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.     

Unresponsiveness of MyD88-deficient mice to endotoxin.

MyD88 is a general adaptor protein that plays an important role in the Toll/IL-1 receptor family signalings. Recently, Toll-like receptors 2 and 4 (TLR2 and TLR4) have been suggested to be the signaling receptors for lipopolysaccharide (LPS). In this study, we demonstrate that MyD88 knockout mice lack the ability to respond to LPS as measured by shock response, B cell proliferative response, and secretion of cytokines by macrophages and embryonic fibroblasts. However, activation of neither NF-kappaB nor the mitogen-activated protein (MAP) kinase family is abolished in MyD88 knockout mice. These findings demonstrate that signaling via MyD88 is essential for LPS response, but the inability of MyD88 knockout mice to induce LPS-dependent gene expression cannot simply be attributed to lack of the activation of MAP kinases and NF-kappaB.     

The role of MyD88 and TLR4 in the LPS-mimetic activity of Taxol

Taxol can mimic bacterial lipopolysaccharide (LPS) by activating mouse macrophages in a cell cycle-independent, LPS antagonist-inhibitable manner. Macrophages from C3H/HeJ mice, which have a spontaneous mutation in Toll-like receptor 4 (TLR4), are hyporesponsive to both LPS and Taxol, suggesting that LPS and Taxol may share a signaling pathway involving TLR4. To determine whether TLR4 and its interacting adaptor molecule MyD88 are necessary for Taxol's LPS mimetic actions, we examined Taxol responses of primary macrophages from genetically defective mice lacking either TLR4 (C57BL/10ScNCr) or MyD88 (MyD88 knockout). When stimulated with Taxol, macrophages from wild-type mice responded robustly by secreting both TNF and NO, while macrophages from either TLR4-deficient C57BL/10ScNCr mice or MyD88 knockout mice produced only minimal amounts of TNF and NO. Taxol-induced NF-kappa B-driven luciferase activity was reduced after transfection of RAW 264.7 macrophages with a dominant negative version of mouse MyD88. Taxol-induced microtubule-associated protein kinase (MAPK) activation and NF-kappa B nuclear translocation were absent from TLR4-null macrophages, but were preserved in MyD88 knockout macrophages with a slight delay in kinetics. Neither Taxol-induced NF-kappa B activation, nor I kappa B degradation was affected by the presence of phosphatidylinositol 3-kinase inhibitors. These results suggest that Taxol and LPS not only share a TLR4/MyD88-dependent pathway in generating inflammatory mediators, but also share a TLR4-dependent/MyD88-independent pathway leading to activation of MAPK and NF-kappa B.     

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.     

SPOP negatively regulates Toll-like receptor-induced inflammation by disrupting MyD88 self-association

Toll-like receptor (TLR) signaling pathways need to be tightly controlled to avoid excessive inflammation and unwanted damage to the host. Myeloid differentiation primary response gene 88 (MyD88) is a critical adaptor of TLR signaling. Here, we identified the speckle-type POZ protein (SPOP) as a MyD88-associated protein. SPOP was recruited to MyD88 following TLR4 activation. TLR4 activation also caused the translocation of SPOP from the nucleus to the cytoplasm. SPOP depletion promoted the aggregation of MyD88 and recruitment of the downstream signaling kinases IRAK4, IRAK1 and IRAK2. Consistently, overexpression of SPOP inhibited the TLR4-mediated activation of NF-κB and production of inflammatory cytokines, whereas SPOP depletion had the opposite effects. Furthermore, knockdown of SPOP increased MyD88 aggregation and inflammatory cytokine production upon TLR2, TLR7 and TLR9 activation. Our findings reveal a mechanism by which MyD88 is regulated and highlight a role for SPOP in limiting inflammatory responses.     

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.     

Deficiency in IRAK4 activity attenuates manifestations of murine Lupus

Interleukin‐1 receptor‐associated kinase (IRAK) 4 mediates host defense against infections. As an active kinase, IRAK4 elicits full spectra of myeloid differentiation primary response protein (MyD) 88‐dependent responses, while kinase‐inactive IRAK4 induces a subset of cytokines and negative regulators whose expression is not regulated by mRNA stability. IRAK4 kinase activity is critical for resistance against Streptococcus pneumoniae, but its involvement in autoimmunity is incompletely understood. In this study, we determined the role of IRAK4 kinase activity in murine lupus. Lupus development in BXSB mice expressing the Y chromosome autoimmunity accelerator (Yaa) increased basal and Toll‐like receptor (TLR) 4/7‐induced phosphorylation of mitogen‐activated protein kinases, p65 nuclear factor‐κB (NF‐κB), enhanced tumor necrosis factor (TNF)‐α and C‐C motif chemokine ligand (CCL) 5 gene expression in splenic macrophages, but decreased levels of Toll‐interacting protein and IRAK‐M, without affecting IRAK4 or IRAK1 expression. Mice harboring kinase‐inactive IRAK4 on the lupus‐prone Yaa background manifested blunted TLR signaling in macrophages and reduced glomerulonephritis, splenomegaly, serum anti‐nuclear antibodies, numbers of splenic macrophages, total and TNF‐α⁺ dendritic cells, activated T‐ and B‐lymphocytes, and lower TNF‐α expression in macrophages compared with lupus‐prone mice with functional IRAK4. Thus, IRAK4 kinase activity contributes to murine lupus and could represent a new therapeutic target.     

Wound healing is impaired in MyD88-deficient mice: a role for MyD88 in the regulation of wound healing by adenosine A2A receptors

Synergy between Toll-like receptor (TLR) and adenosine A2A receptor (A2AR) signaling switches macrophages from production of inflammatory cytokines such as tumor necrosis factor-alpha to production of the angiogenic growth factor vascular endothelial growth factor (VEGF). We show in this study that this switch critically requires signaling through MyD88, IRAK4, and TRAF6. Macrophages from mice lacking MyD88 (MyD88(-/-)) or IRAK4 (IRAK4(-/-)) lacked responsiveness to TLR agonists and did not respond to A2AR agonists by expressing VEGF. Suppression of TRAF6 expression with siRNA in RAW264.7 macrophages also blocked their response to TLR and A2AR agonists. Excisional skin wounds in MyD88(-/-) mice healed at a markedly slower rate than wounds in wild-type MyD88(+/+) mice, showing delayed contraction, decreased and delayed granulation tissue formation, and reduced new blood vessel density. Although macrophages accumulated to higher levels in MyD88(-/-) wounds than in controls, expression of VEGF and HIF1-alpha mRNAs was elevated in MyD88(+/+) wounds. CGS21680, an A2AR agonist, promoted repair in MyD88(+/+) wounds and stimulated angiogenesis but had no significant effect on healing of MyD88(-/-) wounds. These results suggest that the synergistic interaction between TLR and A(2A)R signaling observed in vitro that switches macrophages from an inflammatory to an angiogenic phenotype also plays a role in wound healing in vivo.     

Cellular responses to bacterial cell wall components are mediated through MyD88-dependent signaling cascades

MyD88 is an adaptor molecule essential for signaling via the Toll-like receptor (TLR)/IL-1 receptor family. TLR4 is a member of the TLR family and a point mutation in the Tlr4 gene causes hyporesponsiveness to lipopolysaccharide (LPS) in C3H/HeJ mice. We have previously shown that both TLR4- and MyD88-deficient mice are hyporesponsive to LPS. In this study we examined the responsiveness of these two knockout mice to various bacterial cell wall components. Cells from TLR4-deficient mice responded to several kinds of LPS, peptidoglycan and crude cell wall preparation from Gram-positive bacteria and mycobacterial lysates. In contrast, macrophages and splenocytes from MyD88-deficient mice did not respond to any of the bacterial components we tested. These results show that MyD88 is essential for the cellular response to bacterial cell wall components.     

Key role of proline-rich tyrosine kinase 2 in interleukin-8 (CXCL8/IL-8)-mediated human neutrophil chemotaxis

The signalling pathways leading to CXCL8/IL-8-induced human neutrophil migration have not been fully characterized. The present study demonstrates that CXCL8 induces tyrosine phosphorylation as well as enzymatic activity of proline-rich tyrosine kinase 2 (Pyk2), a non-receptor protein tyrosine kinase (PTK), in human neutrophils. Induction of Pyk2 tyrosine phosphorylation by CXCL8 is regulated by Src PTK activation, whereas it is unaffected by phosphatidylinositol 3-kinase activation. Inhibition of Pyk2 activation by PP1, a Src PTK inhibitor, is paralleled by the inhibition of CXCL8-mediated neutrophil chemotaxis. Among CXCL8 receptors, Src protein tyrosine kinase activation selectively regulates CXCR1-mediated polymorphonuclear neutrophil (PMN) chemotaxis. Overexpression of PykM, the kinase-dead mutant of Pyk2, blocks CXCL8-induced chemotaxis of HL-60-derived PMN-like cells, thus pinpointing the key role of Pyk2 in CXCL8-induced chemotaxis.     

Negative regulation by SHPS-1 of Toll-like receptor-dependent proinflammatory cytokine production in macrophages

SHPS-1 is a transmembrane protein predominantly expressed in macrophages. The possible role of SHPS-1 in regulation of Toll-like receptor (TLR)-dependent production of proinflammatory cytokines by macrophages has remained unknown, however. We now show that expression either of a mutant version of mouse SHPS-1 (SHPS-1–4F) in which the four tyrosine phosphorylation sites in the cytoplasmic region are replaced by phenylalanine or of a chimeric protein comprising the extracellular and transmembrane regions of human CD8 fused to the cytoplasmic region of SHPS-1–4F (CD8–4F) markedly promoted the production of tumor necrosis factor-α (TNF-α) or interleukin-6 (IL-6) induced by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid [poly(I : C)] in RAW264.7 macrophages. In contrast, expression of a mutant form of SHPS-1 that lacks most of the cytoplasmic region did not promote such responses. Expression of SHPS-1–4F promoted the LPS- or poly(I : C)-induced activation of NF-κB. LPS and poly(I : C) each induced the tyrosine phosphorylation of SHPS-1 through a Src family kinase and the association of SHPS-1 with SHP-1 and SHP-2. These results suggest that LPS or poly(I : C) induces tyrosine phosphorylation of SHPS-1 and the association of SHPS-1 with SHP-1 and SHP-2 in a manner dependent on a Src family kinase. SHPS-1 then negatively regulates TLR4- or TLR3-dependent cytokine production through inhibition of NF-κB-dependent signaling.     

Syk kinase is required for collaborative cytokine production induced through Dectin-1 and Toll-like receptors

Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal beta-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF, MIP-1alpha and MIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kappaB (IkappaB), enhancing NFkappaB nuclear translocation. These findings establish the first example of Syk- and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.     

Mechanism of pathogen-specific TLR4 activation in the mucosa: fimbriae, recognition receptors and adaptor protein selection

The mucosal host defence discriminates pathogens from commensals, and prevents infection while allowing the normal flora to persist. Paradoxically, Toll-like receptors (TLR) control the mucosal defence against pathogens, even though the TLR recognise conserved molecules like LPS, which are shared between pathogens and commensals. This study proposes a mechanism of pathogen-specific mucosal TLR4 activation, involving adhesive ligands and their host cell receptors. TLR4 signalling was activated in CD14-negative, LPS-unresponsive epithelial cells by P fimbriated, uropathogenic Escherichia coli but not by a mutant lacking fimbriae. Epithelial TLR4 signalling in vivo involved the glycosphingolipid receptors for P fimbriae and the adaptor proteins Toll/IL-1R (TIR) domain-containing adaptor inducing IFN-beta (TRIF)/TRIF-related adaptor molecule (TRAM), but myeloid differentiation protein 88 (MyD88)/TIR domain-containing adaptor protein were not required for the epithelial response. Substituting the P fimbriae with type 1 fimbriae changed TLR4 signalling from the TRIF to the MyD88 adaptor pathway. In addition, the adaptor proteins and the fimbrial type were found to influence bacterial clearance. Trif(-/-) and Tram(-/-) mice remained infected with P fimbriated E. coli but cleared the type 1 fimbriated strain, while Myd88(-/-) mice became carriers of both the P and the type 1 fimbriated bacteria. Thus, TLR4 may be engaged specifically by pathogens, when the proper cell surface receptors are engaged by virulence ligands.