Toll-like Receptor 4 Endocytosis Research Articles

HIV protease inhibitor saquinavir inhibits toll-like receptor 4 activation by targeting receptor dimerization

ObjectiveToll-like receptor 4 (TLR4) is crucial in induction of innate immune response through recognition of invading pathogens or endogenous alarming molecules. Ligand-triggered dimerization of TLR4 is essential for the activation of NF-κB and IRF3 through MyD88- or TRIF-dependent pathways. Saquinavir (SQV), an FDA-approved HIV protease inhibitor, has been shown to attenuate the activation of NF-κB induced by HMGB1 by blocking TLR4-MyD88 association in proteasome independent pathway. This study aims to define whether SQV is an HMGB1-specific and MyD88-dependent TLR4 signaling inhibitor and which precise signaling element of TLR4 is targeted by SQV.Materials and methodsPMA differentiated human THP-1 macrophages or reconstituted HEK293 cells were pretreated with SQV before stimulated by different TLR agonists. TNF-α level was evaluated through ELISA assay. NF-κB activation was analyzed using NF-κB SEAP reporting system. The levels of MyD88/TRIF pathways-related factors were examined by immunoblot. TLR4 endocytosis was assessed by immunocytochemistry. TLR4 dimerization was determined using immunoprecipitation between different tagged TLR4 and an in silico molecular docking experiment was performed to explore the possible binding site of SQV on its target.ResultsOur data showed that SQV suppresses both MyD88- and TRIF-dependent pathways in response to lipopolysaccharide (LPS), a critical sepsis inducer and TLR4 agonist, leading to downregulation of NF-κB and IRF3. SQV did not suppress MyD88-dependent pathway triggered by TLR1/2 agonist Pam3csk4. In the only TRIF-dependent pathway, SQV did not alleviate IRF3 phosphorylation induced by TLR3 agonist Poly(I:C). Furthermore, dimerization of TLR4 following LPS or HMGB1 stimulation was decreased by SQV.ConclusionWe concluded that TLR4 receptor complex is one of the mammalian targets of SQV, and TLR4-mediated immune responses and consequent risk for uncontrolled inflammation could be modulated by FDA-approved drug SQV.

ACAT1/SOAT1 Blockade Suppresses LPS-Mediated Neuroinflammation by Modulating the Fate of Toll-like Receptor 4 in Microglia.

Cholesterol is stored as cholesteryl esters by the enzymes acyl-CoA:cholesterol acyltransferases/sterol O:acyltransferases (ACATs/SOATs). ACAT1 blockade (A1B) ameliorates the pro-inflammatory responses of macrophages to lipopolysaccharides (LPS) and cholesterol loading. However, the mediators involved in transmitting the effects of A1B in immune cells is unknown. Microglial Acat1/Soat1 expression is elevated in many neurodegenerative diseases and in acute neuroinflammation. We evaluated LPS-induced neuroinflammation experiments in control vs. myeloid-specific Acat1/Soat1 knockout mice. We also evaluated LPS-induced neuroinflammation in microglial N9 cells with and without pre-treatment with K-604, a selective ACAT1 inhibitor. Biochemical and microscopy assays were used to monitor the fate of Toll-Like Receptor 4 (TLR4), the receptor at the plasma membrane and the endosomal membrane that mediates pro-inflammatory signaling cascades. In the hippocampus and cortex, results revealed that Acat1/Soat1 inactivation in myeloid cell lineage markedly attenuated LPS-induced activation of pro-inflammatory response genes. Studies in microglial N9 cells showed that pre-incubation with K-604 significantly reduced the LPS-induced pro-inflammatory responses. Further studies showed that K-604 decreased the total TLR4 protein content by increasing TLR4 endocytosis, thus enhancing the trafficking of TLR4 to the lysosomes for degradation. We concluded that A1B alters the intracellular fate of TLR4 and suppresses its pro-inflammatory signaling cascade in response to LPS.

DOP34 Human MD2 deficiency - an inborn error of immunity predisposing to early onset Inflammatory Bowel Disease

Abstract Background Pattern recognition receptors (PRRs) serve as a hub of immune responses to microbes in the gut and play a critical role in maintaining intestinal homeostasis. Toll-like receptor 4 (TLR4) represents an important PRR that can recognize the gram-negative bacterial cell wall component lipopolysaccharide (LPS). Upon binding to LPS, TLR4 forms a multimeric complex with the indispensable co-receptor myeloid differentiation protein 2 (MD2) facilitating selection of TLR4 as cargo for endocytosis and TLR4-mediated activation of downstream signalling. Despite the critical role of TLR4 in innate immunity, no patients with TLR4 or MD2 deficiency have yet been reported. Methods To investigate potential genetic causes for two related patients presenting with very early onset Inflammatory Bowel Disease (VEO-IBD) and/or pneumonia, we have performed whole exome sequencing (WES). To assess the functional consequences of the identified LY96 (encoding for MD2) variant, we generated a CRISPR/Cas9-mediated knockout (KO) of MD2 or knockin (KI) of the patient mutation in induced pluripotent stem cells (iPSCs) and studied TLR4-mediated signalling, cytokine responses, and bacterial handling in iPSC-derived macrophages. Results Genetic analysis identified a 3 bp homozygous in-frame deletion in the LY96 gene (NM_015364.5, c.347_349delCAA; p.Thr116del) in our index patients following an autosomal recessive inheritance pattern. Immunoblotting revealed an altered protein expression of overexpressed Flag-tagged mutant MD2 protein due to impaired N-linked glycosylation. Correspondingly, iPSC-derived MD2-deficient macrophages showed an impaired LPS-induced TLR4 endocytosis. As a functional consequence, we could detect reduced NF-κB and MAPK signalling (phosphorylation of NF-κB p65 subunit, ERK1/2, and p38 MAPK) and dysregulated inflammasome activation (IL-1b production and secretion) upon challenge with LPS. Gentamycin protection assays and live cell imaging suggested that MD2-deficient macrophages exhibit an impaired phagocytosis of microbial pathogens. In addition, macrophages with mutant MD2 showed decreased cytokine expression (e.g., IFNB, IL6, IL10, and TNF) in response to LPS or gram-negative bacteria (E. coli and Salmonella typhimurium) but not gram-positive bacteria (Listeria monocytogenes). Conclusion Human MD2 deficiency is an inborn error of immunity that may predispose to VEO-IBD associated with altered TLR4-mediated signalling, cytokine responses, and bacterial handling. The first description of patients with MD2 deficiency provides critical insights on human TLR4/MD2 biology and warrants caution on therapeutics strategies targeting TLR4 signalling.

Exposure to hydroxyapatite nanoparticles enhances Toll-like receptor 4 signal transduction and overcomes endotoxin tolerance in vitro and in vivo

Emerging studies indicate hydroxyapatite nanoparticles (HANPs) exhibit modest immunogenicity to elicit innate immune response which might involve Toll-like receptor 4 (TLR4) activation. This study was proposed to elucidate how HANPs direct over TLR4 signal activity in macrophage in response to TLR4 ligand, lipopolysaccharide (LPS). The present study for the first time reveals that HANPs themselves can induce TLR4 endocytosis and activate pathways both of nuclear factor-kappa B (NF-κB) and interferon regulatory factor 3 (IRF3), which potentially trigger the production of inflammatory cytokine by macrophage. Further, HANPs dose-dependently reprogram over LPS driven TLR4 signaling transduction in macrophage, leading to synergistically augmented innate immune response. In particular, HANPs synergize with LPS to promote macrophage polarization toward M1 phenotype. Moreover, HANPs abrogate the endotoxin tolerance in macrophages by restoring the production of inflammatory cytokines from macrophage in response to secondary LPS stimulation, and enhance the responsiveness of the body to LPS re-challenge in the endotoxin tolerance mice model. Therefore, this study sheds a new light on the mechanism by which HANPs drive the innate immune response, and offers a powerful strategy to potentiate LPS mediated TLR4 signaling activation in macrophage. Statement of significanceIn recent years, increasing attention has been given to hydroxyapatite nanoparticles (HANPs) on how they interact with immune cells for achieving appropriate biological effect such as bone tissue repair, soft tissue filler, tumor treatment, vaccine delivery, et al. This study indicated HANPs can induce TLR4 signaling activation. In the further, HANPs dose-dependently synergize with LPS to program over LPS induced TLR4 signaling transduction in macrophage, to favor macrophage polarizing toward M1 phenotype, as well as to abrogate immune tolerance in macrophage in response to repeated LPS stimulation. This work opens a window for the intrinsic mechanism of HANPs to drive immune response and facilitate to direct the rational use or design of HANPs for their better biomedical application.

Structure-activity relationship study of dihydroartemisinin C-10 hemiacetal derivatives as Toll-like receptor 4 antagonists

Dihydroartemisinin (DHA), a natural product isolated from the traditional Chinese herb Artemisia annua and one of the clinical frontline drugs against malarial infections, has recently been discovered as a Toll-like Receptor 4 (TLR4) antagonist. However, the TLR4 antagonistic activity of DHA is modest and it exhibits cellular toxicity. In this work, the structure-activity relationship (SAR) of DHA as TLR4 antagonist was explored. Since destroying the sesquiterpene endoperoxide scaffold substantially compromised the TLR4 antagonistic activity and molecular dynamics analysis showed that the C-10 hydroxyl group formed a hydrogen bond with E72 of myeloid differentiation factor 2 (MD2) to prevent it moving deeper into MD2, SAR of DHA was focused on the C-10 hemiacetal position. With extending the length of the linear alkane chain at C10 position, the TLR4 antagonistic activity of DHA analogs increased first and then decreased with the best TLR4 antagonism occurring at the length of the carbon chain of 3–4 carbons. In contrast, the cellular toxicity of DHA analogs was raised with the increasing length of the linear alkane chain. The TLR4 antagonistic activity of DHA derivatives with substituted halogen as the terminal functional group decreased with the decrease of electronegativity of the substituted halogen, which implies the electron-rich functional group at the end of the alkane chain appears preferred. Therefore, DHA derivative 2k with alkynyl as the end functional group, exhibited 14 times more potent TLR4 antagonistic activity than DHA. Moreover, 2k showed less cellular toxicity than DHA. Cellular signaling characterizations indicated that 2k inhibited LPS-induced TLR4 dimerization and endocytosis and suppressed LPS-induced NF-κB but not MAPKs activation, culminating in blocking LPS-induced TLR4 signaling downstream pro-inflammatory factors NO and IL-1β. Further, 2k was active in vivo; it significantly increased and prolonged morphine analgesia. Collectively, this study provides a structural guidance to reposition DHA derivatives as TLR4 antagonists.

Deficiency of β-arrestin2 exacerbates inflammatory arthritis by facilitating plasma cell formation.

β-arrestin2 (β-arr2) is, a key protein that mediates desensitization and internalization of G protein-coupled receptors and participates in inflammatory and immune responses. Deficiency of β-arr2 has been found to exacerbate collagen antibody-induced arthritis (CAIA) through unclear mechanisms. In this study we tried to elucidate the molecular mechanisms underlying β-arr2 depletion-induced exacerbation of CAIA. CAIA was induced in β-arr2-/- and wild-type (WT) mice by injection of collagen antibodies and LPS. The mice were sacrificed on d13 after the injection, spleen, thymus and left ankle joints were collected for analysis. Arthritis index (AI) was evaluated every day or every 2 days. We showed that β-arr2-/- mice with CAIA had a further increase in the percentage of plasma cells in spleen as compared with WT mice with CAIA, which was in accordance with elevated serum IgG1 and IgG2A expression and aggravating clinical performances, pathologic changes in joints and spleen, joint effusion, and joint blood flow. Both LPS stimulation of isolated B lymphocytes in vitro and TNP-LPS challenge in vivo led to significantly higher plasma cell formation and antibodies production in β-arr2-/- mice as compared with WT mice. LPS treatment induced membrane distribution of toll-like receptor 4 (TLR4) on B lymphocytes, accordingly promoted the nuclear translocation of NF-κB and the transcription of Blimp1. Immunofluorescence analysis confirmed that more TLR4 colocalized with β-arr2 in B lymphocytes in response to LPS stimulation. Depletion of β-arr2 restrained TLR4 on B lymphocyte membrane after LPS treatment and further enhanced downstream NF-κB signaling leading to additional increment in plasma cell formation. In summary, β-arr2 depletion exacerbates CAIA and further increases plasma cell differentiation and antibody production through inhibiting TLR4 endocytosis and aggravating NF-κB signaling.

Noncanonical STAT1 phosphorylation expands its transcriptional activity into promoting LPS-induced IL-6 and IL-12p40 production.

The lipopolysaccharide (LPS)-induced endocytosis of Toll-like receptor 4 (TLR4) is an essential step in the production of interferon-β (IFN-β), which activates the transcription of antiviral response genes by STAT1 phosphorylated at Tyr701 Here, we showed that STAT1 regulated proinflammatory cytokine production downstream of TLR4 endocytosis independently of IFN-β signaling and the key proinflammatory regulator NF-κB. In human macrophages, TLR4 endocytosis activated a noncanonical phosphorylation of STAT1 at Thr749, which subsequently promoted the production of interleukin-6 (IL-6) and IL-12p40 through distinct mechanisms. STAT1 phosphorylated at Thr749 activated the expression of the gene encoding ARID5A, which stabilizes IL6 mRNA. Moreover, STAT1 phosphorylated at Thr749 directly enhanced transcription of the gene encoding IL-12p40 (IL12B). Instead of affecting STAT1 nuclear translocation, phosphorylation of Thr749 facilitated the binding of STAT1 to a noncanonical DNA motif (5'-TTTGANNC-3') in the promoter regions of ARID5A and IL12B The endocytosis of TLR4 induced the formation of a complex between the kinases TBK1 and IKKβ, which mediated the phosphorylation of STAT1 at Thr749 Our data suggest that noncanonical phosphorylation in response to LPS confers STAT1 with distinct DNA binding and gene-regulatory properties that promote both IL12B expression and IL6 mRNA stabilization. Thus, our study provides a potential mechanism for how TLR4 endocytosis might regulate proinflammatory cytokine production.

The ATP-Binding Cassette Gene ABCF1 Functions as an E2 Ubiquitin-Conjugating Enzyme Controlling Macrophage Polarization to Dampen Lethal Septic Shock

Sepsis is a bi-phasic inflammatory disease that threatens approximately 30 million lives and claims over 14 million annually, yet little is known regarding the molecular switches and pathways that regulate this disease. Here, we have described ABCF1, an ATP-Binding Cassette (ABC) family member protein, which possesses an E2 ubiquitin enzyme activity, through which it controls the Lipopolysaccharide (LPS)- Toll-like Receptor-4 (TLR4) mediated gram-negative insult by targeting key proteins for K63-polyubiquitination. Ubiquitination by ABCF1 shifts the inflammatory profile from an early phase MyD88-dependent to a late phase TRIF-dependent signaling pathway, thereby regulating TLR4 endocytosis and modulating macrophage polarization from M1 to M2 phase. Physiologically, ABCF1 regulates the shift from the inflammatory phase of sepsis to the endotoxin tolerance phase, and modulates cytokine storm and interferon-β (IFN-β)-dependent production by the immunotherapeutic mediator, SIRT1. Consequently, ABCF1 controls sepsis induced mortality by repressing hypotension-induced renal circulatory dysfunction.

Core fucose is essential glycosylation for CD14-dependent Toll-like receptor 4 and Toll-like receptor 2 signalling in macrophages.

Core fucosylation, catalysed by α-1, 6 fucosyltransferase (FUT8), regulates growth factor receptors in immune function. Although core fucose regulates many immune cell types, few reports confront the association between core fucose activity and an innate immune reaction. Here, we have investigated the function of core fucose in macrophages in vivo and in vitro using Fut8-deficient mice and cells. Following lipopolysaccharide (LPS) stimulation, inflammatory cytokine production in Fut8-deficient (Fut8-/-) macrophages was suppressed in both in vivo and in vitro experiments. Because LPS is recognized by Toll-like receptor 4 (TLR4), which induces the signalling cascade, TLR4 signalling was assumed to be impaired in Fut8-/- cells. Flow cytometry analyses revealed, however, that a lack of core fucose reduced the expression of, not TLR4, but CD14, which is necessary for TLR4 endocytosis. Because CD14 is necessary for TLR2 signalling, the immune response of TLR2 was also impaired in Fut8-/- macrophages. Moreover, in the dextran sodium sulphate (DSS)-induced murine colitis model, the mice grafted with Fut8-/- bone marrow cells exhibited higher resistance to inflammation than those grafted with Fut8+/+ bone marrow cells. These findings indicate that core fucose is essential for CD14-dependent TLR4 and TLR2 signalling in murine macrophage activity, leading to DSS-induced experimental colitis.

Expression of Siglec-E Alters the Proteome of Lipopolysaccharide (LPS)-Activated Macrophages but Does Not Affect LPS-Driven Cytokine Production or Toll-Like Receptor 4 Endocytosis.

Siglec-E is a murine CD33-related siglec that functions as an inhibitory receptor and is expressed mainly on neutrophils and macrophage populations. Recent studies have suggested that siglec-E is an important negative regulator of lipopolysaccharide (LPS)-toll-like receptor 4 (TLR4) signaling and one report (1) claimed that siglec-E is required for TLR4 endocytosis following uptake of Escherichia coli by macrophages and dendritic cells (DCs). Our attempts to reproduce these observations using cells from wild-type (WT) and siglec-E-deficient mice were unsuccessful. We used a variety of assays to determine if siglec-E expressed by different macrophage populations can regulate TLR4 signaling in response to LPS, but found no consistent differences in cytokine secretion in vitro and in vivo, comparing three different strains of siglec-E-deficient mice with matched WT controls. No evidence was found that the siglec-E deficiency was compensated by expression of siglecs-F and -G, the other murine inhibitory CD33-related siglecs. Quantitative proteomics was used as an unbiased approach and provided additional evidence that siglec-E does not suppress inflammatory TLR4 signaling. Interestingly, proteomics revealed a siglec-E-dependent alteration in macrophage protein composition that could be relevant to functional responses in host defense. In support of this, siglec-E-deficient mice exhibited enhanced growth of Salmonella enterica serovar Typhimurium in the liver following intravenous infection, but macrophages lacking siglec-E did not show altered uptake or killing of bacteria in vitro. Using various cell types including bone marrow-derived DCs (BMDCs), splenic DCs, and macrophages from WT and siglec-E-deficient mice, we showed that siglec-E is not required for TLR4 endocytosis following E. coli uptake or LPS challenge. We failed to see expression of siglec-E by BMDC even after LPS-induced maturation, but confirmed previous studies that splenic DCs express low levels of siglec-E. Taken together, our findings do not support a major role of siglec-E in regulation of TLR4 signaling functions or TLR4 endocytosis in macrophages or DCs. Instead, they reveal that induction of siglec-E by LPS can modulate the phenotype of macrophages, the functional significance of which is currently unclear.

Measurement of TLR4 and CD14 Receptor Endocytosis Using Flow Cytometry.

After recognizing extracellular bacterial lipopolysaccharide (LPS), the toll-like receptor 4 (TLR4)-CD14 signaling complex initiates two distinct signaling pathways-one from the plasma membrane and the other from the signaling endosomes (Kagan et al., 2008). Understanding the early stages of TLR4 signal transduction therefore requires a robust and quantitative method to measure LPS-triggered TLR4 and CD14 receptor endocytosis, one of the earliest events of LPS detection. Here, we describe a flow cytometry-based method that we used recently to study the role of the ion channel TRPM7 in TLR4 endocytosis (Schappe et al., 2018). The assay relies on stimulating the cells with LPS and measuring the cell surface levels of TLR4 (or CD14) at various time points using flow cytometry. Although we detail the method specifically for TLR4 and CD14 from murine bone marrow-derived macrophages, it can be readily adapted to evaluate receptor endocytosis in a variety of other signaling contexts.

Pro-inflammatory hepatic macrophages generate ROS through NADPH oxidase 2 via endocytosis of monomeric TLR4\u2013MD2 complex

Reactive oxygen species (ROS) contribute to the development of non-alcoholic fatty liver disease. ROS generation by infiltrating macrophages involves multiple mechanisms, including Toll-like receptor 4 (TLR4)-mediated NADPH oxidase (NOX) activation. Here, we show that palmitate-stimulated CD11b+F4/80low hepatic infiltrating macrophages, but not CD11b+F4/80high Kupffer cells, generate ROS via dynamin-mediated endocytosis of TLR4 and NOX2, independently from MyD88 and TRIF. We demonstrate that differently from LPS-mediated dimerization of the TLR4–MD2 complex, palmitate binds a monomeric TLR4–MD2 complex that triggers endocytosis, ROS generation and increases pro-interleukin-1β expression in macrophages. Palmitate-induced ROS generation in human CD68lowCD14high macrophages is strongly suppressed by inhibition of dynamin. Furthermore, Nox2-deficient mice are protected against high-fat diet-induced hepatic steatosis and insulin resistance. Therefore, endocytosis of TLR4 and NOX2 into macrophages might be a novel therapeutic target for non-alcoholic fatty liver disease.

Sialic Acids in the Immune Response during Sepsis.

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are a group of cell surface transmembrane receptors expressed on immune cells, and regulate immune balance in inflammatory diseases. Sepsis is a life-threatened inflammatory syndrome induced by infection, and the pathogenesis of sepsis includes immune dysregulation, inflammation, and coagulation disorder. Here, we reviewed the various roles acted by Siglecs family in the pathogenesis of sepsis. Siglec-1, Siglec-5, and Siglec-14 play bidirectional roles through modulation of inflammation and immunity. Siglec-2 regulates the immune balance during infection by modulating B cell and T cell response. Siglec-9 helps endocytosis of toll-like receptor 4, regulates macrophages polarization, and inhibits the function of neutrophils during infection. Siglec-10 inhibits danger-associated molecular patterns induced inflammation, helps the initiation of antigen response by T cells, and decreases B-1a cell population to weaken inflammation. Regulating the Siglecs function in the different stages of sepsis holds great potential in the therapy of sepsis.

Palmitic acid is a toll-like receptor 4 ligand that induces human dendritic cell secretion of IL-1β.

Palmitic acid (PA) and other saturated fatty acids are known to stimulate pro-inflammatory responses in human immune cells via Toll-like receptor 4 (TLR4). However, the molecular mechanism responsible for fatty acid stimulation of TLR4 remains unknown. Here, we demonstrate that PA functions as a ligand for TLR4 on human monocyte derived dendritic cells (MoDCs). Hydrophobicity protein modeling indicated PA can associate with the hydrophobic binding pocket of TLR4 adaptor protein MD-2. Isothermal titration calorimetry quantified heat absorption that occurred during PA titration into TLR4/MD2, indicating that PA binds to TLR4/MD2. Treatment of human MoDCs with PA resulted in endocytosis of TLR4, further supporting the function of PA as a TLR4 agonist. In addition, PA stimulated DC maturation and activation based on the upregulation of DC costimulatory factors CD86 and CD83. Further experiments showed that PA induced TLR4 dependent secretion of the pro-inflammatory cytokine IL-1β. Lastly, our experimental data show that PA stimulation of NF-κB canonical pathway activation is regulated by TLR4 signaling and that reactive oxygen species may be important in upregulating this pro-inflammatory response. Our experiments demonstrate for the first time that PA activation of TLR4 occurs in response to direct molecular interactions between PA and MD-2. In summary, our findings suggest a likely molecular mechanism for PA induction of pro-inflammatory immune responses in human dendritic cells expressing TLR4.

N-glycan branching inhibits pro-inflammatory B cell activity by controlling TLR4 and BCR signaling.

Abstract N-glycosylation is a critical regulator of T cell function. The branching and number of N-glycans on cell surface glycoproteins drive binding to galectins, forming a galectin-glycoprotein lattice that controls T cell growth, differentiation and development by regulating receptor clustering, distribution and endocytosis. N-glycan branching is ~1.5–2 fold higher in B cells compared to T cells, yet effects on B cell function are unknown. Here we report that branching regulates pro-inflammatory signaling via Toll Like Receptor – 4 (TLR4) and the B cell receptor (BCR). Mechanisms controlling pro-inflammatory (cell surface) versus anti-inflammatory (endosomal) TLR4 signaling in B cells is poorly understood. Loss of branching in B cells de-couples LPS induced proliferative from pro-inflammatory responses by inhibiting TLR4 endocytosis. Similar de-coupling is observed with BCR signaling, where loss of branching inhibits BCR driven proliferation and Lyn/Syk/PLCγ2/Ca2+ signaling via reduced CD19 surface expression while concurrently driving pro-inflammatory effector functions and PI3K/Akt/mTOR activation. Together, these data reveal N-glycan branching as a critical negative regulator of TLR4 and BCR triggered pro-inflammatory B cell responses.

Morphine enhances IL-1β release through toll-like receptor 4-mediated endocytic pathway in microglia.

Morphine creates a neuroinflammatory response and enhances release of the proinflammatory cytokines like interleukin-1β (IL-1β), which compromises morphine analgesia as well as induces morphine tolerance. In this study, we attempted to investigate the mechanisms of morphine induced IL-1β synthesis and release. Microglial cells were treated with morphine (100μM) once daily for 3days. Control groups underwent the same procedure but received sterile saline injection instead of morphine. Toll-like receptor 4 (TLR4) and P2X4 receptor (P2X4R) signaling were analyzed using Western blot; immunofluorescence was used to detect the signaling of CD68; real-time RT-PCR and ELISA kit was used to measure the messenger RNA and protein synthesis and release level of IL-1β. Morphine enhanced IL-1β synthesis and P2X4R protein expression. TLR4 were responsible for morphine-induced IL-1β synthesis, while morphine-induced IL-1β release was via P2X4R. Morphine-induced IL-1β release is mediated by endocytosis of TLR4. These results indicated that TLR4 and P2X4R pathways mediated IL-1β synthesis and release in microglia followed chronic morphine. TLR4 internalization is the main mechanism of morphine-induced microglia activation and IL-1β release.

Mechanisms of Toll-like Receptor 4 Endocytosis Reveal a Common Immune-Evasion Strategy Used by Pathogenic and Commensal Bacteria

Microbe-induced receptor trafficking has emerged as an essential means to promote innate immune signal transduction. Upon detection of bacterial lipopolysaccharides (LPS), CD14 induces an inflammatory endocytosis pathway that delivers Toll-like receptor 4 (TLR4) to endosomes. Although several regulators of CD14-dependent TLR4 endocytosis have been identified, the cargo-selection mechanism during this process remains unknown. We reveal that, in contrast to classic cytosolic interactions that promoted the endocytosis of transmembrane receptors, TLR4 was selected as cargo for inflammatory endocytosis entirely through extracellular interactions. Mechanistically, the extracellular protein MD-2 bound to and dimerized TLR4 in order to promote this endocytic event. Our analysis of LPS variants from human pathogens and gut commensals revealed a common mechanism by which bacteria prevent inflammatory endocytosis. We suggest that evasion of CD14-dependent endocytosis is an attribute that transcends the concept of pathogenesis and might be a fundamental feature of bacteria that inhabit eukaryotic hosts.

CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance

Dimerization of Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) heterodimers is critical for both MyD88- and TIR-domain-containing adapter-inducing IFN-β (TRIF)-mediated signaling pathways. Recently, Zanoni et al. [(2011) Cell 147(4):868-880] reported that cluster of differentiation 14 (CD14) is required for LPS-/Escherichia coli- induced TLR4 internalization into endosomes and activation of TRIF-mediated signaling in macrophages. We confirmed their findings with LPS but report here that CD14 is not required for receptor endocytosis and downstream signaling mediated by TLR4/MD2 agonistic antibody (UT12) and synthetic small-molecule TLR4 ligands (1Z105) in murine macrophages. CD14 deficiency completely ablated the LPS-induced TBK1/IRF3 signaling axis that mediates production of IFN-β in murine macrophages without affecting MyD88-mediated signaling, including NF-κB, MAPK activation, and TNF-α and IL-6 production. However, neither the MyD88- nor TRIF-signaling pathways and their associated cytokine profiles were altered in the absence of CD14 in UT12- or 1Z105-treated murine macrophages. Eritoran (E5564), a lipid A antagonist that binds the MD2 "pocket," completely blocked LPS- and 1Z105-driven, but not UT12-induced, TLR4 dimerization and endocytosis. Furthermore, TLR4 endocytosis is induced in macrophages tolerized by exposure to either LPS or UT12 and is independent of CD14. These data indicate that TLR4 receptor endocytosis and the TRIF-signaling pathway are dissociable and that TLR4 internalization in macrophages can be induced by UT12, 1Z105, and during endotoxin tolerance in the absence of CD14.

Anti-Inflammatory Action of an Antimicrobial Model Peptide That Suppresses the TRIF-Dependent Signaling Pathway via Inhibition of Toll-Like Receptor 4 Endocytosis in Lipopolysaccharide-Stimulated Macrophages.

Antimicrobial peptides (AMPs), also called host defense peptides, particularly those with amphipathic helical structures, are emerging as target molecules for therapeutic development due to their immunomodulatory properties. Although the antimicrobial activity of AMPs is known to be exerted primarily by permeation of the bacterial membrane, the mechanism underlying its anti-inflammatory activity remains to be elucidated. We report potent anti-inflammatory activity of WALK11.3, an antimicrobial model peptide with an amphipathic helical conformation, in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. This peptide inhibited the expression of inflammatory mediators, including nitric oxide, COX-2, IL-1β, IL-6, INF-β, and TNF-α. Although WALK11.3 did not exert a major effect on all downstream signaling in the MyD88-dependent pathway, toll-like receptor 4 (TLR4)- mediated pro-inflammatory signals were markedly attenuated in the TRIF-dependent pathway due to inhibition of the phosphorylation of STAT1 by attenuation of IRF3 phosphorylation. WALK11.3 specifically inhibited the endocytosis of TLR4, which is essential for triggering TRIF-mediated signaling in macrophage cells. Hence, we suggest that specific interference with TLR4 endocytosis could be one of the major modes of the anti-inflammatory action of AMPs. Our designed WALK11 peptides, which possess both antimicrobial and anti-inflammatory activities, may be promising molecules for the development of therapies for infectious inflammation.

Gαi1 and Gαi3 regulate macrophage polarization by forming a complex containing CD14 and Gab1

Heterotrimeric G proteins have been implicated in Toll-like receptor 4 (TLR4) signaling in macrophages and endothelial cells. However, whether guanine nucleotide-binding protein G(i) subunit alpha-1 and alpha-3 (Gαi1/3) are required for LPS responses remains unclear, and if so, the underlying mechanisms need to be studied. In this study, we demonstrated that, in response to LPS, Gαi1/3 form complexes containing the pattern recognition receptor (PRR) CD14 and growth factor receptor binding 2 (Grb2)-associated binding protein (Gab1), which are required for activation of PI3K-Akt signaling. Gαi1/3 deficiency decreased LPS-induced TLR4 endocytosis, which was associated with decreased phosphorylation of IFN regulatory factor 3 (IRF3). Gαi1/3 knockdown in bone marrow-derived macrophage cells (Gαi1/3 KD BMDMs) exhibited an M2-like phenotype with significantly suppressed production of TNF-α, IL-6, IL-12, and NO in response to LPS. The altered polarization coincided with decreased Akt activation. Further, Gαi1/3 deficiency caused LPS tolerance in mice. In vitro studies revealed that, in LPS-tolerant macrophages, Gαi1/3 were down-regulated partially by the proteasome pathway. Collectively, the present findings demonstrated that Gαi1/3 can interact with CD14/Gab1, which modulates macrophage polarization in vitro and in vivo.