B4GALT1-Dependent Glycosylation Is Essential to Bone Marrow Reconstitution Following Chemotherapeutic Injury

Abstract

The bone marrow (BM) comprises a three-dimensional (3D) network of branching sinusoids surrounding hematopoietic cells, embedded in an extracellular matrix (ECM) rich in glycans vital for cell maintenance and function. While the interaction between megakaryocytes (MKs) and the components of the environment for BM recovery post-injury becomes more apparent, the role of glycan-dependent thrombopoiesis and nascent platelets in BM maintenance and reconstitution is underexplored. We have found that β-1,4-galactosyltransferase 1 (β4galt1) deficiency results in dysplastic MKs, disrupted proplatelet release, and severe thrombocytopenia. KEGG analysis of SLAM marker sorted hematopoietic stem and progenitor cells (HSPC) bulk RNAseq data, showed downregulation of ECM-receptor interaction, focal adhesion, gap junction, and proteoglycans in cancer pathways in β4galt1 -/- HSPCs compared to controls. We hypothesized that β4galt1-dependent glycosylation regulates the BM microenvironment at steady state and post-injury. To test this, we conducted a study examining the effects of a single dose of 5-fluorouracil (5-FU) treatment (150 µg/g) on BM MKs, c-kit + progenitors, vasculature, ECM, and platelet count in both control and β4GalT1-deficient (B4 -/-) mice. We analyzed femur BM sections using immunofluorescence, targeting the vasculature marker laminin, progenitor marker c-kit, and ECM markers perlecan and fibrinogen, and MK marker GPIbα (CD42b) and CD41. Image processing with Imaris software and MATLAB facilitated cell count, spatial distribution, and 3D reconstruction assessments of the BM. An automated hemoanalyzer quantified platelet counts from days 1-10 post-treatment. Post-5-FU treatment, control mice exhibited a 50% decrease in reticulated and total platelet counts from day 3, persisting until day 8. On day 10, platelet counts increased to 150% above average. As BM cellularity diminished, a marrow sinusoidal cavity emerged at the bone's center, reaching maximum size on day 3 post-injury (mean size = 2.10 ± 0.14 µm 2; p=0.013), contracting from day 4 (mean size = 1.35 ± 0.14 µm 2; p=0.19), and restoring its size by day 8. GPIbα + MKs and c-kit + cell counts decreased by 50% 3 days following the injury while colocalization of c-kit + GPIbα + expressing cells increased preferentially localizing at the sinusoidal cavity, hinting at their role in BM recovery after injury. At day 5 post-injury, c-kit + cellcounts increased but reduced again on day 10, coinciding with the rise of MK number. 3D imaging post-5-FU treatment revealed a preferential localization of GPIbα + MKs and c-kit + cells in the center of the bone by day 10, adjacent to the previously injured area. High-resolution 3D reconstruction revealed that the sinusoidal cavity was filled with ECM components, including perlecan and fibrinogen, and platelets, as determined using GPIbα staining, on day 5 post-injury. Platelets were not released into circulation until the 8th day of reconstitution of BM, suggesting that although platelets are produced by BM MKs and distributed within the sinusoidal cavity, they do not immediately enter circulation. Depleting platelets using a platelet specific antibody before administering 5-FU slowed reconstitution of the BM sinusoidal cavity. In contrast to control mice, B4 -/- mice showed a reduction in platelet count and a notable accumulation of highly dysplastic MKs clusters around the sinusoidal cavity (MK/mm 3= 3598 vs. MK/mm 3=2748) at day 5 post-5FU. Moreover, SLAM marker defined long-term HSCs showed that MK-biased (CD41 + and CD41 +/ GPIbα +) populations notably increased in B4 -/- mice compared to controls (p=0.0043), indicating heightened emergency hematopoiesis in the absence of β4galt1. While the control BM sinusoidal cavity almost returned to normal size at day 8, the B4 -/- BM displayed a lack of organized vasculature, increased sinusoidal cavity size and a heightened expression of GPIbα + dysplastic and clustered MKs, particularly near the sinusoidal cavity. The findings emphasize the crucial role of β4galt1-dependent glycosylation in platelet production and distribution, fundamental for blood clotting and wound healing, during BM reconstitution after injury. Furthermore, the identification of the sinusoidal cavity as a reservoir of ECM and platelets, continuously remodeled during BM recovery, adds unexpected insights to our understanding of the BM reconstitution processes.