Our studies have shed new light on the importance and regulation of homeostatic TLR signals. Recent studies have indicated that homeostatic TLR signals may be chronically triggered by PAMPs from microbes at mucosal surfaces such as the intestinal epithelium. MyD88-dependent TLR signals may be beneficial for intestinal homeostasis under normal circumstances (2
). The appreciation of such homeostatic TLR signals has raised several questions, including whether homeostatic TLR signals are potentially inflammatory. Our finding that unperturbed A20−/−
mice are dramatically less inflamed and survive far longer than A20−/−
mice suggests that homeostatic TLR signals can in fact be profoundly inflammatory and rapidly lethal. We have also elucidated a new function for A20 in regulating TRIF-dependent NF-κB signals but not IRF3 signals.
The idea that homeostatic MyD88 signals can cause spontaneous inflammation is broadly consistent with recent findings that constitutive MyD88 signals drive colitis and eventual mortality in IL-10−/−
). However, although IL-10−/−
mice develop progressive colitis during the first 4–6 mo of life and eventually die from this disease, the majority of A20−/−
mice die within the first 3–6 wk of life from widespread systemic inflammation and cachexia (3
). Thus, our findings indicate that homeostatic MyD88 signals can drive severe perinatal systemic inflammation in addition to intestinal inflammation. Moreover, although IL-10 is particularly important for restricting intestinal inflammation, A20 is critical for restricting systemic inflammation.
Although A20 prevents widespread inflammation, a major source of constitutive, proinflammatory TLR signals in A20−/−
mice may nevertheless be intestinal commensal bacteria. We have found that the removal of many of these bacteria with antibiotics reduced systemic inflammation in chimeric mice bearing A20−/−
hematopoietic cells. Although previous studies have shown that intestinal bacteria can drive intestinal inflammation in several models (33
), our current results link these bacteria and MyD88-dependent signals to A20-mediated regulation of innate immune cells and to systemic immune activation.
The different physiological roles of IL-10 and A20 in restricting MyD88-dependent signals is likely related to the distinct biochemical functions of these immunoregulatory proteins. IL-10 triggers STAT3-mediated transcriptional inhibition of proinflammatory genes, whereas our data indicate that A20 restricts MyD88-dependent TLR signals by regulating TRAF6 ubiquitylation. Thus, the critical physiological role that A20 plays in preventing constitutive homeostatic MyD88 signals from becoming inflammatory is likely caused by A20's biochemical role in directly regulating MyD88-dependent signals.
Proteins such as A20, IRAK-M, ST2, and SIGIRR that restrict TLR signals could regulate the duration and/or intensity of TLR signals and modulate the cellular outcome of TLR signaling, thereby helping to determine whether TLR signals lead to homeostatic or inflammatory responses (14
). Notably, although unperturbed mice lacking IRAK-M, ST2, and SIGIRR exhibit relatively modest inflammation and survive for longer than 9–12 mo of age, A20−/−
mice typically die within the first few weeks of life (13
). Although SOCS-1−/−
mice exhibit perinatal lethality similar to A20−/−
mice are rescued by the absence of IFN-γ signaling in SOCS-1−/−
double-mutant mice (19
). Thus, among proteins known to directly restrict TLR signaling, A20 appears to play a more critical role in regulating homeostatic TLR-driven inflammation in vivo. There are several potential (nonmutually exclusive) reasons why A20 has a greater physiological impact on homeostatic TLR signals. First, A20 enzymatically regulates ubiquitylation of signaling proteins, thereby regulating both the activity and stability of such targets. Thus, A20 may not only deactivate signaling proteins but may also prevent further reactivation of these proteins. Second, A20 may regulate TLR signaling downstream of other known TLR regulating proteins, thus exerting a more definitive impact on the transcriptional outcome of TLR signals. Third, A20's physiological roles in restricting MyD88-dependent TLR signals may be amplified by A20's roles in regulating other non-TLR signals, e.g., TNF signals. However, marked survival differences between A20−/−
mice and A20−/−
mice suggest that restricting MyD88 signals may be the dominant physiological function of A20 during homeostatic conditions. Finally, it is possible that A20 may also regulate IL-1 or IL-18 signals, which also use MyD88 (9
). Although we have not yet evaluated the contribution of aberrant IL-1 or IL-18 signaling to spontaneous inflammation in A20−/−
mice, IL-1 receptor antagonist–deficient mice do not develop spontaneous inflammation within the first 4 mo of life (34
). Thus, restricting basal IL-1 signals may not be critical for immune homeostasis in unperturbed mice. Collectively, A20 appears to be one of the most critical proteins for restricting homeostatic TLR signals in vivo.
Constitutive or basal MyD88-dependent signals could derive from microbial or host ligands. Our experiments with antibiotic-mediated depletion of commensal bacteria reinforce the notion that intestinal bacteria are a major source of MyD88-dependent signals triggering inflammation in unperturbed A20−/− mice. Collectively with previous findings that constitutive MyD88-dependent signals from luminal bacteria are required for maintaining intestinal health, the current results suggest that a moderate but not excessive magnitude of MyD88 signaling in the intestine is essential for intestinal homeostasis.
In the intestine, homeostatic TLR signals might also be distinguished from inflammatory TLR signals by microenvironment- and cell type–specific TLR signals. For example, apical TLR signals on epithelial cells might preferentially induce homeostatic signals, whereas TLR signals on dendritic cells trigger inflammation. Although we have not yet directly addressed the roles of A20 in regulating epithelial cell function, our discoveries that radiation chimera bearing A20−/−
hematopoietic cells exhibit less spontaneous inflammation than chimeric mice bearing A20−/−
cells indicates that A20-mediated restriction of homeostatic TLR signals specifically in hematopoietic cells is important for immune homeostasis. Although it is possible that TLRs on epithelial cells may also be constitutively engaged on mucosal surfaces, our results using radiation chimera indicate that engagement of TLRs on hematopoietic cells occurs in unperturbed mice and must be properly restricted to maintain immune homeostasis. Indeed, one example of how TLRs on hematopoietic cells might tonically bind PAMPs from luminal microbes is via the extrusion of dendrites by mucosal dendritic cells through epithelial tight junctions into the intestinal lumen (35
). Future studies using gene-targeted mice bearing lineage-specific deletions of A20 should facilitate studies of A20's specific role in dendritic cells and other cell types.
The generation and characterization of A20−/−
mice also facilitates further studies of A20's roles in regulating additional signaling pathways. Milder but progressive spontaneous inflammation in older double-mutant A20−/−
mice and triple-mutant A20−/−
mice suggests that A20 regulates additional immune signals besides MyD88-dependent TLR and TNF signals. TRIF-dependent TLR signals may be one of these types of signals, and we have used A20−/−
mice to discover that A20 is also required for restricting TRIF-dependent TLR responses. We have also used A20−/−
cells to decipher the biochemical mechanism by which A20 regulates TLR signaling. Specifically, both A20−/−
mice and BMDMs derived from these mice produce more type I IFNs than A20+/−
mice and BMDMs after LPS stimulation. Previous work demonstrated that TRIF associates with TANK-binding kinase 1, TRAF3, and TRAF6 during the activation of NF-κB and IRF3 signaling (10
). Both NF-κB and IRF3 are required for TRIF-dependent IFN transcription. Receptor-interacting protein (RIP) 1 ubiquitylation, possibly mediated by TRAF6, is also involved in TRIF-dependent NF-κB signaling (39
). Our data suggest that A20 restricts TRIF-dependent NF-κB signaling and not IRF3 phosphorylation. Hence, A20's nonredundant function in regulating TRIF-dependent signaling appears to be restricted to NF-κB signaling. A20 can inhibit TLR3 signals and can bind TRAF6, Nef-associated kinase/TANK-binding kinase 1, and IκB kinase ε (41
). Thus, it is likely that A20 restricts TRIF-dependent signaling by directly regulating proteins in this signaling complex. Our data do not support the previous finding that heterologous A20 expression inhibits IRF3 dimerization, and these differences could be caused by the distinct experimental systems used (43
). Collectively, our data suggest that A20 restricts TRIF-dependent IFN responses by limiting TRIF-dependent NF-κB signaling.
Previous work has shown that conjugation of K63-linked ubiquitin chains to TRAF6 is an essential step in TLR-induced NF-κB activation (30
). Our earlier studies also showed that A20 is a ubiquitin-modifying enzyme that can both deconjugate ubiquitin chains and ligate ubiquitin onto target proteins such as RIP and TRAF6 (18
). Our current data directly demonstrate that A20 is essential for restricting endogenous TRAF6 ubiquitylation after TLR stimulation, further reinforcing this biochemical mechanism. As TRAF6 binds to both MyD88 and TRIF after TLR stimulation, TRAF6 likely activates NF-κB signals in both of these pathways. RIP1 is also involved in TRIF-dependent TLR signaling, and A20 restricts RIP1 ubiquitylation in response to TNF (39
). Thus, it is possible that A20 also restricts TRIF-dependent NF-κB signaling by restricting RIP1 ubiquitylation. However, RIP1 is not involved in MyD88-dependent NF-κB signaling, so it is unlikely that A20's effects on RIP1 can explain all of A20's roles in TLR signaling. In contrast, A20's restriction of TRAF6 ubiquitylation may be a common mechanism by which A20 restricts both MyD88- and TRIF-dependent NF-κB signaling.
TRAF3 has recently been implicated in TRIF-dependent signaling (44
). TRAF3 appears to be more important for IRF signals, whereas TRAF6 is more important for NF-κB signaling. We and others have not been able to obtain evidence that A20 binds to or modifies TRAF3 (unpublished data), and our finding that IRF3 phosphorylation occurs normally in the absence of A20 suggests that A20 may preferentially regulate TRAF6- but not TRAF3-dependent signaling. These two proteins (along with TRAF2, TRAF4, and TRAF5) share structurally homologies, including RING and Zn finger motifs that may mediate E3 ligase activity. Selective regulation of TRAF6- but not TRAF3-dependent signaling by A20 might be explained if A20 preferentially binds TRAF6 rather than TRAF3. Alternatively, although TRAF6 is modified with K63-linked polyubiquitin chains during TLR-initiated signal transduction, it is currently unclear if TRAF3 is similarly ubiquitylated. Thus, A20 may bind K63 polyubiquitylated TRAF6, whereas TRAF3 may not undergo this type of ubiquitylation modification. In either scenario, our findings indicate that A20 is a selective regulator of TRAF6- and not TRAF3-dependent signal transduction and provides new insights into how NF-κB and IRF signaling may be discriminated and differentially regulated.
Our finding that A20−/−
mice virtually all survive to adulthood with modest amounts of inflammation contrasts sharply with our previous findings that A20−/−
, and A20−/−
mice all spontaneously develop severe inflammation, cachexia. and premature death (18
). This result suggests that dysregulated homeostatic TLR signals stimulate downstream innate and adaptive immune signals in the absence of A20 (e.g., TNF, IL-12, IL-6, and chemokine production, T cell activation, B cell activation, etc). Although A20 may play multiple important roles in regulating immune cell signals, its role in restricting homeostatic TLR signals may be physiologically critical because TLR signals are situated at the apex of immune responses. Moreover, our results showing that antibiotics ameliorate inflammation in A20−/−
chimeric mice suggest that commensal intestinal flora trigger these homeostatic MyD88-dependent TLR signals. Finally, we have established a biochemical mechanism by which A20 restricts both MyD88- and TRIF-dependent TLR signals. In summary, our findings demonstrate the profoundly proinflammatory nature of homeostatic MyD88-dependent signals and identify A20 as a critical protein that prevents homeostatic signals from becoming inflammatory. Further studies elucidating how A20 restricts different types of TLR signals should yield important insights into the complex interplay between commensal microbes and host immune cells, as well as the biochemical mechanisms by which signals triggered by microbial molecules are regulated and interpreted.