Involvement of RP105 and Toll-Like Receptors in the Activation of Mouse Peritoneal Macrophages by Staphylococcus aureus

Authors


Correspondence to: Z. Yang, PhD, College of Animal Science and Veterinary Medicine, Jilin University, Xi'an road 5333#, Changchun, Jilin Province 130062, China. E-mail: yzt@jlu.edu.cn

Abstract

Staphylococcus aureus is the aetiological agent of many hospital- and community-acquired infections. Toll-like receptor 2 (TLR2) has been shown to play a crucial role in the host defence against S. aureus infection. The aim of this study is to investigate the roles of the heterogeneous TLR family proteins TLR2, TLR4 and RP105 during S. aureus infection. Peritoneal macrophages from mice were exposed to S. aureus. Their production of inflammatory cytokines and chemokines, their expression of cell-surface markers and interactions between TLR2, TLR4 and RP105 were assessed in the presence or absence of inhibitory antibodies against TLR2, TLR4/MD-2 and RP105/MD-1 complexes. Our results demonstrate that not only TLR2 but also TLR4 and RP105 are involved in the response of macrophages to S. aureus, that TLR2, TLR4 and RP105 physically interact with each other during S. aureus infection, and that TLR2, TLR4 and RP105 both cooperate and play unique roles in the production of inflammatory cytokines (TNF-α, IL-12p40 and IL-10) and chemokine (RANTES) by macrophages after S. aureus infection. This study characterizes the important roles that TLR2, TLR4 and RP105 play in host resistance against S. aureus infection.

Introduction

Staphylococcus aureus is a common gram-positive pathogen that causes wound infections, bacteraemia and sepsis, and it is a major source of mortality in medical facilities [1]. During infection, S. aureus activates cells and induces significant inflammation in the host, which induces the secretion of inflammatory cytokines and chemokines [2-4].

The innate immune system plays essential roles in the host defence against bacterial infection. Toll-like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs), recognize specific pathogen-associated molecular patterns (PAMPs) and serve crucial role in early host responses against invading pathogens [5, 6]. In mammalian species, there are at least 10 TLRs with distinct functions in innate immune recognition, and many TLR ligands have been identified [7, 8]. In previous studies, TLR2 has been shown to play a crucial role in the host defence against S. aureus infection [9, 10]. The cooperation and cross-talk between heterogeneous TLR family proteins in innate immune system play essential roles in host defence against bacterial infection [11, 12]. It has been reported that TLR2 cooperates with TLR1, TLR6 and CD14 during the host response to S. aureus infection [9, 13].

There is a significant body of evidence that supports a broad role for TLR2 as a PRR for a variety of microbes and microbial structures. TLR2 has been reported recognize the S. aureus peptidoglycan (PGN) and lipoteichoic acid (LTA) [13, 14]. Further studies indicate that lipoproteins are the dominant immunobiologically active compounds expressed by S. aureus and that they activate cells via TLR2 [10, 15, 16]. Lipoproteins from pathogens such as mycobacteria, spirochaetes and mycoplasma, in addition to those from S. aureus, have been identified as TLR2 agonists. Blumenthal et al. [17] recently reported that the known TLR2 agonist Mycobacterium tuberculosis 19-kDa lipoprotein stimulated macrophages by both TLR2 and RP105. This result suggests that RP105 might be involved in the host response to S. aureus lipoproteins and might participate in the host defence against S. aureus infections.

RP105 is a member of the TLR family of proteins. Unlike other TLRs, RP105 lacks a Toll-IL-1 receptor domain, possesses only 6–11 intracytoplasmic amino acids and may associate with one or more unidentified signalling molecule(s) [18]. RP105 was originally thought to be expressed specifically in B cells and to regulate B cell proliferation and death [19-21]. B cells from RP105−/− mice exhibit less lipopolysaccharide (LPS)-induced proliferation and antibody production, demonstrating that RP105 enhances the signals downstream of LPS and TLR4 in B cells [22-24]. It has more recently been shown that RP105 expression is not restricted to B cells, but is also found in macrophages and dendritic cells (DCs). RP105 expression on antigen-presenting cells (APC) coincides with TLR4 expression, and RP105 has been identified as a physiological inhibitor of TLR4-mediated responses in macrophages and DCs [25-27]. Phylogenetic analyses placed RP105 and TLR4 into the same subfamily [26]. TLR4 associated with the secreted extracellular protein MD-2, while the surface expression of RP105 is dependent on the coexpression of the MD-2 homologue MD-1 [20, 28, 29].

RP105 enhances and optimizes IL-12p40 secretion and TNF-α production by Mtb-infected macrophages via a TLR2-dependent signalling pathway [17]. B cells from RP105−/− mice respond poorly to TLR4 agonist LPS and the TLR2 agonists lipoproteins from microbial membranes [30]. These studies indicate that the RP105/MD-1 complex is involved in host resistance to invading pathogens and influences inflammatory responses in cooperation with both the TLR4/MD-2 complex and TLR2. We address whether TLR2, TLR4 and RP105 cooperate with each other during S. aureus infection.

In the present study, we demonstrate the following: (1) the surface expression of TLR2, TLR4 and RP105 by macrophages is significantly enhanced by stimulation with S. aureus (2) TLR2, TLR4 and RP105 interact physically with each other during S. aureus infection and (3) TLR2, TLR4 and RP105 both cooperate with each other and play unique roles in the secretion of cytokines and chemokines by macrophages after S. aureus stimulation.

Materials and methods

Ethics statement

All animal studies were conducted according to the experimental practices and standards approved by the Animal Welfare and Research Ethics Committee at Jilin University (Approval ID:20111106-2), and all efforts were made to minimize suffering.

Bacterial strains

Staphylococcus aureus SA113 (ATCC 35556) were obtained from Mikrobielle Genetik, Universität Tübingen, Germany and used in our study. Bacterial strain were cultured in Mueller-Hinton II cation adjusted broth (BD Biosciences, Sparks, MD) at 37 °C for 18 h with constant shaking to an optical density at 600 nm of 2.0.

Culture of primary mouse macrophages and infection by S. aureus

Three to 5 days before peritoneal macrophages were extracted, 8-week-old male C57BL/6J mice that obtained from a licensed bioresource facility (Model Animal Research Center of Nanjing University, China) were injected with 2 ml 3% thioglycollate medium (TG, BD Biosciences, Sparks, MD, USA). The cells were cultured at 37 °C in 5% CO2 in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA). The peritoneal macrophages were identified by flow cytometry using a phycoerythrin (PE)-conjugated specific anti-CD11b antibody (M1/70, Biolegend, San Diego, CA, USA). The macrophages were infected in a total volume of 750 μl of culture medium with 7.5 × 106 or 2.5 × 107 staphylococci, resulting in a multiplicity of infection (MOI) 3:1 or 10:1. After 1 h of incubation at 37 °C, 250 μl of culture medium containing gentamicin (final concentration, 100 μg/ml) was added to kill extracellular bacteria.

Real-time PCR and surface markers expression

Total RNA was isolated at 4 h, 8 h and 12 h post stimulation by Trizol (Invitrogen, Carlsbad, CA, USA). The total RNA was treated with DNase I and reverse transcribed using oligo-dT primers. The total cDNA was used as starting material for real-time PCR with FastStart Universal SYBR Green Master (Roche Applied Science, Mannheim, Germany) on a 7500 real-time PCR System (Applied Biosystems, Foster City, CA, USA). The following primers were used to amplify specific genes: TLR2 (GeneBanK ID: NM_011905), forward, 5′-TTTGCTCCTGCGAACTCC-3′, and reverse, 5′-GCCA CGCCCACATCATTC-3′; TLR4 (GeneBanK ID: NM_021297), forward, 5′-TTCAGAGCCGTTGGTGT ATC-3′, and reverse, 5′- CTCCCATTCCAGGTAGG TGT-3′; RP105 (GeneBanK ID: NM_008533), forward, 5′-TACCCAGTCTCCTCCCCATCTTG-3′, and reverse, 5′-TGCCCACAGCTGCCATACTACA-3′; MD-1 (GeneBanK ID: NM_010745), forward, 5′-GCTGTTTCTGGACATAACTCTGA-3′, and reverse, 5′-TCCTCCTCACAAAGGGGATAG-3′. The gene expression was normalized to the expression of the housekeeping gene GAPDH, using the following primers: GAPDH forward, 5′-AGGTCGGTGTGAACGGATTTG-3′, and reverse, 5′-GGGGTCGTTGATGGCAACA-3′. Gene expression was normalized to expression of GAPDH [2(crossing point GAPDH-crossing point gene)] as described [31].

Macrophages were infected with S. aureus (MOI 3:1) for 6 h or left unstimulated. The cells were stained with PE-conjugated specific anti-RP105 (RP/14), anti-MD-1 (MD-113) antibodies and Alexa Fluor 488-coupled TLR2 (T2.5) or Alexa Fluor 488-coupled TLR4 (UT41) from eBioscience (San Diego, CA, USA). Matched isotype antibodies were used in tests as controls. Data were analysed using Flowjo 7.6 software (Treestar, Ashland, OR, USA).

Because the expression is not increased upon S. aureus infection, TLR3 was added as a control. The following primers were used to amplify TLR3 gene (GeneBanK ID: NM_126166): forward, 5′-GTGAGATACAACGTAGCTGACTG-3′, and reverse, 5′-TCCTGCATCCAAGATAGC AAGT-3′. Macrophages infected with S. aureus (MOI 3:1) for 6 h or left unstimulated were intracellular stained with PE-conjugated anti-TLR3 antibody (11F8, Biolegend) or matched isotype antibody for analysis of TLR3 intracellular expression.

Antibodies, ELISA kits and reagents for function studies

Antibodies that inhibit mouse TLR2 (T2.5), and TLR4/MD-2 complex (MTS510) and MD-1 (MD113), and the corresponding isotype control antibodies were purchased from eBioscience. There is no blocking antibody against RP105. Instead, an inhibitory antibody against MD-1 that had previously been reported to demonstrate a requirement for MD-1 in the RP105-mediated modulation of LPS-driven B cell activation was used to block the function of the RP105/MD-1 complex [17, 22, 32, 33]. All these antibodies contain low endotoxin and azide free. Cytokine or chemokine secretion was not induced in macrophages after antibody incubation. Macrophages were incubated for 1 h at 37 °C with antibodies at a concentration of 1.5 μg/ml before stimulation to block the functions of TLR2, TLR4/MD-2 and the RP105/MD-1 complex.

The concentrations of cytokines and chemokines in the supernatants were measured using ELISA kits for TNF-α, IL-12p40 and IL-10 (Biolegend) and murine RANTES (PeproTech Inc., Rocky Hill, NJ, USA) according to the manufacturer's instructions.

Macrophages were treated with mammalian protein extraction reagent (M-PER, Pierce, Rockford, IL, USA) for total cellular protein extraction. The concentrations of the protein samples were determined using BCA assay kits. For immunoblots (IB), 10 μg of total protein per lane was resolved by SDS-PAGE and blotted onto PVDF membrane. Rabbit anti-phospho-p38, anti-p38, anti-phospho-JNK, anti-JNK monoclonal antibodies, mouse anti-phospho-IκB-α and anti-IκB-α monoclonal antibody (1:1000, Cell Signalling Technology, Beverly, MA, USA) were used for protein detection. GAPDH was detected by a rabbit anti-GAPDH polyclonal antibody (1:1000, GeneTex, San Antonio, TX, USA). Proteins were visualized using secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse antibodies (1:20,000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and Pierce supersignal west pico chemiluminescent substrate. The images were captured using a MicroChemi 4.2 system (DNR Bio Imaging Systems, Jerusalem, Israel).

Microscopy

Macrophages were seeded into 35-mm glass bottom dishes and stimulated with viable S. aureus at 37 °C for 6 h or left unstimulated. The cells were fixed with 4% paraformaldehyde (PFA) and blocked with 5% BSA. The cells were incubated with either anti-mouse TLR2 (T2.5), TLR4/MD-2 (UT12) or the corresponding IgG1 and IgG3 isotype control antibodies (eBioscience), followed by an Alexa Fluor 488-conjugated anti-mouse IgG (H+L) secondary antibody (1:500, Molecular Probes, Inc., Eugene, OR, USA). The cells were cross-linked by incubation with a goat anti-mouse IgG F (ab')2 (Jackson ImmunoResearch Laboratories) at 4 °C for 3 h before being counterstained for RP105 or TLR4/MD-2. The cells were incubated with anti-mouse RP105 (RP/14), TLR4/MD-2 (MTS510) or the corresponding IgG2a isotype control antibody (eBioscience), followed by an Alexa Fluor 568-conjugated anti-rat IgG (H+L) secondary antibody (1:500, Molecular Probes, Inc., Eugene, OR, USA). All samples were counterstained with Hoechst 33258 (blue) to visualize the nuclei. An Olympus FV1000 laser confocal system (Tokyo, Japan) was used for image capture (×180). The fluorescence images from different samples were all captured under identical conditions.

Immunoprecipitation and immunoblot

The RAW264.7 mouse macrophage cell line (ATCC TIB-71) was established from a tumour induced by Abelson murine leukaemia virus and used in our studies. Anti-mouse RP105 (RP/14), anti-mouse TLR2 (T2.5) and anti-mouse TLR4/MD-2 (MTS510) antibodies and the corresponding isotype antibodies were used to immunoprecipitation (IP) lysates from RAW264.7 cells infected with S. aureus. All the antibodies were conjugated to NHS-activated Sepharose beads (GE Healthcare Life Sciences, Little Chalfont, UK). IP precipitates were separated by SDS-PAGE and blotted onto PVDF membrane. For immunoblots, a rabbit anti-mouse RP105 polyclonal antibody (1:1000, raised against a 14 amino acid peptide from near the carboxy terminus of RP105, ProSci Incorporated, Poway, CA, USA) and an anti-TLR2 rabbit polyclonal antibody (1:1000, corresponding to amino acids 375–390 of mouse TLR2, GeneTex) were used for detection; a rabbit anti-mouse TLR4 polyclonal antibody (1:1000, a synthetic peptide made against the C-terminal portion of the TLR4 protein, between residues 650–710, Novus Biologicals, Littleton, CO, USA) was used for TLR4 detection; control experiments were carried out using normal rabbit IgG; followed by a HRP-conjugated goat anti-rabbit secondary antibody (1:20,000); supersignal west femto chemiluminescent substrate (Pierce, Rockford, IL, USA) was used for signal detection.

Data analyses

The ELISA and real-time PCR data were analysed using GraphPad Prism 5 (GraphPad InStat Software, San Diego, CA, USA). Comparisons among the groups were performed with two-way anova. Data were expressed as the mean ± standard deviation (SD). P values ≤ 0.05 are considered to indicate a statistically significant difference (*< 0.05; **< 0.01; *** < 0.001).

Results

S. aureus stimulation increases TLR2, TLR4, RP105 and MD-1 surface expression by macrophages

The expression of mRNA was detected by real-time PCR. Because the mRNA expression by unstimulated cells was changed with time as well, for example, both TLR2 and MD-1 have lower mRNA expression after 12 h than at the beginning of the incubation, mRNA expression of TLR2, TLR4, RP105 and MD-1 was described as stimulus-induced changes as percentage from the untreated cells at the indicated timepoints and labelled in Fig. 1A–D. The expression of TLR2 increased following stimulation with S. aureus both at MOI 3:1 and 10:1 (Fig. 1A). But the expression of TLR4, RP105 and MD-1 decreased significantly in comparison with unstimulated cells (Fig. 1B–D). The surface expression of TLR2, TLR4, RP105 and MD-1 on S. aureus-infected and unstimulated macrophages was examined by immunostaining and flow cytometry. Macrophages expressed higher levels of TLR2 and TLR4 on their surfaces after S. aureus stimulation (Fig. 1E,F). As the change of surface expression was small (Fig. 1G,H), the surface expression of RP105 and MD-1 by macrophages was quantified as median fluorescence intensity. The surface expression of RP105 on macrophages was also enhanced after stimulation, similar to the surface expression of MD-1 (Fig. 1I,J). As mRNA expression for TLR4 and RP105 was discrepant with surface expression, we found that total cellular TLR4 and RP105 expression by macrophages were decreased after S. aureus infection (data not shown). These results are well supported by expression of mRNA for TLR4 and RP105. An explanation was raised that the transfer efficiency of TLR4 and RP105 protein onto cell membrane could be changed after S. aureus infection.

Figure 1.

Toll-like receptor 2 (TLR2), TLR4, RP105 and MD-1 gene and surface expression by macrophages after Staphylococcus aureus infection. (A–D) TLR2, TLR4, RP105 and MD-1 mRNA levels standardized to GAPDH from unstimulated and S. aureus-infected multiplicity of infection (MOI 3:1 and 10:1) macrophages at the indicated timepoints. Data are represented as the mean of triplicates ± SD and compared with unstimulated macrophages. Data from one of three representative experiments are shown. (E–H) TLR2, TLR4, RP105 and MD-1 surface expression by macrophages infected with S. aureus (MOI 3:1) or unstimulated (unst.) for 6 h. Data present one experiment representative of three experiments. Matched isotype control stainings were carried out in each experimental group. (I, J) RP105 and MD-1 surface expression was determined by flow cytometry and expressed as median fluorescence intensity after subtracting the background of the isotype control. Data exhibit the mean of triplicates ± SD from three independent experiments. (K, L) TLR3 mRNA levels and intracellular expression in unstimulated and S. aureus-infected macrophages. Data from one of three representative experiments are shown.

Toll-like receptor 3 mRNA levels and intracellular expression by macrophages after S. aureus stimulation were similar with unstimulated cells (Fig. 1K,L).

TLR2, TLR4 and RP105 interact with each other in S. aureus infection

Macrophages stimulated with S. aureus (MOI 3:1) for 6 h (Fig. 2D–H) or left unstimulated (Fig. 2A–C) were fixed to examine the plasma membrane colocalization of TLR2, TLR4 and RP105 by confocal microscopy. TLR2, TLR4 and RP105 were mainly colocalized in the membrane. These data also indicated that the fluorescence intensities of TLR2, TLR4 and RP105 were increased in stimulated cells in comparison with cells left unstimulated.

Figure 2.

Toll-like receptor 2 (TLR2), TLR4 and RP105 interaction in the macrophages cell membrane after Staphylococcus aureus stimulation. (A–H) Macrophages were stimulated with S. aureus at 37 °C for 6 h or left unstimulated. Then, the cells were fixed and stained with anti-TLR2 (T2.5) or anti-TLR4/MD-2 (UT12) or matched isotype control antibodies, followed by an Alexa Fluor 488-conjugated anti-mouse IgG (H+L) secondary antibody (green). After incubated with a goat anti-mouse IgG F (ab')2 antibody, cells were stained with anti-RP105 (RP/14) or anti-TLR4/MD-2 (MTS510) or matched isotype control antibodies, followed by an Alexa Fluor 568-conjugated anti-rat IgG (H+L) secondary antibody (red). The cells were counterstained with Hoechst 33258 (blue) to visualize the nuclei. (×180, DIC = differential interference contrast in transmitted light images; merge = overlay of images acquired in different fluorescence channels). (I) Immunoprecipitation (IP) and immunoblot (IB) of lysates of RAW264.7 macrophages after stimulation with S. aureus. The interactions between TLR2, TLR4 and RP105 were detected by IB with anti-RP105, anti-TLR2 and anti-TLR4 antibodies, and normal rabbit IgG was used as control. Data show one of four independent representative experiments.

We next analysed whether TLR2, TLR4 and RP105 physically interacted with each other after S. aureus stimulation. In our experiments, RAW264.7 macrophages were infected with S. aureus for 6 h. Then, TLR2, TLR4 and RP105 were bidirectionally coimmunoprecipitated for experimental condition (Fig. 2I). Isotype control antibodies that did not precipitate TLR2, TLR4 and RP105 were used as controls. Our data demonstrate that RP105 physically interacts with both TLR4 and TLR2 during S. aureus infection. Our data also show that TLR2 interacts with TLR4. These results indicated that RP105-TLR2, RP105-TLR4 and TLR2-TLR4 can form heterodimeric complexes in macrophages, which induces their function in host resistance against S. aureus infection.

Involvement of TLR2, TLR4 and RP105 in the phosphorylation of p38, JNK and IκB-α after S. aureus stimulation

Inhibitory TLR2, TLR4/MD-2 and MD-1 antibodies (1.5 μg/ml) were used to determine whether blocking the functions of TLR2, TLR4/MD-2 and RP105/MD-1 alone or in combination affected the phosphorylation of p38, JNK and IκB-α that drive TNF-α mRNA and protein expression. The phosphorylation of p38 was reduced by anti-TLR2 antibody alone. The combined anti-TLR2 and TLR4 antibodies blocked phosphorylation of JNK, but not as significantly as inhibition on p38 and IκB-α phosphorylation. For anti-TLR4 and anti-MD-1, there was no evidence of a reduction of the phosphorylation of p38, JNK and IκB-α (Fig. 3).

Figure 3.

Toll-like receptor 2 (TLR2), TLR4 and RP105 involved in macrophages phosphorylation of p38, JNK and IκB-α after Staphylococcus aureus stimulation. Phosphorylation (P) of p38, JNK and IκB-α in the untreated macrophages lysates, and in the lysates of anti-TLR2, anti-TLR4/MD-2, anti-MD-1 antibodies preincubated (1.5 μg/ml, 1 h) macrophages were analysed by immunoblot after stimulation with S. aureus at multiplicity of infection (MOI) 3:1, or treatment with DMSO (D) for the indicated times. Not phosphorylated p38, JNK and IκB-α were also evaluated. GAPDH was used as a loading control for each condition. Data represent one of three independent experiments.

The roles of TLR2, TLR4 and RP105 in macrophages activation by S. aureus

Because we had confirmed that TLR2, TLR4 and RP105 interacted with each other in macrophages activated by S. aureus, we used inhibitory antibodies to analyse the roles of heterogeneous TLR family proteins TLR2, TLR4 and RP105 in S. aureus-induced macrophage activation (Fig. 4A–D). S. aureus-induced IL-12p40 secretion in macrophages was enhanced by anti-TLR2 antibody preincubation, but TNF-α, IL-10 and RANTES secretion were significantly decreased. We concluded that TLR2 was required for TNF-α, IL-10 and RANTES production by macrophages, and IL-12p40 production was inhibited by TLR2 activation. Macrophages preincubated with anti-TLR4 antibody were decreased in IL-12p40, TNF-α and RANTES secretion. It suggested that the production of these cytokines was TLR4 dependent. The data of IL-10 production by macrophages demonstrated that IL-10 blocking both by anti-TLR4 and by anti-MD-1 was evident at a higher bacterial load, while at a low MOI, these antibodies facilitated IL-10 secretion. The data also exhibited that IL-12p40 secretion was RP105 dependent, but TNF-α and RANTES secretion was inhibited by RP105. To further evaluate the roles of TLR2, TLR4 and RP105 in S. aureus-induced activation, macrophages were preincubated with combined antibodies. Combined antibodies anti-TLR2/TLR4, anti-TLR2/MD-1 and anti-TLR4/MD-1 blocked all investigated cytokines and RANTES secretion. This effect was only evident at a high MOI for IL-12p40 after TLR2/TLR4 blockage and TNF-α after TLR4/MD-1 blockage. At a low MOI, combined anti-TLR4/MD-1 facilitated IL-10 secretion. Altogether, these data suggest that TLR2, TLR4 and RP105 play their unique roles in the secretion of a variety of inflammatory cytokines and chemokines by macrophages after S. aureus infection; they also suggest that TLR2, TLR4 and RP105 are required to shape the inflammatory response against S. aureus infection. In summary, these findings illustrate the functional interaction of these PRRs after S. aureus infection in macrophages.

Figure 4.

Toll-like receptor 2 (TLR2), TLR4 and RP105 cooperate with each other in macrophages inflammatory response to resist against Staphylococcus aureus infection. (A–D) Cytokines (TNF-α, IL-12p40 and IL-10) and chemokine (RANTES) concentrations after S. aureus stimulation for 9 h in supernatants from WT macrophages and macrophages preincubated for 1 h with anti-TLR2, anti-TLR4/MD-2, anti-MD-1 and isotype-matched control antibodies (1.5 μg/ml) alone or in combination. The raw data from three independent wells were compared with control conditions (medium) by two-way anova. Data are the mean ± SD of triplicate wells of one of three representative experiments. The levels of cytokine- and chemokine-secreted macrophages treated with isotype-matched control antibodies did not differ significantly from that of macrophage cultured under the control conditions after stimulation with S. aureus.

Discussion

It was previously demonstrated that S. aureus and the S. aureus cell-surface components LTA and PGN are TLR2 agonists and activate cells through TLR2 signalling [9, 13, 14]. Further studies indicated that one of the predominant lipoproteins in S. aureus, SitC, colocalizes with TLR2, gets internalized by host cells, induces proinflammatory cytokines, and triggers the intracellular accumulation of TLR2 [10]. Lipoproteins in S. aureus can mediate inflammation through TLR2 for better survival of S. aureus in the host [34, 35]. Several earlier studies demonstrate that TLR2 and TLR4 contribute in host MyD88-regulated immune response to S. aureus, which improves bacterial clearing and disease outcome [36, 37]. Although TLR2 has been shown to play a crucial role in the host defence against S. aureus infection, it is not clear whether other TLRs cooperate with TLR2 in this process.

It has been reported that RP105 positively regulates a TLR2-dependent response to Mtb lipoproteins in macrophage [17]. The role of RP105/MD-1 in cellular activation has been studied with focus on the TLR4 agonist LPS. The role of RP105 in TLR4-mediated responses to LPS seems to vary with the cell type. While RP105 is required for B cells to mount a full responsive against LPS, the expression of RP105 by dendritic cells and macrophages negatively regulates TLR4 responses to LPS [26]. Thus, RP105 cooperates directly with both TLR2 and TLR4 in host resistance against microbial pathogens. Because the lipoproteins in S. aureus primarily activate macrophages via TLR2-dependent mechanisms, whether TLR4 and RP105 cooperate with TLR2 in host response to S. aureus infection has not yet been analysed in detail. In addition to TLR2, the surface expression of TLR4, RP105 and MD-1 is also enhanced by S. aureus stimulation of macrophages. The mRNA levels display a very different trend from that of the surface expression. When we measured TLR2, TLR4, RP105 and MD-1 mRNA expression at the indicated times (4 h, 8 h and 12 h post stimulation), we found that TLR2 mRNA expression increased significantly in comparison with unstimulated cells. In contrast, the mRNA expression levels of TLR4, RP105 and MD-1 decreased, especially at 8 h and 12 h after stimulation. The mRNA expression of TLR4, RP105 and MD-1 indicates a tight regulatory interaction between these factors.

Toll-like receptor signalling is initiated following the dimerization of TLRs [8, 12]. TLR4 can form homodimers in response to LPS [38, 39]; TLR2 can form heterodimers with TLR1 or TLR6 and can recognize lipoproteins [5, 40, 41]. A recent study also indicated that TLRs can cooperate with each other in their response to ligands. Good et al. [42] demonstrated that TLR4 and TLR2 interact functionally in the basolateral membrane of medullary thick ascending limb cells to mediate LPS-induced ERK activation. Their findings provide evidence of a novel requirement for TLR2 in LPS-induced TLR4 signalling. In our studies, we showed that TLR2, TLR4 and RP105 physically interact with each other in RAW264.7 macrophages after S. aureus stimulation using bidirectional coimmunoprecipitation. According to our data, TLR4 and RP105 can heterodimerize with TLR2 in recognizing S. aureus infection. The macrophages' TLR2-mediated response to S. aureus infection is optimized and modulated by TLR4 and RP105. As RP105 is a member of the TLR family of proteins and possesses a similar structure to the other TLRs, RP105 could also physically interact with other TLRs and be involved in host resistance against infection with microbial pathogens.

The anti-inflammatory cytokine IL-10, which is induced during infection with bacteria or protozoan parasites, is well known for its ability to downregulate proinflammatory mediators such as IL-12, TNF-α and the RNIs that are triggered through the TLR signalling pathway [43, 44]. IL-10 plays a crucial role in regulating the secretion of proinflammatory cytokines by immune cells. IL-10 production by macrophages after S. aureus infection at either MOI 3:1 or 10:1 was TLR2 dependent. At MOI 3:1, both TLR4 and RP105 inhibited IL-10 production. In contrast, at MOI 10:1, the secretion of IL-10 by macrophages was dependent on both TLR4 and RP105. It is possible that at a lower MOI, the inhibitory action of TLR4/RP105 is to promote proinflammatory cytokines; however, at higher concentrations, an immunosuppressive action is required to down regulate high levels of inflammation. These data indicate that TLR2, TLR4 and RP105 interact with each other differentially at different levels of infectious burden to mediate the secretion of IL-10 and regulate proinflammatory cytokine production to strike a balance between activation and inhibition and avoid detrimental and inappropriate inflammatory responses. This mechanism has physiological relevance for host resistance against pathogens. It has been reported that the IL-10 secretion induced by TLR2 agonists could also suppress the IL-12 production induced by TLR3 and TLR4 signals in human dendritic cells, indicating that cross-talk among TLRs might affect the outcome of the immune response [45]. Our data indicate that macrophages produce IL-12p40 after S. aureus infection in a TLR4-dependent manner and that the activation of TLR2 signalling blocks IL-12p40 production by macrophages. We also found that RP105 is required for macrophages to produce IL-12p40. All these data further support the hypothesis that TLR2, TLR4 and RP105 play crucial roles in regulating the secretion of cytokines by macrophages after infection.

In summary, our results demonstrate that not only TLR2 but also TLR4 and RP105 are involved in response of macrophages to S. aureus infection and affect the character of the immune response. These results extend our understanding of how the innate immune system senses and responds to microbial pathogens and the roles of different TLRs in the innate immune recognition of microbial pathogens, such as S. aureus.

Acknowledgment

This work was supported by a grant from the National Natural Science Foundation of China (No. 30972225, 30771596) and the Research Fund for the Doctoral Program of Higher Education of China (No. 20110061130010). We thank Prof. Friedrich Götz of Mikrobielle Genetik, Universität Tübingen, Germany for providing the bacterial strains. We are grateful to Prof. Yongjun Yang and Qisheng Peng for technical assistance.