Intracellular lipid flux and membrane microdomains as organizing principles in inflammatory cell signaling

Lipid rafts and caveolae play a pivotal role in organization of signaling by TLR4 and several other immune receptors. Beyond the simple cataloguing of signaling events compartmentalized by these membrane microdomains, recent studies have revealed the surprisingly central importance of dynamic remodeling of membrane lipid domains to immune signaling. Simple interventions upon membrane lipid, such as changes in cholesterol loading or crosslinking of raft lipids, are sufficient to induce micrometer-scale reordering of membranes and their protein cargo with consequent signal transduction. In this review, using TLR signaling in the macrophage as a central focus, we discuss emerging evidence that environmental and genetic perturbations of membrane lipid regulate protein signaling, illustrate how homeostatic flow of cholesterol and other lipids through rafts regulates the innate immune response, and highlight recent attempts to harness these insights toward therapeutic development.     

Differential interactions of fimbriae and lipopolysaccharide from Porphyromonas gingivalis with the Toll-like receptor 2-centred pattern recognition apparatus

The lipopolysaccharide (LPS) and fimbriae of Porphyromonas gingivalis play important roles in periodontal inflammation and pathogenesis. We investigated fimbriae and LPS from several P. gingivalis strains in terms of relative dependence on Toll-like receptor (TLR) signalling partners or accessory pattern-recognition molecules mediating ligand transfer to TLRs, and determined induced assembly of receptor complexes in lipid rafts. Fimbriae could utilize TLR1 or TLR6 for cooperative TLR2-dependent activation of transfected cell lines, in contrast to LPS and a mutant version of fimbriae which displayed preference for TLR1. Whether used to activate human cell lines or mouse macrophages, fimbriae exhibited strong dependence on membrane-expressed CD14 (mCD14), which could not be substituted for by soluble CD14 (sCD14). In contrast, sCD14 efficiently substituted for mCD14 in LPS-induced cellular activation. LPS-binding protein was more important for LPS- than for fimbria-induced cell activation, whereas the converse was true for CD11b/CD18. Cell activation by LPS or fimbriae required lipid raft function and formation of heterotypic receptor complexes (TLR1-2/CD14/CD11b/CD18), although wild-type fimbriae additionally recruited TLR6. In summary, TLR2 activation by P. gingivalis LPS or fimbriae involves differential dependence on accessory signalling or ligand-binding receptors, which may differentially influence innate immune responses.     

Mycobacterium abscessus activates the macrophage innate immune response via a physical and functional interaction between TLR2 and dectin-1

Mycobacterium abscessus (Mab) is an emerging and rapidly growing non-tuberculous mycobacterium (NTM). Compared with M. tuberculosis , which is responsible for tuberculosis, much less is known about NTM-induced innate immune mechanisms. Here we investigated the involvement of pattern-recognition receptors and associated signalling in Mab-mediated innate immune responses. Mab activated the extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinases (MAPKs), and induced the secretion of tumour necrosis factor-α, interleukin (IL)-6 and IL-12p40 in murine macrophages via Toll-like receptor (TLR) 2. Notably, the activation of ERK1/2, but not p38, was crucial for Mab-induced pro-inflammatory cytokine production. The ITAM-like motif of dectin-1 critically contributed to Mab internalization and cytokine secretion by macrophages. In addition, dectin-1, in cooperation with TLR2, was required for the efficient phagocytosis of Mab, ERK1/2 activation and pro-inflammatory cytokine secretion. Co-immunoprecipitation and confocal analysis showed the physical interaction and colocalization of dectin-1 with TLR2 following Mab stimulation. Moreover, dectin-1-induced Syk activation was essential for the production of inflammatory cytokines and the release of reactive oxygen species by Mab-infected macrophages. Collectively, these data demonstrate that Mab actively internalizes into and robustly activates innate immune responses in macrophages through a physical and functional interaction between TLR2 and dectin-1.     

Insights into innate immune activation via PS-ASO–protein–TLR9 interactions

Non-CpG PS-ASOs can activate the innate immune system, leading to undesired outcomes. This response can vary—in part—as a function of 2′modifications and sequence. Here we investigated the molecular steps involved in the varied effects of PS-ASOs on the innate immune system. We found that pro-inflammatory PS-ASOs require TLR9 signaling based on the experimental systems used. However, the innate immunity of PS-ASOs does not correlate with their binding affinity with TLR9. Furthermore, the innate immune responses of pro-inflammatory PS-ASOs were reduced by coincubation with non-inflammatory PS-ASOs, suggesting that both pro-inflammatory and non-inflammatory PS-ASOs can interact with TLR9. We show that the kinetics of the PS-ASO innate immune responses can vary, which we speculate may be due to the existence of alternative PS-ASO binding sites on TLR9, leading to full, partial, or no activation of the pathway. In addition, we found that several extracellular proteins, including HMGB1, S100A8 and HRG, enhance the innate immune responses of PS-ASOs. Reduction of the binding affinity by reducing the PS content of PS-ASOs decreased innate immune responses, suggesting that PS-ASO–protein complexes may be sensed by TLR9. These findings thus provide critical information concerning how PS-ASOs can interact with and activate TLR9.     

Human Toll-like receptor 4 responses to P. gingivalis are regulated by lipid A 1- and 4'-phosphatase activities

Signal transduction following binding of lipopolysaccharide (LPS) to Toll-like receptor 4 (TLR4) is an essential aspect of host innate immune responses to infection by Gram-negative pathogens. Here, we describe a novel molecular mechanism used by a prevalent human bacterial pathogen to evade and subvert the human innate immune system. We show that the oral pathogen, Porphyromonas gingivalis , uses endogenous lipid A 1- and 4'-phosphatase activities to modify its LPS, creating immunologically silent, non-phosphorylated lipid A. This unique lipid A provides a highly effective mechanism employed by this bacterium to evade TLR4 sensing and to resist killing by cationic antimicrobial peptides. In addition, lipid A 1-phosphatase activity is suppressed by haemin, an important nutrient in the oral cavity. Specifically, P. gingivalis grown in the presence of high haemin produces lipid A that acts as a potent TLR4 antagonist. These results suggest that haemin-dependent regulation of lipid A 1-dephosphorylation can shift P. gingivalis lipid A activity from TLR4 evasive to TLR4 suppressive, potentially altering critical interactions between this bacterium, the local microbial community and the host innate immune system.     

Candida albicans Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses

Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-β-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated β-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.     

TLR4, ethanol, and lipid rafts: a new mechanism of ethanol action with implications for other receptor-mediated effects

Ethanol (EtOH) is the most widely abused substance in the United States, and it contributes to well-documented harmful (at high dosages) and beneficial (at low dosages) changes in inflammatory and immune responses. Lipid rafts have been implicated in the regulation and activation of several important receptor complexes in the immune system, including the TLR4 complex. Many questions remain about the precise mechanisms by which rafts regulate the assembly of these receptor complexes. Results summarized in this review indicate that EtOH acts by altering the LPS-induced redistribution of components of the TLR4 complex within the lipid raft and that this is related to changes in actin cytoskeleton rearrangement, receptor clustering, and subsequent signaling. EtOH provides an example of an immunomodulatory drug that acts at least in part by modifying lipid rafts, and it could represent a model to probe the relationships between rafts, receptor complexes, and signaling.     

IMPDHII protein inhibits Toll-like receptor 2-mediated activation of NF-kappaB

Toll-like receptor 2 (TLR2) plays an essential role in innate immunity by the recognition of a large variety of pathogen-associated molecular patterns. It induces its recruitment to lipid rafts induces the formation of a membranous activation cluster necessary to enhance, amplify, and control downstream signaling. However, the exact composition of the TLR2-mediated molecular complex is unknown. We performed a proteomic analysis in lipopeptide-stimulated THP1 and found IMPDHII protein rapidly recruited to lipid raft. Whereas IMPDHII is essential for lymphocyte proliferation, its biologic function within innate immune signal pathways has not been established yet. We report here that IMPDHII plays an important role in the negative regulation of TLR2 signaling by modulating PI3K activity. Indeed, IMPDHII increases the phosphatase activity of SHP1, which participates to the inactivation of PI3K.     

Ethanol mimics ligand-mediated activation and endocytosis of IL-1RI/TLR4 receptors via lipid rafts caveolae in astroglial cells

We have recently reported that ethanol-induced inflammatory processes in the brain and glial cells are mediated via the activation of interleukin-1 beta receptor type I (IL-1RI)/toll-like receptor type 4 (TLR4) signalling. The mechanism(s) by which ethanol activates these receptors in astroglial cells remains unknown. Recently, plasma membrane microdomains, lipid rafts, have been identified as platforms for receptor signalling and, in astrocytes, rafts /caveolae constitute an important integrators of signal events and trafficking. Here we show that stimulation of astrocytes with IL-1β, lipopolysaccharide or ethanol (10 and 50 mM), triggers the translocation of IL-1RI and/or TLR4 into lipid rafts caveolae-enriched fractions, promoting the recruitment of signalling molecules (phospho-IL-1R-associated kinase and phospho-extracellular regulated-kinase) into these microdomains. With confocal microscopy, we further demonstrate that IL-1RI is internalized by caveolar endocytosis via enlarged caveosomes organelles upon IL-1β or ethanol treatment, which sorted their IL-1RI cargo into the endoplasmic reticulum–Golgi compartment and into the nucleus of astrocytes. In short, our findings demonstrate that rafts /caveolae are critical for IL-1RI and TLR4 signalling in astrocytes, and reveal a novel mechanism by which ethanol, by interacting with lipid rafts caveolae, promotes IL-1RI and TLR4 receptors recruitment, triggering their endocytosis via caveosomes and downstream signalling stimulation. These results suggest that TLRs receptors are important targets of ethanol-induced inflammatory damage in the brain.     

Compartmentalized DCC signalling is distinct from DCC localized to lipid rafts

Background information. Netrin‐1 is a bi‐functional cue that attracts or repels different classes of neurons during development. The netrin‐1 receptor DCC (deleted in colorectal cancer) acts as a tyrosine kinase‐associated receptor to mediate the attractive response towards netrin‐1. The lipid raft‐localized Src family kinase Fyn is required for DCC‐mediated axon guidance. DCC functions are also dependent on lipid rafts, membrane microdomains corresponding to a low‐density, detergent‐resistant membrane fraction. However, it remains unclear how the association of DCC with lipid rafts controls netrin‐1 signalling. Results. DCC targeted to lipid rafts represented a minor proportion of total DCC inside the cell, but predominated on the cell surface of both IMR‐32 human neuroblastoma cells and embryonic cortical neurons. Netrin‐1 accumulated in lipid rafts, but had no effect on the targeting of DCC to that compartment, with DCC remaining on the cell surface in lipid rafts through 60 min post‐treatment. However, DCC was able to interact with Fyn, both in the lipid rafts and soluble compartments isolated from embryonic E19 rat brains, whereas early downstream signalling components such as Nck‐1, and total and active focal adhesion kinase were mainly localized to the non‐lipid raft compartment. Conclusions. Together, these results suggest that DCC can be found in raft and non‐raft portions of the plasma membrane, with early signalling events propagated by non‐raft associated DCC.     

Reactive oxygen species promote raft formation in T lymphocytes

Lipid rafts are involved in many cell biology events, yet the molecular mechanisms on how rafts are formed are poorly understood. In this study we probed the possible requirement of reactive oxygen species (ROS) for T-cell receptor (TCR)-induced lipid raft formation. Microscopy and biochemical analyses illustrated that blockage of ROS production, by superoxide dismutase-mimic MnTBAP, significantly reduced partitioning of LAT, phospho-LAT, and PLC-gamma in lipid rafts. Another antioxidant N-acetylcysteine (NAC) displayed a similar suppressive effect on the entry of phospho-LAT into raft microdomains. The involvement of ROS in TCR-mediated raft assembly was observed in T-cell hybridomas, T leukemia cells, and normal T cells. Removal of ROS was accompanied by an attenuated activation of LAT and PKCtheta, with reduced production of IL-2. Consistently, treating T cells with the ROS-producer tert-butyl hydrogen peroxide (TBHP) greatly enhanced membrane raft formation, distribution of phospho-LAT into lipid rafts, and increased IL-2 production. Our results indicate for the first time that ROS contribute to TCR-induced membrane raft formation.     

Lipid rafts in T cell signalling and disease

Lipid rafts is a blanket term used to describe distinct areas in the plasma membrane rich in certain lipids and proteins and which are thought to perform diverse functions. A large number of studies report on lipid rafts having a key role in receptor signalling and activation of lymphocytes. In T cells, lipid raft involvement was demonstrated in the early steps during T cell receptor (TCR) stimulation. Interestingly, recent evidence has shown that signalling in these domains differs in T cells isolated from patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Here, we discuss these findings and explore the potential of lipid rafts as targets for the development of a new class of agents to downmodulate immune responses and for the treatment of autoimmune diseases.     

A novel approach to examining compositional heterogeneity of detergent-resistant lipid rafts

Lipid rafts play an important role in cell signalling, cell adhesion and other cellular functions. Compositional heterogeneity of lipid rafts provides one mechanism of how lipid rafts provide the spatial and temporal regulation of cell signalling and cell adhesion. The constitutive presence of some signalling receptors/molecules and accumulation of others in the lipid raft allows them to interact with each other and thereby facilitate relay of signals from the plasma membrane to the cell interior. Devising a method that can analyze these lipid microdomains for the presence of signalling receptors/molecules on an individual raft basis is required to address the issue of lipid raft heterogeneity. SDS-PAGE analysis, currently used for analyses of detergent-resistant lipid rafts, does not address this question. We have designed a cell-free assay that captures detergent-resistant lipid rafts with an antibody against a raft-resident molecule and detects the presence of another lipid raft molecule. Our results suggest that detergent-resistant lipid rafts, also known as detergent-resistant membranes, are heterogeneous populations on an immortalized mouse T-cell plasma membrane with respect to antigen receptor/signalling complex and other signalling/adhesion proteins. This cell-free assay provides a simple and quick way to examine the simultaneous presence of two proteins in the lipid rafts and has the potential to estimate trafficking of molecules in and out of the lipid microdomains during cell signalling on a single detergent-resistant lipid raft basis.     

Toll-like receptor 2 signalling: Significance in megakaryocyte development through wnt signalling cross-talk and cytokine induction

TLR2 is a toll-like receptor protein which is involved in innate immune responses. TLR2 recognize several virus, fungal and bacterial pathogens, upon their uptake cause internalization and cellular activation. During this process several cytokines participate including interleukins, IL6 and IL12. Interestingly, TLR2 is expressed on megakaryocytes (MKs) and platelets, which is crucial for immune mediated platelet activation. The role of TLR2 on MKs is not completely understood. We observed TLR2 induction leads to MK maturation and is involved in production of ROS which is essential for MK development. In Dami cells, TLR2 up-regulation causes increase in the cytokine production, particularly IL-6, which has been shown to stimulate CFU formation and CD41 expression. Additionally, TLR2 ligand induces wnt β-catenin signalling pathway components suggesting a cross talk between wnt and TLR pathway leading to maturation of MKs. This study shows TLR2 signalling induce cytokine production and regulate wnt signalling thereby cause maturation of MKs.     

Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol

We previously showed that macrophages from macrophage-specific ATP-binding cassette transporter A1 (ABCA1) knockout (Abca1^-M/-M) mice had an enhanced proinflammatory response to the Toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS), compared with wild-type (WT) mice. In the present study, we demonstrate a direct association between free cholesterol (FC), lipid raft content, and hyper-responsiveness of macrophages to LPS in WT mice. Abca1^-M/-M macrophages were also hyper-responsive to specific agonists to TLR2, TLR7, and TLR9, but not TLR3, compared with WT macrophages. We hypothesized that ABCA1 regulates macrophage responsiveness to TLR agonists by modulation of lipid raft cholesterol and TLR mobilization to lipid rafts. We demonstrated that Abca1^-M/-M vs. WT macrophages contained 23% more FC in isolated lipid rafts. Further, mass spectrometric analysis suggested raft phospholipid composition was unchanged. Although cell surface expression of TLR4 was similar between Abca1^-M/-M and WT macrophages, significantly more TLR4 was distributed in membrane lipid rafts in Abca1^-M/-M macrophages. Abca1^-M/-M macrophages also exhibited increased trafficking of the predominantly intracellular TLR9 into lipid rafts in response to TLR9-specific agonist (CpG). Collectively, our data suggest that macrophage ABCA1 dampens inflammation by reducing MyD88-dependent TLRs trafficking to lipid rafts by selective reduction of FC content in lipid rafts.     

Multiple MyD88-dependent responses contribute to pulmonary clearance of Legionella pneumophila

MyD88-dependent signalling is important for secretion of early inflammatory cytokines and host protection in response to Legionella pneumophila infection. Although toll-like receptor (TLR)2 contributes to MyD88-dependent clearance of L. pneumophila , TLR-independent functions of MyD88 could also be important. To determine why MyD88 is critical for host protection to L. pneumophila , the contribution of multiple TLRs and IL-18 receptor (IL-18R)-dependent interferon-gamma (IFN-γ) production in a mouse was examined. Mice deficient for TLR5 or TLR9, or deficient for TLR2 along with either TLR5 or TLR9, were competent for controlling bacterial replication and had no apparent defects in cytokine production compared with control mice. MyD88-dependent production of IFN-γ in the lung was mediated primarily by natural killer cells and required IL-18R signalling. Reducing IFN-γ levels did not greatly affect the kinetics of L. pneumophila replication or clearance in infected mice. Additionally, IFN-γ-deficient mice did not have a susceptibility phenotype as severe as the MyD88-deficient mice and were able to control a pulmonary infection by L. pneumophila . Thus, MyD88-dependent innate immune responses induced by L. pneumophila involve both TLR-dependent responses and IL-18R-dependent production of IFN-γ by natural killer cells, and these MyD88-dependent pathways can function independently to provide host protection against an intracellular pathogen.     

MHC Class II Molecules Enhance Toll-Like Receptor Mediated Innate Immune Responses

Background

Major histocompatibility complex (MHC) class II molecules play crucial roles in immune activation by presenting foreign peptides to antigen-specific T helper cells and thereby inducing adaptive immune responses. Although adaptive immunity is a highly effective defense system, it takes several days to become fully operational and needs to be triggered by danger-signals generated during the preceding innate immune response. Here we show that MHC class II molecules synergize with Toll-like receptor (TLR) 2 and TLR4 in inducing an innate immune response.

Methodology/Principal Findings

We found that co-expression of MHC class II molecules and TLR2 or TLR4 in human embryonic kidney (HEK) cells 293 leads to enhanced production of the anti-microbial peptide human-β-defensin (hBD) 2 after treatment with TLR2 stimulus bacterial lipoprotein (BLP) or TLR4 ligand lipopolysaccharide (LPS), respectively. Furthermore, we found that peritoneal macrophages of MHC class II knock-out mice show a decreased responsiveness to TLR2 and TLR4 stimuli compared to macrophages of wild-type mice. Finally, we show that MHC class II molecules are physically and functionally associated with TLR2 in lipid raft domains of the cell membrane.

Conclusions/Significance

These results demonstrate that MHC class II molecules are, in addition to their central role in adaptive immunity, also implicated in generating optimal innate immune responses.     

Lycopene suppresses proinflammatory response in lipopolysaccharide-stimulated macrophages by inhibiting ROS-induced trafficking of TLR4 to lipid raft-like domains

We recently showed that lycopene inhibited lipopolysaccharide (LPS)-induced productions of nitric oxide (NO) and interleukin-6 (IL-6) in murine RAW264.7 macrophages by mechanisms related to inhibition of ERK and nuclear factor-κB. Since the assembly of Toll-like receptor 4 (TLR4) in lipid rafts is a key element in LPS induced signaling, we investigated whether this process would be influenced by lycopene. We found that pretreatment of RAW264.7 cells with lycopene inhibited LPS-induced recruitment of TLR4 into fractions — enriched with lipid raft marker. By the methods of immunoprecipitation and immunoblotting, we also found that lycopene inhibited the subsequent formation of the complex of TLR4 with its adaptors including myeloid differentiation primary-response protein 88 and TIR domain–containing adaptor-inducing IFN-β. We also found that the lycopene induced inhibition was associated with reduced formation of reactive oxygen species (ROS), which was an upstream mechanism for the effects of lycopene, because treating the cells with the antioxidant N-acetyl-l-cysteine and NADPH oxidase inhibitor diphenyleneiodonium chloride significantly inhibited LPS-induced recruitment of TLR4 into lipid raft-like domains as well as the production of proinflammatory molecule NO and IL-6. Thus, our findings suggest that lycopene may prevent LPS-induced TLR4 assembly into lipid rafts through reducing intracellular ROS level.     

CD44 engagement promotes matrix-derived survival through the CD44-SRC-integrin axis in lipid rafts

CD44 is present in detergent-resistant, cholesterol-rich microdomains, called lipid rafts, in many types of cells. However, the functional significance of CD44 in lipid rafts is still unknown. We have previously demonstrated that osteopontin-mediated engagement of CD44 spliced variant isoforms promotes an extracellular matrix-derived survival signal through integrin activation. By using a series of CD44 mutants and pharmacological inhibitors selectively targeted to various cellular pathways, we show in this study that engagement of CD44 induces lipid raft coalescence to facilitate a CD44-Src-integrin signaling axis in lipid rafts, leading to increased matrix-derived survival. Palmitoylation of the membrane-proximal cysteine residues and carboxyl-terminal linkage to the actin cytoskeleton both contribute to raft targeting of CD44. The enrichment of integrin β1 in lipid rafts is tightly coupled to CD44 ligation-elicited lipid raft reorganization and associated with temporally delayed endocytosis. Through the interaction with the CD44 carboxyl-terminal ankyrin domain, Src is cotranslocated to lipid rafts, where it induces integrin activation via an inside-out mechanism. Collectively, this study demonstrates an important role of the dynamic raft reorganization induced by CD44 clustering in eliciting the matrix-derived survival signal.     

Differential initiation of innate immune responses induced by human cytomegalovirus entry into fibroblast cells

Infection of permissive fibroblasts with human CMV (HCMV, AD169) is accompanied by a robust activation of innate immune defense. In this study, we show that inflammatory cytokine (IC) secretion and activation of the type I IFN pathway (alphabeta IFN) are initiated through distinct mechanisms. HCMV is recognized by TLR2 leading to the NF-kappaB activation and IC secretion. However, the IFN response to HCMV is not a TLR2-dependent process, as a dominant negative TLR2 does not affect the antiviral response to infection. Additionally, bafilomycin, an endosomal acidification inhibitor, has no effect on HCMV-induced IFN responses suggesting that IFN signaling is independent of endosomal resident TLRs. By contrast, disruption of lipid rafts by depletion of cellular cholesterol inhibits both HCMV entry as well as IFN responses. Cholesterol depletion had no effect on the induction of ICs by HCMV, illustrating a biological distinction at the cellular level with the initiation of innate immune pathways. Furthermore, HCMV entry inhibitors block IFN responses but not IC signaling. In particular, blocking the interaction of HCMV with beta(1) integrin diminished IFN signaling, suggesting that this virus-cell interaction or subsequent downstream steps in the entry pathway are critical for downstream signal transduction events. These data show that HCMV entry and IFN signaling are coordinated processes that require cholesterol-rich microdomains, whereas IC signaling is activated through outright sensing via TLR2. These findings further highlight the complexity and sophistication of innate immune responses at the earliest points in HCMV infection.