We have identified a unique mechanism by which NOD2-induced signaling and innate immune responses are restricted. Our results suggest that ubiquitylation of RIP2 is a physiological event in macrophages in response to MDP- and NOD2-induced signals, and that the ubiquitin-modifying enzyme A20 is essential for deubiquitylating RIP2 and negatively regulating these signals. These findings provide new insights into how innate immune responses are regulated by non-TLR ligands and signaling.
The recognition of microbial products through non-TLR host proteins such as NOD or CATERPILLER proteins has emerged as an important aspect of innate immunity. The requirement for A20 in restricting MDP- and NOD2-induced signals demonstrates that these signals, like TLR signals, are physiologically restricted by endogenous proteins. A recent study suggests that a short isoform of NOD2, NOD2-S, may also restrict NOD2-induced signals (Rosenstiel et al., 2006
). In addition, certain members of the CATERPILLER family appear to negatively regulate cellular activation of immune cells (Ting et al., 2006
). Hence, the proper regulation of these signaling pathways requires both positive and negative regulators.
Prior studies suggested that both NOD2 and RIP2 are essential for MDP-induced NFκB responses (Girardin et al., 2003b
; Kobayashi et al., 2002
; Chin et al., 2002
; Park et al., 2007
). MDP was thought to activate NFκB signaling by inducing NOD2 oligomerization, RIP2 recruitment, IKKγ recruitment, and IKKγ ubiquitylation (Inohara et al., 2000
; Abbott et al., 2004
). How RIP2 becomes biochemically activated was unknown. Our current studies suggest that RIP2 is physiologically ubiquitylated in primary cells after ligand stimulation. RIP2 may be ubiquity-lated with non-K48-linked polyubiquitin chains that help recruit downstream signaling molecules such as IKKγ. As IKKγ binds polyubiquitylated RIP1 during TNF receptor-induced NFκB signaling, it is possible that IKKγ binds to polyubiquitylated RIP2 in a similar fashion during MDP-induced NFκB signaling (Ea et al., 2006
; Wu et al., 2006
). Recruitment of IKKγ would then lead to IκBα phosphorylation and NFκB transcriptional activity. The identification of RIP2 ubiquitylation has also been shown by others during the review of this study (Hasegawa et al., 2008
; Yang et al., 2007
). Together, these studies provide an important new insight for understanding MDP- and NOD2-induced NFκB signaling.
We have shown that A20 is required for restricting MDP-induced cytokine production and NFκB signaling in primary innate immune cells. These findings demonstrate that NOD2-induced signals are physiologically restricted. Thus, it appears that NOD2 signaling, like TLR signaling, requires negative as well as positive regulation. In addition, we have elucidated a biochemical mechanism by which A20 may restrict NOD2 signals. A20 can directly deubiquitylate non-K48-linked chains on RIP2, and A20 is required for physiologically restricting endogenous RIP2 ubiquitylation after MDP stimulation. As RIP2 may become conjugated with K63-linked polyubiquitin chains after MDP stimulation, A20’s deubiquitylating activity may serve to “deactivate” RIP2. It is also possible that A20 may utilize its E3 ligase activity to remove ubiquitylated RIP2 from active signaling complexes. Both of these activities would serve to terminate NOD2-induced NFκB signaling. These RIP2-targeted activities could also explain A20’s apparent role in restricting NOD1-induced NFκB signaling, which also requires RIP2 (Girardin et al., 2003a
). Although A20 does not stably associate with RIP2 in cells (data not shown), it is not uncommon for ubiquitin-modifying enzymes to associate only transiently with their substrates. The combination of our findings that A20 can directly deubiquitylate RIP2 in cell-free assays and that endogenous RIP2 ubiquitylation is exaggerated in MDP stimulated Tnfaip3−/−
cells provides compelling evidence that RIP2 is the predominant physiological substrate for A20 in the MDP-/NOD2-signaling pathway.
Our current studies show that purified MDP elicit inflammatory responses in vivo independently of TLR signaling. We have used Tnfaip3−/−
mice to establish that MDP elicits proinflammatory responses in the absence of MyD88- and TRIF-dependent TLR signaling and to show that A20 restricts NOD2 signaling independently of its roles in inhibiting TLR signaling. The genetic dissection of TLR- and NOD2-induced signaling is important since NOD2 signals have been suggested to both enhance and restrict MyD88-dependent TLR signals (Netea et al., 2005
; Wolfert et al., 2002
; Watanabe et al., 2004
). Our finding thus provides new avenues for genetically dissecting the physiological roles of A20 in regulating NOD2 versus TLR signaling as well as for understanding the interactions between TLR and NOD2 signaling. Moreover, the ability to elicit MDP responses in intact mice should facilitate further studies interrogating the roles of NOD2-induced signaling in vivo. For example, dissecting the physiological roles of NOD2-induced signals in distinct cell types such as epithelial cells and macrophages can be addressed in chimeric mice or mice bearing lineage-specific gene deletions of NOD2-signaling proteins.
NOD2 signaling is likely to be important to immune homeosta-sis, because polymorphisms in the human NOD2 gene are associated with both Blau’s syndrome and Crohn’s disease in human patients (Miceli-Richard et al., 2001
; Hugot et al., 2001
; Ogura et al., 2001
). However, multiple questions remain in understanding this association. NOD2 polymorphisms have been proposed to exhibit both loss and gain of NOD2 function (Miceli-Richard et al., 2001
; Watanabe et al., 2004
; Maeda et al., 2005
), and it is unclear precisely how hypomorphic or hypermorphic mutations in NOD2 predispose patients to intestinal inflammation. Nevertheless, the clearest physiological function for NOD2 is mediating cellular responses to MDP by activating NFκB signaling. Hence, understanding the molecular mechanisms by which NOD2 activates NFκB signaling and how this process is regulated will likely provide critical insights into how perturbations in NOD2 function affect cellular and ultimately organismal responses. In this context, our discovery that A20 is directly responsible for restricting MDP-triggered signals provides both an important molecular insight into physiological NOD2 function, but also a potential therapeutic target for manipulating or correcting NOD2 functions. Greater understanding of how NOD2-mediated signals regulate immune responses and homeostasis is likely to be biomedically as well as immunologically important.
We have previously shown that A20 restricts TNF and TLR signals by regulating the ubiquitylation of RIP1 and TRAF6, respectively (Lee et al., 2000
; Boone et al., 2004
; Wertz et al., 2004
). Our current studies suggest that A20 is also physiologically required for restricting RIP2 ubiquitylation after MDP stimulation. These findings indicate that A20 enzymatically modifies diverse ubiquitylated target proteins, including RIP1, TRAF6, and RIP2. Hence, like other E3 ligases and deubiquitylating enzymes, A20 appears to act on multiple substrates.
A20’s physiological targets may be dictated partly by the structural nature of the substrate and partly by K63-linked ubiq-uitylation of the substrate. In this scenario, signaling intermediates that share certain structural features and that become decorated with K63-linked polyubiquitin chains in response to a specific ligand may become A20’s preferential enzymatic substrates. Thus, although TRAF6 may be a dominant K63 ubiquity-lated signaling intermediate during TLR-induced NFκB signaling (Deng et al., 2000
) and may also be involved in MDP-induced signals (Abbott et al., 2004
), our current data suggest that RIP2 is conjugated to K63-linked polyubiquitin chains after MDP stimulation and that ubiquitylated RIP2 is the major target for A20 during MDP-triggered signaling. The ability of A20 to recognize both signaling intermediates and their ubiquitin chains is consistent with recent structural data suggesting that A20 may bind ubiquitin chains as well as exert enzymatic activity toward ubiquitylated substrates (Penengo et al., 2006
; Lee et al., 2006
; Komander and Barford, 2008
Finally, A20’s multiple functions may play independent or additive (or perhaps synergistic) roles in restricting immune responses in vivo. These diverse functions suggest that even modest changes in A20 expression or A20 activity may lead to important changes in the intensity and character of immune responses. Such changes may underlie recent genetic suggestions that polymorphisms near or within the TNFAIP3
(A20) gene are associated with inflammatory diseases in human patients (Wellcome Trust Case Control Consortium, 2007
; Plenge et al., 2007
; Thomson et al., 2007
). Therefore, A20 may be an outstanding therapeutic target for inflammatory diseases.