Gene expression profiling of dengue infected human primary cells identifies secreted mediators in vivo

Abstract

We used gene expression profiling of human primary cells infected in vitro with dengue virus (DENV) as a tool to identify secreted mediators induced in response to the infection. Affymetrix GeneChip analysis of human primary monocytes, B cells and dendritic cells infected with DENV in vitro showed strong induction of monocyte chemotactic protein 2 (MCP-2/CCL8), interferon gamma-induced protein 10 (IP-10/CXCL10) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/TNFSF10). The expression of these genes was confirmed in dendritic cells infected with DENV in vitro at mRNA and protein levels. A prospectively enrolled cohort of DENV-infected Venezuelan patients was used to measure the levels of these proteins in serum during three different periods of the disease. Results showed significant increase of MCP-2, IP-10, and TRAIL levels in patients infected with DENV during the febrile period, when compared to healthy donors and patients with other febrile illnesses. MCP-2 and IP-10 levels were still elevated during the post-febrile period while TRAIL levels dropped close to normal after defervescense. Patients with primary infections had higher TRAIL levels than patients with secondary infections during the febrile period of the disease. Increased levels of IP-10, TRAIL and MCP-2 in acute DENV infections suggest a role for these mediators in the immune response to the infection. MCP-2 was identified in this work as a new unreported and important dengue-related protein and IP-10 was confirmed as a novel and strong pro-inflammatory marker in acute disease.

Figure 1

MCP-2 and IP-10 protein levels in DC culture supernatants. Levels of MCP-2 and IP-10 in supernatants from uninfected (white bars) and DENV-infected (black bars) DC (N=3), at 12, 24 and 48 hours post-infection. Mean values ± standard error of mean are shown. Mann–Whitney statistical analysis between uninfected and DENV-infected: MCP-2 (48 hours, p=0.001); IP-10 (48 hours, p=0.022).

Figure 2

Serum levels of MCP-2 (A), IP-10 (B), MIP-1β (C) and TRAIL (D) in DENV-infected patients (solid black) and OFI (solid white); in patients classified as primary (horizontal lines) and secondary infections (solid light gray); and in patients classified according to the absence or presence of hemorrhagic manifestations, as not bleeding (solid dark gray) and bleeding (downward diagonal lines). Results are expressed as a mean value ± standard error of mean, for each patient group and each disease day. Mann–Whitney statistical analysis between OFI and dengue at each disease day: MCP-2 (febrile p=0.015; post-febrile p=0.001); IP-10 (febrile p<0.001; post-febrile p<0.001); MIP-1β (febrile p=0.024); TRAIL (febrile p=0.009); Mann–Whitney statistical analysis between primary and secondary infections: MIP-1β (post-febrile p=0.016); TRAIL (febrile p=0.050); Mann–Whitney statistical analysis between absence and presence of hemorrhagic manifestations: IP-10 (post-febrile p=0.037). Conv.: convalescence.

Figure 3

Effect of rTRAIL pre-treatment of DENV-infected DC. Untreated or r-TRAIL pre-treated DC were infected with DENV for 48 hours (see Methods). The percentage of infected DC was assessed by flow cytometry (A); levels of IFN-α (B), MCP-2 (C) and IP-10 (D) were measured by ELISA in cell culture supernatants. Results are expressed as a mean value ± standard error of mean (N=3-7). Mann–Whitney statistical analysis between untreated and r-TRAIL pre-treated: IFN-α (p=0.025); MCP-2 (p=0.050); and IP-10 (p=0.021).