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. 2010 May;59(5):1182-91.
doi: 10.2337/db09-1071. Epub 2010 Jan 13.

Phagocytosis of enterovirus-infected pancreatic beta-cells triggers innate immune responses in human dendritic cells

Barbara M Schulte  1 , Matthijs Kramer, Marleen Ansems, Kjerstin H W Lanke, Neeltje van Doremalen, Jon D Piganelli, Rita Bottino, Massimo Trucco, Jochem M D Galama, Gosse J Adema, Frank J M van Kuppeveld
Affiliations

Phagocytosis of enterovirus-infected pancreatic beta-cells triggers innate immune responses in human dendritic cells

Barbara M Schulte et al. Diabetes. 2010 May.

Abstract

Objective: Type 1 diabetes is a chronic endocrine disorder in which enteroviruses, such as coxsackie B viruses and echoviruses, are possible environmental factors that can trigger or accelerate disease. The development or acceleration of type 1 diabetes depends on the balance between autoreactive effector T-cells and regulatory T-cells. This balance is particularly influenced by dendritic cells (DCs). The goal of this study was to investigate the interaction between enterovirus-infected human pancreatic islets and human DCs.

Research design and methods: In vitro phagocytosis of human or porcine primary islets or Min6 mouse insuloma cells by DCs was investigated by flow cytometry and confocal analysis. Subsequent innate DC responses were monitored by quantitative PCR and Western blotting of interferon-stimulated genes (ISGs).

Results: In this study, we show that both mock- and coxsackievirus B3 (CVB3)-infected human and porcine pancreatic islets were efficiently phagocytosed by human monocyte-derived DCs. Phagocytosis of CVB3-infected, but not mock-infected, human and porcine islets resulted in induction of ISGs in DCs, including the retinoic acid-inducible gene (RIG)-I-like helicases (RLHs), RIG-I, and melanoma differentiation-associated gene 5 (Mda5). Studies with murine Min6 insuloma cells, which were also efficiently phagocytosed, revealed that increased ISG expression in DCs upon encountering CVB-infected cells resulted in an antiviral state that protected DCs from subsequent enterovirus infection. The observed innate antiviral responses depended on RNA within the phagocytosed cells, required endosomal acidification, and were type I interferon dependent.

Conclusions: Human DCs can phagocytose enterovirus-infected pancreatic cells and subsequently induce innate antiviral responses, such as induction of RLHs. These responses may have important consequences for immune homeostasis in vivo and may play a role in the etiology of type 1 diabetes.

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Figures

FIG. 1.
FIG. 1.
CVB replicates in human and porcine pancreatic islets, and DCs can phagocytose islets, resulting in induction of ISGs. A and B: Human islet cells were infected with CVB3 at an MOI of 10, and at indicated time points replication was analyzed (A) and at 48 h postinfection (p.i.) images were taken (B). C: Human islets were infected with CVB3 at an MOI of 10. After 24-h incubation, islets were adhered onto fibronectin-coated coverslips and stained using 3A (red)- and VP1 (green)-specific antibodies. Hoechst stain is included to visualize cell nuclei. D: Human islets were PKH labeled and infected with CVB3 at an MOI of 10 and cultured for 48 h before addition to CFSE-labeled DCs. Uptake of islets either mock-infected (M) or CVB-infected (CVB) was analyzed by flow cytometry 24 h after co-culture. E: Human islets were PKH labeled and infected as in D and co-cultured with unlabeled DCs for 24 h. Subsequently, DCs were harvested, stained using CD86-specific antibodies (green), and analyzed using confocal microscopy. F: Human islets were infected with CVB3 at an MOI of 10 for 48 h prior to addition to DCs. Expression of RIG-I, Mda5, and PKR mRNA in DCs was analyzed using qPCR. G: Porcine islets were prepared and co-cultured as in F and ISG-induction was analyzed at indicated times. In some experiments (F and G), freeze-thawed preparations of islets were used, but these yielded similar results compared with using viable cells. Experiments are representative of 2, 3, or more than 3 independent experiments. Islet/CVB, CVB-infected human islets; Islet/M, mock-infected human islets; Med, medium, i.e., unstimulated DCs; porc Islet/CVB, CVB-infected porcine islets; porc Islet/M, mock-infected porcine islets. (A high-quality color representation of this figure is available in the online issue.)
FIG. 2.
FIG. 2.
Min6 cells are a good model for primary pancreatic cells, are phagocytosed by DCs, and induce innate antiviral immune responses. A and B: Min6 cells were infected with CVB3 at an MOI of 10, and replication was analyzed at indicated time points (A) and images were taken at 48 h postinfection (p.i.) (B). C: Min6 cells were PKH-labeled and infected with CVB3 at an MOI of 10. After 24-h incubation, cells were harvested and added to CFSE-labeled DCs at a 1:1 ratio. After 24-h co-culture, cells were harvested and uptake was determined using flow cytometry. D: Min6 cells were prepared as in C and co-cultured with unlabeled DCs for 24 h, after which DCs were harvested, stained as in Fig. 1E, and analyzed using confocal microscopy. E and F: Min6 cells were prepared as in C, and after 48-h incubation, cells were harvested and added to DCs at a 1:1 ratio or DCs were stimulated with 20 μg/ml poly (I:C) or left untreated. At 5 h after addition (E), or 5 h and 8 h after addition (F), mRNA induction of ISGs was determined as described. G: Protein expression of RIG-I, Mda5, and PKR was analyzed by Western blot 24 h after stimulation of DCs as described for panel E. H: DCs were stimulated as in E, and after 24-h co-culture cells were harvested and infected with EV9 at an MOI of 1. At indicated times postinfection, EV9 replication was analyzed. Poly (I:C) was used as a positive control (34). In some experiments, freeze-thawed cell populations were used but these yielded similar results compared with using viable cells (supplemental Fig. 1). Data shown are representative (A–D, G, and H) or averages (E and F) of at least three independent experiments. Med, medium, i.e., unstimulated cells; M6/CVB, CVB-infected Min6 cells; M6/M, mock-infected Min6 cells; n.s., not significant. *P < 0.05. (A high-quality color representation of this figure is available in the online issue.)
FIG. 3.
FIG. 3.
Type I IFNs produced by DCs themselves are required for ISG induction. A: DCs were stimulated with cleared supernatants from stimulated DC and DC/Min6 co-cultures (harvested 24 h after co-culture started and used at a 1:2 dilution) and mRNA induction of RIG-I, Mda5, and PKR were determined using qPCR 8 h after stimulation. B: Min6 cells were infected with CVB3 at an MOI of 10 and incubation cells were harvested and added to DCs at a 1:1 ratio after 48 h. Stimulations were performed in the absence or presence of neutralizing antibodies (Iivari, Kaaleppi, and bovine anti–IFN-α/β; see research design and methods). After 8 h, mRNA expression levels of RIG-I, Mda5, and PKR were determined using qPCR. C: DCs were treated as in B and protein expression of RIG-I, Mda5, and PKR was analyzed by Western blotting after 24 h. D: DCs were stimulated with 100 units/ml mIFNα or cleared supernatants from Min6 cells (harvested 48 h postinfection and used at a 1:2 dilution), and ISG mRNA induction was determined after 8 h. Data shown are representative of two (C) or average of three (A, B, and D) independent experiments. IC, poly (I:C); Med, medium, i.e., unstimulated cells; M6/CVB, CVB-infected Min6 cells; M6/M, mock-infected Min6 cells; mIFNa, murine recombinant IFN-α; n.s., not significant; Sup, supernatant; w/o Ab or w Ab, without or with neutralizing anti–IFN-α/β antibodies, respectively. *P < 0.05.
FIG. 4.
FIG. 4.
Induction of innate immune responses in DCs after Min6/CVB uptake requires (viral) RNA within Min6 cells. A: DCs were pretreated with CytD for 30 min prior to stimulation with TLR ligands or Min6 cells. After 8 h, mRNA expression levels of RIG-I, Mda5, and PKR were determined using qPCR. B: DCs were pretreated with CQ for 30 min, stimulated as in A, and mRNA expression was determined as in A. C: Min6 cell preparations were exposed to a mixture of RNase A, RNase VI, and RNase I prior to addition to DC cultures as described. Expression of RIG-I, Mda5, and PKR in DCs was analyzed using qPCR 5 h after addition of Min6-cell preparations. D: DCs were co-cultured with Min6-cell preparations as described for panel A, and protein expression of RIG-I, Mda5, and PKR was analyzed by Western blotting 24 h after the start of co-culture. Data are representative of two (D) or the average of two (A–C) independent experiments. In these experiments, freeze-thawed cell populations were used. IC, poly (I:C); Med, medium unstimulated cells; M6/CVB, CVB-infected Min6 cells; M6/M, mock-infected Min6 cells; n.s., not significant; w/o RNase or w RNase, without or with RNase treatment of Min6 cell preparations prior to co-culture. *P < 0.05.
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