A new TIPE of negative regulator in the immune system

Abstract

Proteins with a death effector domain (DED) are key signal transducers for cell death and immune homeostasis. In this issue of Cell, Sun et al. describe TIPE2, a novel DED protein that controls T cell receptor (TCR) and Toll-like receptor (TLR) signaling (Sun et al., 2008). In mice, genetic ablation causes leukocyte accumulation, hyperresponsiveness, and inflammatory disease indicating that TIPE2 may contribute to the etiology of certain human autoimmune or inflammatory disorders.

Homeostatic balance within the immune system is maintained by a myriad of mechanisms, which include regulation of immune cell activation and programmed cell death (PCD) (Bidere et al., 2006). A subfamily of proteins that contribute to immune homeostasis contain a hexahelical bundle motif called the death effector domain (DED)(Tibbetts et al., 2003). DEDs are structurally related to two other hexahelical bundle structures - “death domains” (DD) and caspase activation and recruitment domains (CARD) - that participate in similar signaling pathways. DEDs were initially identified based on their ability to regulate PCD triggered by Fas and other death receptors. For example, FADD links Fas to the cysteine proteases caspase-8 and -10 through homotypic interaction between their DEDs to initiate PCD. By contrast, cellular and viral anti-apoptotic DEDs, known as FLIPs, inhibit Fas-induced death by interfering with FADD and caspase-8 (Table 1). However, it is now well accepted that several DEDs also critically regulate cell proliferation in addition to PCD (Tibbetts et al., 2003). In this issue, Sun and colleagues have identified TIPE2, a novel DED-containing protein that governs both apoptosis and immune cell function. TIPE2 was found to be abnormally present in spinal cord tissue of mice with experimental autoimmune encephalomyelitis (EAE), and shares 53% amino acid identity with tumor necrosis factor (TNF)-α-induced protein 8 (TNFAIP8), which also contains a DED and may regulate apoptosis.

Table 1.

Cellular and viral DED protein interactions and their functions in cell death and activation.

Protein	Binding to other DED	Role in cell death	Non-apoptotic role
TIPE2	Caspase-8	Positively regulates Fas- and TCR- mediated death, but is not part of the DISC	Negatively regulates TLR2, TLR3, TLR4, and TLR9 signaling pathways in macrophages TIPE2-/- mice are hypersensitive to LPS-induced septic shock
FADD	Caspase-8 MC159 PEA-15	Initiates apoptosis by connecting the death receptors to caspase-8 and caspase-10 to form the DISC	Participates in thymocyte development (arrest at the double negative stage without FADD) Is necessary for proliferation of T cells, and participates in the control of cell cycle Positively regulates TLR3 signaling and modulates TLR4 (positively in B cells and negatively in endothelial cells)
Caspase-8	FADD PEA-15 FLIP TIPE2	Propagates apoptosis once recruited to the DISC through FADD.	Is necessary for T cells, B cells, and NK cells proliferation Controls early NF-κB activation and cooperates with FADD to promote cell cycle progression in T lymphocytes Regulates TLR2, TLR3, and TLR4 signaling in B cells
c-FLIPL	FADD Caspase-8	Competes with caspase-8 for binding to FADD within the DISC and negatively regulates Fas killing	Is necessary for lymphocyte proliferation Promotes ERK, NF-κB and cell proliferation in transgenic animals Is cleaved by caspase-8 into an NF-κB activating fragment
c-FLIPS	FADD Caspase-8	Inhibits Fas killing	Reduces lymphocyte proliferation, caspase-8 activation and NF-κB in T cells from transgenic animals
PEA-15	FADD Caspase-8	Inhibits Fas killing	Sequesters ERK1/2 in the cytosol, and promotes ERK-dependent phosphorylation of RSK
K13 Kaposi’s-sarcoma-associated human herpesvirus-8 FLIP		Blocks Fas in vitro but not in vivo Protects TF-1 leukemic cells from cytokine withdrawal death	Is a potent and selective activator of NF-κB through its binding to IKK Splenocytes from K13 transgenic mice exhibit enhanced proliferation following TCR and TLR4 stimulation K13 transgenic mice present an increased risk of lymphoma

After two months of age, TIPE2-deficient mice develop progressive immune pathology characterized by weight loss, splenomegaly, leukocytosis, and multi-organ inflammation leading to death. If challenged with a bacterial (listeria monocytogenes) or viral (lymphocytic choriomeningitis virus) pathogen prior to development of spontaneous disease, TIPE2-deficient mice show increased numbers of CD8⁺ T cells in the spleen and increased cytokine production, suggesting that TIPE2 regulates the T cell response.

TIPE2 is predominantly expressed in myeloid and lymphoid lineage cells but expression can be induced by TNF-α in at least one fibroblast cell line. Thus, it is plausible that TIPE2 may be expressed in many cell types to establish equipoise during an inflammatory response, or when TNF-α is present. It remains to be determined precisely which cell types contribute to the inflammatory phenotype observed in the TIPE2-deficient mice. Intriguingly, TIPE2 is highly homologous to TIPE1 and TIPE3, both of which are uncharacterized molecules. It will be fascinating to investigate the range of functions of TIPEs 1-3.

Sun et al. went further to show that TIPE2 regulates both adaptive and innate immunity. Although TIPE2^-/- T cells are hyperactivated by TCR stimulation, they do not expand more quickly. Paradoxically, TIPE2 over-expression slightly repressed both lymphocyte activation and proliferation. These finding are intriguing because lymphocyte proliferation is dramatically reduced in the absence of FADD, caspase-8, or c-FLIP_L, and enhanced in animals transgenic for FLIP_L or for the human herpesvirus-8 FLIP K13 (Chugh et al., 2005; Chun et al., 2002; Lens et al., 2002; Zhang et al., 1998). In addition, adaptive immunity occurs normally in TIPE2^-/- B cells, which contrasts with caspase-8 deficiency (Beisner et al., 2005). Altogether, TIPE2 function in T cells is not redundant with the other DEDs studied thus far. Future work will be needed to decipher the molecular pathway used by TIPE2 in lymphocytes.

In addition to altering T cell activation, the authors show that TIPE2 negatively regulates the Toll-like receptor (TLR) pathway. TIPE2^-/- macrophages and B cells stimulated with several TLR ligands produced more IL-6 or TNF-α and IL-1β, respectively, than wild-type cells. Importantly, a dramatic difference in survival was observed when TIPE2^-/- or wild-type mice were treated with low-dose lipopolysaccharide (LPS) to induce septic shock. Interestingly, a common feature of these TLRs is the formation of large multi-protein complexes necessary to convey the signal. Does TIPE2 integrate into these signalosomes? The intracellular localization of TIPE2 remains undefined and it will be interesting to assess whether it changes location after stimulation.

Mechanistically, the authors demonstrate that TIPE2 downmodulates multiple signaling pathways in macrophages stimulated with LPS. How TIPE2 negatively regulates such diverse signaling circuits is currently obscure. TIPE2 represses activation of the c-Jun N-terminal kinase (JNK) and p38 MAP kinase and thus diminishes AP-1 activity. In addition, TIPE2 depletion also leads to an increase in nuclear translocation of NF-κB, subsequent to enhanced phosphorylation and degradation of the inhibitor-of-κBα (IκBα) protein. By contrast, the extracellular signal-related kinase (ERK) pathway is inured to TIPE2. Where does TIPE2 fit in the known signaling pathways? Sun et al., report that a portion of TIPE2 is constitutively associated with caspase-8. It will be important to clarify the binding between these proteins and to assess whether the interaction between caspase-8 and TIPE2 is vital. It is also likely that, similar to other DEDs, TIPE2 associates with non-DEDs proteins. For example, PEA-15 associates with ERK1/2 to prevent its nuclear accumulation (Formstecher et al., 2001). Additionally, K13 strongly binds to the inhibitor of κB kinase (IKK) complex and promotes NF-κB (Liu et al., 2002). Also, odd bedfellows such as FADD and CK1α or caspase-8 and TRAF6 seem to pair in functionally important ways (see Table 1). Defining TIPE2 binding partners will undoubtedly provide insights into how TIPE2 regulates multifarious pathways.

TIPE2 knockdown inhibits Fas-mediated apoptosis. Antigen receptor-induced cell death (AICD), which partially involves Fas, is decreased in TIPE2-deficient cells. Moreover, ectopic expression of TIPE2 enhanced Fas killing. Surprisingly, although TIPE2 binds caspase-8, TIPE2 is not found in the Death Inducing Signaling Complex (DISC) following Fas ligation, and does not impair FADD and caspase-8 recruitment. This clearly differs from the other DEDs, which bind to FADD or caspase-8 and alter the DISC. How TIPE2 inhibits apoptosis by this, and potentially other, death receptors is a mystery for future resolution.

Although much work is required to fully elucidate how TIPE2 works, the identification of this protein and the intriguing phenotype of the TIPE2 deficient mice establish TIPE2 as an important contributor to immune homeostasis.