IL-1 receptor-associated kinase 4 is essential for IL-18-mediated NK and Th1 cell responses

IL-18 is an important cytokine for both innate and adaptive immunity. NK T cells and Th1 cells depend on IL-18 for their divergent functions. The IL-18R, IL-1R, and mammalian Toll-like receptors (TLRs) share homologous intracellular domains known as the TLR/IL-1R/plant R domain. Previously, we reported that IL-1R-associated kinase (IRAK)-4 plays a critical role in IL-1R and TLR signaling cascades and is essential for the innate immune response. Because TLR/IL-1R/plant R-containing receptors mediate signal transduction in a similar fashion, we investigated the role of IRAK-4 in IL-18R signaling. In this study, we show that IL-18-induced responses such as NK cell activity, Th1 IFN-gamma production, and Th1 cell proliferation are severely impaired in IRAK-4-deficient mice. IRAK-4(-/-) Th1 cells also do not exhibit NF-kappaB activation or IkappaB degradation in response to IL-18. Moreover, AP-1 activation which is triggered by c-Jun N-terminal kinase activation is also completely inhibited in IRAK-4(-/-) Th1 cells. These results suggest that IRAK-4 is an essential component of the IL-18 signaling cascade.     

TLR8-mediated NF-kappaB and JNK activation are TAK1-independent and MEKK3-dependent

TLR8-mediated NF-kappaB and IRF7 activation are abolished in human IRAK-deficient 293 cells and IRAK4-deficient fibroblast cells. Both wild-type and kinase-inactive mutants of IRAK and IRAK4, respectively, restored TLR8-mediated NF-kappaB and IRF7 activation in the IRAK- and IRAK4-deficient cells, indicating that the kinase activity of IRAK and IRAK4 is probably redundant for TLR8-mediated signaling. We recently found that TLR8 mediates a unique NF-kappaB activation pathway in human 293 cells and mouse embryonic fibroblasts, accompanied only by IkappaBalpha phosphorylation and not IkappaBalpha degradation, whereas interleukin (IL)-1 stimulation causes both IkappaBalpha phosphorylation and degradation. The intermediate signaling events mediated by IL-1 (including IRAK modifications and degradation and TAK1 activation) were not detected in cells stimulated by TLR8 ligands. TLR8 ligands trigger similar levels of IkappaBalpha phosphorylation and NF-kappaB and JNK activation in TAK1(-/-) mouse embryo fibroblasts (MEFs) as compared with wild-type MEFs, whereas lack of TAK1 results in reduced IL-1-mediated NF-kappaB activation and abolished IL-1-induced JNK activation. The above results indicate that although TLR8-mediated NF-kappaB and JNK activation are IRAK-dependent, they do not require IRAK modification and are TAK1-independent. On the other hand, TLR8-mediated IkappaBalpha phosphorylation, NF-kappaB, and JNK activation are completely abolished in MEKK3(-/-) MEFs, whereas IL-1-mediated signaling was only moderately reduced in these deficient MEFs as compared with wild-type cells. The differences between IL-1R- and TLR8-mediated NF-kappaB activation are also reflected at the level of IkappaB kinase (IKK) complex. TLR8 ligands induced IKKgamma phosphorylation, whereas IKKalpha/beta phosphorylation and IKKgamma ubiquitination that can be induced by IL-1 were not detected in cells treated with TLR8 ligands. We postulate that TLR8-mediated MEKK3-dependent IKKgamma phosphorylation might play an important role in the activation of IKK complex, leading to IkappaBalpha phosphorylation.     

The IRAK-1-BCL10-MALT1-TRAF6-TAK1 cascade mediates signaling to NF-kappaB from Toll-like receptor 4

Our previous studies have revealed that the signaling protein BCL10 plays a major role in adaptive immunity by mediating NF-kappaB activation in the LPS/TLR4 pathway. In this study, we show that IRAK-1 acts as the essential upstream adaptor that recruits BCL10 to the TLR4 signaling complex and mediates signaling to NF-kappaB through the BCL10-MALT1-TRAF6-TAK1 cascade. Following dissociation from IRAK-1, BCL10 is translocated into the cytosol along with TRAF6 and TAK1, in a process bridged by a direct BCL10-Pellino2 interaction. RNA interference against MALT1 markedly reduced the level of NF-kappaB activation stimulated by lipopolysaccharide (LPS) in macrophages, which suggests that MALT1 plays a major role in the LPS/TLR4 pathway. MALT1 interacted with BCL10 and TRAF6 to facilitate TRAF6 self-ubiquitination in the cytosol, which was strictly dependent on the dissociation of BCL10 from IRAK-1. We show that BCL10 oligomerization is a prerequisite for BCL10 function in LPS signaling to NF-kappaB and that IRAK-1 dimerization is an important event in this process.     

Lipopolysaccharide improves cardiomyocyte survival and function after serum deprivation

Toll-like receptor-4 (TLR4) and its signaling molecule interleukin-1 receptor-associated kinase (IRAK-1) play an important role in host defense and tissue inflammation. Intriguingly, systemic administration of lipopolysaccharide (LPS), the agonist for TLR4, confers a cardio-protective effect against ischemic injury. However, the mechanisms leading to the cardiac protection remain largely unknown. The present study was designed to investigate the role of TLR4 activation by LPS in protecting cardiomyocytes (CM) against apoptosis in an in vitro model of ischemia and to explore the downstream mechanisms leading to the protective effect. Incubation with LPS led to activation of IRAK-1 and protected CMs against serum deprivation (SD)-induced apoptosis as demonstrated by DNA laddering, histone-DNA fragment enzyme-linked immunosorbent assay, and activation of caspase-3. Phosphatidylinositol 3-kinase/Akt, extracellular signal-regulated kinase 1/2, and IkappaB kinase beta appear to contribute to the anti-apoptotic effect of LPS since the specific inhibitors, wortmannin, PD98059, and dominant negative IKKbeta transgene expression reversed the LPS effect. To assess whether LPS improves CM function, we examined intracellular Ca(2+) transients and cell shortening in single adult rat CMs. SD for 6 h dramatically inhibited Ca(2+) transients and CM contractility. LPS at 500 ng/ml significantly improved the [Ca(2+)](i) transients and enhanced contractility in control CMs as well as in CMs subjected to SD. Importantly, transient ischemia led to rapid activation of IRAK-1 in cultured CMs and in adult rat myocardium. Adenovirus-mediated transgene expression of IRAK-1 but not its kinase-deficient mutant IRAK-1(K239S) protected CMs against SD-induced apoptosis. Taken together, these data suggest an important role of TLR4 signaling via IRAK-1 in protecting against SD-induced apoptosis.     

Tobacco smoking inhibits expression of proinflammatory cytokines and activation of IL-1R-associated kinase, p38, and NF-kappaB in alveolar macrophages stimulated with TLR2 and TLR4 agonists

Tobacco smoking has been associated with impaired pulmonary functions and increased incidence of infections; however, mechanisms that underlie these phenomena are poorly understood. In this study, we examined whether smokers' alveolar macrophages (AM) exhibit impaired sensing of bacterial components via TLR2 and TLR4 and determined the effect of smoking on expression levels of TLR2, TLR4 and coreceptors, and activation of signaling intermediates. Smokers' AMs exhibited reduced gene expression and secretion of proinflammatory cytokines (TNF-alpha, IL-1beta, IL-6) and chemokines (RANTES and IL-8) upon stimulation with TLR2 and TLR4 agonists, S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH trihydrochloride (Pam(3)Cys), and LPS, whereas expression of anti-inflammatory cytokines (IL-10 and IL-1 receptor antagonist) was not affected. TLR3 activation with polyinosinic-polycytidylic acid led to comparable or even higher cytokine responses in smokers' AMs, indicating that smoking-induced suppression does not affect all TLRs. Comparable expression of cytokines and chemokines was detected in PBMC and purified monocytes obtained from smokers and nonsmokers, demonstrating that the suppressive effect of smoking is restricted to the lung. TLR2/4-inducible IL-1R-associated kinase-1 (IRAK-1) and p38 phosphorylation and NF-kappaB activation was suppressed in smokers' AMs, whereas TLR2, TLR4, CD14, MD-2 mRNA levels, and TLR4 protein expression were not altered. These data suggest that changes in expression and/or activities of signaling intermediates at the postreceptor level account for smoking-induced immunosuppression. Thus, exposure of AMs to tobacco smoke induces a hyporesponsive state similar to endotoxin tolerance as manifested by inhibited TLR2/4-induced expression of proinflammatory cytokines, chemokines, and impaired activation of IRAK-1, p38, and NF-kappaB, resulting in suppressed expression of proinflammatory mediators.     

Tollip regulates proinflammatory responses to interleukin-1 and lipopolysaccharide

Activation of interleukin-1 (IL-1) receptor (IL-1R), Toll-like receptor 2 (TLR2), and TLR4 triggers NF-kappaB and mitogen-activated protein kinase (MAPK)-dependent signaling, thereby initiating immune responses. Tollip has been implicated as a negative regulator of NF-kappaB signaling triggered by these receptors in in vitro studies. Here, deficient mice were used to determine the physiological contribution of Tollip to immunity. NF-kappaB, as well as MAPK, signaling appeared normal in Tollip-deficient cells stimulated with IL-1beta or the TLR4 ligand lipopolysaccharide (LPS). Similarly, IL-1beta- and TLR-driven activation of dendritic cells and lymphocytes was indistinguishable from wild-type cells. In contrast, the production of the proinflammatory cytokines, IL-6 and tumor necrosis factor alpha was significantly reduced after IL-1beta and LPS treatment at low doses but not at lethal doses of LPS. Tollip therefore controls the magnitude of inflammatory cytokine production in response to IL-1beta and LPS.     

The non-provitamin A carotenoid, lutein, inhibits NF-kappaB-dependent gene expression through redox-based regulation of the phosphatidylinositol 3-kinase/PTEN/Akt and NF-kappaB-inducing kinase pathways: role of H(2)O(2) in NF-kappaB activation

Reactive oxygen species (ROS) have been implicated in the regulation of NF-kappaB activation, which plays an important role in inflammation and cell survival. However, the molecular mechanisms of ROS in NF-kappaB activation remain poorly defined. We found that the non-provitamin A carotenoid, lutein, decreased intracellular H(2)O(2) accumulation by scavenging superoxide and H(2)O(2) and the NF-kappaB-regulated inflammatory genes, iNOS, TNF-alpha, IL-1beta, and cyclooxygenase-2, in lipopolysaccharide (LPS)-stimulated macrophages. Lutein inhibited LPS-induced NF-kappaB activation, which highly correlated with its inhibitory effect on LPS-induced IkappaB kinase (IKK) activation, IkappaB degradation, nuclear translocation of NF-kappaB, and binding of NF-kappaB to the kappaB motif of the iNOS promoter. This compound inhibited LPS- and H(2)O(2)-induced increases in phosphatidylinositol 3-kinase (PI3K) activity, PTEN inactivation, NF-kappaB-inducing kinase (NIK), and Akt phosphorylation, which are all upstream of IKK activation, but did not affect the interaction between Toll-like receptor 4 and MyD88 and the activation of mitogen-activated protein kinases. The NADPH oxidase inhibitor apocynin and gp91(phox) deletion reduced the LPS-induced NF-kappaB signaling pathway as lutein did. Moreover, lutein treatment and gp91(phox) deletion decreased the expressional levels of the inflammatory genes in vivo and protected mice from LPS-induced lethality. Our data suggest that H(2)O(2) modulates IKK-dependent NF-kappaB activation by promoting the redox-sensitive activation of the PI3K/PTEN/Akt and NIK/IKK pathways. These findings further provide new insights into the pathophysiological role of intracellular H(2)O(2) in the NF-kappaB signal pathway and inflammatory process.     

TLR2- and TLR4-mediated signals determine attenuation or augmentation of inflammation by acute alcohol in monocytes

Most pathogens express ligands for multiple TLRs that share common downstream signaling. In this study, we investigated the effects of acute alcohol on inflammatory pathways induced by TLR2 or TLR4 ligands and their combination. In human monocytes, alcohol attenuated TLR4- but not TLR2-induced TNF-alpha protein and mRNA levels and NF-kappaB activation. In contrast, acute alcohol augmented TNF-alpha production when both TLR2 and TLR4 ligands were present. IL-1R-associated kinase (IRAK)-1 activity was reduced by alcohol in TLR4, but it was augmented in TLR2- plus TLR4-stimulated cells. IRAK-monocyte, an inhibitor of IRAK-1, was induced in TLR4, but it was reduced in TLR2- plus TLR4-stimulated monocytes by alcohol. This was supported by decreased IRAK-1:TRAF6 association in TLR4 induced but sustained presence of IRAK-1:TRAF6 complexes in TLR2- plus TLR4-stimulated monocytes after alcohol treatment. Phosphorylation of MAPKs such as ERK1/2 was selectively inhibited by acute alcohol in TLR4-stimulated cells. In contrast, JNK phosphorylation as well as AP-1 nuclear binding were augmented by acute alcohol in the presence of combined TLR4 and TLR2 stimulation. Consistent with this result, the JNK inhibitor prevented alcohol-induced augmentation of TNF-alpha production. These results suggest that acute alcohol attenuates TLR4-induced inflammation via inhibition of IRAK-1 and ERK1/2 kinases and increases in IRAK-monocyte levels in monocytes. Conversely, in the presence of TLR2 and TLR4 ligands, acute alcohol augments inflammatory responses via IRAK-1 activation and JNK phosphorylation. Thus, the complexity of TLR-mediated signals may determine attenuation or augmentation of inflammatory responses by acute alcohol.     

MyD88-induced downregulation of IRAK-4 and its structural requirements

IRAK-4 plays an essential role in Toll-like receptor (TLR)/IL-1 receptor signaling. However, its signaling and regulation mechanisms have remained elusive. We have reported previously that stimulation of TLR2, TLR4 or TLR9, but not TLR3, leads to downregulation of IRAK-4 protein. Here, we show that expression of MyD88 leads to downregulation of endogenous as well as exogenously expressed IRAK-4 protein in HEK293 cells. Expression of TRIF did not cause IRAK-4 downregulation although it induced NF-kappaB activation. Expression of either a deletion mutant of MyD88 lacking its death domain or MyD88s, neither of which induced NF-kappaB activation, did not lead to IRAK-4 downregulation. MyD88-induced downregulation was observed in an IRAK-4 mutant lacking the kinase domain, but not in another mutant lacking the death domain. These results demonstrate that downregulation of IRAK-4 requires activation of the MyD88-dependent pathway and that the death domains of both MyD88 and IRAK-4 are important for this downregulation.     

Functional diversity and regulation of different interleukin-1 receptor-associated kinase (IRAK) family members

Interleukin-1 receptor-associated kinase (IRAK) was first described as a signal transducer for the proinflammatory cytokine interleukin-1 (IL-1) and was later implicated in signal transduction of other members of the Toll-like receptor (TLR)/IL-1 receptor (IL-1R) family. In the meantime, four different IRAK-like molecules have been identified: two active kinases, IRAK-1 and IRAK-4, and two inactive kinases, IRAK-2 and IRAK-M. All IRAKs mediate activation of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) pathways. Although earlier observations suggested that IRAKs have redundant functions, this hypothesis is now challenged by knockout studies. Furthermore, recent data imply a role for IRAK-1 in tumor necrosis factor receptor (TNFR) superfamily-induced signaling pathways as well. The scope of this review is to highlight the specific role of different IRAKs and to discuss several mechanisms that contribute to their activation and regulation.     

Lys63-linked polyubiquitination of IRAK-1 is required for interleukin-1 receptor- and toll-like receptor-mediated NF-kappaB activation

Stimulation through the interleukin-1 receptor (IL-1R) and some Toll-like receptors (TLRs) induces ubiquitination of TRAF6 and IRAK-1, signaling components required for NF-kappaB and mitogen-activated protein kinase activation. Here we show that although TRAF6 and IRAK-1 acquired Lys63 (K63)-linked polyubiquitin chains upon IL-1 stimulation, only ubiquitinated IRAK-1 bound NEMO, the regulatory subunit of IkappaB kinase (IKK). The sites of IRAK-1 ubiquitination were mapped to Lys134 and Lys180, and arginine substitution of these residues impaired IL-1R/TLR-mediated IRAK-1 ubiquitination, NEMO binding, and NF-kappaB activation. K63-linked ubiquitination of IRAK-1 required enzymatically active TRAF6, indicating that it is the physiologically relevant E3. Thus, K63-linked polyubiquitination of proximal signaling proteins is a common mechanism used by diverse innate immune receptors for recruiting IKK and activating NF-kappaB.