- Open Access
Targeting of prion-infected lymphoid cells to the central nervous system accelerates prion infection
© Friedman-Levi et al; licensee BioMed Central Ltd. 2012
- Received: 27 December 2011
- Accepted: 21 March 2012
- Published: 21 March 2012
Prions, composed of a misfolded protein designated PrPSc, are infectious agents causing fatal neurodegenerative diseases. We have shown previously that, following induction of experimental autoimmune encephalomyelitis, prion-infected mice succumb to disease significantly earlier than controls, concomitant with the deposition of PrPSc aggregates in inflamed white matter areas. In the present work, we asked whether prion disease acceleration by experimental autoimmune encephalomyelitis results from infiltration of viable prion-infected immune cells into the central nervous system.
C57Bl/6 J mice underwent intraperitoneal inoculation with scrapie brain homogenates and were later induced with experimental autoimmune encephalomyelitis by inoculation of MOG35-55 in complete Freund's adjuvant supplemented with pertussis toxin. Spleen and lymph node cells from the co-induced animals were reactivated and subsequently injected into naïve mice as viable cells or as cell homogenates. Control groups were infected with viable and homogenized scrapie immune cells only with complete Freund's adjuvant. Prion disease incubation times as well as levels and sites of PrPSc deposition were next evaluated.
We first show that acceleration of prion disease by experimental autoimmune encephalomyelitis requires the presence of high levels of spleen PrPSc. Next, we present evidence that mice infected with activated prion-experimental autoimmune encephalomyelitis viable cells succumb to prion disease considerably faster than do mice infected with equivalent cell extracts or other controls, concomitant with the deposition of PrPSc aggregates in white matter areas in brains and spinal cords.
Our results indicate that inflammatory targeting of viable prion-infected immune cells to the central nervous system accelerates prion disease propagation. We also show that in the absence of such targeting it is the load of PrPSc in the inoculum that determines the infectivity titers for subsequent transmissions. Both of these conclusions have important clinical implications as related to the risk of prion disease contamination of blood products.
- immune cells
- spinal cord
Prion diseases are a group of fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease and kuru in humans, bovine spongiform encephalopathy in cattle, scrapie in sheep and goats, and chronic wasting disease in deer . This group of diseases is caused by the accumulation of a misfolded and oxidized isoform of PrPSc, a normal membrane protein believed to play a role in the protection against oxidative insults [2, 3]. All forms of prion diseases are characterized by long incubation periods, which in humans can sometimes amount to decades [3–6]. Although the exact mechanism of prion propagation is unknown, a general sequence of events has been outlined that is consistent with most of the available data . First, and regardless of the route of infection, prions replicate in lymphoid organs, as shown by the fact that both infectivity and accumulation of PrPSc are initially detected in the spleens of the infected animals [5–8]. Prions invade the nervous system by a mechanism believed to involve transmigration of infected lymphoid cells as well as retrograde transport in ascending peripheral neural tracts [4, 9–12]. Once prions enter the brain, prion replication and PrPSc accumulation seems to occur faster than in the lymphoid system, leading to the certain death of the affected individuals [13–15]. In the presence of inflammatory conditions affecting peripheral organs, activated lymphoreticular cells induce deposition of PrPSc and prion infectivity in the sites of infiltration in prion-infected animals . For example, mastitis in sheep results in deposition of PrPSc in the inflamed mammary glands .
While no differences in prion disease incubation time or clinical symptoms were reported in these peripheral infection cases, mice incubating scrapie and induced for experimental autoimmune encephalomyelitis (EAE), an autoimmune inflammatory disease of the central nervous system (CNS) [18, 19], died from a progressive neurological disease long before the control mice succumbed to classical scrapie . Surprisingly, mice affected by the scrapie-EAE syndrome showed almost undetectable to high levels of brain PrPSc, demonstrating that there is no correlation between the clinical status of the affected scrapie-EAE mouse and PrPSc accumulation. Immunostaining studies revealed PrPSc depositions in the demyelinated white matter spinal cord areas of the co-induced mice, mostly co-localized with hematopoietic cell infiltrates. In classical scrapie, PrPSc aggregates are mostly found in the gray matter .
In this project we investigated the mechanism of prion disease acceleration by CNS inflammation. In particular, we asked whether accelerated prion disease in the co-induced animals requires the targeted delivery of PrPSc to the CNS by activated immune cells. To this aim, we compared the effect of EAE induction on prion disease kinetics at several time points before and after infection with scrapie and found that acceleration of disease is the strongest at one month after prion infection, when PrPSc is largely accumulated in immune organs such as the spleen . Next, we inoculated naïve mice with viable or homogenized activated immune cells collected from scrapie-EAE mice and controls. Only the activated viable cells generated CNS inflammation and accelerated prion disease, as compared with non-specific immune activation, or with scrapie brain homogenates with similar PrPSc loads. Our results therefore suggest that EAE-dependent acceleration of fatal prion disease results from the infiltration of PrPSc loaded immune cells into the CNS. Additional results presented here indicate that, in the absence of such infiltration, incubation times of prion disease relate mostly to the levels of the inoculated PrPSc.
Female C57Bl/6 J mice were purchased from Harlan (Hebrew University, Jerusalem, Israel) and housed in the animal care facility in compliance with the standard guidelines for animal care. All experiments were approved by the Institutional Animal Care and Use Committee.
Four- to five-week-old mice were inoculated by intraperitoneal injection with 100 μL or intracerebral injection with 50 μL of 1% scrapie brain homogenate. Mice were followed closely for disease signs until disease manifestation and killed when too sick to reach food and water.
Experimental autoimmune encephalomyelitis induction
Five- to eight-week-old female C57BL/6 J mice were immunized with an emulsion containing 300 μg of MOG35-55 (70% purified; synthesized at the Hebrew University, Jerusalem, Israel) solubilized in saline and an equal volume of complete Freund's adjuvant (CFA; Sigma, Rehovot, Israel) supplemented with 5 mg/mL of heat-killed mycobacteria Tuberculosis H37RA (Difco Laboratories, Detroit, MI, USA). The inoculum (0.2 mL) was injected subcutaneously in both flanks. On the day of inoculation and 48 hours later, 100 ng of pertussis toxin (List Biological Laboratories, Inc. Cambell, CA, USA) in 0.1 mL saline was also administered by intraperitoneal injection. Control groups were inoculated the same way but without MOG35-55.
Passive transfer of experimental autoimmune encephalomyelitis
Nine days post induction mice were killed and cells were obtained from the spleen and lymph nodes. Cells were suspended in Roswell Park Memorial Institute medium (Biological Industries, Beit Haemek, Israel) supplemented with 10% fetal calf serum, 1 mM L-glutamine, antibiotics, MOG35-55 (20 μg/mL) and mouse recombinant IL-2 (50 units/mL; Peprotech, Rocky Hill, NJ, USA) and incubated for three days in a CO2 humidified incubator. Cells were harvested and live cells were separated with Ficoll-Paque (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and injected into naïve mice. In control groups of cells extracts, cells were frozen after separation, thawed three times and then injected into naïve mice. On the day of inoculation and 48 hours later, 100 ng of pertussis toxin in 0.1 mL saline was also administered by intraperitoneal injection. Mice were followed for clinical symptoms of EAE and evaluated according to a 0 to 6 point score (0 represents no clinical signs and 6 represents death as a result of disease).
Western blot: PrP immunoblots of brains and spleens
Brains and spleens from mice in all experimental groups were homogenized in 10 volumes of 10 mM Tris-HCl (pH 7.5) containing 300 mM sucrose. Homogenates were normalized for protein level, digested with 40 μg/mL proteinase K (Sigma), and immunoblotted with anti-PrP monoclonal antibody IPC1 (Sigma) or monoclonal antibody 6H4 (Prionics, Schlieren, Switzerland), 40 μL in each lane for brains and 100 μL in each lane for spleen homogenates.
Histological evaluations were performed on paraffin-embedded sections of brain and spinal cord samples. Sections were stained with luxol fast blue/Periodic acid Schiff (PAS) to assess demyelination. Consecutive sections were used for immunohistochemistry with antibodies against the following targets: macrophages and activated microglia (anti-MAC3; BD Pharmingen, San Diego, CA, USA); T-cells (anti-CD3; Serotec, Oxford, UK); and prion protein (anti-PrP 6H4; Prionics).
The clinical scores of disease in groups of animals with EAE and EAE + scrapie were compared using Kruskal-Wallis one-way analysis of variance on ranks. The survival curves were compared using chi-squared test.
Acceleration of prion disease by experimental autoimmune encephalomyelitis may depend on spleen PrPSc levels
Accelerated disease in mice infected with scrapie-experimental autoimmune encephalomyelitis-activated immune cells
PrPSc in white matter demyelinated areas
Transmission of disease from scrapie-experimental autoimmune encephalomyelitis inoculated brains
We have shown in Figure 3 that while most of the inoculated samples comprised similar levels of PrPSc, the sample consisting of viable scrapie-EAE cells generated disease in the naïve mice significantly earlier than all others, indicating that levels of PrPSc are not the only designator of prion disease incubation time. In our previous work , we showed that mice may succumb to the co-induced scrapie-EAE disease at similar incubation times with very different levels of brain PrPSc, those varying from undetectable levels to the high levels observed in mice with classical scrapie. To this effect, we next investigated whether mice succumbing to the co-induced disease generate a new prion strain that can transmit disease independent of PrPSc inoculum levels, or whether disease transmission from these mice correlate with the accumulated PrPSc levels when in brain homogenates.
We have shown in this work that acceleration of prion disease by CNS inflammation, as is the case for EAE induction, requires the presence of substantial levels of PrPSc in the immune cells of prion-infected mice. Such PrPSc-rich cells may then be targeted by CNS inflammation for infiltration into the CNS, as demonstrated by the presence of PrPSc deposits in white mater areas of brains and spinal cords. This was true for mice induced with EAE a month after scrapie infection  as well as for mice infected directly with viable and activated scrapie-EAE cells (Figures 3 and 4). Our experiments also show that the inflammation-mediated disease does not constitute a more virulent prion strain, since secondary transmission from scrapie-EAE mouse homogenized brain samples indicated that prion titers were dependent mostly on the inoculated levels of PrPSc. These results are consistent with previous observations that suggest inflammation may affect the sites of PrPSc accumulation [16, 17] and, in the case of CNS inflammation, accelerate the presentation of fatal disease , sometimes in the absence of high levels of PrPSc in the brains of the affected animals. It is possible that low levels of prions that have infiltrated into white matter areas are more toxic and facilitate the propagation of disease in a more efficient way. Both mechanisms, retrograde transport of prions and inflammation-dependent neuroinvasion, may occur distinctively or in parallel, explaining how similarly sick mice present different levels of brain PrPSc at the end point of disease. It is also possible that a variable balance between EAE-linked and scrapie symptoms can account for this phenomenon.
The fact that incubation times for second-generation transmission of disease from homogenates of scrapie-EAE brains were independent of the clinical status of donor mice and related only to PrPSc levels indicates that inflammation did not change the virulence properties of the initial prion strain, but rather accelerated the targeting of the infectious agent to vital areas, resulting in a shorter disease. This may explain why endogenous blood prions carry more infectivity than predicted from their low blood PrPSc content [4, 26, 27]. Indeed, our results suggest that infection by viable cells carrying PrPSc may be more rapid and that the infectivity of a specific blood sample may depend not only on the prion incubation status of the blood donor, but also on the inflammatory state of both the donor and recipient on the day of the transfusion.
This work was funded by the Morasha ISF foundation and by the Agnes Ginges foundation.
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