Genetically resistant mice lacking MyD88-adapter protein display a high susceptibility to Leishmania major infection associated with a polarized Th2 response

Host resistance to the intracellular protozoan Leishmania major is highly dependent on IL-12 production by APCs. Genetically resistant C57BL/6 mice develop IL-12-mediated Th1 immune response dominated by IFN-gamma and exhibit only small cutaneous lesions that resolve spontaneously. In contrast, because of several genetic differences, BALB/c mice develop an IL-4-mediated Th2 immune response and a chronic mutilating disease. Myeloid differentiation marker 88 (MyD88) is an adaptator protein that links the IL-1/Toll-like receptor family to IL-1R-associated protein kinase. Toll-like receptors recognize pathogen associated molecular patterns and are crucially implicated in the induction of IL-12 secretion by APC. The role of MyD88 protein in the development of protective immune response against parasites is largely unknown. Following inoculation of L. major, MyD88(-/-) C57BL/6 mice presented large footpad lesions containing numerous infected cells and frequent mutilations. In response to soluble Leishmania Ag, cells from lesion-draining lymph node showed a typical Th2 profile, similar to infected BALB/c mice. IL-12p40 plasma level collapses in infected MyD88(-/-) mice compared with infected wild-type C57BL/6 mice. Importantly, administration of exogenous IL-12 rescues L. major-infected MyD88(-/-) mice, demonstrating that the susceptibility of these mice is a direct consequence of IL-12 deficiency. In conclusion, MyD88-dependent pathways appear essential for the development of the protective IL-12-mediated Th1 response against the Leishmania major parasite. In absence of MyD88 protein, infected mice develop a nonprotective Th2 response.     

Multiple TLRs are expressed in human cholangiocytes and mediate host epithelial defense responses to Cryptosporidium parvum via activation of NF-kappaB

Infection of epithelial cells by Cryptosporidium parvum triggers a variety of host-cell innate and adaptive immune responses including release of cytokines/chemokines and up-regulation of antimicrobial peptides. The mechanisms that trigger these host-cell responses are unclear. Thus, we evaluated the role of TLRs in host-cell responses during C. parvum infection of cultured human biliary epithelia (i.e., cholangiocytes). We found that normal human cholangiocytes express all known TLRs. C. parvum infection of cultured cholangiocytes induces the selective recruitment of TLR2 and TLR4 to the infection sites. Activation of several downstream effectors of TLRs including IL-1R-associated kinase, p-38, and NF-kappaB was detected in infected cells. Transfection of cholangiocytes with dominant-negative mutants of TLR2 and TLR4, as well as the adaptor molecule myeloid differentiation protein 88 (MyD88), inhibited C. parvum-induced activation of IL-1R-associated kinase, p-38, and NF-kappaB. Short-interfering RNA to TLR2, TLR4, and MyD88 also blocked C. parvum-induced NF-kappaB activation. Moreover, C. parvum selectively up-regulated human beta-defensin-2 in directly infected cells, and inhibition of TLR2 and TLR4 signals or NF-kappaB activation were each associated with a reduction of C. parvum-induced human beta-defensin-2 expression. A significantly higher number of parasites were detected in cells transfected with a MyD88 dominant-negative mutant than in the control cells at 48-96 h after initial exposure to parasites, suggesting MyD88-deficient cells were more susceptible to infection. These findings demonstrate that cholangiocytes express a variety of TLRs, and suggest that TLR2 and TLR4 mediate cholangiocyte defense responses to C. parvum via activation of NF-kappaB.     

CD16 regulates TRIF-dependent TLR4 response in human monocytes and their subsets

Blood monocytes recognize Gram-negative bacteria through the TLR4, which signal via MyD88- and TRIF-dependent pathway to trigger an immune-inflammatory response. However, a dysregulated inflammatory response by these cells often leads to severe pathologies such as sepsis. We investigated the role of CD16 in the regulation of human monocyte response to Gram-negative endotoxin and sepsis. Blood monocytes from sepsis patients demonstrated an upregulation of several TRIF-dependent genes as well as a selective expansion of CD16-expressing (CD16(+)) monocytes. Gene expression and biochemical studies revealed CD16 to regulate the TRIF-dependent TLR4 pathway in monocytes by activating Syk, IFN regulatory factor 3, and STAT1, which resulted in enhanced expression of IFNB, CCL5, and CXCL10. CD16 also upregulated the expression of IL-1R-associated kinase M and IL-1 receptor antagonist, which are negative regulators of the MyD88-dependent pathway. CD16 overexpression or small interfering RNA knockdown in monocytes confirmed the above findings. Interestingly, these results were mirrored in the CD16(+) monocyte subset isolated from sepsis patients, providing an in vivo confirmation to our findings. Collectively, the results from the current study demonstrate CD16 as a key regulator of the TRIF-dependent TLR4 pathway in human monocytes and their CD16-expressing subset, with implications in sepsis.     

Protein kinase D1: a new component in TLR9 signaling

Protein kinase D1 (PKD1) is expressed ubiquitously and regulates diverse cellular processes such as oxidative stress, gene expression, cell survival, and vesicle trafficking. However, the presence and function of PKD1 in monocytic cells are currently unknown. In this study, we provide evidence that PKD1 is involved in TLR9 signaling in macrophages. Class B-type CpG DNA (CpG-B DNA) induced activation of PKD1 via a pathway that is dependent on endosomal pH, TLR9, MyD88, and IL-1R-associated kinase 1 in macrophages. Upon CpG-B DNA stimulation, PKD1 interacted with the TLR9/MyD88/IL-1R-associated kinase/TNFR-associated factor 6 complex. Knockdown of PKD1 revealed that PKD1 is required for activation of NF-kappaB and MAPKs, and subsequent expression of cytokines in response to CpG-B DNA. Our findings identify PKD1 as a key signaling modulator in TLR9-mediated macrophage activation.     

The role of intermediary domain of MyD88 in cell activation and therapeutic inhibition of TLRs

Adaptor MyD88 has a pivotal role in TLR and IL-1R signaling and is involved in mediating excessive inflammation. MyD88 is composed of a death domain and a Toll/IL-1R domain connected by an intermediary domain (INT). The alternatively spliced form of MyD88 lacking the INT prevents signaling through MyD88-dependent TLRs. We designed a peptide from the INT and showed that it inhibits TLR4 activation by LPS when linked to a cell-penetrating peptide. As a new approach for the delivery of signaling-inhibitory peptides, INT peptide acylation also provided efficient cell translocation and inhibition of activation. We determined that INT peptide targets IL-1R-associated kinase 4. Furthermore, MyD88 mutant and molecular modeling refines the MyD88- IL-1R-associated kinase 4 interaction model based on the Myddosome structure. In addition to TLR4, INT peptide also inhibited TLR5, TLR2, TLR9, and IL-1R signaling but not TLR3, which uses Toll/IL-1R domain-containing adapter inducing IFN-β signaling adaptor. Inhibition of signaling in murine and human cells was observed by decreased NF-κB activation, cytokine mRNA synthesis, and phosphorylation of downstream kinases. In the endotoxemic mouse model, INT peptide suppressed production of inflammatory cytokines and improved survival, supporting therapeutic application of INT peptides for the suppression of inflammatory conditions mediated by MyD88.     

Modulation of TLR4 signaling by a novel adaptor protein signal-transducing adaptor protein-2 in macrophages

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies have demonstrated that STAP-2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, STAP-2 was found to positively regulate LPS/TLR4-mediated signals in macrophages. Disruption of STAP-2 resulted in impaired LPS/TLR4-induced cytokine production and NF-kappaB activation. Conversely, overexpression of STAP-2 enhanced these LPS/TLR4-induced biological activities. STAP-2, particularly its Src homology 2-like domain, bound to both MyD88 and IkappaB kinase (IKK)-alphabeta, but not TNFR-associated factor 6 or IL-1R-associated kinase 1, and formed a functional complex composed of MyD88-STAP-2-IKK-alphabeta. These interactions augmented MyD88- and/or IKK-alphabeta-dependent signals, leading to enhancement of the NF-kappaB activity. These results demonstrate that STAP-2 may constitute an alternative LPS/TLR4 pathway for NF-kappaB activation instead of the TNFR-associated factor 6-IL-1R-associated kinase 1 pathway.     

Mastoparan, a G protein agonist peptide, differentially modulates TLR4- and TLR2-mediated signaling in human endothelial cells and murine macrophages

Previous studies have implicated a role for heterotrimeric G protein-coupled signaling in B cells, monocytes, and macrophages stimulated with LPS and have shown that G proteins coimmunoprecipitate with membrane-bound CD14. In this study, we have extended these observations in human dermal microvessel endothelial cells (HMEC) that lack membrane-bound CD14 and in murine macrophages to define further the role of heterotrimeric G proteins in TLR signaling. Using the wasp venom-derived peptide, mastoparan, to disrupt G protein-coupled signaling, we identified a G protein-dependent signaling pathway in HMEC stimulated with TLR4 agonists that is necessary for the activation of p38 phosphorylation and kinase activity, NF-kappaB and IL-6 transactivation, and IL-6 secretion. In contrast, HMEC activation by TLR2 agonists, TNF-alpha, or IL-1beta was insensitive to mastoparan. In the murine macrophage cell line, RAW 264.7, and in primary murine macrophages, G protein dysregulation by mastoparan resulted in significant inhibition of LPS-induced signaling leading to both MyD88-dependent and MyD88-independent gene expression, while TLR2-mediated gene expression was not significantly inhibited. In addition to inhibition of TLR4-mediated MAPK phosphorylation in macrophages, mastoparan blunted IL-1R-associated kinase-1 kinase activity induced by LPS, but not by TLR2 agonists, yet failed to affect phosphorylation of Akt by phosphoinositol-3-kinase induced by either TLR2- or TLR4-mediated signaling. These data confirm the importance of heterotrimeric G proteins in TLR4-mediated responses in cells that use either soluble or membrane-associated CD14 and reveal a level of TLR and signaling pathway specificity not previously appreciated.     

Cutting edge: expression of IL-1 receptor-associated kinase-4 (IRAK-4) proteins with mutations identified in a patient with recurrent bacterial infections alters normal IRAK-4 interaction with components of the IL-1 receptor complex

In a patient with recurrent bacterial infections and profound hyporesponsiveness to LPS and IL-1, we previously identified two mutations in IL-1R-associated kinase-4 (IRAK-4) that encoded proteins with truncated kinase domains. Overexpression of either of these mutant IRAK-4 variants in HEK293 cells failed to activate endogenous IRAK-1 and suppressed IL-1-induced IRAK-1 kinase activity, in contrast to wild-type (WT) IRAK-4. In this study, interactions of WT and mutant IRAK-4 species with IL-1R, IRAK-1, and MyD88 in HEK293 transfectants were compared. IL-1 induced a strong interaction among the IL-1R, activated IRAK-1, MyD88, and WT, but not mutant, IRAK-4. Truncated IRAK-4 proteins constitutively interacted more strongly with MyD88 and blunted IL-1-induced recruitment of IRAK-1 and MyD88 to the IL-1R. Thus, decreased IL-1-induced association of IRAK-1 and MyD88 with the IL-1RI may result from sequestration of cytoplasmic MyD88 by IRAK-4 mutant proteins. Therefore, mimetics of these truncated IRAK-4 proteins may represent a novel approach to mitigating hyperinflammatory states.     

Immune complex-mediated cell activation from systemic lupus erythematosus and rheumatoid arthritis patients elaborate different requirements for IRAK1/4 kinase activity across human cell types

IL-1R-associated kinases (IRAKs) are important mediators of MyD88-dependent signaling by the TLR/IL-1R superfamily and facilitate inflammatory responses. IRAK4 and IRAK1 function as active kinases and as scaffolds for protein-protein interactions. We report that although IRAK1/4 kinase activity is essential for human plasmacytoid dendritic cell (pDC) activation, it is dispensable in B, T, dendritic, and monocytic cells, which is in contrast with an essential active kinase role in comparable mouse cell types. An IRAK1/4 kinase inhibitor abrogated TLR7/9-induced IFN-α responses in both mouse and human pDCs, but other human immune cell populations activated via TLR7/9 or IL-1R were refractory to IRAK4 kinase inhibition. Gene ablation experiments using small interfering RNA demonstrated an essential scaffolding role for IRAK1 and IRAK4 in MyD88-dependent signaling. Finally, we demonstrate that autoimmune patient (systemic lupus erythematosus and rheumatoid arthritis) serum activates both pDC and B cells, but IRAK1/4 kinase inhibition affects only the pDC response, underscoring the differential IRAK1/4 functional requirements in human immune cells. These data reveal important species differences and elaborate cell type requirements for IRAK1/4 kinase activity.     

Tumor immunotherapy using bone marrow-derived dendritic cells overexpressing Toll-like receptor adaptors

Myeloid dendritic cells (mDCs) play an important role in the initiation of immune responses to cancer and infectious diseases. Toll-like receptors (TLRs) expressed on mDCs recognize microbial products to elicit signals for mDC maturation, including cytokine production, antigen-presentation and induction of effector cells. TLR agonists work as adjuvants to modulate the function of mDCs. In TLR signaling, MyD88 and TRIF/TICAM-1 are major TLR adaptor molecules, which when overexpressed are able to transduce downstream signals without TLR stimuli. We successfully introduced the adaptors into mouse bone marrow-derived mDCs using lentiviral vectors. Introduction of MyD88 into mDCs in vitro led to the production of IL-6 and IL-12p40 while introduction of TICAM-1 stimulated interferon (IFN)-alpha production. Expression of TICAM-1, but not MyD88, in mDCs slightly induced the co-stimulatory molecule CD86, while significant upregulation of CD86 was observed in response to other TLR stimuli. Both MyD88 and TICAM-1 augmented allogeneic mixed lymphocyte reaction (MLR). Ex vivo mouse spleen cells pre-exposed to tumor antigen exhibited antitumor cytotoxicity when incubated with MyD88- or TICAM-1-expressing mDCs. Using mDC adoptive transfer and a syngeneic mouse tumor implant model, we established an antitumor immunotherapy whereby tumor growth is retarded by adaptor-manipulated mDCs.     

HSP70 as endogenous stimulus of the Toll/interleukin-1 receptor signal pathway

Human heat-shock protein (HSP)70 activates innate immune cells and hence requires no additional adjuvants to render bound peptides immunogenic. Here we tested the assumption that endogenous HSP70 activates the Toll/IL-1 receptor signal pathway similar to HSP60 and pathogen-derived molecular patterns. We show that HSP70 induces interleukin-12 (IL-12) and endothelial cell-leukocyte adhesion molecule-1 (ELAM-1) promoters in macrophages and that this is controlled by MyD88 and TRAF6. Furthermore, HSP70 causes MyD88 relocalization and MyD88-deficient dendritic cells do not respond to HSP70 with proinflammatory cytokine production. Using the system of genetic complementation with Toll-like receptors (TLR) we found that TLR2 and TLR4 confer responsiveness to HSP70 in 293T fibroblasts. The expanding list of endogenous ligands able to activate the ancient Toll/IL-1 receptor signal pathway is in line with the "danger hypothesis" proposing that the innate immune system senses danger signals even if they originate from self.     

MyD88 and TNF receptor-associated factor 6 are critical signal transducers in Helicobacter pylori-infected human epithelial cells

Helicobacter pylori induces NF-kappaB activation, leading to mucosal inflammation via cag pathogenicity island. Although recent studies have implicated several candidate proteins of both H. pylori and host, the molecular mechanism by which H. pylori activates NF-kappaB remains unclear. The aim of this study was to analyze the mechanism of cag pathogenicity island-mediated NF-kappaB activation in epithelial cells. The responses of human cell lines and mouse embryonic fibroblasts to infection with wild-type H. pylori or cagE mutant were investigated. The effect of small interfering RNAs (siRNAs) for several NF-kappaB signaling intermediate molecules was evaluated in H. pylori-induced IkappaBalpha phosphorylation and IL-8 production. Protein interactions of exogenously expressed TNFR-associated factor 6 (TRAF6) and MyD88 or receptor-interacting protein 2 and nucleotide-binding oligomerization domain 1 or those of endogenous IkappaB kinase, TGF-beta-activated kinase 1 (TAK1), and TRAF6 were assessed by immunoprecipitation. Cag pathogenicity island-dependent NF-kappaB activation was observed in human cell lines, but not in mouse fibroblasts. In human epithelial cells, H. pylori-induced IkappaBalpha phosphorylation and IL-8 production were severely inhibited by siRNAs directed against TAK1, TRAF6, and MyD88. In contrast, siRNAs for TRAF2, IL-1R-associated kinases 1 and 4, and cell surface receptor proteins did not affect these responses. H. pylori infection greatly enhanced MyD88 and TRAF6 complex formation in a cag-dependent manner, but did not enhance Nod1 and receptor-interacting protein 2 complex formation. H. pylori also induced TAK1 and TRAF6 complexes. These results suggest that the cag pathogenicity island of H. pylori is a cell type-specific NF-kappaB activator. TAK1, TRAF6, and MyD88 are important signal transducers in H. pylori-infected human epithelial cells.     

MyD88-mediated instructive signals in dendritic cells regulate pulmonary immune responses during respiratory virus infection

Respiratory syncytial virus (RSV) is the leading cause of respiratory disease in infants worldwide. The induction of innate immunity and the establishment of adaptive immune responses are influenced by the recognition of pathogen-associated molecular patterns by TLRs. One of the primary pathways for TLR activation is by MyD88 adapter protein signaling. The present studies indicate that MyD88 deficiency profoundly impacts the pulmonary environment in RSV-infected mice characterized by the accumulation of eosinophils and augmented mucus production. Although there was little difference in CD4 T cell accumulation, there was also a significant decrease in conventional dendritic cells recruitment to the lungs of MyD88(-/-) mice. The exacerbation of RSV pathophysiology in MyD88(-/-) mice was associated with an enhanced Th2 cytokine profile that contributed to an inappropriate immune response. Furthermore, bone marrow-derived dendritic cells (BMDC) isolated from MyD88(-/-) mice were incapable of producing two important Th1 instructive signals, IL-12 and delta-like4, upon RSV infection. Although MyD88(-/-) BMDCs infected with RSV did up-regulate costimulatory molecules, they did not up-regulate class II as efficiently and stimulated less IFN-gamma from CD4(+) T cells in vitro compared with wild-type BMDCs. Finally, adoptive transfer of C57BL/6 BMDCs into MyD88(-/-) mice reconstituted Th1 immune responses in vivo, whereas transfer of MyD88(-/-) BMDCs into wild-type mice skewed the RSV responses toward a Th2 phenotype. Taken together, our data indicate that MyD88-mediated pathways are essential for the least pathogenic responses to this viral pathogen through the regulation of important Th1-associated instructive signals.     

Both hemopoietic and resident cells are required for MyD88-dependent pulmonary inflammatory response to inhaled endotoxin

Inhaled endotoxin induces an inflammatory response that contributes to the development and severity of asthma and other forms of airway disease. Here, we show that inhaled endotoxin-induced acute bronchoconstriction, TNF, IL-12p40, and KC production, protein leak, and neutrophil recruitment in the lung are abrogated in mice deficient for the adaptor molecule MyD88. Bronchoconstriction, inflammation, and protein leak are normal in Toll/IL-1R domain-containing adaptor inducing IFN-beta-deficient mice. MyD88 is involved in TLR, but also in IL-1R-associated kinase 1-mediated IL-1R and -18R signaling. We exclude a role for IL-1 and IL-18 pathways in this response, as IL-1R1 and caspase-1 (ICE)-deficient mice develop lung inflammation while TLR4-deficient mice are unresponsive to inhaled LPS. Significantly, using bone marrow chimera, we demonstrate that both hemopoietic and resident cells are necessary for a full MyD88-dependent response to inhaled endotoxin; bronchoconstriction depends on resident cells while cytokine secretion is mediated by hemopoietic cells.     

Langerhans cells require MyD88-dependent signals for Candida albicans response but not for contact hypersensitivity or migration

Langerhans cells (LC) are a subset of skin-resident dendritic cells (DC) that reside in the epidermis as immature DC, where they acquire Ag. A key step in the life cycle of LC is their activation into mature DC in response to various stimuli, including epicutaneous sensitization with hapten and skin infection with Candida albicans. Mature LC migrate to the skin-draining LN, where they present Ag to CD4 T cells and modulate the adaptive immune response. LC migration is thought to require the direct action of IL-1β and IL-18 on LC. In addition, TLR ligands are present in C. albicans, and hapten sensitization produces endogenous TLR ligands. Both could contribute to LC activation. We generated Langerin-Cre MyD88(fl) mice in which LC are insensitive to IL-1 family members and most TLR ligands. LC migration in the steady state, after hapten sensitization and postinfection with C. albicans, was unaffected. Contact hypersensitivity in Langerin-Cre MyD88(fl) mice was similarly unaffected. Interestingly, in response to C. albicans infection, these mice displayed reduced proliferation of Ag-specific CD4 T cells and defective Th17 subset differentiation. Surface expression of costimulatory molecules was intact on LC, but expression of IL-1β, IL-6, and IL-23 was reduced. Thus, sensitivity to MyD88-dependent signals is not required for LC migration, but is required for the full activation and function of LC in the setting of fungal infection.     

TRAM is involved in IL-18 signaling and functions as a sorting adaptor for MyD88

MyD88, a Toll/interleukin-1 receptor homology (TIR) domain-containing adaptor protein, mediates signals from the Toll-like receptors (TLR) or IL-1/IL-18 receptors to downstream kinases. In MyD88-dependent TLR4 signaling, the function of MyD88 is enhanced by another TIR domain-containing adaptor, Mal/TIRAP, which brings MyD88 to the plasma membrane and promotes its interaction with the cytosolic region of TLR4. Hence, Mal is recognized as the "sorting adaptor" for MyD88. In this study, a direct interaction between MyD88-TIR and another membrane-sorting adaptor, TRAM/TICAM-2, was demonstrated in vitro. Cell-based assays including RNA interference experiments and TRAM deficient mice revealed that the interplay between MyD88 and TRAM in cells is important in mediating IL-18 signal transduction. Live cell imaging further demonstrated the co-localized accumulation of MyD88 and TRAM in the membrane regions in HEK293 cells. These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction. The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal. MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.     

Mammalian target of rapamycin inhibition in macrophages of asymptomatic HIV+ persons reverses the decrease in TLR-4-mediated TNF-α release through prolongation of MAPK pathway activation

TLR-4-mediated signaling is significantly impaired in macrophages from HIV(+) persons, predominantly owing to altered MyD88-dependent pathway signaling caused in part by constitutive activation of PI3K. In this study we assessed in these macrophages if the blunted increase in TLR-4-mediated TNF-α release induced by lipid A (LA) is associated with PI3K-induced upregulation of mammalian target of rapamycin (mTOR) activity. mTOR inhibition with rapamycin enhanced TLR-4-mediated TNF-α release, but suppressed anti-inflammatory IL-10 release. Targeted gene silencing of mTOR in macrophages resulted in LA-induced TNF-α and IL-10 release patterns similar to those induced by rapamycin. Rapamycin restored MyD88/IL-1R-associated kinase interaction in a dose-dependent manner. Targeted gene silencing of MyD88 (short hairpin RNA) and mTOR (RNA interference) inhibition resulted in TLR-4-mediated 70-kDa ribosomal protein S6 kinase activation and enhanced TNF-α release, whereas IL-10 release was inhibited in both silenced and nonsilenced HIV(+) macrophages. Furthermore, mTOR inhibition augmented LA-induced TNF-α release through enhanced and prolonged phosphorylation of ERK1/2 and JNK1/2 MAPK, which was associated with time-dependent MKP-1 destabilization. Taken together, impaired TLR-4-mediated TNF-α release in HIV(+) macrophages is attributable in part to mTOR activation by constitutive PI3K expression in a MyD88-dependent signaling pathway. These changes result in MAPK phosphatase 1 stabilization, which shortens and blunts MAPK activation. mTOR inhibition may serve as a potential therapeutic target to upregulate macrophage innate immune host defense responsiveness in HIV(+) persons.