Lipoteichoic acid from an Enterococcus faecalis clinical strain promotes TNF-α expression through the NF-κB and p38 MAPK signaling pathways in differentiated THP-1 macrophages

  • Authors:
    • Shuai Wang
    • Kun Liu
    • Chaminda Jayampath Seneviratne
    • Xuechen Li
    • Gary Shun Pan Cheung
    • Lijian Jin
    • Chun Hung Chu
    • Chengfei Zhang
  • View Affiliations

  • Published online on: July 27, 2015     https://doi.org/10.3892/br.2015.495
  • Pages: 697-702
Metrics: HTML 0 views | PDF 0 views     Cited By (CrossRef): 0 citations

Abstract

To study the immune-inflammatory response and signaling mechanism of macrophages to purified Enterococcus faecalis (E. faecalis) lipoteichoic acid (LTA), intact LTA was obtained from an E. faecalis clinical strain P25RC using the butanol method and hydrophobic interaction chromatography purification. The fractions containing LTA were determined using phosphate detection. Contaminations with lipopolysaccharide and proteins were excluded using the Limulus amoebocyte lysate assay and sodium dodecyl sulfate‑polyacrylamide gel electrophoresis, respectively. LTA was analyzed using nuclear magnetic resonance. Prior to LTA stimulation assays, THP‑1 monocytes were pretreated with phorbol 12‑myristate 13‑acetate to differentiate into macrophages. Macrophages were treated with LTA in concentration gradients and cells without LTA treatment as the control. Gene expression of TLR2, CD14 and MyD88 were evaluated by quantitative polymerase chain reaction. Tumor necrosis factor‑α (TNF‑α) and interleukin (IL)‑10 were quantified using ELISA. The activated and total nuclear factor‑κB (NF‑κB) p65 and three mitogen‑activated protein kinases (p38, ERK1/2 and JNK) were assessed using western blot analysis. E. faecalis LTA induced the gene expression of TLR2 and MyD88 whilst it downregulated CD14, suggesting a TLR2‑dependent and CD14‑independent immune‑inflammatory activity. LTA stimulated the expression of pro‑inflammatory cytokine TNF‑α (P<0.05), but not the anti‑inflammatory cytokine IL‑10. In conclusion, E. faecalis LTA stimulated the expression of TNF-α in macrophages possibly through the NF-κB and p38 pathways.

Introduction

Enterococcus faecalis (E. faecalis), a Gram-positive bacterium, is a predominant cause of hospital-associated infections (1). E. faecalis is also involved in root canal infections (2) and is considered a major pathogen associated with endodontic treatment failure (3). Several virulence attributes that promote bacterial colonization, the invasion of host tissues and the evasion of host defense mechanisms are indicated in persistent infections caused by E. faecalis (4). The virulence factors include lipoteichoic acid (LTA), peptidoglycan, aggregation substances, cytolysin and lytic enzymes. Among them, LTA is the key virulence factor due to its major role in pathogenicity. It has been demonstrated that E. faecalis LTA can stimulate leukocytes to release certain mediators associated with inflammatory response (5) and plays a critical role in biofilm formation (6). E. faecalis LTA is also known to inhibit the repair mechanism of periapical bone by decreasing the proliferation of human osteoblast-like cells and inducing apoptosis (7).

E. faecalis LTA belongs to classical (type 1) LTA. Its basic structure is comprised of a glycolipid moiety and 1,3-polyglycerolphosphate substituted at position C-2 with D-alanine and kojibiose residues (8). The alanyl esters with D-configured glycerophosphate influence the antibiotic action, pathogenesis, adhesion, biofilm formation and virulence of Gram-positive bacteria (6,9). The dlt operon consists of 4 genes: dltA, dltB, dltC and dltD, which are responsible for D-alanine synthesis (10). The inactivation of the dlt genes resulted in the absence of D-alanyl residues of LTA and further decreased the virulence features of E. faecalis (6).

Bacterial antigens are recognized by the pattern recognition receptors (PRRs) of immune cells and subsequently induce the production of pro-inflammatory cytokines. Toll-like receptors (TLR) are ubiquitous PRRs that play a pivotal role in the innate immune response (11). Of all the TLRs, TLR2 is known to specifically interact with bacterial LTA (12). Baik et al (5) found that E. faecalis LTA stimulates RAW 264.7 cells to express tumor necrosis factor-α (TNF-α). However, molecular mechanisms pertaining to the sensing and signaling of clinically derived E. faecalis LTA and human macrophages have not yet been studied. The cells of the THP-1 human monocytic cell line can differentiate into macrophages under stimulation by phorbol 12-myristate 13-acetate (PMA) (13). The differentiated THP-1 macrophages are widely used as human macrophages in vitro for studying inflammatory diseases (14,15). In order to address this research gap, highly purified, intact LTA was extracted from an E. faecalis clinical strain and its effect on TNF-α production was studied in differentiated THP-1 macrophages. Nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signaling pathways are activated by endodontic infectious contents (16). The present study also investigated the involvement of NF-κB and MAPK signaling pathways in the production of TNF-α.

Materials and methods

Bacteria and cell culture

E. faecalis P25RC was isolated from a patient's root canal at the Peking University Hospital of Stomatology (Beijing, China) by Zhu et al (17). E. faecalis P25RC was grown anaerobically (90% N2, 5% CO2 and 5% H2) overnight at 37°C without agitation in the brain-heart infusion broth (Oxoid Ltd., Basingstoke, Hampshire, UK). Once the bacterial growth reached the mid-exponential phase (A600=0.5), 10 liters of the bacteria were harvested by centrifugation and washed twice with phosphate-buffered saline (PBS; pH 7.4) (Sigma-Aldrich, St. Louis, MO, USA). The bacteria were examined by Gram staining and microscopy to rule out contaminations. The bacteria were stored at −70°C until LTA extraction.

THP-1 cells (ATCC® TIB-202™) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). THP-1 cells were maintained in RPMI-1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT, USA), 50 µM 2-mercaptoethanol (Sigma-Aldrich), 100 IU/ml penicillin and 100 µg/ml streptomycin (Invitrogen Life Technologies, Carlsbad, CA, USA). The cells were incubated at 37°C with 5% CO2. The experiments were performed in 6-well plates at a density of 8×105 cells/ml. Before the LTA stimulation assays, THP-1 monocytes were pretreated with 200 ng/ml PMA (Sigma-Aldrich) for 18 h to differentiate them into macrophages, which facilitates the adhesion of the cells (18).

Isolation and purification of LTA

The processes of isolation and purification of LTA were performed as described previously (19). The bacteria were defrosted and suspended in 100 ml of 0.1 M cold sodium citrate buffer (pH 4.7) (Sigma-Aldrich). Following disruption by Vibra-Cell ultrasonication (Sonic and Materials, Inc., Danbury, CT, USA) at 40% amplitude in pulse mode of 5-sec on and 5-sec off for 30 min on ice, the cell lysate was stirred with 100 ml n-butanol (Sigma-Aldrich) at 4°C for 30 min. Subsequently, centrifugation was carried out at 7,000 × g for 45 min. The lower aqueous phase was collected and 100 ml sodium citrate buffer was added again for re-extraction. The two aqueous phases were pooled and dialyzed overnight in deionized water using a dialysis membrane with MWCO 1000 (Spectrum Laboratories Inc., Rancho Dominguez, CA, USA). The dialyzed sample was treated with vacuum filtration (Millipore, Billerica, MA, USA) and prepared for purification. The dialyzed sample was subjected to hydrophobic interaction chromatography (HIC) on an octyl-sepharose column (2.5×10 cm) (Sigma-Aldrich). The dialyzed sample was prepared and transferred to HIC in 15% n-propanol in 0.1 M ammonium acetate. The bond fractions were eluted with a linear concentration gradient from 15 to 60% n-propanol in 0.1 M ammonium acetate (pH 4.7).

The structure of the LTA was assayed by 1H nuclear magnetic resonance (NMR) spectrometry as described previously (19). The purity of the LTA was determined by measuring the protein content through Coomassie Blue G-250 staining (Bio-Rad, Hercules, CA, USA) following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in gradient gels containing 5–12% wt/vol acrylamide (Bio-Rad). Precision Plus Protein Unstained standards (Bio-Rad) were used as reference markers. The molecular weight of LTA was shown in the SDS-PAGE gel stained with Coomassie Blue dye. Endotoxin contamination was excluded using a Limulus amoebocyte lysate (LAL) assay kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Thus, the endotoxin was quantified and the contamination was evaluated (19,20). DNA or RNA contamination was determined by measuring ultraviolet (UV) absorption at 260 and 280 nm (NanoDrop Spectrophotometer 2000; Thermo Fisher Scientific, Wilmington, DE, USA) (21).

Extraction of the E. faecalis genome and detection of dlt genes

The genome of E. faecalis P25RC was extracted using the Wizard Genome DNA Purification kit (Promega Corp., Madison, WI, USA). The fragments of genes dltA and dltB were amplified using polymerase chain reaction (PCR). The sequences of primers were: dltA forward, 5′-TTGATGAAT GGGCACGTAAA-3′ and reverse, 5′-GCCAATGCGGGA TAAATAGA-3′; and dltB forward, 5′-AGGTGAATGCCA CATGA-3′ and reverse, 5′-GACACCACCAAGATAATGACTGA-3′ (6). The PCR products were visualized on a 1.5% agarose gel stained with Redsafe Nucleic Acid Staining Solution (1:10,000) (iNtRON, Seoul, Korea).

LTA treatment

LTA-containing fractions were pooled and lyophilized. LTA was weighed using an analytical balance (Sartorious, Goettingen, Germany), reconstituted using PBS (pH 7.4) to a concentration of 1 mg/ml and stored at −70°C until use. E. faecalis LTA was added into RPMI-1640 culture medium and mixed thoroughly in plastic tubes. PMA pre-activated THP-1 cells were treated with E. faecalis LTA at a gradient concentration between 0.1 and 50 µg/ml at 37°C for 18 h. Cells without LTA treatment were used as controls. All the experiments were performed in triplicate.

ELISA assays

Following the stimulation of the cells with E. faecalis LTA, supernatants were collected and assayed for inflammatory and anti-inflammatory cytokines using sandwich ELISA with their well-matched antibodies. Cytokines, such as TNF-α and interleukin (IL)-10, were detected using ELISA kits (Invitrogen Life Technologies, Frederick, MD, USA) according to the manufacturer's instructions (22).

Quantitative PCR (qPCR)

The cells were washed twice with PBS and harvested by scraping using cell scrapers (Costar, Corning, NY, USA) following incubation with LTA. The mRNA of each sample was extracted and purified using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany) and quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). The cDNA was obtained through SuperScript® VILO™ MasterMix (Invitrogen Life Technologies) using 1.5 µg mRNA as a template for the final reaction volume of 20 µl. The qPCR was performed using a StepOne qPCR System (Applied Biosystems Inc., Carlsbad, CA, USA) in standard mode and the PCR products were detected with SYBR-Green (Life Technologies, Austin, TX, USA). The signaling pathway messengers, including TLR2, CD14 and MyD88, were amplified using qPCR. Primer sequences were as follows: TLR2 forward, 5′-TTATCCAGCACACGAATACACAG-3′ and reverse, 5′-AGGCATCTGGTAGAGTCATCAA-3′; CD14 forward, 5′-GACCTAAAGATAACCGGCACC-3′ and reverse, 5′-GCAATGCTCAGTACCTTGAGG-3′; MyD88 forward, 5′-GGCTGCTCTCAACATGCGA-3′ and reverse, 5′-CTG TGTCCGCACGTTCAAGA-3′; and GAPDH forward, 5′-ATGTGTCCGTCGTGGATCTGA-3′ and reverse, 5′-ATG CCTGCTTCACCACCTTCT-3′. The qPCR reaction conditions were performed at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The experiment was performed in triplicate. The relative expression of the mRNA was normalized with GAPDH using the 2−ΔΔCT method.

Western blot analysis

The phosphorylation of NF-κB and MAPKs, including ERK1/2, JNK1/2 and p38, was detected by western blot analysis, as previously described (21). The nuclear and cytoplasmic proteins were extracted using NE-PER™ Nuclear and Cytoplasmic Extraction reagents (Thermo Fisher Scientific, Rockford, IL, USA). The protein was quantified with a BCA™ protein assay kit (Thermo Fisher Scientific). Briefly, cell lysates containing 20 µg of protein were boiled for 5 min in Lane Marker Sample buffers (Thermo Fisher Scientific) and the volume ratio of sample buffer in the protein sample was 1:4. The protein was subsequently separated by 5–12% SDS-PAGE (Bio-Rad) and blotted onto polyvinylidene fluoride membranes (GE Healthcare, Uppsala, Sweden). After blocking for 1 h in 20 mmol/l Tris-HCl, 150 mmol/l NaCl and 0.05% Tween-20 (TBS-T) containing 5% skimmed milk, the membranes were washed three times in 20 ml TBS-T and probed overnight at 4°C with rabbit anti-human antibodies for phospho-NF-κB p65 (monoclonal; 3033), NF-κB p65 (monclonal; 4764), phospho-p38 (polyclonal; 9211), p38 (polyclonal; 9212), phospho ERK1/2 (monoclonal; 4377), ERK1/2 (monoclonal; 4695), phospho-SAPK/JNK (monoclonal; 4671), SAPK/JNK (polyclonal; 9252) (Cell Signaling, Beverly, MA, USA) in TBS-T with 0.5% skimmed milk. Following three washes in 20 ml TBS-T, the membranes were incubated with secondary horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (Cell Signaling Technology, Inc., Beverly, MA, USA) for 2 h and washed three times in 20 ml TBS-T. Bands were detected using enhanced chemiluminescence reagents (Thermo Fisher Scientific, Wilmington, DE, USA). The proteins were normalized with β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA). The band densities were measured using Quantity One 4.6.9 software (Bio-Rad).

Statistical analysis

The qPCR, ELISA and western blot analysis data were analyzed by analysis of variance using IBM SPSS software version 20 (IBM Corp., Armonk, NY, USA). The Bonferroni method was used to compare the difference between two groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Extraction, purification and identification of LTA

E. faecalis P25RC was grown and reached an optical density of 0.5 at 600 nm for LTA extraction (Fig. 1A). LTA was extracted using the n-butanol method and purified by HIC on octyl-sepharose. The bond fractions were assayed for total phosphorus (Fig. 1B). The fractions corresponding to the peak on the curve, which were considered the ones containing LTA, were pooled. The structure of LTA was detected using 1H NMR spectrometry (Fig. 1C). The purity of LTA was determined by measuring the protein contamination through Coomassie Blue staining following SDS-PAGE and the molecular weight of LTA showed ~20 kDa (Fig. 1D). Endotoxin contamination was excluded through an LAL assay and endotoxin content was <0.3 EU/mg in the lyophilized LTA. DNA and RNA contaminations were determined by measuring UV absorption at 260 and 280 nm. DNA and RNA accounted for 5.4 and 3.4% (wt/wt), respectively. Conclusively, purified LTA was prepared. The D-alanine residue was confirmed by amplifying the fragments of the dltA and dltB operon genes using PCR (Fig. 1E). The presence of PCR products on the agarose gel showed the dltA (598 bp) and dltB (649 bp) fragments. Preliminarily, LTA from E. faecalis P25RC could be confirmed as D-alanyl LTA.

LTA promotes the gene expression of TLR2 and MyD88

LTA from the E. faecalis P25RC clinical strain was used to treat macrophages that were differentiated from THP-1 cells. Cells without LTA treatment were used as controls. At the concentration of 50 µg/ml LTA, the expression of TLR2 was enhanced ≤1.4-fold compared to the control and additionally, the expression of MyD88 was enhanced ≤1.4-fold compared to 0.1 and 1 µg/ml LTA treatment. The expression of CD14 was decreased by 0.5-fold compared to the control. Based on the results of qPCR, D-alanyl LTA from E. faecalis P25RC promoted the gene expression of TLR2 (P<0.05, Fig. 2A) and MyD88 (P<0.05, Fig. 2B), but LTA downregulated CD14 (P<0.05, Fig. 2C). In particular, LTA from E. faecalis P25RC induced an inflammatory response through TLR2-dependent and CD14-independent signaling transduction.

LTA promotes the expression of TNF-α

The cytokines, such as TNF-α and IL-10, were detected using ELISA sandwich kits. LTA stimulated the expression of pro-inflammatory cytokine TNF-α (P<0.05, Fig. 3A), but not the anti-inflammatory cytokine IL-10 (P>0.05, Fig. 3B).

LTA promotes p38 and NF-κB signaling pathways

The three activated total MAPKs (p38, ERK1/2 and JNK) (Fig. 3C) and NF-κB p65 (Fig. 3D) were assayed using western blot analysis. In the present study, the results also suggest that MAPKs, particularly p38, may play an important role in signal transduction for TNF-α production. There was a dose-dependent increase in p-p38 (P<0.05, Fig. 3E) and p-NF-κB activity (P<0.05 compared to the control, Fig. 3I). By contrast, E. faecalis LTA did not induce ERK1/2 or JNK activity. The total ERK1/2 (Fig. 3G) and JNK (Fig. 3H) were upregulated, but the activation was not detected. Therefore, the p-p38 and p-NF-κB signaling transduction pathways likely play a major role in E. faecalis LTA-induced TNF-α expression in differentiated THP-1 macrophages.

Discussion

The present study examined the molecular mechanism behind the interaction of a highly purified LTA derived from the E. faecalis clinical strain with differentiated THP-1 macrophages. The n-butanol method and HIC on octyl-sepharose were used to extract and purify LTA. The butanol method can be used to extract structurally intact LTA and has been widely used to extract LTA from numerous types of Gram-positive bacteria, such as Staphylococcus aureus, Streptococcus pneumoniae, E. faecalis and Streptococcus gordonii (6,19,23,24). Furthermore, SDS-PAGE and Coomassie Blue G-250 staining were used to detect protein contamination. Coomassie Blue G-250 staining provides a detection limit of 1 ng protein and is sensitive enough to exclude protein contamination. Of note, LTA could be exhibited on the gel following staining by Coomassie Blue, while LTA was previously examined using silver stain (25,26). LTA showed a clear broad band of ~20 kDa. The LTA image was in agreement with a previous study regarding LTA derived from other Gram-positive bacteria (27). Therefore, SDS-PAGE analysis with Coomassie Blue staining could become a rapid and easy way to detect LTA. The NMR spectrum was highly analogous to those reported in a previous study regarding E. faecalis LTA (6). Taken together, highly purified LTA was successfully isolated from an E. faecalis clinical strain.

LTA is an amphiphile that is anchored to the cell membrane via its glycolipid and extends its long chains of glycerophosphate into the wall (20). The dlt genes are involved in the production of D-alanine residues and the inactivation of the dlt genes can lead to the absence of D-alanine linked on the LTA glycerolphosphate backbone. Furthermore, D-alanine is closely associated with the formation of biofilm (9). Fabretti et al (6) found that the dltA deletion mutant of a strong biofilm producer (E. faecalis 12030) was not as potent as the wild-type strain in the production of biofilm. In the present study, the dltA and dltB genes were detected in E. faecalis P25RC. They are responsible for the production of D-alanine, suggesting that E. faecalis P25RC LTA contains D-alanine. The E. faecalis P25RC strain came from an infected root canal of a patient suffering from persistent periapical periodontitis. Therefore, understanding the immune-inflammatory activity of E. faecalis P25RC may enhance our understanding of the apical resorption process observed in endodontic infections.

As a surface molecule of Gram-positive bacteria, LTA plays an important role in the stimulation of inflammatory responses, in vivo and in vitro (21,28). In the present study, gene expression of the signaling mediators was detected using qPCR. LTA from the clinical strain used in the present study induced an inflammatory response through TLR2-MyD88-dependent, but CD14-independent signaling transduction. There are 10 TLRs in human cells, each of which can recognize different pathogen-associated molecular patterns (26). LTA is a ligand against TLR2 and partially dependent on TLR1, TLR6 and CD14 (27). TLR2 may bind easier to E. faecalis LTA than other TLRs in infected dental pulps, as an overwhelming majority of TLR2-positive macrophages are found in pulp inflammation (29).

The interaction of LTA with TLR2-dependent pathways can activate intracellular messengers, such as MyD88 and MAPKs, and subsequently transcription factors, including NF-κB, to stimulate the expression of inflammatory cytokines (28,30). In the present study, the release of cytokines was quantified using ELISA and the protein expression of cells was assessed using western blot analysis. The dose-dependent increment of TNF-α corresponded to the activation of NF-κB at the LTA concentration of 10 µg/ml. NF-κB is an important transcription factor that promotes the rapid release of cytokines (31). The present results show that NF-κB is responsible for the production of TNF-α, in terms of the effect of the E. faecalis clinical strain LTA on macrophages. The p38 was also activated, albeit not simultaneously with NF-κB. Martinho et al (16) also reported that the phosphorylation of NF-κB and p38 did not reach their peaks at the same time in the study of signaling pathways associated with primary endodontic infectious contents. In the signaling pathways, the upstream adaptors could be responsible for this inconsistent phenomenon regarding the activation of NF-κB and p38 (32).

In the present study, highly purified LTA was successfully isolated from a clinical E. faecalis strain derived from the infected root canal of a patient with persistent apical periodontitis. E. faecalis LTA was also demonstrated to promote the expression of pro-inflammatory cytokine TNF-α through activating the p38 MAPK and NF-κB signaling pathways, but did not influence the expression of anti-inflammatory cytokine IL-10. Therefore, the present study provides a new insight into the pathogenic mechanisms of E. faecalis in endodontic infections and may possibly lead to the development of novel therapeutic options targeting signaling pathways.

Acknowledgements

The Research Fund for the Control of Infectious Diseases/Health and Health Services Research Fund (grant no. 12110772) supported the present study. The authors would like to thank Mr. Raymond Tong, Ms. Edith Tong and Ms. Wendy Yu for their technical assistance.

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September 2015
Volume 3 Issue 5

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Wang, S., Liu, K., Seneviratne, C.J., Li, X., Cheung, G.S., Jin, L. ... Zhang, C. (2015). Lipoteichoic acid from an Enterococcus faecalis clinical strain promotes TNF-α expression through the NF-κB and p38 MAPK signaling pathways in differentiated THP-1 macrophages. Biomedical Reports, 3, 697-702. https://doi.org/10.3892/br.2015.495
MLA
Wang, S., Liu, K., Seneviratne, C. J., Li, X., Cheung, G. S., Jin, L., Chu, C. H., Zhang, C."Lipoteichoic acid from an Enterococcus faecalis clinical strain promotes TNF-α expression through the NF-κB and p38 MAPK signaling pathways in differentiated THP-1 macrophages". Biomedical Reports 3.5 (2015): 697-702.
Chicago
Wang, S., Liu, K., Seneviratne, C. J., Li, X., Cheung, G. S., Jin, L., Chu, C. H., Zhang, C."Lipoteichoic acid from an Enterococcus faecalis clinical strain promotes TNF-α expression through the NF-κB and p38 MAPK signaling pathways in differentiated THP-1 macrophages". Biomedical Reports 3, no. 5 (2015): 697-702. https://doi.org/10.3892/br.2015.495