TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells (original) (raw)
TLR2 but not TLR4 is presented on the apical surfaces of airway epithelial cells. The distribution of the major TLRs TLR2 and TLR4 was examined using confocal microscopy in 16HBE human airway cells grown in a polarized fashion at an air-liquid interface with intact tight junctions. TLR2 was found in an apical distribution quite distinct from that of TLR4, which was more diffusely distributed (Figure 1A). AsialoGM1 in these terminally differentiated cells was also apical, and asialoGM1 colocalized with TLR2 but not with TLR4. To establish that these cell lines accurately reflect the properties of cells in vivo, we also imaged the localization of TLR2 and TLR4 in epithelial cells in primary culture and found TLR2 colocalization with asialoGM1 (Figure 1B). Because TLR2 is glycosylated, we confirmed the specificity of the antibodies by using specific blocking peptides and demonstrating that anti-asialoGM1 did not recognize TLR2 when TLR2 was overexpressed in CHO cells (data not shown).
Distribution of asialoGM1, TLR2, and TLR4 in infected airway cells. (A) Confocal microscopy was used to image monolayers of polarized 16HBE cells transfected with Flag epitope–tagged TLR2 or TLR4 and stimulated with P. aeruginosa PAO1. z-sections treated with anti-Flag labeled with TRITC (red) and with anti-asialoGM1 labeled with FITC (green) are shown. AsialoGM1 (aGM1) is apical and colocalizes with TLR2 (yellow) in discrete clusters along the apical surface of the monolayers. TLR4 is more diffuse, and colocalization with asialoGM1 is not appreciable. (B) Human airway cells in primary culture isolated from nasal polyps from a CF patient and stimulated with P. aeruginosa PAO1 were stained for asialoGM1 labeled with TRITC and TLR2 labeled with FITC and show abundant colocalization of these receptors.
More conclusive evidence that TLR2 is available on exposed, apical surfaces of airway epithelial cells was obtained in biotinylation experiments. Polarized 16HBE cells, both unstimulated and after exposure to S. aureus, were biotinylated and, after immunoprecipitation with streptavidin, Western hybridizations were performed to screen for components of the TLR signaling cascade (Figure 2A). TLR2 as well as the kinase TRAF6, which is involved in TLR-mediated responses, were present, as well as asialoGM1. Caveolin-1, the scaffolding protein that is often a component of signaling complexes associated with membrane lipids, was also present (14). Increased amounts of TLR2, caveolin-1, and especially IRAK-1 were surface biotinylated after bacterial stimulation.
Identification of apically exposed components on polarized 16HBE cells exposed to bacteria. (A) Surface-exposed components of the airway cells were biotinylated under control conditions (–) and after a 1-hour exposure to S. aureus (+). After immunoprecipitation with streptavidin, Western hybridizations were done and asialoGM1, TLR2, and caveolin-1 (Cav-1), as well as the kinases IRAK-1 and TRAF6, were detected. (B) Coimmunoprecipitation studies demonstrate TLR2 but not TLR4 in a receptor complex along with asialoGM1. Coimmunoprecipitations of whole-cell lysates from control and _S. aureus_–stimulated cells were done using anti–caveolin-1, anti-TLR2, and anti-asialoGM1 as capture antibodies with screening for expected components of the TLR pathway, MyD88 and IRAK-1, as well as c-Src and TLR4.
A physical association between the receptors and kinases was further established by coimmunoprecipitation studies (Figure 2B). A lysate of 16HBE cells was immunoprecipitated with each of the capture antibodies indicated, including anti-asialoGM1, confirming the observed association of TLR2, caveolin-1, and IRAK-1. In addition, we identified MyD88 and c-Src in this complex. It is of note that caveolin-1 has a binding domain for c-Src, one of the kinases that is activated in asialoGM1-mediated signaling (4, 12), consistent with its close connection with the glycolipid receptor. TLR4 was not associated with asialoGM1. The colocalization of MyD88, IRAK-1, TRAF6, and TLR2 at the apical surface of the airway cells was demonstrated by confocal microscopy (Figure 3).
Surface colocalization of MyD88, IRAK-1, and TRAF6 with TLR2 confirmed by confocal microscopy. After stimulation with S. aureus, permeabilized cells were stained with the kinases, each labeled with an Alexa Fluor 488–tagged secondary antibody (green). All were found at the cell surface, colocalized (yellow) with TLR2, labeled with an Alexa Fluor 594–tagged secondary antibody (red).
TLR2 is mobilized into lipid rafts after bacterial exposure. The association of a ganglioside, caveolin-1, and signaling kinases has been well described in many signaling systems (13). The distribution of both caveolin-1 and GM1 on the apical surface of the polarized airway cells was visualized by confocal microscopy (Figure 4A). When associated with plasma membranes, these components often are compartmentalized into discrete structures called caveolae (14). Caveolae have been demonstrated in pulmonary cells (15, 16), although they have not previously been associated with bacterial infection. Airway epithelial monolayers incubated with P. aeruginosa were examined by electron microscopy for evidence of such structures. The flask-like structure typical of caveolae was readily apparent in the infected monolayers (Figure 4B). While the intact organisms are clearly too large to interact with the caveolae themselves, flagellin (55-kDa subunits) or pilin (15-kDa subunits), both of which bind to the asialoganglioside asialoGM1 (17, 18), could be readily accommodated by these structures. By flow cytometry, an increase in superficial caveolin-1 was readily detected in polarized cells following bacterial exposure (Figure 4C).
Lipid rafts are involved in clustering of receptors and signaling. (A) Confocal z-section images demonstrate caveolin-1 labeled with Alexa Fluor 594 (red) and GM1, identified with cholera toxin β-subunit (CTB) conjugated to Alexa Fluor 488 (green), on the apical surfaces of polarized 16HBE cells permeabilized after stimulation with Pam3Cys-Ser-Lys4. (B) CF nasal polyp cells were infected with P. aeruginosa PAO1 (PA) and grown on semipermeable supports, and transmission electron micrograph were obtained after 3 hours of bacterial exposure. Arrow (Cav) indicates the flask-shaped electron-dense structures typical of caveolae (magnification, ∞30,000). (C) Flow cytometry was used to detect superficial caveolin-1 on polarized 16HBE cells after exposure to S. aureus. Unstim, unstimulated. (D) Aliquots of Triton-insoluble lysates of 16HBE cells obtained before (–) and after (+) stimulation with P. aeruginosa PAO1 were fractionated on discontinuous sucrose gradients (4–40%) and were immunoblotted with anti-flotillin, anti-caveolin, anti-TLR-2, anti-IRAK-1, or anti-asialoGM1. Downward arrow indicates raft fraction containing all the components after stimulation. (E) Aliquots of sucrose gradient fractions from cells treated with filipin prior to stimulation with P. aeruginosa PAO1 were immunoblotted with anti-flotillin, anti-TLR2, and anti-asialoGM1.
Biochemical evidence of lipid rafts in airway epithelial cells was obtained by the isolation of Triton-insoluble material from 16HBE cells before and after exposure to bacteria (19). This material was then fractionated on a discontinuous sucrose gradient and aliquots were screened for components of the receptor-signaling complex (Figure 4D). Flotillin was used as a marker for raft fractions (20). Caveolin-1 was distributed throughout the fractions, including those expected to represent lipid rafts. TLR2 was specifically mobilized into the raft fraction in cells stimulated with bacteria, as was IRAK-1 and asialoGM1. To confirm the compartmentalization of TLR2 into lipid rafts, we treated cells with filipin, which intercalates into lipid moieties disrupting lipid raft structures (21). Sucrose gradient fractions were similarly screened for TLR2, asialoGM1, and flotillin (Figure 4E). In filipin-treated cells, the partitioning of both flotillin and TLR2 in the sucrose fractions expected to include lipid rafts was lost. Instead, they were distributed throughout the Triton-insoluble material. AsialoGM1, as a lipid, was dispersed by the filipin treatment.
Given that superficial TLR2 has an active role in signaling from an apical lipid-raft complex, we postulated that both TLR2 and asialoGM1 would be co-mobilized to the cell surface in response to ligands with specificity for either receptor. There is a limited amount of surface asialoGM1 on normal human airway cells (18). Using flow cytometry, we quantified superficial asialoGM1 and TLR2 on human nasal polyp (HNP) airway cells before and after exposure to bacteria (Figure 5). In response to P. aeruginosa or S. aureus, there was a substantial increase in asialoGM1 as well as TLR2 on epithelial cells in primary culture (Figure 5A). Mobilization of these receptors in 16HBE cells was less substantial but was evident. No changes were detected in surface-accessible TLR4. We postulated that there may be reciprocal mobilization of both asialoGM1 and TLR2 induced by ligands with specificity for either receptor. Pretreatment of airway cells with monoclonal anti-asialoGM1 also resulted in increased surface expression of TLR2 and asialoGM1. Much greater mobilization of both asialoGM1 and TLR2 was observed after stimulation with the TLR2 agonist Pam3Cys-Ser-Lys4. There was no increase in superficial TLR4 in response to any of the ligands tested, including LPS (Figure 5B).
Mobilization of TLR2 and asialoGM1 in response to bacteria or bacterial ligands. Flow cytometry was used to quantify exposed asialoGM1, TLR2, and TLR4 on primary (HNP) cells (A) or 16HBE cells (B) after stimulation with P. aeruginosa or S. aureus or with monoclonal anti-asialoGM1, Pam3Cys-Ser-Lys4 (Pam3Cys), a TLR2 ligand, or LPS, a TLR4 ligand. Peaks outlined with a thin black line indicate binding by secondary antibody alone; gray-shaded peaks represent the labeled population under control conditions; and peaks demarcated by the heavy black line represent the population after stimulation.
AsialoGM1-associated ligands signal through TLR2. The availability of TLR2 on the apical surface of the airway epithelium suggests that TLR2-associated ligands released from lysed organisms, such as lipoproteins or peptidoglycan fragments, could readily activate the epithelial cells via interactions with caveolae. However, the presence of asialoGM1 in caveolae provides an additional receptor for bacterial components, including pili and flagella, that potently activate epithelial NF-κB and proinflammatory gene expression. The association of asialoGM1 and TLR2 receptors, along with the accessory proteins and kinases, suggested that ligands that recognize asialoGM1 and activate NF-κB or IL-8 expression do so through the TLR components MyD88 and TRAF6 (22). IL-8 expression induced by ligands with specificity for asialoGM1 should be inhibited by dominant negative (DN) mutations that interfere with TLR signaling. In human airway cells expressing a DN form of TLR2, activation of IL-8 by P. aeruginosa, S. aureus, or anti-asialoGM1 was inhibited by 48%, 49%, and 59%, respectively, compared with that of cells transfected with a vector expressing a wild-type TLR2 plasmid construct (P < 0.001 for each) (Figure 6A). Activation of an NF-κB–luciferase reporter in cells transfected with plasmids expressing TLR2, MyD88, or TLR4 DN mutations was similarly compared with that of cells transfected with an empty vector control plasmid (Figure 6B). Activation mediated by P. aeruginosa, S_. aureus_, anti-asialoGM1, or the TLR2 agonist Pam3Cys-Ser-Lys4 was significantly inhibited by the TLR2 DN and the MyD88 DN mutants (P < 0.001). A TLR4 DN mutation had no effect on the activation of NF-κB activity, and LPS (alone or in the presence of serum) failed to stimulate sufficiently to test the effects of the DN mutations (data not shown).
Activation of NF-κB and IL-8 by bacteria or bacterial agonists is inhibited by DN mutations in TLR2 and MyD88. (A) IL-8 expression in 1HAEo- cells transfected with plasmids containing wild-type, DN TLR2, or a vector control was quantified by ELISA after exposure to P. aeruginosa PAO1, S. aureus RN6390, or anti-asialoGM1. Values represent the fold increase in IL-8 compared with that of unstimulated cells. IL-8 in cells stimulated by PAO1 was 1.9–2.5 ng/ml (*P < 0.001). (**B**) NF-κB luciferase activity in 1HAEo- cells transfected with plasmids expressing TLR2 DN or MyD88 DN constructs compared with that of cells transfected with the corresponding empty vector control was significantly inhibited after stimulation with _P. aeruginosa_ PAO1, _S. aureus_ RN6390, anti-asialoGM1, or Pam3Cys-Ser-Lys4 (*_P_ < 0.001 for each). The TLR4 DN construct did not inhibit NF-κB luciferase activity. NF-κB luciferase activity for cells expressing the control vector was normalized for each stimulus and represents three- to fivefold increases over that of unstimulated cells. (**C**) Inhibition of IL-8 activation in the presence of filipin. IL-8 production induced by _P. aeruginosa_, _S. aureus_, anti-asialoGM1 (aGM1), or Pam3Cys-Ser-Lys4, but not TNF-α, was significantly reduced by filipin (_P_ < 0.05, _P_ < 0.001, _P_ < 0.05, and _P_ > 0.05, respectively).
We confirmed the involvement of the lipid raft structure in signaling by testing the effects of filipin, which intercalates into lipid moieties and interrupts ganglioside-dependent signaling (21). Bacterial activation of IL-8, as stimulated by P. aeruginosa, S. aureus, Pam3Cys-Ser-Lys4, or anti-asialoGM1, was significantly inhibited in the presence of filipin compared with that of untreated controls (P < 0.05, P < 0.001, P < 0.05, and P < 0.05, respectively). In contrast, expression of IL-8 induced by TNF-α was not significantly inhibited (Figure 6C). Nystatin and methyl-β-cyclodextrin had similar effects (data not shown).