Plasmacytoid dendritic cells sequester high prion titres at early stages of prion infection

Abstract

In most transmissible spongiform encephalopathies prions accumulate in the lymphoreticular system (LRS) long before they are detectable in the central nervous system. While a considerable body of evidence showed that B lymphocytes and follicular dendritic cells play a major role in prion colonization of lymphoid organs, the contribution of various other cell types, including antigen-presenting cells, to the accumulation and the spread of prions in the LRS are not well understood. A comprehensive study to compare prion titers of candidate cell types has not been performed to date, mainly due to limitations in the scope of animal bioassays where prohibitively large numbers of mice would be required to obtain sufficiently accurate data. By taking advantage of quantitative in vitro prion determination and magnetic-activated cell sorting, we studied the kinetics of prion accumulation in various splenic cell types at early stages of prion infection. Robust estimates for infectious titers were obtained by statistical modelling using a generalized linear model. Whilst prions were detectable in B and T lymphocytes and in antigen-presenting cells like dendritic cells and macrophages, highest infectious titers were determined in two cell types that have previously not been associated with prion pathogenesis, plasmacytoid dendritic (pDC) and natural killer (NK) cells. At 30 days after infection, NK cells were more than twice, and pDCs about seven-fold, as infectious as lymphocytes respectively. This result was unexpected since, in accordance to previous reports prion protein, an obligate requirement for prion replication, was undetectable in pDCs. This underscores the importance of prion sequestration and dissemination by antigen-presenting cells which are among the first cells of the immune system to encounter pathogens. We furthermore report the first evidence for a release of prions from lymphocytes and DCs of scrapie-infected mice ex vivo, a process that is associated with a release of exosome-like membrane vesicles.

Figure 1. Isolation of splenic cell types by magnetic-activated cell sorting.

A: Schematic representation for the isolation of specific splenic cell types from mice. Splenocytes were released by repeated collagenase digestion from freshly dissected spleens, followed by removal of erythrocytes and purification of splenocytes on Lympholyte M gradients. Splenic cell types are isolated by positive selection with magnetic beads coated with cell type-specific mAbs as specified. B: The purities of MACS-isolated cells were analysed by FACS using cell-type specific mAbs and isotype controls as specified in Materials and Methods. One representative out of three experiments is shown. (Bv) CD11^low B220⁺ pDCs, isolated with murine plasmacytoid dendritic antigen-1 (mPDCA-1) showed a purity of about 90% in three independent experiments. (Bvi) The macrophage population, isolated with CD11b microbeads after depletion of CD11c⁺ cells was contaminated with CD11c⁺ CD11b⁺ mDCs. Macrophages were therefore isolated by FACS instead (C). C: Splenocytes labelled with mAbs against anti-CD11b (M1/70) and anti-CD11c (HL3) were isolated by FACS using a DAKO cell sorter.

Figure 2. Regression analysis of SCEPA endpoint titration assay using GLM.

For the estimation of prion titers by SCEPA, prion-susceptible cells were infected with serially diluted RML I6200 brain homogenate and the proportions of scrapie-positive wells were analysed using a GLM with binomial family and complementary log-log link. A: Observed and estimated proportions of scrapie-positive wells with 95% confidence intervals. B: Linearized link-transformed proportions of scrapie-positive wells. Here the four zero values at dilution 10⁻⁸ were replaced by 0.5 in order to plot the observed loglog values. Data represent eight technical assay repeats of serially diluted RML I6200 brain homogenate.

Figure 3. High splenic prion titers at early stages after prion infection.

Groups of four 129/Sv×C57BL/6 and Prnp ^0/0 mice were inoculated i.p. with 100 µl 1% (w/v) RML I6200 (9.3 log LD₅₀ units/g brain). At various time points after inoculation spleens and mesenteric lymph nodes were dissected and prion titers and PrP^Sc levels determined. (A) Infectious titers of spleens (closed circles) and mesenteric lymph nodes (open circles) of 129/Sv×C57BL/6 mice. To account for residual inoculum infectious titers of spleens of RML-inoculated Prnp^0/0 mice (closed square) were determined. Prion titers were estimated by a GLM with binomial family complementary log-log link. Data represent mean infectious titers ± SD of four repeats. (B) Spleen homogenates were analyzed for levels of PK-resistant PrP (PrP^Sc) by Western blotting after NaPTA precipitation as described in Materials and Methods. (C) Detection of PrP^Sc-positive deposits in spleens of prion-infected 129/Sv×C57BL/6 mice. At 3 dpi less than 5% of the total number of follicles was weakly PrP^Sc-positive. An increase in the number of immunopositive follicles, but overall a weak immunostaining for abnormal PrP was observed at 7 dpi. At 14 dpi, the number of positive follicles was similar, but the staining intensity increased to ‘moderate’ in two animals. At 30 dpi 90% of lymphoid follicles showed moderate or strong labeling. The scale bar corresponds to 100 µm (20 µm in the inserts).

Figure 4. Exosomes are released from scrapie-infected B cells ex vivo.

Spleens were dissected from 129/Sv×C57BL/6 mice 30 days after i.p. inoculation with 1% (w/v) RML I6200. MACS-isolated B lymphocytes were cultured under passive leakage (A) and basal (B) conditions essentially as described in Table 4 and tissue culture supernatants were isolated by sequential centrifugation (Materials and Methods). After centrifugation at 100,000× g for 2 h pellets were resuspended in PBS, absorbed onto carbon-coated grids and negatively stained with 1% uranyl acetate. Cup-shaped exosome-like membrane particles of different sizes (see arrows) are shown in Figure 1B. Twenty randomly recorded images (surface area: 2.82 µm²) from each condition were counted and the number of exosome-like particles (1.7±1.2 (A) and 22.8±6.5 (B) per surface area, p≪0.001) determined in a blinded manner. Scale bar: 0.2 µm.