Local Irradiation Induces Systemic Inflammatory Response and Alteration of the Hematopoietic Stem Cell Niche
Authors: Kawano Yuko , Byun Daniel K , Frisch Benjamin J. , Kawano Hiroki , LaMere Mark W , Johnston Carl J. , Williams Jaqueline P. , Calvi Laura M. 
Published: November 2019

Radiotherapy is used in the treatment of ~50% of tumors. We and others have reported long-term suppression of hematopoietic stem and progenitor cells (HSPCs) in the setting of total body irradiation; however, it has been shown that even relatively small irradiation volumes can result in systemic adverse events, such as myeloablation and secondary malignancies. The mechanisms underlying these effects are unclear. We hypothesize that localized radiation may activate a systemic inflammatory response that can acutely alter HSPCs and bone marrow microenvironment (BMME) components, including marrow stromal cells (MSCs), thereby contributing to late effects. We therefore established a murine model of targeted irradiation (TR) using a small animal radiation research platform (SARRP).
Methods: We administered local irradiation to a single tibia of 6-8 week old C57BL/6 male mice using a single dose of 15 Gy. Subsequently, we analyzed peripheral blood, BM, BM extracellular fluid (BMEF), collagenase-1 digested bone associated cells of both the irradiated (TR) and non-irradiated, contralateral (CONT) tibiae at 2, 6, 48 hours, 1 and 3 weeks post-TR, performing phenotypic (flow cytometry) and cytokine analyses. For all studies, n = 10-13 mice/time point.
Results: In the TR tibia at 2 hours, although total cell numbers were unchanged, there was a significant upregulation of inflammatory cytokines (interleukin 1β (IL1b), IL18), chemokines (CXCL2, CXCL10, CCL2, CCL3) and macrophage colony stimulating factor (M-CSF). Of note, most of these changes normalized by 48 hours (M-CSF at 1 week). Changes in mediator expression were followed, at 6 hours post-TR, by significant increases in macrophage (macs) numbers, including CD206 phagocytic macs, neutrophils (PMNs) and cytotoxic lymphocytes, including CD8+ cells expressing CXCR3+, the receptor for CXCL9 and CXCL10. Interestingly, similar to the TR tibia, CXCL2 expression was also increased significantly in the CONT at 2 hours, followed (6 hours) by significant increases in macs and CD8+ cells, suggesting a systemic or abscopal effect.
With respect to the effects of radiation on HSPCs, by 6 hours, most of the stem and progenitor cell (HSPC) populations in the TR marrow were significantly decreased; the decrease in long-term-HSCs was delayed until 48 hours post-TR. All populations remained severely depleted until 3 weeks post RT, demonstrating a rapid and sustained effect of TR on all HSPCs within the irradiation volume. In comparison, in the CONT tibia at 6 hours, CD41+ HSCs were expanded; this is consistent with previous demonstrations that CD41+ LT-HSCs expand with inflammatory signals and suggests that TR-induced signals induced a systemic impact on the non-irradiated HSPCs. By 1 week post-radiation, short term-HSCs were significantly decreased in the CONT marrow, likely due to mobilization since CFU-Cs were correspondingly significantly increased in the circulation. Finally, MSCs, previously shown to support HSCs, were found to be significantly increased in the TR tibia starting at 6 hours and peaking at 48 hours post-radiation. Surprisingly, MSCs were also expanded in the CONT marrow at 48 hours; this expansion was likely associated with the increased CXCL12 levels seen in both TR and CONT marrow, although the CXCL12 levels were higher in the irradiated tibia. Taken together, these changes indicate TR-induced global disruption of the HSC niche. Furthermore, in addition to the transient effects of localized irradiation, we observed a second wave of inflammatory signals, including a significant increase in CCL3, at 1 week post-TR and increased IL1b in the CONT marrow at 3 weeks, changes that may have contributed to the sustained loss of HSPC populations.
Conclusions: We present the effects of local irradiation on global hematopoiesis, showing that, in addition to the anticipated acute local changes in the irradiated bone marrow, TR-induced persistent and, more importantly, systemic inflammation. We believe that using this murine model will allow us to dissect the contribution of direct (local) and indirect (systemic) responses to radiation on treatment effects, such as marrow failure and secondary malignancies.

Disclosures
No relevant conflicts of interest to declare.