We found that the intensity and distribution of PrP
CWD deposits in brain and peripheral tissues of
PRNP polymorphic (i.e. different PrP
C primary structures) white-tailed deer was distinct from Q95G95 (wt) homozygous deer exposed to the same prion strain (i.e. Wisc-1). We have previously shown that H95 and S96
PRNPpolymorphisms play a key role in CWD susceptibility, increasing survival periods and having dramatic effects on the propagation of CWD strains [
16, 17, 18, 19, 20,
37].
Our results show that deer expressing the H95-PrP
C presented a more limited peripheral distribution of PrP
CWD compared to wt/wt and S96/wt deer. Under identical experimental conditions and disease stage, the number of organs with positive immunolabeling was reduced in deer with H95-PrP
C allelotypes [
18]. The most significant differences in PrP
CWD deposition between deer with different
PRNP genotypes were found in pancreas, heart, kidney and intestine samples. Both deer expressing the H95-PrP
C showed no immunolabeling or reduced accumulation of PrP
CWD aggregates in these tissues.
The presence of PrP
CWD in endocrine tissues has been previously described in the adrenal medulla, the pituitary gland and islets of Langerhans in the pancreas of CWD-affected cervids [
31,
32], results that agree with our observations in CWD-infected wt/wt and S96/wt deer. Only deer of these genotypes (Fig.
3a,
b) showed moderate to strong chromagen deposition in islets of Langerhans, which are innervated by the vagus nerve [
32]. Differences in adrenal glands immunolabeling were minor between deer expressing different PrP
Cpolymorphisms. Consistent with wt-PrP
C being the cognate substrate for Wisc-1 homologous prion conversion, wt/wt deer presented higher and widespread PrP
CWD deposition compared to deer of other genotypes.
The distribution of PrP
CWD aggregates was also limited in hearts of animals expressing the H95-PrP
C allelotype. Cardiac tissues were collected from D6 (S96/wt), D9 (H95/wt) and D10 (H95/S96). PrP
CWD accumulation was detected in multiple heart samples from D6, affecting separated groups of cardiac myocytes (Fig.
4a). Conversely, no immunolabeling was observed in any heart sample from deer expressing the H95-PrP
C (Fig.
4b,
c).
A distinct pattern of distribution of PrP
CWD was also observed in intestinal tissues for H95 carriers. It is not surprising that strong immunolabeling was observed in gut-associated lymphoid and nervous tissues as these are known to be the first sites of PrP
d accumulation following oral infection [
26,
30,
31,
38]. Nevertheless, H95/wt and H95/S96 deer, which had the longest incubation periods [
18], showed more restricted or localized PrP
CWD accumulation (Fig.
5). These findings however, do not necessarily indicate that these animals excrete a lower amount of prions or present a lack of infectivity in intestinal tissues.
PrP
CWD deposits were observed in renal tissues of all wt/wt and S96/wt deer evaluated in the present study, whereas no immunopositive material was found in any of the kidney samples collected from deer expressing the H95 allele (Fig.
6). Immunohistochemical detection of PrP
CWD in kidneys of CWD-infected white-tailed deer has previously only been reported in ectopic lymphoid follicles [
31,
39]. PrP
CWD in renal tissues has, however, been demonstrated by sPMCA [
40] and it has been shown that CWD-infected cervids can shed infectious prions in urine [
41, 42, 43], although the proximal source of PrP
CWD in urine is not known [
40]. In the present study, PrP
CWD positive immunolabeling of kidney samples was detected along the wall of the renal arteries, especially in the main renal artery and the wall of arcuate arteries (Fig.
6a,
b). In scrapie-affected sheep, prion deposition has been found in renal papillae and renal corpuscles [
33,
44]. Periarterial and periarteriolar immunolabeling could be due to spread of prions through peripheral nerves [
44] since the wall of these renal arteries is strongly innervated and sympathetic nerve fibers from the renal plexus enter the kidney accompanying the branches of the main renal artery. To our knowledge, this is the first description of PrP
CWD deposition, detected by conventional techniques, in renal arteries of CWD-infected deer.
D1 also presented intense PrP
CWD immunolabeling in the renal cortex associated with accumulations of inflammatory cells (Fig.
7a). Inflammatory processes affect prion pathogenesis and peripheral accumulation [
45,
46], and chronic nephritis triggers prionuria in prion-infected mice [
47]. In addition, PrP
CWD shedding has been reported in CWD infected deer presenting with inflammatory kidney disease [
41]. We cannot predict the effect of prion accumulation in the arterial walls on prionuria, however, we have observed that inflammatory kidney conditions greatly increase PrP
CWD deposition in renal tissues from deer with CWD, which might increase shedding of PrP
CWD within urine. This deer also showed PrP
CWDaccumulation in the lungs associated with inflammatory cell foci and in the bronchiolar epithelium (Fig.
7b). PrP accumulation in the lung related to inflammatory conditions and in the epithelium of the bronchioles has been previously described in scrapie-affected sheep [
33,
48]. Deer in our study were housed indoors with ample access to clean food and water [
18]. It is likely that free-ranging deer would be at greater risk of coincident infections and commensurate inflammation that may influence the effect of protective alleles on susceptibility to CWD, tissue colonization by CWD prions and shedding.
In organs related to excreta production, differences in PrP
CWD deposition were found in salivary glands between deer genotypes. PrP
d in salivary glands, detected by conventional techniques, has been described in scrapie-affected sheep [
49] and in the serous epithelial cells of the submandibular salivary gland in experimentally infected red deer [
50]. Likewise, considerable PrP
CWD amplifying activity, similar to that observed in brain, accumulates in salivary glands of cervids with CWD [
40]. Comparison of single salivary gland sections identified PrP
CWD immunostaining in wt/wt or S96/wt deer but not in deer expressing H95-PrP
C (Table
2). Intense PrP
CWD immunolabeling in ganglia cells immersed in the salivary gland tissue was observed in animal D4 (Fig.
6c).
Our observations suggest that deer expressing H95-PrP
C have reduced centrifugal trafficking via descending nerves into peripheral tissues (i.e. salivary glands, pancreas, heart and kidney). This is consistent with previous observations in different experimental prion infections [
32,
33,
36,
44,
51]. However, it has been suggested that, in initial stages of CWD infection, PrP
CWD may be trafficked via blood [
26], and infectivity has been demonstrated in blood components [
52,
53]. Therefore, we cannot exclude the hematogenous route as a complementary pathway of prion dissemination.
None of the clinically affected deer presented positive immunolabeling in any of the skeletal muscle samples evaluated, including the tongue. The absence of PrP
daccumulation in skeletal muscles detectable by IHC techniques has been reported in both naturally and experimentally prion infected deer [
54,
55]. Nevertheless, although we did not detect PrP
CWD deposition in skeletal muscle samples in this experimental Wisc-1 transmission to white-tailed deer, we cannot assume the complete absence of prion accumulation. Others have demonstrated the presence of PrP
CWD in skeletal muscles by bioassay [
56], Western Blot, PMCA and tissue-blotting [
34].
The presence of prion deposits in skeletal muscles has previously been reported in neuromuscular spindles, which are highly innervated structures [
33,
35] and, in CWD-infected white-tailed deer, in nerve fascicles [
34]. Although most of the vagus nerve samples evaluated in the present study showed positive immunolabeling, sciatic nerve and brachial plexus samples presented sparse or no PrP
CWD deposits (Table
2). Our results are similar to those in mule deer naturally infected with CWD [
32]. We did not detect PrP
CWD deposition in forelimb and hindlimb skeletal muscle samples, not even in the neuromuscular spindles. Due to the fact that brachial plexus and sciatic nerve innervate, respectively, the forelimb and hindlimb muscles, and taking into account that prions can spread to these muscles via these neural pathways [
32], we can suggest that the scant or absent PrP
CWD immunolabeling in brachial plexus and sciatic nerve samples from deer in our study may relate to the absence of deposits in these groups of muscles.
Cumulative evidence supports the limiting effect of H95 and S96 PrP
Cpolymorphisms on natural CWD infection and disease progression [
16, 17, 18,
37]. These PrP
C allelic variants modulate CWD propagation and the efficiency of intraspecies CWD transmission [
19,
57]. The passage of CWD prions in white-tailed deer expressing the H95-PrP
C led to the emergence of the novel CWD strain H95
+ [
19]. This strain presented distinct biochemical and transmission properties, efficiently propagating in transgenic mice expressing deer S96-PrP
C and in non-transgenic C57BL/6 mice [
19,
20]. Wisc-1 propagation in deer expressing the H95-PrP
C also presented limited peripheral PrP
CWD accumulation.
As demonstrated in sheep with scrapie,
PRNP genotype strongly influences the tropism and distribution of PrP deposits [
23,
25,
30]. In the present study, the observed effects of the H95 allele in prion distribution resemble those described for sheep expressing the resistance-associated allele ARR at codons 136, 154 and 171 of the prion protein. ARR/VRQ sheep, despite developing disease and accumulating PrP
Sc in the brain, present a much more limited and infrequent PrP
Sc distribution in lymphoid tissues compared to those with other susceptible genotypes [
30,
58,
59]. This effect is likely due to a modulation of the prion pathogenesis [
25,
60] and it is not necessarily associated with the prolonged incubation period [
25].
The similarities in peripheral PrP
CWD accumulation between H95/wt and H95/S96 deer indicate a limiting role for the H95 amino acid substitution on the production and/or accumulation of PrP
CWD. Similar observations have been made in goats expressing methionine at codon 142, which show reduced incidence of scrapie infection and a lower tendency to accumulate PrP
Sc outside the brain compared to 142 isoleucine homozygotes [
50,
61]. In contrast, our findings suggest that H95-PrP
C does not affect LRS involvement in CWD-affected deer (Table
1).
Influences of genotype on PrP
CWD deposition pattern have been described in experimentally infected mule deer, with F225/S225 deer presenting with milder PrP
CWD accumulation and limited tissue distribution compared to S225 homozygotes at identical intervals post-inoculation. This suggests the F225 amino acid variant limits prion conversion and delays PrP
CWD tissue accumulation [
31,
57]. Similar observations have been made in white-tailed deer expressing the S96 PrP
C, which, compared to wt/wt deer, show reduced PrP
CWD in brain and lymphoid tissues, consistent with slower disease progression [
17,
26]. Deer of this genotype have also been reported to present lower PrP
CWD immunostaining scores in the obex than wt/wt deer [
62,
63]. However, observations were made in naturally infected animals in different stages of the disease. Although we observed certain differences between wt/wt and S96/wt deer in particular brain regions (Fig.
2), the overall intensity and peripheral distribution of PrP
CWD was similar for both genotypes at the terminal stage of the disease (Tables
1 and
2). By contrast, the H95 polymorphism was a stronger driver of disease phenotype. The interference exerted by H95-PrP
C in the replication and tissue accumulation of Wisc-1 prions relates to the biology of cervid PrP
C polymorphisms and the evolution of CWD prion strains [
19,
20].
Given that the diversity of CWD agents can be expanded in deer expressing PrP
Cpolymorphisms [
19,
20,
64], our findings cannot be generalized to include all potentially existing CWD strains which will likely have distinct host specific interactions. Prion disease characteristics, including the lesion distribution and the IHC phenotype of PrP
d accumulation, are strongly influenced by the infecting prion strain [
21,
65, 66, 67]. Thus, it is possible that the reduced PrP
CWDimmunolabeling observed in areas of the H95/S96 brain (Figs.
1 and
2) could be due to intra-species transmission barrier determined by the compatibility between the invading strain and the PrP
C sequence of the host [
68,
69]. Likewise, heterozygous deer presented lower levels of PrP
CWD deposits in certain rostral brain areas, as compared to wt/wt deer (Fig.
2). These observations are consistent with the reduced amounts of PrP
CWD as detected by Western blottimg [
18]. Deer in the present study were orally inoculated with Wisc-1 prions [
19], a CWD source obtained from animals homozygous for the wt-PrP
C [
18]. Therefore, transmission of the Wisc-1 strain into H95/S96 deer involves the adaptation of the infectious agent to this new host microenvironment, which could partially explain the reduced PrP
CWD accumulation in particular brain regions, considering that the expression of wt-PrP
C favors the propagation of the Wisc-1 strain [
19].
The PrP
CWD accumulated in the H95/S96 animal (H95-PrP
CWD) [
19] is more PK-sensitive than PrP
CWD from deer with at least one wt allele [
18]. Differences in PrP resistance to proteolytic degradation can lead to variable results in diagnostic tests. For example, atypical/Nor98 scrapie isolates are highly sensitive to PK digestion compared with classical scrapie strains, which leads to inconsistent diagnosis by PrP
d IHC [
70]. Therefore, this particular characteristic of H95-PrP
CWDmay also explain the lower immunolabeling observed in the brain of H95/S96 deer. Nevertheless, prion disease neuropathological phenotypes, which include the PrP
d profile, may depend on complex interactions between the infecting prion strain and host factors (e.g. the
PRNP genotype) [
23]. This host-pathogen interaction might explain the differences observed between deer genotypes with respect to the PrP
CWD deposition in the cerebellum (Fig.
2). However, as mentioned, other CWD strains could differentially interact with PrP primary sequence and those effects should be further explored to understand how PrP
Cpolymorphisms modulate the propagation of CWD infectious agents.
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