Toll-like receptor 2 agonists inhibit human fibrocyte differentiation
© Maharjan et al; licensee BioMed Central Ltd. 2010
Received: 29 July 2010
Accepted: 24 November 2010
Published: 24 November 2010
In healing wounds, some monocytes enter the wound and differentiate into fibroblast-like cells called fibrocytes. Since Toll-like receptors (TLRs) are present on monocytes, and pathogens that can infect a wound have and/or release TLR agonists, we examined whether TLR agonists affect fibrocyte differentiation.
When human peripheral blood mononuclear cells (PBMCs) were cultured with TLR3, TLR4, TLR5, TLR7, TLR8 or TLR9 agonists, there was no significant effect on fibrocyte differentiation, even though enhanced extracellular tumor necrosis factor (TNF)-α accumulation and/or increased cell surface CD86 or major histocompatibility complex (MHC) class II levels were observed. However, all TLR2 agonists tested inhibited fibrocyte differentiation without any significant effect on cell survival. Adding TLR2 agonists to purified monocytes had no effect on fibrocyte differentiation. However, some TLR2 agonists caused PBMCs to secrete a factor that inhibits the differentiation of purified monocytes into fibrocytes. This factor is not interferon (IFN)-α, IFN-γ, interleukin (IL)-12, aggregated immunoglobulin G (IgG) or serum amyloid P (SAP), factors known to inhibit fibrocyte differentiation. TLR2 agonist-treated PBMCs secrete low levels of IL-6, TNF-α, IFN-γ, granulocyte colony-stimulating factor and tumor growth factor β1, but combinations of these factors had no effect on fibrocyte differentiation from purified monocytes.
Our results indicate that TLR2 agonists indirectly inhibit fibrocyte differentiation and that, for some TLR2 agonists, this inhibition involves other cell types in the PBMC population secreting an unknown factor that inhibits fibrocyte differentiation. Together, these data suggest that the presence of some bacterial signals can inhibit fibrocyte differentiation and may thus slow wound closure.
Following injury, circulating peripheral blood cells such as neutrophils, monocytes, dendritic cells and lymphocytes leave the bloodstream and enter the injured site. Once monocytes are in the injured site, they can differentiate into fibroblast-like cells called fibrocytes [1–8]. Fibrocytes have a distinct spindle-shaped appearance. Fibrocytes express hematopoietic markers, including CD45, major histocompatibility complex (MHC) class II, and CD34, along with stromal markers including collagen I, collagen III, and fibronectin [1, 9, 10]. Fibrocytes help to rebuild injured tissue by secreting angiogenic and fibrogenic growth factors as well as matrix metalloproteinases . Fibrocytes are also contractile cells, which further helps to close wounds by pulling the wound edges together .
We found that the serum protein serum amyloid P (SAP) can directly inhibit monocytes from differentiating into fibrocytes . Purified SAP inhibits fibrocyte differentiation, but other serum proteins such as serum amyloid A and C-reactive protein (CRP) are unable to inhibit the differentiation of monocytes into fibrocytes [5, 12]. The profibrotic cytokines interleukin (IL)-4 and IL-13 directly activate monocytes to differentiate into fibrocytes, while cross-linked immunoglobulin G (IgG) and the proinflammatory cytokine interferon (IFN)-γ directly inhibit the differentiation of monocytes into fibrocytes [6, 7]. Another proinflammatory cytokine, IL-12, activates some cells in the peripheral blood mononuclear cell (PBMC) population, possibly natural killer (NK) cells, to indirectly inhibit fibrocyte differentiation . In human PBMC culture, IFN-α2b inhibits fibrocyte differentiation, but whether this acts directly on monocytes is unknown . Other regulators of fibrocytes include the adenosine A2A receptor and cysteinyl leukotriene receptor 1 (CysLT1) [14, 15]. The adenosine A2A receptor regulates cell proliferation and cytokine production, and blocking this receptor inhibits the recruitment of fibrocytes in bleomycin-treated mouse skin . CysLT1 is a receptor for lipid mediators which promote fibroblast proliferation, fibroblast chemotaxis and collagen synthesis . In mice with fluorescein isothiocyanate (FITC)-induced lung fibrosis, blocking CysLT1 inhibits the appearance of fibrocytes .
It is unclear why some of the above factors affect fibrocyte differentiation. However, we hypothesized that SAP prevents fibrocyte differentiation in the circulation and that aggregated IgGs prevent fibrocyte differentiation because aggregated IgGs signify the presence of infection. We hypothesized that after monocytes detect an infected wound, monocytes do not differentiate into fibrocytes, since closing an infected wound could cause further damage such as gangrene, an infectious wound closure that results in further decay of the surrounding cells [16, 17]. The immune system can also recognize pathogens using Toll-like receptors (TLRs) [18–24]. TLR agonists include pathogen-specific molecules such as lipopolysaccharides (LPSs) from gram-negative bacteria, lipotechoic acid (LTA) from gram-positive bacteria, flagellin from bacteria, single-stranded DNA (ssDNA) from viruses and unmethylated DNA from bacteria [24, 25]. TLR signaling pathways trigger innate immune responses through nuclear factor (NF)-κB-dependent and IFN-regulatory factor-dependent pathways . Since TLRs are present on monocytes, we examined whether TLR agonists could also affect the differentiation of monocytes into fibrocytes.
Culturing PBMC with TLR agonists or IFN-α
Blood was collected from healthy adult volunteers in accordance with specific approval of Rice University's Institutional Review Board. Written consent was received from all volunteers, and all samples were deidentified before analysis. PBMCs were isolated and incubated in serum-free media (SFM) as described previously [5–7, 26]. TLR agonists (Invivogen, San Diego, CA, USA) were reconstituted in endotoxin-free water (Invivogen). All experiments were done with at least three different batches of agonists. IFN-α was obtained from EMD-Calbiochem (Darmstadt, Germany). A dilution series of TLR agonists (or the same volume of water as a control) was made in serum-free medium. A quantity of 100 μl of serum-free medium, TLR agonist or IFN-α dilution, or water dilution, was added to duplicate wells of a 96-well tissue culture plate (BD Biosciences, San Jose, CA, USA). A quantity of 100 μl of human PBMCs at a concentration of 5 × 105 cells/ml in serum-free medium was then added to each well. On day 5, fields of PBMCs were photographed using a phase-contrast microscope, and the number of cells per image was counted. Cells were then fixed and stained, and fibrocytes were counted as previously described [5–7, 26].
Preparation of monocytes
Monocytes were purified from 5 × 107 PBMCs using an EasySep Monocyte Depletion Kit (catalog no. 19059; StemCell Technology, Vancouver, BC, Canada) according to the manufacturer's instructions. To determine the purity of the monocytes, cells were analyzed using flow cytometry (FACScan, BD Biosciences; or Accuri C6 cytometer, Accuri Cytometers Inc., Ann Arbor, MI, USA) as described previously . A sample of each monocyte preparation was stained with 5 μg/ml primary antibodies against the T cell marker CD3, the monocyte marker CD14, the NK cell marker CD16, the B cell marker CD19 and the leukocyte marker CD45 as previously described . Monocytes obtained were greater than 95% pure as determined by the expression of CD14. Monocytes were 99% CD45-positive, 0.93% CD3-positive, 0.93% CD16-positive and 1% CD19-positive. To assess the effect of TLR2 agonists on monocytes, 5 μl of TLR2 agonist or water were added to 245 μl of serum-free medium. A quantity of 100 μl of serum-free medium, TLR2 agonist dilution or water dilution was added to a well of a 96-well plate, with each condition represented in duplicate wells. A quantity of 100 μl of purified human monocytes at a concentration of 5 × 105 cells/ml in serum-free medium was then added to each well. Fibrocytes were fixed, stained and counted after monocytes were cultured for 5 days.
Treating monocytes with conditioned media from PBMC stimulated with TLR2 agonists
To make conditioned medium, a dilution of TLR2 agonists was made as described above. A quantity of 100 μl of either SFM, the TLR2 agonist dilution or the corresponding water dilution was added to duplicate wells of a 96-well plate along with 100 μl of PBMCs at a concentration of 5 × 105 cells/ml in SFM. After 3 days of incubation, 180 μl of the conditioned media from one well representing each condition were transferred into an Eppendorf tube, snap-frozen in liquid nitrogen and stored at -20°C. For the well from which the medium was not removed, at day 5 the fibrocytes were fixed, stained and counted as previously described [5–7]. Also on day 5, monocytes were prepared from the same donor as described above. A quantity of 50 μl of monocytes at a concentration of 1 × 106 cells/ml was incubated with 50 μl of the day 3 conditioned medium. After 5 days, cells were fixed and stained, and the number of fibrocytes was counted.
Detection of cytokines and SAP by enzyme-linked immunosorbent assay
The day 3 conditioned media were analyzed for IFN-α, IFN-γ and IL-12 using enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Peprotech, Rocky Hill, NJ, USA). The day 3 conditioned media were also analyzed for IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17A, IFN-γ, TNF-α, granulocyte colony-stimulating factor (G-CSF) and tumor growth factor (TGF)-β1 using a multi-analyte profiler ELISArray kit according to the manufacturer's instructions (SABiosciences, Frederick, MD, USA).
The day 3 conditioned media were also analyzed for SAP as described previously [5, 27], with the exception that the ELISA plates were coated overnight at 4°C with mouse antihuman SAP antibody (SAP-5; Sigma, St. Louis, MO, USA) diluted 1:1,000 in phosphate-buffered saline (PBS) instead of 50 mM sodium carbonate buffer, and undiluted day 3 conditioned media were assayed.
Staining PBMC with CD86 or MHC Class II
Human PBMCs were cultured in the presence or absence of 8.9 μg/mL TLR3 agonist Poly (I:C), 0.89 μg/mL TLR7 agonist imiquimod (IMIQ), or 2.0 μg/ml nucleotide oligomerization domain (NOD)-like receptor (NLR) agonist peptidoglycan (PGN) in 1 ml in the well of a 48-well plate. On day 1 or 3, 900 μl of the conditioned media were carefully pipetted out and transferred into an Eppendorf tube, snap-frozen in liquid nitrogen and stored at -20°C. A quantity of 500 μl of ice-cold 50 mM ethylenediaminetetraacetic acid (EDTA) in PBS was then added to the PBMCs for 5 minutes at 4°C. The cells were vigorously resuspended with a plastic transfer pipette. The cells were transferred into an Eppendorf tube, collected by centrifugation at 300 × g for 5 min and the supernatant was discarded. The remaining cells on the plates were washed with 1 ml of ice-cold PBS, and this solution was added to the first pellet of cells. The cells were again collected by centrifugation at 300 × g for 5 min and then resuspended in 200 μl of 4% bovine serum albumin (BSA) in PBS. Cells treated with TLR agonists were divided into two separate Eppendorf tubes. The cells were incubated with 5 μg/ml antihuman CD86 or 5 μg/ml antihuman Human leukocyte antigen-DR/DP/DQ (HLA-DR/DP/DQ) (MHC class II) (both from BD Biosciences) for 30 min at 4°C as described previously . Meanwhile, untreated cells were divided into three separate Eppendorf tubes. These cells were incubated with 5 μg/ml antihuman CD86 or 5 μg/ml antihuman HLA-DR/DP/DQ, or they were kept in 4% BSA in PBS for 30 min at 4°C. All of the cells were washed three times in 1 ml of ice-cold PBS and then incubated with 2.5 μg/ml goat antimouse FITC (Southern Biotechnology, Birmingham, AL, USA) for 30 min at 4°C as described previously . After washing the cells three times in 1 ml of ice-cold PBS, the cells were resuspended in 100 μl of 4% BSA-PBS, and the staining was analyzed using flow cytometry with an Accuri C6 cytometer (Accuri Cytometers Inc.).
Detection of Ig molecules by Western blot analysis
Human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was diluted to 10 μg/ml, 1 μg/ml and 0.1 μg/ml in PBS. A quantity of 10 μl of conditioned media from day 3 or diluted human IgG was mixed with 2.5 μl of sodium dodecyl sulfate (SDS) sample buffer containing 20 mM dithiothreitol (DTT) and heated to 100°C for 5 min. After electrophoresis of the samples on 4-15% Tris-glycine polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA, USA), proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA) in Tris-NaCl-SDS buffer containing 20% methanol. Western blot staining was performed as described previously , with the exception that the detection antibody was 0.05 μg/ml biotinylated goat Fab'(2) antihuman Ig (H+L) (Southern Biotechnology) followed by 1:5,000 ExtrAvidin-Peroxidase staining (Sigma).
Treating monocytes with different combinations of cytokines
After analyzing the day 3 conditioned media with multi-analyte ELISArray kits, we calculated the concentrations of IL-6, IFN-γ, TNF-α, G-CSF, and TGF-β1 in LTA (TLR2 agonist)-treated conditioned media, LPS (TLR4 agonist)-treated conditioned media and control conditioned media. Cocktails of cytokines corresponding to twice the observed concentrations were made in SFM. A quantity of 100 μl of purified human monocytes at a concentration of 5 × 105 cells/ml were incubated in 100 μl of SFM, 100 μl of the above cytokine cocktails or 100 μl of the cytokine cocktail with all five of the above cytokines at either 2,000 pg/ml, 1,000 pg/ml, 200 pg/ml or 20 pg/ml. After 5 days, cells were fixed and stained, and fibrocytes were counted.
Statistical analysis was performed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Statistical significance was determined using either analysis of variance (ANOVA) or a t-test, and significance was defined as P < 0.05.
TLR3, TLR4, TLR5, TLR7, TLR8 and TLR9 agonists do not inhibit the differentiation of PBMCs to fibrocytes
To investigate the role of TLR agonists on fibrocyte differentiation, human PBMCs were cultured in the presence of various TLR agonists. Since immune cells can affect each other, we used PBMCs instead of purified monocytes to more closely mimic a human immune system. All of the TLR agonists were reconstituted in endotoxin-free water, and a control series of water dilutions had no discernible effect on fibrocyte differentiation (data not shown).
TLR agonists stimulate different cytokines and effectors in various cell typesa
Induction of IL-6
Lipomannan M. smegmatis (LM-MS)
Heat-killed Listeria monocytogenes (HKLM)
3 × 107 cells/ml
Induction of type I and type II IFN
Lipotechoic acid from S. aureus (LTA)
Increased expression of IL-1β, TNF-α, IL-6, and IP-10
Lipopolysaccharide from P. gingivalis (PG-LPS)
Secretion of IL-6
Induction of Type I and Type II IFN
Poly (I:C) (synthetic)
Production of IL-8, MCP-1, and TNF-α
E. coli K12 lipopolysaccharide (LPS)
0.01, 0.1, and 1 μg/ml
Production of TNF-α, IL-6, and IL-10
S. typhimurium flagellin
Activation of NF-κB
Expression of IFN, TNF-α, IL-6, and IL-8 increased
Plasmacytoid dendritic cells
Expression of IFN-α, IFN-β, and RANTES increased
RAW264.7 mouse macrophage cell line
Expression of IL-23 p19 increased
Activation of NK cells
Activation of TNF-α
E. coli ssDNA/LyoVec
Production of nitrite
Increased production of IFN-γ, IFN-α, IL-6, IL-8, and IL-12
Decreased production of IFN-α
Peptidoglycan from S. aureus (PGN)
Activation of NF-κB
TLR2 agonists inhibit the differentiation of PBMCs to fibrocytes
Some TLR2 agonists inhibit fibrocyte differentiation indirectly
Some TLR2 agonists cause PBMCs to secrete an unknown factor that inhibits fibrocyte differentiation
Conditioned media from LTA-treated PBMC, LPS-treated PBMC, and control conditioned media contain low levels of IL-6, TNF-α, IFN-γ, G-CSF and TGF-β1
Concentration in control CM (pg/ml)
Concentration in LPS-treated CM (pg/ml)
Concentration in LTA-treated CM (pg/ml)
4 ± 12
530 ± 250
750 ± 250
4.6 ± 1.6
29 ± 13
33 ± 6
1.0 ± 0.5
2.4 ± 0.9
4.5 ± 3.5
2.5 ± 0
17 ± 0
9.3 ± 1.8
13 ± 1.8
3.5 ± 0
4.5 ± 0
We found that a variety of TLR2 agonists inhibit fibrocyte differentiation. However, the TLR2 agonists do not directly inhibit the differentiation of monocytes to fibrocytes, but rather cause some other cell type in the PBMC population to inhibit monocytes from differentiating into fibrocytes. There are five known factors (IFN-α, IFN-γ, IL-12, SAP and aggregated IgG) which inhibit fibrocyte differentiation [5–7, 13]. Our data suggest that PBMCs incubated with some TLR2 agonists secrete an unknown sixth factor that inhibits fibrocyte differentiation.
The TLR2 agonist LTA induces PBMCs to increase the extracellular accumulation of IL-6, TNF-α, IFN-γ and G-CSF and to decrease the extracellular accumulation of TGF-β1. Both TLR2 agonists and TLR4 agonist induce primary adrenocortical cells to secrete IL-6 [37, 38]. However, we previously found that IL-6 has no effect on fibrocyte differentiation . Like the above five factors, this combination of cytokines at the concentrations found in the conditioned media, or at higher concentrations, was unable to inhibit fibrocyte differentiation and is thus not the factor in LTA-stimulated conditioned medium that inhibits fibrocyte differentiation.
Monocytes express TLR1-TLR9, but not TLR10 [39, 40]. They have a high expression of TLR1, TLR2 and TLR4; intermediate expression of TLR 5, TLR6 and TLR8; and low expression of TLR7 and TLR9 [39, 40]. Once activated, TLR agonists trigger downstream signaling events by one of four adaptor molecules: MyD88, MyD88-like adaptor protein (Mal), Toll/Interleukin-1 receptor (TIR) domain-containing adaptor protein-inducing IFN-β (TRIF) or TRIF-related adaptor molecule (TRAM) [41–45]. MyD88 activates mitogen-activated protein kinases (MAPKs) through IL-1R-associated kinase (IRAK) and tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF-6) [41, 45]. The signaling eventually causes the translocation of transcription factors such as NF-κB and activator protein 1 (AP-1), which then induces the production of inflammatory cytokines such as TNF-α, IL-6, IL-1β and IL-12 . TLR3 does not require the MyD88 pathway; instead it induces the production of IFN-β via TRIF [41, 46]. TLR4 can cause NF-κB activation by either a MyD88-dependent pathway or a MyD88-independent pathway . TLR4 activates a MyD88-independent pathway via TRIF, which complexes with TRAM and leads to NF-κB activation . Since none of the TLR agonists appeared to directly inhibit the differentiation of monocytes to fibrocytes, our data suggest that none of the above signal transduction pathways in monocytes affects their ability to differentiate into fibrocytes.
TLR2 recognizes various bacterial components such as bacterial lipoproteins and LTA [41, 47]. PBMCs incubated with the TLR2 agonists LM-MS, PG-LPS and LTA secrete an unknown factor that inhibits fibrocyte differentiation. However, for unknown reasons, the TLR2 agonists Pam3CSK4 (a synthetic triacylated lipopeptide), heat-killed Listeria monocytogenes (HKLM) and FSL-1 (a synthetic lipoprotein) do not appear to cause other cells in the PBMC population to secrete factors that inhibit fibrocyte differentiation. This suggests that these agonists indirectly inhibit fibrocyte differentiation by either cell-cell contact or a labile secreted factor.
We previously found that aggregated IgG inhibits fibrocyte differentiation . We hypothesized that since aggregated IgG (that is, IgG bound to something such as a bacterium) signifies the presence of bacterial infection, and since closing an infected wound and thus forming an anaerobic pocket can be detrimental, the inhibition of fibrocyte differentiation by aggregated IgG might have evolved to prevent closure of septic wounds by fibrocytes. An interesting possibility is that the indirect inhibition of fibrocyte differentiation by TLR2 agonists allows other cells in a wound environment to check for the presence of bacteria, and if bacteria are present, to relay this information to monocytes and prevent fibrocyte differentiation and the possible closure of an infected wound.
Conflict of interest
The authors declare that they have no competing interests.
We thank Varsha Vakil for assistance with blood collection. This work was supported by National Institutes of Health grant HL083029.
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