Productive replication of Middle East respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response

Abstract

The Middle East respiratory syndrome coronavirus (MERS-CoV) closely resembled severe acute respiratory syndrome coronavirus (SARS-CoV) in disease manifestation as rapidly progressive acute pneumonia with multi-organ dysfunction. Using monocyte-derived-dendritic cells (Mo-DCs), we discovered fundamental discrepancies in the outcome of MERS-CoV- and SARS-CoV-infection. First, MERS-CoV productively infected Mo-DCs while SARS-CoV-infection was abortive. Second, MERS-CoV induced significantly higher levels of IFN-γ, IP-10, IL-12, and RANTES expression than SARS-CoV. Third, MERS-CoV-infection induced higher surface expression of MHC class II (HLA-DR) and the co-stimulatory molecule CD86 than SARS-CoV-infection. Overall, our data suggests that the dendritic cell can serve as an important target of viral replication and a vehicle for dissemination. MERS-CoV-infection in DCs results in the production of a rich combination of cytokines and chemokines, and modulates innate immune response differently from that of SARS-CoV-infection. Our findings may help to explain the apparent discrepancy in the pathogenicity between MERS-CoV and SARS-CoV.

Keywords: Antigen-presentation; Cytokine and chemokine response; MERS-CoV; Pathogenesis; SARS-CoV; Viral replication.

Fig. 1

Detection of MERS-CoV nucleocapsid protein (NP) in MERS-CoV-infected Mo-DCs with immunofluorescence staining. Mo-DCs were seeded on glass coverslips in 24-well plates at a density of 1×10⁵ cells per well and infected with MERS-CoV at 2 TCID₅₀/cell for 1  h. Infected Mo-DCs were fixed at 12 hpi (A) and 48 hpi (B) and processed for immunostaining. MERS-CoV NP was detected with guinea pig anti-NP antibody followed by FITC-conjugated rabbit-anti-guinea pig IgG. The slides were mounted with a mounting buffer that contained DAPI and examined with a Carl Zeiss LSM 710 microscope. Mock-infected Mo-DCs that were fixed at 48 hpi are shown in panel (C).

Fig. 2

Replication kinetics of MERS-CoV and SARS-CoV in infected Mo-DCs. Mo-DCs were infected with MERS-CoV or SARS-CoV at 2 TCID₅₀ per cell for 1 h. Supernatants and cell lysates were harvested at the indicated time points and processed for RNA extraction, reverse transcription, and quantitative PCR. The copy number of the positive-sense viral genomic RNA are illustrated for cell lysate (A) and supernatant (B). The number of infectious particles was quantified with TCID₅₀ assays (C). Data from three representative experiments are shown.

Fig. 3

Quantitations of cytokine and chemokine induction in MERS-CoV- or SARS-CoV-infected Mo-DCs. Mo-DCs were infected with MERS-CoV or SARS-CoV at 2 TCID₅₀ per cell for 1 h. At the indicated time points post infection, cells were lysed for RNA extraction, RT and qPCR to detect the mRNA expression level of antiviral and proinflammatory cytokines (A) as well as a number of representative chemokines (B). Results were normalized to GAPDH and presented as fold change in gene expression in relation to mock-infected Mo-DCs. Data represented the mean and SEM from at least five donors. Statistical analysis was performed between MERS-CoV- and SARS-CoV-infected Mo-DCs for all time points in all panels using Student׳s t test. p<0.05 was considered to be statistically significant and is indicated by asterisk marks.

Fig. 4

Surface expression of antigen-presentation and co-stimulatory molecules in Mo-DCs infected with MERS-CoV or SARS-CoV. Mo-DCs were infected with MERS-CoV or SARS-CoV at 2 TCID₅₀ per cell for 1 h. At 48 hpi, cells were detached, fixed, and labeled for specific cellular surface markers. The expression of surface markers was assessed by flow cytometry using a BD FACSCanto II flow cytometer and the data was analyzed using FlowJo. The numbers in each panel represent mean fluorescent intensity. The filled curve represents the staining of isotype controls. Results in the right panels represent mean %MFI and SEM summarized from four independent experiments. Statistical analysis was performed between all groups using Student׳s t test. p<0.05 was considered to be statistically significant and is indicated by asterisk marks.