Functional complementation between FADD and RIP1 in embryos and lymphocytes

Abstract

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(-/-) mice die in utero and conditional deletion of FADD leads to impaired lymphocyte proliferation. 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-κB activation. Here we show that Fadd(-/-) 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(-/-) mice. Notably, 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 T cells are resistant to death induced by Fas or TNF-α and show 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.

Figure 1. RIP1 deficiency rescues FADD^−/− mice from embryonic necrosis and lethality

a, FADD^−/− embryos exhibit massive necrosis and altered RIP1 and RIP3 expression. E12.5 wild type (WT, top panels) or FADD^−/− embryos (bottom panels) were fixed in formalin. Left panels of hematoxylin and eosin (H&E) staining show extensive cell loss and pyknotic nuclei in the FADD^−/− fetal liver. Middle and right panels indicate staining for RIP1or RIP3. b, E14.5 DKO embryos appear normal, contrasting the defective FADD^−/− embryos.

Figure 2. FADD deficiency partially corrects the RIP1^−/− T cell developmental defect by blocking apoptosis

Lymphocytes (a-b) in the chimeras of the indicated genotypes were analyzed. E18.5 fetal thymocytes of the indicated genotypes were treated with anti-Fas antibodies (c) or TNFα (d) and death responses determined 12 hours post stimulation. Error bars represent mean ± SEM of triplicates. T cells (e) and B cells (f) from NSG chimeras of the indicated genotypes were stimulated with anti-CD3 and anti-CD28 antibodies or with LPS, respectively. MEFs were stimulated with TNFα (g). NF-κB activation was indicated by the induction of p65 phosphorylation. β-actin, loading controls.

Figure 3. RIP1 deficiency rescues the FADD^−/− T cell proliferation defect

a, T cells of the indicated wild type and chimera genotypes were stimulated with anti-CD3 and anti-CD28 antibodies. Proliferation was measured by [³H]-thymidine incorporation. b, Splenocytes of the indicated genotypes were transferred into TCRαβ^−/− hosts and challenged with Pichinde virus (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 ± SEM of triplicates.