Differential susceptibility and response of primary human myeloid BDCA1(+) dendritic cells to infection with different Enteroviruses

Abstract

Coxsackie B viruses (CVBs) and echoviruses (EVs) form the Human Enterovirus-B (HEV-B) species within the family Picornaviridae. HEV-B infections are widespread and generally cause mild disease; however, severe infections occur and HEV-B are associated with various chronic diseases such as cardiomyopathy and type 1 diabetes. Dendritic cells (DCs) are the professional antigen-presenting cells of our immune system and initiate and control immune responses to invading pathogens, yet also maintain tolerance to self-antigens. We previously reported that EVs, but not CVBs, can productively infect in vitro generated monocyte-derived DCs. The interactions between HEV-B and human myeloid DCs (mDCs) freshly isolated from blood, however, remain unknown. Here, we studied the susceptibility and responses of BDCA1(+) mDC to HEV-B species and found that these mDC are susceptible to EV, but not CVB infection. Productive EV7 infection resulted in massive, rapid cell death without DC activation. Contrary, EV1 infection, which resulted in lower virus input at the same MOI, resulted in DC activation as observed by production of type I interferon-stimulated genes (ISGs), upregulation of co-stimulatory and co-inhibitory molecules (CD80, CD86, PDL1) and production of IL-6 and TNF-α, with a relative moderate decrease in cell viability. EV1-induced ISG expression depended on virus replication. CVB infection did not affect DC viability and resulted in poor induction of ISGs and CD80 induction in part of the donors. These data show for the first time the interaction between HEV-B species and BDCA1(+) mDCs isolated freshly from blood. Our data indicate that different HEV-B species can influence DC homeostasis in various ways, possibly contributing to HEV-B associated pathology.

Figure 1. Replication of human enteroviruses in primary human myeloid BDCA1⁺ dendritic cells.

A) Freshly isolated BDCA1⁺ mDCs were infected with indicated viruses at an MOI of 2 and replication was assessed at indicated time points by endpoint titration. B) Freshly isolated mDCs were stained with indicated antibodies or corresponding isotypes and analyzed using flowcytometry. C) mDCs were infected as indicated (MOI 50) and after 18 h infection 3D polymerase protein expression was assessed by western blot analysis. D) mDCs infected as indicated were harvested 18 h after infection using cold PBS and amount of dsRNA was analyzed using flowcytometry. E) mDCs infected as in D) were tested for cell viability using Annexin V/7-AAD double staining. Statistical significance was determined using ANOVA and post-hoc Tukey test. Shown are representative experiments of more than 3 independent experiments using different donors (A–D) or average of 3 independent experiment (E) (mean values+ SEM). * p<0.05; ** p<0.001.

Figure 2. ISG induction in HEV-B infected primary human myeloid BDCA1⁺ dendritic cells is dependent on virus replication.

A) Freshly isolated BDCA1⁺ mDCs were infected (EV1, MOI 50), stimulated with poly I:C (20 µg/ml) or left untreated and at indicated times RNA was isolated and qPCR was performed as described. Statistical significance versus mock-infected cells determined by Students T-test, *P<0.05. B) mDCs were infected with indicated viruses (MOI 50) or stimulated as for A) and after 18 h protein expression was assessed by western blot analysis. The intensity given below the image (Mda5 Int.) represents Mda5 intensity relative to actin calculated as described in materials & methods section. C) Freshly isolated BDCA1⁺ mDCs were infected (CVB3, MOI 50), stimulated with poly I:C (20 µg/ml) or left untreated and at 6 h p.i. RNA was isolated and qPCR was performed as described. D) mDCs were infected with EV1 (MOI 50), washed in PBS and plated out. Directly after infection rupintrivir (50 µM) (indicated as w Rupin) was added and replication was analyzed as for Fig. 1A) E) cells were infected as in D), stimulated with polyI:C (20 µg/ml) or left unstimulated and after 20 h protein expression was analyzed by western blot. The intensity given below the image represents Mda5 or 3D intensity relative to actin calculated as described in materials & methods section.+rupin indicates that rupintrivir was added to the DC cultures directly after plating the cells following infection. Shown are representative experiments of 3 (B), individual donor results+average from 3 (A) or 7 (C), or representative of 2 (D, E) independent experiments using different donors.

Figure 3. EV1 infection results in phenotypic maturation and production of IL6 and TNF-α.

A) Freshly isolated BDCA1⁺ mDCs were infected as indicated (MOI 50) and after 18 h expression of cell surface markers was determined using flowcytometry. B) Supernatant taken from mDCs infected as in A) was analyzed for production of IL6 and TNF-α. Data shown (mean+SEM) are averages of at least 8 different experiments using different donors. Statistical significance determined by Students T-test, * p<0.05; **p<0.01. MFI; mean fluorescence intensity.

Figure 4. EV, but not CVB infection impairs TLR-induced responses in BDCA1+ mDCs.

A) Freshly isolated BDCA1⁺ mDCs were infected as indicated (MOI 50) and after 16 h all cells were stimulated with poly I:C (20 µg/ml). After an additional 24 h expression of cell surface markers was determined using flowcytometry. Data is shown as mean fluorescent intensity minus isotype control. B) Supernatant taken from mDCs infected and stimulated as in A) was analyzed for production of IL6 and TNF-α. Shown are averages of 2 experiments using different donors. Statistical significance determined by Students T-test, * p<0.05. **p<0.01.

Figure 5. BDCA1⁺ mDC respond faster and more pronounced than moDC to EV1 infection.

A) Freshly isolated BDCA1⁺ mDCs or day 6 immature moDC generated from monocytes from the same donor were infected as indicated (MOI 5) and replication was analyzed at indicated time points. B) Freshly isolated BDCA1⁺ mDCs or moDCs from the same donor were infected as indicated (MOI 50) and after 18 h cell surface marker expression was determined using flowcytometry. C) As for B) but analyzed at 40 h p.i. Shown are representative (A) or averages of 2 experiments using different donors. Expression of surface markers in mock-infected DCs is set to 100 in all subsets for comparison. Statistical significance determined by Students T-test, *P<0.05.