IRAK-4 kinase activity is required for IRAK-4-dependent innate and adaptive immune responses

Interleukin-1 receptor-associated kinase (IRAK)-4 is a serine-threonine kinase that plays an important role in innate and adaptive immune responses. While the requirement of IRAK-4 kinase activity has been studied in the context of IL-1R signaling, it is not clear whether IRAK-4 requires its kinase function for all of its roles in the immune system. IRAK-4 kinase-dead knock-in (IRAK-4KD/KD) mice were generated to further elucidate whether IRAK-4 kinase activity is required for IRAK-4 to induce cytokine production. IRAK-4KD/KD mice were impaired in their ability to produce cytokines in response to in vivo challenge with lipopolysaccharide (LPS), a potent TLR4 ligand. Cytokine production was also reduced in macrophages and dendritic cells from IRAK-4KD/KD mice in response to LPS and other TLR ligands. In addition, adaptive immune responses were impaired in IRAK-4KD/KD mice. Although in vitro T cell proliferation in response to TCR activation was unaffected in IRAK-4-deficient mice, in vivo T cell responses to lymphocytic choriomeningitits virus infection were significantly impaired in IRAK-4-knockout mice or mice expressing the kinase-dead mutant of IRAK-4. Collectively, these results indicate that IRAK-4 kinase activity is required for IRAK-4-dependent signaling in innate and adaptive immunity.     

Interleukin-1 receptor-associated kinase 4 is essential for initial host control of Brucella abortus infection

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. Recent studies have revealed that Toll-like receptor (TLR)-initiated immune response to Brucella spp. depends on myeloid differentiation factor 88 (MyD88) signaling. Therefore, we decided to study the role of the interleukin-1 receptor-associated kinase 4 (IRAK-4) in host innate immune response against B. abortus. After Brucella infection, it was shown that the number of CFU in IRAK-4(-/-) mice was high compared to that in IRAK-4(+/-) animals only at 1 week postinfection. At 3 and 6 weeks postinfection, IRAK-4(-/-) mice were able to control the infection similarly to heterozygous animals. Furthermore, the type 1 cytokine profile was evaluated. IRAK-4(-/-) mice showed lower production of systemic interleukin-12 (IL-12) and gamma interferon (IFN-γ). Additionally, a reduced percentage of CD4(+) and CD8(+) T cells expressing IFN-γ was observed compared to IRAK-4(+/-). Further, the production of IL-12 and tumor necrosis factor alpha (TNF-α) by macrophages and dendritic cells from IRAK-4(-/-) mice was abolished at 24 h after stimulation with B. abortus. To investigate the role of IRAK-4 in mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways, macrophages were stimulated with B. abortus, and the signaling components were analyzed by protein phosphorylation. Extracellular signal-regulated kinase 1 (ERK1) and ERK2 and p38 as well as p65 NF-κB phosphorylation was profoundly impaired in IRAK-4(-/-) and MyD88(-/-) macrophages activated by Brucella. In summary, the results shown in this study demonstrated that IRAK-4 is critical to trigger the initial immune response against B. abortus but not at later phases of infection.     

IL-1beta-induced phosphorylation of PKB/Akt depends on the presence of IRAK-1

IL-1, IL-18 and LPS are recognized by specific receptor complexes of the Toll/IL-1R family, characterized by a common intracellular domain indispensable for downstream signaling. Upon ligand binding, these receptors activate the central MyD88-IRAK-TRAF6 signaling module, resulting in the activation of NF-kappaB. Ligated receptors also induce activation of other signaling cascades, suchas the PI3-kinase (PI3-K) and the p38 mitogen-activated protein kinase (MAPK) pathways. Unlike the p38MAPK pathway, which couples to the central signaling module, the PI3-K pathway seems to directly interact with the receptor molecules. Thus, activation of the PI3-K pathway is thought to be independent of the IRAK-containing signaling module. Employing two cell lines, we show that the PI3-K pathways can be activated by IL-1, IL-18 or LPS with comparable, but cell type specific kinetics, which can be correlated to biological consequences. This indicates that activation of the PI3-K pathways may be regulated by an element common for all three receptor types, the MyD88-IRAK-TRAF6 module being a candidate for this function. Using an IRAK-1-deficient cell line, we demonstrate that the IRAK-1-containing signaling module is essential for the IL-1-induced activation of the PI3-K pathway. Possible models of the interaction between IRAK-1 and the PI3-K pathway are discussed.     

IRAK-4 as the central TIR signaling mediator in innate immunity

Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns and members of the proinflammatory interleukin-1 receptor (IL-1R) family, share homologies in their cytoplasmic domains. Engagement of members of both of these families initiates a common intracellular signaling cascade, in which MyD88 and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) are key adaptor proteins. Signaling between MyD88 and TRAF6 is mediated by members of the IL-1R-associated kinase (IRAK) family; however, the exact function of each IRAK protein remains controversial. IRAK-1 is required for the optimal transduction of IL-1R- and TLR-mediated signals, but IRAK-1 can be replaced by other IRAKs. Surprisingly, gene targeting studies show that the newest IRAK protein, IRAK-4, has an essential role in mediating signals initiated by IL-1R and TLR engagement. The kinase activity of IRAK-4 might be necessary to functionally modify IRAK-1 and perhaps other signal transducing substrates. Understanding the role of IRAK-4 in innate immunity will enable us to design novel strategies for therapeutic intervention in human infectious disease.     

Minimal structure of IRAK-1 to induce degradation of TRAF6

Excessive activation of Toll-like receptor (TLR) leads to sepsis. Inflammatory responses to various microbiological components are initiated via different TLR proteins, but all TLR signals are transmitted by TRAF6. We reported that TRAF6 associated with ubiquitinated IRAK-1 undergoes proteasome-mediated degradation, suggesting that IRAK-1 has a negative regulatory role in TLR signaling. Here, we investigated the minimal structural region of IRAK-1 needed for degradation of TRAF6. The IRAK-1 protein contains an N-terminal death domain (DD; amino acids 1–102), a serine/proline/threonine-rich ProST domain (amino acids 103–197), a central kinase domain with an activation loop (amino acids 198–522), and the C-terminal C1 and C2 domains (amino acids 523–712), which contain two and one putative TRAF6-binding (TB) sites, respectively. TRAF6 degradation was severely impaired by deletion of the DD or C1 domain, and a mutant (DC1) containing only the DD and C1 domains could induce TRAF6 degradation. IRAK-1 mutants lacking the N- or C-terminal amino acids of DD induced little degradation. Deletion or mutation of TB2 (amino acids 585–591) in the C1 domain also inhibited TRAF6 degradation. An IRAK-1 mutant possessing only DD and TB2 did not induce TRAF6 degradation, although a mutant in which a short spacer was inserted between DD and TB2 induced TRAF6 degradation, which and DC1-induced degradation were inhibited by proteasome inhibitors. All IRAK-1 mutants that induced TRAF6 degradation could be immunoprecipitated with TRAF6. Meanwhile, NF-κB activation was observed for all IRAK-1 mutants–including those that failed to induce degradation and was severely impaired only for a mutant carrying mutations in both TBs of C1. These results demonstrate that only DD and TB2 separated by an appropriate distance can induce TRAF6 degradation. Conformational analysis of this minimal structural unit may aid development of low molecular compounds that negatively regulate TLR signaling.     

Identification of IRAK-4 in grouper (Epinephelus coioides) that impairs MyD88-dependent NF-κB activation

Interleukin 1 (IL-1) receptor-associated kinase (IRAK) family members are crucial signal transducer in the Toll-like receptor/IL-1R signal pathway, which mediates downstream signal cascades involved in the innate and adaptive immune responses. In this study, we identified an IRAK-4 protein (EcIRAK-4) in the orange-spotted grouper (Epinephelus coioides), with an N-terminal death domain, a proST domain, and a central kinase domain, similar to that of other fishes and mammals. A sequence alignment and phylogenic analysis demonstrated that full-length EcIRAK-4 shares a high degree of sequence identity with those of other fishes, especially the roughskin sculpin, and their death domains and kinase domains share greater identity than their proST domains. A conservation analysis indicated that most of the functional sites in mammalian IRAK-4 are conserved in IRAK-4 of the grouper and other fishes, with the exception of the sites of interaction with IRAK-2 and one autophosphorylation site within the activation loop. EcIRAK-4 is broadly expressed in all the tissues examined, with highest expression in the head kidney and liver. After infection with Cryptocaryon irritans, EcIRAK-4 expression was significantly upregulated, especially in the skin, which suggests that this molecule is involved in the host’s defense against parasitic infection. Surprisingly, after cotransfection with grouper MyD88, EcIRAK-4 significantly impaired the NF-κB activity induced by MyD88. EcIRAK-4 was uniformly distributed throughout the cytoplasm in HeLa cells. These findings suggest that although IRAK-4 is evolutionarily conserved between fish and mammals, its signal transduction function is markedly different.     

IRAK-4 kinase activity-dependent and -independent regulation of lipopolysaccharide-inducible genes

IRAK-4 kinase inactive (IRAK-4 KD) knock-in mice display defects in TLR- and IL-1 receptor signaling and are resistant to LPS-induced shock. In the present study we examined the LPS-induced response in IRAK-4 KD mice in more detail. We show that IRAK-4 kinase activity is required for certain aspects of TLR-mediated signaling but not for others. We found that IRAK-4 KD cells displayed reduced JNK and p38 signaling, while NF-kappaB was activated to a normal level but with delayed kinetics compared to wild-type cells. TLR4-mediated IRF3 activation was intact in these cells. Comprehensive analysis of expression of LPS-inducible genes by microarray demonstrated that IRAK-4 KD cells were severely impaired in the expression of many pro-inflammatory genes, suggesting their dependence on IRAK-4 kinase activity. In contrast, the expression of a subset of LPS-induced genes of anti-viral response was not affected by IRAK-4 kinase deficiency. Additionally, we demonstrate that LPS-activated early expression and production of some cytokines, e.g., TNF-alpha, is partially induced in the absence of IRAK-4 kinase activity. This suggests that the partially unaffected TLR4-mediated signaling could still drive expression of these genes in early phases and that IRAK-4 kinase activity is important for a more sustained anti-bacterial response.     

The interleukin-1 receptor associated kinase 1 contributes to the regulation of NFAT

IRAK-1 is a critical modulator regulating innate immunity signaling processes. However, the physiological substrates for IRAK-1 remain poorly defined. In this report, we have demonstrated that IRAK-1 is a kinase responsible for the constitutive phosphorylation and inactivation of the Nuclear Factor of Activated T-cell (NFAT). Expression of IRAK-1 suppressed NFAT reporter activity. Correspondingly, the levels of both nuclear NFATc1 and NFATc4 were constitutively elevated in IRAK-1(-/-) cells. Furthermore, the phosphorylation of NFATc4 at the S(168)PS(170)P site was significantly diminished in IRAK-1(-/-) cells. Mechanistically, we observed that IRAK-1 interacted with NFATc4 via the C-terminus of IRAK-1 and the N-terminal NHR region of NFATc4. IRAK-1 mutants that ablated either its kinase activity or its interaction with NFATc4 failed to suppress NFAT reporter activity. The expression level of COX2, which is under the control of NFAT, was elevated in IRAK-1(-/-) cells. Functionally, ApoE(-/-)/IRAK-1(-/-) mice were protected from high-fat-diet-induced hypertension and atherosclerosis. Taken together, our findings reveal NFAT molecules as novel physiological targets for IRAK-1.     

Toll-Like Receptors’ Pathway Disturbances are Associated with Increased Susceptibility to Infections in Humans

Toll-like receptors (TLRs) sense microbial products and play an important role in innate immunity. Currently, 11 members of TLRs have been identified in humans, with important function in host defense in early steps of the inflammatory response. TLRs are present in the plasma membrane (TLR1, TLR2, TLR4, TLR5, TLR6) and endosome (TLR3, TLR7, TLR8, TLR9) of leukocytes. TLRs and IL-1R are a family of receptors related to the innate immune response that contain an intracellular domain known as the Toll-IL-1R (TIR) domain that recruits the TIR-containing cytosolic adapters MyD88, TRIF, TIRAP and TRAM. The classical pathway results in the activation of both nuclear factor κB and MAPKs via the IRAK complex, with two active kinases (IRAK-1 and IRAK-4) and two non-catalytic subunits (IRAK-2 and IRAK-3/M). The classical pro-inflammatory TLR signaling pathway leads to the synthesis of inflammatory cytokines and chemokines, such as IL-1β, IL-6, IL-8, IL-12 and TNF-α. In humans, genetic defects have been identified that impair signaling of the TLR pathway and this may result in recurrent pyogenic infections, as well as virus and fungi infections. In this review, we discuss the main mechanisms of microbial recognition and the defects involving TLRs.     

Bacillus Calmette Guerin triggers the IL-12/IFN-gamma axis by an IRAK-4- and NEMO-dependent, non-cognate interaction between monocytes, NK, and T lymphocytes

The IL-12/IFN-gamma axis is crucial for protective immunity to Mycobacterium in humans and mice. Our goal was to analyze the relative contribution of various human blood cell subsets and molecules to the production of, or response to IL-12 and IFN-gamma. We designed an assay for the stimulation of whole blood by live M. bovis Bacillus Calmette-Guerin (BCG) alone, or BCG plus IL-12 or IFN-gamma, measuring IFN-gamma and IL-12 levels. We studied patients with a variety of specific inherited immunodeficiencies resulting in a lack of leukocytes, or T, B, and/or NK lymphocytes, or polymorphonuclear cells, or a lack of expression of key molecules such as HLA class II, CD40L, NF-kappaB essential modulator (NEMO), and IL-1 receptor-associated kinase-4 (IRAK-4). Patients with deficiencies in IL-12p40, IL-12 receptor beta1 chain (IL-12Rbeta1), IFN-gammaR1, IFN-gammaR2, and STAT-1 were used as internal controls. We showed that monocytes were probably the main producers of IL-12, and that NK and T cells produced similar amounts of IFN-gamma. NEMO and IRAK-4 were found to be important for IL-12 production and subsequent IFN-gamma production, while a lack of CD40L or HLA class II had no major impact on the IL-12/IFN-gamma axis. The stimulation of whole blood by live BCG thus triggers the IL-12/IFN-gamma axis by an IRAK-4- and NEMO-dependent, non-cognate interaction between monocytes, NK, and T lymphocytes.     

TRAF6 distinctively mediates MyD88- and IRAK-1-induced activation of NF-kappaB

MyD88 and IL-1R-associated kinase 1 (IRAK-1) play crucial roles as adaptor molecules in signal transduction of the TLR/IL-1R superfamily, and it is known that expression of these proteins leads to the activation of NF-kappaB in a TNFR-associated factor 6 (TRAF6)-dependent manner. We found in this study, however, that a dominant-negative mutant of TRAF6, lacking the N-terminal RING and zinc-finger domain, did not inhibit IRAK-1-induced activation of NF-kappaB in human embryonic kidney 293 cells, although the TRAF6 mutant strongly suppressed the MyD88-induced activation. The dominant-negative mutant of TRAF6 did not affect the IRAK-1-induced activation, regardless of the expression level of IRAK-1. In contrast, small interfering RNA silencing of TRAF6 expression inhibited MyD88-induced and IRAK-1-induced activation, and supplementation with the TRAF6 dominant-negative mutant did not restore the IRAK-1-induced activation. Expression of IRAK-1, but not MyD88, induced the oligomerization of TRAF6, and IRAK-1 and the TRAF6 dominant-negative mutant were associated with TRAF6. These results indicate that TRAF6 is involved but with different mechanisms in MyD88-induced and IRAK-induced activation of NF-kappaB and suggest that TRAF6 uses a distinctive mechanism to activate NF-kappaB depending on signals.     

The IL-17F signaling pathway is involved in the induction of IFN-gamma-inducible protein 10 in bronchial epithelial cells

BACKGROUND: IL-17F is involved in airway inflammation, but its biologic activity and signaling pathway remain incompletely defined. Interferon-gamma-inducible protein 10 (IP-10) is widely expressed and plays a role in airway inflammatory diseases. OBJECTIVE: We sought to investigate the functional linkage between IL-17F and IP-10 expression in bronchial epithelial cells. METHODS: Bronchial epithelial cells were cultured in the presence or absence of IL-17F, and/or a T(H)1 cytokine, T(H)2 cytokines, proinflammatory cytokines, various kinase inhibitors, or a Raf1 dominant-negative mutant to analyze the expression of IP-10. Moreover, the involvement of p90 ribosomal S6 kinase (p90RSK) and cyclic AMP response element-binding protein (CREB) in IL-17F-induced IP-10 expression were investigated. RESULTS: IL-17F induces the gene and protein expression of IP-10. The addition of IFN-gamma, IL-1beta, and TNF-alpha augmented IL-17F-induced IP-10 expression. The mitogen-activated protein kinase kinase (MEK) inhibitors PD98059, U0126, and Raf1 kinase inhibitor I significantly inhibited its production. In contrast, a p38 inhibitor, a JNK inhibitor, protein kinase C inhibitors, and a phosphatidylinositol 3-kinase inhibitor, showed no inhibitory effect. Furthermore, overexpression of a Raf1 dominant-negative mutant inhibited its expression. Of interest, IL-17F phosphorylated p90RSK and CREB, and transfection of the cells with a short interfering RNA for p90RSK or CREB inhibited its expression, suggesting p90RSK and CREB as novel signaling molecules of IL-17F. CONCLUSION: IL-17F is a potent inducer of IP-10 in bronchial epithelial cells through the activation of the Raf1-MEK1/2-extracellular signal-regulated kinase 1/2-p90RSK-CREB pathway, supporting its regulatory role in airway inflammation. CLINICAL IMPLICATIONS: The IL-17F-IP-10 axis might be a novel and critical therapeutic target for airway inflammatory diseases.     

Activation of the extracellular signal-regulated kinase pathway is differentially required for TCR-stimulated production of six cytokines in primary T lymphocytes

The extracellular signal-regulated kinase (ERK) signaling pathway is strongly activated in response to TCR stimulation in normal T cells. However, the extent to which activation of the ERK pathway is necessary for TCR-stimulated cytokine production is not clear. We have addressed this question by use of two separate methods to interfere with TCR activation of the ERK cascade. The first approach utilized transient expression of a catalytically inactive form of mitogen-activated/ERK 1 (CI-MEK1), while the second involved using the MEK1- and MEK2-specific inhibitor PD98059 to block ERK activation by the TCR. In order to assess the requirement for ERK activation in T cell cytokine production, we have measured the effect of ERK inhibition upon the production of six cytokines, IL-3, IL-4, IL-5, IL-10, granulocyte macrophage colony stimulating factor (GM-CSF) and IFN-gamma, by newly activated normal mouse T cells in response to TCR stimulation. The results of experiments using both methods to block ERK activation have revealed a requirement for intact ERK signaling for the full elicitation of TCR-stimulated cytokine production. Dose-response analyses using the MEK inhibitor PD98059 showed that the TCR-stimulated production of all cytokines measured was affected by this treatment. However, the production of IL-3 and IL-4 was only partially dependent upon ERK activation, whereas IL-5, IL-10, IFN-gamma and GM-CSF production was severely affected by diminished ERK activation. We conclude that the ERK pathway is differentially involved in the activation of different cytokine genes in normal T cells.      

Heat shock protein 90 (Hsp90) regulates the stability of transforming growth factor beta-activated kinase 1 (TAK1) in interleukin-1beta-induced cell signaling

Heat shock protein 90 (Hsp90) is an abundantly and ubiquitously expressed chaperone with majority of client proteins which act as signal molecules. Transforming growth factor beta-activated kinase 1 (TAK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK), and is essential in interleukin-1beta (IL-1beta) triggered signaling pathways. In the present study, we found that Hsp90 plays an important role in regulating IL-1beta signaling by keeping TAK1 stability. The results showed that the specific inhibitor geldanamycin (GA) of Hsp90 dramatically inhibited IL-1beta stimulated TAK1-MAPKs and TAK1-nuclear factor-kappaB (NF-kappaB) activation, resulting in the decrease of cyclooxygenase-2 (COX-2) protein expression. Silencing Hsp90 expression through RNA interference (RNAi) also down-regulated TAK1, as well as attenuated IL-1beta induced phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and p38 MAPKs, and degradation of IkappaBalpha. The same results were obtained in T6RZC stable cells which initiated IL-1beta-induced cell signaling at the level of the oligomerization and ubquitination of TNF receptor-associated factor 6 (TRAF6). We further found that Hsp90 formed a complex with TAK1 via its N-terminal domain and GA destabilized TAK1 and induced TAK1 degradation through proteasome pathway. Taken together our results demonstrate that Hsp90 regulates IL-1beta-induced signaling by interacting with TAK1 and maintaining the stability of TAK1, suggesting that Hsp90 might act as the chaperone of TAK1 in immune and inflammatory responses related with IL-1 signal cascades.     

Regulation of Lck activity by CD4 and CD28 in the immunological synapse

Although the Src family tyrosine kinase Lck is essential for T cell receptor (TCR) signaling, whether or how Lck is activated is unknown. Using a phosphospecific antiserum to Lck, we show here that Lck becomes autophosphorylated when T cells are stimulated by antigen-presenting cells (APCs). We found that TCR cross-linking alone could not stimulate Lck autophosphorylation and CD45 was not required for this process. Instead, the T cell accessory molecules CD4 and CD28 cooperated to induce autophosphorylation of Lck. CD4 recruited Lck to the T cell--APC interface, whereas CD28 sustained Lck activation. These data show how the multiple interactions afforded by the immunological synapse drive efficient and highly specific signaling.