation experiments using flow-cytometry-sorted populations revealed the negative effect of high levels of ROS on HSCs, suggesting a causal role of elevated ROS levels in TNF-α-mediated impairment of stem cell ability.

Accordingly we examined the hypothesis that specific inhibition of the TNF-α-ROS signaling could preserve graft HSCs' functions. The TNF receptor 1 (TNFR1) blocking peptide (PepTNFR1) that specifically blocks NADPH oxidase-mediated ROS production in cells after stimulation with TNF-α was evaluated for this purpose. Pre-incubation of murine HSPCs with PepTNFR1, but not with a scrambled control peptide, inhibited TNF-α-mediated ROS accumulation. Eventually, to determine if reduction of TNF-α-mediated ROS accumulation in graft-HSCs by PepTNFR1 could improve transplantation outcomes, we compared reconstitution kinetics of highly purified HSCs pre-incubated for 2 hours either with PepTNFR1 or a scrambled control peptide. Pre-treatment with PepTNFR1 successfully protected transplanted HSCs from an inflammatory BM environment, showing higher donor-cell chimerism in recipients of "protected" HSCs than in those of non-protected HSCs.

Conclusion

We here provide a proof of concept that stem cell protection measures through specific TNF-α signal blockade in the context of HSCT will eventually lead to better engraftment and reconstitution kinetics in transplantation, thereby ameliorating outcomes of HSCT.