High secretion of interferons by human plasmacytoid dendritic cells upon recognition of Middle East respiratory syndrome coronavirus

Abstract

The Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012 as the causative agent of a severe respiratory disease with a fatality rate of approximately 30%. The high virulence and mortality rate prompted us to analyze aspects of MERS-CoV pathogenesis, especially its interaction with innate immune cells such as antigen-presenting cells (APCs). Particularly, we analyzed secretion of type I and type III interferons (IFNs) by APCs, i.e., B cells, macrophages, monocyte-derived/myeloid dendritic cells (MDDCs/mDCs), and by plasmacytoid dendritic cells (pDCs) of human and murine origin after inoculation with MERS-CoV. Production of large amounts of type I and III IFNs was induced exclusively in human pDCs, which were significantly higher than IFN induction by severe acute respiratory syndrome (SARS)-CoV. Of note, IFNs were secreted in the absence of productive replication. However, receptor binding, endosomal uptake, and probably signaling via Toll-like receptor 7 (TLR7) were critical for sensing of MERS-CoV by pDCs. Furthermore, active transcription of MERS-CoV N RNA and subsequent N protein expression were evident in infected pDCs, indicating abortive infection. Taken together, our results point toward dipeptidyl peptidase 4 (DPP4)-dependent endosomal uptake and subsequent infection of human pDCs by MERS-CoV. However, the replication cycle is stopped after early gene expression. In parallel, human pDCs are potent IFN-producing cells upon MERS-CoV infection. Knowledge of such IFN responses supports our understanding of MERS-CoV pathogenesis and is critical for the choice of treatment options.

Importance: MERS-CoV causes a severe respiratory disease with high fatality rates in human patients. Recently, confirmed human cases have increased dramatically in both number and geographic distribution. Understanding the pathogenesis of this highly pathogenic CoV is crucial for developing successful treatment strategies. This study elucidates the interaction of MERS-CoV with APCs and pDCs, particularly the induction of type I and III IFN secretion. Human pDCs are the immune cell population sensing MERS-CoV but secrete significantly larger amounts of IFNs, especially IFN-α, than in response to SARS-CoV. A model for molecular virus-host interactions is presented outlining IFN induction in pDCs. The massive IFN secretion upon contact suggests a critical role of this mechanism for the high degree of immune activation observed during MERS-CoV infection.

FIG 1

Inoculation of murine cells with MERS-CoV. (A) Type I IFN secretion by murine immune cells. Cells were inoculated with MERS-CoV (MOIs as indicated), SARS-CoV, or the indicated positive controls. Single dots, individual experiments; horizontal lines, means. IFNs were measured at 24 hpi. b.d., below detection. Limits of detection were 12.5 pg/ml for IFN-α and 15.6 pg/ml for IFN-β. (B) Growth kinetics of MERS-CoV on Vero cells or murine APCs (MOI of 0.01). Indicated cell types were inoculated with virus, and sampled supernatants were titrated. Filled symbols, MERS-CoV; open symbols, SARS-CoV; ◇, PECs; ○, mDCs; □, pDCs. Values are means of three independent experiments; error bars indicate standard deviations. conc, concentration.

FIG 2

Inoculation of human immune cells with MERS-CoV. (A) Type I and III IFN secretion by human immune cells. Human cells were inoculated with MERS-CoV (MOIs indicated), SARS-CoV (MOI of 1), or the indicated positive control (CpG 2006, VSV-M2, or CpG 2216). Supernatants were sampled at 24 hpi, and secreted IFNs were determined by specific ELISAs. (B) Isolated RNA was used for qRT-PCR. MERS RNA signals were normalized to cellular GAPDH mRNA [ΔC_T = C_{T(MERS RNA)} − C_{T(GAPDH mRNA)}]. ΔC_T values and respectively calculated x-fold amounts of RNA normalized to an MOI of 0.1 were determined. (C) Titers of MERS-CoV or SARS-CoV (control) in human immune cells (APCs) infected at an MOI of 0.01 or on pDCs infected at an MOI of 5. (D) IL-6 secretion by human B cells upon inoculation with stimulating CpG 2006. Supernatants were sampled at 24 h after inoculation with CpG 2006, and secreted IFNs were determined by ELISA. Individual donors are displayed as single dots, and horizontal lines indicate means. Limits of detection were 7 pg/ml for IFN-α, 50 pg/ml for IFN-β, and 8 pg/ml for IFN-λ. b.d., below detection; filled symbols, MERS-CoV; open symbols, SARS-CoV; ◇, B cells; △, M1 macrophages; ▽, M2 macrophages; ○, MDDCs; □, pDCs. Growth in human APCs (MOI of 0.01) (i, left), or in human pDCs (MOI of 5) (ii, right) was determined. Values are means of three independent experiments; error bars indicated standard deviation. *, P < 0.05.

FIG 3

Dissecting type I and III IFN induction in human pDCs. Impact of different parameters on IFN induction in pDCs after inoculation of MERS-CoV (MOI of 1). (A to C) Live virus. pDCs were incubated with UV-inactivated virus (UV) or live virus (MERS-CoV). (D to F) Entry receptor. Infection was performed in the presence or absence of the MERS-CoV S receptor binding domain (RBD) or IgG1-Fc control protein (Ctrl). (G to I) Endosomal maturation. Infection was performed in the presence of chloroquine. (J to L) TLR recognition. Infection was performed in the presence of TLR7 inhibitor (IRS661). IFNs were sampled at 24 hpi. Mock and MERS-CoV data for live virus experiments are the same as displayed in Fig. 2. Mock, uninfected cells; sham, infected but untreated cells. Single dots, individual donors; horizontal lines, means. *, P < 0.05; **, P < 0.01; ns, not significant.

FIG 4

CD26 expression and functionality of human pDCs. (A) Expression of MERS-CoV receptor DPP4 on human pDCs. pDCs were stained with anti-DPP4 antibody and analyzed via flow cytometry. ITC, isotype control antibody. (B) Viability of pDCs of three different donors (D1 to D3) treated with inhibitors. pDCs were treated as indicated. At 24 hpi, cells were stained for viability and analyzed via flow cytometry. Dead, cells killed by osmotic shock. (C) Block of CpG 2216-induced IFN secretion by MERS-CoV RBD. Secretion of the indicated IFNs was determined in the presence or absence of the MERS-CoV S receptor binding domain (RBD) or IgG1-Fc control protein (Ctrl) upon MERS-CoV infection or the DPP4-independent stimulus CpG 2216. IFNs were sampled at 24 hpi. Single dots, individual donors; horizontal lines, means. b.d., below detection.

FIG 5

Infection of human pDCs by MERS-CoV. (A) Quantification of viral N RNA in human or murine pDCs by qRT-PCR in the presence or absence of chloroquine, normalized to cellular GAPDH mRNA [ΔC_T = C_{T(MERS vRNA)} − C_{T(GAPDH mRNA)}] at indicated time points after inoculation. (B to D) Immunoblot analysis of N protein expression in murine pDCs (B) or human pDCs (C and D) after inoculation with MERS-CoV (MOI of 3). pDCs of three different donors (D1 to D3 and D4 to D6) were infected in the presence of blocking anti-DPP4 serum (DPP4), the receptor binding domain of MERS-CoV S protein (RBD), or respective controls (Ctrl) or with UV-inactivated (UV) or live MERS-CoV in the presence (Chl) or absence (sham) of chloroquine. Cells were lysed at the indicated time points and subjected to analyses.

FIG 6

Model for MERS-CoV-induced type I IFN secretion in human pDCs. The figure schematically depicts the life cycle of MERS-CoV in human pDCs and events triggering secretion or infection of type I IFNs. Successful inhibition of IFN secretion at single steps is indicated. Inhibitors or proteins which have been analyzed are depicted in bold. Question marks point out steps during assembly or release of viral particles, the block of which could be responsible for absence of significant viral replication in pDCs. α, anti.