Role of CD14 in a Mouse Model of Acute Lung Inflammation Induced by Different Lipopolysaccharide Chemotypes

Background

Recognition of lipopolysaccharide (LPS) is required for effective defense against invading gram-negative bacteria. Recently, in vitro studies revealed that CD14 is required for activation of the myeloid differentiation factor (MyD)88-dependent Toll-like receptor (TLR)4 signaling pathway by smooth (S)-LPS, but not by rough (R)-LPS. The present study investigated the role of CD14 in induction of lung inflammation in mice by these different LPS chemotypes.

Methodology/Results

Neutrophil accumulation and tumor necrosis factor (TNF) release in bronchoalveolar lavage fluid were determined 6 hours after intranasal treatment of wild type (WT) and CD14 knock-out (KO) mice with different doses S-LPS or R-LPS. The contribution of CD14 to lung inflammation induced by S-LPS or R-LPS depended on the LPS dose. At low doses, S-LPS and R-LPS induced neutrophil influx in a CD14-dependent manner. Low dose S-LPS-induced cytokine release also depended on CD14. Strikingly, neutrophil influx and TNF release induced by high dose S-LPS or R-LPS was diminished in the presence of CD14. Intranasal administration of sCD14 to CD14 KO mice treated with S-LPS partially reversed the inflammatory response to the response observed in WT mice.

Conclusions

In conclusion, CD14 modulates effects of both S-LPS and R-LPS within the lung in a similar way. Except for R-LPS-induced TNF release, S-LPS and R-LPS at low dose induced acute lung inflammation in a CD14-dependent manner, while the inflammatory response triggered by high dose S-LPS or R-LPS was diminished by CD14.     

Liposomal Lipopolysaccharide Initiates TRIF-Dependent Signaling Pathway Independent of CD14

Lipopolysaccharide (LPS) is recognized by CD14 with Toll-like receptor 4 (TLR4), and initiates 2 major pathways of TLR4 signaling, the MyD88-dependent and TRIF-dependent signaling pathways. The MyD88-dependent pathway induces inflammatory responses such as the production of TNF-α, IL-6, and IL-12 via the activation of NFκB and MAPK. The TRIF-dependent pathway induces the production of type-I IFN, and RANTES via the activation of IRF-3 and NFκB, and is also important for the induction of adaptive immune responses. CD14 plays a critical role in initiating the TRIF-dependent signaling pathway response to LPS, to support the internalization of LPS via endocytosis. Here, we clearly demonstrate that intracellular delivery of LPS by LPS-formulated liposomes (LPS-liposomes) initiate only TRIF-dependent signaling via clathrin-mediated endocytosis, independent of CD14. In fact, LPS-liposomes do not induce the production of TNF-α and IL-6 but induce RANTES production in peritoneal macrophages. Additionally, LPS-liposomes could induce adaptive immune responses effectively in CD14-deficient mice. Collectively, our results strongly suggest that LPS-liposomes are useful as a TRIF-dependent signaling-based immune adjuvant without inducing unnecessary inflammation.     

Absence of TRIF signaling in lipopolysaccharide-stimulated murine mast cells

In macrophages, two signaling pathways, dependent on MyD88 or TIR domain-containing adaptor-inducing IFN-β (TRIF) signaling, emanate from the LPS receptor TLR4/MD-2. In this study, we show that in murine bone marrow-derived mast cells (BMMCs), only the MyD88-dependent pathway is activated by LPS. The TRIF signaling branch leading both to NF-κB activation and enhanced proinflammatory cytokine production, as well as to IRF3 activation and subsequent IFN-β production, is absent in LPS-stimulated BMMCs. IRF3 activation is also absent in peritoneal mast cells from LPS-injected mice. We observed strongly diminished TRAM expression in BMMCs, but overexpression of TRAM only moderately enhanced IL-6 and did not boost IFN-β responses to LPS in these cells. A combination of very low levels of TRAM and TLR4/MD-2 with the known absence of membrane-bound CD14 are expected to contribute to the defective TRIF signaling in mast cells. We also show that, unlike in macrophages, in BMMCs the TRIF-dependent and -independent IFN-αβ responses to other recognized IFN inducers (dsRNA, adenovirus, and B-DNA) are absent. These results show how the response to the same microbial ligand using the same receptor can be regulated in different cell types of the innate immune system.     

Genome-wide expression profiling and mutagenesis studies reveal that lipopolysaccharide responsiveness appears to be absolutely dependent on TLR4 and MD-2 expression and is dependent upon intermolecular ionic interactions

Lipid A (a hexaacylated 1,4' bisphosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. In this article, we define the role of TLR4 and MD-2 in LPS signaling by using genome-wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulation with peptidoglycan-free LPS and synthetic Escherichia coli lipid A. Of the 1396 genes significantly induced or repressed by any one of the treatments in the wild-type macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin and that both are required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. According to our murine TLR4/MD-2-activation model, the two phosphates on lipid A were predicted to interact extensively with the two positively charged patches on mouse TLR4. When either positive patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were nearly abrogated. However, the MyD88-dependent and -independent pathways were impaired to the same extent, indicating that the adjuvant activity of monophosphorylated lipid A most likely arises from its decreased potential to induce an active receptor complex and not more downstream signaling events. Hence, we concluded that ionic interactions between lipid A and TLR4 are essential for optimal LPS receptor activation.     

Toll-like receptor 2 is required for LPS-induced Toll-like receptor 4 signaling and inhibition of ion transport in renal thick ascending limb

Previously we demonstrated that basolateral LPS inhibits HCO(3)(-) absorption in the renal medullary thick ascending limb (MTAL) through TLR4-dependent ERK activation. Here we report that the response of the MTAL to basolateral LPS requires TLR2 in addition to TLR4. The basolateral addition of LPS (ultrapure Escherichia coli K12) decreased HCO(3)(-) absorption in isolated, perfused MTALs from wild-type mice but had no effect in MTALs from TLR2(-/-) mice. In contrast, inhibition of HCO(3)(-) absorption by lumen LPS was preserved in TLR2(-/-) MTALs, indicating that TLR2 is involved specifically in mediating the basolateral LPS response. LPS also did not increase ERK phosphorylation in MTALs from TLR2(-/-) mice. TLR2 deficiency had no effect on expression of TLR4, MD-2, or MyD88. However, LPS-induced recruitment of MyD88 to the basolateral membrane was impaired in TLR2(-/-) MTALs. Inhibition of HCO(3)(-) absorption by LPS did not require CD14. Co-immunoprecipitation studies demonstrated an association between TLR4 and TLR2. Inhibition of HCO(3)(-) absorption by TLR2-specific ligands was preserved in MTALs from TLR4(-/-) mice. These results indicate that the effect of basolateral LPS to inhibit HCO(3)(-) absorption in the MTAL through MyD88-dependent ERK activation depends on a novel interaction between TLR4 and TLR2. TLR2 plays a dual role in the induction of intracellular signals that impair MTAL function, both through cooperation with TLR4 to mediate ERK signaling by LPS and through a TLR4-independent signaling pathway activated by Gram-positive bacterial ligands. Regulation of TLR2 expression and its interaction with TLR4 may provide new mechanisms for controlling and therapeutic targeting of TLR4-mediated LPS responses.     

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.     

The MyD88-dependent, but not the MyD88-independent, pathway of TLR4 signaling is important in clearing nontypeable haemophilus influenzae from the mouse lung

TLRs are important for the recognition of conserved motifs expressed by invading bacteria. TLR4 is the signaling receptor for LPS, the major proinflammatory component of the Gram-negative cell wall, whereas CD14 serves as the ligand-binding part of the LPS receptor complex. Triggering of TLR4 results in the activation of two distinct intracellular pathways, one that relies on the common TLR adaptor MyD88 and one that is mediated by Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF). Nontypeable Haemophilus influenzae (NTHi) is a common Gram-negative respiratory pathogen that expresses both TLR4 (LPS and lipooligosaccharide) and TLR2 (lipoproteins) ligands. To determine the roles of CD14, TLR4, and TLR2 during NTHi pneumonia, the following studies were performed: 1) Alveolar macrophages from CD14 and TLR4 knockout (KO) mice were virtually unresponsive to NTHi in vitro, whereas TLR2 KO macrophages displayed a reduced NTHi responsiveness. 2) After intranasal infection with NTHi, CD14 and TLR4 KO mice showed an attenuated early inflammatory response in their lungs, which was associated with a strongly reduced clearance of NTHi from the respiratory tract; in contrast, in TLR2 KO mice, lung inflammation was unchanged, and the number of NTHi CFU was only modestly increased at the end of the 10-day observation period. 3) MyD88 KO, but not TRIF mutant mice showed an increased bacterial load in their lungs upon infection with NTHi. These data suggest that the MyD88-dependent pathway of TLR4 is important for an effective innate immune response to respiratory tract infection caused by NTHi.     

Differential effects of a Toll-like receptor antagonist on Mycobacterium tuberculosis-induced macrophage responses

We previously showed that viable Mycobacterium tuberculosis (Mtb) bacilli contain distinct ligands that activate cells via the mammalian Toll-like receptor (TLR) proteins TLR2 and TLR4. We now demonstrate that expression of a dominant negative TLR2 or TLR4 proteins in RAW 264.7 macrophages partially blocked Mtb-induced NF-kappa B activation. Coexpression of both dominant negative proteins blocked virtually all Mtb-induced NF-kappa B activation. The role of the TLR4 coreceptor MD-2 was also examined. Unlike LPS, Mtb-induced macrophage activation was not augmented by overexpression of ectopic MD-2. Moreover, cells expressing an LPS-unresponsive MD-2 mutant responded normally to Mtb. We also observed that the lipid A-like antagonist E5531 specifically inhibited TLR4-dependent Mtb-induced cellular responses. E5531 could substantially block LPS- and Mtb-induced TNF-alpha production in both RAW 264.7 cells and primary human alveolar macrophages (AM phi). E5531 inhibited Mtb-induced AM phi apoptosis in vitro, an effect that was a consequence of the inhibition of TNF-alpha production by E5531. In contrast, E5531 did not inhibit Mtb-induced NO production in RAW 264.7 cells and AM phi. Mtb-stimulated peritoneal macrophages from TLR2- and TLR4-deficient animals produced similar amounts of NO compared with control animals, demonstrating that these TLR proteins are not required for Mtb-induced NO production. Lastly, we demonstrated that a dominant negative MyD88 mutant could block Mtb-induced activation of the TNF-alpha promoter, but not the inducible NO synthase promoter, in murine macrophages. Together, these data suggest that Mtb-induced TNF-alpha production is largely dependent on TLR signaling. In contrast, Mtb-induced NO production may be either TLR independent or mediated by TLR proteins in a MyD88-independent manner.     

Wolbachia endosymbiotic bacteria of Brugia malayi mediate macrophage tolerance to TLR- and CD40-specific stimuli in a MyD88/TLR2-dependent manner

Lymphatic filarial nematodes are able to down-regulate parasite-specific and nonspecific responses of lymphocytes and APC. Lymphatic filariae are reliant on Wolbachia endosymbiotic bacteria for development and survival. We tested the hypothesis that repeated exposure to Wolbachia endosymbionts would drive macrophage tolerance in vitro and in vivo. We pre-exposed murine peritoneal-elicited macrophages to soluble extracts of Brugia malayi female worms (BMFE) before restimulating with BMFE or TLR agonists. BMFE tolerized macrophages (in terms of IFN-beta, IL-1beta, IL-6, IL-12p40, and TNF-alpha inflammatory cytokine production) in a dose-dependent manner toward self, LPS, MyD88-dependent TLR2 or TLR9 ligands (peptidoglycan, triacyl lipopeptide, CpG DNA) and the MyD88-independent/TRIF-dependent TLR3 ligand, polyinosinic-polycytidylic acid. This was accompanied with down-regulation in surface expression of TLR4 and up-regulation of CD14, CD40, and TLR2. BMFE tolerance extended to CD40 activation in vitro and systemic inflammation following lethal challenge in an in vivo model of endotoxin shock. The mechanism of BMFE-mediated macrophage tolerance was dependent on MyD88 and TLR2 but not TLR4. Evidence that desensitization was driven by Wolbachia-specific ligands was determined by use of extracts from Wolbachia-depleted B. malayi, aposymbiotic filarial species, and a cell line stably infected with Wolbachia pipientis. Our data promote a role for Wolbachia in contributing toward the dysregulated and tolerized immunological phenotype that accompanies the majority of human filarial infections.     

Sphingomyelin synthase activity affects TRIF-dependent signaling of Toll-like receptor 4 in cells stimulated with lipopolysaccharide

Bacterial lipopolysaccharide (LPS) is recognized by CD14 protein and the Toll-like receptor (TLR)4/MD2 complex localized in the plasma membrane of immune cells. TLR4 triggers two signaling pathways engaging the MyD88 and TRIF adaptor proteins which lead to production of various pro-inflammatory cytokines. These processes are likely to be modulated by sphingomyelin, as the CD14 - TLR4 interaction takes place in plasma membrane rafts enriched in this lipid. To verify this assumption, we analyzed the influence of tricyclodecane-9-yl xanthogenate (D609), which was proven here to be an SMS inhibitor, and silencing of sphingomyelin synthase (SMS) 1 and/or SMS2 on LPS-induced signaling in macrophages. LPS up-regulated the expression and activity of SMS while exposure to D609 or silencing of SMS1 and SMS2 counteracted this action and led (except for SMS2 silencing) to a depletion of sphingomyelin in cells. Concomitantly, the MyD88- and TRIF-dependent signaling pathways of TLR4 were inhibited with the latter being especially sensitive to the reduction of the SMS1 and/or SMS2 activity. The D609 treatment and SMS1 and/or SMS2 depletion all reduced the level of CD14 protein in cells, which likely was an important determinant of the reduction of the LPS-induced pro-inflammatory responses.     

Synthetic glycolipid-based TLR4 antagonists negatively regulate TRIF-dependent TLR4 signalling in human macrophages

TLRs, including TLR4, play a crucial role in inflammatory-based diseases, and TLR4 has been identified as a therapeutic target for pharmacological intervention. In previous studies, we investigated the potential of FP7, a novel synthetic glycolipid active as a TLR4 antagonist, to inhibit haematopoietic and non-haematopoietic MyD88-dependent TLR4 pro-inflammatory signalling. The main aim of this study was to investigate the action of FP7 and its derivative FP12 on MyD88-independent TLR4 signalling in THP-1 derived macrophages. Western blotting, Ab array and ELISA approaches were used to explore the effect of FP7 and FP12 on TRIF-dependent TLR4 functional activity in response to LPS and other endogenous TLR4 ligands in THP-1 macrophages. A different kinetic in the inhibition of endotoxin-driven TBK1, IRF3 and STAT1 phosphorylation was observed using different LPS chemotypes. Following activation of TLR4 by LPS, data revealed that FP7 and FP12 inhibited TBK1, IRF3 and STAT1 phosphorylation which was associated with down-regulation IFN-β and IP-10. Specific blockage of the IFN type one receptor showed that these novel molecules inhibited TRIF-dependent TLR4 signalling via IFN-β pathways. These results add novel information on the mechanism of action of monosaccharide FP derivatives. The inhibition of the TRIF-dependent pathway in human macrophages suggests potential therapeutic uses for these novel TLR4 antagonists in pharmacological interventions on inflammatory diseases.     

Signaling of apoptosis through TLRs critically involves toll/IL-1 receptor domain-containing adapter inducing IFN-beta, but not MyD88, in bacteria-infected murine macrophages

TLRs are important sensors of the innate immune system that serve to identify conserved microbial components to mount a protective immune response. They furthermore control the survival of the challenged cell by governing the induction of pro- and antiapoptotic signaling pathways. Pathogenic Yersinia spp. uncouple the balance of life and death signals in infected macrophages, which compels the macrophage to undergo apoptosis. The initiation of apoptosis by Yersinia infection specifically involves TLR4 signaling, although Yersinia can activate TLR2 and TLR4. In this study we characterized the roles of downstream TLR adapter proteins in the induction of TLR-responsive apoptosis. Experiments using murine macrophages defective for MyD88 or Toll/IL-1R domain-containing adapter inducing IFN-beta (TRIF) revealed that deficiency of TRIF, but not of MyD88, provides protection against Yersinia-mediated cell death. Similarly, apoptosis provoked by treatment of macrophages with the TLR4 agonist LPS in the presence of a proteasome inhibitor was inhibited in TRIF-defective, but not in MyD88-negative, cells. The transfection of macrophages with TRIF furthermore potently promoted macrophage apoptosis, a process that involved activation of a Fas-associated death domain- and caspase-8-dependent apoptotic pathway. These data indicate a crucial function of TRIF as proapoptotic signal transducer in bacteria-infected murine macrophages, an activity that is not prominent for MyD88. The ability to elicit TRIF-dependent apoptosis was not restricted to TLR4 activation, but was also demonstrated for TLR3 agonists. Together, these results argue for a specific proapoptotic activity of TRIF as part of the host innate immune response to bacterial or viral infection.     

Signaling events leading to peroxiredoxin 5 up-regulation in immunostimulated macrophages

Peroxiredoxins (PRXs) are thiol peroxidases associated with many cellular functions including proliferation, cell cycle, apoptosis, and differentiation. There is also increasing evidence that these ubiquitous antioxidant enzymes control H₂O₂ signaling in eukaryotes. Here, we provide evidence that the LPS/TLR4 and the Th1 cytokine IFN-γ pathways induce expression of PRX5, a potent peroxide and peroxynitrite reductase, in primary macrophages. Furthermore, deletion of TRIF, MyD88, or type I IFN receptor revealed that the LPS/TLR4-dependent increase in PRX5 expression is mediated by a TRIF-dependent/IFN-β-independent pathway. IFN-γ-dependent induction of the PRX5 gene was markedly reduced in MyD88^−/− and TNF^−/− macrophages. Moreover, addition of exogenous TNF allowed the recovery of full PRX5 expression in both MyD88^−/− and TNF^−/− cells stimulated with IFN-γ, suggesting that basal TNF produced in an MyD88-dependent manner contributes to PRX5 induction. Downstream of the TLR pathways, we have explored the role of MAPK activation and found that p38 and JNK mainly contribute to PRX5 up-regulation in immunostimulated macrophages. Expression of PRX5 is thus responsive to innate immunity signals, and we propose that PRX5 is an additional host defense weapon of activated macrophages.     

Overlapping and distinct roles of GRK5 in TLR2-, and TLR3-induced inflammatory response in vivo

G-protein coupled receptor kinase-5 (GRK5) is a recently described NFκB regulator in TLR4 signaling pathway. To determine whether the role of GRK5 is MyD88- or TRIF-dependent, we injected wild type and GRK5 knockout mice with Pam3CSK4 (MyD88-dependent TLR1/2 ligand) and Poly(I:C) (TRIF-dependent TLR3 ligand) and examined the in vivo systemic inflammatory response. Our results demonstrate that GRK5 regulates IL-12p40 and G-CSF via a mechanism that is common to both MyD88 and TRIF. However, GRK5 regulates IL-5 and MCP-1 in a MyD88-dependent but TNFα in a TRIF-dependent manner. Together, our results demonstrate multiple roles of GRK5 in TLR signaling.     

Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the toll-like receptor 4-MD-2-CD14 complex in a process that is distinct from the initiation of signal transduction

Mammalian responses to LPS require the expression of Toll-like receptor 4 (TLR4), CD14, and MD-2. We expressed fluorescent TLR4 in cell lines and found that TLR4 densely localized to the surface and the Golgi. Similar distributions were observed in human monocytes. Confocal imaging revealed rapid recycling of TLR4-CD14-MD-2 complexes between the Golgi and the plasma membrane. Fluorescent LPS followed these trafficking pathways in CD14-positive cells. The TLR4- adapter protein, MyD88, translocated to the cell surface upon LPS exposure, and cross-linking of surface TLR4 with antibody induced signaling. Golgi-associated TLR4 expression was disrupted by brefeldin A, yet LPS signaling was preserved. We conclude that LPS signaling may be initiated by surface aggregation of TLR4 and is not dependent upon LPS trafficking to the Golgi.     

Chlamydia pneumoniae-induced foam cell formation requires MyD88-dependent and -independent signaling and is reciprocally modulated by liver X receptor activation

Chlamydia pneumoniae is detected by macrophages and other APCs via TLRs and can exacerbate developing atherosclerotic lesions, but how that occurs is not known. Liver X receptors (LXRs) centrally control reverse cholesterol transport, but also negatively modulate TLR-mediated inflammatory pathways. We isolated peritoneal macrophages from wild-type, TLR2, TLR3, TLR4, TLR2/4, MyD88, TRIF, MyD88/TRIF, and IFN regulatory factor 3 (IRF3) KO mice, treated them with live or UV-killed C. pneumoniae in the presence or absence of oxidized LDL, then measured foam cell formation. In some experiments, the synthetic LXR agonist GW3965 was added to macrophages infected with C. pneumoniae in the presence of oxidized LDL. Both live and UV-killed C. pneumoniae induced IRF3 activation and promoted foam cell formation in wild-type macrophages, whereas the genetic absence of TLR2, TLR4, MyD88, TRIF, or IRF3, but not TLR3, significantly reduced foam cell formation. C. pneumoniae-induced foam cell formation was significantly reduced by the LXR agonist GW3965, which in turn inhibited C. pneumoniae-induced IRF3 activation, suggesting a bidirectional cross-talk. We conclude that C. pneumoniae facilitates foam cell formation via activation of both MyD88-dependent and MyD88-independent (i.e., TRIF-dependent and IRF3-dependent) pathways downstream of TLR2 and TLR4 signaling and that TLR3 is not involved in this process. This mechanism could at least partly explain why infection with C. pneumoniae accelerates the development of atherosclerotic plaque and lends support to the proposal that LXR agonists might prove clinically useful in suppressing atherogenesis.     

Toll-like receptor 4 signaling regulates cytosolic phospholipase A2 activation and lipid generation in lipopolysaccharide-stimulated macrophages

Inflammatory lipid mediators such as prostaglandins and leukotrienes play crucial roles in the pathogenesis of bacterial lipopolysaccharide (LPS)-induced inflammation. Cytosolic phospholipase A(2) (cPLA(2)) is a key enzyme in the generation of pro-inflammatory lipid mediators. Here, we found that Toll-like receptor 4 (TLR4) is essential for LPS-induced cPLA(2) activation and lipid release. Inhibition of TLR4 protein expression by TLR4 small interfering RNA or neutralization of TLR4 by the specific antibody against TLR4/MD2 blocked cPLA(2) phosphorylation and cPLA(2)-hydrolyzed arachidonic acid release. Furthermore, activation of the TLR4 signaling pathway by LPS regulated cPLA(2) activation and lipid release. cPLA(2) phosphorylation and cPLA(2)-hydrolyzed lipid release were significantly impaired when TLR4 adaptor protein, either MyD88 or TRIF, was knocked down in LPS-stimulated macrophages. Similarly, LPS-induced arachidonate release was inhibited in cells transfected with a dominant-negative MyD88 or TRIF construct. Subsequently, cPLA(2) activation could be suppressed by inhibition of the TLR4 adaptor protein-directed p38 and ERK MAPK pathways. These findings suggest that, in LPS-induced inflammation, the TLR4-mediated MyD88- and TRIF-dependent MAPK pathways result in cPLA(2) activation and production of pro-inflammatory lipid mediators.     

Dysregulation of LPS-induced Toll-like receptor 4-MyD88 complex formation and IL-1 receptor-associated kinase 1 activation in endotoxin-tolerant cells

Prior exposure to LPS induces a transient state of cell refractoriness to subsequent LPS restimulation, known as endotoxin tolerance. Induction of LPS tolerance has been reported to correlate with decreased cell surface expression of the LPS receptor complex, Toll-like receptor 4 (TLR4)/MD-2. However, other results have underscored the existence of mechanisms of LPS tolerance that operate downstream of TLR4/MD-2. In the present study we sought to delineate further the molecular basis of LPS tolerance by examining the TLR4 signaling pathway in endotoxin-tolerant cells. Pretreatment of human monocytes with LPS decreased LPS-mediated NF-kappaB activation, p38 mitogen-activated protein kinase phosphorylation, and TNF-alpha gene expression, documenting the induction of endotoxin tolerance. FACS and Western blot analyses of LPS-tolerant monocytes showed increased TLR2 expression, whereas TLR4 expression levels were not affected. Comparable levels of mRNA and protein for myeloid differentiation factor 88 (MyD88), IL-1R-associated kinase 1 (IRAK-1), and TNFR-associated factor-6 were found in normal and LPS-tolerant monocytes, while MD-2 mRNA expression was slightly increased in LPS-tolerant cells. LPS induced the association of MyD88 with TLR4 and increased IRAK-1 activity in medium-pretreated cells. In LPS-tolerant monocytes, however, MyD88 failed to be recruited to TLR4, and IRAK-1 was not activated in response to LPS stimulation. Moreover, endotoxin-tolerant CHO cells that overexpress human TLR4 and MD-2 also showed decreased IRAK-1 kinase activity in response to LPS despite the failure of LPS to inhibit cell surface expression of transfected TLR4 and MD-2 proteins. Thus, decreased TLR4-MyD88 complex formation with subsequent impairment of IRAK-1 activity may underlie the LPS-tolerant phenotype.     

Toll-like receptor (TLR) signaling in response to Aspergillus fumigatus

Aspergillus fumigatus causes life-threatening infections in patients with qualitative and quantitative defects in phagocytic function. Here, we examined the contribution of Toll-like receptor (TLR)-2, TLR4, the adapter protein MyD88, and CD14 to signaling in response to the three forms of A. fumigatus encountered during human disease: resting conidia (RC), swollen conidia (SC), and hyphae (H). Compared with elicited peritoneal macrophages obtained from wild-type and heterozygous mice, TLR2(-/-) and MyD88(-/-) macrophages produced significantly less tumor necrosis factor-alpha (TNFalpha) following A. fumigatus stimulation. In contrast, following stimulation with RC, SC, and H, TLR4(-/-) and CD14(-/-) macrophages exhibited no defects in tumor necrosis factor-alpha release. TLR2(-/-), TLR4(-/-), MyD88(-/-), and CD14(-/-) macrophages bound similar numbers of RC and SC compared with wild-type macrophages. RC, SC, and H stimulated greater activation of a nuclear factor kappa B (NFkappaB)-dependent reporter gene and greater release of tumor necrosis factor-alpha from the human monocytic THP-1 cell line stably transfected with CD14 compared with control cells stably transfected with empty vector. A. fumigatus stimulated NFkappaB-dependent reporter gene activity in the human embryonic kidney cell line, HEK293, only if the cells were transfected with TLR2. Moreover, activity increased when TLR2 and CD14 were co-transfected. Taken together, these data suggest that optimal signaling responses to A. fumigatus require TLR2 in both mouse and human cells. In contrast, a role for CD14 was found only in the human cells. MyD88 acts as a central adapter protein mediating signaling responses following stimulation with RC, SC, and H.