Irradiated Bone Marrow Environment Impairs Hematopoietic Stem Cells by Exposure to Tumor Necrosis Factor a.
Authors: Suzuki Sachie , Okabe Motohito , Otsu Makoto , Ema Hideo , Nakauchi Hiromitsu 
Published: October 2019

Abstract
Abstract 3724

Background:
Hematopoietic stem cell transplantation (HSCT) represents a curative treatment for various disorders including hematopoietic malignancies. Most HSCT requires cytoreductive conditioning such as total body irradiation (TBI) and/or chemotherapy to ensure engraftment of HSCs by emptying recipients' marrow niches. Our preliminary experiments, however, revealed that TBI could induce local inflammation peaking around days 2–3 within marrow environment. Of note is that conditioning regimens widely used in the current HSCT settings are mostly compatible with HSC exposure to inflammatory storm in post-irradiation bone marrow (BM). Although certain inflammatory cytokines have been shown to affect HSC functions in in vitro, it remains unknown whether in vivo exposure to inflammatory BM environment can alter the characteristics of transplanted HSCs. We therefore sought to investigate what effects irradiated BM environment would have on transplanted donor HSCs using murine systems.

Methods and Results:
We first tested whether infusion of HSCs at varying timing post TBI would affect transplantation outcomes and HSC functions. To this end, fifty HSCs (CD34-negative/low, cKit+, Sca-1+, lineage marker-negative cells) obtained from Ly5.1-B6 mice were transplanted into lethally irradiated Ly5.2-B6 mice with 1 × 106 competitor Ly5.1/5.2-B6 BM cells at day 0, 3, or 5 post irradiation. No mice survived long-term in the group that received transplants 5 days post irradiation, indicating insufficient HSC engraftment and hematopoietic reconstitution. Although hematopoiesis reconstituted in long term survivors was comparable between another two groups (d0 and d3), we found significant difference in donor HSC ability when tested in a competitive repopulation assay using Ly5.1-KSL cells sorted from primary recipients: Test HSCs obtained from mice transplanted 3 days after irradiation showed poor secondary reconstitution ability, suggesting alteration of pre-engraftment HSC functions depending on transplantation protocols. We then tested whether in vivo exposure of HSCs to irradiated BM environment would have negative effects on HSC functions. Test HSCs (400 cells) from Ly5.1-B6 mice were transplanted into Ly5.2-B6 primary recipients at varying time points (day 0, 1, 2, or 3) after lethal-dose TBI. Approximately 24 h later, BM samples were subjected to a competitive repopulation assay to test secondary reconstitution ability in test HSCs that homed to irradiated BM environment. Consequently, test HSCs that were exposed for ≂f24 h to BM environment at day 2 post TBI showed marked impairment in their long-term reconstitution ability. When “BM-homed” HSCs were enumerated 24 h after infusion, we found modestly impaired HSC homing to BM irradiated 2 days prior to transplantation, compared to BM irradiated on day 0, indicating negative effects on HSCs transplanted during a peak phase of inflammatory storm. Next we examined whether inflammatory cytokines were capable of impairing HSC functions. When tested in liquid culture using purified HSCs, both IFN-g and IL-1 had little effects on colony formation. In contrast, TNF-a inhibited colony formation in a dose-dependent manner. When in vivo HSC exposure to irradiated BM environment was tested using TNF-a KO mice as primary recipients, significant improvement of HSC ability was observed, indicating a major role for this inflammatory cytokine in the HSC-inhibitory effect observed within the irradiated host tissues. We finally sought to test if blocking of TNF-a signaling in HSCs at a peak phase of inflammation could lead to better transplantation outcomes. We utilized the peptide previously shown to block TNFR signaling, and confirmed that pre-incubation of HSCs with this molecule did suppress TNF-a induced reactive oxygen species production in HSCs. Studies are ongoing to test if HSCs transplanted in a peak-phase of TBI-mediated inflammation will benefit from this shielding measure.

Conclusion:
We demonstrated that inflammatory response in BM environment 2–3 days after irradiation could have a negative effect on donor HSCs regarding both homing efficiency and secondary reconstitution ability. These findings provide important implications for developing the measures that enable HSCs to escape from this inhibitory effect to achieve far more improvement in clinical HSCT outcomes.

Disclosures:
No relevant conflicts of interest to declare.