Abstract
Toll–interleukin-1 receptor (TIR) domain–containing proteins are best known as critical players in vertebrate immune defense against pathogens. Four of the five members of this family are required for the activation of immune cells downstream of the engagement of Toll-like receptors (TLRs) by microbial molecules. Mice deficient in any one of these four molecules show greatly enhanced susceptibility to specific classes of pathogens. However, the physiological function of the fifth mammalian protein, sterile alpha and TIR motif–containing 1 [SARM1, also known as myeloid differentiation marker 88-5 (MyD88-5)], has remained elusive. Now, the study of the SARM1 reporter and SARM1-deficient mice has uncovered an unanticipated function of this molecule in the regulation of neuronal survival in response to metabolic stress. Together with pioneering observations on the functions of TIR-1, the worm ortholog of SARM1, and other reports on the role of TLRs in neuronal development and responses to injury in mammals, these exciting results suggest that further studies of SARM1-deficient animals may uncover unexpected similarities between the ways in which neurons and immune cells sense and respond to danger.
In evolutionary terms, SARM1 [sterile alpha and Toll–interleukin-1 receptor (TIR) motif–containing 1, also known as myeloid differentiation marker 88-5 (MyD88-5)] is the best-conserved TIR domain–containing protein, because it is the only one for which an ortholog has been identified in Caenorhabditis elegans (1–4). SARM1 can be further distinguished from the four other mammalian adaptor proteins [MyD88; MyD88–adaptor-like (MAL, also known as TIRAP); TIR domain–containing adaptor inducing interferon-β (TRIF); and TRIF-related adaptor molecule (TRAM, also known as TICAM2)] because it contains sterile alpha motifs (SAMs), which are potentially involved in protein-protein interactions (5). Although ectopic transfection of MyD88, MAL, TRIF, or TRAM leads to the activation of the nuclear factor κB (NF-κB) pathway in reporter cells, that of SARM1 does not (2, 6). Taken together, these findings suggest that SARM1 activates a different signaling pathway from that of the other MyD88 family members. Indeed, in C. elegans, TIR-1, the homolog of SARM1, signals through a p38 mitogen-activated protein kinase (MAPK) signal transduction cascade. TIR-1 interacts directly with the MAPK kinase kinase (MAP3K) neuronal symmetry family member-1 (NSY-1), which activates the downstream MAPK kinase (MAP2K) SEK-1 [stress-activated protein kinase (SAPK) and extracellular signal-regulated kinase (ERK) family member-1], which in turn activates p38 MAPK (2, 7). Part of this MAPK signaling pathway is conserved and contributes to innate immunity in mammals (8). Whether mammalian SARM1 activates the same MAPK cascade as does TIR-1 remains to be established.
TIR-1 Controls Epithelial Immune Responses and Neuronal Olfactory Receptor Expression
In C. elegans, TIR-1 induces the expression of genes that encode antimicrobial peptides in the epidermis in response to fungal infection, and it is required for resistance to intestinal bacterial infection (1, 2). There are five alternatively spliced isoforms of TIR-1: TIR-1a to TIR-1e (2). Using interfering RNA (RNAi) against each tir-1 isoform, Kurz et al. (9) showed that depletion of the short tir-1b isoform alone specifically increases the susceptibility of C. elegans to Pseudomonas aeruginosa infection. TIR-1 also controls the asymmetrical expression in neurons of a gene that encodes an olfactory receptor that is important for the discrimination of environmental cues (7). The role of TIR-1 in this function is cell-autonomous to neurons. Moreover, it does not require the expression of tir-1b, in contrast to its role in immunity (7). Thus, in the worm, TIR-1 is clearly endowed with at least two independent tissue-specific functions for sensing and responding to danger: (i) antimicrobial immune responses in the epidermis and intestine and (ii) control of olfactory receptor gene expression in neurons. In both instances, however, TIR-1 ultimately signals through similar MAPK pathways (2, 7).
SARM1 Downmodulates TLR3 and TLR4 Responses in Human Leukocytes
SARM1 is low in abundance in human peripheral blood mononuclear cells (PBMCs) under steady-state conditions but is more abundant after the activation of PBMCs by lipopolysaccharide (6). Treatment of human PBMCs with small inhibitory RNA (siRNA) specific for SARM1 enhances the production of the chemokine RANTES [regulated upon activation, normal T cell–expressed and –secreted, also known as CC chemokine ligand 5 (CCL5)] and the cytokine tumor necrosis factor–α (TNF-α) in response to TLR3 and TLR4 ligands, respectively (6). Transfection of HEK293-TLR3 cells with a truncated SARM1 that is essentially reduced to its TIR and SAM domains is sufficient to inhibit TLR3-dependent signaling. Thus, as in C. elegans, the corresponding natural splicing isoform of SARM1 may be endowed with potent immune regulatory functions in mammals. A direct interaction between SARM1 and TRIF occurs when they are overexpressed in the same cells (6). Ectopic transfection of reporter cells with SARM1 does not appear to alter either the MyD88-dependent responses of the cells to TLR3 or TLR4 stimulation or their responses to other TLRs. Thus, SARM1 specifically inhibits the TRIF-dependent responses to TLR3 or TLR4 activation in human PBMCs (6). The precise molecular mechanisms underlying this immunoregulatory function of SARM1, as well as the cell type in which it takes place, are unknown. However, new insights into these questions have been brought through the generation and study of SARM1-deficient mice and of reporter mice that express several copies of a bacterial artificial chromosome–derived transgene that allows the expression of enhanced green fluorescent protein under the control of regulatory sequences from Sarm1 (10).
Macrophages derived from the bone marrow of SARM1-deficient mice show no alterations, as compared to macrophages from wild-type mice, in their responses to the activation of TLR2, TLR3, TLR4, TLR7, or TLR9 (10). This is consistent with the lack of detection of SARM1 protein in myeloid cells, as assessed in SARM1 reporter mice (10). The study of these mice revealed that, within leukocytes, SARM1 was detected only in lymphocytes and more specifically in CD8+ T cells (Fig. 1). T cells are known to contain TLRs, but they respond only weakly to stimulation of these receptors (11, 12). Thus, it is possible that SARM1 functions in CD8+ T cells to prevent the triggering of cytolytic effector functions solely by the direct recognition of microbial products, while allowing it to substitute for other costimulation signals during antigen-specific engagement of the T cell receptor. It is also unknown whether SARM1 in CD8+ T cells signals through apoptosis-stimulated kinase 1 (ASK1, also known as MAP3K5), the mammalian ortholog of NSY-1. This MAPK cascade is also activated in myeloid cells upon stimulation of TLR4, in a MyD88-dependent manner, through the recruitment of ASK1 by TRAF6 (13). The availability of SARM1-deficient mice will be instrumental in testing these hypotheses. It will be especially interesting to study the impact of SARM1 deficiency under conditions in which T cell–dependent life-threatening inflammatory reactions can develop, such as during concomitant bacterial and viral infections (14).
Potential roles for SARM1 in mammalian immune responses to danger. Among human and mouse leukocytes, SARM1 is selectively found in lymphocytes, especially in CD8+ T cells. SARM1 may shape CD8+ T cell responses to the activation of TLR3 (not shown in the figure) or TLR4 by inhibiting the canonical TRIF-dependent pathway that leads to NF-κB activation and by promoting the use of an alternative ASK1-MAPK cascade, which leads to the recruitment of p38 and JNK. TIR domains in the relevant proteins are represented by circles; SAM domains in SARM1 are represented by hexagons. ZAP, T cell receptor zeta-chain–associated protein kinase; IRAK, interleukin-1 receptor–associated kinase; TRAF, tumor necrosis factor receptor–associated factor; MKK4, -6, and -7, mitogen-activated protein kinase kinases; NFAT, nuclear factor of activated T cells.
SARM1 Contributes to Neuronal Apoptosis in Response to Metabolic Stress
The generation and study of SARM1 reporter mice led to the discovery that SARM1 was preferentially expressed in neurons where it colocalized, in part, with mitochondria and c-Jun N-terminal kinase 3 (JNK3), a member of the MAPK family (10) (Fig. 2). Hippocampal neurons from JNK3-deficient mice are protected from death during ischemia (15), and SARM1-deficient mice exhibited a similar phenotype (10). Thus, an unanticipated function of SARM1 in the regulation of neuronal survival in response to metabolic stress in mammals was uncovered. Human neurons contain TLR3 and produce type I interferons upon infection with viruses (16). Injury or infection inhibits neurite outgrowth and induces neuronal death in a TLR3-dependent but an MyD88-independent manner (17, 18). Thus, it is possible that SARM1 modulates the responses of neurons to virus infections by preventing excessive and detrimental cytokine production and promoting apoptosis of infected cells so as to slow down local replication of virus, independently of systemic immune responses.
Modeling the role of SARM1 in the tuning of mammalian neuronal responses to danger. SARM1 is highly abundant in neurons, where it is associated with mitochondria and recruits JNK3 to induce apoptosis upon metabolic stress. The transduction cascade downstream of JNK3 in this pathway remains to be deciphered but may include translocation of the proapoptotic molecule Bax to mitochondria, which results in the triggering of apoptosis. In C. elegans, the MAP kinase NSY-1 binds to TIR-1, the ortholog of SARM1, and mediates downstream signaling for both immune function and neuronal development. In mammals, the ortholog of NSY-1, ASK1, is also involved in neuronal apoptosis upon metabolic stress. TIR domains in the relevant proteins are represented by circles; SAM domains in SARM1 are represented by hexagons. IRF, interferon regulatory factor.
SARM1: A Common Regulator of Neuronal and Immune Responses to Danger?
It has been hypothesized that SARM1 is implicated in genetic susceptibility both to infection with Mycobacterium tuberculosis and to the development of multiple sclerosis, an autoimmune disease of the central nervous system (CNS) (19). These hypotheses remain to be rigorously tested by family-based human linkage disequilibrium studies. Thanks to the generation of SARM1-deficient mice (10), a new era is opening for the understanding of the physiological role of this molecule in vivo. It will be possible to test, under physiologic conditions, the role of SARM1 in the regulation of systemic immune responses to infection and also in the local control of viral replication and inflammatory responses in neurons. Studying the role of SARM1 and its potentially associated MAPK signaling pathways in the susceptibility to blood-borne pathogens and in the development of infection-induced pathologies in the CNS may uncover unexpected and evolutionarily conserved similarities between the regulation of immune (Fig. 1) (20) and neuronal (Fig. 2) (21) responses to danger. In particular, it will be important to investigate whether, and how, ASK1 may contribute to the SARM1/JNK3 pathways that are responsible for neuronal apoptosis in mice. It also remains to be determined whether SARM1 and ASK1 tune mammalian neuronal responses to TLR3 stimulation, how the TLR3 pathway eventually connects to that of SARM1-dependent JNK3 recruitment to mitochondria for the induction of apoptosis, and how this affects the pathophysiology of viral infections that target the CNS.
