Non-myeloablative busulfan chimeric mouse models are less pro-inflammatory than head-shielded irradiation for studying immune cell interactions in brain tumours

A Saam Youshani,Kaye Williams,Claire O’Leary,Gabriella Forte,Aiyin Liao,Brian Telfer,Helen Parker,Samuel Rowlston,Ian D Kamaly-Asl,Brian W Bigger

doi:10.1186/s12974-019-1410-y

Abstract

BackgroundChimeric mouse models generated via adoptive bone marrow transfer are the foundation for immune cell tracking in neuroinflammation. Chimeras that exhibit low chimerism levels, blood-brain barrier disruption and pro-inflammatory effects prior to the progression of the pathological phenotype, make it difficult to distinguish the role of immune cells in neuroinflammatory conditions. Head-shielded irradiation overcomes many of the issues described and replaces the recipient bone marrow system with donor haematopoietic cells expressing a reporter gene or different pan-leukocyte antigen, whilst leaving the blood-brain barrier intact. However, our previous work with full body irradiation suggests that this may generate a pro-inflammatory peripheral environment which could impact on the brain’s immune microenvironment. Our aim was to compare non-myeloablative busulfan conditioning against head-shielded irradiation bone marrow chimeras prior to implantation of glioblastoma, a malignant brain tumour with a pro-inflammatory phenotype.MethodsRecipient wild-type/CD45.1 mice received non-myeloablative busulfan conditioning (25 mg/kg), full intensity head-shielded irradiation, full intensity busulfan conditioning (125 mg/kg) prior to transplant with whole bone marrow from CD45.2 donors and were compared against untransplanted controls. Half the mice from each group were orthotopically implanted with syngeneic GL-261 glioblastoma cells. We assessed peripheral blood, bone marrow and spleen chimerism, multi-organ pro-inflammatory cytokine profiles at 12 weeks and brain chimerism and immune cell infiltration by whole brain flow cytometry before and after implantation of glioblastoma at 12 and 14 weeks respectively.ResultsBoth non-myeloablative conditioning and head-shielded irradiation achieve equivalent blood and spleen chimerism of approximately 80%, although bone marrow engraftment is higher in the head-shielded irradiation group and highest in the fully conditioned group. Head-shielded irradiation stimulated pro-inflammatory cytokines in the blood and spleen but not in the brain, suggesting a systemic response to irradiation, whilst non-myeloablative conditioning showed no cytokine elevation. Non-myeloablative conditioning achieved higher donor chimerism in the brain after glioblastoma implantation than head-shielded irradiation with an altered immune cell profile.ConclusionOur data suggest that non-myeloablative conditioning generates a more homeostatic peripheral inflammatory environment than head-shielded irradiation to allow a more consistent evaluation of immune cells in glioblastoma and can be used to investigate the roles of peripheral immune cells and bone marrow-derived subsets in other neurological diseases.

Highlights

Adoptive transfer can be used to generate bone marrow chimeras and track immune cell populations in both homeostatic and inflammatory states
Non-myeloablative conditioning achieves equivalent blood chimerism to head-shielded irradiation Having shown previously [19] that the non-myeloablative conditioning (NMC) model can generate high levels of chimerism without damaging the blood-brain barrier (BBB), we compared our chimeric model with the commonly used HIR myeloablative technique to determine if either model was superior in terms of peripheral blood chimerism of donor haematopoietic cells
An UnTx control group and full conditioning (FC) busulfan group were used as comparative controls throughout the study (Fig. 1)

Summary

Introduction

Adoptive transfer can be used to generate bone marrow chimeras and track immune cell populations in both homeostatic and inflammatory states. After HSCT, high levels of chimerism must be achieved to accurately distinguish between recipient brain populations and donor peripheral immune cells and reliably determine respective contributions in a pathological state without concern about the negative effects of pre-conditioning. Chimeras that exhibit low chimerism levels, blood-brain barrier disruption and pro-inflammatory effects prior to the progression of the pathological phenotype, make it difficult to distinguish the role of immune cells in neuroinflammatory conditions. Our aim was to compare nonmyeloablative busulfan conditioning against head-shielded irradiation bone marrow chimeras prior to implantation of glioblastoma, a malignant brain tumour with a pro-inflammatory phenotype

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