Page 106 - 2021_05-Haematologica-web

N. Luebbering et al.
Figure 4.Vitamin D binding protein function after hematopoietic stem cell transplant. Schematic of pro- posed vitamin D binding pro- tein (VDBP) activity during hematopoietic stem cell transplant, with predominant effect early after transplant being actin scavenging with later effects on macrophage polarization and tissue heal- ing.
followed by binding of monomeric actin to VDBP. The actin-VDBP complex is then removed from the circulation by the reticulo-endothelial system with a reported half-life of 30 minutes. We first examined actin-VDBP complex formation by western blot analysis of serum VDBP after running a native gel to identify mobility changes of serum VDBP due to binding of actin released by cell lysis. Results for analysis of serum VDBP from one patient (unique patient number [UPN] 198) showed the presence of a lower molecular weight band corresponding to VDBP (unbound to F-actin) in all samples including pre-trans- plant serum (baseline [BL]). However, in post-transplanta- tion serum a larger slower migrating band was also observed corresponding to VDBP bound to actin (Figure 2A). There was variability in the presence of complex seen after transplant, with large amounts of complex seen in some cases, while less or none was seen in others. For example one patient, UPN 198, had a large amount of complex present post-transplant, while a second patient, UPN 436, had modest and similar amounts of complex present at all timepoints examined (Figure 2A).
We considered whether the presence of a large amount of complex on day 0 might be associated with inferior outcomes, as has been suggested by a prior animal study.24 Western blot analysis of VDBP was carried out using serum from 93 transplant recipients, and each blot was scored as positive (as seen in UPN 198, Figure 2A left panel) or negative (as seen in UPN 436, Figure 2A right panel) for the presence of elevated-actin-VDBP complex. We found no association between the presence of elevat- ed actin- VDBP complex and TA-TMA, (35% vs. 40%, P=0.67), GvHD (35% vs. 26%, P=0.39) or NRM (5% vs. 10%, P=0.66). We also considered whether the amount of complex simply reflected the level of VDBP but found no such association (P=0.17).
VDBP bound to F-actin is removed from the circulation by the reticuloendothelial system, so that serum levels of actin-VDBP complex are likely to change post-transplant. We therefore examined the kinetics of clearance of com- plex in a patient with a large amount of complex further by examining later timepoints after infusion of stem cells
(Figure 2B). Data show that levels of actin-VDBP complex returned to baseline by 21-28 days after transplant. Further analysis showed a temporal decline in serum VDBP levels at day 14 post-transplantation, common to all patients and all VDBP genotypes, with a rebound back to previous levels by day 30 post HSCT (Figure 2C).
Gelsolin also participates in actin scavenging by VDBP, serving to depolymerize F-actin to monomeric G-actin. We measured plasma gelsolin levels on day 7 after trans- plant, and found that levels above the median were asso- ciated with increased risk of TA-TMA (53% vs. 32%, P=0.03), but gelsolin levels were not associated with any other outcome. We also hypothesized that endothelial toxicity may also be related to the release of ATP into the circulation together with F-actin, but found no evidence of an increase in circulating ATP after HSCT. Extracellular ATP increased in only 1 of 4 HSCT recipients, in contrast to our hypothesis that extracellular ATP would increase markedly in most cases at the time of absolute neutrophil count nadir (time of maximum cell lysis) after HSCT (Online Supplementary Figure S1). The main cause of death in those with no detectable F-actin was relapse, in contrast to those with detectable F-actin in whom the main causes of death was GvHD (in which endothelial injury is impli- cated) (Online Supplementary Table S2).
Vitamin B binding protein functions as a macrophage-activation factor
Our findings regarding clearance of F-actin by VDBP provide a plausible mechanism for a role for VDBP in pro- tection against endothelial injury and early toxicities of transplant such as TA-TMA and GvHD. However, as VDBP can also function as a macrophage activating factor, we investigated whether higher levels of VDBP func- tioned to modify cytokine production by immune cells later after transplant.18,19 PBMC from a normal healthy vol- unteer were incubated overnight with serum from 18 dif- ferent HSCT recipients at four different timepoints. Supernatants from these cells were then assayed for key cytokines and the concentrations of these cytokines were correlated with serum VDBP, total 25-hydroxyvitamin D
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haematologica | 2021; 106(5)