The immunobiology of prion diseases

REVIEWS The immunobiology of prion diseases Adriano Aguzzi1, Mario Nuvolone1,2 and Caihong Zhu1 Abstract | Individuals infected with prions succumb to brain damage, and prion infections continue to be inexorably lethal. However, many crucial steps in prion pathogenesis occur in lymphatic organs and precede invasion of the central nervous system. In the past two decades, a great deal has been learnt concerning the cellular and molecular mechanisms of prion lymphoinvasion. These properties are diagnostically useful and have, for example, facilitated preclinical diagnosis of variant Creutzfeldt–Jakob disease in the tonsils. Moreover, the early colonization of lymphoid organs can be exploited for post-exposure prophylaxis of prion infections. As stromal cells of lymphoid organs are crucial for peripheral prion infection, the dedifferentiation of these cells offers a powerful means of hindering prion spread in infected individuals. In this Review, we discuss the current knowledge of the immunobiology of prions with an emphasis on how basic discoveries might enable translational strategies. Protein-only hypothesis Introduced by Griffith and formally enunciated by Prusiner, it states that prions are unconventional infectious agents that are devoid of informational nucleic acids and that uniquely consist of an infectious, pathogenic protein. Prion The aetiological agent of prion disease; prion is short for proteinaceous infectious particle. 1 Institute of Neuropathology, University Hospital of Zurich, Schmelzbergstrasse 12, CH‑8091 Zurich, Switzerland. 2 Amyloidosis Research and Treatment Centre, Foundation Istituto di Ricovero e Cura a Carattere Scientifico San Matteo Hospital and Department of Molecular Medicine, University of Pavia, Institute for Advanced Study, Pavia I‑27100, Italy. Correspondence to A.A. e‑mail: adriano.aguzzi@usz.ch doi:10.1038/nri3553 Published online 5 November 2013 Diseases caused by prions are fatal neurodegenerative conditions that affect humans and several other mammals (TABLE 1). The transferral of brain extracts from affected individuals into permissive host species can transmit the disease 1. Transmission among humans occurred during the kuru epidemic in Papua New Guinea through cannibalistic rituals2. In addition, more than 450 cases of iatrogenic Creutzfeldt– Jakob disease (iCJD) have occurred following pituitary hormone treatment or surgical procedures3. Finally, bovine spongiform encephalopathy (BSE) has affected more than 180,000 cattle worldwide (see the BSE Portal on the World Organisation for Animal Health website) and has caused variant CJD (vCJD) in humans4. vCJD was shown to be transmitted through blood or blood derivatives, even from subclinical donors5,6. According to the protein-only hypothesis, the infectious agent — that is, the prion itself — consists of scrapie prion protein (PrPSc), which is an assembly of conformers of cellular prion protein (PrPC)7. A PrPSc aggregate can recruit PrPC proteins and can perpetuate its own amplification7 in a similar way to crystal growth and fragmentation8. When this cycle occurs within the central nervous system (CNS) and involves membraneanchored PrPC at the neuronal surface, a neurotoxic signal is triggered, plausibly through PrPC itself 9. This results in the typical spongiform changes that are seen in diseased brains. The recognition of the infectious potential of prion diseases resulted in their designation as transmissible spongiform encephalopathies (TSEs). Prion diseases are interesting to immunologists for three main reasons. First, numerous studies have suggested that there are physiological roles for PrPC in cells of the immune system10, which suggests that clarifying such roles might help us to understand the molecular mechanisms of prion pathogenesis. However, the physiological roles of PrPC in the immune system, and elsewhere, remain unclear 10. Second, the immune system has a crucial role in prion pathogenesis: prions can escape immune surveillance, colonize the immune system of their hosts, hijack immune components (a stage known as peripheral replication) and gain access to the CNS (the neuroinvasion stage). Although our understanding of the underlying peripheral replication and neuroinvasion stages is advanced, we lack a similar comprehension of the mechanisms underlying prion toxicity after the invasion of the CNS. Neuroimmunological phenomena also have an important role in this final phase of prion diseases11. Third, manipulation of the immune system might represent a valid therapeutic strategy for prion diseases12. Indeed, current antiprion interventions have prominently focused on immunological strategies, including immunoprophylactic, immunosuppressive and immunostimulatory approaches12. In this Review, we discuss the current state of prion immunobiology. We examine the role of PrPC in the immune system, the mechanisms of peripheral prion replication and neuroinvasion by prions, and the neuroinflammatory changes that are associated with prion 888 | DECEMBER 2013 | VOLUME 13 www.nature.com/reviews/immunol © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS Table 1 | Prion diseases* Disease Natural host species Route of transmission or Other susceptible species disease-induction mechanism Sporadic CJD Humans Unknown Primates, hamsters, guinea pigs, bank voles, humanized and chimeric human–mouse transgenic mice, and wild-type mice Iatrogenic CJD Humans Accidental medical exposure to CJD-contaminated tissues, hormones or blood derivatives Primates, humanized and chimeric human–mouse transgenic mice, and wild-type mice Familial CJD Humans Genetic (germline PRNP mutations)‡ Primates, bank voles, chimeric human–mouse transgenic mice and wild-type mice Variant CJD Humans Genetic (germline PRNP mutations) Primates, guinea pigs, humanized transgenic mice and wild-type mice Kuru Humans Ritualistic cannibalism Primates and humanized transgenic mice Fatal familial insomnia Humans Genetic (germline PRNP mutations) Humanized and chimeric human–mouse transgenic mice, and wild-type mice Sporadic fatal insomnia Humans Unknown Chimeric human–mouse transgenic mice Gerstmann–Sträussler– Scheinker syndrome Humans Genetic (germline PRNP mutations) Primates, guinea pigs, mutated Prnp transgenic mice and wild-type mice Scrapie Sheep, goat and mouflon Horizontal and possibly vertical Primates, elk, hamsters, raccoons, bank voles, ovinized transgenic mice (which express sheep PrPC) and wild-type mice Atypical scrapie Sheep and goat Unknown Ovinized transgenic mice and porcinized transgenic mice (which express pig PrPC) Chronic wasting disease Mule deer, white-tailed Horizontal and possibly vertical deer, Rocky Mountain elk and moose Primates, ferrets, cattle, sheep, cats, hamsters, bank voles, cervidized transgenic mice (which express deer PrPC) or murine Prnp-overexpressing transgenic mice BSE Cattle Ingestion of BSE-contaminated food Primates, guinea pigs, humanized and bovinized transgenic mice, and wild-type mice Atypical BSE Cattle Unknown Primates, humanized and bovinized transgenic mice, and wild-type mice Feline spongiform encephalopathy Zoological and domestic felids Ingestion of BSE-contaminated food Wild-type mice Transmissible mink encephalopathy Farmed mink Ingestion of BSE-contaminated food Primates, cattle, hamsters and raccoons Ingestion of BSE-contaminated food Wild-type mice Spongiform encephalopathy Zoological ungulates of zoo animals and bovids BSE, bovine spongiform encephalopathy; CJD, Creutzfeldt–Jakob disease; PRNP, gene encoding prion protein; PrPC, cellular prion protein. *Data from REF. 12. ‡One case of somatic mosaicism175. diseases. We also address the immune responses that can be initiated against prions and the immunological intervention strategies under investigation for the treatment of prion diseases. Scrapie prion protein (PrPSc). The pathological version of prion protein that is present in the central nervous system and other tissues of patients with transmissible spongiform encephalopathies. It is believed to differ from cellular PrP only in terms of post-translational modifications. Cellular prion protein (PrPC). The physiological version of prion protein, which is present in the central nervous system and other tissues under normal circumstances. Physiological functions of PrPC Immune functions of PrPC. Mice represent a powerful experimental model for prion research (BOX 1). Despite the availability of mice that are deficient in PrP (encoded by Prnp) since 1992 (REF. 13), the elucidation of the physiological functions of PrPC is rudimentary. In peripheral nerves, PrPC contributes to myelin maintenance14. Many other functions have also been ascribed to PrPC, including immunological ones10. It has been suggested that PrPC is involved in T cell development, activates and interacts with dendritic cells (DCs), inhibits phagocytosis in macrophages and contributes to haematopoietic stem cell self-renewal. Similarly, PrPC has been shown to be involved in the internalization of Brucella abortus in macrophages, in the replication of different viruses and in the modulation of neuroinflammatory changes (reviewed in REF. 10) (BOX 2). Recently, PrPC has been implicated in the pluripotency and differentiation of embryonic stem cells15, in intestinal barrier function16 and in the uptake of B. abortus in intestinal microfold cells (M cells)17. None of these functions has been unambiguously elucidated at a molecular level and conflicting results have often been reported. We have recently identified a crucial caveat to the above claims. All currently available Prnp–/– mouse lines were generated in embryonic stem cells from the 129 mouse strain. Hence, any loci that are linked to Prnp and that are polymorphic between 129 and the backcrossing strain may represent a confounder when comparing Prnp–/– and Prnp+/+ mice. For example, the polymorphic signal regulatory protein-α (Sirpa) is linked to Prnp and Prnp–/– mice were shown to carry the Sirpa allele from the 129 strain (Sirpa129) despite extensive backcrossing18. Indeed, the increased phagocytosis of apoptotic cells, which was previously reported in Prnp–/– mice and NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | DECEMBER 2013 | 889 © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS Box 1 | Mice in prion research Prion diseases Also known as transmissible spongiform encephalopathies (TSEs). These are a group of transmissible neurodegenerative diseases that affect humans and various mammals. Prion strains Natural sources or isolates of prions that, when inoculated into genetically homogeneous hosts, induce a prion disease with peculiar clinical, histological and biochemical features. Propagons Proteinaceous aggregates that are capable of seeding a self-perpetuating reaction of templated nucleation within a biological system. Propagons are not necessarily identical to scrapie prion proteins but might represent a subset of prion protein conformations, some of which might not be resistant to proteolysis. Propagons could, in principle, have specific post-translational modifications. Prionoids Self-aggregating proteins that are capable of transmitting between cells within one organism, but not from one organism to another. Amyloid-β, tau, huntingtin and amyloid A protein are examples of prionoids. Synuclein was thought to be a prionoid, but recent evidence suggests that it might behave like a bona fide prion. • Small rodents, including mice, can be infected with prions from various natural sources. Prion inoculation of mice results in bona fide transmissible spongiform encephalopathies (TSEs). Therefore, prion-infected mice have represented and still represent an important paradigm to increase our understanding of prion biology. • Early studies identified quantitative trait loci (QTLs) that influence the incubation period of scrapie in mice. These QTLs were later shown to coincide with Prnp, which is the gene encoding cellular prion protein (PrPC)157. Prnp–/– mice, which lack PrPC, were shown to be healthy13 and resistant to prion infection59 and propagation158. Prnp+/– mice had longer prion incubations than wild-type mice158, which indicates that Prnp gene dosage controls the speed of disease onset. • Numerous Prnp–/– strains have been generated. Although most of the Prnp–/– strains seem to be essentially healthy, some were shown to develop a neurodegenerative disease of the cerebellum159. However, it was found that the disease was caused by the incidental upregulation of the prion protein dublet prion-like protein doppel (Prnd) gene, which is located immediately downstream of Prnp160. • Several subtle phenotypes have been reported in Prnp–/– mice, with inconsistencies across different laboratories and mouse lines. Differences in genetic background, gut microbiota and experimental methodologies might explain some of the reported incongruities. • Reciprocal brain-graft experiments between mice lacking or expressing PrPC (REF. 9) and mice with a conditional deletion of Prnp161 showed that neuronal PrPC mediates prion toxicity. • PrPC-overexpressing mice show accelerated disease, thus expediting bioassays to determine prion titres162,163. • Transgenic mice expressing xenogeneic Prnp sequences can display lowered species-transmissibility barriers, thus facilitating transmission studies of prions from these species164,165. • Certain deletion mutants of PrPC induce neurodegeneration, which is rescued by wild-type PrPC, confirming that PrPC can mediate neurotoxicity166. • Finally, mice expressing select point mutants of PrPC spontaneously form prions167, corroborating the protein-only hypothesis. which was initially attributed to the absence of Prnp, has recently been shown instead to be caused by differences in the Sirpa alleles, indicating that the inhibition of phagocytosis was previously misattributed to PrPC (REF. 19). PrPC to PrPSc conversion. PrPC and PrPSc differ in their tertiary and quaternary structure. Although PrPC contains mostly disordered and α-helical structures, PrPSc has a high β-sheet content20. The supramolecular arrangement of PrPSc is thought to determine the specific features of different prion strains. PrPC itself is an innocuous constituent of many cell types. On infection, the disease-causing PrPSc functions as a template that incorporates PrPC into aggregates. The conversion might require additional chaperoning molecules, such as lipids21. This self-perpetuating feature of prion propagons is shared by several other proteins, some of which (termed prionoids) were found to transmit within, but not necessarily between, individuals. Prion entry sites Routes of infection. With the exception of the unfortunate cases in which prions have been inadvertently introduced into the brain3, in acquired prion diseases the infectious agent replicates in the periphery before reaching the brain. But how do prions spread from their portals of entry? The answer crucially depends on the type of exposure (FIG. 1). For oral exposure, which is the most relevant route for acquired prion diseases in nature, prions must first cross the wall of the digestive tract. Prions resist exposure to digestive enzymes, and gastric acidity affords only limited protection against oral prion challenge. Intragastric inoculation of prions in mice led to prion disease, and prions showed rapid accumulation in Peyer’s patches before colonizing the spleen22. Accordingly, susceptibility to prion infection following oral challenge in mice positively correlates with the number of Peyer’s patches that are present in the small intestine23. These observations suggest that the follicle-associated epithelium (FAE) of the Peyer’s patches is a plausible prion entry site. Another indication that immune mechanisms are implicated in this process comes from the observation that experimentally induced bacterial colitis enhanced prion susceptibility on oral exposure24. Entry of prions into Peyer’s patches. Scattered and intercalated between classical enterocytes, M cells continuously sample the intestinal lumen to facilitate immunosurveillance. This property can be hijacked by several pathogens, including prions, to invade the intestinal mucosa. Co-culture systems using differentiated cell lines with morphological and functional features of bona fide M cells showed efficient transcytosis of prions25. These observations were corroborated by in vivo studies that showed that M cells can uptake orally administered prions and that oral prion pathogenesis can be inhibited when M cells are depleted through administration of receptor activator of NF-κB ligand (RANKL; also known as TNFSF11)26. Although M cells have been indicated in prion transport, alternative mechanisms, including transcytosis of prions through enterocytes, could also take place27. After crossing the FAE, prions spread — possibly through a cell-mediated mechanism. Follicle-associated lymphocytes are unlikely to be involved in this process23. Macrophages have been shown to inactivate prion infectivity in vitro28, but residual infectivity could persist within these cells. Bisphosphonate-mediated 890 | DECEMBER 2013 | VOLUME 13 www.nature.com/reviews/immunol © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS Box 2 | PrPC as a receptor for amyloid‑β oligomers A genome-wide unbiased screen has identified cellular prion protein (PrPC) as a potential receptor for amyloid-β oligomers, which are believed to be the main cause of Alzheimer’s disease pathogenesis168. This report generated much attention because it implied that similar mechanisms might be involved in prion disease and Alzheimer’s disease, as in both conditions PrPC seems to be the key mediator of toxicity. Binding of oligomeric amyloid-β to PrPC results in a poorly understood signalling pathway, which possibly includes the phosphorylation of FYN, microtubule-associated protein tau (MAPT) and the N-methyl-d-aspartate receptor subunit 2B, ultimately leading to amyloid-β-mediated neurotoxicity168,169. Genetic ablation of PrPC or the administration of PrPC-specific antibodies or PrPC-mimetic compounds that can interfere with PrPC–amyloid-β binding reduces or prevents amyloid-β-mediated toxicity in a plethora of in vitro and in vivo experimental systems168–171. However, in other situations, PrPC was shown to be dispensable for amyloid-β-mediated toxicity172–174, which indicates a more complex scenario. Although differences in experimental conditions might explain some of the inter-experimental variation, the discrepancy observed in amyloid-β-mediated toxicity could reflect a context-dependent involvement of PrPC in amyloid-β-dependent neurotoxicity, and this deserves further analysis. Currently, it is unclear whether interference with PrPC–amyloid-β binding is of any therapeutic value for Alzheimer’s disease. macrophage depletion in mice that were challenged orally or intraperitoneally with prions resulted in increased PrPSc levels in lymphoid tissues, which suggests that macrophages limit the amount of prions that initiate the infection29. However, whether this effect is truly dependent on macrophage depletion or whether it is an indirect effect remains to be established29. Further studies to determine whether macrophages represent important prion carriers are also needed. Uptake of prions by DCs. DCs patrol gut-associated lymphoid tissue (GALT) and sample luminal or transcytosed antigens for presentation to and priming of B cells and T cells. DCs can acquire intestinally administered prions and transfer them to mesenteric lymph nodes after their migration through the lymphatic system30. Depletion of CD11c+ DCs in vivo impaired prion accumulation in GALT and spleen and reduced the susceptibility to orally administered prions, pointing towards a role for DCs in promoting the spread of prions at these sites31. However, CD11c is also expressed by mononuclear phagocytes other than DCs within GALT, and the respective contribution of DCs and other mononuclear phagocytes to prion uptake is currently unclear. Transmission through blood. Prions can be efficiently transmitted through blood or blood derivatives. BSE and scrapie were transmitted to sheep through whole-blood transfusion or buffy coats, even from subclinical donors, which implies that blood represents an efficient vehicle of infection32. Similarly, blood transfusion efficiently transmitted the TSE chronic wasting disease (CWD) to deer33, and vCJD was transmitted from blood donors who subsequently developed vCJD5,6. These tragic episodes were not previously anticipated on the basis of epidemiological evidence from case–control studies34 and resulted in the creation of blood-donor deferral criteria and quality-control measures. Extensive experimental work has also indicated that prions can be transmitted through the skin35–37 and aerosols38–40 and colonize draining lymph nodes soon after prion exposure. In aerosol transmission, M cells and epithelial cells of the nasal mucosa seem to be involved in prion transport 41. Whether these routes are relevant to naturally occurring TSEs remains to be established. Peripheral replication of prions Prions and SLOs. In many TSEs, prions accumulate and replicate within secondary lymphoid organs (SLOs) before neuroinvasion occurs. This is most apparent in natural scrapie 42, CWD43, transmissible mink encephalopathy (TME)44 and vCJD45, which are regarded as lymphotropic prion diseases. Neurotropic prions directly invade the CNS without requiring a peripheral replicative phase46. This dichotomy of lymphotropic and neurotropic prions is likely to be an oversimplification because numerous observations indicate that the extent of lymphotropism of a prion is the result of a combination of the prion strain, inoculation route, the host species and the gene sequence encoding the prion protein. It has also been observed that lymphotropic prions seem to have increased host ranges when compared with neurotropic prions 47. Owing to the early colonization of lymphoid tissues by prions, a tonsil biopsy can be used for preclinical diagnosis of vCJD 48 and in nationwide prevalence screening studies15. Targets of prion infectivity: haematopoietic or stromal cells? The detection of prion infectivity within lymphoid organs of prion-infected mice 49 has encouraged the identification of the cells and molecules that are involved in this process. Genetic asplenia or splenectomy, but not athymia or thymectomy, extend the survival of mice that are peripherally inoculated with prions, thereby excluding a relevant role for T cells in peripheral prion pathogenesis50. However, if splenectomy is carried out in peripherally inoculated mice when prions have already invaded the spinal cord, survival is not extended51. Also, constitutive or acquired lymphocyte deficiency impairs prion replication following peripheral, but not intracerebral, inoculation of rodentadapted prions52,53. This indicates that splenic replication of prions is crucial only in the early phases of the disease if prions are peripherally administered. Of note, severe combined immunodeficient (SCID) mice proved to be partially resistant against intracerebral inoculation of a BSE isolate, which suggests that the peripheral immune system plays a part in the species-barrier phenomenon54. NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | DECEMBER 2013 | 891 © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS Established route of infection Potential route of infection Intracranial Corneal grats Airway Ingestion Skin Intramuscular Intravenous Figure 1 | Entry sites for acquired prion diseases. The solid arrows indicate the recognized routes of prion transmission. Cases of iatrogenic Creutzfeldt–Jakob disease (iCJD) have occurred through corneal and dura mater transplantations from diseased cadaveric donors, through the use of prion-contaminated electroencephalography electrodes and neurosurgical instruments, and through the intramuscular administration of contaminated pituitary-derived hormones. Ingestion of meat from cows with bovine spongiform encephalopathy and cannibalistic rituals cause variant CJD (vCJD) and kuru, respectively. vCJD can also be transmitted through blood products. The dashed arrows indicate potential routes of prion transmission that have been suggested on the basis of experimental studies in animal models; their clinical relevance is currently unknown. Fractionation of splenocytes identified subpopulations of cells with high prion infectivity and suggested that most infectivity is present in the stromal compartment 55,56. Supporting these observations, sublethal ionizing irradiation — which depletes mitotically active haematopoietic cells but not mitotically quiescent stromal cells — had no effect on prion pathogenesis in mice that were challenged by different inoculation routes, doses and strains of rodent-adapted scrapie prions57. Follicular dendritic cells (FDCs). Stromal cells derived from platelet-derived growth factor receptor-β (PDGFRβ)+ perivascular precursors and localized in lymphoid follicles. FDCs trap and retain immune complexes to stimulate an immune response. FDCs also express milk fat globule epidermal growth factor 8 (MFGE8) to facilitate the removal of apoptotic cells in secondary lymphoid organs. Role of B cells in prion disease. The recognition that B cells are required for neuroinvasive scrapie58 was surprising and triggered a plethora of follow-up studies. Expression of PrPC is necessary to sustain prion replication59, and this requirement has been exploited to identify the cells that enable prion replication in SLOs. PrPC is expressed at moderate levels in circulating lymphocytes, including in B cells. However, PrPC expression in B cells is not required for prion neuroinvasion60, and PrPC expression solely in B cells is not sufficient for prion replication61. In addition, chimeric Prnp–/– mice that have been reconstituted with wild-type bone marrow or fetal liver cells can support the replication of the Rocky Mountain laboratory (RML) prion strain in the spleen62,63, even though optimal replication requires PrPC expression in both the haematopoietic and the stromal compartments63. This property could be prion strain-dependent because similar experiments carried out with the ME7 strain of rodent-adapted scrapie prions showed no replication in chimeric Prnp–/– mice that were reconstituted with wildtype bone marrow64. However, other reports indicate substantial splenic ME7 prion infectivity in Prnp–/– mice that were reconstituted with wild-type or Prnp-overexpressing bone marrow 65. Collectively, these data indicate that splenic prion replication requires a PrPC-expressing cell of stromal origin that depends, directly or indirectly, on B cells, and this is irrespective of PrPC expression in B cells. Role of FDCs. Early observations had identified follicular dendritic cells (FDCs) as a site of PrPSc accumulation in the lymphoid tissues of prion-infected mice52. In addition, SCID mice lacking functional FDCs succumb to intracerebral, but not to intraperitoneal, infection with the Fukuoka-1 prion strain52. As FDCs are derived from ubiquitous stromal perivascular precursor cells66 and they express high levels of PrPC, they were thought to represent a candidate for peripheral prion replication (FIG. 2). Indeed, treatment with a soluble lymphotoxin-β receptor immunoglobulin (LTβR-Ig) results in the ablation of mature FDCs from the spleen, abolishes splenic prion accumulation and slows neuroinvasion after intraperitoneal scrapie prion inoculation67, but it does not alter prion pathogenesis after intracerebral inoculation68. Similar effects were obtained using an inhibitor of tumour necrosis factor receptor (TNFR)69. However, dedifferentiation of FDCs following treatment with LTβR-Ig was efficient at interfering with prion infection only when applied before, but not after, intraperitoneal or oral challenge with prions70. Also, mice lacking LTα, LTβ, LTβR, LTα and TNF, all resisted intraperitoneal prion infection and contained no prion infectivity in their spleens and, if present, in their lymph nodes71. Collectively, these data indicate that FDCs have a crucial role within lymphoid tissues for early peripheral retention and replication of lymphotropic strains of prions. Dedifferentiating FDCs might represent a valid option in post-exposure prophylaxis against prion infections12. Role of complement in prion disease. As FDCs trap and retain opsonized antigens within SLOs through the CD21 and/or CD35 complement receptors, several studies have investigated the effect of ablation of complement components on prion pathogenesis. Genetic ablation of complement receptors and the complement components C3, C1q, C2 and factor B, individually or in combination, or the temporary depletion of C3 led to increased resistance to peripheral prion infection in mice72,73. Ablation of stromal cell, but not of haematopoietic cell, CD21 and/ or CD35 resulted in increased resistance to intraperitoneal prion inoculation74. These data indicate an important role for opsonizing complement components in prion pathogenesis. There might also be other retention mechanisms for prions72. 892 | DECEMBER 2013 | VOLUME 13 www.nature.com/reviews/immunol © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS a Tonsils Spleen Lymph nodes Peyer’s patches PrPSc B cell FDC B cell zone T cell zone T cell b Normal ontogenesis of SLOs Chronic inflammation leading to tertiary lymphoid organs Prion replication and accumulation PrPC Marginal sinus pre-FDC MFGE8 PDGFRβ Blood vessel Perivascular pre-FDC TNFR LTβR LTα1β2 PrPSc PrPSc aggregate PrPC TNF Mature FDC FcγRIIB B cell B cell or LTi cell CD21 and/or CD35 Figure 2 | Peripheral prion replication and the involvement of FDCs. a | Peripherally acquired prions replicate in lymphoid follicles of secondary lymphoid organs (SLOs; such as tonsils, spleen, Peyer’s patches in the intestines and lymph nodes) and are mainly associated with follicular dendritic cells (FDCs). b | During normal ontogenesis of SLOs, FDCs emerge from platelet-derived growth factor receptor-β (PDGFRβ)-expressing ubiquitous perivascular pre-FDCs through an intermediate cell termed the marginal sinus pre-FDC. FDC maturation requires exposure to B cell- or lymphoid tissue inducer (LTi) cell-derived lymphotoxin-αβ heterotrimers (LTα1β2) for the first transition from the perivascular pro-FDC stage to the marginal sinus pre-FDC stage, and exposure to B cell-derived LTα1β2 and tumour necrosis factor (TNF) for the second transition from the marginal sinus pre-FDC stage to the mature FDC. This process is accompanied by upregulation and downregulation of numerous transcripts. Similarly, during chronic lymphocytic inflammation, perivascular pre-FDCs can differentiate into mature FDCs, thereby favouring the formation of tertiary lymphoid organs (TLOs), potentially at any site of the body. Mature FDCs express high levels of cellular prion protein (PrPC) and are involved in peripheral prion replication and accumulation. FcγRIIB, low affinity immunoglobulin-γ Fc region receptor II-B; MFGE8, milk fat globule epidermal growth factor 8; LTβR, LTβ receptor; PrPSc, scrapie prion protein; TNFR, TNF receptor. FDCs: prion factory or trap? An important question is do FDCs contain replicating prions or are they just trapping prions that are produced in close proximity to them? Mabbott and colleagues75 devised an elegant way to study the effect of Prnp expression or ablation specifically in FDCs on splenic prion replication. Complement receptor type 2 (Cr2), which encodes the complement receptor component CD21, is known to be expressed in FDCs and mature B cells. Reciprocal bone-marrow transplantation between mice with the appropriate genotypes (Cd21– Cre × Prnpstop/stop or Prnpflox/flox mice) generates mice in which Prnp is selectively expressed or deleted either in FDCs or in mature B cells. Interestingly, PrPC expression on FDCs turned out to be necessary and sufficient to sustain splenic replication of ME7 prions75. Studies identifying genes that are specific for FDCs will be instrumental in increasing our understanding of the contributions of FDCs to health and disease, including to prion diseases. NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | DECEMBER 2013 | 893 © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS Ectopic FDCs and ectopic prions. Chronic lymphocytic inflammation, resulting in FDC-containing lymphoid follicles in the parenchyma of affected organs, can enable ectopic prion replication — a process that is dependent on the presence of LTα and its receptor 76. When kidneys are affected, prion replication can be associated with prion excretion in urine (known as prionuria), even in pre-symptomatic animals77. The relevance of these findings, which were originally made in transgenic mice and were subsequently reproduced in experimentally inoculated deer 78, was further confirmed by the observation that coincident mastitis and scrapie infection in field sheep can result in PrPSc deposition within mammary glands79. Furthermore, it has been shown that prions are present in the colostrum and milk of sheep80 and can thereby be vertically transmitted81, a process that can be favoured by mastitis82. Overall, these observations suggest that chronic lymphocytic inflammation can function as a modifier of prion diseases by extending the affected tissue distribution. Another important implication is that prion excretion in milk, which is probably exacerbated by concomitant mastitis, could represent a natural route of scrapie transmission within flocks. It remains unclear whether this applies also to BSE and, if so, whether dairy products represent a risk to public health80. Despite the key role of FDCs during peripheral replication of prions, scenarios have been identified in which prions can replicate and accumulate in the absence of mature FDCs. These include the lymph nodes of Tnf–/– and Tnfr1–/– mice71,83 as well as soft-tissue granulomas84. Neuroinvasion by prions Peripheral nerves facilitate neuroinvasion. After replication and accumulation within SLOs, prions enter the CNS, where they ultimately cause neurotoxicity. The innervation pattern of SLOs is primarily sympathetic, and experimental models show that prion agents spread from SLOs to the CNS through the autonomic nervous system56,85–87. Chemical or immunological sympathectomy prevented or significantly delayed peripheral prion pathogenesis88. Conversely, sympathetic hyperinnervation of SLOs in transgenic mice shortened prion incubation, which shows that sympathetic innervation of SLOs is rate limiting for prion neuroinvasion88. Disease-associated PrP and prion infectivity have been found in the enteric nervous system of sheep with scrapie89, deer with CWD90 and humans with vCJD91, which suggests that this could represent another portal of entry after peripheral exposure. Mastitis Inflammation occurring in the mammary gland. It can be caused by infection or by blockage of milk ducts. From FDCs to nerves: mind the gap. Manipulation of the distance between FDCs and splenic nerve endings has led to the conclusion that the relative positioning of FDCs to nerves controls the speed of neuroinvasion92. However, the sessile nature of FDCs and the differential localization of FDCs and nerve endings in microanatomical compartments within SLOs raise the question of how prions spread from FDCs to the nerves. Different scenarios have been envisaged, including direct cell-to-cell contact, vesicle-associated infectivity (for example, prion transmission through exosomes93), tunnelling nanotubes94 and free-floating infectious particles95. After nerves have been invaded, prions travel through the spinal cord to ultimately reach the brain. The mechanisms underlying this process are not fully understood. Incoming PrPSc might convert PrPC at the axolemma surface, thereby initiating a domino-like cascade or, alternatively, prions could be internalized at the nerve endings and be transported in a retrograde manner96. Peripheral immune responses to prions From the discussion above, it is clear that components of the immune system can contribute to the spread of prions. However, an important question is whether the immune system can actually mount a protective response to prions. Toll-like receptors (TLRs) are key mediators of innate immune responses. Prions or their components might be able to trigger TLRs. Following prion infection, TLR signalling seems to be protective under certain conditions. Mice deficient in functional TLR4 signalling or in interferon-regulatory factor 3 (IRF3), which is a myeloid differentiation primaryresponse protein 88 (MYD88)-independent transcription factor that is activated downstream of TLRs, showed accelerated prion pathogenesis following intraperitoneal infection97,98. Interestingly, Myd88–/– mice, which are defective in most TLR-mediated responses, succumb to disease to a similar extent to wild-type mice following intraperitoneal inoculation99. Prion infection usually does not trigger apparent adaptive immune responses. This is probably due to selftolerance caused by the similar immunogenicity of PrPSc with PrPC; PrPC is ubiquitously expressed by the host. Immune tolerance prevents robust immune responses to prions, and PrP-specific antibodies are not detected in animals infected with prions100. Although subtle alterations in cellular homeostasis were observed in the lymphoid organs of animals that are infected with prions101, immune system function was not compromised102. The effect of these alterations on prion pathogenesis might be negligible, as interleukin-6 (IL-6)-deficient mice or CD40 ligand (CD40L)-deficient mice, which have impaired germinal centre development, succumbed to disease at the same rate as wild-type mice following intraperitoneal inoculation103,104. CNS immune responses to prions Progressive deposition of prions in the brain leads to fatal spongiform encephalopathies, which manifest as synaptic and neuronal loss, vacuolation and neuroinflammation. Neuroinflammation that is associated with prion infection is characterized by the activation of astrocytes and microglia, which are the principal immune cells in the CNS11. Microglial cell activation. Microglial cell activation is evident in patients with TSEs105 and in animal models of prion diseases106, but investigations into the function of microglia in prion diseases have been hampered by technical limitations. The cerebellar organotypic 894 | DECEMBER 2013 | VOLUME 13 www.nature.com/reviews/immunol © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS A Astrocyte MFGE8 promotes phagocytosis of MFGE8 apoptotic neurons PrPC Microglial cell PrPSc accumulation Microglia clear apoptotic neurons Damaged neuron Excessive prion accumulation leads to neurodegeneration Healthy neuron B a Prnp knockdown PrPC production Prnp DNA Prnp mRNA b Antibodies prevent prion conversion PrPC → PrPSc conversion c Antibodies prevent prion aggregation PrPSc aggregate formation PrPSc Prion fibril d Compounds stabilize prion fibrils f Compounds interfere Stabilized prion fibril e Antibodies or compounds promote protein clearance Degraded prion fibril with neurotoxicity PrPC-mediated PrPSc toxicity Neurotoxicity Figure 3 | Prion-induced neurodegeneration and potential therapeutic targets. A | Prion infection elicits neuronal damage and glial activation. Astrocyte-released milk fat globule epidermal growth factor 8 (MFGE8) facilitates phagocytosis of apoptotic neurons by microglia. Overall, microglia function as scavengers and have neuroprotective roles in prion pathogenesis. However, microglia-mediated clearance might become overwhelmed by the progressive accumulation of scrapie prion protein (PrPSc). It is possible that excess prion accumulation in the brain could reprogramme microglia into a pro-inflammatory phenotype, which might facilitate the spread of prions within the central nervous system, ultimately leading to worsening of the disease and neurodegeneration. B | Select stages of prion pathogenesis can offer therapeutic targets for treating or preventing transmissible spongiform encephalopathies. Knockdown of the gene encoding prion protein (Prnp) or pharmacological downregulation of cellular prion protein (PrPC) can interfere with prion conversion and neurotoxicity (part a). Antibodies or other compounds can specifically capture prions or can otherwise prevent the conversion of PrPC into PrPSc (part b) or the formation of higher-order aggregates (part c). Compounds can stabilize prion fibrils, thereby interfering with the process of prion replication and neurotoxicity (part d). Antibodies or other compounds can promote natural protein-aggregate-clearing mechanisms, thereby interfering with the process of prion replication and neurotoxicity (part e). Finally, compounds can interfere with the neurotoxic pathway mediated by PrPC at the neuronal cell membrane (part f). cultured slice (COCS) system, in which in vivo prion pathogenesis can be faithfully reproduced, provides a powerful tool for studying microglial cell functions in prion infections 107. Depletion of microglia resulted in markedly enhanced PrPSc deposition and augmented prion infectivity 108. Similar effects were recorded in mice that were deficient for milk fat globule epidermal growth factor 8 (MFGE8; also known as lactadherin), which is secreted by astrocytes and promotes phagocytic engulfment and clearance. MFGE8-deficient mice showed accelerated prion pathogenesis and increased levels of apoptotic cell remnants, which suggests that MFGE8-mediated apoptotic cell clearance by microglia quells prion accumulation109. The effect of MFGE8 depletion was visible only in certain mouse strains, which implies that there are further polymorphic determinants of prion removal. These experiments revealed a protective function for microglia in prion disease and potential insights about the crosstalk between microglia and astrocytes in neuroinflammation (FIG. 3). Ultimately, microglia-mediated prion clearance in vivo is insufficient, even after intracerebral lipopolysaccharide (LPS) treatment to prime the immune system110. The failure to clear prions might convert microglia from the phagocytic M2 phenotype into the pro-inflammatory M1 phenotype, consequently contributing to disease pathobiology by the spreading of prions or the secretion of cytotoxic mediators. Perhaps manipulating microglia to adopt the M2 phenotype might lower prion levels and ameliorate pathology. Cytokines induced by prion infection. Prion infection induces the production of pro-inflammatory cytokines, such as IL-1α, IL-1β, TNF and IL-6, in patients with TSEs111 and in some mouse models of prion disease112,113 but not in ME7-infected C57BL/6 mice114. Intriguingly, the anti-inflammatory cytokine transforming growth factor-β (TGFβ) was also significantly induced in mice that were infected with prions115. Similarly, in patients with CJD, levels of the anti-inflammatory cytokines IL-4 and IL-10 are increased in the cerebrospinal fluid116. To elucidate whether the induction of these cytokines is involved in prion pathogenesis, various mouse models deficient for or overexpressing certain cytokines have been challenged with prions. Although most of the cytokines do not seem to be important contributors to prion pathogenesis in the CNS (TABLE 2), depletion of IL-1 receptor 1 (IL-1R1), which is the receptor for IL-1α and IL-1β, resulted in a small but significant incubation prolongation117,118. This effect is probably due to delayed astrocytosis, although augmented microglial activation might contribute by enhancing phagocytosis of prions in Il1r1–/– mice. The role of the IL-1R pathway in prion pathogenesis was also indicated by an association study on the polymorphisms in the Il1r1 locus and the incubation time of mouse prion disease119. Conversely, deficiency of IL-10 rendered mice on a 129/Sv background much more susceptible to prions following both intraperitoneal and intracerebral inoculation120, which implies that IL-10 has a neuroprotective NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | DECEMBER 2013 | 895 © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS Table 2 | Role of cytokines and chemokines in prion pathogenesis in the central nervous system Mouse line Genetic background Tnf–/– mice 129/Sv × C57BL/6 ME7 No 103 C57BL/6 RML No 71,118 Tnfr1–/– mice Prion strain Effects (in comparison with wild-type mice with the same genetic background) Refs 129/Sv RML No 58 129/Sv × C57BL/6 RML No 71 B6;129S7/SvEvBrd RML No 118 B6;129S7/SvEvBrd RML No 118 Il6 mice 129/Sv × C57BL/6 ME7 No 103 Il1r1–/– mice C57BL/6 139A Delayed astrocytosis, augmented microglial activation and prolonged incubation time by 22–25 days 117 B6;129S1/Sv RML Prolonged incubation time by 21 days 118 Tgfb1 mice NIH/Ola RML No 118 Tgfb1 transgenic mice SJL/J RML No 118 Tgfbr2Δk × tTA × Prnp mice* C57BL/6J × FVB/N RML No 118 Il4–/– mice BALB/c RML Shortened incubation time by 29 days at low-dose inoculum 120 Il13–/– mice BALB/c RML Shortened incubation time by 39 days at low-dose inoculum 120 Il10–/– mice 129/Sv RML Accelerated inflammation and shortened incubation time by 41–82 days 120 129S6 RML No 118 C57BL/6J RML Shortened incubation time by 34 days 118 C57BL/6J ME7 Unaltered early behavioural dysfunction, but delayed incubation time by 2–3 weeks 121 Tnfr2–/– mice –/– +/– +/– –/– Ccl2 mice +/– +/– C57BL/6J RML No 122 –/– Ccr1 mice C57BL/6 RML Enhanced ERK1 and ERK2 activation, shortened incubation time by 15 days 123 Ccr2–/– mice C57BL/6J RML No 118 Ccr5–/– mice C57BL/6J RML No –/– 118 Sc Cxcr3 mice C57BL/6 139A Accelerated PrP accumulation, reduced microglial activation and pro-inflammatory cytokine production and delayed incubation time by 20–30 days Cxcr5–/– mice 129/Sv RML No 125 92 Ccl, CC-chemokine ligand; Ccr, CC-chemokine receptor; Cxcr, CXC-chemokine receptor; ERK, extracellular signal-regulated kinase; Il, interleukin; Il1r1, IL-1 receptor 1; Prnp, gene encoding prion protein; PrPSc, scrapie prion protein; RML, Rocky Mountain laboratory; Tgfb1, transforming growth factor-β1; Tnf, tumour necrosis factor; Tnfr, TNF receptor. *These mice neuronally express a transdominant-negative kinase-deficient mutant of TGFβ receptor 2. M2 phenotype Activated macrophages or microglia that show phagocytic behaviour and express factors such as interleukin-4 (IL-4), IL-10 and arginase 1. M1 phenotype Activated macrophages or microglia that show pro-inflammatory features and express factors such as interleukin-1β, tumour necrosis factor and inducible nitric oxide synthase. function in prion disease. Nevertheless, the role of IL-10 in prion pathogenesis seems to be context dependent because mild prion disease acceleration was observed in Il10–/– mice on a C57BL/6J background but not on a 129S1/SvImJ background118. Chemokines induced by prion infection. Increased chemokine expression occurs in various neurodegenerative disorders, including prion diseases. CC-chemokine ligand 2 (CCL2; also known as MCP1) is progressively upregulated in ME7-infected C57BL/6 mice. However, survival time following prion infection was only slightly prolonged in CCL2-deficient mice, and deletion of this chemokine had no effect on the levels of microglial cell activation or neuronal damage observed in response to prion infection 121. This effect might also be prion-strain dependent because CCL2-deficient mice did not show any delay in disease progression after intracerebral inoculation with RML prions122. Similarly, mice deficient for the CCL2 receptor CC-chemokine receptor 2 (CCR2) had an unaltered disease course compared with wild-type mice after RML prion infection118. CCL5 (also known as RANTES) and its receptors CCR1, CCR3 and CCR5 are also upregulated in mouse prion models 115. Following intracerebral 896 | DECEMBER 2013 | VOLUME 13 www.nature.com/reviews/immunol © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS inoculation with RML prions, Ccr1–/– mice showed more robust induction of CCR5 and CCL3 than wildtype mice, which suggests a compensatory mechanism. This induction resulted in enhanced activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2, and accelerated disease progression 123. However, mice deficient for CCR5 developed prion disease to a similar extent to wild-type mice following intracerebral inoculation of RML118. Hence, it is still unclear how much these components influence prion pathogenesis. Levels of the chemokines CXC-chemokine ligand 9 (CXCL9) and CXCL10, which signal through CXCchemokine receptor 3 (CXCR3), are also increased in prion diseases117,124. Cxcr3–/– mice intracerebrally infected with 139A prions showed prolonged incubation time but enhanced PrPSc accumulation125. Further analysis revealed that deletion of CXCR3 resulted in attenuated microglial activation and consequently decreased phagocytosis and degradation of PrPSc after prion infection, which possibly explains the enhanced deposition of PrPSc. Absence of CXCR3 caused more pronounced astrocytosis but reduced the production of pro-inflammatory cytokines in prion disease, which possibly accounts for the prolonged incubation time. The chemokine CXCL13 (also known as BLC) is also upregulated in prion diseases124,126, but mice deficient for its receptor, CXCR5, had similar incubation time compared to wild-type mice following intracerebral inoculation with RML prions92. Finally, the chemokine axis CX 3C-chemokine ligand 1 (CX 3CL1)–CX 3Cchemokine receptor 1 (CX 3CR1) — which has an important role in microglial activation and amyloid-βand tau protein-mediated pathology127 — is altered in prion diseases 128,129, although the relevance of these changes remains to be determined. NF‑κB signalling in prion infection. The nuclear factor-κB (NF-κB) signalling pathway is involved in numerous physiological and pathological conditions, including in the induction of inflammatory cytokines, and chemokines, and in the regulation of apoptosis. NF-κB is activated in astrocytes of mice infected with prions130. Moreover, enhanced binding, but no transcriptional activity, of NF-κB was observed in neuroblastoma cells after prion infection, leading to mitochondria-mediated apoptosis that is associated with decreased expression of the anti-apoptotic protein B cell lymphoma-XL (BCL-XL)131. To investigate the role of NF-κB in prion diseases, mice deficient for components of the canonical NF-κB pathway (Nfkb1–/–, p65CNS−KO, inhibitor of NF-κB kinase subunit-β (Ikkb)CNS−KO and IkkgCNS−KO mice), of the non-canonical NF-κB pathway (Nfkb2–/– and IkkaAA/AA mice), or of NF-κB target genes (Bcl3–/– mice) were intracerebrally inoculated with prions. Surprisingly, only the mice with impairments in the non-canonical pathway showed any reduction in disease incubation time. Therefore, in vivo data suggest that NF-κB-mediated signalling is not a major determinant of prion pathogenesis131,132. Immunotherapy for prion diseases As discussed above, mice with various states of immunodeficiency are resistant to peripheral prion infection58,71. Conversely, pro-inflammatory conditions increase the susceptibility to prion infection and promote peripheral prion deposition24,76,77,79,133. Furthermore, the lymphoid tissue might be more permissive than the brain to cross-species transmission47. As it precedes neuroinvasion, the lymphoid replication phase provides a window for post-exposure prophylactic and therapeutic interventions against peripherally acquired prions. Disease progression can be impeded through dedifferentiating FDCs by blocking LTβR67,68,70 or TNFR1 (REF. 69) signalling, inhibiting prion trapping by FDCs through the modulation of the complement system72–74,134, or by sympathectomy 88. Encouragingly, when applied early following infection, these therapeutics can significantly decrease splenic PrPSc accumulation and/or prolong the intraperitoneally inoculated prion incubation time in mice. Meanwhile, numerous strategies targeting different stages of prion diseases are currently being explored (FIG. 3). Targeting innate versus adaptive immune responses. The role of the innate immune system in prion pathology remains unclear. Although some reports suggested that TLR stimulation might provide some benefits during prion infection97,98, others reported contradictory findings99. An early study found repeated TLR9 stimulation to be protective against prion pathogenesis135, but ultimately this was attributable to iatrogenic alterations in the morphology and function of lymphoid structures136. The potential of antibody-mediated therapy for prion disease was first reported in a cell-free model showing that PrP-specific antisera could neutralize prion infectivity 137. In cell culture models, the monoclonal antibody 6H4 or the monovalent antibody fragments (D13, D18, R1 and R2) that are specific for PrP efficiently suppressed prion replication in mouse neuroblastoma cells that were chronically infected with prions 138,139. These results, together with the initial success of amyloid-β immunotherapy in mouse models of Alzheimer’s disease140, have encouraged the exploration of antibody-mediated immunotherapy for prion disease, as discussed below. Active immunization against prions. Active immunization against prions is challenging because immune responses are stifled by tolerance to PrP. To provide proof-of-principle for immunotherapy, transgenic mice expressing the μ-chain of the PrP-specific antibody 6H4 (6H4μ) were generated. Encouragingly, expression of this transgene antagonized prion pathogenesis 141. Although transgenesis is impractical as a clinical strategy for human prion diseases, this pioneering study indicates the potential value of antibodies as prophylactic and therapeutic treatment strategies for prion diseases. Various vaccination strategies have attempted to break self-tolerance to prions with limited success (TABLE 3). Synthetic PrP peptides (PrP31–50 and PrP211–230) elicited immune responses and reduced PrPSc levels in NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | DECEMBER 2013 | 897 © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS Table 3 | Active immunization for prion disease* Mouse strain Immunogen Adjuvant and immunization strategy Prion strain and Effects on delay infection route development Refs CD1 Recombinant mouse PrP23–230 CFA and IFA, s.c. 139A, i.p. 16-day delay 143 NMRI Peptide PrP105–125 covalently linked to KLH Montanide IMS1313, i.p. 139A, oral 23-day delay 176 NMRI Recombinant mouse PrP90–230 Montanide IMS1313, i.p. 139A, oral Ineffective 176 C57BL/6 × 129/Sv Dimeric mouse recombinant PrP23–231 CFA and IFA, in combination with OX40-specific antibodies; s.c. for CFA and i.p. for IFA RML, i.p. No effect 144 CD1 Salmonella spp. vaccine strain expressing mouse PrP In combination with sodium bicarbonate and alum, oral 139A, oral Full protection of mice with high mucosal anti-PrP titres 177 BALB/c Recombinant mouse fragment PrP90–230 In sodium bicarbonate buffer with cholera toxin, intranasal 139A, oral 9-day delay 178 C57BL/6 PrP141–159 or PrP165–178 conjugated with KLH Mycobacterium avium subsp. avium-based adjuvant (AdjuVac; National Wildlife Research Center), intramuscular RML, i.p. 21–25-day delay 179 BALB/c Recombinant bovine PrP25–242 CFA and IFA, i.p. Fukuoka-1, i.p. 31-day delay 180 BALB/c Recombinant mouse PrP23–231 CFA and IFA, i.p. Fukuoka-1, i.p. No effect 180 C57BL/6 pCG plasmid containing mouse PrP cDNA fused with tetanus toxin (P30) CpG, intradermal and s.c. RML, i.p. No effect 181 C57BL/6 Peptide PrP98–127 or PrP158–187 CpG and IFA, s.c. 139A, i.p. 15–20-day delay 182 Sc FVB/N Dynabeads adsorbed-native PrP CFA and IFA, s.c. RML, i.c. and i.p. 22-day delay in i.p., and no effect in i.c. 183 C57BL/6 Dendritic cells expressing human PrP together with adenovirus Intramuscular injection 139A, i.p. 37-day delay 184 C57BL/6 Dendritic cells loaded with peptides PrP98–127 i.p. 139A, i.p. 40-day delay 185 BALB/c 6H4-epitope mimicking bacterial succinylarginine dihydrolase CFA and IFA, i.p. Fukuoka-1, i.p. 31-day delay 186 C57BL/6 Aggregated PrP CFA and IFA, s.c. RML, i.p. 28-day delay 145 129/Ola Plasmid pCMV–UbPrP or pCMV–PrPLII Anterior tibial muscle BSE, i.c. 2-week delay 146 BSE, bovine spongiform encephalopathy; CFA, complete Freund’s adjuvant; i.c, intracerebral; IFA, incomplete Freund’s adjuvant; i.p, intraperitoneal; KLH, keyhole limpet haemocyanin; pCMV–PrPLII, plasmid containing prion protein fused to the lysosomal-targeting signal from lysosomal membrane protein 2 under the control of the cytomegalovirus promoter; pCMV–UbPrP, plasmid containing prion protein fused to ubiquitin under the control of the cytomegalovirus promoter; PrP, prion protein; RML, Rocky Mountain laboratory; s.c., subcutaneous. *Adapted from REF. 187. prion-infected mouse neuroblastomas transplanted into A/J mice142. Only a slight delay (16 days) in disease onset was observed in mice that were immunized with recombinant mouse PrP (recPrP23–230)143. The incubation time correlated with PrP-specific antibody titres, which suggests that the beneficial effect was immune mediated. Other active immunization attempts resulted in neither a considerable PrP-specific antibody titre nor a significant increase in survival time. These discrepancies might pertain to differences in immunogens, regimens, mouse strains or prion strains — but the prospects of active immunization are dim 144. Interestingly, the effect of active immunization might extend beyond PrP to have consequences on immune cell status. Immunization of C57BL/6 mice with aggregated PrP and complete Freund’s adjuvant resulted in an acute depletion of mature FDCs from the spleen and consequently a prolongation of incubation time (28 days) after peripheral prion challenge145. Active immunization typically failed to mitigate prion disease in cases in which it is caused by intracerebral challenge or in cases in which neuroinvasion had already occurred. This might be due to the blood– brain barrier limiting penetrance of antibodies into the CNS. However, immunizing 129/Ola mice with a DNA construct expressing mouse PrP fused with lysosome membrane protein 2 (LIMP2; also known as SCARB2) or ubiquitin resulted in a breakage of host tolerance to PrP and an induction of PrP-specific antibodies. Surprisingly, this DNA vaccination delayed disease onset by 2 weeks in mice that had been intracerebrally inoculated with mouse-adapted BSE prions146. Passive immunization. Even if it were effective, active immunization carries potential risks, including the remote possibility of converting immunogens into infectious prions. However, numerous antibodies against various PrP epitopes have been generated by immunizing 898 | DECEMBER 2013 | VOLUME 13 www.nature.com/reviews/immunol © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS Prnp–/– mice with synthetic PrP peptides, recombinant PrP or native PrP purified from tissues. Peripheral administration of the PrP-specific monoclonal antibodies ICSM18 (which recognizes PrP146–159) and ICSM35 (which recognizes PrP91–110) to FVB/N mice147 inhibited prion accumulation in the spleen. Continuous treatment with these antibodies prolonged mouse survival times to 500 days post inoculation. By contrast, mice treated with a control antibody succumbed at 197 days post inoculation. However, antibody treatment did not prevent disease when it was started at clinical disease onset (129–136 days post inoculation) or in mice intracerebrally inoculated with RML prions147. When CD1 mice were intraperitoneally given the PrP-specific antibodies 8B4 (which is specific for PrP34–52) or 8H4 (which is specific for PrP175–185) immediately after intraperitoneal inoculation with ME7 prions, the incubation time was delayed by 10% (REF. 148). Moreover, after intraperitoneal inoculation with 22L prions, peripheral administration of the PrP-specific antibody 6D11 (which recognizes the PrP97–100 epitope) to CD1 mice for 4 or 8 weeks suppressed PrPSc replication in lymphoid tissues and prolonged incubation times149. Disappointingly, a recent study in which C57BL/6 mice infected with RML prions were administered a high dose of the PrP-specific antibody W226 or its recombinant single-chain variable fragment (scW226) found them to have only a minor protective effect150. As W226 and ICSM18 antibodies recognize the same PrP epitope (PrP145–153), these divergent effects are difficult to explain. Clearly, more research is needed to understand the role of passive immunization in prion disease. Risks associated with immunotherapy. Passive immunization has so far been ineffective in slowing disease progression in mice following intracerebral inoculation or in mice already showing clinical signs. Unfavourable pharmacokinetics might account for the lack of success, but even the intracerebral delivery of PrP-specific reagents through osmotic pumps151 or adeno-associated virus (AAV) vectors152 has had disappointing results. In addition, the intracerebral injection of certain PrPspecific antibodies, even in their monovalent form, is reported to be neurotoxic153,154. Interestingly, the toxicity of these PrP-specific antibodies is dependent on two distinct modules of PrPC; the ‘regulatory’ globular domain of PrPC, which is bound by the antibody, and the ‘executive’ flexible tail of PrPC, which mediates toxicity. The binding of PrP-specific antibodies to the 1. 2. 3. 4. 5. Cuille, J. & Chelle, P. L. Experimental transmission of trembling to the goat. Comptes Rendus Séances Acad. Sci. 208, 1058–1160 (1939). Gajdusek, D. C. & Zigas, V. Degenerative disease of the central nervous system in New Guinea; the endemic occurrence of kuru in the native population. N. Engl. J. Med. 257, 974–978 (1957). Duffy, P. et al. Letter: possible person-to-person transmission of Creutzfeldt–Jakob disease. N. Engl. J. Med. 290, 692–693 (1974). Will, R. G. et al. A new variant of Creutzfeldt–Jakob disease in the UK. Lancet 347, 921–925 (1996). Llewelyn, C. A. et al. Possible transmission of variant Creutzfeldt–Jakob disease by blood transfusion. Lancet 363, 417–421 (2004). globular domain triggers a cascade of events and eventually leads to neuronal cell death that is mediated by the flexible tail154. Therefore, any clinical trials of passive immunization will require great caution because PrP-specific antibodies might cause neurotoxicity and exacerbate clinical deterioration. PrP–Fc2 mediated therapy. Soluble dimeric receptors, also termed immunoadhesins, consist of the Fc portion of IgG fused to various binding domains155. Transgenic mice expressing a prion immunoadhesin (PrP‒Fc2) showed resistance against prion disease, and PrP‒Fc2 was not converted into a self-propagating, protease-resistant isoform. These properties encouraged the investigation of the interaction of PrP–Fc2 with PrPC and the role of PrP‒Fc2 in prion pathogenesis. Mice expressing both PrPC and PrP–Fc2 showed a decrease of PrPSc accumulation and a delay in the onset of disease156. PrP‒Fc2 binds to PrPSc and functions as a dominant-negative prion antagonist. The peculiar features of PrP–Fc2 indicate that soluble PrP derivatives might represent a novel category of antiprion molecules. Future directions Despite intense investigations, some fundamental aspects of the immunobiology of prions are still unclear. The physiological function of PrPC in the immune system remains enigmatic. The absence of Prnp–/– mice on a pure background and that are devoid of the shortcomings that are caused by gene targeting in embryonic stem cells has so far hampered research in this field. However, recent advancements in genome-editing technologies as well as the implementation of rigorous standards in carrying out and reporting animal experiments leave grounds for optimism. The elucidation of the role of the CNS innate immune system in prion pathogenesis has just started. As for other aspects of neurobiology, there are reasons to believe that progress will continue at a rapid pace. However, research into immunotherapy against prions will have to proceed cautiously in light of the recent realization that certain PrP-specific antibodies, also in monovalent form, can trigger specific PrPC-mediated neurotoxic pathways. 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Microglia: scapegoat, saboteur, or something else? Science 339, 156–161 (2013). 12. Aguzzi, A. & Sigurdson, C. J. Antiprion immunotherapy: to suppress or to stimulate? Nature Rev. Immunol. 4, 725–736 (2004). 13. Bueler, H. et al. Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 356, 577–582 (1992). 14. Bremer, J. et al. Axonal prion protein is required for peripheral myelin maintenance. Nature Neurosci. 13, 310–318 (2010). This study shows that neuronal PrPC and its regulated proteolysis are required to maintain peripheral myelination. VOLUME 13 | DECEMBER 2013 | 899 © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS 15. Miranda, A., Pericuesta, E., Ramirez, M. A. & Gutierrez-Adan, A. Prion protein expression regulates embryonic stem cell pluripotency and differentiation. PLoS ONE 6, e18422 (2011). 16. Petit, C. S. et al. 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A.A. is the recipient of an Advanced Grant of the European Research Council and is supported by grants from the European Union (PRIORITY and NEURINOX), the Swiss National Foundation, the Foundation Alliance BioSecure, the Novartis Research Foundation and the Clinical Research Priority Program (KFSP) of the University of Zurich, Switzerland. M.N. received grants from Collegio Ghislieri (Pavia, Italy) and the Foundation for Research at the Medical Faculty of the University of Zurich. Competing interests statement The authors declare no competing interests. FURTHER INFORMATION BSE Portal on the World Organisation for Animal Health website: http://www.oie.int/en/animal-health-in-the-world/ bse-portal/ ALL LINKS ARE ACTIVE IN THE ONLINE PDF www.nature.com/reviews/immunol © 2013 Macmillan Publishers Limited. All rights reserved