TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling

The mammalian family of Toll-like receptors (TLR) consists of thirteen members with TLR4 being the most extensively studied one. TLRs are representatives of pattern recognition receptors (PRR), so named for their ability to recognize evolutionarily conserved components of microorganisms, including bacteria, viruses, fungi and parasites, collectively called pathogen-associated molecular patterns (PAMPs). The recognition of a PAMP by a PRR triggers rapid inflammatory reactions essential for the innate immunity, as discussed in several previous exhaustive reviews [ 1 – 5 ]. TLR4 is activated by lipopolysaccharide (LPS, endotoxin), a major component of the outer membrane of Gram-negative bacteria. During infection, TLR4 responds to the LPS present in tissues and the bloodstream and triggers pro-inflammatory reactions facilitating eradication of the invading bacteria [ 6 ]. It has been indicated that TLR4 can also be activated by endogenous compounds called damage-associated molecular patterns (DAMPs), including high mobility group box protein 1 (HMGB1) and hyaluronic acid. These compounds are released during tissue injury and can activate TLR4 in non-infectious conditions to induce tissue repair [ 7 , 8 ]. Altogether, apart from LPS and its derivatives, up to 30 naturally occurring agonists of TLR4 with various chemical structures have been postulated. However, only three of them, Ni 2+ , the plant secondary metabolite paclitaxel, and disulfide HMGB1 have been demonstrated to be direct activators of TLR4, while the others can act as chaperones for TLR4 or promoters of LPS internalization [ 7 , 9 , 10 ]. Nevertheless, the impact of endogenous DAMPs on the TLR4 activity broadens the spectrum of pathophysiological conditions involving the TLR4-induced pro-inflammatory responses far beyond infectious diseases. TLR4 binds LPS with the help of LPS-binding protein (LBP) and CD14, and an indispensable contribution of the MD-2 protein stably associated with the extracellular fragment of the receptor (Fig. 1 ). The requirement for MD-2 for TLR4 activation by LPS was established shortly after the identification of TLR4 as the LPS receptor, since virtually no responses to LPS were detected in macrophages derived from MD-2 −/− mice [ 11 , 12 ]. The binding of an LPS molecule to the TLR4/MD-2 complex involves acyl chains and phosphate groups of lipid A, the conserved part of LPS and the main inducer of pro-inflammatory responses to LPS [ 13 , 14 ]. Hexa-acylated and diphosphorylated LPS, like Escherichia coli LPS (O111:B4), is one of the most potent agonists of TLR4 whereas under-acylated LPS and dephosphorylated LPS species have a weaker pro-inflammatory activity especially in human cells [ 15 ]. Structural determinants of this phenomenon are found in the TLR4/MD-2 complex and also in CD14 protein [ 13 , 16 ], as discussed in the following sections. Fig. 1 Pro-inflammatory signaling pathways of TLR4. TLR4 activates the MyD88-signaling pathway at the plasma membrane and after a CD14-dependent endocytosis initiates the TRIF-dependent cascade. Via activated NF-κB TLR4 also contributes to the activation of the cytosolic NLRP3 inflammasome. See text for details Under-acylated and dephosphorylated LPS is synthesized by commensal bacteria which colonize human intestines, like Bacteroides thetaiotaomicron , which evade recognition by PRR [ 17 ] and are crucial for the maintenance of the intestinal immune balance [ 15 ]. In addition, the impermeability of the intestine epithelium to LPS is an important factor preventing its egress into the bloodstream [ 18 ]. On the other hand, during infection, deacylation and dephosphorylation of bacterial LPS is important for the termination of inflammatory responses, as discussed in detail below in the section concerning LPS detoxification. An exaggerated and uncontrolled pro-inflammatory signaling triggered by TLR4 during infection can lead to sepsis, septic shock, and death [ 19 ]. Infections with Gram-negative bacteria, including E. coli and Pseudomonas aeruginosa , are the prevailing cause of severe sepsis in humans [ 20 ]. In addition, low doses of LPS derived from the gut microbiota can in certain conditions enter the bloodstream and evoke so-called metabolic endotoxemia leading to a chronic low-grade TLR4-dependent inflammation which contributes to the development of metabolic diseases, such as type 2 diabetes [ 18 , 21 – 23 ]. Prolonged activation of TLR4 is also linked with several human hereditary and neurodegenerative diseases and also with autoimmune diseases and cancer [ 24 , 25 ]. On the other hand, an experimental exclusion of TLR4-triggered signaling during low-grade polymicrobial sepsis resulted in an impaired bacterial clearance and thereby worsened organ injury leading to a higher mortality of mice [ 26 , 27 ]. Thus, the cellular level of TLR4 and its signaling activity have to be tightly regulated to be beneficial rather than harmful to the host. TLR4 is expressed in immune c

Endocytosis of TLR4 is required for the TRIF-dependent pro-inflammatory signaling to occur and also for the following degradation of the receptor and termination of the signaling [ 40 , 41 ]. Somewhat surprising, it has been established that the LPS-induced internalization of TLR4 is independent of its signaling activity. Thus, a TLR4 mutant lacking the intracellular part, hence the TIR domain, underwent internalization despite being unable to trigger myddosome formation in murine bone marrow-derived macrophages (BMDM) stimulated with LPS. The amount of the truncated receptor in the plasma membrane progressively declined, similarly as in the case of wild type TLR4 [ 17 ]. On the contrary, the extracellular domain of TLR4 was indispensable for its LPS-induced endocytosis and further studies have indicated that in fact the interaction of MD-2 with CD14 drives the uptake of the receptor. Thus, the impact of MD-2 on LPS-induced signaling stems from its role in the dimerization of the TLR4/MD-2 complexes during LPS binding and the contribution to the endocytosis of TLR4 [ 17 ]. In most cases, the LPS-induced internalization of TLR4 is controlled by CD14 (Tab. 1 ) [ 32 , 33 ]. Exceptions include TLR4 endocytosis followed by the TRIF-dependent signaling induced by a TLR4/MD-2 agonistic antibody (UT12) or a synthetic small-molecule TLR4 ligand (1Z105) [ 141 ]. And while phagocytosis of the Gram-negative E. coli did occur in DC derived from CD14-knock-out mice [ 33 ], no LPS-induced endocytosis of TLR4 took place in DC or BMDM derived from those animals. Accordingly, the TRIF-dependent signaling was abolished, while the MyD88-dependent one was not affected by the CD14 depletion especially in cells stimulated with so-called rough chemotype (devoid of the O -polysaccharide chain) of LPS [ 31 , 32 , 34 ]. In cells poor in CD14, such as murine splenic B lymphocytes, TLR4 does not undergo endocytosis [ 33 ]. The dependence of TLR4 internalization on CD14 has been confirmed by recent studies on CD14 glycosylation. Inhibition of CD14 core fucosylation caused by depletion of α-(1,6)-fucosyltransferase impaired CD14 and TLR4 endocytosis and thereby the TRIF-dependent signaling. It was found that the interference with CD14 fucosylation led to a reduction of its cell surface level which was considered the main reason of the impaired TLR4 endocytosis [ 142 , 143 ]. Conversely, the increase of CD14 level during maturation of murine DC accelerates the LPS-induced endocytosis of TLR4 [ 33 ]. Table 1 Effects of down-regulation of selected proteins involved in TLR4 endocytosis and degradation on TLR4 internalization and signaling Protein a Effect on Cells References TLR4 internalization MyD88-dependent pathway TRIF-dependent pathway CD14 ↓ ↓ ↓ macrophages, DC [ 17 , 33 , 160 ] TRAM ↓ nd ↓ HEK293, macrophages [ 40 , 93 ] PLC-γ2/Ca 2+ ↓ ↑ ↓ macrophages [ 33 ] ↓ – ↓ DC [ 33 , 150 ] Syk ↓ – ↓ macrophages [ 33 ] PI3K p110δ ↓ ↑ ↓ DC [ 74 ] Arf6 ↓ ↓ ↓ J776, Raw264.7, macrophages, DC [ 73 , 152 , 153 ] Dynamin b ↓ – ↓ DC, macrophages, Raw264.7 [ 40 , 74 , 155 ] ↓ ↓ ↓ J774, actrocytes [ 159 , 160 ] Dynamin nd ↑ nd HEK293 [ 41 ] Clathrin b ↓ ↓ ↓ Raw264.7, J774, macrophages [ 155 , 160 , 265 ] ↓ – ↓ astrocytes [ 159 ] Dab2 ↑ – ↑ Raw264.7 [ 166 ] Hrs nd ↑ nd HEK293, monocytes [ 41 ] Lyst c – – ↓ macrophages, DC [ 183 ] Vps33B – ↑ ↑ macrophages [ 180 ] GMFG d ↓ ↑ ↑ macrophages [ 196 ] Rab7a nd nd ↓ macrophages [ 183 ] Rab7b d – ↑ ↑ Raw264.7, macrophages [ 184 ] Rab11 nd – ↓ monocytes, HEK293 [ 95 ] Rab10 ↓ ↓ ↓ macrophages, Raw264.7 [ 179 ] TMED7 nd – ↑ HEK293, THP-1, macrophages, PBMCs [ 182 ] TAG nd – ↑ HEK293, THP-1, PBMCs [ 181 ] ↓ Down-regulation, ↑ upregulation, nd no data a In most cases, proteins were down-regulated by knock-out or knock-down of encoding genes b Inhibition of the protein caused by a drug treatment c Deleterious mutation d Overexpression of the protein had opposite effects on the MyD88- and TRIF-dependent pathways Notably, it has been established that CD14 undergos low-rate constitutive endocytosis also in unstimulated cells [ 17 ]. The decreasing pool of cell-surface CD14 is replenished by the newly synthesied protein. Binding of LPS to TLR4/MD-2 and the concomitant interaction of MD-2 with CD14 converts the TLR4/MD-2/LPS complex into a cargo of CD14. Simultanously, the rate of CD14 internalization is accelerated [ 17 , 144 ]. On the basis of those data, CD14 has earned the name of “transporter associated with the execution of inflammation” (TAXI) [ 17 ]. The mechanism of the CD14-dependent internalization of TLR4 remains to be revealed, but it is likely linked with the raft localization of CD14, activation of the Syk tyrosine kinase, and local turnover of PI(4,5)P 2 [ 33 , 50 , 145 ]. CD14 triggers biphasic generation of PI(4,5)P 2 in LPS-stimulated macrophages and the newly generated PI(4,5)P 2 accumulates in the raft fraction of these cells. The PI(4,5)P 2 generation correlated with a biphasic activation of NF-κB and w

Progressive acidification of the endosome interior provides an optimal environment for the dissociation of ligands from their receptors and for the activity of hydrolytic enzymes. In agreement, acidification of endosomes was found to induce splitting of TLR4/MD-2 dimers and dissociation of LPS from the receptor [ 188 , 189 ], albeit the pH optimum for those processes has not been determined. In general, activation of endosomal proteases facilitates degradation of the internalized proteins, but for endosomal TLRs a limited proteolysis catalyzed by cathepsins and asparaginyl endopeptidase is necessary for their dimerization and signal transduction [ 152 , 190 ]. Notably, TLR4 does not undergo such modification and binding of LPS overcomes the repulsion of two receptor ectodomains and forces TLR4 dimerization [ 69 ]. The acidic pH of endosomes could facilitate induction of the TRIF-dependent signaling by TLR4. Such hypothesis has been put forward by Ganglioff and co-workers based on their analysis of the crystallographic structure of TLR3, a receptor residing in endosomes and triggering the TRAM/TRIF-dependent signaling pathway. By analogy with TLR3, the acidic environment of endosomes (pH 5.5) would induce changes in the position of TLR4 ectodomains which after internalization face the endosome lumen. Concomitant conformational changes of the transmembrane and cytosolic regions of the receptor together with specific features of the endosome membrane, such as its high curvature and the presence of distinct species of PIs, could facilitate recruitment of TRAM to TLR4 [ 191 ]. Two approaches have been used to examine the role of endosome acidification in LPS-induced signaling of TLR4. Treatment of Raw264.7 cells with chloroquine, which neutralized the pH of the endosome interior, enhanced the depletion of LPS-activated TLR4 from the cell surface [ 155 ]. In agreement, chloroquine was shown to induce intracellular accumulation of LPS-activated TLR4 instead of its degradation, thus the stability of the TLR4/MD-2 complex was also prolonged [ 41 , 189 ]. In chloroquine-treated Raw264.7 cells, the production of the MyD88- and TRIF-dependent cytokines was decreased, which was interpreted as a result of impaired recycling of TLR4 from endosomes to the plasma membrane [ 155 ]. On the other hand, knock-down of a ATP6V0D2, a subunit of the V-ATPase responsible for acidification of endosomes, attenuated the TRIF-dependent signaling pathway in Raw264 cells as reflected by a decreased expression of Ifnb1 and upregulated expression of the MyD88-dependent Tnfa, Il6 and Il12p40 [ 152 ]. In this case, the enhancement of the MyD88-dependent signaling and the reduction of the TRIF-dependent one correlated with reduced endocytosis of TLR4 and its prolonged maintenance on the cell surface. At the basis of this effect was an impaired interaction of the V-ATPase complex with Arf6 caused by the silencing of ATP6V0D2 [ 152 ], indicating that the regulation of TLR4 signaling via V-ATPase goes beyond controlling the endosome acidification and also involves its influence on the Arf6 activity. Ample data indicate that several other proteins which determine the functionality of the endo-lysosomal compartment, such as Lyst, Vps33B, GMFG and Rab7b, affect the TLR4-triggered signaling and the receptor degradation (Table 1 ). Lyst is a lysosomal trafficking regulator contributing to endo-lysosomal biogenesis and also takes part in terminal maturation of secretory vesicles [ 192 ]. Structurally, Lyst belongs to BEACH domain-containing proteins which are large scaffolding proteins associated with cellular membranes due to the interaction of their PH domain with phospholipids, mainly PIs [ 193 ]. In murine BMDM and bone marrow-derived DC (BMDC) bearing a mutation in the Lyst gene leading to the production of a dysfunctional Lyst, neither the LPS-induced MyD88-dependent activation of MAP kinases and IκB nor the TLR4 endocytosis were affected; however, the TRIF-dependent IRF3 phosphorylation and its translocation to the nucleus were impaired. In agreement, that deleterious mutation of Lyst decreased the production of IFN-β, TNFα, and IL-12. Similar effects were observed in THP-1 cells of human origin. In vivo studies of pulmonary inflammation in mice showed that the Lyst dysfunction led to a lower production of TNF-α and IFN-β after administration of LPS, and protected the animals from endotoxin-induced lethal shock. This suggests that the dysfunction of the endo-lysosomal compartment observed in cells expressing the mutated form of Lyst inhibited the TRIF-dependent signaling. One can only speculate whether this impairment resulted from a faster degradation of TLR4 or an inefficient formation of its endosomal signaling complex. Later studies on the phagocytosis of E. coli and LPS-coated beads by BMDC expressing the mutated Lyst indicated that the recruitment of Rab7 (also called Rab7a) to maturing phagosomes, but not their acidification, was affected

The participation of CD14 in the activation of TLR4 by LPS and in the endocytosis of LPS-activated TLR4 followed by the TRIF-dependent pro-inflammatory signaling is well established, as discussed above. Notably, CD14 is also involved in the internalization of high doses of LPS which engages scavenger receptors and leads to LPS detoxification by J774 cells [ 160 ], as described in the following section. However, it has to be emphasized that apart from LPS, CD14 binds a broad spectrum of other molecules, including PAMPs, like peptidoglycan, lipoteichoic acid, CpG DNA, and phospholipids [ 51 , 207 ], and also DAMPs such as β-amyloid [ 208 ]. Thus, CD14 can affect the activity of several PRRs. The binding of phospholipids by CD14 is interesting in the context of the LPS-induced pro-inflammatory signaling of TLR4. Among the phospholipids bound by CD14 are 1-palmitoyl-2-glutaroyl- sn -glycero-3-phosphorylcholine and 1-palmitoyl-2-(5-oxovaleroyl)- sn -glycero-3-phosphorylcholine, two forms of oxidized 1-palmitoyl-2-arachidonyl- sn -glycero-3- phosphorylcholine (oxPAPC) released from dying cells at the site of tissue injury. The oxPAPC-induced internalization of CD14 depletes its cell surface pool and as a consequence makes these cells less sensitive to LPS. However, in LPS-primed murine macrophages and DC, the CD14-dependent delivery of oxPAPC into the cells leads to the activation of caspase-11 and caspase-1 followed by IL-1β release. Notably, oxPAPC did not induce pyroptosis of such cells leaving them hyperactive to produce IL-1β without the lethal outcome of an inflammatory response and sepsis in mice [ 144 , 209 ]. These data support the TAXI name given to CD14 as it is able to capture various cargo in addition to TLR4/MD-2/LPS and deliver them to signaling-competent locations [ 17 ]. Only scarce data allow speculation on the pathway(s) involved in the internalization of CD14 carrying LPS and other ligands, like oxPAPC, and also in the constitutivie endocytosis of CD14 in resting murine macrophages [ 17 ]. The latter is of importance since a down-regulation the cell surace level of CD14 and TLR4 in resting cells should prevent excessive LPS-induced pro-inflammatory signaling of TLR4 [ 161 , 210 ]. This mechanism can also contribute to the antagonistic activity of R. spheroides LPS toward human and murine TLR4. Studies performed on murine BMDM showed that this LPS species induced CD14 endocytosis preventing subsequent binding of the pro-inflammatory E. coli LPS to CD14 and consequently to TLR4. The ability of R. spheroides LPS to induce CD14 endocytosis was determined by diphosphorylation of its lipid A [ 17 ]. The LPS-induced endocytosis of CD14 is independent of the signaling activity of TLR4, but it was reduced by a Syk inhibitor or a knocdown of PLCγ2 [ 33 ]. This suggests that, upon LPS binding, CD14 and TLR4 are internalized by the same route discussed above for TLR4 endocytosis. Since this LPS-induced uptake of CD14/TLR4/MD-2 does not require the signaling activity of TLR4, it has been proposed to be controled by CD14 itself [ 17 ]. However, it is still unknown whether the endocytic pathway triggered by LPS is the same as that involved in the constitutive internalization of CD14 in unstimulated cells or in the CD14-mediated uptake of other ligands. Studies on the endocytosis of other GPI-anchored proteins (GPI-APs) may shed light on the LPS-independent internalization of CD14. The majority of GPI-APs are internalized via the dynamin- and clathrin-independent carrier (CLIC) endocytic pathway which also contributes markedly to fluid phase uptake. The internalization of CD14 detected in resting J774 cells displayed properties of a CLIC-mediated uptake; however, the apparent similarities between the CD14 and TLR4 uptake found in those inhibitor-based studies do not allow unequivocal identification of the endocytic pathway involved [ 161 ]. The CLIC-mediated endocytosis is sensitive to actin depolymerization and requires activation of the Arf1 and Cdc42 GTPases [ 211 ]. Cdc42 interacts with PS in the inner leaflet of the plasma membrane. Depletion of CDC50, the α-subunit of the flippase complex, and the following inhibition of PS transport from the outer to the inner monolayer of the plasma membrane increased the surface level of CD14 in unstimulated THP-1 macrophages [ 212 ]. These data suggest that CD14 can be internalized by CLIC-mediated endocytosis involving Cdc42 and PS. An attempt has been undertaken to assess whether the CLIC-mediated endocytosis is also involved in the internalization of CD14 upon LPS binding. It was found that, depletion of galectin-3, a protein triggering CLIC-mediated endocytosis via clustering of cell-surface glycosylated proteins with glycosphingolipids, did not affect the LPS-induced internalization of either CD14 or TLR4 [ 17 , 213 ]. Those results spoke against a role of the galectin-3-facilitated endocytosis of CD14 in LPS-stimulated cells; however, a hypothetical involvemen

The multiple pathways of TLR4 trafficking in diverse cells require tight regulation to ensure an optimal magnitude and duration of pro-inflammatory responses induced by LPS and DAMPs. For this reason, several human hereditary and neurodegenerative diseases caused by disturbances in the functioning of the endo-lysosmal compartment are linked with TLR4 hyperactivation and inflammation contributing substantially to their pathogenesis. This fatal relationship is exemplified by lysosomal storage disorders (LSD) caused by hereditary dysfunctions of lysosomes which are accompanied by an abnormal activation of TLR4 and production of pro-inflammatory cytokines [ 238 , 239 ]. These responses can be triggered by an extracellular accumulation of undegraded substances which act as DAMPs and also by excessive TLR4 stability and persistent signaling caused by an impaired lysosomal proteolysis in cells of LSD patients. In particular, TLR4 has been shown to be involved in the development of mucopolysaccharidosis (MPS), a heterogenous group of LSD resulting from deficiencies in lysosomal enzymes degrading glycosaminoglycans (GAGs). Various MPS subtypes affect different tissues and organs, including central nervous system, bones, muscles, and the connective tissue [ 238 ]. Under physiological conditions, hyaluronian of the GAG family is transported to the extracellular matrix while other GAGs, like heparan sulphate, are exposed on the cell surface and remain bound to the plasma membrane via a proteoglycan core. Thereby heparan sulphate of epithelial cells affects the adhesion of immune cells during inflammation. Several GAGs also induce production of pro-inflammatory chemokines and cytokines. Studies conducted on animal models of MPS VI and/or VII showed an increased level of IL-1β and TNF-α and also an upregulated expression of several other inflammatory markers in synovial fluid, fibroblast-like synoviocytes and serum. Those changes were linked with TLR4 activity as a knock-down of TLR4 in MPS VII animals normalized the serum level of TNF-α and phosphorylation of chondrocyte STAT1 and also alleviated some of the pathological effects of the disease in bones and joints [ 240 , 241 ]. A similar line of data indicated a contribution of TLR4-induced pro-inflammatory signaling of microglia to the onset of neurodegenerative MPS III [ 242 ]. The pro-inflammatory signaling of TLR4 is considered to be triggered by GAG—DAMPs which accumulate in the extracellular space. These include hyaluronian which stimulates TLR4 in cooperation with CD44, and also heparan sulphate and its derivatives resulting from an incomplete degradation of this GAG in lysosomes and subsequent exocytosis of its products. However, an increased level of TLR4 has also been detected in fibroblast-like synoviocytes of MPS VI animals. In addition, these cells have increased mRNA levels of LBP, CD14, and MyD88. The upregulation of TLR4 level was caused by its accumulation in dysfunctional lysosomes with an unchanged expression of the Tlr4 gene [ 240 , 241 ]. Collectively, these results indicate that an impaired lysosomal degradation of TLR4 contributes to the inflammation in MPS. An increased activity of TLR4 is also linked with the pathogenesis of another LSD, Niemann–Pick's type C (NPC) disease leading to progressive neurological dysfunctions in children. NPC is caused by mutations in the NPC1 or NPC2 genes causing malfunctioning of the encoded proteins, which results in an aberrant endosomal membrane flow and accumulation of cholesterol and other lipids in endo-lysosomes. TLR4 level is increased in fibroblasts and glial cells of NPC patients and in Npc1 knock-out mice, respectively, as a result of its impaired degradation. This leads to the accumulation of TLR4 in endosomes and upregulation of its signaling pathways enhancing the production of pro-inflammatory cytokines, such as IL-6, IL-8, and IFN-β. The increased inflammatory response, particularly the glial production of IL-6, can contribute to the pathogenesis of NPC via both the autocrine and paracrine effects of this cytokine [ 243 ]. A participation of the TLR4 receptor has also been noted in the development of cystic fibrosis (CF)—an autosomal recessive disorder caused by mutations in the CFTR gene encoding cystic fibrosis transmembrane conductance regulator, a chloride channel expressed in airway epithelial cells and, to a lower extent, also in other cells. The disease is manifested chiefly by a dysfunction of the airway epithelium, which leads to a dehydrated and acidic airway surface liquid favoring chronic bacterial infections [ 244 , 245 ]. A characteristic feature of cystic fibrosis is an excessive inflammatory response leading to lung damage [ 244 , 245 ]. Data concerning the role of bronchial epithelial cells in the inflammatory response in cystic fibrosis are inconsistent. Early studies reported a decreased or unchanged expression of TLR4 in those cells in comparison with the cells of healthy donors