Prions, Cytokines, and Chemokines: A Meeting in Lymphoid Organs

Immunity, Vol. 22, 145–154, February, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.immuni.2004.12.007

Prions, Cytokines, and Chemokines: A Meeting in Lymphoid Organs Adriano Aguzzi* and Mathias Heikenwalder Institute of Neuropathology University Hospital of Zürich Schmelzbergstrasse 12 CH-8091 Zürich Switzerland

Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative diseases of humans and animals. The underlying infectious agent, the prion, accumulates not only in the central nervous system (CNS) but also in secondary lymphoid organs. This review revisits the role of the immune system in peripheral prion pathogenesis while focusing on the mechanisms by which extraneural and extralymphatic prion infectivity develops. Interestingly, the same proinflammatory cytokines and homeostatic chemokines that are involved in lymphoid neogenesis and compartmentalization of immune cells appear to represent the crucial molecular switches responsible for the establishment of extraneural prion reservoirs. Prion diseases continue to be inexorably fatal, and no therapy other than palliation is available. On the other hand, prion infections represent a fascinating biological phenomenon, which has elicited a tremendous interdisciplinary research effort at the interface between neuroscience and immunology. It has long been known that prions not only invade the CNS but also colonize the lymphoid compartment of infected individuals. This finding raises a number of intriguing questions concerning the interface between prions and the immune system. Some of the most inspiring results have been obtained by studying prion pathogenesis in mice whose lymphoid organogenesis was manipulated by genetic or pharmacological means. Significant titers of prion infectivity can be detected in the lymphoreticular system (LRS) in most transmissible spongiform encephalopathies (TSEs). However, the extent of involvement is variable and depends on the tropism of specific prion strains and also on ill-defined host factors (Aguzzi and Sigurdson, 2004). In many instances prion infectivity is detected in the LRS before it arises in the CNS. LRS-associated prion infectivity, or proteinase K (PK)-resistant prion protein (termed PrPSc), was found in human patients suffering from variant and sporadic Creutzfeldt-Jakob disease (vCJD or sCJD), scrapie-affected sheep and goats, terminally BSE-sick cattle, as well as elk and deer suffering from chronic wasting disease (CWD) (Aguzzi and Polymenidou, 2004; Bruce et al., 2001; Glatzel et al., 2003; Mabbott and Bruce, 2003; Sigurdson et al., 2002). However, many fundamental questions regarding the tropism of prions for lymphoid tissues remain unanswered. Also, the mechanisms by which prions are transported from the site of prion replication to peripheral nerves are un-

*Correspondence: [email protected]

Review

known (Aguzzi and Heikenwalder, 2003; Mabbott et al., 1998; Weissmann et al., 2001). In the central nervous system (CNS), prion infections cause characteristic neuropathological lesions with spongiform vacuolation, accumulation of abnormally folded PrPC, designated PrPSc, and neuronal cell loss in the CNS accompanied by proliferation of astrocytes. Prion pathology also includes the brain’s intrinsic macrophages, with large-scale activation and proliferation of microglial cells. One essential requirement for prion pathogenesis has been established beyond all reasonable doubt: if prions multiply by imparting their conformation onto normal cellular host-encoded prion protein (PrPC), organisms devoid of PrPC should be resistant to scrapie. Indeed, lack of the Prnp gene confers absolute resistance against intracerebral and peripheral scrapie infection (Büeler et al., 1993). PrPC is essential for neuronal damage in infected individuals (Brandner et al., 1996; Mallucci et al., 2003). Quite surprisingly, high prion titers in lymphoid organs are not accompanied by significant histopathological changes (Clarke and Haig, 1971; Dickinson et al., 1972), although murine scrapie infection was recently reported to cause an abnormal germinal center reaction in the spleen (McGovern et al., 2004). The Early Days of Prion Immunology Genetic asplenia or splenectomy of mice prior or shortly after peripheral prion challenge significantly prolongs the life span of scrapie-infected mice, whereas thymectomy or genetic athymia has no effect (Clarke and Haig, 1971). Splenectomy after intraperitoneal (i.p.) prion inoculation at various time points has revealed that peripheral prion pathogenesis becomes independent of the spleen once it has reached the spinal cord (Kimberlin and Walker, 1989). However, a splenic replication phase is not obligatory in all rodent TSE models (Kimberlin and Walker, 1986). For example, Tateishi and coworkers have found no effect of splenectomy on incubation times for the Fukuoka-1 strain, a mouse-adapted CJD prion (Manuelidis et al., 1978; Mohri et al., 1987). This is one of several hints that the various prion strains appear to differ in their tissue tropisms (Aguzzi, 2004). Before the invention of fluorescence-activated cell sorting, the cellular distribution of prions in the LRS was studied by separation of lymphocyte cells into various subpopulations based on buoyant density of different adherence of cells to plastic (Lavelle et al., 1972). In these early experiments, it was reported that cells with relatively high specific infectivity had a density characteristic of lymphoblasts, myeloblasts, and macrophages. Enrichment of macrophages did not enhance scrapie infectivity. Already in these relatively crude fractionation attempts it became clear that the stroma contained w10 times more prion infectivity than the pulp (Clarke and Kimberlin, 1984). Furthermore, sublethal doses of γ irradiation, affecting mitotically active hematopoietic cells (but not resident postmitotic cells), administered as a single dose or fractionated dose before or after inocula-

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Figure 1. Necessity of LT Signaling in Lymphoid Organ Development, Maintenance of Lymphoid Microarchitecture, and Prion Replication (A) LTα1β2-expressing CD3−CD4+CD45+ cells bind vascular adhesion molecule 1 (VCAM-1)+ stromal cells through activated α4β1 integrin. This binding ensures LTßR engagement of stromal cells, and induces the production of homeostatic chemokines CCL19, CCL21, CXCL12 and CXCL13, driving lymphoid organogenesis and upregulation of VCAM-1.

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tion, failed to alter the incubation period of the disease (Fraser and Farquhar, 1987). These findings suggested that scrapie replication or accumulation in the LRS depends on radioresistant, postmitotic cells localized within the stroma. However, these experiments did not address the exact cellular and molecular preconditions for prion transport from their entry portals to the peripheral sites of replication, and transmigration of infectivity into the CNS. Interestingly, PrPC itself is involved in transporting prion infectivity from peripheral sites to the CNS: titration experiments indicate that adoptive transfer of wildtype (wt) bone marrow into Prnpo/o mice reconstitutes the capability of the spleen to accumulate prions of the RML strain (Blättler et al., 1997; Kaeser et al., 2001). These results were taken to suggest that PrPC-expressing hematopoietic cells transport prions from the entry site to the LRS, accumulating and replicating prions. Further, reconstitution experiments with wt mice into Prnpo/o mice were insufficient to restore neuroinvasion. Therefore, hematopoietic cells (e.g., B and T cells and dendritic cells) facilitate the transport prions from the peripheral entry site to secondary lymphoid organs, where prions accumulate and/or replicate. However, the elemental compartment for prion neuroinvasion appears to be nonhematopoietic, because it cannot be adopted by bone marrow reconstitution (Blättler et al., 1997; Kaeser et al., 2001; Klein et al., 1997). In conclusion, after peripheral exposure—may this be by ingestion or by peripheral infection—prion pathogenesis is a dynamic process that can be split spatially and temporally into: (1) infection and peripheral replication, (2) transmigration from the periphery to the central nervous system (CNS), and (3) progressive neurodegeneration leading to death. A Chain of Events Leading to Brain Which cells and which molecules, besides PrPC, are needed for efficient prion replication in the LRS and subsequent neuroinvasion? There is no doubt that B cells are of crucial importance for peripheral prion spread and neuroinvasion (Klein et al., 1997). However, PrPC expression on B cells is not required for prion neuroinvasion (Klein et al., 1998; Montrasio et al., 2001). The latter result, combined with the fact that a stromal compartment is the essential mediator of prion inva-

sion, indicates that B cells themselves are unlikely to represent a major replicative compartment for prions. Instead, the apparent requirement for B lymphocytes in peripheral pathogenesis is more likely to derive from indirect effects, such as the expression of cytokines. A prime candidate mediator is the proinflammatory cytokine lymphotoxin α (LTα), which was originally recognized for its cytotoxic effects on normal and transformed cells in vitro and therefore termed cytotoxin (Ruddle and Waksman, 1967; Ruddle and Waksman, 1968). Today, lymphotoxins (LTs) are known to belong to a growing family of structurally related cytokines, the TNF superfamily, along with various ligands such as TNF, CD40L, LIGHT, FasL, BAFF, and their corresponding receptors (Fu and Chaplin, 1999; Locksley et al., 2001). Members of the TNF superfamily serve pleiotropic functions in proliferation, costimulation, regulation of the immune response, apoptosis of particular cell lineages, control of lymphoid organogenesis, as well as maintenance of secondary lymphoid microarchitecture (Fu and Chaplin, 1999; Fu et al., 1997; Locksley et al., 2001). LTα, LTβ, and TNF share structural and functional similarities, including their assembly into functionally active homotrimers and/or heteotrimers. Interestingly, the genes encoding for LTα, LTβ, and TNF are also found in close proximity to each other, within the major histocompatibilty locus (Muller et al., 1987). LTs are mainly expressed on the surface of B cells and activated T cells and exist as heterotrimeric LTα1β2 or LTα2β1, both triggering LTβ receptor (LTßR), expressed by stromal cells (Browning et al., 1993). In addition to T and B cells, LT can be produced by recently characterized CD3−CD4+CXCR5+ precursor cells, which are essential for the development of lymph nodes and Peyer’s Patches (PP) (Mebius et al., 1997; Ware et al., 1992) (Figure 1). The cellular and molecular requirements for the establishment of lymph nodes and tertiary structures are remarkably similar, and hyperactivated lymphocytes can fulfill the role of lymphoid tissue inducers during inflammatory responses (Cupedo et al., 2004a). Although the participation of LTβ leads to membrane anchoring of the ternary complex, LTα can also be secreted as soluble LTα3 homotrimer, signaling via TNFR1, TNFR2, and most likely the herpes simplex virus entry site mediator (HVEM) (Browning et al., 1995,

(B) Cell cluster formation through hematopoietic cells, in contact with various stromal cells, allows transdifferentiation of CD3−CD4−CD45+ cells toward CD3−CD4+CD45+. This leads to subsequent upregulation of LTα1β2 with increased LTßR engagement, and thereby establishes a positive feedback loop. (C) Formation of splenic B cell follicles (left) is mediated through CXCR5 signaling through its ligand CXCL13, which upregulates LTα1β2 expression on B cells. These activated B cells trigger LTßR on stromal cells thereby upregulating CXCL13, again generating a positive feedback loop. (right) At neonatal stage CD3−CD4+CD45+ cells are the predominant source of LTα1β2 expression and respond to CXCL13, whereas B cells do not at this stage of development. CD3−CD4+CD45+ cells induce chemokine expression in the developing lymph nodes thereby initiating organization of nodal microenvironment (e.g., B cell follicles) (Cupedo et al., 2004b). (D and E) Peripheral infection with the prion agent (e.g., orally or intraperitoneally). The prion agent might (with the help of the complement system) be captured and transported by B cells, dendritic or other cells via the afferent lymph or blood system into secondary lymphoid organs, where it accumulates in germinal centers. There, it is mainly replicated by CD35+/FDC-M1+/PrPC high mature FDCs, leading to neuroinvasion and terminal disease of the host. (F) Pharmacological blockade of LTßR signaling via administration of soluble dimeric LTßR-Ig profoundly impairs peripheral prion pathogenesis (Mabbott et al., 2000; Montrasio et al., 2000). (G) Passive immunization with PrP-specific antibodies after prion exposure delays disease and markedly increased the incubation period compared with nonimmunized mice (White et al., 2003).

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1993). LTα mRNA expression is inducible in B cells, whereas LTβ mRNA is constitutively produced: the productive generation of surface LTβ depends on the formation of complexes with LTα (Browning et al., 1993; Worm and Geha, 1994). Surface LT expression enhances production of homeostatic chemokines by stromal cells within the spleen and other secondary lymphoid organs (Ngo et al., 1999; Shakhov et al., 2000). Homeostatic chemokines (e.g., CXCL13), expressed by LT responsive cells (e.g., FDCs), upregulate surface expression of the LT complex on B cells (Ansel et al., 2000), providing a positive feedback loop essential for the organization of lymphoid follicles and for the generation of tertiary follicles in nonlymphoid organs. Moreover, ectopic expression of LTα in kidney and pancreas was shown to induce chronic inflammation with characteristics of organized lymphoid tissue (Kratz et al., 1996). Lymphoid Microarchitecture and Prion Replication Within lymphoid organs, B cells localize to morphologically and functionally distinct compartments, depending on the status of their development. A majority of B cells can be either found in the splenic white pulp or in B cell zones of Peyer’s patches (PP) and lymph nodes (LN). There, B cells not only initiate and control immune responses but also provide LT, TNF, and other factors, contributing to maturation and maintenance of preexisting mature FDCs and microarchitecture of secondary lymphoid organs (Cyster et al., 1999; Fu and Chaplin, 1999). The histogenesis of FDCs is still quite unclear, but there is a near consensus that they are of stromal origin and express high levels of PrPC. The impairment of signals provided by B cells leads to the failure of maintenance of mature FDCs, to abrogation of germinal centers, and to a generally disorganized microarchitecture, as documented in LTα−/−, LTβ−/−, LTβR−/−, and TNFR1−/− mice (Bluethmann et al., 1994; De Togni et al., 1994; Futterer et al., 1998; Koni et al., 1997). Inhibition of the LTßR pathway in mice and nonhuman primates by treatment with LTβR-immunoglobulin fusion protein (LTβßR-Ig) leads to the disappearance and dedifferentiation of mature, functional FDCs, in spleen, in lymph nodes, and also in inflammatory foci containing tertiary follicles (e.g., models of lupus, nephritis, or pancreatitis) (Gommerman and Browning, 2003; Gommerman et al., 2002; Mackay and Browning, 1998). Because FDCs accumulate PrPSc after scrapie infection (Kitamoto et al., 1991) and prion replication in spleen depends on PrPC-expressing FDCs (Brown et al., 1999), it appeared worthwhile to investigate whether depletion of FDCs inhibits prion pathogenesis. Indeed, mice devoid of FDCs and lacking an organized microarchitecture (e.g., LTα−/−, LTβ−/−, and LTßR−/− mice) showed highly reduced efficiency of prion replication after peripheral exposure (Prinz et al., 2002). Encouragingly, even pharmacological blockade of LTβR signaling via administration of soluble dimeric lymphotoxin β receptor (LTβR-Ig) was found to significantly impair peripheral prion pathogenesis (Mabbott et al., 2000; Montrasio et al., 2000). In a recent series of experiments aimed at determining dose-response relationships, we found that treatment of mice with

LTβR-Ig for 1 week before a relatively low-dose i.p. exposure to prions and then every week for only 2 weeks after inoculation completely protected mice from scrapie, whereas control injection of pooled immunoglobulin did not prevent disease (M.H. and A.A., unpublished data, and Figure 1F). FDCs are bifunctional cells: they support the formation and maintenance of the lymphoid microarchitecture but also play a role in antigen trapping, in the capturing of immune complexes by Fcγ receptors, and in the binding of opsonized antigens to the CD21/CD35 complement receptors. For this reason, functional FDCs are believed to express CD21/CD35 in addition to their positivity for “FDC-specific” markers (e.g., FDCM1). These facts made it interesting to investigate the role of the complement cascade in prion diseases. Two independent studies demonstrated that the complement system is highly relevant to prion pathogenesis. Mice genetically engineered to lack complement factors (Klein et al., 2001) as well as mice depleted for the C3 complement component by administration of cobra venom factor (Mabbott et al., 2001) exhibited enhanced resistance to peripheral prion inoculation. In future experiments, it will be exciting to study whether FDC-associated complement receptors have a role in promoting the efficiency and transport of prion infectivity in extraneural compartments. From the Periphery into the Lymphoid System: The First Portal of Entry An early rise in prion infectivity is observed in the distal ileum of orally infected organisms. This applies to several species but was most extensively investigated in sheep and deer (Heggebo et al., 2003; Sigurdson et al., 2002). Western blot analyses and bioassays have shown that PPs accumulate PrPSc and contain high titers of prion infectivity. Similarly, after administration of mouse-adapted scrapie prions (RML strain) mice experience a surge in intestinal prion infectivity as early as a few days after inoculation (Mabbott et al., 2003; Prinz et al., 2003c). Immune cells are crucially involved in the process of neuroinvasion after oral administration: mature FDCs, located in PPs, may be critical for the transmission of scrapie from the gastrointestinal tract (Aguzzi, 2003; Mabbott et al., 2003; Prinz et al., 2003c). However, the cellular basis for prion transmigration from the gut into the lymphoid system is still poorly understood. Membranous epithelial cells (M cells) are believed to be the key sites of antigen sampling for the mucosal-associated lymphoid system (MALT) and have been recognized as major ports of entry for enteric pathogens in the gut via transepithelial transport (Neutra et al., 1996). Interestingly, maturation of M cells is dependent on signals transmitted by intraepithelial B cells. Efficient in vitro systems have been developed in which epithelial cells can be instructed to undergo differentiation to cells that resemble M cells by morphological and functional physiological criteria (Kerneis et al., 1997). This led to the presumption that M cells may represent a site of prion entry—a fact substantiated in coculture models (Heppner et al., 2001a)—but not yet established in vivo.

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From Spleen to Brain: Neuroinvasion Proper Even though the results discussed above clearly show that FDCs are of great importance for peripheral prion pathogenesis, it is counterintuitive that a sessile cell of stromal origin, stably localized in germinal centers of the LRS, should be involved in transporting prions to the CNS. In the past, many studies focused on the temporal and spatial dynamics of neuroinvasion, suggesting that the autonomic nervous system might be responsible for transport from lymphoid organs to the CNS (Clarke and Kimberlin, 1984; Cole and Kimberlin, 1985; McBride and Beekes, 1999). The innervation pattern of lymphoid organs is primarily sympathetic (Felten et al., 1988). Sympathectomy delays the onset of scrapie upon i.p. inoculation of the infectious agent, whereas sympathetic hyperinnervation enhances splenic prion replication and neuroinvasion, suggesting that innervation of secondary lymphoid organs is the rate limiting step to neuroinvasion (Glatzel et al., 2001). However, there is no direct physical synapse between FDCs and sympathetic nerve endings (Heinen et al., 1995). There is strong evidence that the distance between FDCs and splenic nerves affects the velocity of neuroinvasion (Prinz et al., 2003a). FDC positioning was manipulated by ablation of the CXCR5 chemokine receptor, which directs lymphocytes toward specific microcompartments (Forster et al., 1996). In this model the distance between germinal center-associated FDCs and nerve endings was greatly reduced (Forster et al., 1996; Prinz et al., 2003a). CXCR5 deficiency did not affect any aspect of prion pathogenesis within the CNS. However, although after peripheral administration of high doses of prion inocula velocity of pathogenesis was similar in CXCR5−/− and wild-type mice, delivery of smaller inocula resulted in a dose-dependent increase in incubation periods in wt mice. Measurement of the kinetics of prion infectivity titers in the thoracic spinal cord showed that increased velocity of prion entry into the CNS in CXCR5−/− mice is due to repositioning of FDCs in juxtaposition with highly innervated, splenic arterioles. This was validated by prolongation of incubation periods in CXCR5−/− mice depleted for mature FDCs. It remains to be determined whether increased velocity of neuroinvasion results from passive diffusion of prions or whether, alternatively, mobile cells (e.g., germinal center B cells) are involved in an active process. This study also raises the possibility that the spread of infection to peripheral nerves occurs more rapidly in lymphoid tissues, where FDCs are in near proximity to nerves than in the spleen of wt mice, such as PP (Beekes and McBride, 2000; Mabbott and Bruce, 2003). Only recently was it shown that FDCs are crucial to disease progression for only a very short time window after oral scrapie challenge: if FDCs are depleted in this time window, mice will not succumb to scrapie (Mabbott and Bruce, 2003; Mabbott et al., 2003). Because lymphoid infectivity is found in most prion diseases and proinflammatory cytokines and immune cells are involved in lymphoid prion replication (Brown et al., 1999; Kitamoto et al., 1991; Mabbott et al., 2002; Montrasio et al., 2000; Prinz et al., 2003a, 2002), it seemed reasonable to test whether chronic inflammatory conditions

affect prion pathogenesis. We therefore administered prions to mice with various naturally occurring or transgenetically induced inflammatory or autoimmune diseases of kidney, pancreas, or liver. In each of these paradigms, chronic inflammation enabled prion replication in otherwise prion-free organs, and inflammatory foci consistently correlated with LT upregulation and ectopic induction of PrPC-expressing FDC-M1+ cells (Heikenwalder et al., 2005). It is therefore plausible that chronic inflammatory conditions may act as modifiers of natural and iatrogenic prion transmission by expanding the tissue distribution of prions. Interestingly, inflamed organs of prion-inoculated mice lacking LTα or LTβR were devoid of PrPSc and prion infectivity. This indicated that inflammation and prion replication competence are hierarchically linked by LT signaling. It is important to note that as recently shown, dendritic cells resembling mobile cells of the immune system can act as a cellular bridge between the gut lumen and the lymphoid TSE replicative machinery (Huang et al., 2002). As a further extension of this, one could postulate that DCs could therefore also be a cargo machinery for directed transport from the site of prion replication to peripheral nerves within lymphoid organs, thereby enabling neuroinvasion. Moreover, other mobile “elements” (e.g., exosoms; budding endogenous viruses) could serve as transport vehicles of prion infectivity. Additionally, it was shown that splenic CD11c+ cells isolated from scrapie-infected donors and injected intravenously into RAG1−/− mice induced scrapie without accumulation of prions in the spleen (Aucouturier et al., 2001). Idiosyncrasies of the Proposed Model for Neuroinvasion The findings described above have led us and others to hypothesize a linear model of prion dissemination in which prions accumulate primarily in the FDCs, get to the neighboring nervous endings, and from there progress in a neuroretrograde way up to the CNS. Although this scenario is supported by good experimental evidence, it shouldn’t go unmentioned that it does not quite explain how prions reach FDC sites at first hand, how they disseminate secondarily throughout the lymphoid system, and how they travel from FDCs to nerve endings. Each of these questions will need to be addressed in order to achieve a thorough understanding of peripheral prion pathogenesis. Most importantly, the accumulating evidence of blood-borne infectivity in various forms of TSE (Houston et al., 2000; Llewelyn et al., 2004; Peden et al., 2004) implies that mobile cells participate to the spreading of the infection. The nature of such mobile cells is most likely hematopoietic, as wt bone marrow restores prion replication in Prnpo/o spleens (Blättler et al., 1997). Lymphocytes may well contribute to the prion load in blood, as they were shown to acquire infectivity when residing in lymphoid organs (Raeber et al., 1999). If so, the crucial role of B cells for peripheral prion pathogenesis (Klein et al., 1997) may transcend their function as lymphotoxin producers. Increasing evidence suggests that dendritic cells may also represent a candidate “prion transporter” (Aguzzi, 2001; Aucouturier et al., 2001; Hu-

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ang et al., 2002). Finally, micellar membrane components, be they actively extruded from prion-infected cells membranes (“exosomes”) or simply representing residues of dead cells, may represent a further vehicle of prion spread (Fevrier et al., 2004). The Fight against the Prion One may wonder how to translate the impressive body of accrued knowledge of the molecular underpinnings of prion neuroinvasion into practical applications. As in any other infectious disease, three major long-tems goals need to be achieved: (1) refined, sensitive prion diagnostics, (2) methods for postexposure prophylaxis of prion infections, and (3) efficacious therapeutic interventions. The realization that the agent is present in lymphoid organs has indeed paved the way to a variety of lowly invasive diagnostic methods. Whereas 10 years ago the diagnosis of Creutzfeldt-Jakob disease could only be confirmed ante mortem by brain biopsy, it is now possible to unambiguously detect PrPSc in tonsils and/or spleen of patients with vCJD (Hill et al., 1997) and sCJD (Glatzel et al., 2003). However, although a tonsil biopsy is preferable to a brain biopsy, neither method is suitable to mass screening, which would be most desirable; e.g., for identifying prion-tainted blood units. Unfortunately, the diagnostic holy grail of recognizing prion infection by testing blood remains elusive. Early claims of identification of prions in urine (Shaked et al., 2001) have stirred considerable interest but were subsequently proven to derive from trivial artifacts (Furukawa et al., 2004; Serban et al., 2004). However, the scientific, economic, and public health interest in prion diagnostics on body fluid is red hot, and we have little doubt that the accrued knowledge on peripheral prion pathogenesis will pave the way to advances in this field. There continue to be no efficacious therapeutic options against prion diseases. Various drugs were screened for possible prion-curing properties in vitro. Substances included Congo red (Caughey and Race, 1992), amphotericin B, anthracyclins (Tagliavini et al., 1997), sulfated polyanions (Caughey and Raymond, 1993), porphyrins (Priola et al., 2000), branched polyamines (Supattapone et al., 2001), “β-sheet breakers” (Soto et al., 2000), the spice curcumin (Caughey et al., 2003), and recently even siRNAs downregulating PrPC (Daude et al., 2003), as well as dominant-negative mutants of PrPC (Crozet et al., 2004). Most of these molecules exert their biological effects by directly or indirectly interfering with conversion of PrPC to PrPSc, thereby also aiding clearance of PrPSc. Yet none of these compounds have proved very effective for actual therapy (Aguzzi et al., 2004). After results obtained in mice, cytidyl-guanyl oligodeoxynucleotides (CpG-ODN), which stimulate the innate immune system via Toll-like receptor 9 (TLR9) signaling receptors on a variety of immune cells, have been proposed as a feasible treatment regimen to delay prion disease (Sethi et al., 2002). It was shown that prion disease incubation period was extended in mice repeatedly treated with CpG-ODN for 20 days, and it was speculated that stimulation of innate immunity accounts for this apparent antiprion effect, possibly through induc-

tion of anti-PrP antibodies. However, this is difficult to reconcile with several studies that indicate that immune deficiencies of various sorts inhibit prion pathogenesis (Frigg et al., 1999; Klein et al., 1997, 1998, 2001). In addition, prion pathogenesis is unhampered in MyD88−/− mice, in which TLR9 signaling is impaired (Prinz et al., 2003b). In fact, repeated CpG-ODN treatment has severe side effects, ranging from lymphoid follicle destruction and impaired antibody class switch to ascites, hepatotoxicity, and thrombocytopenia (Heikenwalder et al., 2004). Besides, exhaustive studies have failed to reveal the induction of anti-PrP antibodies in CpGODN-treated mice (Heikenwalder et al., 2004; Polymenidou et al., 2004). It is therefore more likely that the antiprion effects of repeated CpG-ODN treatment arise via indiscriminate and undesired destruction of lymphoid tissue. From all of the above, it would appear that LTβR-Ig may presently represent the only antiprion compound that may be realistically exploited for postexposure treatment of humans. As an important qualification, however, treatment with LTβR-Ig could be considered as possible postexposure prophylaxis at early time points, and only in those rare cases in which prion exposure is well documented and punctual. These cases might include recipients of blood transfusions from patients with CJD, and possibly pathologists, neurologists, neurosurgeons, researchers, and technicians after accidental CJD injection. In contrast, the presumed mechanism of action of LTβR-Ig allows us to predict that it will not provide any relief to patients with overt CJD for whom neural entry has already taken place. The Quest for a Prion Vaccine Strategies for immunization against prion diseases continue to represent a field of intense efforts (Aguzzi and Sigurdson, 2004). Mice transgenic for the µ-chain of a PrP-specific antibody (6H4µ mice) were found to resist prion disease after exposure by a peripheral route (Heppner et al., 2001b). Of course, 6H4µ mice are not wt, and this may represent a caveat (Mallucci and Collinge, 2005). However, 6H4␮ mice mount fully protective immune responses against vesicular stomatitis virus and lymphocytic choriomeningitis virus (Heppner et al., 2001b). Also, they have normal lymphocyte subpopulations in thymus, spleen, and blood; normal immunoglobulin levels; and normal splenic and lymph nodal microarchitecture. By contrast, expression of a nonimmunoreactive transgenic heavy chain did not afford antiprion protection. We find it therefore difficult to argue that antiprion protection came about by anything else than anti-PrPC immunity in this mouse model. Prion-specific antibodies were not shown to be generated during the course of prion infections, but artificial induction of humoral immune responses to PrPC and/or PrPSc might be protective. However, efficient vaccination against the prion protein has proven extremely challenging. First, wt mice are highly tolerant to PrP as an immunogen, because PrPC is expressed almost ubiquitously and found at the surface of both B and T cells (Cashman et al., 1990; Manson et al., 1992). Second, it was generally thought that antibodies specific for PrPC might lead to severe systemic immune-

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mediated diseases, because PrP is expressed by many cell types outside the CNS. Third, PrP-specific antibodies would be unlikely to cross the blood brain barrier in therapeutic concentrations. In parallel, many other publications addressed prion immunoprophylaxis, both in vitro and in vivo (Enari et al., 2001; Peretz et al., 2001). Additionally, passive immunization with PrP-specific antibodies after prion exposure delayed disease and markedly increased the incubation period compared with nonimmunized mice (White et al., 2003) (Figure 1G). However, so far active immunization efforts have not succeeded in eliciting protective PrP-specific antibodies, most likely due to immune tolerance to self-antigens (Polymenidou et al., 2004). Recently, a transgenic mouse was developed that expresses full-length mouse PrP rendered soluble and dimeric by fusion with the Fc portion of human IgG1 (known as PrP-Fc2) (Meier et al., 2003). After prion inoculation, these mice were surprisingly found to exhibit delayed onset of disease after intracerebral or intraperitoneal prion inoculation, which was equivalent to an w100-fold reduction in titer of the prion-infected inoculum (Meier et al., 2003). The PrPFc2 was not converted to a prion disease-causing isoform. This remarkable antiprion effect occurred in two distinct lines of PrP-Fc2-expressing transgenic mice. Therefore, it seems that PrP-Fc2 effectively antagonizes prion accumulation in the spleen and brain. If one were to translate these findings into a therapeutical concept, the crucial question would be whether PrP-Fc2 needs to be produced within the cell to be protected or whether it can act from outside (“non-cell-autonomously”). Preliminary data (N. Genoud and A.A., unpublished data) appear to suggest that the antiprion effect of PrP-Fc2 is not necessarily cell autonomous. If this finding were to be robustly confirmed in a few independent paradigms, one might envisage that injection of PrP-Fc2 protein, or gene transfer of PrP-Fc2 via viral vectors, could have therapeutic potential. Conclusions Prion immunology is not an entirely novel branch of science. The first seminal experiments identifying prion replication in lymphoid organs were performed by the prion research community of Edinburgh in the 1960s. In those days, the term “prion” had not yet been coined and the animal bioassay was the only available analytical tool. Considering the technical limitation of that time, it is amazing how clairvoyant the pioneers of prion immunology, including Hugh Fraser and Richard Kimberlin, have been. Many molecular experiments that were performed with transgenic and knockout mice in recent days have essentially confirmed several of the observations and predictions that were put forward 30 years ago. Spurred not only by the BSE disaster and by public health needs, but also by the fundamental biological challenges posed by the “prion puzzle,” the pace of prion research has picked up considerably in the meantime. Much of the mechanics of prion lymphoinvasion and neuroinvasion has been elucidated in considerable molecular detail. In the next phase, it will be exciting to see these theoretical advances being

translated into medical progress toward the establishment of efficient diagnostic, prophylactic, and therapeutic antiprion regimens.

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