Excessive proinflammatory cytokine and chemokine responses of human monocyte-derived macrophages to enterovirus 71 infection

Abstract

Background: The levels of proinflammatory cytokine or chemokine in blood and cerebrospinal fluid are thought to be one of predictors for clinical severity of enterovirus 71 (EV71) infection, yet the cellular sources or signalling mechanism remain undefined. Here, we focused on the response of human primary monocyte-derived macrophages (MDMs) to EV71 virus and its possible mechanisms.

Methods: Human primary MDMs were infected by EV71 virus in vitro. Infectivity and viral replication were assayed, and cytokine responses were determined by Cytometric Bead Array(CBA) analysis. The relative changes of Toll-like receptors, retinoic acid-inducible gene I (RIG-I) and melamoma differentiation associated gene 5 (MDA5) mRNA expression were detected by real-time RT-PCR.

Results: Effective infection and viral replication were detected in EV71-infected MDMs. The titters of progeny virus released from EV71-infected MDMs gradually increased from 6-h to 48-h point of infection (POI.). Proinflammatory cytokines: IL-1, IL-6, TNF-α but not IFN-α and γ were induced in MDMs by EV71. EV71 infection significantly increased the release of IL-8, IP-10 and RANTES at 12-h or 24-h POI. Upregulation of TLR2, TLR7 and TLR8 mRNA expression rather than TLR3, TLR4, TLR6, TLR9, TLR10, RIG-I, MDA5 were found at different time points in EV71-infected MDMs.

Conclusions: Our findings suggested that macrophages are not only the important target cells but also the effectors during EV71 infection, and they may play an important role in the pathogenesis of EV71 infection. And the proinflammatory cytokine and chemokine responses in EV71-infected MDMs may be mediated by the activation of differential pattern of TLRs.

Figure 1

A dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.

Figure 2

Viral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 10⁴copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.

Figure 3

Proinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).

Figure 4

Chemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).

Figure 5

A higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).