Allogeneic Transplantation, Fas Signaling, and Dysregulation of Hepcidin

Abstract

Hepatic iron overload is common in patients undergoing hematopoietic cell transplantation. We showed previously in a murine model that transplantation of allogeneic T cells induced iron deposition and down-regulation of hepcidin (Hamp) in hepatocytes. We hypothesized that hepatic injury was related to disrupted iron homeostasis triggered by the interaction of Fas-ligand, expressed on activated T cells, with Fas on hepatocytes. In the current study, we determined the effects of modified expression of the Flice inhibitory protein (FLIP long [FLIPL]), which interferes with Fas signaling, on the impact of Fas-initiated signals on the expression of IL-6 and Stat3 and their down-stream target, Hamp. To exclude a possible contribution by other pathways, we used agonistic anti-Fas antibodies (rather than allogeneic T cells) to trigger Fas signaling. Inhibition of FLIPL by RNA interference resulted, as expected, in enhanced hepatocyte apoptosis in response to Fas signals, but also in decreased levels of IL-6, Stat3 and Hamp. In contrast, over-expression of FLIPL protected hepatocytes against agonistic anti-Fas antibody-mediated apoptosis, and increased the levels of IL-6 and Stat3, thereby maintaining the expression of Hamp in an NF-κB-dependent fashion. In vivo over-expression of FLIPL in the liver via hydrodynamic transfection, similarly, interfered with Fas-initiated apoptosis and prevented down-regulation of IL-6, Stat3 and Hamp. These data indicate that Fas-dependent signals alter the regulation of iron homeostasis and suggest that signals initiated by Fas may contribute to peri-transplant iron accumulation.

Disruption of Iron Regulation after Radiation and Donor Cell Infusion

“…In particular, after injection of gld cells, levels of IL-6 increased considerably less, Hamp expression was decreased (rather than increased) and Fpn1 expression declined progressively. These observations were consistent with our previous results, which showed a profound effect of expression of the Fas signal-modulating factor FLIPL on iron regulation [11], and support the model of Fas-mediated dysregulation of iron homeostasis.…”

“…In allogeneic transplant models this was accompanied by reduced levels of IL-6, Stat3 and Hamp [11]. The present experiments, using the anti-Fas antibody JO2, confirmed those observations, showing a significant increase in liver iron content following JO2 injection (see below).…”

“…As in previous studies [3,11], serum iron levels increased after T cell injection, in parallel with a decrease in hepcidin and an increase of ferroportin at the RNA level. Figure 2 illustrates changes in serum iron after injection of 1 × 10 6 T lymphocytes.…”

“…Since allo-activated T cells up-regulate expression of the cognate Fas ligand, CD178, these observations were consistent with a T cell-mediated effect on iron homeostasis via Fas/Fas-ligand interactions. In further support of this hypothesis, over-expression of the Fas adaptor molecule FLIP-long, which interferes with Fas signaling, allowed for maintenance of hepcidin levels [11]. Based on that model, we characterized in the present study, interactions between the effects of TBI conditioning and donor cell-dependent signals on iron homeostasis in wild type mice.…”

“…The data suggested that activated donor-derived T lymphocytes expressing CD 178, the cognate ligand for Fas (CD95), interacted with Fas-expressing hepatocytes in the liver [17,18], an organ central to iron regulation [19] . We then characterized transplant-associated alterations in gene expression triggered by Fas-signaling in a wild type parent (P) into F1 hybrid model (not requiring transplant conditioning), which further supported a role for Fas-mediated signals in modifying the expression of IL-6, Stat3 and Hamp [11,20]. …”