TREM-1 is a positive regulator of TNF-α and IL-8 production in U 937 foam cells

Th e purpose of our study was to investigate the expression levels of TREM- (triggering receptor expressed on myeloid cells-) in U foam cells and determine whether TREM- regulates the production of tumor necrosis factor-alpha and interleukin- in these cells. Human U cells were incubated with phorbol -myristate -acetate and then oxidized human low-density lipoprotein to induce foam cell formation. Oil red O staining was used to identify the foam cells. Th e production of IL- and TNF-α by U foam cells was assayed by enzyme-linked immunosorbent assay. Th e expression of TREM- mRNA in U foam cells was detected by reverse transcription-polymerase chain reaction. Moreover, U foam cells were transfected by small interfering RNA using Lipofectamine  to knockdown TREM-. Western blot was performed to assay protein expression of TREM- and ELISA was used to examine the eff ect of TREM- knockdown on IL- and TNF-α production. PMA and ox-LDL induced U cells to form foam cells. Th e production of TNF-α and IL- was found to be signifi cantly elevated in U foam cells, concomitant with a signifi cant up-regulation of TREM- mRNA. TREM- siRNA was able to partially silence the expression of TREM- protein and remarkably inhibited TNF-α and IL- production in U foam cells, suggesting that TREM- is a positive regulator of TNF-α and IL- production in U foam cells. Our fi nding that TREM- controls the production of IL- and TNF-α in U foam cells defi nes a potentially critical role of TREM- in the pathogenesis of atherosclerosis and implicates TREM- as a potential therapeutic target for the disease. ©  Association of Basic Medical Sciences of FBIH. All rights reserved


INTRODUCTION
Atherosclerosis is a complex, progressive disease that is newly recognized to have a strong infl ammatory component [, ].In the early stage of atherosclerosis, monocytes migrate into the arterial intima and diff erentiate into macrophages that can take up oxidized low-density lipoprotein (ox-LDL) to stimulate diff erentiation into foam cells [, ].Th e accumulation of foam cells in the artery wall causes the formation of fatty streak(s), the earliest visible lesion of atherosclerosis [].The uptake of ox-LDL by macrophages results in the production of numerous proinfl ammatory cytokines that can amplify the infl ammatory response and promote the evolution of atheroma [].Interleukin- (IL-) and tumor necrosis factor-alpha (TNF-α) are two important proatherosclerotic cytokines secreted by ox-LDL-activated macrophages [-].Both of these molecules have been found to be up-reg-ulated at the sites of formation of atherosclerotic plaques [, ].IL- may induce the fi rm adhesion of monocytes to endothelial cells and mediate the intimal accumulation of macrophages in atherosclerotic lesions [, ], while TNF-α can enhance macrophage foam cell formation by inhibition of intracellular lipid catabolism and, therefore, contribute to atherosclerotic development and progression [].Accumulating evidence indicates that infectious processes may contribute to atherosclerosis [].Infectious agents, such as cytomegalovirus [], Chlamydop hila pneumoniae [], Helicobacter pylori [], and parvovirus [], have been shown to augment the production of cytokines in macrophages and provide inflammatory stimuli that can accelerate atherogenesis.TREM- (triggering receptor expressed on myeloid cells-) is an activating receptor that is selectively expressed on neutrophils and monocytes/macrophages and can be up-regulated by bacterial and fungal stimuli [].Engagement of TREM- on monocytes can trigger the release of large amounts of proinfl ammatory cytokines, including IL- and TNF-α, and amplify infl ammatory responses [, ].At present, it remains unclear whether TREM- is upregulated during foam cell formation and, if it is, whether TREM- regulates the production of proin- In the present study, we established an in vitro foam cell formation model by stimulating human myelomonocytic U cells with phorbol -myristate -acetate (PMA) and ox-LDL to investigate the expression of TREM- in macrophage-derived foam cells and its relationship with the secretion of TNF-α and IL-.Furthermore, small interfering RNA (siRNA) was employed to knockdown TREM- in order to examine TREM- effects on the production of TNF-α and IL- in U foam cells.

Cell culture and induction of foam cell formation
Human myelomonocytic cell line U was purchased from KeyGen Biotech (Nanjing, China) and was maintained in RPMI- medium (Gibco, USA) containing  fetal bovine serum (FBS) in a  CO incubator at °C.U cells during the logarithmic growth phase (at a density of . ×   cells/L) were stimulated with  nmol/L of PMA (Sigma, USA) for  h to induce the formation of macrophage-like U cells.After  hours of culture in serum-free medium, PMA-induced U cells were divided into three groups and incubated with RPMI- medium containing  FBS (PMA group),  mg/L of LDL (PMA+LDL group) or  mg/L of ox-LDL (PMA + ox-LDL group; Yuanyuan Biotechnology, Guangzhou, China).Experiments were performed in quintuplicate.After  h of culture, supernatants were collected to measure the contents of TNF-α and IL-, and the cells were harvested for detection of TREM- mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR).
Identifi cation of U foam cells U foam cells were identifi ed by oil red O staining.Briefl y, adherent U foam cells were stained with freshly prepared . oil red O solution for  min.Cell nuclei were then counterstained with hematoxylin solution for  min.After rinsing with  ethanol, cells were mounted onto glass slides with an aqueous mounting reagent.Stained cells were observed under an inverted light microscope (TE; Nikon, Japan).

RT-PCR
Total RNA was isolated from cells using the TRIzol Reagent (Gibco) according to the manufacturer's protocol.Reverse transcription was performed using M-MLV reverse transcriptase and oligo-dT primers (Fermentas, USA) following the manufacturer's instructions.PCR was then carried out to determine the expression levels of TREM- and glyceraldehyde--phosphate dehydrogenase (GAPDH, control) mRNAs using the following parameters: pre-denaturation at °C for  min;  cycles of denaturation at  °C for  s, annealing at  °C for  s, and extension at  °C for  s; and, a fi nal extension at  °C for  min.Th e sequences of the TREM- and GAPDH primers were as follows: TREM- forward, '-TGCTGTGGATGCTCTTTGTC-' and reverse, '-CACAGTTCTGGGGCTGGTAT-'; GAPDH forward, '-ACCACAGTCCATGCCATCAC-' and reverse, '-TCCACCACCCTGTTGCTGTA-'.The PCR products were resolved by electrophoresis on a  agarose gel containing ethidium bromide (. μg/ml).Th e expression level of TREM- mRNA relative to GAP-DH transcripts was determined by densitometric scanning (Gel Image system-; Tanon, Shanghai, China).
Cytokine measurement TNF-α and IL- in culture supernatants were assayed by enzyme-linked immunosorbent assay (ELISA) using commercial kits (Westang, Shanghai, China), according to the manufacturer's instructions.Briefl y,  μl culture supernatants were added into the -well plate.After incubation for  h at  °C, the plate was washed three times with cleaning solution [TBST:  mmol/L Tris-HCl,  mmol/L NaCl , . (V/V) Tween ].With the exception of the negative control, all the wells were supplemented with  μl of horseradish peroxidase (HRP)-antibody working solution and incubated at  °C for  h.After washing as above, a drop of substrate A and B were added sequentially.After incubation at  °C for  min, a drop of end solution was added into each well, and the absorbed density (OD) was detected at  nm.
siRNA transfection Th e following two oligonucleotides encoding a TREM- siR-NA were obtained from Dharmacon (Lafayette, USA): sense, '-CCGGAAGTGTATGTGATCAGAGTAAT TCAAGA-GATTACTCTGATCACATACACTTTTTTTG-'; antisense, '-AATTCAAAAAAAGTGTATGTGATCAGAG-TAATCTCTTGAATTACTCTGATCACATACACTT-'.Macrophage-like U cells induced with PMA as described above were used for siRNA transfection.In addition to the TREM- siRNA group (TREM- siRNA), an empty liposome group (empty liposome) and a random siRNA group (negative siRNA) were used as negative controls.Transfection was performed using Lipofectamine  according to the manufacturer's protocol.After  h of transfection, cells were treated with  mg/L of ox-LDL as described above and subjected to detection of TREM- protein expression by Western blot, and TNF-α and IL- production by ELISA.

Statistical analysis
Numerical data have been expressed as mean ± standard deviation (SD).Statistical analysis was performed using the SSPS . software package.Intergroup differences were compared using one-way analysis of variance (ANOVA) and p<. was considered to be significantly different.

PMA and ox-LDL induced the diff erentiation of U cells into foam cells
As shown in Figure A, the untreated U cells exhibited the characteristic round shape.After incubation with PMA for  h, the majority of U cells shifted to an ameboid shape and began to aggregate and adhere, indicating the differentiation from monocytes into macrophage-like cells (Figure B).In the presence of ox-LDL, PMA-induced macrophage-like cells showed obvious foamy changes as many oil red O-positive lipid droplets were observed in the cytoplasm.Some cells showed an enlarged size due to uptake and accumulation of an excessive amount of ox-LDL (Figure C).In contrast, no obvious lipid droplets were observed in PMA-induced macrophage-like cells with (data not shown) or without (Figure B) LDL treatment.

TNF-α and IL- levels were elevated in U foam cells
To determine the effects of ox-LDL treatment on the levels of TNF-α and IL- in PMA-induced U cells, ELI-SA was performed (Figure ).The levels of both TNF-α and IL- in PMA+ox-LDL U cells were found to be significantly higher than those in PMA and PMA+LDL groups (all p <.): TNF-α, . ± . (PMA+ox-LDL) vs. .±.(PMA) and . ± . (PMA+LDL); IL-, . ± . (PMA+ox-LDL) vs. .± . (PMA) and . ± . (PMA+LDL).The levels of both TNF-α and IL- in PMA+LDL group were slightly higher than those in the PMA group, but there were no significant differences observed (p>.).These results indicated that the levels of both TNF-α and IL- were elevated in U foam cells.

TREM- mRNA expression was up-regulated in U foam cells
To examine the impact of ox-LDL treatment on the expression of TREM- mRNA in PMA-induced U cells, the level of TREM- mRNA relative to GAPDH was determined by RT-PCR (Figure ).The relative level of TREM- mRNA in PMA+ ox-LDL U cells was significantly higher than those in PMA and PMA+LDL groups: . ± . (PMA+ox-LDL) vs. .± . (PMA) and . ± . (PMA+LDL) (both p<.;Figure A and B).However, there was no signifi cant diff erence observed in the relative level of TREM- mRNA between PMA and PMA+LDL groups (p>.).These data clearly indicated that TREM- mRNA expression was up-regulated in U foam cells.

TREM- siRNA partially silenced the expression of TREM- protein in PMA-induced U cells
To examine whether TREM- is involved in the regulation of TNF-α and IL- production in U foam cells, siRNA-me-diated silencing of the TREM- gene was performed.Western blot analysis indicated that the expression of TREM- protein was signifi cantly down-regulated in PMA-induced U cells transfected with TREM- siRNA, as compared to untransfected cells or cells transfected with empty liposome or negative siRNA (p<., Figure  and Table ).In contrast, no signifi cant diff erences were noted in the expression levels of TREM- protein among untransfected U cells and U cells transfected with empty liposome or negative siRNA.Thus, TREM- was partially silenced by the corresponding siRNA in PMA-induced U cells.

TREM- knockdown suppressed TNF-α and IL- production in U foam cells
After PMA-induced U cells transfected with TREM- siR-NA were treated with ox-LDL, the production of TNF-α and IL- in U foam cells was detected by ELISA.As shown in Figure  and Table , TREM- knockdown signifi cantly inhibited ox-LDL-induced TNF-α and IL- secretion in U foam cells.In contrast, the levels of TNF-α and IL- in foam U cells transfected with empty liposome or negative siR-NA were not signifi cantly diff erent from those detected in untransfected cells.Apparently, siRNA-mediated silencing of the TREM- gene was capable of signifi cantly suppressing the production of TNF-α and IL- in U foam cells.

TREM- knockdown suppressed ox-LDL-induced up-regulation of PKC expression in U foam cells
To determine whether ox-LDL treatment alters the expres-sion of PKC protein in PMA-induced U cells, Western blot was performed (Figure ).Th e expression level of PKC protein in the PMA+ox-LDL group was signifi cantly higher than that in the PMA group (p<.).However, TREM- knockdown significantly inhibited ox-LDLinduced up-regulation of PKC expression in U foam cells.In contrast, the expression levels of PKC protein in foam U cells transfected with empty liposome or negative siRNA were not significantly different from that detected in untransfected cells.Collectively, these results indicated that TREM- knockdown suppressed ox-LDLinduced up-regulation of PKC expression in U foam cells.

DISCUSSION
Infectious agents can contribute to the pathogenesis of atherosclerosis by promoting macrophage production of infl ammatory cytokines [].Moreover, TREM- plays a critical role in monocyte/macrophage-mediated inflammatory responses to microbial infection [].Based on these observations, we hypothesized that TREM- may be involved in regulating the production of proinfl ammatory cytokines during

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foam cell formation.In the present study, we demonstrated that ox-LDL treatment signifi cantly up-regulate the expression of TREM- mRNA in PMA-induced macrophage-like cells, and TREM- knockdown remarkably inhibited ox-LDL-induced TNF-α and IL- secretion, suggesting that TREM- is a positive regulator of TNF-α and IL- production in U foam cells.Given that TNF-α and IL- are proinfl ammatory cytokines that play an important role during macrophage foam cell formation and atherosclerotic development and progression [-], these findings suggest a possible critical role of TREM- in the pathogenesis of atherosclerosis.
Multiple studies have demonstrated that TNF-α and IL- are up-regulated in macrophage foam cells in atherosclerotic plaques and involved in the potentiation of atherosclerosis [-].Th e activation of p mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) may be implicated in the secretion of IL- in lipid-laden macrophages [], while TNF-α production may depend on the activation of activator protein  (AP-) [], phosphoinositide -kinase (PIK) and extracellular signal-regulated kinase (ERK) [].
In the present study, we found that TREM- could control the release of TNF-α and IL- in U foam cells.Similarly, previous studies also showed that TREM--mediated TNF-α and IL- secretion was observed in monocytes/macrophages [].Although the mechanism underlying TREM--mediated release of IL- and TNF-α in U foam cells is still unclear, the connection between TREM- and the cytokineinducing Toll-like receptor  (TLR)/NF-κB pathway may provide a clue to this mechanism [].Yang et al. [] found that blocking of TLR was able to substantially inhibit NF-κB activity and ox-LDL-induced IL- expression.Ornatowska et al. [] demonstrated that TREM- silencing in macrophages resulted in decreased expression of key proteins in the TLR signaling pathway.Th us, it is reasonable that the TLR/NF-κB signaling pathway might be involved in TREM--mediated TNF-α and IL- production in U foam cells.
Protein kinase C (PKC) is a family of serine/threonine protein kinases that play a key role in the regulation of the inositol phosphate/diacylglycerol signalling pathway.Upon activation, PKC enzymes are translocated from the cytosol to the plasma membrane and exert diverse biological functions by phosphorylating target proteins.Feng et al. [] found that PKC could promote the expression of scavenger receptor CD and lipid droplet-associated PAT-proteins in monocytes, thus contributing to foam cell formation.Oso et al. [] demonstrated that inhibition of PKCbeta prevents LDL uptake and foam cell formation by reducing scavenger receptor an expression in human macrophages.Another study [] indicated that PKC activation could modulate adipophilinmediated lipid accumulation in THP- macrophages.In the present study, we discovered that the expression pattern of PKC in U foam cells was similar to that of TREM-, suggesting that PKC activation may be involved in TREM--mediated IL/TNF-α production in U foam cells.However, the mechanisms behind the link between PKC activation and TREM- link remain to be further studied.Of note, the PKC family consists of at least  isozymes that are divided into three subfamilies.Th erefore, elucidation of the roles of diff erent PKC isoforms in foam cell formation will lead to a better understanding of the pathogenesis of atherosclerosis.At present, the mechanism underlying ox-LDL-induced TREM- up-regulation remains elusive.Although a natural ligand for TREM- has not yet been identifi ed, previous studies have shown that TREM- expression can be modulated in vitro and in vivo by various TLR ligands, including the gram-negative bacterial lipopolysaccharide, the primary ligand of TLR [].Th is raises the possibility that the TLR signaling pathway may also be involved in ox-LDL-induced TREM- up-regulation.Interestingly, TREM- and TLR ligands have been shown to induce infl ammatory responses via intersecting and mutually stimulating pathways [].In addition, LDL oxidation is capable of inducing the formation of a number of highly reactive substances, some of

A B
which may also directly mediate TREM- up-regulation.As stated above, macrophage foam cell formation is a characteristic feature of atherosclerotic plaques.Inhibition of foam cell formation and reduction of infl ammatory cytokine production may be of therapeutic value in the treatment of atherosclerosis [].Our fi nding that TREM- controls the release of IL- and TNF-α in U foam cells defi nes a possible critical role of TREM- in the pathogenesis of atherosclerosis and implicates TREM- as a potential therapeutic target for the disease.In animals with LPS-induced endotoxemia, blockage of TREM- signaling by administration of either a soluble form of the TREM- molecule, small-molecule inhibitor or siRNA have been found to be able to improve animal survival [, , ].If our results are verifi ed in vivo, these approaches might also be applicable to the treatment of atherosclerosis.

CONCLUSION
In conclusion, the present study provides evidence that, in U foam cells, up-regulated expression of TREM- is concomitant with an increase in the levels of TNF-α and IL-, and siRNA-mediated down-regulation of TREM- expression suppresses the production of TNF-α and IL-.Apparently, TREM- can stimulate the release of TNF-α and IL- in U foam cells.These results suggest a critical role of TREM- in foam cell formation and the pathogenesis of atherosclerosis and may aid in the design and development of new strategies to combat atherosclerosis.

FIGURE 1 .
FIGURE 1. PMA and ox-LDL induced the diff erentiation of U937 cells into foam cells.(A).Inverted microscopy showed the characteristically round U937 cells prior to treatment.(B).After incubation with PMA for 72 h, the U937 cells shifted to an ameboid shape, and aggregated and adhered, indicating their diff erentiation from monocytes into macrophage-like cells.(C).In the presence of ox-LDL, PMA-induced macrophage-like cells showed obvious foamy changes: many oil red O-positive lipid droplets were observed in the cytoplasm; some cells showed an enlarged size due to uptake and accumulation of excessive ox-LDL (magnifi cation ×200).

FIGURE 4 .BFIGURE 5 .
FIGURE 4. TREM-1 siRNA partially silenced the expression of TREM-1 protein in PMA-induced U937 cells.After PMA-induced U937 cells were transfected with TREM-1 siRNA (TS), negative siRNA (NS) or empty liposome (EL), and then treated with ox-LDL, the expression levels of TREM-1 protein were measured by Western blot.Untransfected U937 cells untreated (PM) or treated with ox-LDL (PO) were used as controls.Data have been shown as mean ± SD (n = 5).(A).Representative image showing the expression of TREM-1 and GAPDH proteins in U937 cells; (B).Quantitative analysis of the expression level of TREM-1 protein relative to GAPDH protein.*p < 0.05 vs. the PMA group (PM); # p < 0.05 vs. the PMA+ox-LDL group (PO), Negative siRNA group (NS) or Empty liposome group (EL), respectively.

FIGURE 6 .
FIGURE 6. TREM-1 knockdown suppressed ox-LDL-induced up-regulation of PKC expression in U937 foam cells.After PMAinduced U937 cells were transfected with TREM-1 siRNA (TS), negative siRNA (NS) or empty liposome (EL), and then treated with ox-LDL, the expression levels of PKC in these cells were determined by Western blot.Untransfected U937 cells untreated (PM) or treated with ox-LDL (PO) were used as controls.Data have been shown as mean ± SD (n = 5).(A).Representative image showing the expression of TREM-1 and GAPDH proteins in U937 cells; (B).Quantitative analysis of the expression level of TREM-1 protein relative to GAPDH protein.*p < 0.05 vs. the PMA group (PM); # p < 0.05 vs. the PMA+ox-LDL group (PO), Negative siRNA group (NS) or Empty liposome group (EL), respectively.