Processing of the Bovine Spongiform Encephalopathy-Specific Prion Protein by Dendritic Cells

Animals.Six- to 8-week-old C57BL/6J (Janvier, Le Genest-Saint-Isle, France), PrP⁰ (CDTA, France), B6129SF1/J (the F1 cross between C57BL/6J and 129S, stock number 101043; Jackson ImmunoResearch Laboratories), and recombination-activated gene 2⁰/common cytokine γ chain-deficient (RAG γc⁰) immunodeficient mice were used for this study (7, 11, 12). RAG γc⁰ mice originated on a 129SF1 background were backcrossed on the C57BL/6 background. All animals were housed in level 3 care facilities of the Pasteur Institute, officially registered for prion experimental studies with rodents (Ministry agreement number A 75-15-27 for animal care facilities and agreement number 75-585 for animal experimentation). RAG γc⁰ immunodeficient mice were kept in isolators under specific-pathogen-free conditions.

Infectious material.The mouse-adapted BSE strain 6PB1 was kindly provided by D. Dormont (CEA, Fontenay-aux-Roses, France). It was stabilized and propagated in C57BL/6 mice (24). The inoculum was a brain homogenate at 10% (wt/vol) in 5% glucose solution from BSE-infected mice at the terminal stage of disease, routinely titrating 5 × 10⁸ to 5 × 10⁹ mean 50% lethal doses (LD₅₀) per gram in intracerebral-infection challenges. The mean (±standard deviation) survival time was around 181 ± 5.4 days after intracerebral infection or 315 ± 27.3 days after intraperitoneal challenge (23). Healthy mouse brain homogenate was used as a negative control.

SAF isolation.Scrapie-associated fibrils (SAF) were purified from infected brains according to the method developed by Vilette et al. (38), which was slightly modified (38). Briefly, 1 volume of 20% NaCl (Sigma, St. Louis, MO) was added to 1 volume of 20% brain homogenate (BSE or control). After homogenization and addition of 1 volume of a mix containing 20% Sarkosyl (Interchim, Montluçon, France), 2% SB3-14 (Calbiochem, VWR, Fontenay-sous-Bois, France), and 2 mM Tris-HCl (pH 7.4), samples were incubated for 15 min at room temperature. They were then centrifuged at 26,500 × g for 2 h at 20°C on a 20% sucrose cushion. Pelleted material was resuspended in Tris-borate-sodium buffer and heated at 80°C for 10 min before use for cell infections.

Preparation of spleen DC.DC were purified by magnetic cell sorting as previously described (30). Briefly, spleens were removed from mice and treated for 45 min at 37°C with 400 U/ml of collagenase type IV and 50 μg/ml of DNase I (Roche, Mannheim, Germany) in RPMI 1640. After inhibition of collagenase activity with 6 mM EDTA in phosphate-buffered saline (PBS), spleens were dissociated in Ca2⁺- and Mg2⁺-free PBS in the presence of 2.5 mM EDTA, 0.5% fetal calf serum (Invitrogen, Cergy-Pontoise, France), and 1% antibiotics (penicillin, 100 U/ml; streptomycin, 100 μg/ml). Single spleen cell suspensions were prepared and incubated with colloidal superparamagnetic microbeads conjugated to anti-CD11c monoclonal antibody (MAb) (MACS-anti-CD11c, N418 clone; Miltenyi Biotec, Bergisch-Gladbach, Germany) by following the manufacturer's instructions. CD11c⁺ cells were positively selected by two serial passages with miniMACS magnetic cell sorting (Miltenyi Biotec). The purity of DC preparations was superior to 70% after the first column and superior to 93% after the second one.

Preparation of T and B spleen cells.T and B cells were purified from mouse spleen by magnetic cell sorting with pan-T and -B cell isolation kits, respectively, following the manufacturer's instructions (Miltenyi Biotec). The purity of both T and B populations was superior to 97%.

Culture medium.Complete medium consisted of RPMI 1640 containing l-alanyl-l-glutamine dipeptide (Invitrogen, Cergy-Pontoise, France) supplemented with 10% fetal calf serum (ICN Pharmaceuticals France S.A., Orsay, France), 50 μM 2-mercaptoethanol, and antibiotics (penicillin, 100 U/ml; streptomycin, 100 μg/ml).

Generation of BMM, BMDC, and BMpDC.BMM, BMDC, and BMpDC were generated from bone marrow precursors, as previously described (17). Briefly, bone marrow cells from C57BL/6 or PrP-null mice were harvested and plated at 2 × 10⁵ cells/ml in 60-mm cell culture dishes (BD Falcon, Bedford, Mass.) in complete medium with 5% of a supernatant containing granulocyte-macrophage colony-stimulating factor (GM-CSF), 5% of a supernatant containing fms-like tyrosine kinase 3 ligand (Flt3-L), or 10% of a supernatant with macrophage colony-stimulating factor for the generation of BMDC, BMpDC, or BMM, respectively. J558 GM-CSF (a kind gift from T. Rolink, Basel, Switzerland), Sp210 Flt3-L (a kind gift from R. Rottapel, Toronto, Canada), and L929 (ATCC, Teddington, Middlesex, United Kingdom) cell lines were used to produce GM-CSF, Flt3-L, and macrophage colony-stimulating factor, respectively. BMpDC were purified prior to infection by magnetic cell sorting using a murine plasmacytoid dendritic cell antigen 1 isolation kit following the manufacturer's instructions (Miltenyi Biotec). The purity of BMpDC preparations was superior to 86%.

Prion infection of cultured cells.T and B splenic cells were infected just after purification by magnetic cell sorting. Infections of BMDC and BMM were performed on day 6. Infection of BMpDC was performed on day 6, after purification by magnetic cell sorting. For in vitro activation, BMM or BMDC were incubated at day 5 for 24 h with 10 μg/ml of lipopolysaccharide (LPS) (Invivogen, San Diego, Calif.) prior to infection. The murine SAF from 1 mg of brain homogenate of uninfected or BSE terminal-stage mice or 1 mg of direct brain homogenate was incubated with 4 × 10⁶ cells in 60-mm cell culture dishes (BD Falcon, Bedford, Mass.). Inocula were previously heated at 80°C for 10 min to avoid bacterial contamination. The nonadherent and semiadherent cells were recovered by flushing the plates with PBS containing 5 mM EDTA and washed in PBS before fluorescence-activated cell sorter (FACS) and Western blot analysis or before injection into wild-type or RAG γc⁰ mice.

Western blot analysis.Briefly, tissue homogenates (5% [wt/vol] in 5% glucose) or cell lysates (8 × 10⁵ cells in 0.5% sodium deoxycholate [Sigma] and 0.5% NP-40 [Coger, France]) were treated or not for 1 h at 37°C with 10 or 40 μg/ml of proteinase K (PK) (Sigma, France), respectively. Laemmli buffer (Bio-Rad, Ivry sur Seine, France) containing 3 M (final concentration) urea was added, and 20 μl of each extract, corresponding to 100 μg of brain or to 1.6 × 10⁵ cultured cells, was used for PrP^bse detection. Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 12% polyacrylamide gels and electroblotted onto nitrocellulose membranes. Immunodetection was done by using a pool at 1 μg/ml of two or three mouse monoclonal anti-PrP antibodies, all raised against hamster PrP (SAF32, recognizing the octo-repeat region located in the N-terminal part of PrP; SAF84, recognizing an epitope located between 160 to 170 residues; and SAF83, with an unknown recognized epitope [all from Spi-Bio, Massy, France]), followed by a peroxidase-conjugated goat anti-mouse antibody (Jackson ImmunoResearch Laboratories). Immunoreactivity was visualized by enhanced chemiluminescence (ECL kit; Pierce, France).

PrP immunostaining of DC.Immunocytochemistry was run on fixed, frozen DC as previously described, with minor modifications (37). Cells were fixed for 15 min in 4% formaldehyde in PBS, incubated in cold acetone for 10 min, and cryoprotected in 15% sucrose for 15 min before freezing. Two combined monoclonal anti-PrP antibodies were used: SAF84 (1 μg/ml) and SAF83 (1 μg/ml) from SpiBio. SAF84 gave a weaker immunostaining than SAF83. Slides were treated with 5 M guanidine for 5 min before PrP staining. Staining was performed with a mouse ABC staining system (Santa Cruz Biotechnology). Images were taken with a Leica DMLBHC microscope by using a Leica DC 100 charge-coupled-device camera and Leica IM50 image manager software (Leica Microsystems, Rueil-Malmaison, France).

Flow cytometry analysis.Cells were washed and resuspended in PBS containing 1% bovine serum albumin and 0.2% sodium azide (both purchased from Sigma), incubated with anti-CD16/CD32 Fc Block (2.4G2 clone; BD Pharmingen, San Diego, Calif.) to avoid antibody binding via Fc, and labeled at 4°C for 20 min with different combinations of the following fluorescein isothiocyanate-, phycoerythrin-, or allophycocyanin-conjugated MAbs: anti-B220 (RA3-6B2), anti-CD11c (HL-3), anti-CD11b (M1/70), anti-CD3 (17A2), polyclonal goat anti-mouse immunoglobulin (Ig), and appropriate Ig isotype controls. All MAbs were purchased from BD Pharmingen, except anti-PrP (SAF83; SpiBio, France). SAF83 was conjugated to fluorescein isothiocyanate (Sigma) or to Alexa 488 by use of a Zenon mouse IgG labeling kit (Molecular Probes, PoortGebouw, The Netherlands) prior to staining. Cells were analyzed with CellQuestPro software (BD Biosciences) on a FACSCalibur flow cytometer (BD Biosciences, San Diego, Calif.) situated in our level 3 care facilities.

BSE diagnosis.Animals were examined three times per week for clinical signs of BSE, which could include bradykinesia, waddling gait, poor coat condition, terminal incoordination, and weight loss. BSE-infected mice, animals affected by injuries, and surviving mice were euthanized by cervical dislocation at the terminal stage of the disease or at 650 days postinoculation by following the Pasteur Institute's animal experimentation regulations. BSE diagnosis was confirmed by detection of PrP^bse by Western blotting after PK digestion. The survival period was calculated as the interval between the first inoculation and the death, or in extremis, stage. The BSE incidence and the mean survival time were determined for each group of mice. Statistics were performed by use of Prism software (GraphPad Software, San Diego, Calif.).

Infectivity measurements.End point titration was performed by intracerebral inoculations of serial decimal dilutions of the infectious inoculum, ranging from 10⁻² to 10⁻¹⁰. Twenty microliters of inoculum was injected into groups of four to six C57BL/6 mice. The infectious titer was determined by the calculation of the LD₅₀ according to the formula of Reed and Muench (35).

Adoptive cell transfer.At day 1 or 6 postinfection, wild-type or PrP-null BMDC semiadherent cells were washed and suspended in PBS. Live BMDC (4 × 10⁶) in 500 μl PBS were immediately injected by the intraperitoneal route into groups of four to six RAG γc⁰ mice. In parallel, as a control, four to six RAG γc⁰ mice were injected by the same route with the same amount of BMDC that had been killed by three cycles of freezing and thawing.

Detection of PrP^bse in spleen DC of BSE-infected mice.To unravel the role of DC in BSE pathology, we investigated the expression of both normal and misfolded PrP isoforms in spleen DC of mice inoculated or not with BSE prions. For noninfected C57BL/6 mice, FACS analysis showed that splenic CD11c⁺ DC express membrane PrP (Fig. 1A). A high expression of PrP was noted in 35% of splenic CD11c⁺ DC. As expected, no PrP expression was detected in PrP⁰ mice (Fig. 1A). To determine which subsets of splenic DC express PrP, spleen DC were analyzed by flow cytometry for the expression of CD11c, CD11b, B220, and PrP. As shown in Fig. 1B, myeloid (CD11c⁺ CD11b⁺), lymphoid (CD11c⁺ CD11b⁻), and plasmacytoid (CD11c⁺ B220⁺) DC express PrP on their surfaces. The highest expression of PrP was detected in lymphoid DC and the lowest in plasmacytoid DC. Similarly, we found that cultured BMDC, BMpDC, and BMM express PrP on their surfaces (data not shown).

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FIG. 1.

Expression of PrP on spleen DC. Spleen DC of C57BL/6 or PrP⁰ mice were analyzed by flow cytometry for the expression of CD11c, CD11b, B220, and PrP. (A) Dot plot analysis of CD11c versus PrP labeling. No PrP was detected in PrP⁰ DC. (B) Expression of PrP on myeloid DC (CD11c⁺ CD11b⁺), lymphoid DC (CD11c⁺ CD11b⁻), and plasmacytoid DC (CD11c⁺ B220⁺) is shown by histograms. Gray shading indicates appropriate isotype control. The results are representative of three experiments.

In BSE-infected C57BL/6 mice, at 45 days after intraperitoneal inoculation, PrP^bse in total spleen cells was detectable by Western blotting as well as in CD11c⁺ DC purified by magnetic cell sorting (Fig. 2A). DC from noninfected control mice were negative for PrP^bse. These data were confirmed by PrP immunostaining of splenic DC purified by magnetic cell sorting (Fig. 2B). A strong intracellular labeling of PrP was observed with DC purified from spleens of mice with preclinical BSE but not with those from PrP⁰ or noninfected C57BL/6 mice. Altogether, these data strongly suggest that murine splenic DC express normal PrP and PrP^bse, and thus these cells could be infected in vivo in the BSE preclinical stage.

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FIG. 2.

PrP^bse is expressed in spleen DC of BSE-infected mice at 45 days postinjection. (A) Immunoblots show the accumulation of PK-resistant PrP^bse in total spleen cells and purified CD11c⁺ DC from C57BL/6 mice intraperitoneally inoculated with BSE and analyzed at day 45 postinfection. The treatment of samples in the presence (+) or the absence (−) of PK is indicated. C, control (C57BL/6 noninfected mice). (B) PrP immunostaining of CD11c⁺ DC purified from PrP⁰, C57BL/6 noninfected (Control), and BSE-infected (45 days post-intraperitoneal infection) mice. Magnification, ×40.

In vitro capture and processing of PrP^bse by BMM, BMDC, and BMpDC.Cell cultures were exposed at day 6 to BSE brain homogenate or to PrP^bse purified in SAF. The control inoculum was LPS, brain homogenate, or SAF from healthy mice.

Western blot analysis showed that PrP^bse of the BSE inoculum was taken up within 2 h by BMDC (Fig. 3A). PrP^bse was then cleared in 2 to 3 days. Then, long-term cultures showed no PrP^bse expression, strongly suggesting that BMDC did not multiply BSE prions in vitro (data not shown). BMDC from PrP⁰ mice also acquired and cleared PrP^bse in vitro with a similar time course (Fig. 3B). Similarly, SAF were taken up and cleared in vitro (Fig. 3A and B). Normal PrP of the control inoculum was processed and rapidly degraded by BMDC from normal or PrP⁰ mice (Fig. 3A and B). Similarly, BMM and BMpDC acquired and rapidly cleared PrP of the control or BSE inoculum (Fig. 3C and D). LPS activation of BMDC or BMM before BSE infection resulted in an accelerated breakdown of PrP^bse (Fig. 3G). Splenic T or B cells acquired only small amounts of PrP^bse and did not multiply it (Fig. 3E and F). To verify that PrP^bse was processed by BMDC, PrP immunocytochemistry was run on BMDC of PrP⁰ mice after their exposure to the BSE inoculum (Fig. 4). A strong intracellular labeling of PrP in PrP⁰ BMDC was detected after 24 h of exposure to the BSE inoculum (Fig. 4). As no staining was detected in BMDC treated or not with control inoculum, these data indicate that only PrP^bse, but not normal PrP, was detected in BSE inoculum-treated BMDC, confirming the Western blot analysis results. Furthermore, these immunocytochemistry data also indicate that PrP^bse was internalized by BMDC before clearing. Altogether, these findings highlight the specific high capacity of DC and macrophages to capture and clear PrP^bse in vitro.

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FIG. 3.

Uptake and degradation of PrP^bse by BMM, BMDC, and BMpDC. Immunoblots show levels of PrP in BMDC (A and B), BMM (C), BMpDC (D), and splenic B (E) and T (F) lymphocytes untreated or treated with LPS, SAF, control, or BSE inoculum. Panel G shows PrP^bse levels in LPS-activated BMDC or BMM following 2 h of exposure to the BSE inoculum, a brain homogenate from noninfected or terminally BSE-sick mice. SAF were extracted from control or BSE brain homogenate. The period of treatment (2, 24, 48, 72, or 144 h) is indicated. BMDC, BMpDC, and BMM were prepared from C57BL/6 (A, C, and D) or PrP⁰ (B) mice. Control samples included untreated BMDC (labeled C), BMDC treated with SAF from control homogenate (labeled Control), and BMDC treated with SAF from 20 μg undigested terminally BSE-infected brain (labeled BSE SAF). Samples were digested (+) or not (−) with 40 μg/ml PK. Immunoblots with undigested or PK-digested samples were revealed with anti-PrP antibodies SAF32 and SAF84.

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FIG. 4.

Detection of PrP^bse in BMDC of PrP-null mice after in vitro infection with BSE prions. Immunocytochemical analysis detected the presence of PrP in BMDC treated with BSE inoculum for 24 h (lower right panel) but not in cells exposed to control uninfected inoculum (lower left panel). A control untreated BMDC is shown in the upper left panel. Antibody (Ab) control staining of BSE inoculum-treated BMDC is shown in the upper right panel. Magnification, ×40.

Prion infectivity in BMDC incubated with BSE prions.To quantify prion infectivity in DC cultures, a time course of in vitro infection was performed by in vivo titration of cell lysates (Table 1). After 1 or 6 days of BMDC culture with the BSE inoculum, adherent cells were washed twice before being scraped in PBS, frozen, and injected without filtration into the brains of C57BL/6 mice. We found that following 1 or 6 days of in vitro incubation with BSE brain homogenate, PrP-expressing or PrP⁰ BMDC were highly infectious in vivo. Following 1 day of BSE prion incubation, BMDC from C57BL/6 or PrP⁰ mice have an infectious titer of LD₅₀ of 10^4.7 and 10^3.9 infectious units per 4 × 10⁶ injected cells, respectively. BMDC were 10-fold less infectious after 6 days of culture than after 1 day.

Fate of RAG γc⁰ mice after intraperitoneal injection with BSE-infected BMDC.To further investigate whether DC have the ability to propagate prion infectivity to the central nervous system, we tested, in RAG γc⁰ immunodeficient mice, the infectivity of BMDC exposed in vitro to BSE prions. These mice are resistant to murine BSE inoculum (brain homogenate) by the intraperitoneal route, although they are susceptible to the same inoculum after intracerebral inoculation (Table 2). While infected immunocompetent mice accumulated PrP^bse in spleen and brain, RAG γc⁰ mice intracerebrally infected with BSE brain homogenate accumulated PrP^bse in brain but not in spleen (data not shown). These findings confirm that RAG γc⁰ immunodeficient mice lack splenic target cells supporting BSE prion multiplication.

Four million live or dead BMDC from normal or PrP⁰ mice were incubated for 1 or 6 days with BSE prions and then intraperitoneally injected into RAG γc⁰ mice. At 6 months after injection, FACS analysis of blood showed that these mice still lacked B220 cells (data not shown). Seven hundred days after injection, no animal had developed BSE (Table 3), suggesting that BMDC did not directly release infection to nerve terminals.