Type I interferon negatively controls plasmacytoid dendritic cell numbers in vivo

Abstract

Plasmacytoid dendritic cells (pDCs) specialize in the secretion of type I interferons (IFN-I) and thus are considered critical mediators of antiviral responses. We recently reported that pDCs have a very early but limited and transient capacity to curtail viral infections. Additionally, pDC numbers are not sustained in human infections caused by Hepatitis B or C viruses (HBV and HCV) and HIV. Thus, the numbers and/or function of pDCs appear to be regulated during the course of viral infection. In this study, we show that splenic pDCs are reduced in vivo during several systemic viral infections and after administration of synthetic toll-like receptor ligands. We demonstrate that IFN-I, regardless of the source, contributes to this decline and mediates pDC death via the intrinsic apoptosis pathway. These findings demonstrate a feedback control mechanism by which IFN-I modulates pDC numbers, thus fine-tuning systemic IFN-I response to viruses. IFN-I-mediated control of pDCs may explain the loss of pDCs during human infections caused by HBV, HCV, or HIV and has important therapeutic implications for settings in which IFN-I is used to treat infections and autoimmune diseases.

Figure 1.

Decline of splenic pDCs during systemic viral infections. Mice were infected with RNA or DNA viruses and absolute numbers of pDCs and serum IFN-α levels were determined at different time points p.i. (A) C57BL/6 mice were infected with 10⁷ pfu HSV-1. Data are representative of two experiments with four mice per time point. (B) C57BL/6 mice were infected with 5 × 10⁶ pfu VSV-OVA. Data are representative of two experiments with four mice per time point. (C) C57BL/6 mice were infected with 5 × 10⁴ pfu MCMV. Data are combined from two experiments with five to six mice per time point. (D) Spleen sections from uninfected (Ctrl) and MCMV-infected C57BL/6 mice were stained for Siglec-H (blue) and CD3 (brown). A magnification is shown in the inset. Bar, 100 µm. The graph shows mean pDC numbers per high power field (HPF) in Ctrl and MCMV-infected spleen sections (n = 8). (E) pDC numbers in spleens and serum IFN-α levels were determined from C57BL/6 (5 × 10⁴ pfu MCMV; 10⁴ pfu Δm157) and 129/SvJ (10⁴ pfu MCMV) mice infected or not for 48 h (n = 4–5 mice per group). (F) C57BL/6 mice were infected with 3 × 10⁶ pfu LCMV clone 13. Data are combined from two experiments with six to eight mice per time point. (G) C57BL/6 mice were infected with 2 × 10⁶ pfu VACV. Data are representative of two experiments with three to five mice per time point. (A–C and E–G) Error bars represent means ± SEM. (A, F, and G) pDC numbers, black bars; IFN-α levels, white bars. (B and C) pDC numbers, open squares; IFN-α levels, closed circles. (D) Statistical significance is indicated by **, P < 0.001. Error bars represent means ± SD.

Figure 2.

pDCs undergo cell death during systemic HSV-1 infection in an IFN-I–dependent manner. (A and B) C57BL/6 (A) and IFNAR^−/− (B) mice were infected with 10⁷ pfu HSV-1 for 8 h. Control mice were uninfected. pDC frequencies in spleens, expression of PDC-TREM, and caspase activation in pDCs were measured by flow cytometry. Dot plots and histograms show data from three to five mice per genotype representative of three experiments. (C) pDC numbers in spleens from C57BL/6 and IFNAR^−/− mice infected or not with 10⁷ pfu HSV-1 for 8 h. Data are representative of three experiments with three to five mice per group. Percentages denote the reduction in pDC numbers in infected mice relative to controls. (D) C57BL/6 mice were treated with an isotype control mAb or anti-IFNAR mAb 4 h before infection with 10⁷ pfu HSV-1. Bar graphs show absolute numbers of pDCs (left) and frequencies of caspase⁺ pDCs (right) in control and infected mice 8 h p.i. Data are representative of two experiments with three to four mice per group. Percentages indicate the reduction in pDC numbers in infected mice relative to controls. Statistical significance is indicated by ***, P < 0.0001. (E) C57BL/6 and IFNAR^−/− mice were infected or not with 10⁷ pfu HSV-1 for 8 h. Caspase⁺ and Annexin V⁺ pDCs were determined by flow cytometry. Data are representative of two experiments with three to four mice per group. Statistical significance is indicated by ***, P ≤ 0.0006. (F) MyD88^−/− mice were infected or not with 10⁷ pfu HSV-1. pDC frequencies in spleens, expression of PDC-TREM, and caspase activation in pDCs were measured by flow cytometry 8 h p.i. Dot plots and histograms show data from three to five mice per group representative of two experiments. (G and H) Mice of the indicated genotype were infected or not with 10⁷ pfu HSV-1 for 8 h. Bar graphs show frequencies of caspase⁺ pDCs in spleens (G) and serum IFN-α levels (H). Data are combined from two experiments with four to eight mice per genotype. Statistical significance is indicated by **, P < 0.007; ***, P < 0.0007. (I) C57BL/6 mice were infected or not with different doses of HSV-1 (10⁷, 10⁶ or 10⁵ pfu) for 8 h. pDC numbers in spleens, serum IFN-α levels, and Annexin V⁺ pDCs were determined. Data are from representative of two experiments with three to four mice per group. (C–E and G–I) Error bars represent means ± SEM.

Figure 3.

IFN-I–mediated death of pDCs does not depend on the IFN-I source. (A) BDCA2-DTR Tg mice were injected with PBS or DT before 10⁷ pfu HSV-1. Serum IFN-α levels were measured by ELISA 6 h p.i. Data are combined from two experiments with five mice per group. (B) BDCA2-DTR Tg mice were injected with PBS or DT before CpGA or poly(I:C). Serum IFN-α levels were determined by ELISA 8 h after CpGA or 8 and 24 h after poly(I:C). Data are representative of two experiments with four to five mice per group. (C) BDCA2-DTR Tg mice were injected with CpGA or poly(I:C) and pDC frequencies in spleens, expression of PDC-TREM, and caspase activation were measured by flow cytometry 8 h after CpGA or 16 h after poly(I:C) administration. Dot plots and histograms show data from three to four mice per group representative of two experiments. Statistical significance is indicated by **, P < 0.008. (D and E) C57BL/6 and IFNAR^−/− mice were injected with recombinant mouse IFN-I. (D) Dot plots show frequencies of pDCs in spleens 12 h after injection. (E) Absolute numbers of pDCs (left) as well as caspase⁺ and Annexin V⁺ pDCs in spleens (right) 12 h after injection. Data are representative of two experiments with three mice per group. Statistical significance is indicated by **, P < 0.006; ***, P ≤ 0.0008. (A, B, and E) Error bars represent means ± SEM.

Figure 4.

pDCs up-regulate extrinsic and intrinsic apoptotic proteins in an IFN-I–dependent manner during systemic HSV-1 infection. (A–I) Mice were infected or not with 10⁷ pfu HSV-1. (A) Expression of Fas and DR5 on pDCs from spleens was determined 8 h p.i. Histograms show data from three to five mice per group from two experiments. (B) FasL expression on NK, NKT, and T cells from spleens of control and HSV-1–infected C57BL/6 mice 8 h p.i. Histograms show data from three to five mice per group from three experiments. (C) C57BL/6 mice were treated or not with anti-FasL mAb before HSV-1 infection. Bar graphs show serum IFN-α levels (left) and frequencies of caspase⁺ pDCs (right) in spleens 8 h p.i. (D) C57BL/6 mice were treated or not with anti-TRAIL mAb before HSV-1 infection. Bar graphs show serum IFN-α levels (left) and frequencies of caspase⁺ pDCs (right) in spleens 8 h p.i. (E) Granzyme B (GrB) expression on NK, NKT, and T cells from spleens of control and HSV-1–infected C57BL/6 mice 8 h p.i. Histograms show data from three to five mice per group from two experiments. (F) Mice were depleted of NK1.1⁺ cells before HSV-1 infection. Dot plots show frequencies of pDCs and histograms show frequencies of caspase⁺ pDCs in spleens 8 h p.i. Data are representative of two experiments with four mice per group. (G and H) C57BL/6 and IFNAR^−/− mice were infected or not with HSV-1, RNA was isolated from pDCs sorted from spleens 4 h p.i., and relative levels of pro- and antiapoptotic molecules were measured in duplicate by qPCR. (G) Relative expression in naive (left) and HSV-1–infected (right) pDCs. (H) Fold change in WT and IFNAR^−/− mice. (I) Absolute numbers of pDCs in spleens were determined in C57BL/6, IFNAR^−/−, and Bim/Bid^−/− mice 8 h p.i. The bar graph shows the percent reduction in pDCs in HSV-1–infected mice compared with controls. Data are combined from two experiments with four mice per group. Statistical significance is indicated by **, P < 0.005. (C and D) Data are representative of two experiments with three to five mice per group. (G and H) Data are representative of two experiments where pDCs were sorted and pooled from three mice per genotype. (C, D, and G–I) Error bars represent means ± SEM.