In summary, Tollip regulates the magnitude and kinetics of IL-6 and TNF-α production upon stimulation with IL-1β and low doses or physiological doses of LPS. We did not observe any effect of Tollip deficiency in response to lethal doses of LPS, suggesting that Tollip acts in “fine tuning” or coordinating optimal signaling through IL-1RI and TLR4. In this way, Tollip resembles a scaffolding protein that is required for optimal responses. As such, high concentrations of Tollip may titrate out critical signaling proteins in IL-1R and TLR4 pathways, thereby blocking signaling. This would accommodate the apparently conflicting observations (i.e., repressor versus activator) and predicts that the cellular concentration of Tollip may be crucial for its capacity to regulate signaling. Further it could explain why overexpression or upregulation of Tollip in intestinal epithelial cells correlates with a hyporesponsive state, after a second challenge with a TLR ligand (12
Quantitative PCR suggests that Tollip deficiency affects IL-6 and TNF-α production at the transcriptional level. NF-κB, JNK, and p38 signaling cascades are crucial both for the transcription and translation of IL-6 and TNF-α (1
). However, surprisingly, activation of NF-κB, JNK, and p38 signaling appeared to be normal in Tollip−/−
cells. This could easily be explained if Tollip deficiency resulted in subtle defects in the activation of the membrane proximal signaling complex (Tollip is rapidly and transiently recruited to this complex) and thereby ultimately in downstream activation of these signaling cascades. Interestingly, IRAK-1-deficient macrophages have a similar phenotype in that TNF-α secretion is only impaired upon low doses of LPS (16
). The Tollip-IRAK-1 interaction may thus be important for optimal responses to these stimuli.
Although we cannot exclude that Tollip does not contribute entirely or partly to the initial activation of NF-κB, JNK, and p38 signaling, it is also possible that Tollip modulates signaling via alternative means. Several recent observations suggest that Tollip may participate in the recognition and trafficking of ubiquitinated proteins within endocytic/biosynthetic pathways. Tollip partially localizes to late endosomes or lysosomes (B. Brissoni, L. Agostini, M. Kropf, H. Everett, N. Aebi, V. Swoboda, S. Janssens, E. Meylan, F. Martinen, M. Felderbaum-Corti, H. Hirling, J. Gruenberg, J. Tschopp, and K. Burns, submitted for publication), binds Tom1 and Tom1L1 (molecules likely to have a role in endocytic trafficking of proteins to the lysosome) (6
), interacts with phosphatidylinositol-3-phosphate [PtdIns(3)P] and phosphatidylinositol-3,4,5-phosphate (phospholipids abundant on endocytic vesicles) (9
), and binds ubiquitin, an internalization and sorting tag on cargo proteins of the endocytic or biosynthetic pathways (14
Endosomes, via associated proteins, can function as platforms for the formation and activation of specific signaling complexes (15
). Interestingly, overexpression of a Tollip mutant, which does not bind PtdIns(3)P and is presumably not endosomally localized, does not block LPS signaling (9
). Several TLRs are known to be endosomally localized (reviewed in reference 1
). We have recently shown that IL-1RI traffics via the late endosome to lysosomes for degradation (Brissoni et al., submitted). It thus conceivable that Tollip acts in the recruitment of a signalosome at the late endosome that is required for optimal responses to IL-1β, LPS, and other TIR ligands that signal via MyD88-dependent pathways. Future studies are now required to address the precise molecular mechanisms by which Tollip modulates cytokine production and/or secretion. Given that the physiological concentrations of IL-1β or LPS at the sites of infection/inflammation most likely do not exceed the low nanomolar range, Tollip is undoubtedly an important mediator of these responses.