1. Introduction
Toll-like receptor (TLR) is a pattern recognition receptor for various ligands, and it is widely expressed in immune cells [
1]. The ligand stimulation for each TLR induces activation in the immune cells which is characterized by upregulation of cytokine production, surface marker expression and cell proliferation [
2,
3]. TLR signaling is an indispensable factor to induce innate immune response against pathogen to prevent serious infectious disease [
4]. Dendritic cell (DC), one of the myeloid origin immune cells, expresses several types of TLRs and these receptors are working for the regulation of DCs activity [
5]. As a professional antigen presenting cells (APCs), DCs are activated by antigenic substances which captured by both phagocytotic and pinocytotic manner, then the cells present the processed antigen into T cells to activate adaptive immune response against the target with high specificity [
6]. DCs activation status can be augmented by TLR stimulation so that the expression of the molecules directory related with antigen presentation, such as major histocompatibility complex class II (MHCII), CD80 and CD86, are upregulated to promote antigen presentation in activation of T cells [
7,
8]. The TLR signaling is generally thought as an unidirectional event which means the signal works only for activation in the cells. However, other suppressive mechanism via TLR with specific ligand has gradually been revealed recently. Lipoteichoic acid (LTA) from gram-positive bacteria recognized by TLR2, however, the recognition itself didn’t induce inflammatory response in the keratinocytes [
9]. On the contrary, the LTA signal suppressed another TLR signaling, such as TLR3 by poly (I:C) stimulation which characterized by suppression of inflammatory cytokine production in the cells [
9]. It was an interesting finding of LTA’s anti-inflammatory function in TLR-mediated manner, however, no other clear evidence hasn’t been reported in the immune cells yet. In addition, it has been expected to confirm that the efficiency of LTA’s immunosuppressive effect in other TLR ligands mediated inflammation.
In this report, we show that LTA, originated from Staphylococcus aureus (S. aureus), suppressed TLR signal dependent inflammatory response in the DCs. The inflammatory cytokine production was dramatically suppressed by LTA treatment in TLR ligand stimulated murine bone marrow derived dendritic cells (BMDCs). In this response, CD80, CD86 and MHC class II expressions were all downregulated by LTA treatment in the cells. LTA treatment itself was no-inflammatory event confirmed as fewer cytokine production, which was same level as vehicle control, than other ligands stimulation in the BMDCs. The LTA based suppressive effect against lipopolysaccharide (LPS)-induced cytokine production and surface molecule upregulation were TLR2 dependent manner which confirmed by in vitro TLR2 blocking assay. The antigen specific IFN-γ+CD4+ T (Th1) cells generation was suppressed by LTA treatment in BMDCs. Imiquimod (IMQ)-induced acute skin inflammation was suppressed by co-treatment of LTA on the mice ears. In this suppressive effect, TNF-α production was decrease in the skin accumulated DCs.
These findings provide a novel understanding of immunosuppressive effect by LTA against other TLRs-induced inflammation in the immune cells such as DCs.
3. Discussion
TLR signaling has an important role in immune cells to control the inflammatory response [
14]. The response is not only for exogenous ligands, such as those produced from bacteria and fungus, but also for auto-antigens derived from their own tissue [
15,
16]. TLR signaling triggered in the recognition of a ligand leads to the activation of downstream factors, such as MyD88, TNF receptor associated factor (TRAF) and protein kinase B (PKB as well as Akt), and eventually, several transcription factors are driven to induce cytokine production and other inflammatory responses [
17]. The signaling pathway has been well characterized after the identification of TLRs, and the causes of some of inflammatory diseases were revealed as TLR-dependent inflammation [
18]. In DCs, TLR signaling is also an important factor to induce various immunological responses [
19]. As one of the gatekeepers in the immune system, DCs frequently come into contact with many endogenous and exogenous simulators which can be antigens to activate T cells [
20]. In this process, the TLRs are activated by several ligands so that the function is enhanced as a similar mechanism to immunization using an adjuvant in the DCs [
21].
However, our immune system requires a suppressive function to prevent overactivation and autoreactivity. We hypothesized that an exogenous substance may be able to attenuate or regulate DC-based immune responses especially in a TLR-dependent manner. Therefore, we performed a BMDCs stimulation assay with various combinations of ligands, and eventually found that LTA suppressed the TLR-mediated cytokine production and cellular activation in the DCs (
Figure 1,
Figure 2 and
Figure 3). LTA dramatically suppressed TNF-α production in the TLR ligand-stimulated BMDCs (
Figure 1). The suppressive effect was obvious in LPS, PGN and IMQ stimulation because TNF-α production was dramatically suppressed by co-treatment of LTA, even though the dose was relatively low in the culture (
Figure 1A,B,E). The suppressive signal of LTA was TLR2-dependent in the BMDCs, because the TLR2 blocking suppressed TNF-α production which was induced by LPS or IMQ stimulation (
Figure 2A and
Supplementary Figures S1 and S2). Our data on the determination of the responsive receptor for LTA are strongly supported by the evidence that LTA is dominantly recognized by TLR2 [
22]. However, the possibility that LTA works through other receptors except TLRs must be considered. In fact, we previously reported that C-type lectin receptors (CLRs) may be bound and activated by LTA in the high dose treatment [
23]. LTA treatment suppressed the downstream signaling pathway of TLR 4 in LPS-stimulated BMDCs (
Figure 2B,C). We analyzed MyD88, pErk1/2, pp38 and pp65 as indispensable elements in the pathway of TLR4-related cytokine production, however, other factors, such as mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), also must be analyzed to complete the characterization of the LTA-based suppressive effect in DCs.
The suppressive effect of LTA in the immune response was also confirmed in antigen presentation to CD4+ T cells activation in BMDCs (
Figure 3 and
Figure 4). The BMDCs treated with LPS or IMQ upregulated the generation of Th1 cells in an antigen-specific manner. The responses were both strongly suppressed by co-treatment of LTA in the TLR ligand stimulation, so that the percentage of Th1 cells was significantly decreased in the culture (
Figure 4,
Supplementary Figure S4). The underlying mechanism might be based on the downregulation of MHC class II, CD80 and CD86 in the LTA-treated BMDCs (
Figure 3,
Supplementary Figure S4). This phenomenon was remarkably interesting and may be understood as a substantial mechanism in the regulation of adaptive immune response by LTA treatment.
To confirm the immunosuppressive effect of LTA in a practical inflammation model, we generated acute skin inflammation in mice eras by using IMQ as well as other TLR ligands (
Figure 5,
Supplementary Figure S7). The IMQ-induced skin inflammation was dramatically suppressed in the ears which received co-treatment of LTA. The ear thickness was consequently decreased in the mice (
Figure 5A,B). We obtained the same results in other TLR ligand-induced skin inflammation models (
Supplementary Figure S7). As a consistent phenomenon with an in vitro study, the inflammatory response, such as TNF-α production, in the skin-accumulated DCs was significantly suppressed by LTA treatment (
Figure 5D). This is strong evidence that our initial finding of a LTA-originated suppressive effect in BMDCs can be adapted into an in vivo environment. While we must consider the possibility of a comprehensive immunological event in the LTA-mediated suppressive effect in the inflamed lesion, because Th1 and Th17 cells as well as neutrophils accumulation were completely parallel to the accumulation of DCs and inflammatory grade in the ears (
Figure 5E–G). These data suggest that both adaptive and innate immune responses were regulated by LTA treatment in the TLR ligand-induced skin inflammation with DC-based manner. However, the suppression of DCs activity by LTA treatment should be an initial step in the immunosuppressive event in our hypothesis, because neither mice CD4+ T cells nor neutrophils were influenced directly in the LTA treatment (unpublished data).
These findings can be also adapted to the concept of symbiosis of commensal bacteria.
Staphylococcus aureus (
S. aureus) is one of the commensal bacteria and LTA is the major component in the bacterial cell wall [
23]. LTA may suppress or regulate the host immune response, which is activated by several substances produced from the bacteria itself. As a consequence, the immunosuppressive effect of LTA may let the bacteria survive in the host without critical inflammation. A previous study showed that LTA has immunogenicity and induces an inflammatory response by the activation of immune cells [
24], however, this concept is recently denied. The inflammatory responses observed in the study was originated from the contamination of lipopeptide in the LTA, therefore, a purified LTA did not show any inflammatory response in immune cells [
25]. Our study used a pre-treated LTA with lipoprotein lipase, and the result also showed that LTA treatment did not induce cytokine production and cellular activation in the BMDCs (
Figure 1F and
Figure 3E and
Supplementary Figures S1, S3 and S4).
To confirm the interesting immunosuppressive effect of LTA, further experiments must be performed using another DC model, such as primary DC. In vivo studies of other inflammatory diseases are necessary to reveal the effectiveness of LTA’s anti-inflammatory effect not only for TLR ligand-induced inflammation but also for that derived from other factors. In addition, we are interested in whether this anti-inflammatory response of LTA is conserved in other immune cells, especially myeloid lineages such as macrophages. Once all the mechanisms in the LTA-based anti-inflammatory response were revealed in immune cells, LTA may be used as a natural anti-inflammatory agent not only for daily care but also for clinical treatment. The bacterial ligand has potential in immune regulation. The study to identify an effective substance in bacterial components must be continued in both basic and clinical immunology.
4. Materials and Methods
4.1. Reagents and Antibodies
Lipopolysaccharide (LPS; E. coli origin), Pam3CSK4, peptidoglycan (PGN; S. aureus origin), Poly (I:C), imiquimod (IMQ) and lipoteichoic acid (LTA; S. aureus origin) were all purchased from Invivogen (San Diego, CA, USA). LTA was treated with lipoprotein lipase to inactivate contaminated lipoprotein before use in each experiment. Phorbol 12-myristate 13-acetate (PMA), ionomycin and ovalbumin (OVA) peptide (OVA323–339) were all purchased from Sigma Aldrich (St. Louis, MO, USA). In addition, 5% IMQ cram (Aldara) was purchased from 3M Health Care Limited (Loughborough, UK). Recombinant murine granulomacropahge colony stimulating factor (rmGM-CSF) was purchased from Peprotech (Rocky Hill, NJ, USA). Cytofix/Cytoperm kit with GoldiStopTM was purchased from BD Bioscience (Franklin Lakes, NJ, USA). PerFix-p Kit was purchased from Beckman Coulter (Indianapolis, IN, USA); anti-TLR2 (QA16A01), anti-TLR3 (11F8), anti-TLR4 (MTS510), anti-CD80 (16-10A1), anti-CD86 (GL-1), anti-CD3 (17A2), anti-CD4 (GK1.5), anti-CD8a (53-6.7), CD69 (H1.2F3) and 7-amino-actinomycin D (7-AAD) were all purchased from BioLegend (San Diego, CA, USA). Anti-TLR1 (eBioTR23 (TR23)), Anti-TLR7 (4G6), anti-I-A/I-E (M5/114.15.2), anti-interferon gamma (IFN-γ) (XMG1.2), anti-CD16/CD32 (2.4G2) (93) were all purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-MyD88 (4D6) was purchased from Novus Biologicals (Centennial, CO, USA). Anti-pErk (pT202/pY204) (20A), anti-p38 (pT180/pY182) (36/p38) and anti-NF-κB p65 (pS529) (K10-895.12.50) were purchased from BD Bioscience (Franklin Lakes, NJ, USA). The isotype-matched control for each antibody was purchased from the same company.
4.2. Mice
C57BL/6J and OT-II mice (B6.Cg-Tg (TcraTcrb) 425Cbn/J) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All mice were maintained under specific pathogen-free (SPF) conditions with free access to water and food in 12 h day/night cycle. Gender-matched mice between 8 and 12 weeks of age were used for each experiment. All the animal experiments were carried out in accordance with the guidelines of the animal welfare committee and the ethics committee of the institutes (Northwest A&F University, Protocol No. 15-10-874 and Central South University, Protocol No. 201503302).
4.3. Acute Skin Inflammation Model
To establish IMQ-induced acute skin inflammation model, the mice ears were treated with 5% IMQ cream or control cream for 5 days. The mice received intradermal (ID) injection of LTA (100 ug) or saline daily during the experimental period. At day 5, the mice were sacrificed, and the ears were used for analysis. To establish acute skin inflammation model using other TLR ligands, the mice ears were treated with TLR ligand (LPS: 1 μg, PGN: 100 μg, Pam3CSK4: 1 μg or Poly (I:C): 100 μg) by intradermal (ID) injection. The mice ears received ID injection of LTA (100 μg) or vehicle control twice (in the meantime and post-24 h of TLR ligand treatment) during the experimental period. After 48 h, the mice were sacrificed, and the ears were used for analysis.
4.4. Mouse Primary Cell Isolation
Splenocytes were prepared from spleen by following a method described in a previous report [
26]. Briefly, the extracted spleen was crushed on a 70 μm cell strainer with cell culture medium (RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin). The cell suspension was filtered through a 70 μm cell strainer again and then washed with cell culture medium. After centrifugation at 300 g for 5 min, the cell pellet was resuspended in 1 × RBC lysis buffer at RT for 10 min. After being washed twice with the cell culture medium, the cells were precipitated by centrifugation at 300 g for 5 min. The cells were resuspended in cell culture medium and used as splenocytes. Skin leukocytes were isolated by following a method described in a previous report, with modifications [
27]. Briefly, the extracted ear piece was washed with tissue washing buffer (RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 100 U/mL penicillin, 100 mg/mL streptomycin) at 37 °C for 30 min with gentle shaking. The ear piece was incubated at 37 °C for 30 min with dispase working solution (tissue washing buffer containing 0.25 mg/mL of dispase) to separate the epidermal and dermal sheets from cartilage. These sheets were cut with scissors into the smallest possible pieces (−1 mm) and then the skin fragments were incubated at 37 °C for 30 min in collagenase working solution (tissue washing buffer containing 1 mg/mL collagenase D and 0.01% DNase). The digested sample was filtered through a 70 μm cell strainer, then undigested skin pieces were crushed on the strainer. The strainer was washed with cell culture medium, then all flow through was passed through a 5 mL syringe with a 22 G needle to make single cell suspension. Mouse bone marrow leukocytes were obtained from the tibia and femur by following a method described in a previous report [
28]. Briefly, the cells were flushed out from the tibia and femur by a 10 mL syringe with a 27 G needle containing cell culture medium. The cell suspension was filtered through a 70 μm cell strainer and washed once with cell culture medium, then the cells were treated with the 1 × RBC lysis buffer at RT for 10 min. After being washed twice with cell culture medium, the cells were used as bone marrow leukocytes. Splenic CD4+ T cells were isolated from OT-II mice-originated splenocytes by using MagniSort
TM Mouse CD4 T cell Enrichment Kit (Thermo Fisher Scientific, Waltham, MA, USA). The whole procedure for the cell isolation kit was performed by following the manual. The cell purity was checked by flow cytometry and the purified cell suspension with over 90% of CD4+ T cells was used for the experiment.
4.5. Mouse Bone Marrow Dendritic Cells (BMDCs) Preparation
Mouse BMDCs were prepared by following a method described in a previous report, with minor modifications [
18]. At day 0, 2.0 × 10
6 of bone marrow leukocytes were suspended in 10 mL of DC culture medium (cell culture medium containing 20 ng/mL of rmGM-CSF), and the cells were seeded on a 100 mm dish. At day 3, 10 mL of the fresh DC culture medium was added to the culture. At day 6 and 8, half of the cultured medium was collected and centrifuged, then the cell pellets were resuspended in 10 mL of fresh DC culture medium. The cell suspension was put back into the original plate. At day 10, cells were ready to use for each experiment. The differentiated BMDCs condition was checked by flow cytometry in every culture. The cell suspension with over 90% of CD11c+ and CD80low, CD86low and I-A/I-E+ in the CD11c+ population was used for experiment.
4.6. BMDCs Stimulation Assay
BMDCs (1.0 × 106) were seeded on 12-well plate with cell culture medium. The cells were simulated with TLR ligand (LPS: 100 ng/mL, PGN: 10 ug/mL, Pam3CSK4: 100 ng/mL, Poly (I:C): 10 ug/mL or IMQ: 10 ug/mL) or vehicle control. Some cultures were further treated with LTA (0.1, 0.5, 1 and 5 ug/mL) or vehicle control at 37 °C for 24 h. After the stimulation, the cultured medium was harvested for cytokine ELISA and was kept at −80 °C until use. In some cultures, the treated cells were harvested for flow cytometry.
4.7. TLR2 Blocking in TLR Ligand-Stimulated BMDCs
BMDCs (1.0 × 106) were seeded on 12-well plate with cell culture medium. The cells were pre-treated with anti-TLR2 mAb (10 ug/mL) or isotype antibody at 37 °C for 1 h. Then, the cells were treated with TLR ligand (LPS: 100 ng/mL or IMQ: 10 μg/mL) combined with LTA (5 μg/mL) or vehicle control at 37 °C for further 24 h. The cultured medium was harvested for cytokine ELISA and was kept at −80 °C until use. In some cultures, the treated cells were harvested for flow cytometry.
4.8. In Vitro Antigen Presentation and T Cell Activation
BMDCs (1.0 × 105) were mixed with OT-II CD4+ T cells (5.0 × 105) on 96-well plate in cell culture medium. OVA peptide (OVA323–339: 10 ug/mL) was added into cultures and then samples were treated with single or combination TLR ligands (LPS or IMQ LPS or IMQ+LTA, LPS). The ligand concentrations were LPS: 100 ng/mL, IMQ: 10 μg/mL and LTA: 5 ug/mL. Some cultures were treated with LTA only or vehicle control (no-Ag negative control). The culture was incubated at 37 °C for 72 h. At the last 5 h, the cells were re-stimulated with PMA (500 ng/mL) and ionomycin (1 ug/mL) in the presence of GolgiStopTM. The IFN-γ+CD4+ T (Th1) cells and CD69 expression in Th1 cell populations were analyzed by flow cytometry.
4.9. Flow Cytometry
Cell surface markers and intracellular proteins were analyzed by a flow cytometer (FACSCanto and LSR-II; BD Biosciences) with the fluorochrome-conjugated monoclonal antibodies described in reagents and antibodies. The cells were initially incubated with FcγRII/III blocker (anti-CD16/32; 2.4G2) at 4 °C for 10 min. For surface marker staining, the cells were incubated with the antibody at 4 °C for 30 min. Intracellular protein staining was performed by using a Cytofix/CytoPerm Kit or PerFix-p Kit by following the manual. Briefly, the cells stained with the antibody for the surface marker were fixed and permeabilized at 4 °C for 20 min. The cells were incubated with the antibody for staining of the target proteins at 4 °C for 30 min. The dead cells were excluded by forward scatter, side scatter and 7-AAD gating. All data were analyzed by BD FACS Diva (BD bioscience, Franklin Lakes, NJ, USA) or FlowJo (Tree Star; Ashland, OR, USA).
4.10. Cytokine Measurement by Enzyme-Linked Immuno Sorbent Assay (ELISA)
The cytokine (TNF-α) produced from the stimulated cell was measured by ELISA Ready-SET-Go!™ Mouse TNF-α Kit (Thermo Fisher Scientific, Waltham, MA, USA). The whole procedure was performed by following the manual.
4.11. Statistical Analyses
Student’s t-test was used to analyze the data for significant differences. Values of p < 0.05, p < 0.01 and p < 0.001 were regarded as significant.