RPMI 1640 medium and fetal bovine serum (FBS) were obtained from Gibco-BRL (Gaithersburg, MD, USA). Tunicamycin (TM), dexamethasone (DEX), 3-(4,5-dimethylthiazol-2-y-1)-2,5-diphenyl-2H-tetrazolium bromide (MTT), phorbol 12-myristate 13-acetate (PMA) and monoclonal antibody to β-actin were from Sigma-Aldrich (St. Louis, MO, USA). IMQ was obtained from InvivoGen (San Diego, CA, USA). Monoclonal antibodies to GRP78 and CHOP were from Cell Signaling Technology (Beverly, MA, USA). The horseradish peroxidase (HRP)-conjugated anti-rabbit/mouse secondary antibodies were acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Polyvinylidene fluoride (PVDF) membranes were purchased from Millipore (Bedford, MA, USA). TRIzol reagent was from Invitrogen (Carlsbad, CA, USA). Interleukin (IL)-6 and monocyte chemotactic protein (MCP)-1 enzyme-linked immunosorbent assay (ELISA) kits were from R&D Systems (Minneapolis, MN, USA). Enhanced chemiluminescence (ECL) kits were from Thermo Scientific Pierce (Rockford, IL, USA). The reagent kits for cDNA synthesis and quantitative reverse transcription PCR (qRT-PCR) were from Bioneer Corp. (Daejeon, Korea); the reagents for reverse trancription PCR (RT-PCR) reagents were from Tiangen Biotech (Beijing, China). The Annexin V/PI apoptosis kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA). oxLDL was obtained from Union Biological Chemistry (Beijing, China). The reagents for western blot analysis were from Beyotime Institute of Biotechnology (Nantong, China). The other reagents were of analytical grade and were obtained from commercial sources.

The THP-1 monocyte cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin, and were maintained at (3–5)x10⁵ cells/ml at 37°C in a humidified incubator containing 5% CO₂. The THP-1 cells were seeded at 5×10⁵ cells/ml in tissue culture dishes, and differentiation was induced by 5 ng/ml PMA for 48 h with maintenance medium (2% FBS). Our observation that 5 ng/ml of PMA stimulated the THP-1 cells to differentiate into macrophages and did not induce undesirable gene upregulation was in accordance with the results presented in the sudy by Park et al (22). Subsequently, the THP-1-derived macrophages were pre-incubated with or without oxLDL (5 μg/ml) for 24 h prior to the addition of the TLR7 agonist, IMQ, for 24 h at 1 μg/ml or infected with IVA at the indicated concentrations in the presence or absence of 1 μg/ml dexamethasone (DEX) for 24 h. TM was used as a positive control (10 μg/ml) for 24 h. The IVA infection model was as follows: cells were prepared in dishes and inoculated with the indicated viral titers (1×10³ TCID50/ml – 1×10⁴ TCID50/ml) for 1 h. The inoculums were removed after 1 h of viral adsorption, followed by washing the monolayer 3 times with PBS. The monolayer cells were maintained in a virus isolation medium for the indicated periods of time and used for the subsequent experiments.

The influenza A strain PR/8/34 (H1N1) was provided by the Department of Epidemiology of Harbin Medical University, Harbin, China. The virus was propagated in MDCK cells and stored in a −80°C freezer, following the standard procedure. To evaluate the presence of the virus in the cell culture, we used RT-PCR and a hemagglutination assay as previously described by Mehrbod et al (23) and tested the median (50%) tissue culture infective dose (TCID50).

The cells were plated in 48-well plates at 2×10⁴ cells per well overnight and treated with IVA or IMQ as described above. The supernatant was harvested to detect the levels of IL-6 and MCP-1 by ELISA. All assays were performed according to the manufacturer’s instructions. The absorbance of the reaction solution was measured at 450 nm using a microplate autoreader (LUmo microplate luminometer; PHOmo microplate reader; Autobio Diagnostics, Henan, China).

The cells were seeded in 6-well plates, and after being subjected to the different treatments, whole cell protein extracts were prepared using RIPA lysis buffer following the manufacturer’s instructions. The lysates were sonicated for 20 sec and kept at 4°C for 15 min. Following 10 min of centrifugation (12,000 × g, 4°C), the supernatant was saved as a whole-cell lysate and measured using the Micro BCA™protein assay reagent kit (Thermo Scientific Pierce). Loading buffer was added to adjust the lysate followed by boiling for 10 min. The samples were resolved on SDS-polyacrylamide gels and transferred onto PVDF membranes. Following blocking with 5% non-fat dry milk for 1 h at room temperature and washing with Tris-buffered saline with 0.05% Tween-20 (TBST), the PVDF membranes were incubated overnight at 4°C with primary antibody in TBST containing 5% non-fat dry milk. The HRP-conjugated secondary antibody (1:5,000 dilution) was subsequently incubated with the membranes for 1 h at room temperature and washed extensively 3 times, 8–10 min each time with TBST at room temperature. The blots were probed using the ECL western blot detection system and visualized using X-ray film.

Total RNA was extracted using TRIzol reagent. Equal amounts of total RNA were reverse transcribed using a cDNA kit following the manufacturer’s instructions. The primers used for PCR are listed in Table I. For RT-PCR, initial denaturation was performed at 95°C for 3 min, followed by 40 cycles of denaturation at 94°C for 20 sec, then annealing at 60°C for 30 sec and extension at 72°C for 30 sec, and a final extension at 72°C for 10 min. The PCR products were visualized after electrophoresis on a 2% agarose gel containing ethidium bromide. qPCR was performed using Bioneer Corporation SYBR-Green qRT-PCR Master Mix kits. The incubation conditions were as follows: 95°C for 20 sec, followed by 40 cycles of 15 sec at 95°C, annealing/extension for 45 sec, at 60°C. The data were calculated using the 2^−ΔΔCt method and normalized against GAPDH. For each sample, qRT-PCR was performed in triplicate for both the target genes and GAPDH control.

The THP-1 cells were seeded at 5×10³ cells per well in 96-well flat-bottomed plates and incubated in 2% FBS-supplemented 1640 medium and treated with 5 ng/ml PMA for 48 h. Following treatment, MTT solution was added to a final concentration of 0.5 mg/ml, and the cells were incubated in a CO₂ incubator at 37°C for 4 h. After discarding the supernatant, the reduced MTT was solubilized in 150 μl per well of dimethylsulfoxide (DMSO), and the absorbance was measured at 490 nm using a PHOmo microplate reader. Cell viability was expressed as the percentage of the untreated controls.

Annexin V-FITC/PI double-staining was used to quantify the number of apoptotic cells by flow cytometry. Following treatment as described above, the cells harvested and washed 3 times with PBS. The concentration was adjusted to 1×10⁶ cells/ml, and 100 μl of cell suspension were stained with 5 μl Annexin V and 5 μl propidium iodide (PI) for 15 min in the dark at room temperature; the cells were then re-suspended in 400 μl calcium-binding solution and 1×10⁴ cells per sample were analyzed immediately using a BD FACSCantoII flow cytometer (BD Biosciences, Oxford, UK) and relevant software (BD FACSDiva). The identification of the different cell population patterns was as follows: live cells (FITC⁻ PI⁻), early apoptotic cells (FITC⁺ PI⁻), late apoptotic cells (FITC⁺ PI⁺) and necrotic cells (FITC⁻ PI⁺).

All data are presented as the means ± SD. One-way ANOVA was applied in order to detect statistically significant differences. Statistically significant differences between the treatment groups were calculated using SPSS 17.0 software for Windows. Values of P<0.05 were considered to indicate statistically significant differences.

The mRNA expression of the influenza matrix protein (M1) was detected after the cells were infected with growing concentrations of IVA for 24 h to prove that IVA can infect THP-1-derived macrophages in our cell model of IVA infection. M1 is a conservative protein among the influenza viruses. The results of RT-PCR revealed that IVA infected the THP-1-derived macrophages as M1 mRNA expression was observed in the IVA-infected groups and not in the untreated controls (Fig. 1A).

To confirm the effects of IVA or IMQ on TLR7 expression, we examined the mRNA expression of TLR7 in macrophages co-incubated with IVA or IMQ by RT-PCR. As shown in Fig. 1B, the mRNA expression of TLR7 was detected in all groups. In addition, both IVA infection (1×10³ TCID50/ml) and IMQ treatment (1 μg/ml) for 24 h increased TLR7 mRNA expression. At the same time, the production of IL-6 and MCP-1 was determined by ELISA following treatment with IVA or IMQ. The expression of IL-6 and MCP-1 was significantly increased following treatment with IVA or IMQ (Fig. 1C and D). These findings indicate that TLR7 is actually activated during IVA infection or IMQ treatment.

The THP-1 macrophages were co-incubated with increasing IVA concentrations (1×10³, 2×10³, 5×10³, 1×10⁴ TCID50/ml) as described above and collected at 8, 24 and 48 h post-inoculation (pi). The results revealed a pronounced decrease in cell viability in the infected groups, as determined by MTT assay (Fig. 2A). In early infection (8 h pi), cell viability fluctuated slightly, and the difference was not statistically significant (P>0.05). The decline in viability was more pronounced at 24 h pi. For further confirmation of this phenomenon, we detected the percentage of apoptotic cells (FITC⁺ PI⁻ and FITC⁺ PI⁺) by flow cytometry at 24 h pi. Similar results were observed, except that the number of apoptotic cells following treatment with 1×10⁴ TCID50/ml IVA was significantly higher compared with the apoptosis observed at the other IVA concentrations (Fig. 2B), suggesting that flow cytometry was more sensitive in detecting apoptotic cells than MTT assay.

A diversity of concentrations of IMQ (0.1, 1, 10 μg/ml) incubated with the THP-1 macrophages for 24 h induced cell apoptosis in a dose-dependent manner (Fig. 2C and D). These results suggest that TLR7 activation is important in cell apoptosis, which represents a host defense mechanism to limit viral replication.

It has been proven that IMQ has no direct antiviral activity in vitro and is not virucidal; rather, the antiviral activity is indirect through cytokine induction and immune activation (24). To investigate whether pro-inflammatory cytokines play a role in cell apoptosis, we used DEX to decrease inflammation as described above. As shown in Fig. 1C and D, DEX inhibited pro-inflammatory cytokine secretion to a large extent, although apoptosis did not decline (Fig. 3). These data indicate that cell apoptosis is independent of pro-inflammatory cytokine secretion in vitro.

oxLDL is a potent atherogenic factor in the progression of AS. It has been proven that a high concentration of oxLDL (200 μg ApoB/ml) triggers a prolonged ER stress activation that switched towards apoptosis, as supported by the increased expression of the pro-apoptotic mediator, CHOP, and JNK (25). Our results, which suggest that oxLDL had contradictory pro-apoptotic effects that depended on the oxLDL concentrations, are consistent with those of Hundal et al (26). In our study, oxLDL at 5 μg/ml did not influence cell viability and foam cells formed from macrophages (data not shown). To mimic IVA infection in obese individuals who are in a sensitive pro-atherogenic situation, we pre-treated the THP-1 macrophages with oxLDL (5 μg/ml) prior to treatment with IVA or IMQ. As shown in Fig. 4, oxLDL pre-treatment increased cell apoptosis as compared with IVA or IMQ treatment alone. This phenomenon demonstrates that oxLDL has a potential pro-apoptotic effect and acts as a ‘second hit’ to promote cell apoptosis with other detrimental stimulations.

Previous studies have produced controversial results on ER stress occurring in influenza infection (27–29). To investigate this, in this study, GRP78 and CHOP mRNA were detected by RT-PCR. As shown in Fig. 5A, the mRNA level of GRP78 and CHOP markedly increased. In agreement with this finding, the results produced by qRT-PCR (Fig. 5B) showed that the GRP78 levels markedly increased at first. It is interesting to note however that the GRP78 levels declined following treatment with 1×10⁴ TCID50/ml IVA. In contrast to GRP78, CHOP mRNA expression was induced in a dose-dependent manner and paralleled with the number of apoptotic cells (Fig. 2B). Collectively, these data verified that IVA infection induced ER stress and that CHOP played a role in IVA-induced apoptosis. Additionally, it is generally accepted that GRP78 expression suppresses cell apoptosis. In the present study, we found that GRP78 was inhibited when the cells were exposed to strong stimuli (1×10⁴ TCID50/ml); this resulted in the ratio of CHOP/GRP78 becoming much higher. The same phenomenon was observed in the cells treated with TM, a strong inducer of ER stress. It faintly induced GRP78 (10-fold) expression, but significantly increased CHOP (>2,000-fold) mRNA expression (data not shown). This phenomenon indicated that CHOP/GRP78 was a more sensitive index in predicting cell apoptosis. We also observed an induction in GRP78 and CHOP protein levels in the IVA-infected cell lysates, thus confirming the qRT-PCR results (Fig. 5C).

Having established that the caspase-dependent mitochondrial pathway was activated in IMQ-induced cell apoptosis (30,31), we wished to determine whether ER stress also participates in IMQ-induced apoptosis. To examine this hypothesis, GRP78 and CHOP expression was detected by RT-PCR, qRT-PCR and western blot analysis. As shown in Fig. 6A and B, the levels of GRP78 and CHOP mRNA were increased following treatment with IMQ, particularly at a high concentration (10 μg/ml). The protein levels of GRP78 and CHOP were consistent with the mRNA levels (Fig. 6C). These results suggest that ER stress plays a pro-apoptotic role in IMQ treatment, particularly with a potent stimulation.