Previously, we demonstrated that macrophages from thrombospondin 1 (TSP1)-deficient mice have a reduced inflammatory phenotype, suggesting that TSP1 plays a role in macrophage activation. In this study, we determined how TSP1 regulates macrophage function. We found that recombinant or purified platelet human TSP1 treatment stimulated tumor-necrosis factor (TNF)-α expression in bone marrow-derived macrophages in a time- and dose-dependent manner. Toll-like receptor 4 (TLR4) expression (at the mRNA and protein levels) and nuclear factor-kappaB (NF-κB) activity were also stimulated by TSP1 treatment. The TSP1-mediated increase in TNF-α production was abolished in TLR4-deficient macrophages, suggesting that TSP1 activates macrophages through a TLR4-dependent pathway. TSP1 also stimulated TLR4 activation in macrophages in vivo. Furthermore, TSP1-mediated macrophage activation was attenuated by using a peptide or an antibody to block the association between TSP1 and CD36. Taken together, these data suggest that the stimulation of the macrophage TLR4 pathway by TSP1 is partially mediated by the interaction of TSP1 with its receptor, CD36.
Thrombospondin 1 (TSP1) is a major component of platelet alpha granules.1,2 It is also expressed in many cell types, including adipocytes and macrophages, and exists as both a component of the extracellular matrix and as a soluble molecule in cell culture-conditioned medium.3,4,5,6,7 TSP1 is a multifunctional protein, and its diverse biological activities have been mapped to specific domains of the molecule by interaction with different cell surface receptors.8,9,10,11,12,13,14,15,16,17
Recently, TSP1 has been shown to play a role in inflammation and obesity. TSP1 is upregulated in the developing adipose tissue of mice with diet- or genetically induced obesity.18 Importantly, TSP1 has been identified as an adipokine that is upregulated in obese human subjects and is correlated with adipose inflammation and insulin resistance.19 By using TSP1-deficient mice, our previous studies revealed a novel role for TSP1 in regulating macrophage recruitment and activation in adipose tissue that contributes to inflammation and insulin resistance in the high-fat diet-induced obese mouse model.20 We found that high-fat diet-fed TSP1-deficient mice had improved glucose tolerance and insulin sensitivity, which were accompanied by reduced adipose tissue macrophages and decreased adipose inflammation.20 Importantly, these protective effects of TSP1 deficiency were observed even though TSP1-deficient mice exhibited levels of obesity similar to those of the wild-type controls.20 In vitro data demonstrated that macrophages isolated from TSP1-deficient mice had decreased chemotactic activity and a reduced inflammatory phenotype. However, how TSP1 regulates macrophage function is largely unknown.
In the current study, we determined the mechanisms by which TSP1 activates macrophages. We demonstrated that TSP1 activates Toll-like 4 pathways in macrophages and induces tumor-necrosis factor (TNF)-α production partially via an interaction with its receptor, CD36.
Materials and methods
Purified human platelet thrombospondin 1 (native TSP1) was purchased from Creative BioMart (Shirley, NY, USA). Recombinant human TSP1 (rTSP1) was purchased from R&D Systems (Minneapolis, MN, USA). Macrophage colony-stimulating factor, polymyxin B sulfate, lipopolysaccharide (LPS) and phorbol-12-myristate-13-acetate were purchased from Sigma (St Louis, MO, USA). All other cell culture reagents, including media and serum, were purchased from Life Technologies (Carlsbad, CA, USA). The blocking peptide (CD36 peptide (93–110), YRVRFLAKENVTQDAEDN) and the scrambled peptide (RFAYLRKNVTENDEQAVCD) were purchased from American Peptide Company (Sunnyvale, CA, USA). TNF-α and nuclear factor-kappaB (NF-κB) ELISA kits were purchased from eBioscience (San Diego, CA, USA).
Macrophage isolation and functional analysis
To prepare bone marrow-derived macrophages, bone marrow-derived cells were isolated from femurs and tibias of male mice (wild-type, TSP1−/− and TLR4−/− mice were purchased from Jackson Laboratory; CD36−/− mice were generously provided by Dr Denys van der Westhuyzen, University of Kentucky) as described previously.21 First, these cells were cultured in RPMI-1640 medium containing 20% fetal bovine serum, 25 ng/ml macrophage colony-stimulating factor and penicillin/streptomycin for 7 days to allow proliferation and differentiation into mature macrophages. Then, the macrophages were plated at a density of 3×104–5×104 cells/ml in cell culture plates and treated with different concentrations of TSP1 (0–10 µg/ml, 10 µg/ml=approximately 22 nM) in serum-free RPMI-1640 medium for different time periods. After treatment, the conditioned media and cells were harvested.
The TNF-α level in the conditioned medium was measured using an ELISA. The levels of TNF-α transcripts were determined by real-time PCR. In some experiments, prior to TSP1 treatment, a blocking peptide, a control scrambled peptide, a CD36 blocking antibody (clone FA6-152 IgG1, from Abcam (Cambridge, MA, USA))22,23 or an isotype control anti-mouse IgG1 (from Sigma) was used to pre-treat the macrophages, and the above experiment was repeated. In addition, in some experiments, recombinant human TSP1 was incubated with polymyxin B sulfate (10 µg/ml) for 30 min and then added to macrophages. After 24 h of incubation, the conditioned media and cells were harvested for TNF-α measurements as described above. To isolate peritoneal macrophages, 8-week-old male wild-type and TLR4−/− mice were intraperitoneally injected with saline (control group) or purified TSP1 (10 µg/day). Each group contained 3–4 mice.
Twenty-four hours after the injection, the mice were euthanized. Blood was collected, and platelet-poor plasma was prepared as described previously.24 The TSP1 level in the plasma was measured using an ELISA kit from TSZ ELISA (Framingham, MA, USA). In addition, macrophages were harvested by lavage of the peritoneal cavity with sterile phosphate-buffered saline.25 The expression of TNF-α and the NF-κB activity in these cells were measured. LPS (5 ng/ml) was used as a positive control in the experiment. To obtain human macrophages, human U937 monocytes were purchased from the American Type Culture Collection (Manassas, VA, USA). These cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and penicillin/streptomycin and differentiated into macrophages by treatment with phorbol-12-myristate-13-acetate (5 µg/ml) as described previously.26 These human macrophages were treated with TSP1 (10 µg/ml) for 24 h, and then the expression of Toll-like receptor 4 (TLR4) in these cells was determined.
A Limulus Amebocyte Lysate Assay Kit from Thermo Scientific Pierce (Pittsburgh, PA, USA) was used to evaluate endotoxin contamination in the samples. The measurement of the endotoxin levels in the samples was performed according to the manufacturer's protocol.
Total RNA was isolated from cells using TRIzol reagent (Life Technologies) and treated with DNase I (Roche, Indianapolis, IN, USA). The treated RNA was purified using an RNeasy kit (Qiagen, Valencia, CA, USA). Total RNA (2 µg) was used for cDNA synthesis using a High Capacity cDNA Reverse Transcription Kit (Life Technologies, Grand Island, NY, USA). Real-time PCR analyses were performed using a SYBR Green PCR Master Mix kit with a MyiQ Real-time PCR Thermal Cycler (Bio-Rad, Hercules, CA, USA). All reactions were performed in triplicate in a final volume of 25 µl. Dissociation curves were analyzed to detect nonspecific amplification, and we confirmed that a single product was amplified in each reaction. The quantities of each test gene and the internal control (18S rRNA) were then determined from the standard curve using the MyiQ system software. The mRNA expression levels of the test genes were normalized to the 18S rRNA level. The primer sequences for the 18S rRNA and TNF-α have been described previously.20 The primer sequences for TLR2 were forward, 5′-ATG CTTCGTTGTTCCCTGTGTTGC-3′, and reverse, 5′-AACAAAGTGGTTGTCGCCTGCTTC-3′. The primer sequences for TLR4 were forward, 5′-AACCAGCTGTATTCCCTCAGCACT-3′, and reverse, 5′-ACTGCTTCTGTTCCTTGACCCACT-3′. The primer sequences for CD36 were forward, 5′-ACTGGTGGATGGTTTCCTAGCCTT-3′, and reverse, 5′-TTTCTCGCCAACTCCCAGGTACAA-3′.
Western blot analysis
Macrophages were cultured and treated with TSP1 for 24 h. After treatment, the cells were harvested, and cell lysates were prepared. Equal amounts of protein were subjected to SDS–PAGE (10%) under reducing conditions. After the proteins had been electrophoretically transferred to nitrocellulose membranes and the membranes had been blocked, the membranes were incubated with an anti-TLR4 antibody (Santa Cruz Biotechnology, Inc. Dallas, TX, USA) for 1 h at room temperature. After extensive washing, a secondary antibody was used for the detection of immunoreactive bands with an enhanced chemiluminescence detection system (Pierce, Rockford, IL, USA).
NF-κB activity measurement
Macrophages were treated with recombinant TSP1 for different time periods. After treatment, macrophages were washed with ice-cold phosphate-buffered saline and lysed. The NF-κB activity in the whole-cell lysates was measured using an NF-κB Pathway Activation InstantOne ELISA kit from eBioscience according to the instruction manual. The NF-κB activity in the nuclear extracts was not specifically examined in this study.
The data are expressed as the mean±s.e. Differences between groups were assessed by analysis of variance followed by Turkey's post hoc test or Student's t-test as appropriate. The significance level was P<0.05.
TSP1 treatment stimulates TNF-α production in macrophages
Our previous studies demonstrated that macrophages from TSP1-deficient mice have a reduced inflammatory phenotype,20 suggesting that TSP1 plays a role in macrophage activation. To determine how TSP1 regulates macrophage activation, recombinant or purified human platelet TSP1 was used to treat bone marrow-derived macrophages from wild-type mice. The production of TNF-α was determined. We found that rTSP1 dose- and time-dependently induced the expression/production of TNF-α in macrophages (Figure 1a–c). To exclude the possibility of a confounding effect of LPS contamination in the rTSP1-mediated activation of macrophages, rTSP1 was pre-incubated with polymyxin B and then added to the cell culture for 24 h. We found that polymyxin B pre-treatment did not significantly reduce the TSP1-induced TNF-α production (data not shown). Similarly, purified human platelet TSP1 (native TSP1) also stimulated TNF-α production in macrophages (Figure 1d).
TSP1 treatment stimulates TNF-α production in macrophages through a TLR4-dependent pathway
The results demonstrated that TSP1 treatment upregulated the TLR4 mRNA and protein levels in wild-type bone marrow-derived macrophages (WT BMDMs) (Figure 2a and b). The TLR2 levels were not affected by TSP1 treatment (TLR2 mRNA levels/18S RNA: 0.63±0.19 (control) vs. 0.59±0.08 (TSP1 treatment), P=0.39). In addition, the NF-κB activity also increased in TSP1-treated WT BMDMs (Figure 2c). TSP1-stimulated TLR4 expression was also confirmed in TSP1−/− BMDMs after TSP1 treatment (Figure 2e) and in human macrophages (Figure 2d).
To determine whether the TLR4/NF-κB pathway is involved in TSP1-induced TNF-α production, bone marrow-derived macrophages were isolated from TLR4−/− mice. These cells were treated with rTSP1, and the level of TNF-α production was determined. The data demonstrated that TSP1-stimulated TNF-α production was abolished in TLR4−/− macrophages, suggesting the involvement of a TLR4-dependent pathway (Figure 3).
TSP1 stimulates TLR4 activation in macrophages in vivo
To determine whether TSP1 stimulates TLR4 activation in macrophages in vivo, rTSP1 was injected into wild-type and TLR4−/− mice. Saline-injected mice were used as vehicle controls. After 24 h, the mice were euthanized. The plasma TSP1 levels were measured by ELISA (mean±s.e.: 0.17±0.02 µg/ml for the control group vs. 0.38±0.05 µg/ml for the TSP1 injection group, P=0.007). Peritoneal macrophages were harvested, and the expression of TNF-α and the NF-κB activity were determined. As shown in Figure 4, macrophages from TSP1-injected wild-type mice had increased TNF-α expression and increased NF-κB activity compared with those from saline-injected wild-type mice. In contrast, macrophages from TSP1-injected TLR4−/− mice did not show elevated levels of either TNF-α expression or NF-κB activity. These data suggest that TSP1-stimulated macrophage activation in vivo is also TLR4-dependent.
The interaction between TSP1 and its receptor, CD36, partially mediates the TSP1-induced activation of macrophages
CD36 is a multiligand scavenger receptor and is expressed on many cell types. It has been implicated in multiple biological processes, including cellular adhesion, fatty acid and lipid transport, antigen presentation and the clearance of apoptotic cells.27 CD36 is a receptor for TSP1.27 Recently, studies have demonstrated that signals from CD36 are required for TLR4–TLR6 activation in macrophages as well as microglial cells.28 Whether CD36 is involved in TSP1-mediated macrophage activation is unknown. First, we determined whether CD36 expression is regulated by TSP1 treatment in BMDMs from wild-type mice or TLR4−/− mice. As shown in Figure 5, TSP1 treatment similarly stimulated CD36 expression in these two types of macrophages. Second, we determined whether CD36−/− BMDMs are resistant to TSP1-mediated increases in TLR4 expression. As shown in Figure 6, TSP1-simulated TLR4 expression (at both the mRNA and protein levels) was abolished in CD36−/− BMDMs, suggesting that CD36 is involved in TSP1-mediated macrophage activation.
It is known that CD36 interacts with TSP1 type 1 repeats. The residues of the CD36 molecule that specifically interact with TSP1 are referred to as the CD36 peptide (p93–110).29,30 We further determined the involvement of the TSP1–CD36 interaction in mediating the TSP1-induced activation of macrophages by two approaches. WT BMDMs were treated with TSP1 in the presence of a CD36 blocking antibody (which blocks the binding of TSP1 to CD36) or an isotype control IgG, as described previously.22,23 In addition, WT BMDMs were treated with a blocking peptide (CD36 peptide) or a scrambled peptide. After 24 h of treatment, the TNF-α concentration in the conditioned media was measured. As shown in Figure 7, the CD36 blocking antibody (Figure 7a) or blocking peptide (Figure 7b) significantly reduced the TSP1-induced increase in the TNF-α level. Taken together, these data indicate that the interaction between TSP1 and CD36 partially mediates the TSP1-induced activation of macrophages.
Previously, we demonstrated that macrophages from thrombospondin 1 (TSP1)-deficient mice have a reduced inflammatory phenotype, suggesting that TSP1 plays a role in macrophage activation. In the present study, we determined the mechanisms by which TSP1 regulates macrophage activation. We first demonstrated that TSP1 treatment stimulated macrophages to produce TNF-α through the activation of the TLR4/NF-κB pathway. Furthermore, other experiments demonstrated that CD36, a known receptor of TSP1, was involved in the TSP1-mediated TLR4 activation in macrophages.
TSP1 is expressed by many cell types and is the most abundant protein in the alpha granules of platelets. The normal plasma levels of TSP1 are very low (typically 0.1–0.2 µg/ml).31 However, in obese and diabetic individuals, the plasma TSP1 levels are significantly increased.19,32 To determine the effect of obesity-induced TSP1 elevation on macrophage function and to mimic the conditions of obesity, we used purified recombinant human TSP1 at a series of concentrations from 0–10 µg/ml to treat murine BMDMs or human macrophages. Recombinant human TSP1 stimulated macrophages to produce TNF-α in a dose-dependent manner. The maximum stimulatory effect was achieved at 10 µg/ml recombinant TSP1. To exclude the possibility of a confounding effect of LPS contamination in rTSP1-mediated activation of macrophages, rTSP1 was pre-incubated with polymyxin B and then used to treat macrophages.
The results showed that polymyxin B pre-treatment did not significantly reduce the TSP1-induced TNF-α production. Importantly, purified human platelet TSP1 similarly stimulated macrophages to produce TNF-α, with the concomitant activation of TLR4 and NF-κB. These results suggest that the activation of the TLR4 pathway by TSP1 in macrophages was not attributable to contamination of reagents with LPS. To confirm this conclusion, the LPS levels in the recombinant TSP1 were measured using the limulus amebocyte lysate assay. We found that the recombinant TSP1 used in this study had significantly lower LPS levels (<0.04 ng/ml) than those required to activate TLR4.33 In addition, the macrophage TLR4 pathway was activated in vivo by TSP1 injection into mice (Figure 4), although saline injection might not be a good control in this experiment. Together, these data indicate that TSP1 specifically activates the TLR4 pathway in macrophages.
TSP1 is a large homotrimer. Each monomer consists of an N-terminal domain; type I, type II and type III repeats; and a C-terminal domain. The diverse biological activities of TSP1 have been mapped to specific domains of the molecule by interaction with different cell surface receptors.8,9,10,11,12,13,14,15,16,17 CD36 is a receptor for TSP1. It binds to the type I repeat of TSP1 and is actively involved in signal transduction. The TSP1–CD36 interaction has been shown to contribute to the adhesive and anti-angiogenic functions of TSP1.5 Previous studies have demonstrated that signals from CD36 are required for TLR4–TLR6 activation in macrophages as well as microglial cells.28 CD36–TLR4–TLR6 activation serves as a mechanism by which atherogenic lipids and amyloid-beta stimulate sterile inflammation.28 CD47 is another receptor for TSP1 and binds to the C-terminal domain of TSP1. The TSP1–CD36–CD47 complex has been shown to be involved in T-cell expansion and inflammatory responses to amyloid-beta.34 In the present study, we found that TSP1 stimulated CD36 expression in macrophages. When a peptide or a CD36 blocking antibody was used to block the TSP1–CD36 interaction, the TSP1-induced stimulation of TNF-α production was significantly reduced, suggesting that the interaction between TSP1 and CD36 at least in part mediates the TSP1-induced activation of the TLR4 pathway in macrophages. Whether CD47 is also involved in this TSP1-mediated macrophage activation is unknown and warrants further investigation.
In this study, we demonstrated that TSP1 induces inflammatory signals in BMDMs in vitro and in peritoneal macrophages in vivo, suggesting that TSP1 has a pro-inflammatory effect on macrophages. However, in TSP1-null mice, a phenotype of acute pneumonia (lung inflammation) with neutrophil and macrophage infiltration was observed.35 This phenotype may be explained by the other functions of TSP1. In addition to regulating angiogenesis, inflammation and blood flow, TSP1 is a major activator of TGF-β.36,37,38 TGF-β suppresses the immune system.39 In TSP1-deficient animals, little active TGF-β may be produced in the lungs, resulting in exaggerated inflammatory responses.38 Other unknown mechanisms may also contribute to the lung inflammation observed in TSP1−/− mice.
In summary, our studies demonstrated that TSP1 activates the TLR4 pathway and induces TNF-α production in macrophages. TSP1 plays important roles in obesity, chronic inflammation and insulin resistance.19,20 Our findings may provide a mechanism by which TSP1 promotes inflammation and insulin resistance in obese animals, thereby aiding in the identification of therapeutic targets.
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This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK 081555 to SW), a Veterans Affairs merit award (BX 001204 to SW) and the National Institutes of Health (P20RR021954).
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Li, Y., Qi, X., Tong, X. et al. Thrombospondin 1 activates the macrophage Toll-like receptor 4 pathway. Cell Mol Immunol 10, 506–512 (2013). https://doi.org/10.1038/cmi.2013.32
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