Induction of IFNα or IL-12 depends on differentiation of THP-1 cells in dengue infections without and with antibody enhancement

Phorbol 12-myristate 13-acetate (PMA), and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO). RPMI 1640 culture medium was obtained from Gibco BRL (Grand Island, NY, USA). Fetal bovine serum (FBS), gentamicin, and L-glutamine were purchased from Gibco BRL, Inc. (Grand Island, N.Y., USA).

Monocytic THP-1 cells were grown in RPMI 1640, 10% FBS (endotoxin level <0.25 EU/mL; Gibco BRL, Grand Island, N.Y., USA), 2 mM L-glutamine (Gibco BRL, Grand Island, N.Y., USA) at 37°C, and 5% CO₂ incubator. THP-1 cells (2×10⁵ cells/ml) were subcultured every 3 days, and PMA (8 nM) was used to induce THP-1 cell differentiation. To study time dependent effect, cells (2×10⁶ cells/ml) were used for studies with dengue-2 infection at multiplicity of infection (MOI) = 1.0 for 6 to 72 hours as indicated. For studying different infection dose, we used MOI from 0.1, 0.5, 1, 5 and 10 to study its dose-dependent effect.

Dengue virus type 2 (DEN-2, New Guinea C strain, ATCC) was obtained from the Institute of Preventive Medicine, National Defense Medical Center, Taipei. Viruses were propagated in C6/36 mosquito cells in Eagle's minimal essential medium (MEM) (Gibco BRL, Grand Island, N.Y., USA) containing nonessential amino acids (Gibco BRL, Grand Island, N.Y., USA), 1% sodium pyruvate, 0.2% sodium bicarbonate and supplemented with 1% antibiotic (Gibco BRL, Grand Island, N.Y., USA) and 10% heat-inactivated FBS at 28°C for 5 days. Baby hamster kidney cells (BHK-21) were grown in MEM as described above. A large collection of virus culture was pooled and showed a titer of 1.0 × 10⁷ PFU/ml determined by real-time quantitative RT-PCR as previously described [30]. All experiments about DEN infection were set at MOI = 1.

In order to characterize the change of cell differentiation markers on THP-1 cells, we measured pDC-specific and mDC-specific markers on THP-1 cells by flow cytometry. These cells (2 × 10⁶ cells/ml) are treated with and without 8 nM PMA for 72 hours. Cell surface stainings were performed by direct immunofluorescent assay with fluorescence-conjugated mAbs (CD14-PE, CD11b-PE, CD11c-PE, and CD123-FITC) and corresponding isotype control antibodies for 30 minutes. After washing in PBS twice, cells were fixed in 2% paraformaldehyde for 20 min, washed, and resuspended at ~10⁶ cells per milliliter before acquisition.

We subjected total RNA extracted from THP-1 cells with and without differentiation treatment to quantitative analysis of mRNA expression of IL12B and IFNα. In brief, the cell pellet was mixed with 0.5 ml of Tri-Zol solution (Invitrogen, California, USA). After thorough vortexing, samples were added 0.1 ml of chloroform (Scharlau, sa, Barcelona, European Union) for phase separation. After centrifugation, the upper aqueous phase was transferred to a fresh DEPC-treated eppendorf and the same volume of isopropanol (Merck KGaA, Darmstadt, Germany) was added for RNA precipitation at −20°C for 1 hour. The RNA was harvested by centrifugation at 12,000 × g for 10 minutes at 4°C, followed by 75% ethanol (Merck KGaA, Darmstadt, Germany) precipitation. Finally, the RNA was subjected to the real-time RT-PCR detection with SYBR Green PCR reagents (RealQ-PCR Master Mix Kit, Ampliqon) using the ABI PRISM 7500 instrument (Applied Biosystems, Foster City, CA) as previously described [46]. Primers for the quantitative detection of target mRNAs were designed by using Primer Express computer software (Applied Biosystem, Foster City, CA). For the IL12B gene, the primer sequences were 5’- acctccacctgccgagaat-3’ (forward) and 5’- acctccacctgccgagaat-3’ (reverse). For the IFNα. gene, the primer sequences were 5’-atttctgctctgacaacctc-3’ (forward) and 5’-tgacagagactcccctgatg-3’ (reverse) [47]. In order to evaluate PCR efficiency, a 3 replicates and 5-log dilution series were performed. A slope of −3.3 ±10% reflects an efficiency of 100% ±10%. Samples were analyzed in three independent duplicate experiments. The RT-PCR cycling parameters were set as follows: the RT reaction at 50°C, 2 minutes; 60°C, 30 minutes; and 95°C, 5 minutes; followed by 40 cycles of PCR reactions at 94°C, 20 seconds, and 60°C, 1 minute. The results were detected in a real-time fashion and recorded on a plot showing fluorescence vs. time. RT-PCR products were also visualized on ethidium bromide-stained 1.5% agarose (Pierce Co., Rockford, IL, USA) gel with a 100-bp ladder (Pharmacia Biotech, Piscataway, NJ, USA) as a reference. The increase of the IL12B and IFNα mRNA expression were therefore calculated assuming 100% efficient PCR where each C_t was normalized to β-actin mRNA expression as shown by the equation at 2 ^{[Ct1(target) − Ct1(actin)][Ct2(target) − Ct2(actin)]}. The C_t1 (target) and C_t2 (target) represent the C_t values for the IL12B or IFNα gene expression in treated and mock samples, respectively. C_t1 (actin) and C_t2 (actin) represent the C_t values for the β-actin gene.

To assess ADE, DEN-2 viruses were preopsonized with and without DEN-1 or DEN-2 immune sera at 4°C for 60 min as previously described [32], PMA-differentiated THP-1 cells (2 x 10⁵cells/ml) were incubated with DEN-2 virus with and without DEN immune sera at MOI of 1 at 37°C for 60 min. The infected cells were washed to remove extracellular viruses before culture in RPMI 1640. The infected PMA-differentiated THP-1 cells were cultured for 24 hours before the culture supernatants were harvested for determination of DEN-2 virus titers by real-time RT-PCR assay for 40 cycles using TaqMan technology (Applied Biosystems, Foster City, CA). The forward primer, reverse primer and nested fluorescent probe sequence for detecting DEN-2 were 5'-GGC TTA GCG CTC ACA TCC A-3', 5'-GCT GGC CAC CCT CTC TTC TT-3', and FAM-CGC CCA CCA CTA TAG CTG CCG GA-TAMRA, respectively [30]. The intracellular viruses were harvested from the cell pellets after washing twice with PBS to remove extracellular virus. The cell pellets were frozen and thawed for three times to release intracellular DEN-2 virus, then resuspended to 20 μl and subjected to real-time RT-PCR assay.

In order to characterize the expression of cell markers on THP-1 cells, we assessed the CD123 levels, a pDC-specific marker, on THP-1 cells by flow cytometry. As shown in Figure 1A, CD123 was expressed on the surface of THP-1 and down-regulated on the PMA-differentiated THP-1 cells. To investigate whether THP-1 cells elicited Th1 promoting cytokines under DEN-2 infection, cellular RNA was extracted and assessed using real-time RT-PCR after 24 h infection. Higher levels of IFNα mRNA but not IL12B mRNA were induced in post-infected THP-1 cells by DEN-2 (Figure 1B).

THP-1 cells grew as a single-cell suspension, and only a few cells spread onto the flask substrate. Approximately 80% of the cells attached to the substrate as early as 3 h after the addition of PMA (8 nM). After a 72-h treatment with PMA, THP-1 spread onto the substrate to be irregular and flattened in shape. Addition of DMSO only at the same dilutions required for obtaining the given concentrations of PMA in culture system did not induce adherence of the cells to the substrate.

The effect of PMA treatment on the expression of surface markers of THP-1 cells was examined by flow cytometry. THP-1 treated with 8 nM PMA expressed more mDC cell markers such as CD14, CD11b, and CD11c (Figure 2A-C), but not CD123, pDC cell markers (Figure 1A). To analyze whether PMA-treated THP-1 cells elicited Th1 promoting cytokines under DEN-2 infection, cellular RNA was extracted and assessed using real-time RT-PCR after 24 h DEN-2 infection. Higher levels of IL12B mRNA but not IFNα mRNA were induced in PMA-treated THP-1 cells after 24 h DEN-2 infection (Figure 2D).

Undifferentiated THP-1 cells elicited higher production of IFNα mRNA (Figure 1B); PMA-treated THP-1 cells after DEN-2 infection expressed IL12B mRNA (Figure 2D). To investigate whether Th1 promoting cytokines induced in undifferentiated THP-1 cells were dose dependent, cells were infected by different DEN-2 titers (MOI = 0.1 ~ 5) and RNA were assessed by real-time RT-PCR. As shown in Figure 3A, THP-1 elicited IFNα mRNA levels when cells infected by DEN-2 even at lower MOI, and induced IFNα mRNA reached the maximum at MOI = 5. To analyze whether Th1 promoting cytokines were kinetically induced in THP-1, cells were infected by DEN-2 at MOI = 1 at different time from 30 min, 1 h, 2 h, 6 h, and 24 h. Results showed that the IFNα mRNA expressing levels were kinetically increased in undifferentiated THP-1 cells and reached the maximum at 24 h post-infection of DEN-2 (Figure 3B). Interestingly, the IL12B mRNA expressing levels were also kinetically induced and dose-dependent in PMA-differentiated THP-1 cells and reached its maximum induction at 24 h post-infection of DEN-2 (Figure 3C, D). In order to understand whether the induction of Th1 promoting cytokines were limited to transcriptional level, we assessed the cytokine levels by ELISA in the same reactions. However, we could not detect the IFNα production in the supernatants of DEN-2 infected undifferentiated THP-1 cells. However, only IL-12 p40 proteins were kinetically and dose-dependently detected in PMA differentiated THP-1 cells Results showed that IL-12 p40 production could be induced at 1 h post-infection of DEN-2 and also reached its maximum at 24 h post-infection of DEN-2 in differentiated THP-1 cells, but not in undifferentiated THP-1 cells (Figure 3E, F).

Antibody-dependent enhancement of DEN-2 infection occurred when sub-neutralizing antibodies facilitated DEN-2 infection via Fc receptors (FcR). To investigate the role of IL-12 p40 in ADE of DEN-2 infection, we compared the viral replication and IL12B expression in the differentiated THP-1 cell using real-time RT-PCR. As shown in Figure 4A, we found that the sub-neutralizing DEN-1 antiserum could enhance DEN-2 replication in differentiated THP-1 cells from 2.64×10⁵ copies/ml to 1.49×10⁶ copies/ml in the presence of antiserum at 1:1250 dilution. However, anti-DEN2 antiserum at 1:1250 dilution did not enhance DEN-2 replication in differentiated THP-1 cells (2.64×10⁵ copies/ml vs. 3.28×10⁵ copies/ml). To measure the expression of IL-12 p40 levels in the ADE of DEN-2 infection of differentiated THP-1 cells, we found that DEN-2 infection induced a higher IL12B expression (Figure 4B). Presence of anti-DEN1 antiserum at 1:1250 dilution, but not anti-DEN2 antiserum, significantly suppressed the DEN-2 induced IL12B expression in differentiated THP-1 cells.