FADD Deficiency Impairs Early Hematopoiesis in the Bone Marrow Stephen Rosenberg, Haibing Zhang and Jianke Zhang This information is current as J Immunol 2011; 186:203-213; Prepublished online 29 of September 27, 2021. November 2010; doi: 10.4049/jimmunol.1000648 http://www.jimmunol.org/content/186/1/203 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2010/11/29/jimmunol.100064 Material 8.DC1 References This article cites 77 articles, 37 of which you can access for free at: http://www.jimmunol.org/content/186/1/203.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology FADD Deficiency Impairs Early Hematopoiesis in the Bone Marrow Stephen Rosenberg, Haibing Zhang, and Jianke Zhang Signal transduction mediated by Fas-associated death domain protein (FADD) represents a paradigm of coregulation of apoptosis and cellular proliferation. During apoptotic signaling induced by death receptors including Fas, FADD is required for the recruit- ment and activation of caspase 8. In addition, a death receptor-independent function of FADD is essential for embryogenesis. In previous studies, FADD deficiency in embryonic stem cells resulted in a complete lack of B cells and dramatically reduced T cell numbers, as shown by Rag12/2 blastocyst complementation assays. However, T-specific FADD-deficient mice contained normal numbers of thymocytes and slightly reduced peripheral T cell numbers, whereas B cell-specific deletion of FADD led to increased peripheral B cell numbers. It remains undetermined what impact an FADD deficiency has on hematopoietic stem cells and progenitors. The current study analyzed the effect of simultaneous deletion of FADD in multiple cell types, including bone Downloaded from marrow cells, by using the IFN-inducible Mx1-cre transgene. The resulting FADD mutant mice did not develop lymphoprolifera- tion diseases, unlike Fas-deficient mice. Instead, a time-dependent depletion of peripheral FADD-deficient lymphocytes was observed. In the bone marrow, a lack of FADD led to a dramatic decrease in the hematopoietic stem cells and progenitor- enriched population. Furthermore, FADD-deficient bone marrow cells were defective in their ability to generate lymphoid, myeloid, and erythroid cells. Thus, the results revealed a temporal requirement for FADD. Although dispensable during lym- phopoiesis post lineage commitment, FADD plays a critical role in early hematopoietic stages in the bone marrow. The Journal of http://www.jimmunol.org/ Immunology, 2011, 186: 203–213. poptosis plays a critical role in mammalian development downstream caspases and other cellular proteins, leading to cell and homeostasis (1, 2). The intrinsic apoptotic signaling death. A is mediated by the mitochondrion, involving cytochrome The importance of apoptosis in development is exemplified by C and the Bcl-2 family proteins (3). The extrinsic apoptotic the embryonic defects caused by a lack of Bcl-x or Mcl-1, members pathways are initiated by ligation of death receptors (DRs) by of the Bcl-2 family that mediate the intrinsic pathway (12, 13). either their cognate ligands or cross-linking Abs (4, 5). DRs, in- Deficiencies in proapoptotic Bcl-2 family members such as Bax cluding TNF-R1, Fas/Apo-1, and TRAIL-Rs (DR4 and DR5), and Bak resulted in interdigital webbing due to insufficient death by guest on September 27, 2021 activate a caspase cascade through the adaptor protein Fas- of superfluous cells (14, 15). Deletion of another proapoptotic Bcl- associated death domain protein (FADD), which recruits procas- 2 family member, Bim, leads to autoimmune diseases caused by pase 8 to form the death-inducing signaling complex (DISC) (6– impaired death of autoreactive lymphocytes (16). The extrinsic 11). The assembly of the DISC promotes the activation of caspase pathways are essential for maintaining homeostasis in the immune 8 by self-processing, and the resulting active caspase 8 cleaves system. In particular, a systemic loss of Fas leads to the devel- opment of an age-dependent lymphoproliferative (lpr) and auto- immune disease (17). Although expressed in a wide range of tissues, DRs do not appear Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas to play an overt role in mouse development (18–22). Interestingly, Jefferson University, Philadelphia, PA 19107 mice deficient in FADD or caspase 8 die in utero by day 9.5–10.5 Received for publication February 24, 2010. Accepted for publication October 26, 2010. of gestation (23–25). Conditional deletion of FADD or caspase 8 This work was supported in part by National Institutes of Health Grants C0A95454, following lineage commitment in double-negative thymocytes or AI083915, and AI076788, a W. W. Smith Charitable Trust grant, a Thomas Jefferson pro-B cells resulted in no significant defects in the maturation of T University Enhancement grant, and a Concern Foundation grant to J.Z. S.R. was or B cells within primary lymphoid organs (26–30). When tested supported by a National Research Service Award Training grant (T32-AI07492). in vitro, FADD2/2 or caspase 82/2 lymphocytes are defective in Address correspondence and reprint requests to Dr. Jianke Zhang, Department of 2/2 Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson Univer- death receptor-induced apoptosis. Additionally, FADD and 2/2 sity, 233 South 10th Street, Room 731 BLSB, Philadelphia, PA 19107. E-mail address: caspase 8 T cells failed to expand efficiently upon stimulation [email protected] through the TCR, whereas FADD2/2 and caspase 82/2 B cells are The online version of this article contains supplemental material. impaired in TLR-induced proliferation. The effect of FADD de- Abbreviations used in this paper: BFU-E, erythroid burst-forming unit; CFU-GEMM, ficiency on the development and function of myeloid cells and granulocytic-erythrocytic-megakaryocytic-macrophagic CFU; CFU-GM, granuloma- hematopoietic progenitors has not been determined. crophagic CFU; CLP, common lymphoid progenitor; CMP, common myeloid pro- genitor; DC, dendritic cell; DISC, death-inducing signaling complex; DR, death The process of hematopoiesis commences with hematopoietic receptor; EPO, erythropoietin; ES, embryonic stem; FADD, Fas-associated death stem cells (HSCs) (31). HSCs are located in the endosteal niche domain protein; GMP, granulocytic-macrophage progenitor; HSC, hematopoietic 2 + 2 + 2 stem cell; LCCM, L-cell conditioned media; LK, Lin c-Kit Sca-1 ; lpr, lymphopro- within the bone marrow and are characterized as Lin Sca-1 c- 2 + liferative; LSK, Lin Sca-1+c-Kit+; MEP, megakaryocytic-erythroid progenitor; MPP, Kit (32). HSCs proceed to differentiate into multipotent pro- multipotent progenitor; Mut, mutant; poly(I:C), polyinosinic-polycytidylic acid; rm, genitor (MPP) cells, which subsequently develop into either recombinant mouse; SCF, stem cell factor; WT, wild-type. common lymphoid progenitors (CLPs) or common myeloid pro- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 genitors (CMPs) (33, 34). The differentiation of MPPs indicates www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000648 204 FADD FUNCTION IN EARLY HEMATOPOIESIS the branching point at which developing hematopoietic cells on a MoFlo cell sorter (DakoCytomation, Carpinteria, CA). To analyze commit to the lymphoid or myeloid lineage. CLPs develop into lymphocyte populations, cells were washed once with staining buffer precursors for the T, B, and NK cell populations, whereas CMPs and labeled with the following lineage-specific fluorochrome-conjugated Abs: CD4-PE, CD8-TriColor, B220-TriColor, streptavidin-R670 (Caltag- further differentiate into the granulocytic-macrophage progenitors Invitrogen), CD5-PE (eBioscience), CD3-PE, CD19-Bio, c-Kit–PE, CD25- (GMPs) and megakaryocytic-erythroid progenitors (MEPs) (33, PE, IgD-PE (BD Pharmingen), IgM-FITC (Jackson ImmunoResearch 35). GMPs differentiate into macrophages and granulocytes, Laboratories), IgM-PE, IgD-Bio (Southern Biotechnology Associates, whereas MEPs are capable of differentiation into megakaryocytes Birmingham, AL). Cells were analyzed using a Coulter Epics XL cy- tometer (Beckman Coulter, Fullerton, CA). FlowJo (Tree Star, Ashland, and erythrocytes (36, 37). Both CLPs and CMPs lead to dendritic OR) was used for the generation of histograms and dot plots. For sorting, cell (DC) differentiation; therefore, DCs can be of either myeloid bone marrow cells were resuspended in sorting medium (1:1 PBS/RPMI or lymphoid origin (34, 38, 39). 1640). GFP2 bone marrow cells were isolated using a MoFlo high-speed Apoptosis, proliferation, and differentiation of HSCs and pro- cell sorter (DakoCytomation) in the Flow Cytometry Facility at Thomas genitors are tightly