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

Abstract

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.

Keywords: Eritoran; TLR4 endocytosis; agonistic antibody; endotoxin tolerance; small-molecule ligands.

Fig. 1.

CD14 is not required for UT12-induced TLR4 endocytosis and downstream signaling in PMs. WT and CD14^−/− mouse PMs were medium-treated or treated with LPS (100 ng/mL), UT12 (1,000 ng/mL), and UT12 istoype (1,000 ng/mL) for the indicated times, and TLR4 surface expression was analyzed by flow cytometry (A and B). A representative histogram was shown after 90 min of treatment (A), and TLR4 internalization was quantitated by mean fluorescence intensity (MFI) at each time point (B). Total cell lysates from WT and CD14^−/− PMs were prepared at 30 min (A, Left) and 60 min (A, Right) after treating with LPS, UT12, and UT12-isotype control antibody and activating signaling molecules analyzed by Western blotting (C). For cytokine/chemokine secretion, WT and CD14^−/− PMs were treated with LPS, UT12, and UT12-isotype control antibody for 16 h, and culture supernatants were analyzed by ELISA (D). CD14 expression in WT and CD14^−/− PMs was analyzed by flow cytometry (E, Left). WT PMs were treated with medium only, LPS, UT12, and UT12-isotype control antibody for indicated times, and CD14 surface expression was analyzed by flow cytometry and quantified by MFI (E, Right). Data represent the mean ± SEM from two to three independent experiments. ^#P < 0.05, nontreated vs. treated groups; *P < 0.05, treated WT vs. treated CD14^−/− groups. (NT, not treated; Iso, isotype).

Fig. S1.

Dose-dependent TLR4 internalization in WT and CD14^−/− PMs. (A) WT and CD14^−/− PMs were treated with medium only or with increasing doses of LPS (0.1–1,000 ng/mL), UT12 (1–1,000 ng/mL), UT12-istoype antibody (1,000 ng/mL), or (B) synthetic TLR4 ligands 1Z105 and 1Z204 (0.1–10 μM) and a control compound, 1Y88 (10 μM) for 90 min, and TLR4 surface expression was analyzed by flow cytometry and quantified by MFI at each dose.

Fig. S2.

FcR α-chain and γ-chain deficiency does not affect UT12- and LPS-induced TLR4 internalization. WT and FcR α-chain^−/− and γ-chain^−/− BMDMs were untreated or treated with UT12 or LPS for indicated times, and TLR4 surface expression was analyzed by flow cytometry and quantified by MFI at each time point.

Fig. S3.

Syk and PLC-γ2 inhibitors reversed LPS- and UT12-induced TLR4 endocytosis and IRF3 activation. PMs from WT mice were pretreated with either DMSO (0.1%) or the Syk inhibitor, piceatannol (75 μM), or the PLC-γ2 inhibitor, U73122 (5 μM) for 30 min and then treated with LPS (50 ng/mL) and UT12 (100 ng/mL) for 90 min. TLR4 internalization was analyzed by flow cytometry; a representative histogram is shown (A and B). TLR4 surface expression was quantified using MFI (C and D). For signaling studies, PMs were pretreated with piceatannol (E) or U73122 (F) for 30 min and stimulated with LPS or UT12 for 60 min as described above, and total cell lysates were subjected to Western blotting (E and F). Data represent mean ± SEM from three independent experiments. (NT, not treated; Iso, Isotype).

Fig. S4.

MyD88 and TRIF deficiency did not affect TLR4 internalization induced by LPS and UT12. WT, MyD88^−/−, and TRIF^−/− PMs were treated with medium only or with LPS (100 ng/mL), UT12 (1000 ng/mL), or 1Z105 (5 μM) for the indicated times, and TLR4 surface expression was analyzed by flow cytometry and quantified by MFI at each time point (A and C). Culture supernatants were collected after 16 h of treatment, and ELISAs were performed for secreted cytokines/chemokines (B and D).

Fig. S5.

CD14 deficiency minimally affected pI:C-induced IFN-β. WT and CD14^−/− PMs were treated with medium or with pI:C (20 μg/mL) for the indicted times, and surface expression of TLR4 and CD14 was analyzed by flow cytometry (A and B) (NT, not treated; Iso, Isotype). (A) A representative histogram of surface TLR4 at 3 h (Left). TLR4 internalization quantified by MFI at each time point (Right). (B) A representative histogram for surface CD14 is shown at the indicated times (Left). CD14 internalization quantified by MFI at each time point (Right). (C) Culture supernatants from cells analyzed in B were harvested at 16 h and analyzed for secreted cytokines/chemokines.

Fig. 2.

TLR4 synthetic small-molecule ligands induce receptor endocytosis and related signaling in a CD14-independent manner. WT and CD14^−/− PMs were medium-treated or treated with LPS (100 ng/mL) and different synthetic ligands (5 μM) for the indicated times, and TLR4 internalization was analyzed by flow cytometry and quantified by MFI at each time point (A) as described in the legend to Fig. 1. Total cell lysates from WT and CD14^−/− PMs were prepared 60 min after treating cells with LPS or synthetic ligands, and activation of signaling molecules was analyzed by Western blotting (B). For cytokine/chemokine secretion, WT and CD14^−/− PMs were treated with LPS and synthetic ligands for 16 h, and culture supernatants were analyzed by ELISA (C). Data represent the mean ± SEM from two to three independent experiments. ^#P < 0.05, nontreated vs. treated groups; *P < 0.05, treated WT vs. treated CD14^−/− groups.

Fig. S6.

CD14-independent up-regulation of B7 costimulatory molecules by UT12 and 1Z105. WT and CD14^−/− mouse PMs were medium-treated (NT) or treated with LPS (100 ng/mL), UT12 (1,000 ng/mL), or 1Z105 (5 μM) for 18 h, and surface expression of CD80 (A) and CD86 (B) was analyzed by flow cytometry. (A and B) A representative histogram after 90 min treatment. (C and D) CD80/CD86 up-regulation quantified by MFI. Data represent mean ± SEM from three independent experiments. (NT, not treated; Iso, Isotype).

Fig. S7.

CD14 is not required for UT12- and 1Z105-induced TLR4 endocytosis and downstream signaling in BMDMs. WT and CD14^−/− BMDMs were medium-treated or treated with LPS (100 ng/mL), UT12 (1,000 ng/mL), or 1Z105 (5 μM) for the indicated times, and TLR4 surface expression was analyzed by flow cytometry (A and B) (NT, not treated; Iso, Isotype). A representative histogram is shown after 90 min treatment (A), and TLR4 internalization was quantified by MFI at each time point (B). Total cell lysates from WT and CD14^−/− BMDMs were prepared at 60 min after treatment with medium only or with LPS, UT12, or 1Z105, and activation of signaling molecules was analyzed by Western blotting (C). For secreted cytokines/chemokines, WT and CD14^−/− BMDMs were treated with LPS, UT12, or 1Z105 for 16 h, and culture supernatants were collected and analyzed by ELISA (D). WT BMDMs were treated with medium only, LPS, UT12, and 1Z105 for the indicated times, and CD14 surface expression was analyzed by flow cytometry and quantified by MFI (E). Data represent the mean ± SEM from two to three independent experiments.

Fig. S8.

UT12 and 1Z105 did not induce CD14 colocalization with endosome in BMDMs. WT BMDMs were treated with medium (Med), LPS (100 ng/mL), UT12 (1 μg/mL), and 1Z105 (5 μM) for 30 min. Cells were fixed, permeabilized, blocked, and stained with anti–CD14-Alexa488 (green), anti–EEA-Alexa647 (red), and DAPI (blue) and analyzed using confocal microscopy. Merged images of CD14, EEA1, and DAPI are shown. Original magnification: 40×. (Scale bar: 20 μm.)

Fig. S9.

Dynasore inhibited LPS- and 1Z105-driven not UT12-induced TLR4 endocytosis but inhibited LPS-, UT12-, and 1Z105-induced IRF3 activation. BMDMs from WT and CD14^−/− mice were pretreated with either DMSO or dynasore (80 μM) for 60 min and then treated with medium only, LPS (50 ng/mL), UT12 (100 ng/mL), or 1Z105 (5 μM) for 90 min. TLR4 internalization was analyzed by flow cytometry; and a representative histogram is shown (A and B) (NT, not treated; Iso, Isotype). TLR4 surface expression was quantified by MFI (C). For signaling studies, BMDMs were stimulated with LPS or UT12 or 1Z105 in the absence or presence of dynasore for 60 min, and total cell lysates were subjected to Western analysis (D). Culture supernatants were collected after 16 h of treatment described above and cytokines were analyzed by ELISA (E). Data represent mean ± SEM from two experiments.

Fig. 3.

The TLR4 antagonist E5564 failed to inhibit UT12-induced receptor internalization, but inhibited MyD88- and TRIF-dependent signaling. WT PMs were pretreated with 10 ng/mL E5564 for 60 min and then treated with LPS, UT12, or 1Z105 for the indicated times. TLR4 internalization was analyzed by flow cytometry, and surface expression was quantitated using MFI (A). TLR4 internalization induced by 1Z105 was analyzed after 90 min of treatment in CD14^−/− PMs in the absence or presence of E5564 as described above (B). PMs were stimulated with LPS, 1Z105, UT12, or R848 in the absence or presence of E5564 (10 ng/mL) for 60 min, and total cell lysates were subjected to Western blotting (C). PMs were treated with different TLR4 ligands as described above, and RNA was isolated after 1 and 5 h. Gene expression was analyzed by quantitative real-time PCR (qRT-PCR) (D). HEK293T cells were treated with different TLR4 ligands in the absence or presence of Eritoran for 30 min, and induction of TLR4 dimerization was analyzed by immunoprecipitation followed by Western analysis (E). Data represent the mean ± SEM from two to three independent experiments. *P < 0.05, treated without vs. with E5564 groups.

Fig. 4.

Endotoxin tolerance induced TLR4 endocytosis in CD14^−/− PMs. WT and CD14^−/− PMs were tolerized overnight for 18 h with LPS or UT12. Cells were washed to remove endotoxin or UT12 and restimulated with either medium or LPS or UT12 for indicated times. Surface expression of TLR4 (A and B) was analyzed by flow cytometry as described in Fig. 1. Similarly, WT and CD14^−/− PMs were tolerized for 18 h as described above, and total cell lysate was prepared after 30 min of restimulation with medium only, LPS, or UT12; activation of signaling molecules was analyzed by Western blotting (C). For cytokine secretion, WT and CD14^−/− PMs were tolerized for 18 h as described above and restimulated with LPS or UT12 for 16 h, and culture supernatants were analyzed for cytokine/chemokine level by ELISA (D). For each graph, data represent the mean ± SEM from two to three independent experiments. Percentage surface TLR4 in WT and CD14^−/− was normalized using medium-treated WT macrophages as 100%. ^#P < 0.05, medium treated vs. nontolerized and tolerized groups; *P < 0.05, M/L (nontolerized) vs. L/L and U/L (tolerized); ^‡P < 0.05, M/U (nontolerized) vs. U/U and L/U (tolerized).

Fig. S10.

Hypothetical model of CD14-dependent and -independent TLR4 internalization and TRIF-dependent signaling and their dissociation. Unlike LPS that requires CD14 for stabilization of the TLR4/MD2 complex, UT12 and/or 1Z105 bind directly to TLR4/MD2 and form a stable TLR4/MD2 dimer complex, even in the absence of CD14, that leads to NF-κB activation whereas TLR4/MD2 internalization leads to IRF3 activation (A). UT12-induced TLR4 endocytosis is not affected in the presence of E5564, but both NF-κB and IRF3 signaling are blocked (B). E5564 blocks 1Z105-induced TLR4/MD2 dimer complex formation that inhibits both TLR4 endocytosis and NF-κB and IRF3 signaling (C). In tolerized macrophages, TLR4 endocytosis is not affected, but both NF-κB and IRF3 signaling are blocked (D).