Toll-like Receptor 4 Endocytosis Research Articles

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.

Rab8a interacts directly with PI3Kγ to modulate TLR4-driven PI3K and mTOR signalling.

Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS) to mount innate immune responses. The TLR4-induced release of pro- and anti-inflammatory cytokines generates robust inflammatory responses, which must then be restrained to avoid disease. New mechanisms for the critical regulation of TLR-induced cytokine responses are still emerging. Here we find TLR4 complexes localized in LPS-induced dorsal ruffles on the surface of macrophages. We discover that the small GTPase Rab8a is enriched in these ruffles and recruits phosphatidylinositol 3-kinase (PI3Kγ) as an effector by interacting directly through its Ras-binding domain. Rab8a and PI3Kγ function to regulate Akt signalling generated by surface TLR4. Rab8a and PI3Kγ do not affect TLR4 endocytosis, but instead regulate mammalian target of rapamycin signalling as a mechanism for biasing the cytokine profile to constrain inflammation in innate immunity.

LPS or ethanol triggers clathrin‐ and rafts/caveolae‐dependent endocytosis of TLR4 in cortical astrocytes

Toll-like receptor 4 (TLR4) activation and signalling in glial cells play critical roles in neurological disorders and in alcohol-induced brain damage. TLR4 endocytosis upon lipopolysaccharide (LPS) stimulation regulates which signalling pathway is activated, the MyD88-dependent or the TIR-domain-containing adapter-inducing interferon-β (TRIF)-dependent pathway. However, it remains elusive whether ethanol-induced TLR4 signalling is associated with receptor internalization and trafficking, and which endocytic pathway(s) are used in cortical astrocytes. Using the adenoviral over-expression of TLR4(GFP) , confocal microscopy and the imagestream technique, we show that upon ethanol or LPS stimulation, TLR4 co-localizes with markers of the clathrin and caveolin endocytic pathways, and that this endocytosis is dependent on dynamin. Using chlorpromazin and filipin as inhibitors of the clathrin and rafts/caveolae endocytic pathways, respectively, we demostrate that TRIF-dependent signalling relies on an intact clathrin pathway, whereas disruption of rafts/caveolae inhibits the MyD88- and TRIF-dependent signalling pathways. Immunofluorescence studies also suggest that lipid rafts and clathrin cooperate for appropriate TLR4 internalization. We also show that ethanol can trigger similar endocytic pathways as LPS does, although ethanol delays clathrin internalization and alters TLR4 vesicular trafficking. Our results provide new insights into the effects of ethanol or LPS on TLR4 signalling in cortical astrocytes, events that may underlie neuroinflammation and brain damage. The results demonstrate that ethanol or lipopolysaccharide (LPS) triggers toll-like receptor 4 (TLR4) endocytosis by caveolae and clathrin-dependent pathways in astrocytes. We proposed that while clathrin is the protein responsible for TLR4 internalization, caveolin-1/lipid rafts membrane microdomains are required for TLR4 signaling. The results provide new insights into the effects of ethanol on TLR4 signalling in astrocytes, events that may underlie neuroinflammation.

CD14 Mediates Toll-like Receptor 4 (TLR4) Endocytosis and Spleen Tyrosine Kinase (Syk) and Interferon Regulatory Transcription Factor 3 (IRF3) Activation in Epithelial Cells and Impairs Neutrophil Infiltration and Pseudomonas aeruginosa Killing in Vivo

In the current study, we examined the role of CD14 in regulating LPS activation of corneal epithelial cells and Pseudomonas aeruginosa corneal infection. Our findings demonstrate that LPS induces Toll-like receptor 4 (TLR4) internalization in corneal epithelial cells and that blocking with anti-CD14 selectively inhibits TLR4 endocytosis, spleen tyrosine kinase (Syk) and IRF3 phosphorylation, and production of CCL5/RANTES and IFN-β, but not IL-8. Using a murine model of P. aeruginosa corneal infection, we show that although infected CD14(-/-) corneas produce less CCL5, they exhibit significantly increased CXC chemokine production, neutrophil recruitment to the corneal stroma, and bacterial clearance than C57BL/6 mice. We conclude that CD14 has a critical role in mediating TLR4 signaling through IRF3 in resident corneal epithelial cells and macrophages and thereby modulates TLR4 cell surface activation of the MyD88/NF-κB/AP-1 pathway and production of CXC chemokines and neutrophil infiltration to infected tissues.

Phospholipase Cγ-2 and Intracellular Calcium Are Required for Lipopolysaccharide-induced Toll-like Receptor 4 (TLR4) Endocytosis and Interferon Regulatory Factor 3 (IRF3) Activation

Toll-like receptor 4 (TLR4) is unique among the TLRs in its use of multiple adaptor proteins leading to activation of both the interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB) pathways. Previous work has demonstrated that TLR4 initiates NF-κB activation from the plasma membrane, but that subsequent TLR4 translocation to the endosomes is required for IRF3 activation. Here we have characterized several components of the signaling pathway that governs TLR4 translocation and subsequent IRF3 activation. We find that phospholipase C γ2 (PLCγ2) accounts for LPS-induced inositol 1,4,5-trisphosphate (IP(3)) production and subsequent calcium (Ca(2+)) release. Blockage of PLCγ2 function by inhibitors or knockdown of PLCγ2 expression by siRNAs in RAW 264.7 macrophages lead to reduced IRF3, but enhanced NF-κB activation. In addition, bone marrow-derived macrophages from PLCγ2-deficient mice showed impaired IRF3 phosphorylation and expression of IRF3-regulated genes after LPS stimulation. Using cell fractionation, we show that PLCγ2-IP(3)-Ca(2+) signaling cascade is required for TLR4 endocytosis following LPS stimulation. In conclusion, our results describe a novel role of the PLCγ2-IP(3)-Ca(2+) cascade in the LPS-induced innate immune response pathway where release of intracellular Ca(2+) mediates TLR4 trafficking and subsequent activation of IRF3.

An Inside Job?

Toll-like receptor (TLR) 4 interacts with the TIRAP-MyD88 pair of adaptor proteins to stimulate the production of inflammatory cytokines and with the TRAM-TRIF pair to stimulate the production of type I interferons (see Watts). Curious about how TLR4 coordinates activation of these two signaling pathways, Kagan et al . used tagged constructs to analyze TLR4 location in macrophages and found that it was present both at the plasma membrane and in early endosomes. Inhibition of dynamin-dependent endocytosis prevented lipopolysaccharide (LPS)-induced internalization of endogenous TLR4 and blocked TRAM-TRIF-dependent phosphorylation of the transcription factor IRF3 (and the expression of target genes), whereas TIRAP-MyD88-dependent phosphorylation of p38 mitogen-activated protein kinase and IκBα degradation were unaffected. Whereas fluorescently labeled TIRAP localized to the plasma membrane, fluorescently labeled TRAM was present at both the plasma membrane and in early endosomes. Overall TRAM localization depended on the first 20 residues, a bipartite motif consisting of a previously characterized myristoylation motif (the first 7 residues) followed by a region enriched in basic and aromatic residues, with the myristoylation motif alone sufficient for localization to endosomes. Analyses of LPS-induced cytokine production by TRAM localization mutants indicated that TLR4 signaling through TRAM-TRIF took place in endosomes. Noting that no known TLRs stimulate type I interferon production from the plasma membrane (TLR4 had been thought to be the lone exception), the authors determined that localization of TRAF3 (an adaptor critical for type I interferon production) was solely cytosolic. Moreover, targeting of TRAF3 to the plasma membrane enabled interferon-β induction by TLR2, which isn’t usually involved in this pathway. The authors thus propose that TLR4 stimulates TIRAP-MyD88 signaling from the cell surface and initiates TRAM-TRIF signaling only after internalization. J. C. Kagan, T. Su, T. Horng, A. Chow, S. Akira, R. Medzhitov, TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-β. Nat Immunol. 9 , 361-368 (2008). [PubMed] C. Watts, Location, location, location: Identifying the neighborhoods of LPS signaling. Nat Immunol . 9 , 343-345 (2008). [PubMed]

Membrane-anchored CD14 is required for LPS-induced TLR4 endocytosis in TLR4/MD-2/CD14 overexpressing CHO cells

Lipopolysaccharide (LPS) induces inflammatory activation through TLR4 (toll-like receptor-4)/MD-2 (myeloid differentiation-2)/CD14 (cluster of differentiation-14) complex. Although optimal LPS signaling is required to activate our innate immune systems against gram-negative bacterium, excessive amount of LPS signaling develops a detrimental inflammatory response in gram-negative bacterial infections. Downregulation of surface TLR4 expression is one of the critical mechanisms that can restrict LPS signaling. Here, we found that membrane-anchored CD14 is required for LPS-induced downregulation of TLR4 and MD-2 in CHO cells. Moreover, pretreatment of the cells with sterol-binding agent filipin reduced LPS-induced TLR4 downregulation, suggesting the involvement of caveolae-mediated endocytosis pathway. Involvement of caveolae in LPS-induced TLR4 endocytosis was further confirmed by immunoprecipitation. Thus, our data indicate that caveolae-dependent endocytosis pathway is involved in LPS-induced TLR4 downregulation and that this is dependent on membrane-anchored CD14 expression.