Analysis of the function of FADD in hematopoietic stem cells, lineage development and hematopoietic function
FADD is an adaptor protein responsible for the transduction of apoptotic signaling through multiple death receptors, through the recruitment and activation of caspase 8 and c-Flip to the DISC, initiating a caspase cascade, culminating in cell death. However, systemic deletion of FADD led to embryonic lethality, implicating a role for FADD in non-apoptotic processes. FADD -/- ES cells→Rag1-/- blastocyst chimeric mice displayed a severe blockage of both T and B lymphopoiesis, and isolated T cells from these mice were defective in proliferation following αCD3 or ConA stimulation. This led us to hypothesize that a deletion of FADD negatively impacted both hematopoietic development and function. Conditional deletion of FADD in T cells occurring following T lineage commitment revealed no overt requirement for FADD in T cell development following lineage commitment. However, FADD-/- T cells displayed decreased αCD3-induced proliferative responses. As the FADD-/- ES cells→Rag1 -/- chimeras displayed both a blockage of T and B development, we sought to determine the impact of FADD on B cell development. As with T cells, deletion of FADD following B lineage commitment caused no significant developmental defects. Analysis of FADD-/- B cells revealed that, unlike with TCR signaling, a FADD deficiency had no impact on BCR signaling but did impaired signaling through TLR 3 and 4. Additionally, aged B-cell specific FADD-/- mice contained enlarged spleens and lymph nodes when compared to similarly aged controls. This appears to be due to an accumulation of activated lymphocytes. This led to the disruption of the normal splenic architecture along with co-localization of T and B cells, indicating that while dispensable following lymphocytic lineage commitment, FADD is required for TLR signaling and the maintenance of peripheral homeostasis. While defective lymphopoiesis was observed in FADD-/- ES cells→Rag1-/- chimeric mice, no determination of FADD's role in myelopoiesis was determined. Additionally, although present on B cells, TLRs are more abundantly expressed by macrophages. Thus, FADD was deleted during macrophage development in order to study its function in macrophages. Deletion of FADD during macrophage maturation had no impact on development both in vivo and in vitro. Analysis of FADD -/- macrophages revealed decreased cytokine expression following TLR 4 ligation, ostensibly due to decreased phosphorylation of NF-κB regulatory proteins. TLR 3-mediated signaling pathways were shown to be similarly impaired. These results reveal that FADD is required for NF-κB activation downstream of TLRs 3 and 4, but not for the maturation of macrophages from monocytic precursors. While FADD-/- ES cells→Rag1-/- blastocyst chimeras displayed severe defects in lymphopoieses, deletion of FADD following lineage commitment had no apparent impact. Deletion of FADD in maturing macrophages similarly did not inhibit their development. This suggests a requirement for FADD at earlier, less-committed stages of hematopoiesis. Furthermore, excision of FADD in multiple cell types concurrently led to a time-dependant decrease in the number of peripheral FADD-/- cells, further implicating FADD in the replenishment of the immune system. Analysis revealed decreased numbers of hematopoietic stem and progenitor cells present in the bone marrow and FADD-/- progenitors displayed defective differentiation into cells of the myeloid, lymphoid and erythroid lineages. These results reveal a temporal requirement for FADD during hematopoiesis, where FADD is dispensable after lineage commitment, but plays a critical role in hematopoietic stem cells and early progenitors. Cumulatively, the data presented in this thesis reveal roles for FADD in the regulation of hematopoiesis, as well as hematopoietic function and peripheral homeostasis.
Rosenberg, Stephen M, "Analysis of the function of FADD in hematopoietic stem cells, lineage development and hematopoietic function" (2010). ETD Collection for Thomas Jefferson University. AAI3426293.