Total Marrow Irradiation Reduces Organ Damage in a Bone Marrow Transplant Model of Sickle Cell Disease

Abstract

Sickle cell disease (SCD) is associated with significant organ pathology. Currently, the only curative option is bone marrow transplantation (BMT) after myeloablative conditioning. However, significant organ damage from myeloablative total body irradiation (TBI) remains one of the major side effects of BMT. To reduce exposure to vital organs, but efficient enough to maintain engraftment, we developed 3D image guided-total marrow irradiation (TMI) for targeted delivery to the bone marrow (BM) and spleen of humanized BERK sickle mice while sparing the vital organs. We hypothesized that TMI will prevent organ damage compared to that induced by TBI during BMT. Sickle mice were treated with: [i] TBI (4:4), 4 Gy to the BM & 4 Gy to the whole body; [ii] TMI (4:0), 4 Gy to BM, 0 Gy to the body, and [iii] TMI (8:2), 8 Gy to BM & 2 Gy to the whole body. After 24h of irradiation, SS mice were transplanted with 5 million donor BM cells from HbAA BERK mice expressing normal human hemoglobin A (n=3/group). Thirty days after BMT mice were euthanized, and organs were necropsied for histopathology. Formalin-fixed paraffin-embedded sections of the lung, liver, kidney, spleen, and bone marrow (femur & tibia) were stained with H&E and Prussian blue (iron deposits). Morphologic findings of vascular congestion, inflammatory infiltrate, and iron deposits were scored from '0' for no lesion up to '6' for severe lesions occupying 90-100% of the field (Manci et al., Blood 2006). Tubular brush border thickness was assessed on periodic acid Schiff's (PAS)-hematoxylin-stained kidney sections using a 0-4 grading scale: no changes ='0'; lesion area <25%=1; 25%-50% = 2; >50% = 3 and 100% = 4 (Kasztan et al. J Am Soc Nephrol. 2017). We observed significantly increased vascular congestion in the liver (p=0.003), and spleen (p=0.013), and inflammatory infiltrates in the lungs (p=0.031) of TBI-treated mice compared to TMI (8:2). Total body irradiation has been shown to induce inflammation with granulocyte infiltration into tissues in association with endothelial damage, vascular dilatation, and congestion. Moreover, increase iron content in BM and serum and disrupted iron metabolism is another known sequala of total body irradiation, which contributes to hemosiderin formation in tissues. We found significant increase in iron deposits in TBI (4:4) vs TMI (8:2) in liver (p=0.001), spleen (p=0.014), and kidney (p=0.043). TBI (4:4) led to more renal tubular lesions (p=0.003) marked by a significant loss in the tubular brush border on PAS-stained kidney sections compared to TMI (8:2). Thus, TMI shows lesser damaging effect on vital organs compared to TBI, while TMI (8:2) showed donor chimerism similar to TBI on days 7 and 14 but higher than TMI (4:0) treated mice. However, the engraftment failed (less than 10% chimerism) in all groups by day 30. To improve engraftment, we transplanted 10- and 25-million donor HbAA BERK BM cells into TMI (4:0) and TMI (8:2) treated sickle mice. TMI (8:2) showed sustained donor chimerism but TMI (4:0) did not. Histopathological analysis 90 days post-BMT showed that TMI (8:2) treatment (n=3) results in significantly lesser organ pathology Vs untreated sickle mice (n=2), as evinced by reduction in: vascular congestion (lung, p=0.014; liver, p=0.014; spleen p= 0.014), inflammatory infiltrate (lung, p= 0.031; liver p= 0.005), liver infarctions (p=0.003) and liver and kidney iron deposits (p=0.041 & 0.021, respectively). Untreated mice showed a significant loss in the tubular brush border compared to TMI (8:2) treatment (p=0.008). BM in TMI (8:2) (cellularity~70%) showed improvement in cellular differentiation and topography in comparison to the untreated (cellularity ~90% ) that were packed with minimal fat spaces, thin trabeculae, and marked erythroid hyperplasia. These data suggest recovery from sickle cell phenotype with TMI-8:2 without significant organ pathology. Additionally, toluidine blue stained skin sections showed that the number (~45 Vs ~15) and % degranulating mast cells (~85 % Vs 50 %) were significantly lower in TMI (8:2) Vs untreated sickle mice (p<0.05), suggesting that TMI (8:2) treatment and successful engraftment of healthy donor cells may reduce inflammation and pain phenotype. We show that dose escalated TMI (8:2) is well tolerated with reduced organ pathology. Therefore, TMI has the potential to improve BMT-related outcomes without an adverse effect on organ damage.