Systemic Ceramide Accumulation Causes Dysregulated Hematopoiesis

Abstract 3461

Ceramide is a bioactive lipid that exists in all cells of the body. Its role in hematopoiesis has not been elucidated. We recently generated the first viable mouse model of systemic ceramide accumulation due to acid ceramidase (ACDase) deficiency (Farber disease). Mice homozygous for the ACDase P362R mutation (Hom) accumulate ceramide, have a reduced lifespan of 7–13 weeks, and are smaller than wild-type (WT) or heterozygous (Het) littermates. Peripheral blood analysis revealed that Hom ACDase-deficient mice had severe leukocytosis, mostly due to increased neutrophils. Lymphocyte and erythrocyte counts were normal. Hematopoietic and accessory organs were larger normalized to body weight in Hom compared to Het and WT animals: spleen (4× larger), thymus (3×), lymph nodes (LN) (8–12x), liver (1.5×). Kidneys and heart were not enlarged. IHC of the enlarged organs and bone marrow (BM) revealed massive infiltration by large, lipid-filled “foamy macrophages”. Infiltration was so severe that hematopoietic niches were disrupted: germinal center architecture was destroyed in the spleen of Hom ACDase mice, for example. In contrast, total cell numbers from the BM, spleen, thymus, and LN were reduced in Hom ACDase-deficient mice. Flow cytometry analyses revealed that T and B cell intermediates were most affected. By 9 weeks thymic CD4 CD8 double positive T cells were dramatically reduced to 5% of cells in Hom animals from 80% in WT. BM pro-B cells were reduced to 3% of cells in Hom (8% in WT) with pre-B cells reduced to <1% (14% in WT). Consequently, there were fewer IL-7 responsive cells. Responsiveness to LPS and anti-μ were similar in Hom and WT mice. In Dexter assays, an in vitro system where a monolayer of stromal cells supports myelopoiesis through direct cell contact, cultures from Hom ACDase-deficient mice failed to establish the typical foci formation seen in WT after successful attachment of myeloid precursors to the stroma. In cellulose based media, hematopoietic stem and progenitor cells (HSPC) were able to properly differentiate down the myeloid lineage: in colony-forming cell (CFC) assays, BM and spleen cells from 5, 7, and 9 week-old Hom, Het, and WT mice demonstrated similar differentiation capacities. Interestingly, there was an increase in the total number of CFC colonies in Hom ACDase mice at 5 weeks that normalized by 9 weeks. Additional CFC assays and IHC of the liver revealed extramedullary hematopoiesis, which may also contribute to the over-production of leukocytes and macrophages observed.

In preliminary BMT experiments, lethally-irradiated Het animals transplanted with cells derived from Hom ACDase deficient mice lived as long as Het animals transplanted with WT cells (10 months; study end-point) and had normal peripheral blood cell counts. The reverse transplants of WT cells into Hom ACDase-deficient mice are underway. Together with the Dexter assay results these data suggest that the BM niche is unable to support and control HSPC differentiation. Briefly, to begin to elucidate the mechanism through which systemic ceramide accumulation is exerting its effects, cytokine analyses and ELISAs were performed on organs and serum. Serum cytokine analyses revealed an increase in MCP-1 and IL-12(p40) levels along with a trend towards an increase in MIP-1α and RANTES expression. No differences were observed in TNF-α, INF-γ, MIP-1β, eotaxin, IL-13, IL-12(p70), IL-10, IL-9, and IL-1b levels. MCP-1 levels were also higher in the organs of Hom ACDase-deficient mice than in WT animals; further, MCP-1 levels were higher in the enlarged organs of Hom mice than the serum, possibly creating a gradient to attract macrophages. Follow-up RT-PCR analyses revealed an increase in the expression of MCP-1 (in BM, LN, thymus, spleen), MIP-1α (LN, thymus, spleen), IL-6 (thymus, spleen), GM-CSF (thymus), iNOS (BM, spleen), and galectin-3 (LN, thymus, spleen) mRNA. No change was found in CCR2 (MCP-1 receptor), TGF-β, M-CSF, TNF-α, IL-1β, and IL-10 mRNA levels.

This study reveals previously unappreciated consequences of ceramide accumulation on hematopoiesis. Our results suggest that ceramide accumulation due to ACDase deficiency perturbs hematopoiesis: the interaction between support cells and HSPC is disrupted and foamy macrophages infiltrate organs. HSPC themselves appear to be functional. Further studies will elucidate the mechanism through which ceramide hinders support cells from interacting with HSPCs.