Analysis of TLR4 polymorphic variants: new insights into TLR4/MD-2/CD14 stoichiometry, structure, and signaling

TLR4 is the signal-transducing receptor for structurally diverse microbial molecules such as bacterial LPS, respiratory syncytial virus fusion (F) protein, and chlamydial heat shock protein 60. Previous studies associated two polymorphic mutations in the extracellular domain of TLR4 (Asp(299)Gly and Thr(399)Ile) with decreased LPS responsiveness. To analyze the molecular basis for diminished responsiveness, site-specific mutations (singly or coexpressed) were introduced into untagged and epitope (Flag)-tagged wild-type (WT) TLR4 expression vectors to permit a direct comparison of WT and mutant signal transduction. Coexpression of WT TLR4, CD14, and MD-2 expression vectors in HEK293T cells was first optimized to achieve optimal LPS-induced NF-kappaB reporter gene expression. Surprisingly, transfection of cells with MD-2 at high input levels often used in the literature suppressed LPS-induced signaling, whereas supraoptimal CD14 levels did not. Under conditions where WT and polymorphic variants were comparably expressed, significant differences in NF-kappaB activation were observed in response to LPS and two structurally unrelated TLR4 agonists, chlamydial heat shock protein 60 and RSV F protein, with the double, cosegregating mutant TLR4 exhibiting the greatest deficiency. Overexpression of Flag-tagged WT and mutant vectors at input levels resulting in agonist-independent signaling led to equivalent NF-kappaB signaling, suggesting that these mutations in TLR4 affect appropriate interaction with agonist or coreceptor. These data provide new insights into the importance of stoichiometry among the components of the TLR4/MD-2/CD14 complex. A structural model that accounts for the diminished responsiveness of mutant TLR4 polymorphisms to structurally unrelated TLR4 agonists is proposed.     

The lipopolysaccharide-recognition mechanism in cells expressing TLR4 and CD14 but lacking MD-2

We analysed the lipopolysaccharide (LPS)-recognition mechanism in cells expressing TLR4 and CD14 but lacking MD-2. When TLR4 and CD14 were transiently expressed in HEK293 cells, cell-surface expression of TLR4 was observed, although the expression level was lower than that in cells coexpressing MD-2. We found that membrane CD14-TLR4 complexes were formed in these cells in response to LPS stimulation even in the absence of MD-2 expression, although NF-kappaB-dependent reporter activity was not induced. A strong activation of NF-kappaB was observed when these cells were stimulated with LPS followed by soluble MD-2 in this order, even when excess LPS was removed after formation of the CD14-TLR4 complex by washing cells prior to sMD-2 addition. From these results, we propose an additional LPS-recognition mechanism. In cells expressing TLR4 and CD14 but lacking MD-2, LPS is first transferred to membrane CD14 with the aid of LPS binding protein, which leads to the formation of the TLR4-CD14 complex. Then, the binding of soluble MD-2 to this complex triggers the transmembrane signal transduction. Cells expressing TLR4 and CD14 but lacking MD-2, such as airway epithelial cells, may be activated in response to LPS by this mechanism.     

CD36 Differently Regulates Macrophage Responses to Smooth and Rough Lipopolysaccharide

Lipopolysaccharide (LPS) is the major pathogen-associated molecular pattern of Gram-negative bacterial infections, and includes smooth (S-LPS) and rough (R-LPS) chemotypes. Upon activation by LPS through CD14, TLR4/MD-2 heterodimers sequentially induce two waves of intracellular signaling for macrophage activation: the MyD88-dependent pathway from the plasma membrane and, following internalization, the TRIF-dependent pathway from endosomes. We sought to better define the role of scavenger receptors CD36 and CD204/SR-A as accessory LPS receptors that can contribute to pro-inflammatory and microbicidal activation of macrophages. We have found that CD36 differently regulates activation of mouse macrophages by S-LPS versus R-LPS. The ability of CD36 to substitute for CD14 in loading R-LPS, but not S-LPS onto TLR4/MD-2 allows CD14-independent macrophage responses to R-LPS. Conversely, S-LPS, but not R-LPS effectively stimulates CD14 binding to CD36, which favors S-LPS transfer from CD14 onto TLR4/MD-2 under conditions of low CD14 occupancy with S-LPS in serum-free medium. In contrast, in the presence of serum, CD36 reduces S-LPS binding to TLR4/MD-2 and the subsequent MyD88-dependent signaling, by mediating internalization of S-LPS/CD14 complexes. Additionally, CD36 positively regulates activation of TRIF-dependent signaling by both S-LPS and R-LPS, by promoting TLR4/MD-2 endocytosis. In contrast, we have found that SR-A does not function as a S-LPS receptor. Thus, by co-operating with CD14 in both R- and S-LPS loading onto TLR4/MD-2, CD36 can enhance the sensitivity of tissue-resident macrophages in detecting infections by Gram-negative bacteria. However, in later phases, following influx of serum to the infection site, the CD36-mediated negative regulation of MyD88-dependent branch of S-LPS-induced TLR4 signaling might constitute a mechanism to prevent an excessive inflammatory response, while preserving the adjuvant effect of S-LPS for adaptive immunity.     

Monocytic thrombomodulin triggers LPS- and gram-negative bacteria-induced inflammatory response

Sepsis results from the host hyperinflammatory response to bacterial infection, causing multiple organ failure and high mortality. We previously demonstrated that LPS binds to monocytic membrane-bound thrombomodulin (TM), but the role of monocytic TM in LPS-induced inflammation remains unknown. In this study, we demonstrated that TM knockdown in human monocytic cells attenuated LPS-induced signaling pathways and cytokine production. Coimmunoprecipitation and immunofluorescence assays showed that monocytic TM interacted with the LPS receptors, CD14 and TLR4/myeloid differentiation factor-2 (MD-2) complex, indicating that it binds to LPS and triggers an LPS-induced inflammatory response by interacting with the CD14/TLR4/MD-2 complex. We also found that monocytic TM knockdown reduced cytokine production induced by gram-negative bacteria Klebsiella pneumoniae, suggesting that monocytic TM plays an important role in gram-negative bacteria-induced inflammation. To further investigate the function of monocytic TM in vivo, myeloid-specific TM-deficient mice were established and were found to display improved survival that resulted from the attenuation of septic syndrome, including reduced systemic inflammatory response and resistance to bacterial dissemination, after K. pneumoniae infection or cecal ligation and puncture surgery. The inhibition of bacterial dissemination in mice with a deficiency of myeloid TM may be caused by the early increase in neutrophil infiltration. Therefore, we conclude that monocytic TM is a novel component in the CD14/TLR4/MD-2 complex and participates in the LPS- and gram-negative bacteria-induced inflammatory response.     

Interaction of soluble form of recombinant extracellular TLR4 domain with MD-2 enables lipopolysaccharide binding and attenuates TLR4-mediated signaling

TLRs have been implicated in recognition of pathogen-associated molecular patterns. TLR4 is a signaling receptor for LPS, but requires MD-2 to respond efficiently to LPS. The purposes of this study were to examine the interactions of the extracellular TLR4 domain with MD-2 and LPS. We generated soluble forms of rTLR4 (sTLR4) and TLR2 (sTLR2) lacking the putative intracellular and transmembrane domains. sTLR4 consisted of Glu(24)-Lys(631). MD-2 bound to sTLR4, but not to sTLR2 or soluble CD14. BIAcore analysis demonstrated the direct binding of sTLR4 to MD-2 with a dissociation constant of K(D) = 6.29 x 10(-8) M. LPS-conjugated beads precipitated MD-2, but not sTLR4. However, LPS beads coprecipitated sTLR4 and MD-2 when both proteins were coincubated. The addition of sTLR4 to the medium containing the MD-2 protein significantly attenuated LPS-induced NF-kappaB activation and IL-8 secretion in wild-type TLR4-expressing cells. These results indicate that the extracellular TLR4 domain-MD-2 complex is capable of binding LPS, and that the extracellular TLR4 domain consisting of Glu(24)-Lys(631) enables MD-2 binding and LPS recognition to TLR4. In addition, the use of sTLR4 may lead to a new therapeutic strategy for dampening endotoxin-induced inflammation.     

Induction of tolerance to lipopolysaccharide and mycobacterial components in Chinese hamster ovary/CD14 cells is not affected by overexpression of Toll-like receptors 2 or 4

Down-regulation of cell surface expression of Toll-like receptor (TLR) 4 following LPS stimulation has been suggested to underlie endotoxin tolerance. In this study, we examined whether overexpression of TLR2 or TLR4 would affect the ability of cells to become tolerant to LPS or the mycobacterial components, arabinose-capped lipoarabinomannan (LAM) and soluble tuberculosis factor (STF). To this end, Chinese hamster ovary/CD14 cells stably transfected with a NF-kappaB-dependent reporter construct, endothelial leukocyte adhesion molecule CD25 (the 3E10 clone), were engineered to overexpress either human TLR2 or TLR4. Transfected TLRs exhibited proper signaling functions, as evidenced by increased LPS responsiveness of 3E10/TLR4 cells and acquisition of sensitivity to TLR2-specific ligands upon transfection of TLR2 into TLR2-negative 3E10 cells. Pretreatment of cells with LPS, LAM, or STF did not modulate TLR2 or TLR4 cell surface expression. Following LPS exposure, 3E10, 3E10/TLR2, and 3E10/TLR4 cells exhibited comparable decreases in LPS-mediated NF-kappaB activation and mitogen-activated protein (MAP) kinase phosphorylation. Likewise, LPS pretreatment profoundly inhibited LPS-induced NF-kappaB translocation in Chinese hamster ovary cells that concomitantly overexpressed human TLR4 and myeloid differentiation protein-2 (MD-2), but failed to modulate TLR4 or MD-2 cell surface expression. Pretreatment of 3E10/TLR2 cells with LAM or STF decreased their NF-kappaB responses induced by subsequent stimulation with these substances or LPS. Conversely, prior exposure of 3E10/TLR2 cells to LPS led to hyporesponsiveness to LPS, LAM, and STF, indicating that LPS and mycobacterial products induce cross-tolerance. Thus, tolerance to LPS and mycobacterial components cannot be attributed solely to a decrease in TLR/MD-2 expression levels, suggesting inhibition of expression or function of other signaling intermediates.     

Kinetics of binding of LPS to recombinant CD14, TLR4, and MD-2 proteins

TLR4 together with CD14 and MD-2 forms a pattern recognition receptor that plays an initiating role in the innate immune response to Gram-negative bacteria. Here, we employed the surface plasmon resonance technique to investigate the kinetics of binding of LPS to recombinant CD14, MD-2 and TLR4 proteins produced in insect cells. The dissociation constants (KD) of LPS for immobilized CD14 and MD-2 were 8.7 microM, and 2.3 microM, respectively. The association rate constant (Kon) of LPS for MD-2 was 5.61 x 10(3) M-1S-1, and the dissociation rate constant (Koff) was 1.28 10 2 S 1, revealing slow association and fast dissociation with an affinity constant KD of 2.33 x 10-6 M at 25 degreesC. These affinities are consistent with the current view that CD14 conveys LPS to the TLR4/MD-2 complex.     

MD-2 and TLR4 N-linked glycosylations are important for a functional lipopolysaccharide receptor.

The lipopolysaccharide (LPS) receptor is a multi-protein complex that consists of at least three proteins, CD14, TLR4, and MD-2. Because each of these proteins is glycosylated, we have examined the functional role of N-linked carbohydrates of both MD-2 and TLR4. We demonstrate that MD-2 contains 2 N-glycosylated sites at positions Asn(26) and Asn(114), whereas the amino-terminal ectodomain of human TLR4 contains 9 N-linked glycosylation sites. Site-directed mutagenesis studies showed that cell surface expression of MD-2 did not depend on the presence of either N-linked site, whereas in contrast, TLR4 mutants carrying substitutions in Asn(526) or Asn(575) failed to be transported to the cell surface. Using a UV-activated derivative of Re595 LPS (ASD-Re595 LPS) in cross-linking assays, we demonstrated a critical role of MD-2 and TLR4 carbohydrates in LPS cross-linking to the LPS receptor. The ability of the various glycosylation mutants to support cell activation was also evaluated in transiently transfected HeLa cells. The double mutant of MD-2 failed to support LPS-induced activation of an interleukin-8 (IL-8) promoter-driven luciferase reporter to induce IL-8 secretion or to activate amino-terminal c-Jun kinase (JNK). Similar results were observed with TLR4 mutants lacking three or more N-linked glycosylation sites. Surprisingly, the reduction in activation resulting from expression of the Asn mutants of MD-2 and TLR4 can be partially reversed by co-expression with CD14. This suggests that the functional integrity of the LPS receptor depends both on the surface expression of at least three proteins, CD14, MD-2, and TLR4, and that N-linked sites of both MD-2 and TLR4 are essential in maintaining the functional integrity of this receptor.     

Chaperone insufficiency links TLR4 protein signaling to endoplasmic reticulum stress

Inflammation plays an important pathogenic role in a number of metabolic diseases such as obesity, type 2 diabetes, and atherosclerosis. The activation of inflammation in these diseases depends at least in part on the combined actions of TLR4 signaling and endoplasmic reticulum stress, which by acting in concert can boost the inflammatory response. Defining the mechanisms involved in this phenomenon may unveil potential targets for the treatment of metabolic/inflammatory diseases. Here we used LPS to induce endoplasmic reticulum stress in the human monocyte cell-line, THP-1. The unfolded protein response, produced after LPS, was dependent on CD14 activity but not on RNA-dependent protein kinase and could be inhibited by an exogenous chemical chaperone. The induction of the endoplasmic reticulum resident chaperones, GRP94 and GRP78, by LPS was of a much lower magnitude than the effect of LPS on TLR4 and MD-2 expression. In face of this apparent insufficiency of chaperone expression, we induced the expression of GRP94 and GRP78 by glucose deprivation. This approach completely reverted endoplasmic reticulum stress. The inhibition of either GRP94 or GRP78 with siRNA was sufficient to rescue the protective effect of glucose deprivation on LPS-induced endoplasmic reticulum stress. Thus, insufficient LPS-induced chaperone expression links TLR4 signaling to endoplasmic reticulum stress.     

Lipopolysaccharide needs soluble CD14 to interact with TLR4 in human monocytes depleted of membrane CD14

Toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. TLR4 recognizes lipopolysaccharide (LPS) in monocytes/macrophages with the help of other molecules like CD14 and MD-2, which indicates that the functional LPS receptor forms a large complex. The functional relationship between the components has been the subject of debate, as have the modifications induced by the ligand in the expression of some of these components. Moreover, as for other members of this family of receptors, the possible direct interaction of receptors and their ligands is a matter of discussion. In this paper we address the question of whether the expression of some of the components influences the expression of the rest. Human monocytes in which CD14 has been downregulated through interference in the turnover of the molecule at the Golgi level, show normal membrane TLR4 expression, when compared with control cells. On the other hand, LPS alters membrane TLR4 expression by monocytes devoid of membrane CD14 only in the presence of human serum. The effect of serum is blocked by anti-CD14 monoclonal antibodies, which strongly suggests a functional role for soluble CD14/LPS complexes in the interaction with TLR4. Our data add information on the relationship between the components of the LPS receptor and the characteristics of the interaction of LPS and TLR4 in cells devoid of membrane CD14.     

Regulatory roles for CD14 and phosphatidylinositol in the signaling via toll-like receptor 4-MD-2

The complex consisting of Toll-like receptor 4 (TLR4) and associated MD-2 signals the presence of lipopolysaccharide (LPS) when it is expressed in cell lines. We here show that normal human mononuclear cells express TLR4 and signal LPS via TLR4. CD14 is a molecule that binds to LPS and facilitates its signaling. Little is known, however, about the relationship of CD14 with TLR4-MD-2. We show that CD14 helps TLR4-MD-2 to sense and signal the presence of LPS. CD14 has also been implicated in recognition of apoptotic cells, which leads to phagocytosis without activation. Membrane phospholipids such as phosphatidylserine (PS) or phosphatidylinositol (PtdIns) are thought to serve as the ligands for CD14 in apoptotic cells. We find that PtdIns acts as an LPS antagonist in the signaling via TLR4-MD-2. TLR4-MD-2 seems to discriminate LPS from phospholipids. The signaling via TLR4-MD-2 is thus regulated by CD14 and phospholipid such as PtdIns.     

CD14 controls the LPS-induced endocytosis of Toll-like receptor 4

The transport of Toll-like Receptors (TLRs) to various organelles has emerged as an essential means by which innate immunity is regulated. While most of our knowledge is restricted to regulators that promote the transport of newly synthesized receptors, the regulators that control TLR transport after microbial detection remain unknown. Here, we report that the plasma membrane localized Pattern Recognition Receptor (PRR) CD14 is required for the microbe-induced endocytosis of TLR4. In dendritic cells, this CD14-dependent endocytosis pathway is upregulated upon exposure to inflammatory mediators. We identify the tyrosine kinase Syk and its downstream effector PLCγ2 as important regulators of TLR4 endocytosis and signaling. These data establish that upon microbial detection, an upstream PRR (CD14) controls the trafficking and signaling functions of a downstream PRR (TLR4). This innate immune trafficking cascade illustrates how pathogen detection systems operate to induce both membrane transport and signal transduction.     

MD-2 mediates the ability of tetra-acylated and penta-acylated lipopolysaccharides to antagonize Escherichia coli lipopolysaccharide at the TLR4 signaling complex

We have demonstrated previously that tetra-acylated LPS derived from the oral bacterium, Porphyromonas gingivalis, and penta-acylated msbB LPS derived from a mutant strain of Escherichia coli can antagonize the ability of canonical hexa-acylated E. coli LPS to signal through the TLR4 signaling complex in human endothelial cells. Activation of the TLR4 signaling complex requires the coordinated function of LPS binding protein (LBP), CD14, MD-2, and TLR4. To elucidate the specific molecular components that mediate antagonism, we developed a recombinant human TLR4 signaling complex that displayed efficient LPS-dependent antagonism of E. coli LPS in HEK293 cells. Notably, changes in the expression levels of TLR4 in HEK293 cells modulated the efficiency of antagonism by P. gingivalis LPS. Both soluble (s) CD14 and membrane (m) CD14 supported efficient P. gingivalis LPS-dependent and msbB LPS-dependent antagonism of E. coli LPS in the recombinant TLR4 system. When cells expressing TLR4, MD-2, and mCD14 were exposed to LPS in the absence of serum-derived LBP, efficient LPS-dependent antagonism of E. coli LPS was still observed indicating that LPS-dependent antagonism occurs downstream of LBP. Experiments using immunoprecipitates of sCD14 or sMD-2 that had been pre-exposed to agonist and antagonist indicated that LPS-dependent antagonism occurs partially at sCD14 and potently at sMD-2. This study provides novel evidence that expression levels of TLR4 can modulate the efficiency of LPS-dependent antagonism. However, MD-2 represents the principal molecular component that tetra-acylated P. gingivalis LPS and penta-acylated msbB LPS use to antagonize hexa-acylated E. coli LPS at the TLR4 signaling complex.     

CD11b/CD18 acts in concert with CD14 and Toll-like receptor (TLR) 4 to elicit full lipopolysaccharide and taxol-inducible gene expression

Overproduction of inflammatory mediators by macrophages in response to Gram-negative LPS has been implicated in septic shock. Recent reports indicate that three membrane-associated proteins, CD14, CD11b/CD18, and Toll-like receptor (TLR) 4, may serve as LPS recognition and/or signaling receptors in murine macrophages. Therefore, the relative contribution of these proteins in the induction of cyclooxygenase 2 (COX-2), IL-12 p35, IL-12 p40, TNF-alpha, IFN-inducible protein (IP)-10, and IFN consensus sequence binding protein (ICSBP) genes in response to LPS or the LPS-mimetic, Taxol, was examined using macrophages derived from mice deficient for these membrane-associated proteins. The panel of genes selected reflects diverse macrophage effector functions that contribute to the pathogenesis of septic shock. Induction of the entire panel of genes in response to low concentrations of LPS or Taxol requires the participation of both CD14 and TLR4, whereas high concentrations of LPS or Taxol elicit the expression of a subset of LPS-inducible genes in the absence of CD14. In contrast, for optimal induction of COX-2, IL-12 p35, and IL-12 p40 genes by low concentrations of LPS or by all concentrations of Taxol, CD11b/CD18 was also required. Mitigated induction of COX-2, IL-12 p35, and IL-12 p40 gene expression by CD11b/CD18-deficient macrophages correlated with a marked inhibition of NF-kappa B nuclear translocation and mitogen-activated protein kinase (MAPK) activation in response to Taxol and of NF-kappa B nuclear translocation in response to LPS. These findings suggest that for expression of a full repertoire of LPS-/Taxol-inducible genes, CD14, TLR4, and CD11b/CD18 must be coordinately engaged to deliver optimal signaling to the macrophage.     

Folimycin (concanamycin A) inhibits LPS-induced nitric oxide production and reduces surface localization of TLR4 in murine macrophages

Lipopolysaccharide (LPS) is a major cell wall component of Gram-negative bacteria and signals through a receptor complex which consists of TLR4, MD-2 and CD14. LPS signaling in macrophages induces the production of many pro-inflammatory molecules, including nitric oxide (NO). In this study, we have shown that folimycin, a macrolide antibiotic and a specific inhibitor of vacuolar ATPase (V-ATPase), inhibits LPS-induced NO production, but not TNFalpha production, in murine elicited peritoneal macrophages. However, folimycin did not affect interferon-gamma induced NO production. LPS-induced iNOS mRNA and protein expression and NF-kappaB activation were also inhibited by folimycin. Interestingly, folimycin-treated cells showed reduced surface expression of TLR4 molecules and dilated Golgi apparatus. These findings suggest that folimycin, by inhibiting V-ATPases, alters intra-Golgi pH, which in turn causes defective processing and reduced surface expression of TLR4 reducing the strength of LPS signaling in murine macrophages.     

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.     

Myeloid differentiation factor-2 interacts with Lyn kinase and is tyrosine phosphorylated following lipopolysaccharide-induced activation of the TLR4 signaling pathway

Stimulation with LPS induces tyrosine phosphorylation of numerous proteins involved in the TLR signaling pathway. In this study, we demonstrated that myeloid differentiation factor-2 (MD-2) is also tyrosine phosphorylated following LPS stimulation. LPS-induced tyrosine phosphorylation of MD-2 is specific; it is blocked by the tyrosine kinase inhibitor, herbimycin A, as well as by an inhibitor of endocytosis, cytochalasin D, suggesting that MD-2 phosphorylation occurs during trafficking of MD-2 and not on the cell surface. Furthermore, we identified two possible phospho-accepting tyrosine residues at positions 22 and 131. Mutant proteins in which these tyrosines were changed to phenylalanine had reduced phosphorylation and significantly diminished ability to activate NF-κB in response to LPS. In addition, MD-2 coprecipitated and colocalized with Lyn kinase, most likely in the endoplasmic reticulum. A Lyn-binding peptide inhibitor abolished MD-2 tyrosine phosphorylation, suggesting that Lyn is a likely candidate to be the kinase required for MD-2 tyrosine phosphorylation. Our study demonstrated that tyrosine phosphorylation of MD-2 is important for signaling following exposure to LPS and underscores the importance of this event in mediating an efficient and prompt immune response.     

CD14, TLR4 and TRAM Show Different Trafficking Dynamics During LPS Stimulation

Toll‐like receptor 4 (TLR4) is responsible for the immediate response to Gram‐negative bacteria and signals via two main pathways by recruitment of distinct pairs of adaptor proteins. Mal‐MyD88 [Mal (MyD88‐adaptor‐like) ‐ MYD88 (Myeloid differentiation primary response gene (88))] is recruited to the plasma membrane to initiate the signaling cascade leading to production of pro‐inflammatory cytokines while TRAM‐TRIF [TRAM (TRIF‐related adaptor molecule)‐TRIF (TIR‐domain‐containing adapter‐inducing interferon‐β)] is recruited to early endosomes to initiate the subsequent production of type I interferons. We have investigated the dynamics of TLR4 and TRAM during lipopolysaccharide (LPS) stimulation. We found that LPS induced a CD14‐dependent immobile fraction of TLR4 in the plasma membrane. Total internal reflection fluorescence microscopy (TIRF) revealed that LPS stimulation induced clustering of TLR4 into small punctate structures in the plasma membrane containing CD14/LPS and clathrin, both in HEK293 cells and the macrophage model cell line U373‐CD14. These results suggest that laterally immobilized TLR4 receptor complexes are being formed and prepared for endocytosis. RAB11A was found to be involved in localizing TRAM to the endocytic recycling compartment (ERC) and to early sorting endosomes. Moreover, CD14/LPS but not TRAM was immobilized on RAB11A‐positive endosomes, which indicates that TRAM and CD14/LPS can independently be recruited to endosomes.      

Mycobacterial PIMs inhibit host inflammatory responses through CD14-dependent and CD14-independent mechanisms

Mycobacteria develop strategies to evade the host immune system. Among them, mycobacterial LAM or PIMs inhibit the expression of pro-inflammatory cytokines by activated macrophages. Here, using synthetic PIM analogues, we analyzed the mode of action of PIM anti-inflammatory effects. Synthetic PIM(1) isomer and PIM(2) mimetic potently inhibit TNF and IL-12 p40 expression induced by TLR2 or TLR4 pathways, but not by TLR9, in murine macrophages. We show inhibition of LPS binding to TLR4/MD2/CD14 expressing HEK cells by PIM(1) and PIM(2) analogues. More specifically, the binding of LPS to CD14 was inhibited by PIM(1) and PIM(2) analogues. CD14 was dispensable for PIM(1) and PIM(2) analogues functional inhibition of TLR2 agonists induced TNF, as shown in CD14-deficient macrophages. The use of rough-LPS, that stimulates TLR4 pathway independently of CD14, allowed to discriminate between CD14-dependent and CD14-independent anti-inflammatory effects of PIMs on LPS-induced macrophage responses. PIM(1) and PIM(2) analogues inhibited LPS-induced TNF release by a CD14-dependent pathway, while IL-12 p40 inhibition was CD14-independent, suggesting that PIMs have multifold inhibitory effects on the TLR4 signalling pathway.     

Regulatory roles for MD-2 and TLR4 in ligand-induced receptor clustering

LPS, a principal membrane component in Gram-negative bacteria, is recognized by a receptor complex consisting of TLR4 and MD-2. MD-2 is an extracellular molecule that is associated with the extracellular domain of TLR4 and has a critical role in LPS recognition. MD-2 directly interacts with LPS, and the region from Phe(119) to Lys(132) (Arg(132) in mice) has been shown to be important for interaction between LPS and TLR4/MD-2. With mouse MD-2 mutants, we show in this study that Gly(59) was found to be a novel critical amino acid for LPS binding outside the region 119-132. LPS signaling is thought to be triggered by ligand-induced TLR4 clustering, which is also regulated by MD-2. Little is known, however, about a region or an amino acid in the MD-2 molecule that regulates ligand-induced receptor clustering. MD-2 mutants substituting alanine for Phe(126) or Gly(129) impaired LPS-induced TLR4 clustering, but not LPS binding to TLR4/MD-2, demonstrating that ligand-induced receptor clustering is differentially regulated by MD-2 from ligand binding. We further show that dissociation of ligand-induced receptor clustering and of ligand-receptor interaction occurs in a manner dependent on TLR4 signaling and requires endosomal acidification. These results support a principal role for MD-2 in LPS recognition.