Toll-like receptor interactions: tolerance of MyD88-dependent cytokines but enhancement of MyD88-independent interferon-beta production

Toll-like receptors (TLRs) signal through two main pathways: a myeloid differentiation factor (MyD)88-dependent pathway that acts via nuclear factor kappaB (NF-kappaB) to induce proinflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and a MyD88-independent pathway that acts via type I interferons to increase the expression of interferon-inducible genes. Repeated signalling through TLR4 and a number of other TLRs has been reported to result in a reduction in the subsequent proinflammatory cytokine response, a phenomenon known as TLR tolerance. In this study we have shown that, whilst NF-kappaB activation and production of TNF-alpha and interleukin-12 by murine RAW264.7 and J774.2 cells in response to stimulation by TLR4, -5, -7 or -9, was reduced by prior stimulation with TLR4, -5, -7 or -9 ligands, the primary stimulation of TLR3, which does not use the MyD88 pathway, did not reduce the TNF-alpha or interleukin-12 responses to subsequent TLR stimulation. The response to TLR3 stimulation was not diminished by prior TLR ligand exposure. Furthermore, the production of interferon-beta (IFN-beta) following stimulation of TLR3 or -4, which is MyD88-independent, was increased by prior activation of TLR4, -5, -7 or -9. In contrast, TLR9 ligand-induced IFN-beta production, which is MyD88-dependent, was tolerized by prior TLR stimulation. These results are consistent with differential regulation of MyD88-dependent and MyD88-independent cytokine production following serial activation of TLRs.     

Identification of a TLR4- and TRIF-dependent activation program of dendritic cells

Dendritic cell activation by Toll-like receptors (TLR) is crucial for the generation of protective immune responses. In addition to the common myeloid differentiation factor 88 (MyD88)-dependent signaling pathway, TLR4 engages the adaptor protein Toll/IL-1 receptor (TIR)-domain-containing adaptor inducing IFN-beta (TRIF), leading to interferon regulatory factor 3 (IRF-3) activation and type I interferon production. Using microarray expression profiling we now identify TRIF as a major regulator of the TLR4-triggered activation program of dendritic cells. We show that the expression of 47% of the genes that are responsive to TLR4 stimulation in wild-type dendritic cells is significantly altered in cells carrying a loss-of-function mutation of TRIF. Specifically, expression of IL-12, IL-18, and IL-23 was impaired in the absence of functional TRIF, suggesting that TLR4-promoted Th1 responses are TRIF-dependent. Furthermore, we provide evidence that TRIF regulates TLR4-mediated gene expression both by type I IFN-dependent and -independent mechanisms. Whereas dendritic cell production of CXCL10 and CCL12 was dependent on both TRIF and the type I interferon receptor, expression of IL-6 required TRIF but not type I interferon activity. Functional TRIF was also required for the normal induction of numerous genes considered important for host defense against diverse pathogens.Together, these data therefore identify TRIF as a crucial regulator of TLR4-dependent dendritic cell responses.     

Bacterial Cell Wall Constituents Induce Hepcidin Expression in Macrophages Through MyD88 Signaling

Hepcidin is a key regulator of iron recycling by macrophages that is synthesized mainly by hepatocytes but also by macrophages. However, very little is known about the molecular regulation of hepcidin in macrophages. In the present study, we investigated hepcidin regulation in the RAW264.7 macrophage cell line and in murine peritoneal macrophages stimulated with different Toll-like receptor (TLR) ligands. We found that TLR-2 and TLR-4 ligands activated hepcidin expression in RAW264.7 cells and in wild-type murine peritoneal macrophages, but not in murine peritoneal macrophages isolated from TLR2(-/-), TLR-4-deficient or MyD88(-/-) mice. IL-6 production by RAW264.7 cells stimulated with lipopolysaccharide (LPS, TLR4 ligand) was enhanced by high amounts of iron present in the culture medium. We conclude that hepcidin expression in macrophages is regulated mainly through TLR2 and TLR4 receptors via the MyD88-dependent signaling pathway and that autocrine regulation of iron accumulation in macrophages by hepcidin may affect the levels of proinflammatory cytokine production.     

A variety of microbial components induce tolerance to lipopolysaccharide by differentially affecting MyD88-dependent and -independent pathways

Exposure of macrophages to lipopolysaccharide (LPS) induces a hypo-responsive state to a second challenge with LPS that is termed LPS tolerance. LPS tolerance is also induced by pre-exposure to lipopeptides and lipoteichoic acid, which trigger Toll-like receptor (TLR) 2-mediated signaling. LPS signaling involves at least two pathways: a MyD88-dependent cascade that is essential for production of inflammatory cytokines and a MyD88-independent cascade that mediates the expression of IFN-inducible genes. We analyzed the induction of LPS tolerance by several microbial components in mouse peritoneal macrophages. Pre-exposure to LPS led to impaired activation of both the pathways. In contrast, mycoplasmal lipopeptides did not affect the MyD88-independent pathway, but impaired the MyD88-dependent signaling by inhibiting LPS-mediated activation of IL-1 receptor-associated kinase (IRAK) 1. The induction of LPS tolerance by recently identified TLR ligands was analyzed. Pretreatment with double-stranded RNA, which triggers the activation of TLR3, led to defective activation of the MyD88-independent, but not the MyD88-dependent, pathway. Imidazoquinoline compounds, which are recognized by TLR7, had no effect on the MyD88-independent pathway, but inhibited LPS-induced activation of MyD88-dependent signaling through down-regulation of IRAK1 expression. Thus, each microbial component induced LPS tolerance in macrophages.     

TLR4/CD14-mediated PI3K activation is an essential component of interferon-dependent VSV resistance in macrophages

Phosphatidylinositol-3-phosphate kinase (PI3K) has been reported to exhibit anti-inflammatory roles as a negative modulator of the NF-κB pathway (MyD88- and Mal-dependent) triggered upon Toll-like receptor (TLR)4 activation by lipopolysaccharide (LPS). Here, we investigated the role of PI3K on the TLR4-dependent, MyD88-independent signaling cascade which is activated in macrophages infected by Vesicular Stomatitis Virus (VSV) and leads to interferon production, thus conferring antiviral protection. We show that VSV induces TLR4 (and CD14)-dependent Akt phosphorylation. We observed hypersusceptibility to viral infections after pharmacological inactivation of the PI3K pathway in macrophages, which indicates that normal PI3K functions are critical for type I interferon synthesis and viral resistance. Conversely, we noticed increased resistance in macrophages isolated from genetically modified mice in which the PI3K pathway is constitutively active. Our data, which demonstrate that PI3K-Akt axis is an important component of the TLR4-dependent antiviral mechanism, also indicate that pharmacological modulation of this pathway to regulate the inflammatory response could promote viral susceptibility.     

Contribution of interferon-beta to the immune activation induced by double-stranded DNA

Introducing double-stranded DNA (dsDNA) into the cytoplasm of macrophages and dendritic cells triggers the activation of these professional antigen-presenting cells (APCs). This process is characterized by the up-regulation of costimulatory molecules and the production of various cytokines, chemokines, and antibacterial/viral factors. Current findings indicate that interferon-beta (IFN-beta) plays a key role in the stimulatory cascade triggered by dsDNA. Both immune and non-immune cells respond to intracytoplasmic dsDNA by up-regulating IFN-beta) expression, a process that reduces host susceptibility to infection. The immune activation induced by dsDNA is independent of MyD88, TRIF and DNA-PKcs, indicating that a Toll-like receptor-independent mechanism underlies the cellular activation mediated by intracytoplasmic dsDNA.     

Differential induction of the toll-like receptor 4-MyD88-dependent and -independent signaling pathways by endotoxins

The biological response to endotoxin mediated through the Toll-like receptor 4 (TLR4)-MD-2 receptor complex is directly related to lipid A structure or configuration. Endotoxin structure may also influence activation of the MyD88-dependent and -independent signaling pathways of TLR4. To address this possibility, human macrophage-like cell lines (THP-1, U937, and MM6) or murine macrophage RAW 264.7 cells were stimulated with picomolar concentrations of highly purified endotoxins. Harvested supernatants from previously stimulated cells were also used to stimulate RAW 264.7 or 23ScCr (TLR4-deficient) macrophages (i.e., indirect induction). Neisseria meningitidis lipooligosaccharide (LOS) was a potent direct inducer of the MyD88-dependent pathway molecules tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 3alpha (MIP-3alpha), and the MyD88-independent molecules beta interferon (IFN-beta), nitric oxide, and IFN-gamma-inducible protein 10 (IP-10). Escherichia coli 55:B5 and Vibrio cholerae lipopolysaccharides (LPSs) at the same pmole/ml lipid A concentrations induced comparable levels of TNF-alpha, IL-1beta, and MIP-3alpha, but significantly less IFN-beta, nitric oxide, and IP-10. In contrast, LPS from Salmonella enterica serovars Minnesota and Typhimurium induced amounts of IFN-beta, nitric oxide, and IP-10 similar to meningococcal LOS but much less TNF-alpha and MIP-3alpha in time course and dose-response experiments. No MyD88-dependent or -independent response to endotoxin was seen in TLR4-deficient cell lines (C3H/HeJ and 23ScCr) and response was restored in TLR4-MD-2-transfected human embryonic kidney 293 cells. Blocking the MyD88-dependent pathway by DNMyD88 resulted in significant reduction of TNF-alpha release but did not influence nitric oxide release. IFN-beta polyclonal antibody and IFN-alpha/beta receptor 1 antibody significantly reduced nitric oxide release. N. meningitidis endotoxin was a potent agonist of both the MyD88-dependent and -independent signaling pathways of the TLR4 receptor complex of human macrophages. E. coli 55:B5 and Vibrio cholerae LPS, at the same picomolar lipid A concentrations, selectively induced the MyD88-dependent pathway, while Salmonella LPS activated the MyD88-independent pathway.     

Role of the Toll-like receptor pathway in the recognition of orthopedic implant wear-debris particles

The inflammatory response to prosthetic implant-derived wear particles is the primary cause of bone loss and aseptic loosening of implants, but the mechanisms by which macrophages recognize and respond to particles remain unknown. Studies of innate immunity demonstrate that Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPS). All TLRs signal through myeloid differentiation factor 88 (MyD88), except TLR3 which signals through TIR domain containing adapter inducing interferon-beta (TRIF), and TLR4 which signals through both MyD88 and TRIF. We hypothesized that wear-debris particles may act as PAMPs/DAMPs and activate macrophages via TLRs. To test this hypothesis, we first demonstrated that inhibition of MyD88 decreases polymethylmethacrylate (PMMA) particle-induced production of TNF-α in RAW 264.7 macrophages. Next we compared particle-induced production of TNF-α among MyD88 knockout (MyD88(-/-)), TRIF knockout (TRIF(-/-)), and wild type (WT) murine macrophages. Relative to WT, disruption of MyD88 signaling diminished, and disruption of TRIF amplified the particle-induced production of TNF-α. Gene expression data indicated that this latter increase in TNF-α was due to a compensatory increase in expression of MyD88 associated components of the TLR pathway. Finally, using an in?vivo model, MyD88(-/-) mice developed less particle-induced osteolysis than WT mice. These results indicate that the response to PMMA particles is partly dependent on MyD88, presumably as part of TLR signaling; MyD88 may represent a therapeutic target for prevention of wear debris-induced periprosthetic osteolysis.     

MyD88 is critical for the development of innate and adaptive immunity during acute lymphocytic choriomeningitis virus infection

We investigated the roles of Toll-like receptor 2 (TLR2) and myeloid differentiation factor 88 (MyD88) in the course of a lymphocytic choriomeningitis virus (LCMV) infection and revealed the following: (i) studies of transfected cells and murine peritoneal macrophages demonstrated that TLR2 and MyD88 are essential for the initial pro-inflammatory cytokine response (human IL-8, mouse IL-6) to LCMV; (ii) TLR2 knockout (KO) mice and MyD88 KO mice challenged with LCMV produced less IL-6 and monocyte chemotactic protein-1 in the serum than wild-type mice; (iii) in contrast to inflammatory cytokines, the production of type 1 IFN (IFN-alpha) in response to LCMV was MyD88 independent; (iv) MyD88 plays an essential role in antiviral CD8(+) T cell responses, CD8(+) T cells in MyD88 KO mice were defective in their expression of intracellular antiviral cytokines; and (v) the failure of MyD88 KO mice to activate CD8(+) T cells was accompanied by persistent viral infection in MyD88 KO mice. We demonstrate that TLR-mediated responses are important in the innate immune response to LCMV and that MyD88 is essential for the control of the LCMV infection and the maturation/activation of virus-specific CD8(+) T cells.     

Unexpected regulatory roles of TLR4 and TLR9 in experimental autoimmune encephalomyelitis

Innate immune mechanisms essential for priming encephalitogenic T cells in autoimmune neuroinflammation are poorly understood. Experimental autoimmune encephalomyelitis (EAE) is a IL-17-producing Th (Th17) cell-mediated autoimmune disease and an animal model of multiple sclerosis. To investigate how upstream TLR signals influence autoimmune T cell responses, we studied the role of individual TLR and MyD88, the common TLR adaptor molecule, in the initiation of innate and adaptive immune responses in EAE. Wild type (WT) C57BL/6, TLR-deficient and MyD88-deficient mice were immunized with myelin oligodendrocyte glycoprotein (MOG) in CFA. MyD88(-/-) mice were completely EAE resistant. Purified splenic myeloid DC (mDC) from MyD88(-/-) mice expressed much less IL-6 and IL-23, and serum and T cell IL-17 were absent. TLR4(-/-) and TLR9(-/-) mice surprisingly exhibited more severe EAE symptoms than WT mice. IL-6 and IL-23 expression by mDC and Th17 responses were higher in TLR4(-/-) mice, suggesting a regulatory role of TLR4 in priming Th17 cells. IL-6 expression by splenocytes was higher in TLR9(-/-) mice. Our data suggest that MyD88 mediates the induction of mDC IL-6 and IL-23 responses after MOG immunization, which in turn drives IL-17-producing encephalitogenic Th17 cell activation. Importantly, we demonstrate that TLR4 and TLR9 regulate disease severity in MOG-induced EAE.     

Toll样受体4信号转导研究进展

Toll样受体（Toll-like-receptors,TLRs）是一个主要分布于炎症细胞的识别病源分子的受体超家族,其中TLR4主要识别革兰阴性细菌细胞壁成分脂多糖（lipopolysaccharide,LPS）。LPS与TLR4结合后活化髓样分化因子88 (myeloid differentiation factor 88, MyD88)依赖性和非依赖性两条信号途径;前者活化丝裂原激活的蛋白激酶（mitogen-activated protein kinase,MAPK）和核因子-κB（nuclear factor kappa B,NF-κB）信号通路,后者活化NF-κB和干扰素调节因子-3(IFN-regulated factor-3,IRF3)信号通路。通过这些信号途径TLR4诱导炎症细胞释放炎症因子介导炎症反应;同时TLR4通过活化树突状细胞促进抗原递呈,介导先天性免疫向获得性免疫的转化。此外,TLR4能诱导磷脂酰肌醇-3激酶-蛋白激酶B（PI3K-AKT）的信号转导,LPS介导的细胞存活和增殖与TLR4活化 PI3K-AKT途径有关。     

GM-CSF regulates a PU.1-dependent transcriptional program determining the pulmonary response to LPS

Alveolar macrophages (AMs) normally respond to lipopolysaccharide (LPS) by activating Toll-like receptor (TLR)-4 signaling, a mechanism critical to lung host defense against gram-negative bacteria such as Pseudomonas aeruginosa. Because granulocyte macrophage colony-stimulating factor (GM-CSF)-deficient (GM(-/-)) mice are hyporesponsive to LPS, we evaluated the role of GM-CSF in TLR-4 signaling in AMs. Pulmonary TNF-alpha levels and neutrophil recruitment 4 h after intratracheal administration of Pseudomonas LPS were reduced in GM(-/-) compared with wild-type (GM(+/+)) mice. Secretion of TNF-alpha by AMs exposed to LPS ex vivo was also reduced in GM(-/-) mice and restored in mice expressing GM-CSF specifically in the lungs (SPC-GM(+/+)/GM(-/-) mice). LPS-dependent NF-kappaB promoter activity, TNF-alpha secretion, and neutrophil chemokine release were reduced in AM cell lines derived from GM(-/-) mice (mAM) compared with GM(+/+) (MH-S). Retroviral expression of PU.1 in mAM cells, which normally lack PU.1, rescued all of these AM defects. To determine whether GM-CSF, via PU.1, regulated expression of TLR-4 pathway components, mRNA and protein levels for key components were evaluated in MH-S cells (GM(+/+), PU.1(Positive)), mAM cells (GM(-/-), PU.1(Negative)), and mAMPU.1+ cells (GM(-/-), PU.1(Positive)). Cluster of differentiation antigen-14, radioprotective 105, IL-1 receptor-associated kinase (IRAK)-M mRNA, and protein were dependent upon GM-CSF and restored by expression of PU.1. In contrast, expression of other TLR-4 pathway components (myeloid differentiation-2, TLR-4, IRAK-1, IRAK-2, Toll/IL-1 receptor domain containing adapter protein/MyD88 adaptor-like, myeloid differentiation primary-response protein 88, IRAK-4, TNF receptor-associated factor-6, NF-kappaB, inhibitor of NF-kappaB kinase) were not GM-CSF or PU.1-dependent. These results show that GM-CSF, via PU.1, enables AM responses to P. aeruginosa LPS by regulating expression of a specific subset of components of the TLR-4 signaling pathway.     

MYD88‐dependent and ‐independent activation of TREM‐1 via specific TLR ligands

Triggering receptor expressed on myeloid cells (TREM)‐1 plays an important role in myeloid cell‐activated inflammatory responses. Although TLR ligands such as LPS and lipoteichoic acid have been shown to upregulate TREM‐1 expression in macrophage and neutrophils, the role of specific TLR in inducing the expression of TREM‐1 remains unclear. In this study, we investigated whether the presence of TLR is necessary for the expression of TREM‐1. We show that BM‐derived macrophages from TLR4 and TLR2 KO mice failed to induce expression of TREM‐1 message and protein in response to their specific ligands. Interestingly, the expression of TREM‐1 in response to LPS is not altered in myeloid differentiation factor 88 (MyD88) KO macrophages, suggesting that downstream of TLR a MyD88‐independent pathway induces the expression of TREM‐1. Inhibiting toll/IL‐1R domain‐containing adaptor‐inducing IFN‐β (TRIF) expression by siRNA decreased TREM‐1 expression in response to LPS, suggesting that the expression of TREM‐1 in response to LPS was mediated by the TRIF signaling pathway. On the other hand, the expression of TREM‐1 in response to lipoteichoic acid is dependent on MyD88 expression. These data indicate that the expression of TREM‐1 in response to TLR ligands occurs secondary to downstream signaling events and that the presence of TLR is necessary for the expression of TREM‐1 in response to their specific ligands. However, the downstream signaling required for the expression of TREM‐1 is dependent on the stimulus and the surface receptor through which the signaling is initiated.     

The Toll-IL-1 receptor adaptor family grows to five members

Toll-like receptor (TLR) signal transduction is mediated by an adaptor protein termed MyD88. In the case of TLR2 and TLR4, another adaptor related to MyD88 called Mal also participates in signalling. Two recent papers have added a third adaptor to the family, called Toll-interleukin-1 receptor (TIR) domain-containing adaptor inducing interferon-beta (IFN-beta) (TRIF) or TIR-containing adaptor molecule-1 (TICAM-1), which is particularly important for IFN regulatory factor-3 (IRF-3) activation by antiviral TLR3. Two additional adaptors are present in humans, termed Trif-related adaptor molecule (TRAM) and sterile alpha and HEAT-Armadillo motifs (SARM). It is probable that differential use of adaptors will help explain the distinct pathways activated by TLRs during host defence.     

Toll-like receptor signal adaptor protein MyD88 is required for sustained endotoxin-induced acute hypoferremic response in mice

Hypoferremia, associated with immune system activation, involves a marked reduction in the levels of circulating iron, coupled with iron sequestration within macrophages. Toll-like receptor (TLR) signaling plays an important role in the development of the hypoferremic response, but how downstream signaling events affect genes involved in iron metabolism is incompletely understood. We investigated the involvement of MyD88-dependent (MyD88) and MyD88-independent (TRIF) TLR signaling in the development of hypoferremia. Using MyD88-deficient and TRIF-deficient mice, we show that MyD88 and TRIF signaling pathways are critical for up-regulation by lipopolysaccharide (LPS) of the iron regulator hepcidin. In addition, MyD88 signaling is required for the induction of lipocalin 2 secretion and iron sequestration in the spleen. Activation of TLR4 and TLR3 signaling through LPS and polyinosinic:polycytidylic acid [poly(I:C)] treatments resulted in rapid down-regulation of HFE protein [encoded by the hemochromatosis gene (Hfe)] and ferroportin [encoded by solute carrier family 40 (iron-regulated transporter), member 1 (Slc40a1)] expression in the spleen, independent of MyD88 or TRIF signaling and proinflammatory cytokine production. However, lack of MyD88 signaling significantly impaired the hypoferremic response triggered by LPS, indicating that ferroportin and HFE protein down-regulation alone are insufficient to maintain hypoferremia. The extent of the hypoferremic response was found to be limited by initial, basal iron levels. Together, these results suggest that targeting specific TLR signaling pathways by affecting the function of adaptor molecules may provide new strategies to counteract iron sequestration within macrophages during inflammation.