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Functional complementation between FADD and RIP1 in embryos and lymphocytes

Summary FADD is a common adaptor shared by several death-receptors (DRs) for signaling apoptosis through recruitment and activation of caspase 81-3. DRs are essential for immune homeostasis, but dispensable during embryogenesis. Surprisingly, FADD−/− mice die in utero4-5 and conditional deletion of FADD leads to impaired lymphocyte proliferation6-7. How FADD regulates embryogenesis and lymphocyte responses has been a long standing enigma. FADD could directly bind to RIP1, a serine/threonine kinase which mediates both necrosis and NF-κB activation. Here we show that FADD−/− embryos contain elevated levels of RIP1 and exhibit massive necrosis. To investigate potential in vivo functional interaction between RIP1 and FADD, null alleles of RIP1 were crossed into FADD−/− mice. Strikingly, RIP1 deficiency allowed normal embryogenesis of FADD−/− mice. Conversely, the developmental defect of RIP1−/− lymphocytes was partially corrected by FADD deletion. Furthermore, RIP1 deficiency fully restored normal proliferation in FADD−/− T cells but not in FADD−/− B cells. FADD−/−RIP1−/− double knockout (DKO) T cells are resistant to death induced by Fas or TNFα and display reduced NF-κB activity. Therefore, our data demonstrate an unexpected cell type-specific interplay between FADD and RIP1, which is critical for the regulation of apoptosis and necrosis during embryogenesis and lymphocyte function.

FADD is a common adaptor shared by several death receptors for signalling apoptosis through recruitment and activation of caspase 8 (refs 1-3). Death receptors are essential for immune homeostasis, but dispensable during embryogenesis. Surprisingly, Fadd 2/2 mice die in utero 4,5 and conditional deletion of FADD leads to impaired lymphocyte proliferation 6,7 . How FADD regulates embryogenesis and lymphocyte responses has been a long-standing enigma. FADD could directly bind to RIP1 (also known as RIPK1), a serine/threonine kinase that mediates both necrosis and NF-kB activation. Here we show that Fadd 2/2 embryos contain raised levels of RIP1 and exhibit massive necrosis. To investigate a potential in vivo functional interaction between RIP1 and FADD, null alleles of RIP1 were crossed into Fadd 2/2 mice. Notably, RIP1 deficiency allowed normal embryogenesis of Fadd 2/2 mice. Conversely, the developmental defect of Rip1 2/2 lymphocytes was partially corrected by FADD deletion. Furthermore, RIP1 deficiency fully restored normal proliferation in Fadd 2/2 T cells but not in Fadd 2/2 B cells. Fadd 2/2 Rip1 2/2 double-knockout T cells are resistant to death induced by Fas or TNF-a and show reduced NF-kB activity. Therefore, our data demonstrate an unexpected cell-type-specific interplay between FADD and RIP1, which is critical for the regulation of apoptosis and necrosis during embryogenesis and lymphocyte function.
Programmed cell death-including apoptosis and necrosis-is a fundamental biological process that is essential during embryonic development and for homeostasis in somatic tissues. Death receptors can signal apoptotic cell death when engaged by their cognate ligands 8 . This extrinsic death pathway requires the adaptor protein FADD, which couples the signal generated by death receptors to the apical caspase 8 (refs 3, 9, 10). The resulting activation of caspase 8 triggers a battery of downstream caspases, leading to apoptosis. Recently, death receptors were shown to induce necrosis-like cell death in the presence of caspase inhibitors 11,12 . Death-receptor-induced necrosis is blocked in cells lacking the protein serine/threonine kinases RIP1 and RIP3 [11][12][13][14][15] . FADD and RIP1 have indispensable roles in development, as Fadd 2/2 mice die during mid-gestation stages 4,5 , and Rip1 2/2 mice die at birth 16 . Whereas the developmental defect in Rip1 2/2 mice is presumably due in part to defective NF-kB activation and increased cell death 17 , the mechanism that underlies the developmental defect of Fadd 2/2 mice has remained elusive.
Developmental retardation of Fadd 2/2 mouse is apparent at embryonic day (E)11.5 to 13.5 ( Supplementary Fig. 1a, b). Histological analysis revealed extensive necrotic cell death and cell loss in Fadd 2/2 embryos (Fig. 1a). Absence of FADD sensitizes human Jurkat T lymphoma cells to tumour necrosis factor (TNF)-induced necrosis 11,12 . Furthermore, Fadd 2/2 mouse embryonic fibroblasts (MEFs) were hypersensitive to reactive oxygen species (ROS)-induced necrosis 18 . In contrast, necrosis induced by ROS was blocked in Rip1 2/2 MEF cells. Examination of wild-type E12.5 embryos showed that RIP1 was expressed at low levels whereas RIP3 was readily detected in multiple tissues including the nervous system, heart and lung ( Fig. 1a and Supplementary Fig. 2). Interestingly, RIP1 expression is highly elevated in Fadd 2/2 embryos (Fig. 1a). Furthermore, punctate RIP3 staining was observed in cells of Fadd 2/2 embryos, which is indicative of RIP3 aggregation and activation 14 . These results indicate that induction of RIP1 expression might have a role in the necrosis observed in Fadd 2/2 embryos.
To investigate a potential in vivo molecular interplay between FADDand RIP1-mediated signalling, we crossed the Rip1 knockout alleles into Fadd 2/2 mice. Notably, Fadd 2/2 Rip1 2/2 double-knockout (DKO) embryos were detected at E14.5 at the expected Mendelian frequencies (Table 1, Fig. 1b and Supplementary Fig. 1c-f). In sharp contrast to the highly deformed E14.5 Fadd 2/2 embryos, E14.5 DKO embryos were indistinguishable from wild-type control embryos (Fig. 1b). DKO embryos of normal morphology were also found at later gestation stages E15.5, E16.5, E17.5 and E18.5 at the expected Mendelian frequencies (Supplementary Fig. 1c and Table 1). Importantly, live DKO neonates were also detected at birth (Supplementary Fig. 1d). No Fadd 2/2 embryos were detected at E15.5 or later stages. Histological examination did not reveal obvious defects in Rip1 2/2 and DKO E18.   Table 1), 30 died within 4 days after birth, and this group contained 6 DKO and 19 Rip1 2/2 genotypes. No death was observed after postnatal day 4 and DKO mice were not present in the remaining 74 mice that survived beyond 3 weeks (Table 1). These results demonstrate that RIP1 deficiency fully restores embryonic development of Fadd 2/2 mice. However, loss of FADD does not prevent neonatal lethality of Rip1 2/2 mice. ROS is an important effector mechanism for necrotic cell death. Fadd 2/2 MEFs were hypersensitive to ROSinduced death (Supplementary Fig. 4a, b). In contrast, Rip1 2/2 MEFs were highly resistant to ROS-induced necrosis 18 . Addition of the RIP1specific inhibitor necrostatin 1 (Nec1) 19 greatly reduced ROS hypersensitivity in Fadd 2/2 MEFs (Supplementary Fig. 4a, b). Importantly, DKO MEFs were resistant to ROS-induced death. Collectively, these results indicate that FADD deficiency primes embryonic cells to ROSand RIP1-dependent necrosis, which might cause embryonic lethality. Although important at early haematopoietic stages 20 , FADD has a minor role in post-lineage commitment lymphopoiesis 4,6,7 . Although Rip1 2/2 neonates contain normal thymocyte numbers 16 , Rip1 2/2 fetal liver cells failed to reconstitute the peripheral T-cell compartment 17 . The rescue of embryonic development in DKO mice prompted us to examine whether a similar FADD-RIP1 interaction might regulate lymphocyte development. To this end, we adoptively transferred fetal liver cells containing haematopoietic progenitor cells into immunodeficient NSG recipient mice. In agreement with previous results 17 , NSG chimaeras reconstituted with Rip1 2/2 fetal liver cells contained markedly reduced CD4 1 CD8 1 double-positive immature and CD4 1 or CD8 1 single-positive mature thymocytes (Fig. 2a). In contrast, the thymic population profile of DKO fetal liver cell chimaeras was similar to that of the wild-type control thymus (Fig. 2a). Reconstitution of the peripheral lymphoid compartment by DKO fetal liver cells was apparent, as indicated by the spleen sizes of the recipients of DKO fetal liver cells, which were similar to the size of the control spleens receiving wild-type fetal liver cells ( Supplementary Fig. 5a). In contrast, the spleen of Rip1 2/2 chimaeras was smaller than that of wild-type or DKO chimaeras. Flow cytometric analyses showed that Rip1 2/2 chimaeras contained few CD3 1 T cells in the periphery ( Fig. 2b and Supplementary Fig. 5b, c). In contrast, DKO chimaeras contained significantly higher numbers of T cells in the spleen, lymph nodes and blood ( Fig. 2b and Supplementary 5b, c). Similarly, FADD deficiency partially rescued Rip1 2/2 B-cell development ( Supplementary  Fig. 5c). Rip1 2/2 thymocytes were readily killed by treatments with anti-Fas antibodies or TNF (Fig. 2c, d). Interestingly, DKO thymocytes were highly resistant to these death stimuli. Although FADD deficiency fully reversed the hypersensitivity to Fas-and TNF-a-induced killing, it only partially corrected the NF-kB activation defect in Rip1 2/2 T cells, B cells and MEFs (Fig. 2e-g). These results indicate that the partial rescue of lymphocyte development in the DKO chimaeras is due to inhibition of FADD-mediated apoptosis rather than rescue of NF-kB activation.
Although T-cell-specific deletion of FADD had no effect on thymic development, the resulting mature Fadd 2/2 T cells were highly defective in T-cell antigen receptor (TCR)-induced proliferation 6 (Fig. 3a). When compared to Fadd 2/2 and wild-type controls, DKO T cells stimulated through the TCR/CD28 showed a remarkable rescue in their proliferative responses (Fig. 3a and Supplementary Table 1a). When transferred to hosts with the genes encoding TCR-a and TCR-b deleted (Tcra/ b 2/2 ), DKO T cells were functionally competent to expand and produce IFN-c in response to challenge with Pichinde virus (PV) (Fig. 3b) . Acute  Timed pregnancy analyses were set up to determine the frequencies of each genotype of offspring from intercrosses of Fadd 1/2 Rip1 1/2 mice. The actual and expected numbers of each or groups of genotypes were shown. Neonates were killed and analysed at birth (Day 0) or monitored for survival for up to 6 months (.Day 0). Asterisks indicate numbers of DKO embryos or neonates. Postnatal Rip1 2/2 and DKO neonates (.Day 0) died within 4 days after birth.

RESEARCH LETTER
CD8 1 T-cell responses to the immunodominant epitope NP38 and subdominant epitope NP205 were similar between wild-type and DKO donor cells (Fig. 3b). Moreover, challenge of wild-type hosts adoptively transferred with DKO lymphocytes with lymphocytic choriomenigitis virus (LCMV) showed that the DKO T cells could generate a productive antiviral response to the immunodominant epitope NP396 (Supplementary Fig. 6a). Collectively, these results indicate that RIP1-dependent necrosis underlies the defective proliferation in Fadd 2/2 T cells and that inactivation of RIP1 restores the proliferative capacity of Fadd 2/2 T cells. Although FADD does not have a significant role in B-cell antigen receptor (BCR)-or CD40-induced proliferation responses, it is required for TLR3 and TLR4 signalling in B cells 7 . Consistent with these observations, wild-type, Rip1 2/2 , Fadd 2/2 and DKO B cells responded similarly to stimulation with anti-IgM or anti-CD40 antibodies (Supplementary Fig. 6b and data not shown). In contrast to the rescue of T-cell proliferation, DKO B cells remained defective in proliferative responses to the TLR3 and TLR4 agonists poly(I:C) and lipopolysaccharide (LPS), respectively (Fig. 3c, Supplementary Fig. 6c and Supplementary Table 1b). The differential effect of RIP1 deletion on Fadd 2/2 T-and B-cell proliferation was confirmed with the RIP1specific inhibitor Nec1 (Supplementary Fig. 6d, e). Although caspase inhibition did hamper LPS-induced B-cell proliferation (Supplementary Fig. 7a, b), RIP1 cleavage, which inactivates the pro-necrotic activity of RIP1, was only observed in TCR-treated T cells but not LPS-induced B cells or ROS-treated MEFs (Supplementary Fig. 8a-c). Therefore, the FADD-RIP1 axis is preferentially required for controlling proliferation in T cells, but not in B cells.
This study demonstrates that absence of RIP1 restores normal embryogenesis of Fadd 2/2 mice, and that FADD deficiency partially corrects the developmental defect in Rip1 2/2 T cells. This finding provides compelling genetic evidence that a critical in vivo role for FADD during embryogenesis is to inhibit RIP1-mediated necrosis. In T cells, RIP1 is required to help suppress FADD-mediated apoptosis. Interestingly, caspase 8 2/2 mice showed embryonic and T-cell defects similar to that of Fadd 2/2 mice 4,5,21 . Moreover, Nec1 rescued the proliferative defect of caspase 8 2/2 and Fadd 2/2 T cells 22,23 . Hence, FADD and caspase 8 probably act in concert to keep RIP1-mediated necrosis in check by cleavage and inactivation of RIP1. Such a regulatory mechanism is crucial to ensure proper embryogenesis and to prevent abortive expansion of T cells during immune responses. Our results also reveal an unexpected role for RIP1 in keeping FADD-mediated apoptosis at bay during T-cell development. This regulation is not entirely dependent on RIP1-mediated activation of the NF-kB pathways, unlike that proposed previously 16,17 . The reduced NF-kB activation had no major effect on DKO T-cell proliferations. In addition to lymphocytes, impairment of the NF-kB pathway is also present in Rip1 2/2 and DKO MEFs and probably other cell types, which might lead to postnatal lethality as seen in Rip1 2/2 and DKO mice. In summary, our results reveal a complex functional interaction between FADD and RIP1 that is context and cell-type dependent.

METHODS SUMMARY
Heterozygous Fadd 1/2 mutant mice have previously been described 4 . Heterozygous Rip1 1/2 mutant mice 16 were provided by M. Kelliher and crossed into Fadd 1/2 mice. Timed pregnancy was set up with the resulting Fadd 1/2 Rip1 1/2 mouse intercrosses. Embryos were isolated and genotyped by PCR using tissue genomic DNA templates and confirmed by western blots. Fetal liver cells were isolated from E14.5 embryos and adoptively transferred into irradiated (200 rad) NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ) mice purchased from Jackson Laboratory. All animal studies were approved by Institutional Animal Care and Use Committees. Tento-twelve weeks after transfer, cells were isolated from the indicated organs and were analysed by flow cytometry. T cells and B cells were purified from the spleen and lymph nodes by high-speed sorting and proliferative responses were analysed as described previously 6,7 . Fadd 2/2 mutant T cells and B cells were isolated by sorting from T-cell-specific and B-cell-specific Fadd 2/2 mice as described previously 6,7 . Western blotting was performed according to standard protocols. For embryonic cell death assays, primary MEFs were prepared following the NIH 3T3 protocol. Fadd 2/2 MEFs were prepared from E8.5 embryos and DKO MEFs from E14.5 MEFs. MEF cells were cultured in complete DMEM to 80% confluence, and treated with 0.5 mM H 2 O 2 with or without Nec1 (50 mM) for 12 h, and cell death was determined by propidium iodide staining and flow cytometry. Images were taken by using a Nikon inverted light microscope. For virus infections, after adoptive transfer of lymphocytes, mice were challenged with 5 3 10 4 p.f.u. of LCMV or 1 3 10 7 p.f.u. of PV. Peptide-specific CD8 T-cell responses were measured 8 days after infection by intracellular IFN-c staining.
Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.     a, T cells of the indicated wild-type and chimaera genotypes were stimulated with anti-CD3 and anti-CD28 antibodies. Proliferation was measured by [ 3 H]thymidine incorporation (c.p.m., counts per minute). b, Splenocytes of the indicated genotypes were transferred into Tcra/b 2/2 hosts and challenged with PV. Acute responses of the donor CD8 T cells to the indicated epitopes were determined. Anti-CD3 antibodies were used as control. c, B cells of the indicated genotypes were stimulated with LPS and proliferation was measured as in a. Results shown (a and c) are mean 6 s.e.m. of triplicates.

LETTER RESEARCH
In Fig. 3b of this Letter, the plot for the NP205 epitope response in the Fadd 2/2 Rip1 2/2 double-knockout (DKO) splenocytes was a duplicate of that for the NP38 epitope response. The correct figure panel is shown below. The panel has been corrected in the HTML and PDF versions online.