Genetic Depletion of Complement Receptors CD21/35 Prevents Terminal Prion
Disease in a Mouse Model of Chronic Wasting Disease
Brady Michel*, Adam Ferguson*, Theodore Johnson*, Heather Bender*, Crystal
Meyerett-Reid*, Bruce Pulford*, Adriana von Teichman†, Davis Seelig*, John H.
Weis‡, Glenn C. Telling*, Adriano Aguzzi† and Mark D. Zabel*
+ Author Affiliations
*Department of Microbiology, Immunology and Pathology, College of
Veterinary Medicine and Biomedical Sciences, Colorado State University Prion
Research Center, Fort Collins, CO 80523; †Institute for Neuropathology,
University Hospital of Zürich, CH-8091 Zürich, Switzerland; and ‡Department of
Pathology, University of Utah, Salt Lake City, UT 84132
Address correspondence and reprint requests to Prof. Mark Zabel, Department
of Microbiology, Immunology and Pathology, College of Veterinary Medicine and
Biomedical Sciences, Colorado State University Prion Research Center, 1619
Campus Delivery, Fort Collins, CO 80523-1619. E-mail address: mark.zabel@colostate.edu
Abstract
The complement system has been shown to facilitate peripheral prion
pathogenesis. Mice lacking complement receptors CD21/35 partially resist
terminal prion disease when infected i.p. with mouse-adapted scrapie prions.
Chronic wasting disease (CWD) is an emerging prion disease of captive and
free-ranging cervid populations that, similar to scrapie, has been shown to
involve the immune system, which probably contributes to their relatively facile
horizontal and environmental transmission. In this study, we show that mice
overexpressing the cervid prion protein and susceptible to CWD (Tg(cerPrP)5037
mice) but lack CD21/35 expression completely resist clinical CWD upon peripheral
infection. CD21/35-deficient Tg5037 mice exhibit greatly impaired splenic prion
accumulation and replication throughout disease, similar to CD21/35-deficient
murine prion protein mice infected with mouse scrapie. TgA5037;CD21/35−/− mice
exhibited little or no neuropathology and deposition of misfolded,
protease-resistant prion protein associated with CWD. CD21/35 translocate to
lipid rafts and mediates a strong germinal center response to prion infection
that we propose provides the optimal environment for prion accumulation and
replication. We further propose a potential role for CD21/35 in selecting prion
quasi-species present in prion strains that may exhibit differential zoonotic
potential compared with the parental strains.
Footnotes
This work was supported by National Institute of Neurological Disorders and
Stroke Grant R01 NS56379. Received June 8, 2012. Accepted August 22, 2012.
Copyright © 2012 by The American Association of Immunologists, Inc.
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Discussion
We investigated the role of the complement receptors CD21/35 in CWD prion
accumulation, replication, and disease progression.We observed a complete rescue
from terminal CWD of Tg5037 mice lacking CD21/35. Only 3 of 11 nonclinical
Tg5037;CD21/352/2 mice displayed detectable, yet reduced, prion neuropathology
and PrPRES deposition in their brains. These results reveal a more dramatic
outcome than earlier studies showing only a partial rescue of CD21/35-deficient
mice from scrapie infection, despite those mice expressing only WT (i.e., 5-fold
less) PrPC levels. This could reflect differences between mouse and cervid CD21
expression, as are apparent between mouse and human CD21. However, little is
known about cervid CD21. The gene has yet to be cloned, so comparative analyses
with murine CD21/35 are impossible at present. We can, however, compare CD21
sequence homology and phylogeny among other species that are susceptible to
TSEs. For example, sheep, which are susceptible to scrapie, a TSE that closely
resembles CWD in transmission efficiency, and lymphotropism, express a CD21
molecule that shares 65% sequence identity with murine CD21/35, including their
ligand binding domains (Fig. 5A). This may explain the similar lymphotropic
characteristics of murine and ovine scrapie. Ovine CD21 also shares 65% identity
with human CD21/35. Overall, CD21/35 from these three species share 52% identity
and 64% similarity. In contrast, bovine CD21, which is 40% larger than the other
three CD21/35 molecules (∼1400 compared with ∼1000 aa, respectively), shares
,20% similarity to the other three CD21/35 molecules. Phylogenetic analysis
reveals a clustering of murine, ovine, and human CD21/35 proteins, with bovine
CD21 much more distantly related (Fig. 5B). Interestingly, bovine spongiform
encephalopathy has been shown to have little or no lymphotropic characteristics
(44–47), perhaps owing to the vastly different CD21 molecule that bovids
express.
These results indicate a significant role in prion pathogenesis for
CD21/35, the importance of which may vary by prion strain. Complement components
C1q and C3 have recently been shown to exhibit similar strain preferences in
vitro and in vivo (48). We are currently investigating other prion strains to
determine the contribution of CD21/35 to prion pathogenesis in those infection
models. Interestingly, cross-species prion transmission was recently shown to
result in a higher infection rate of the lymphoreticular system than the CNS in
the xenohost (49). This crossspecies infection resulted in distinct lymphotropic
and neurotropic strains with differential host ranges. These strains may result
from tissue-specific strain selection or mutation. The higher efficiency of
prion infection in the spleen (which harbors CD21/35- expressing FDCs and B
cells) compared with the brain (which lacks them) alludes to a critical role for
CD21/35 in prion retention, replication, and possibly strain selection in
trans-species prion infection. The lack of CD21/35 that delays peripheral prion
accumulation might further limit the lymphoid replication of neurotropic prion
strains, resulting in delayed or abrogated disease progression. If so, this
would have profound implications for prion xenotransmission and possible
therapeutic approaches aimed at CD21/35. For example, targeting CD21/35 to slow
the spread of neurotropic prions could be an attractive alternative to most
prion disease therapeutics developed to date that target the CNS, which can
complicate drug delivery. Interfering with CD21/35-mediated prion strain
selection could also mitigate emergence of new prion strains with expanded host
ranges and prevent a breach of the species barrier similar to the one that
likely caused the bovine spongiform encephalopathy and subsequent new-variant
Creutzfeldt- Jakob disease outbreak 15 y ago in the United Kingdom.
To study the kinetics of extraneural CWD prion accumulation, we amplified
PrPRES from spleens of CWD prion-infected Tg5037 and Tg5037;CD21/352/2 mice at
various time points throughout infection. At 15, 70, and 140 dpi and at terminal
disease, prion accumulation was significantly lower in CD21/35-deficient mice.
The extremely high prion load detected at 15 dpi most likely reflects increased
retention of prion inocula early after infection. This delay in extraneural
prion accumulation strongly correlates with abrogation of prion neuropathology
and terminal disease. These results coincide with our previous data from scrapie
mouse models (17), further strengthening evidence that CD21/35 play an integral
part in prion accumulation in peripheral lymphoid organs that ultimately
facilitates neuroinvasion.
Furthermore, we show that CD21/35 are present in prion preparations
enriched from spleen homogenates by NaPTA precipitation. We also demonstrate GC
formation in spleens during prion infection primarily dependent on CD21/35 and
PrPC expression on FDCs. It may seem surprising that CD21/35 expression on FDCs,
rather than B cells, correlates with prion-induced GC formation, because
CD21/CD19/CD81 B cell coreceptor ligation helps activate B cells to form GCs.
However, maximal B cell activation and GC formation require signaling from both
the BCR and B cell coreceptor (32, 33). In this study, we show that although
prion infection stimulates CD21/35 translocation to lipid rafts on B cells,
signaling appears to be suboptimal for GC formation in the absence of
concomitant BCR translocation. We observed a strong dependence on both PrP and
CD21/35 expression on FDCs for a strong GC response. Paradoxically, CD21/35
translocation did not occur on FDCs, which are the major prion trappers and
replicators but lack other B cell coreceptor components required for CD21/35
movement. One could therefore argue that GC formation represents an artifact,
rather than being a driver of splenic prion replication. Elimination of GCs had
no effect on peripheral prion replication and disease progression in mice
infected i.p. with RML5 (50), supporting this interpretation. However,
GC-deficient mice infected intracranially with 139A mouse-adapted scrapie prions
exhibited a significant delay to terminal disease (51). Thus, distinct prion
strains may differentially influence GC formation and subsequent prion
pathogenesis. Additionally, this discrepancy further highlights potential
preferences of distinct tissues for different prion strains. CD21/35- expressing
cells within GCs may facilitate this selection process in the lymphoid system.
Increased retention of prions on FDCs could induce a persistent state of prion
presentation to adjacent B cells sufficient to cause an atypical GC response
(40). FDCs may coax B cells to linger there, providing increased lymphotoxin
signaling to FDCs that may promote formation of hypertrophic dendrites that
efficiently retain and replicate prions. Consistent with their role as
long-lived, long-term APCs, FDCs may also present prions to neighboring
PrPC-expressing B cells that could induce CD21/35 translocation and move prions
proximal to PrpC in lipid rafts and promote further prion replication and GC
formation.
Taken together, these data support a principal role of CD21/35 in
peripheral prion pathogenesis by trapping PrPRES on both B cells and FDCs.
CD21/35 expression on FDCs remains of paramount importance in this process, with
B cells playing a lesser, but still important, role. We have recently shown that
few prion-bearing B cells transport prions from infection sites to draining
lymph nodes, but their presence increased dramatically within lymph nodes,
indicating a prominent role for B cells in intranodal prion trafficking (52). We
propose that CD21/35 mediate this and other crucial processes in lymph node
prion trapping and replication and we are currently testing this
hypothesis.
Acknowledgments
We thank Ed Hoover and Steve Dow for helpful advice and discussion of the
project and data.
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