dynamin-dependent internalization interferes with LPS-mediated TRAM–TRIF-dependent signaling pathway

Cellular Immunology 274 (2012) 121–129

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Inhibition of clathrin/dynamin-dependent internalization interferes with LPS-mediated TRAM–TRIF-dependent signaling pathway Yanyan Wang a,c,1, Yang Yang a,1, Xin Liu a, Ning Wang a, Hongwei Cao a, Yongling Lu a, Hong Zhou b,⇑⇑, Jiang Zheng a,⇑ a b c

Medical Research Center, Southwest Hospital, The Third Military Medical University, Chongqing 400038, China Department of Pharmacology, College of Pharmacy, The Third Military Medical University, Chongqing 400038, China Department of Clinical Lab, Chengdu Military General Hospital, Chengdu 610083, China

a r t i c l e

i n f o

Article history: Received 26 April 2011 Accepted 15 December 2011 Available online 2 January 2012 Keywords: LPS TLR4 Internalization MDC Dynasore Chloroquine Cytokines Chemokines TRAM–TRIF-dependent pathway MyD88-dependent pathway

a b s t r a c t Recognition of lipopolysaccharide (LPS) by Toll-like receptor 4 (TLR4) activates two district proinflammatory signaling pathway and initiates LPS internalization. To investigate roles of LPS internalization, a traditionally regarded metabolic pathway for LPS, in regulation of these two pathways, three internalization inhibitors, monodansylcadaverine (MDC, a clathrin inhibitor), dynasore (DS, a dynamin inhibitor) and chloroquine (CQ, an endosome acidifying maturation inhibitor) were applied to induce internalization dysfunction in macrophages. Results showed MDC and DS affected LPS internalization but did not interfere with their colocalization. Additionally, they decreased cytokines and chemokines release and inhibited signaling molecules activation mediated by TRAM–TRIF-dependent pathway as determined by protein array. In contrast, CQ did not inhibit LPS internalization but affected the colocalization. It also suppressed macrophage activation mediated by both MyD88-dependent and TRAM–TRIF-dependent pathways. The above data indicated that LPS internalization was clathrin/dynamin dependent and it was essential for activation of TRAM–TRIF-dependent signaling pathway. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction The mammalian immune system evolves an elaborately regulated network response to protect the host against infection. In this network, detection of the microbes is mainly mediated by a group of germline encoded pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are best characterized PRRs and play critical roles in the frontier of immune defense. TLRs recognize microbes and recruit TIR domain-containing adaptors which link with kinases to propagate intracellular signal. There are two distinct signaling pathways downstream of TLRs, each with unique adaptor molecules called MyD88 and TRAM–TRIF pairs, respectively. Studies have identified detailed features of the way MyD88 involved in signal transduction included by different TLRs. In contrast, it remains to be better elucidated for mechanisms of TRAM–TRIF pathway and relations of the two signaling pathways.

⇑ Corresponding author. Fax: +86 23 6876 5468. ⇑⇑ Co-corresponding author. Fax: +86 23 6875 2266. E-mail addresses: [email protected] (H. Zhou), [email protected] (J. Zheng). 1 These authors contributed equally to this work. 0008-8749/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2011.12.007

Toll-like receptor 4 (TLR4) is the PRR for lipopolysaccharide (LPS), the major components of the outer membrane of gram negative bacteria [1,2]. TLR4 is also a unique TLR as it recruits both MyD88 and TRAM–TRIF pairs for signal transduction. Herein, it was also the best candidate to study the detailed regulatory mechanism of engagement of these two signaling pathway in LPS–TLR4 activation. It was formerly regarded that LPS was recognized by TLR4 solely from the cell surface, and then initiated MyD88-dependent and TRAM–TRIF-dependent pathway [3]. The internalization of LPS/ TLR4 complex (which has also been identified for decades) was considered to be mainly involved in LPS detoxication and TLR4 recycling [4]. However, recent evidences demonstrated that TLR4 recognized LPS on the cell surface and induced MyD88-dependent signaling. Then the LPS/TLR4 complex was internalized in endosome and triggered the macrophage activation via the TRAM– TRIF-dependent pathway [5]. Internalization of LPS/TLR4 complex is a smart receptor mediated process, requiring either clathrin or not [6]. However, recent results using siRNA for clathrin clearly demonstrated that the early phase (up to 40 min) of LPS internalization is predominantly clathrin-dependent [7]. During clathrin-dependent pathway there are

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three key steps (Fig. S1): cell membrane infolding that depends on clathrin, vesicle formation that relies on dynamin and endosome maturation [8,9]. After the process, the ligands will be degraded in late endosome or lysosome subsequently, whereas the receptors via clathrin/dynamin-dependent internalization are recycled to the cell membrane and re-used to several hundred times. The recycling pathway is essential for returning essential receptor that carries out specific functions for the cells. Specific inhibitors to disturb clathrin/dynamin dependent internalization or endosome maturation or receptor recycling pathway would inhibit specific signaling pathway and then cells functions. Although TLR4 is proposed to firstly induce MyD88-dependent signaling at the plasma membrane and is then internalized and activates MyD88-independent (TRAM–TRIF) signaling from early endosome [8], the role of endocytic pathway for macrophage activation mediated by LPS internalization remains to be elucidated. Therefore, in the present study, three inhibitors such as clathrin inhibitor (monodansylcadaverine), dynamin inhibitor (dynasore) and endosome acidifying maturation inhibitor (chloroquine) were used to make artificial internalization dysfunction cell model at the different stages in order to reveal the role of endocytic pathway for macrophage activation mediated by LPS internalization.

2. Materials and methods 2.1. Materials LPS purified from Escherichia coli O111:B4 (LPS), monodansylcadaverine (MDC), chloroquine (CQ) and 3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dynasore (DS) was purchased from Tocris Bioscience (Bristol, UK). LPS conjugated with fluorescein isothiocyanate (FITC-LPS) was made in our lab. Primary antibodies for TLR4, p-ERK, ERK, p-p38, p38, p-JNK, JNK, p-ERK1/2, ERK1/2, IRF-3 and b-actin and the peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology, Inc. (CA, USA). Phycoerythrin (PE) anti-TLR4 antibody, mouse TNF-a and IL-6 ELISA kits were purchased from eBioscience Inc. (CA, USA). PCR primers for MyD88, TRAM and GAPDH were synthesized from Sangon Biotech Co., Ltd. (Shanghai, China). siRNA for MyD88 and TRAM as well as transfect reagents lipofectamin was synthesized by Invitrogen life technology (CA, USA).

2.2. Cell culture and internalization inhibitors treatment RAW 264.7 cells, the murine macrophage-like cell line (purchased from ATCC, Manassas, VA, USA), were cultured at 37 °C in a 5% CO2 humidified incubator and maintained in a Dulbecco’s modified Eagle’s minimum essential medium (DMEM) supplemented with 10% endotoxin free fetal bovine serum (FBS) (GIBCO, Grand Island, NY, USA), penicillin (100 U/ml), and streptomycin sulfate (100 lg/ml). The cells were diluted with 0.4% trypan blue in phosphate buffered saline (PBS, 0.1 mM, pH 7.4) and live cells were counted by a hemacytometer. The concentration of the cells was adjusted to 1.0  106/ml (except specially mentioned) and culture medium was replaced with FBS free DEME medium. Cells grown in different culture plates were pre incubated with 100 lM MDC, 80 lM DS or 20 lg/ml CQ for 1 h separately and stimulated by LPS or FITC-LPS (100 ng/ml) for indicated time. Cells or supernatants were then harvested for further study.

2.3. MyD88 and TRAM siRNA treatment RAW 264.7 cells (5  104/ml) were cultured in a 24-well plate. After growth of cell fusion, 20 pmol of siRNA was mixed with lipofectamin and added into the cells. The cells were cultured for another 48 h for transfection. The efficiency of specific knockdown was detected by western blot. Then cells were treated with LPS for 24 h. and supernatants were collected for protein array analysis. 2.4. Flow cytometry analysis Cells pretreated with inhibitors in 96-well plates were stimulated with or without LPS for 1 h. Cells were washed twice with PBS and then incubated with the mouse Fluorescent labeled antimouse TLR4 monoclonal antibody (10 lg/ml) for 30 min on ice. Cells were washed twice and resuspended in PBS. Each sample was examined on a FACScalibur flow cytometer (Becton Dickinson, USA). Fluorescence intensity was detected and calculated using the Cellquest Software. 2.5. Immunofluorescence imaging Cells pretreated with inhibitors on glass cover slips were incubated in the presence or absence of FITC-LPS for 1 h, followed by rinse with PBS, fix with 4% paraformaldehyde, block in 5% normal goat serum and incubation with primary antibody of TLR4 at a 1:50 dilution in PBS overnight at 4 °C. Cells were further treated with fluorescent labeled secondary antibody at 1:200 dilution in PBS for 1 h at 37 °C. Nucleuses were stained with nucleic acid dye 4,6-diamino-2-phenylindole (DAPI; Molecular Probes). Each sample was examined under a 510 Meta confocal microscope (Zeiss, Germany) at appropriate wavelengths. Images were captured and processed using the LSM Image Examiner software. 2.6. Cytokines quantification Cells pretreated with inhibitors in 96-well plates were incubated with or without LPS for 0, 3, 6, 12, 24 and 48 h. TNF-a and IL-6 in supernatants harvested at each indicated time-point were detected with mouse TNF-a and IL-6 ELISA kit. 2.7. Cytokines and chemokines protein array analysis Cells pretreated with inhibitors in 96-well plates were incubated with or without LPS for 24 h. The supernatants were collected and cytokines and chemokines analysis was performed on mouse cytokines and chemokines antibody arrays (RayBiotech, Atlanta, GA) according to the manufacturer’s instructions. Briefly, the cell-free culture supernatants containing 50 lg of total proteins were incubated with the array membrane, followed by an additional incubation with Alexa Flour 555-conjugated streptavidin, signals were detected by a laser scanner (Genpix4000B). The intensity of the signals was quantified by Image Analysis Software. The signal intensities were normalized against the positive controls on each array membrane. Fold change in each sample was then calculated and compared. 2.8. Real-time PCR analysis Cells (2  106 cells/ml, 2 ml) pretreated with inhibitors in 6-well plates were incubated with or without LPS ml for 4 h. Total RNA was extracted from the harvested cells using a Trizol reagent (Roche, US) and reverse transcribed into cDNA with a ReverTra Ace-a-RNA easy kit (TOYOBO, Japan). Real-time PCR was performed according to the manufacturer’s instructions. Transcribed cDNA template was mixed with SYBR Green PCR

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mastermix (TOYOBO, Osaka, Japan) and the following primers: MyD88, forward (50 -ACTCGCAGTTTGTTGGATG-30 ) and reverse (50 -CACCTGTAAAGGCTTCTCG-30 ), product, 183 bp; TRAM, forward (50 -AGCCAGAAAGCAATAAGC-30 ) and reverse (50 -CAAACCCAAAGAACCAAG-30 ), product, 188 bp; mouse b-actin, forward (50 -GGGAAATCGTGCGTGACATCAAAG-30 ) and reverse (50 - CATACCCAAGAAG GAAGGCTGGAA-30 ), product, 191 bp. Quantitative real-time PCR was performed using an iCycler Thermal Cycler (Bio-Rad, Hercules, CA, USA), with the reaction conditions as follows: 94 °C hold for 3 min, then 40 cycles of 94 °C for 15 s, 58 °C for 15 s, 72 °C for 40 s, and 72 °C for 5 min. The comparative Ct (threshold cycle) method with arithmetic formulae (2DDCt) was used to determine relative quantitation of gene expression of both target and housekeeping genes (b-actin). 2.9. Western blot analysis Cells (2  106/ml) pretreated with inhibitors in 6-well were treated with or without LPS for 4 h. Cells were collected by centrifugation and the washed cell pellets were resuspended in extraction lysis buffer and incubated 20 min at 4 °C. Cell debris was removed by centrifugation, followed by quick freezing of the supernatants. The protein concentration was determined using the pierce protein assay reagent according to the manufacturer’s instructions. Cellular protein (40 lg) from treated and untreated cell extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Blots on the membranes were blocked in 5% dry skim milk and probed with primary antibodies at 4 °C. The blots were then incubated with secondary goat anti-mouse or-rabbit IgG antibodies (1:10000 dilutions) and developed with SuperSignal West Femto Maximum Sensitivity Substrate kit (ThermoPierce) for chemiluminescence assay under a ChemiDoc XRS gel imaging system (Bio-Rad). 2.10. Trans-AM NF-jB ELISA Cells (2  106/ml) pretreated with inhibitors in 6-well plates were treated with or without LPS for 4 or 12 h. Nuclear proteins were then extracted. The DNA binding activity of NF-jB in the nucleic protein of each sample was quantified by ELISA using the Trans-AM NF-jB p50 Transcription Factor Assay Kit (Active Motif, Tokyo, Japan) according to the instructions of the manufacturer. Briefly, each protein sample was incubated in 96-well plates

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coated with immobilized oligonucleotide containing a consensus (50 -GGGACTTTCC-30 ) binding site. The active NF-jB binding to the target oligonucleotide was detected by incubation with a primary antibody specific for the activated form of p50 or p65, visualized by anti-IgG horseradish peroxidase conjugate and Developing Solution, and detected and quantified at 450 nm. 2.11. Cytotoxicity assay Cells (5  105/ml) were plated in 96-well plates and cultured overnight, and then washed twice and incubated with indicated concentrations of MDC, DS and CQ for 24 h. Subsequently, after the culture medium was removed, 20 ll of the MTT solution (5 mg/ml) was added to the medium to a total volume of 200 ll. After the cells were incubated for 4 h. the supernatants were removed and 150 ml of dimethyl sulfoxide was added to each well for the dissolution of the formazan crystals, the optical densities of which was assayed at 550 nm in a Model 550 microplate reader (Bio-Rad). 2.12. Statistical analysis All experiments were performed at least three times and representative data were presented. Cytokine concentrations were expressed as mean ± S.D. one-way ANOVA test was used for multiple comparisons. A p value less than 0.05 was considered statistically significant. 3. Results 3.1. MDC/DS and CQ affect the decrease of cell-surface TLR4 distribution induced by presence of LPS in a different way To investigate the relations between LPS induced macrophage activation and its endocytic pathway, three specific inhibitors for receptor mediated cellular endocytosis, MDC (disturbing cell membrane infolding) [9], DS (inhibiting vesicle formation [10–12] and CQ (blocking endosome maturation) [13], were used. In order to exclude the possibility that their effects were due to cytotoxicities, we examined the cytotoxic effects of these three agents on RAW 264.7 cells. The MTT results showed that they had no influence on the cell viability used when used in indicated concentrations in this study (Fig. S2).

Fig. 1. Cell surface expression of TLR4 on RAW 264.7 cells. After RAW 264.7 cells were, respectively incubated with MDC (100 lM), DS (80 lM) and CQ (20 lg/ml) for 1 h, and then treated with or without FITC-LPS (100 ng/ml) for 1 h, cell-surface TLR4 level was tested by flow cytometry. The cells without any treatment and incubated with only secondary mAb was used as negative control. The cells without any treatment and incubated with first and secondary mAb was used as control. The data were the representative of triplicate.

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Herein, effects of these inhibitors on cell surface TLR4 distribution were detected by flow cytometry. The results showed the cell-surface TLR4 level decreased quickly after LPS treatment, suggesting a rapid internalization of the receptor. Pretreatment of MDC or DS, which interfered with the endocytic process, markedly suppressed such a decrease of cell surface TLR4 induced by LPS. In contrast, pretreatment of CQ by which the endosome maturation was blocked, the distribution of surface TLR4 decreased even more dramatically compared with LPS alone (Fig. 1). 3.2. MDC and DS prevent internalization of LPS–TLR4 complex while CQ interferes with the colocalization of LPS and TLR4 internalized into the cells To study the effects of the three inhibitors on colocalization of LPS and TLR4, confocal microscopy was used for direct observation of surface or intracellular distribution of the two conjugated with fluorescence. Results showed TLR4 (red fluorescence) distributed both on cell surface and in cytoplasm around the inner part of membranes in cells treated with FITC-LPS alone. After FITC-LPS (green fluorescence) treatment for 1 h, the red and green fluorescence are more concentrated in the cytoplasm and merged as yellow fluorescence, indicating that LPS and TLR4 were internalized and colocalized as a complex. However, in cells which had been pre-incubated with MDC or DS before treatment of FITC-LPS, the merged yellow fluorescence accumulated near the inner part of cell membranes and seldom was observed in the plasma. Interestingly, in CQ pretreated cells the red and green fluorescence became strong and separate dots within the cells, none of which could be merged as yellow fluorescence (Fig. 2). Above results suggested that MDC or DS prevented the internalization of LPS and TLR4 but did not influence LPS and TLR4 colocalization. In contrast, CQ had no effect on the internalization of LPS and TLR4 but led to separation of LPS and TLR4 internalized in the 264.7 cells. 3.3. CQ inhibits cytokines release triggered by both MyD88- and TRAM–TRIF-dependent pathways while MDC and DS only downregulate cytokines production induced by TRAM–TRIF-dependent pathway

Fig. 2. Extracellular and intracellular localization of FITC-LPS and TLR4 (63). After RAW 264.7 cells were, respectively treated with MDC (100 lM), DS (80 lM) and CQ (20 lg/ml) for 1 h, and then treated with or without FITC-LPS (100 ng/ml) for 1 h, the distribution of LPS/TLR4 complex was observed by confocal microscope. The cells were stained with FITC-LPS as green color and stained with anti-TLR4 antibody that was detected by a secondary antibody coupled to PE as red color (PE-TLR4). Nuclear DNA was stained with DAPI (blue).

Release of proinflammatory cytokines like TNF-a and IL-6 is regarded as the marker of macrophages activation mediated by interaction of LPS and TLR4 [14–16]. On the other hand, LPS-induced TLR4 internalization is traditionally thought to result in downregulation of TLR4 signaling. However, its role as a requisite step in TLR4 signaling has been realized recently. To assess how interference of LPS and TLR4 internalization affected macrophages activation, we first detected LPS induced release of TNF-a and IL-6 in RAW 264.7 cells treated with the three inhibitors. The results showed pretreatments of MDC and DS only significantly decreased IL-6 release while CQ inhibited both TNF-a and IL-6 secretion, as compared to LPS treatment alone. Recent published data have established that TNF-a and IL-6 were mediated by TLR4/MyD88dependent pathway and TRAM–TRIF-dependent pathway, respectively [17,18]. Therefore, our results suggested that MDC and DS affected TRAM–TRIF-dependent pathway while CQ plays inhibitory impacted both pathways (Fig. 3), suggesting LPS/TLR4 complex was internalized via clathrin-dependent pathway and endosome maturation had more important role during LPSmediated signaling pathways. To further assess this issue that inhibition of endocytosis (MDC/ DS) and inhibition of endosome maturation (CQ) affected different signaling pathways, a cytokines and chemokines antibody array method was used to detect a wide range of proinflammatory cytokines and chemokines of TLR4 activation (Figs. S3 and S4). Herein, MDC and DS significantly decreased IL-6, IL-1a, sTNF-R1, TIMP-1

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A

B 4000

3000

LPS MDC+LPS DS+LPS CQ+LPS

2500 2000

LPS MDC+LPS DS+LPS CQ+LPS

3000 2000 1000

1500 1000

IL-6 (pg/ml)

TNF-α (pg/ml)

3500

0

3

6

12

24

0

48

Time (h)

0

3

6 12 Time (h)

24

48

Fig. 3. Effects of MDC, DS and CQ on LPS-induced TNF-a and IL-6 release. RAW 264.7 cells (1.0  106 cells/ml) were, respectively pre-incubated with MDC (100 lM), DS (80 lM) and CQ (20 lg/ml) for 1 h, and then incubated with or without LPS (100 ng/ml) for 0, 3, 6, 12, 24, and 48 h. The supernatants were collected for TNF-a and IL-6 assay using respective ELISA kit. The experiment was repeated three times. The cytokine levels were expressed as means ± S.D. ⁄p < 0.05 and ⁄⁄p < 0.01 as compared to LPS. Other details are as described under Section 2.

Table 1 Effects of internalization inhibitors (MDC, DS and CQ) as well as siRNA (MyD88 and TRAM) on cytokines and chemokines release from RAW 264.7 cells treated by LPS. Name

(MDC + LPS)/LPS

(DS + LPS)/LPS

(CQ + LPS)/LPS

(TRAMsiRNA + LPS)/LPS

(MyD88siRNA + LPS)/LPS

IL-12p40a IL-6a IL-2a IL-3a IL-4a sTNF RIa RANTESb SDF-1b TECKb TIMP-1 b IL-1a a IL-1ba IL-17 a Fas Ligand a TCA-3 b MCP-1 b TNF-a a IFN-c a Leptin a IL-10 a IL-13 a sTNF R II a TIMP-2b Lymphotactinb MIGb MIP-1cb Eotaxinb

1.755 0.754 0.744 0.632 0.749 0.624 0.709 0.709 0.733 0.250 0.873 0.850 0.773 0.799 0.837 1.007 1.159 0.873 0.857 0.852 0.899 0.789 1.006 0.959 0.963 0.883 0.884

1.669 0.324 0.743 0.541 0.649 0.641 0.668 0.745 0.534 0.087 0.641 0.680 0.755 0.721 0.647 0.754 0.835 0.964 0.811 0.985 0.808 0.784 0.903 0.892 0.928 0.892 1.008

0.773 0.103 0.472 0.204 0.438 0.544 0.732 0.295 0.246 0.036 0.478 0.266 0.352 0.288 0.326 0.569 0.331 0.724 0.482 0.710 0.360 0.579 0.611 0.717 0.527 0.766 0.493

1.403 0.503 0.452 0.540 0.563 0.909 0.294 1.354 0.697 0.731 0.894 N/A 0.527 0.064 0.765 0.710 0.777 0.825 N/A 0.844 N/A 0.852 1.139 1.111 0.940 0.887 0.998

0.689 0.755 0.716 0.835 0.702 0.910 0.812 0.812 0.920 1.020 0.261 N/A 0.680 0.547 1.021 0.700 0.327 0.765 N/A 0.700 N/A 0.986 0.872 1.011 0.340 1.018 0.471

RAW 264.7 cells (1  106/ml) for inhibition tests were, respectively incubated with MDC (100 lM), DS (80 lM) and CQ (20 lg/ml) for 1 h and then incubated with or without LPS (100 ng/ml) for 24 h. The supernatants were collected for cytokines and chemokines assay using antibody array method. RAW 264.7 cells (5  105/ml) for siRNA tests were cultured in 24-well plates. After growth of cell fusion, 20 pmol of siRNA was mixed with lipofectamin and added into the cells. Then the cells were cultured for another 48 h. The culture media were treated with LPS for 24 h. Supernatants were collected for protein array analysis. The intensities of the signals were quantified by a laser scanner for fluorescence detection and Image Analysis Software. The signal intensities were normalized against the positive control on each array membrane, which were given the arbitrary unit of 1. The ratio of treated groups/LPS group, greater than 1.3 or less than 0.77, was considered significant. The ratios with significance were highlighted in bold and italic form. a Cytokines. b Chemokines.

and MCP-1 release, while CQ inhibited almost all of cytokines and chemokines release (Table. 1). Above data suggested there was a close relation of the release of IL-6, IL-1a, sTNF-R1, TIMP-1, MCP-1 and IL-12p40 to clathrin-dependent endocytic pathway (data got from MDC and DS). These cytokines/chemokines were also identified to be related to TRAM–TRIF-dependent pathway with siRNA knockdown results. Direct transfection with specific siRNA for MyD88 and TRAM could significantly inhibit the expression of the two adaptor molecules separately (Fig. S5). Knockdown of TRAM by its specific siRNA led to decreased release of the above cytokines or chemokines. In contrast, knockdown of MyD88 did not affect above cytokines or chemokines’ expressions (Table. 1).

3.4. MDC and DS inhibit TRAM mRNA expression and CQ affects both MyD88 and TRAM mRNA expressions To determine the direct consequence of disrupted internalization process on the adaptors, MyD88 and TRAM mRNA expressions were examined using real-time PCR analysis. The results were in accordance with those from the cytokines assays. First, MDC and DS significantly decreased TRAM mRNA expression induced by LPS whereas MyD88 mRNA expression was not interrupted. Second, CQ significantly decreased both MyD88 and TRAM mRNA expressions (Fig. 4). Above results further demonstrated disruption of clathrin-dependent internalization would decrease TRAM mRNA

*

**

Medium

LPS

MDC

DS

CQ

Fold change in TRAM mRNA levels

Y. Wang et al. / Cellular Immunology 274 (2012) 121–129 Flod chang in MyD88 mRNA levels

126

* * **

**

Medium

LPS

MDC

DS

CQ

Treatment

Treatment

Fig. 4. Effect of MDC, DS or CQ on mRNA expressions of MyD88 and TRAM. RAW 264.7 cells (2  106/ml) were respectively treated with MDC (100 lM), DS (80 lM) and CQ (20 lg/ml) for 1 h, and then treated with 100 ng/ml LPS for 4 h. Total RNA was extracted, and real-time PCR was programmed according to the manufacturer’s instructions. The comparative Ct (threshold cycle) method with arithmetic formulae (2DDCt) was used to determine relative quantitation of gene expression of both target and housekeeping genes (b-actin). ⁄p < 0.05 and ⁄⁄p < 0.01 as compared to LPS treatment.

expression, leading to inhibition of TRAM–TRIF-dependent pathway and disruption of endosome maturation affected both TRAM–TRIF-dependent and TLR4/MyD88-dependent pathways.

3.5. MDC, DS and CQ inhibit LPS-induced MAPKs phosphorylation and IRF3 degradation Upon recognition of LPS, the cytoplasm domains of TLR4 recruits two adaptors MyD88 [3,19] and TRAM [20–22] to activate two signaling pathways, leading to a cascade of signaling events involving activation of MAPKs and IjB kinases (IKKs) as well as the transcription factor like AP-1, NF-jB and IRF3 [23–26]. MAPKs are activated downstream of recruitment of both MyD88 and TRAM–TRIF, whereas IRF3 is only stimulated by activation of TRAM–TRIF [27–29]. In order to determine the consequence effects of disrupted internalization process, phosphorylations of MAPK family member JNK1/2, ERK1/2, p38 as well as degradation of IRF3 were investigated. The results showed MDC, DS and CQ significantly inhibited MAPKs phosphorylation and IRF3 degradation induced by LPS (Fig. 5), suggesting not only MDC and DS but also CQ inhibited LPS-induced phosphorylation of MAPKs and the degradation of IRF3. Above results further demonstrated disruption of clathrin-dependent internalization and endosome maturation would inhibit LPS induced MAPKs phosphorylation and IRF3 degradation. MAPKs phosphorylation and IRF3 degradation were required for both TRAM–TRIF-dependent and TLR4/MyD88-dependent signaling pathways in macrophages activated by LPS.

3.6. MDC and DS only inhibits late NF-jB activation but CQ inhibits both early and late NF-jB activation The transcription factor NF-jB is an important downstream regulator of the expression of proinflammatory cytokines induced by LPS [30]. Early and late NF-jB activation induced by LPS lead to different cytokines or chemokines release [31]. In order to investigate the consequences that inhibitors of clathrin/dynamin and inhibitor of endosome acidification inhibited different cytokines or chemokines release, the effects of MDC, DS and CQ on early and late NFjB activation induced by LPS were investigated using ELISA method. The results showed LPS could induce early and late NF-jB activation. MDC and DS had no influence on early NF-jB activation but significantly inhibited late NF-jB activation. In contrast, CQ significantly inhibited both early and late NF-jB activation (Fig. 6). Above results further demonstrated disruption of clathrin-dependent internalization would affect LPS-induced early NF-jB activation and endosome maturation would affect LPS-induced both early and late NF-jB activation.

4. Discussion In this study, we identified consequences of interference of the major endocytic pathway on LPS and TLR4 distribution as well as LPS induced cell activation in mouse macrophages. Prevention of clathrin/dynamin-dependent internalization (MDC and DS) only inhibited cytokines/chemokines release and signaling molecules activation mediated by TRAM–TRIF dependent pathway. Inhibition of endosome acidifying maturation (CQ) affected cytokines and chemokines signaling molecules activation mediated by both MyD88 and TRAM–TRIF-dependent pathways. LPS stimulation has been found contributing to downregulation of the cell surface TLR4, as a result of rapid endocytosis. After LPS and TLR4 enter the endosomes, the two form a complex and colocalize at the same site [7]. Herein, we obtained similar results and identified preliminary the entering route of LPS and TLR4. Inhibition of clathrin-dependent pathway using clathrin and dynamin inhibitors (MDC and DS) was found to increase the cell surface TLR4 level and decrease amounts of LPS/TLR4 complex within the cells alternatively. But these two inhibitors did not affect LPS/ TLR4 colocalization. We thought MDC and DS inhibited clathrin/ dynamin and then the cell-surface TLR4 could not enter the cells, leading to higher cell-surface TLR4 level and less LPS/TLR4 complex within the cells. In this case, MyD88-dependent signaling pathway was not affected. However, TRAM–TRIF-dependent pathway that requires activation in endosome is significantly impaired. Pretreatment of CQ affected the internalization and colocalization of LPS and TLR4 in a way different from MDC and DS. CQ markedly decreased the cell-surface TLR4 level and increased the amounts of LPS and TLR4 within cells. Interestingly, the increased LPS and TLR4 neither formed complex nor colocalized at same compartment in the cells treated with CQ. As an endosome acidifying maturation inhibitor which does not influence LPS/TLR4 internalization, CQ may take effect by disturbing the regular recycling of TLR4 [32]. Therefore, the cell-surface TLR4 could enter the cells, displaying the phenomenon of cell-surface TLR4 level decrease. However, CQ inhibited endosome acidifying maturation, which is necessary for degradation of LPS and recycling of TLR4 within cells. The failure of TLR4 recycling further contributes to the incompetence of macrophages to be activated by LPS stimulation. Based on the above analysis, it was reasonable that CQ could increase amounts of LPS and TLR4 and inhibited LPS induced macrophage activation. Nevertheless, the reason that LPS and TLR4 neither formed complex nor colocalized at same compartment within cells treated with CQ needs further investigation. After recognized by TLR4, LPS triggers MyD88-dependent and TRAM–TRIF-dependent pathway to induce cytokines and chemokines release [18]. TNF-a release was generally mediated by MyD88-dependent pathway [17]. IL-6, IL-12p40, IFN-b and IP-10

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LPS

MDC+LPS DS+LPS

Medium

LPS

CQ+LPS

P-JNK

46/54KD

JNK

46/54KD

p-ERK

42/44KD

ERK

42/44KD

p-p38

38KD

p38

38KD

IRF3

50KD

β-actin

40KD

Relative Intensity

0.5 0.4 *

0.3

‡ ‡

0.2

Relative Intensity

p-JNK

†

0.1 0.0 Medium

LPS MDC+LPS Treatment

DS+LPS

Medium

LPS

CQ+LPS

Treatment

p-ERK1/2

2.0

**

0.4 ‡

0.3

†

0.2 0.1

Relative Intensity

Relative Intensity

0.5

0.0 Medium

LPS MDC+LPS Treatment

** 1.6 †

1.2 0.8 0.4 0.0

DS+LPS

Medium

10.0

CQ+LPS

1.2

**

7.5

‡

5.0

‡ 2.5

Relative Intensity

Relative Intesity

LPS Treatment

p-p38

0.0 Medium

LPS MDC+LPS Treatment

**

1.0

‡

0.8 0.6 0.4 0.2 0.0

DS+LPS

Medium

LPS

CQ+LPS

Treatment IRF3

Relative Intensity

‡ 0.2

†

0.1 **

0.1 0.0

Relative Intensity

1.0

0.2

‡

0.8 0.6

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0.4 0.2 0.0

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MDC+LPS

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Medium

LPS

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Fig. 5. Effects of MDC, DS and CQ on LPS-induced phosphorylation of MAPKs and degradation of IRF3. RAW 264.7 cells (2  106/ml) were, respectively treated with MDC (100 lM), DS (80 lM) and CQ (20 lg/ml) for 1 h, and then treated with 100 ng/ml LPS for 4 h. Whole cell extracts or cytoplasmic fractions were prepared from the cells, and examined for phosphorylated JNK, ERK1/2, p38 and the degradation of IRF3 by western blotting. b-Actin was used as an equal loading and transfer control. The relative intensity of the specific band was calculated and shown in the right figures. Each column represents the mean ± SD of three independent experiments. ⁄p < 0.05 and ⁄⁄p < 0.01 as compared to medium;  p < 0.05 and àp < 0.01 as compared to LPS.

release were predominately mediated by TRAM–TRIF-dependent pathway (Table 2) [9,33,34]. In our study, we first found that IL-6 release was impaired in internalization dysfunctional

macrophages but the secretion of TNF-a was as normal. This phenomenon suggests that inhibition of LPS internalization only affects TRAM–TRIF-dependent pathway. However, there was no

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Appendix A. Supplementary data

2.00 **

NF-κ B activity

1.60

NF-κB 4h

NF-κB 12h

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cellimm.2011.12.007.

† ‡

1.20

‡

References

0.80 †

** 0.40 0.00 Medium

LPS

MDC+LPS

DS+LPS

CQ+LPS

Treatment Fig. 6. Effects of MDC, DS and CQ on NF-jB activation. RAW 264.7 cells (2  106 cells/ml, 2 ml) were, respectively incubated in the absence or presence of MDC (100 lM), DS (80 lM) and CQ (20 lg/ml) for 1 h at 37 °C in 6-well polystyrene plates. After cells were re-cultured with LPS (100 ng/ml) for 4 or 12 h, nuclear proteins were extracted and NF-jB activation in RAW 264.7 cells was determined by TransAM™ NF-jB p50 Transcription Factor Assay Kits. Data shown were the results of one experiment that was representative of three independent experiments. ⁄⁄p < 0.01 as compared to medium;  p < 0.05 and àp < 0.01 as compared to LPS.

report of detailed cytokines and chemokines profiles which were probably related to TRAM–TRIF dependent pathway or LPS internalization. So we conducted a protein arrays analysis in our present experiments and outlined a draw that IL-6 and other cytokines and chemokines like IL-2, IL-3, IL-4, sTNF RI, RANTES, TECK, and TIMP-1, etc. (Table 1) were released via TRAM–TRIFdependent pathway. These proteins are also dependent on internalization of LPS as MDC and DS also downregulate their release. This result further identified our assumption that inhibition of LPS internalization only affects TRAM–TRIF-dependent pathway. The MyD88-dependent signaling pathway induces the rapid activation of serine-threonine kinases such as p38 and IKKb [23,24], whereas the TRAM–TRIF-dependent pathway induces the activation of transcription factor IRF3, and the late-phase activation of NF-jB and MAPK [33]. IKK and MAPK are kinases for both TLR4/MyD88-dependent and TRAM–TRIF-dependent pathways while IRF3 is only required for TRAM–TRIF-dependent pathway. Herein, MDC and DS could significantly inhibit phosphorylation of MAPK (p-JNK, p-ERK1/2 and p-p38), leading to decreased late phase NF-jB activation and IRF3 degradation. CQ could inhibit phosphorylations of MAPKs and degradation of IRF3 much more significantly, leading to dramatic decrease of both early and late NF-jB activation as well as IRF3 degradation. Above results further demonstrated clathrin/dynamin-dependent internalization was only associated with the TRAM–TRIF-dependent pathway but the endosome maturation was associated with both of MyD88dependent and TRAM–TRIF-dependent pathways. In conclusion, our results demonstrated internalization of LPS/ TLR4 complex was clathrin/dynamin-dependent process, which was essential for TRAM–TRIF-dependent signaling pathway. Disruption of clathrin/dynamin-dependent internalization would interfere with TRAM–TRIF-dependent signaling pathway meanwhile inhibition of endosome acidification would interfere with both of MyD88-dependent and TRAM–TRIF-dependent pathways.

Acknowledgments This work was supported by a Grant from the National Natural Science Foundation of China 30872681 to Jiang Zheng.

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