NATURE IMMUNOLOGY VOLUME 7 NUMBER 2 FEBRUARY 2006 123

Mal-function of TLRs by SOCSTakashi Kobayashi, Giichi Takaesu & Akihiko Yoshimura

Suppressor of cytokine signaling 1 has been linked to the negative regulation of Toll-like receptor signaling. The mechanism for this seems to be suppressor of cytokine signaling 1–mediated degradation of the adaptor Mal, which is required for Toll-like receptor signaling.

The innate immune system is triggered by various signaling receptors, including

the Toll-like receptor (TLR) family, which includes more than ten members. Each TLR responds precisely to molecular pat-terns associated with pathogens. However, TLR signaling must be properly regulated to avoid excessive damage to the host1. The increasing number of known TLR-negative regulators indicates that multiple pathways are needed to dampen or reduce signaling through various TLRs. In this issue of Nature Immunology, Mansell et al.2 describe a new target of suppressor of cytokine signaling 1 (SOCS-1)–mediated negative regulation. They demonstrate that SOCS-1 is required for the rapid degradation of Mal, an adap-tor protein involved in TLR2 and TLR4 sig-naling. Characterization of this previously unknown pathway of regulating TLR signal-ing increases understanding of the manifold ways in which innate signals are kept in check.

All members of the TLR family consist of cytoplasmic, transmembrane and extra-cellular domains. The cytoplasmic portion of all TLRs is characterized by a common motif called the Toll–interleukin 1 recep-tor (TIR) domain. After stimulation with cognate ligands, the cytoplasmic domains recruit signal adaptor molecules, via TIR-TIR interactions, that relay signals to activate transcription factors NF-κB and/or IRF3. The four TIR adapter molecules are MyD88, Mal (also called TIRAP), TRIF and TRAM. There are two main signal pathways, medi-ated by MyD88 and TRIF, and individual TLRs use either one pathway or both path-ways. The lipopolysaccharide (LPS) receptor, the TLR4 complex, uses both MyD88- and TRIF-mediated pathways associated with the additional adapter molecules Mal and

TRAM, respectively (Fig. 1a). TLR2, which forms in heterodimers with TLR1 or TLR6 also activates the MyD88-mediated pathway with help from Mal. TLR5, TLR7 and TLR9 use only the MyD88-dependent pathway but do not require Mal. After stimulation with TLR ligands, the MyD88-mediated pathway leads to activation of NF-κB and mitogen-associated protein kinases, whereas the TRIF-mediated pathway results in acti-vation of NF-κB and IRF3. Subsequently, interferon-β (IFN-β) induced through IRF3 activates transcription factor STAT1 through IFN-αβ receptors (Fig. 1a) via an autocrine mechanism.

Although TLR-mediated signal pathways are indispensable for the eradication of microbes, their excessive and/or prolonged activation is itself harmful and sometimes causes pathogenesis. Downregulation of LPS signaling, called ‘LPS tolerance’, as well as cross-tolerance among various TLR ligands might have developed to prevent fatal responses to systemic infection. Although the molecular mechanisms underlying these types of tolerance remain mostly unknown, studiesof the molecular basis of TLR-mediatedsignal pathways now allow investigation of the mechanisms of self- and cross-tolerance.Most likely TLR tolerance is achieved

Takashi Kobayashi, Giichi Takaesu and Akihiko

Yoshimura are with the Division of Molecular

and Cellular Immunology, Medical Institute of

Bioregulation, Kyushu University, Fukuoka 812-

8582, Japan.

e-mail: yakihiko@bioreg.kyushu-u.ac.jp

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Figure 1 The function of SOCS-1 in inhibition of TLR4 signaling and of Mal in mediating p65 Ser 536 phosphorylation of NF-κB. (a) SOCS-1 expression is induced by LPS probably through the transcription factor Egr-1. SOCS-1 binds to Mal, p65 and Jak molecules and induces ubiquitination and degradation of these molecules. Mal is tyrosine-phosphorylated by Btk, which allows SOCS-1 to bind. Tyrosine-phosphorylated Mal induces phosphorylation of p65 Ser 536, which activates NF-κB transcriptional activity. The mechanism for phosphorylating p65 Ser 536 through Mal is not known. SOCS-1 also binds to and degrades p65 when Thr 254 is dephosphorylated. SOCS-1 also inhibits Jak activation by IFN-β induced by the TRIF-IRF3 pathway. (b) SOCS-1 mediates polyubiquitination (Ub) of Mal. SOCS-1 binds to tyrosine-phosphorylated Mal through the SH2 domain. The SOCS box of SOCS-1 recruits the elongin B–elongin C–Cullin–Rbx2 complex (Elongin B–C–Cul–Rbx2). Rbx2 binds to the E2 ubiquitin-transferase and induces polyubiquitination of Mal. Ubiquitinated Mal will be degraded by the 26S proteasome.

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124 VOLUME 7 NUMBER 2 FEBRUARY 2006 NATURE IMMUNOLOGY

through multiple mechanisms involving var-ious negative regulators such as A20, IRAK-M, MyD88s, RIP3. SIGIRR, ST2, Triad3A, IRF4, CYLD and FLN29 as well as SOCS-1 (refs. 3,4).

SOCS and cytokine-inducible Src homo-logy 2 (SH2) protein (CIS) comprise a family of intracellular proteins, several of which regulate the responses of immune cells to cytokines that use the Janus kinase (Jak)–STAT pathway4. All CIS-SOCS family members have a central SH2 domain and a C-terminal SOCS box and bind to target molecules through the SH2 domain. SOCS-1 binds to Jak molecules through the SH2 domain and inhibits Jak tyrosine kinase activity by a kinase inhibitory region in its N-terminal domain4. The SOCS box inter-acts with elongins B and C and recruits the ubiquitin-transferase system composed of Cullin-5 or Cullin-2, Rbx2 and E2 (ref. 5). CIS-SOCS family proteins, as well as other SOCS box–containing molecules, thus probably function as E3 ubiquitin ligases and mediate the degradation of proteins associated through their SH2 and/or N-terminal regions. The SOCS box of SOCS-1is necessary for degradation of activated Jak2, oncogenic TEL-Jak2 and adaptor molecules Vav and IRS1-2 as well as papillomavirus oncoprotein E7 (ref. 4).

The discovery of the SOCS proteins has defined an important mechanism for nega-tive regulation of the cytokine-Jak-STAT pathway; however, studies using gene-disrupted (knockout) mice have unexpectedlydemonstrated profound functions for SOCS-1 in innate immunity. SOCS-1 knockout mice die within 3 weeks of birth with severe inflammation, depending on IFN-γ signal-ing4. However, SOCS-1 also has important regulatory functions in IFN-γ-independent inflammatory diseases, as Socs1–/–Ifng–/– as well as Socs1–/–Stat1–/– mice are hypersensi-tive to LPS-induced death6,7.

SOCS-1 is strongly induced by TLR ligand stimulation in macrophages. Macrophages from Socs1–/– mice produce more proin-flammatory cytokines such as tumor necro-sis factor and interleukin 12 in response to the TLR4 ligand LPS or the TLR2 ligand Pam3Cys. One important mechanism for suppression of LPS-induced macrophage activation by SOCS-1 is inhibition of IFN-β signaling, which uses the Jak-STAT pathway (Fig.1a). However, direct effects of SOCS-1 on the NF-κB pathway have also been sug-gested.

It has been proposed7 that TLR regula-tion occurs via interaction between SOCS-1 and the kinase IRAK-1, which is degraded

through a phosphorylation-induced ubiquitin-proteasome pathway8. Direct binding of SOCS-1 to the p65 subunit of NF-κB induces proteasomal degradation of p65, which is one potential mechanism of NF-κB suppression by SOCS-1 (ref. 9; Fig.1a). The peptidyl-prolyl isomerase Pin1 binds and stabilizes the Thr 254–phosphorylated p65 NF-κB subunit, whereas unphosphory-lated p65 is rapidly degraded by binding to SOCS-1.

Here, Mansell et al. have identified a pre-viously unknown target of SOCS-1 in TLR signaling2. Mal was originally described as a MyD88 homolog because of its homology with the C-terminal TIR domain. MyD88 has an N-terminal death domain that homotypi-cally recruits IRAK1 and IRAK4, whereas the N-terminal region of Mal contains a putative ‘PEST’ domain (rich in Pro, Glu, Ser and Thr residues). Many short-lived proteins contain PEST domains that undergo phosphoryla-tion and polyubiquitination. Therefore, the authors have investigated the half-life of Mal in response to various TLR ligands. They find that Mal, but not MyD88, undergoes rapid degradation within 15–30 min of acti-vation by TLR2 or TLR4 stimulation but not by stimulation with TLR7 or TLR9, which do not use Mal. Coimmunoprecipitation experiments show interaction between Mal and SOCS-1, and overexpression of SOCS-1 induces ubiqutin-proteasome–dependent degradation of Mal. Notably, Mal degrada-tion is not seen in Socs1–/– bone marrow–derived macrophages.

Among eight conserved Lys residues in Mal, the Lys residues at positions 15 and 16 of Mal are shown to be targets of SOCS-1-induced ubiquitin incorporation. SOCS-1-dependent Mal polyubiquitination requires TLR stimulation and the SH2 domain of SOCS-1. Thus, Mal may be tyrosine-phosphorylated by TLR stimulation. Indeed, Mansell et al. demonstrate that the Bruton’s tyrosine kinase (Btk) inhibitor LFM-A13 inhibits LPS-induced Mal degradation. Furthermore, LPS-induced Mal degrada-tion does not occur in splenocytes derived from mice with X-linked immunodeficiency, which have an inactivating mutation of the gene encoding Btk. Therefore, Mansell et al. conclude that Mal undergoes tyrosine phos-phorylation by Btk, then binds to the SH2 domain of SOCS-1, leading to polyubiqui-tination by the SOCS box (Fig. 1b).

In addition, Mansell et al. show that SOCS-1 affects Mal-specific signaling. Other studies have suggested that Mal is necessary for TLR2- and TLR4-induced p65 transac-tivation through increased phosphorylation

of p65 Ser 536, which is dependent on Btk10. In the study reported here2, LPS induces transient phosphorylation of p65 in wild-type MEFs but not in Mal-deficient MEFs. Notably, TLR4- and TLR2-mediated p65 phosphorylation but not p38 mito-gen–associated protein kinase phosphory-lation is prolonged in SOCS-1-deficient macrophages. Thus, the authors conclude that SOCS-1 specifically downregulates the Mal–p65 Ser 536 phosphorylation pathway but not other Myd88–TRAF6–dependent pathways.

This study provides a very clear view of how SOCS-1 regulates TLR signaling. Furthermore, it emphasizes the importance of the Btk–Mal–p65 Ser 536 phosphorylation pathway in NF-κB transactivation activity. Degradation of Mal could be an important mechanism for TLR2 and TLR4 tolerance. However, several unresolved issues remain. Mal degradation by SOCS-1 may explain TLR2 and TLR4 tolerance; however, SOCS-1 is involved in tolerance of TLR9, which does not use Mal. Enhanced LPS-induced IκB phosphorylation has also been noted in SOCS-1-deficient cells5,6. Those discrepan-cies can probably be explained by the involve-ment of SOCS-1 in multiple mechanisms of TLR-tolerance, and defects in SOCS-1 may be compensated for by other mechanisms in different experimental conditions.

Finally, abundant SOCS-1 mRNA but lit-tle SOCS-1 protein is induced in response to LPS in wild-type macrophages. Similarly, SOCS-1 protein has not been detected in mouse and human inflammatory diseases even though there is often abundant SOCS-1 mRNA. One explanation for those find-ings is that SOCS-1 protein is very unstable and may be degraded very rapidly; alterna-tively, macrophages may have a mechanism that induces inactivation or degradation of SOCS-1. An important future study, there-fore, will be to identify possible suppressors of SOCS-1 in physiological and pathological conditions.

1. Akira, S. & Takeda, K. Nat. Rev. Immunol. 4, 499–511 (2004).

2. Mansell, A. et al. Nat. Immunol. 7, 148–155 (2006).

3. Han, H. & Ulevitch, R. Nat. Immunol. 6, 1198–1205 (2005).

4. Naka, T., Fujimoto, M., Tsutsui, H. & Yoshimura, A. Adv. Immunol. 87, 61–122 (2005).

5. Kamura, T. et al. Genes Dev. 18, 3055–3065 (2004).

6. Kinjyo, I. et al. Immunity 17, 583–591 (2002).7. Nakagawa, R. et al. Immunity 17, 677–687

(2002).8. Yamin, T.T. & Miller, D.K. J. Biol. Chem. 272,

21540–21547 (1997).9. Ryo, A. et al. Mol. Cell 12, 1413–1426 (2003).10. Doyle, S.L. Jefferies, C.A. & O’Neill, L.A. J. Biol.

Chem. 280, 23496–23501 (2005).

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