Background
The cellular prion protein (PrP
C) has been extensively studied for decades, but its normal function is still not fully understood. However, expression of this highly conserved protein across tissues in vertebrates suggests that it may have roles in a variety of physiological functions [
1]. Accumulation of the misfolded isoform (PrP
Sc) occurs in all prion disorders, and it has been postulated that loss of PrP
C function participates in the progression of these diseases [
2]. Thus, identifying the normal function of PrP
C is considered an essential step in understanding the pathogenesis of prion disorders.
PrP
C is abundantly expressed not only in the central nervous system (CNS) but also in non-neural tissues such as gonads, the pregnant uterus, and the immune system [
3‐
5]. Several roles for PrP
C in immunological processes have been suggested (reviewed in [
6]). Lack of PrP
C seems to exacerbate inflammation, both in the periphery [
7] and in the CNS [
8], as well as ischemic [
9‐
11] and traumatic [
12] brain lesions. Likewise, PrP
C has been linked to regulation of pro- and anti-inflammatory cytokines upon systemic lipopolysaccharide (LPS) challenge [
13].
Systemic administration of LPS activates the Toll-like receptor 4 signaling cascade in a range of immune cells, resulting in synthesis and release of a variety of pro-inflammatory cytokines [
14]. This, in turn, induces characteristic signs of sickness behavior, which includes depression, periods of shivering, and reduced appetite and locomotor activity [
15]. We recently demonstrated that LPS is a potent activator of innate immunity in goats, describing a dynamic regulation of leukocyte genes involved in immunological processes [
16]. Because only small amounts of LPS and cytokines cross the blood-brain barrier (BBB) [
17], information from the periphery is transmitted to the CNS through neuronal and humoral communication routes. Pro-inflammatory cytokines and LPS can stimulate the vagus nerve, directly initiating afferent signaling to the brain [
18]. The humoral route is characterized by circulating cytokines that activate endothelial cells of the BBB or act on tissues that lack BBB, such as the circumventricular organs and choroid plexus (ChP) [
19]. Consequently, a mirror image of peripheral cytokines is created within the brain. The ChP is localized within the brain ventricular system and is composed of vascularized stroma surrounded by a monolayer of epithelial cells. The epithelial cells are responsible for the production of cerebrospinal fluid and can release cytokines into the ventricular system. Thus, the ChP plays a key role in transmitting signals into the brain during inflammatory conditions [
20,
21]. The cellular composition of the stroma can be dynamically altered through recruitment of circulating immune cells, such as lymphocytes, neutrophils, and monocytes [
22,
23]. Although the hippocampus is considered more immunoprivileged than the ChP, systemic LPS challenge may also impair hippocampal function [
24,
25]. Certainly, cytokine receptors such as IL1R, which is fundamental in the response to inflammatory signals, are expressed in the hippocampus [
26].
Recently, a nonsense mutation early in the gene encoding PrP
C (
PRNP) in Norwegian dairy goats was discovered [
27]. The mutation terminates PrP
C synthesis only seven amino acids into the mature protein. Goats homozygous for the mutation (
PRNP
Ter/Ter) are devoid of PrP
C and postulated to be scrapie-resistant [
27,
28]. Physiological and immunological studies have not identified major disturbances under normal herd conditions, which is in agreement with studies in transgenic animals without PrP
C [
29,
30]. However, closer phenotypic characterization indicates a small increase in red blood cell count of PrP
C-deficient goats compared both with normal animals and with goats heterozygous for the mutation [
28]. These outbred, non-transgenic goats provide a new model for studying PrP
C physiology.
We hypothesized that goats without PrPC are more susceptible to inflammation or stressful stimuli. To investigate this, we performed a longitudinal LPS study in normal (PRNP
+/+) and PrPC-deficient goats (PRNP
Ter/Ter) comprising clinical, biochemical, and hematological responses, as well as end-point tissue transcriptional profiles and characterization of morphological changes. In the current paper, we focus on the PrPC-rich hippocampus, which is important in behavior and memory, as well as the ChP, an essential tissue in the interplay between the periphery and the brain.
Methods
Animals
A total of 26 Norwegian dairy goat kids, 13
PRNP
Ter/Ter and 13
PRNP
+/+ animals, were included in the study. The goats were kept under a 16-h light/8-h dark cycle, housed in groups of two to four, and acclimatized at least 21 days before the experiment. Hay and water were provided ad libitum, and they were fed a commercial goat pellet concentrate. During the acclimatizing period, clinical examinations were performed three times, and fecal and blood samples were analyzed to ensure that the animals were healthy before the experiment. An overview of the study groups including treatment, animal number, age, weight, and gender can be found in Additional file
1a.
LPS challenge
The goats were split in groups as follows: 16 goats (8
PRNP
Ter/Ter and 8
PRNP
+/+
) that received LPS intravenously and a control group of 10 goats (5
PRNP
Ter/Ter and 5
PRNP
+/+) that were given corresponding volumes of sterile saline. Based on existing literature [
31,
32] and a pilot titration study (data not included), the LPS group received a dual dose of LPS (
Escherichia coli O26:B6, L2654 Sigma-Aldrich, USA) with a 24-h time interval between doses; 0.1 μg/kg (day 1) and 0.05 μg/kg (day 2). As goats are very sensitive to LPS, the second dosage was reduced to avoid the risk of sensitization and mortalities. The animals were euthanized by an overdose of pentobarbital 5 h after the second LPS challenge. An overview of the study protocol is given in Additional file
1b.
Clinical examination
Clinical examination, including rectal temperature, heart and respiratory rate, and rumen contraction frequency was performed by veterinary surgeons at 12 time points during the first 7 h of day 1 and at 9 time points after the second LPS injection. Measurements of rectal temperature were repeated three times at each time point. Clinical examination was performed correspondingly, but at fewer time points, in control animals.
The clinical examination and evaluation of sickness behavior were scored blinded with respect to genotype. Signs of sickness behavior were recorded by evaluating body position (standing, lying, head and ear position), locomotor activity, social interaction, appetite, and shivering. Based on this, goats were scored as presenting “sickness behavior” (S) or “no sickness behavior” (N) every 15 min. The animals were evaluated until three consecutive “N” scorings were recorded, and the total duration of sickness behavior was calculated.
Blood sampling, hematology and biochemistry
Blood samples (EDTA and whole blood) were drawn from v. jugularis using a vacutainer system (BD Company, USA). Baseline samples (0 h) were taken within 30 min before LPS challenge. The other sampling times were 1, 2, 5, and 24 h after the day 1 LPS administration. Hematology, including a complete blood count, was performed immediately by using the ADVIA 120 Hematology system (caprine analyzing program). Whole blood tubes were centrifuged, and serum stored at −20 °C until biochemical analysis. Serum total protein, albumin, and glucose were analyzed by ABX Pentra 400 (Horiba, France) and ceruloplasmin by Cobas Mira Plus (Roche). Copper was quantified by AAnalyst 300 atomic absorption spectrometer (PerkinElmer, USA).
Histological examination
The left half of the brain was removed immediately from euthanized goats and immersion-fixed in 4% formaldehyde for 1 week. Defined brain slices were then dehydrated in graded ethanol and paraffin embedded. Morphological changes, including neuronal chromatolysis, single-cell necrosis, and inflammatory cell infiltration, were evaluated by analysis of hematoxylin and eosin-stained 4-μm-thick tissue sections. Brain regions, including hippocampus, ChP in the lateral ventricle, and obex, were investigated.
Immunohistochemistry and semi-quantitative scoring
Paraffin sections (4 μm thick) from the abovementioned areas were mounted on Superfrost® Plus slides (Menzel-Gläser, Thermo Scientific). The distribution and morphological appearance of the astrocyte marker, GFAP (Dako, Z0334), and the microglia/macrophage marker, Iba1 (Wako, 019-19741), were investigated by immunohistochemistry. The sections were dried overnight at 58 °C, deparaffinized in xylene, and rehydrated through decreasing concentrations of graded ethanol. For Iba1 analysis, epitope retrieval was performed by trypsinization (10 mg/ml, 1:10 0.1 M Tris/HCl-buffer, 0.1% CaCl2) for 30 min at 37 °C. Endogenous peroxidase activity was blocked by incubation in 3% H2O2 in methanol for 10 min at room temperature. The sections were then blocked in normal goat serum (1:50) diluted in 5% bovine serum albumin (BSA) for 20 min and incubated with the primary antibodies anti-Iba1 (1.0 μg/ml) or anti-GFAP (1.9 μg/ml) for 1 h at room temperature. Further steps were performed with EnVison+ kit (Dako, K4009). The sections were counterstained with hematoxylin for 40 s. Washing between steps was in Tris-buffered saline (TBS). All runs included a negative control section where the primary antibody was replaced with 1% BSA/TBS.
The sections were examined by light microscopy and a blinded, semi-quantitative evaluation was performed by an investigator. The labeling intensity of the Iba1 and GFAP signals, the number of and localization of cells, and the appearance of primary and secondary processes were scored as follows: 0 = minimal, 1 = little, 2 = moderate, 3 = strong, including half-step grading.
Tissue samples were collected from the right half of the brain within 15 min after euthanasia. The samples were dissected into small pieces, immediately immersed in RNAlater and stored at −80 °C. RNA extraction was carried out using RNeasy Lipid Tissue Mini Kit (Qiagen, 74804) according to the manufacturer’s instruction. The isolated RNA was quantified at optical density (OD)
260, and purity was assessed by OD
260/280 and OD
260/230 absorbance readings with a DeNovix DS-11 spectrophotometer (Wilmington, USA). RNA integrity was assessed by RNA 600 Nano chips in compliance with the Agilent Bioanalyzer 2100 system in all individual samples before pooling. RNA quality data are summarized in Additional file
1c.
Extracted RNA was diluted to 500 ng/μl and then re-measured three times. Equal amounts (ng) of RNA from individual samples were pooled, reaching a final amount of 15,000 ng. The samples were pooled according to tissue, treatment, and genotype making a total of eight pools. RNA samples were shipped on dry ice to Novogene (Hong Kong) for RNA sequencing. As the transcriptome profile might be sensitive to gender, one buck (LPS, PRNP
Ter/Ter) was excluded from the material, leaving only female samples.
RNA sequencing
After quality control, messenger RNA (mRNA) was enriched using oligo (dT) beads and then randomly fragmented. First-strand complementary DNA (cDNA) was synthesized using random hexamers and reverse transcriptase. Second-strand synthesis was done by nick-translation using a buffer containing dNTPs, RNase H, and
E. coli polymerase I (Illumina). The cDNA fragments were processed using an end-repair reaction after the addition of a single “A” base, followed by adapter ligation. These products were then purified and amplified using PCR to generate the final cDNA library. The quality of each library was evaluated by 2100 Bioanalyzer (Agilent), followed by paired-end 150-bp sequencing on an Illumina HiSeq2000. The quality control summary can be found in Additional file
1d.
Differential expression analysis
Raw reads (FASTQ) were clipped and trimmed of adapter contamination, and those of low quality were removed. Quality-controlled FASTQ files were mapped to the
Capra hircus (domestic goat) reference genome using the TopHat2 (v2.0.12) software with two mismatches. Mapping status is summarized in Additional file
1e. Differential gene expression analysis (DEA) was performed using DEGSeq2 (1.12.0) with the following criteria: Log2 ratio ±0.59 (fold change ±1.5) and a false discovery rate (FDR) adjusted
q-value (
q < 0.05). For each tissue, four DEAs were performed. Differences in basal transcriptome levels were assessed by comparing
PRNP
Ter/Ter (saline) to
PRNP
+/+ (saline). The genomic response to LPS in each genotype was assessed by comparing the LPS groups with the saline-treated control of the matching genotype. Finally, DEAs between
PRNP
Ter/Ter (LPS) and
PRNP
+/+
(LPS) were performed to identify differences between the genotypes during acute inflammation. FPKM (fragments per kilobase of exon per million fragments mapped) values, which take into account the effects of both sequencing depth and gene length, were used to estimate gene expression levels. Genes encoding ribosomal subunit proteins are not included in the tables.
Gene ontology enrichment analysis
To characterize the overall LPS effect, gene ontology (GO) analysis was performed on genes that were differentially expressed (DEGs) in at least one of the
PRNP genotypes. We used the online PANTHER classification system to identify over-represented biological processes among the DEGs [
33,
34]. Because the
C. hircus genome was not available, and the
Bos taurus genome resulted in fewer mapped genes, the well-annotated
Homo sapiens genome was used as reference. The fold enrichment displays the over-representation of genes in a given biological process, compared with the expected number in the reference genome.
p values <0.05 represents a statistical significant over-representation and are calculated by the binomial test as described in [
33]. In total, eight genes (
SAA3,
OAS1L,
MHCI,
IFI203,
VCAM,
ADGRG6,
C4, and
C21H14orf132) were not mapped to the GO reference genome.
Validation of RNA sequencing by qPCR
First, 600 ng total RNA from each individual sample was converted into first-strand cDNA using QuantiTect Reverse Transcription Kit (Qiagen, Germany) according to the manufacturer’s instructions. A non-reverse transcriptase control (NoRT) and no template control (NTC) were included.
The expression of
PRNP,
IFI6,
CXCL10, and
SAA3 genes was investigated by Light cycler 480 qPCR using SYBR Green PCR Master Mix under the following conditions: initial denaturation for 5 min at 95 °C, followed by 40 amplification cycles (10 s at 95 °C, 10 s at 60 °C, and 15 s at 72 °C) and construction of melting curves. For each primer assay, a pool of cDNA samples was used to make three separate series with the following dilutions: 1:2, 1:10, 1:50, 1:250, and 1:1250. Standard curves were constructed to obtain primer amplification efficiencies, correlations, and dynamic range. Internal normalization was performed against the
ACTB reference gene, and relative expression was calculated using the 2
−ΔΔCq method [
35]. Primer sequences are given in Additional file
1f.
Descriptive and statistical analyses
Clinical, biochemical, hematological, and qPCR expression data are presented as mean ± standard error of the mean (SEM). Graphical and statistical analyses were performed in GraphPad Prism 6 (GraphPad software Inc., USA) and Microsoft Excel 2013. Comparisons between two groups were performed using Student’s t test, assuming equal variance.
Discussion
The high degree of conservation of the
PRNP gene across species [
37] suggests that the protein possesses important biological functions. These have, however, proven difficult to pin-point even after the creation of
Prnp-KO mice [
38]. It has been proposed that compensatory mechanisms could mask loss-of-function phenotypes under normal conditions and become evident during stress such as inflammation. For instance,
Prnp-KO mice displayed an exacerbated disease progression of experimental autoimmune encephalomyelitis [
8] and colitis [
7]. Here, we report the first study of the inflammatory response in a unique, non-rodent model naturally devoid of PrP
C. Considering the high
PRNP expression in the hippocampus and the role of the choroid plexus (ChP) in responding to inflammatory signals at the blood-brain boundary, we investigated both these tissues by full-scale transcriptome analysis. Our data suggest a role for PrP
C in modulating the innate immune response.
Systemic LPS challenge induced characteristic signs of sickness behavior that was prolonged by about 2 h in
PRNP
Ter/Ter goats after the initial high dose of LPS (0.1 μg/kg). This is a novel clinical loss-of-function phenotype, pointing to a more potent inflammatory response in the absence of PrP
C. When the dosage was halved on day 2, the mean duration of sickness behavior was only about 1–2 h in both groups. The difference between genotypes was similar as day 1, but not statistically significant (
p = 0.1). This suggests that the lower dose of LPS did not induce a sufficient amount of inflammatory stress to clearly separate the two genotypes. In the ChP, a clear acute phase response as well as activation of a range of interferon-stimulated genes was observed, which underlines the widespread role of these genes in the host defense to bacterial endotoxin [
16]. Interestingly, several genes associated with the immune response were differentially expressed between the
PRNP genotypes after LPS challenge. This included acute phase proteins genes and multiple chemokines, as well as
COCH that has an anti-bacterial role by regulating local cytokine production [
39]. Based on previous reports of PrP
C regulating cytokines [
13], we compared the 23 genes characterized as cytokine-responsive (GO:0034097) between the two genotypes. Most of these genes are primarily induced by type I and/or type II IFNs [
40,
41]. Notably, there was a relatively more pronounced type I IFN response and a weaker type II response in PrP
C-deficient goats, compared with the normal group. A potential role of PrP
C in regulating type II IFN response has been previously suggested, as IFN-γ levels were decreased in ConA-treated PrP 0/0 splenocytes [
42], but type I interferon signaling has received less attention. Type I interferons are key modulators of innate immunity and may affect the manifestation of sickness behavior by facilitating the immune activation of other cytokines [
43]. Indeed, interferon signaling is involved in many of the effects previously attributed PrP
C, such as apoptosis [
44], protection against oxidative stress [
45], DNA repair [
46], and depressive-like behavior immediately after stress [
47,
48]. Taken together, our data indicate that PrP
C contributes as a modulator of innate immunity signaling, particularly downstream of type I interferons, which might affect the duration of sickness behavior.
The substantial activation of the ChP transcriptome, including upregulation of
AIF1 expressed by activated macrophages/microglia, corresponded with a parallel increase in Iba1 signal. Markers of classical activation M1 (
CXCL9,
CXCL10) and alternative activation M2 (
TGM2,
MRC1) increased [
49,
50], suggesting a combination of M1 and M2 phenotype of activated macrophages/Iba1-positive cells. Moreover, cytokines involved in leukocyte migration were upregulated, as well as genes involved in collagen catabolism and extracellular matrix organization, indicating that the integrity of the blood-cerebrospinal fluid barrier was altered. These findings agree with the observation of increased numbers of Iba1-positive cells in the ChP stroma, some of which were presumably migrating through the epithelium. The stromal cells could represent antigen-presenting cells as dendritic cells [
51], recently blood-derived monocytes, or residing macrophages [
52].
Not surprisingly, alterations in the hippocampus transcriptome were modest compared with those observed in the ChP, yet a somewhat similar cytokine response was present. However, it is possible that the filtration of single genes strictly by fold change and
q-value might exclude biologically relevant pathways characterized by a subtle increase in a subset of genes. The two most upregulated genes in the hippocampus were
CXCL9 and
CXCL10, which are primarily induced by IFN-γ signaling [
41]. Recently,
CXCL10 expression in hippocampus was traced to activated astrocytes and cells lining the blood vessels [
53]. This suggests that endothelial cells within the BBB, as well as nearby glial cells, react to circulating LPS and cytokines by releasing IFN-γ, which, in turn, stimulates expression of
CXCL9 and
CXCL10. Despite the important role of these chemokines in recruiting immune cells into the brain [
54], no inflammatory cell infiltration was observed in our study. Although the overall LPS response in the hippocampus was similar in the two
PRNP genotypes, two metallothioneins (MT) were significantly upregulated in PrP
C-deficient goats. Metallothioneins bind metals and scavenge free radicals and participate in reducing the inflammatory and oxidative stress [
55]. In the brain, MT-I and MT-II are primarily expressed by activated astrocytes [
55]. We further found that
GFAP transcription increased significantly in
PRNP
Ter/Ter goats, indicating an early activation of astrocytes as previously described [
53,
56]. This was confirmed by an increased GFAP labeling after LPS treatment. Given the role of MTs [
55] and PrP
C [
57] in neuroprotection, it is tempting to speculate that upregulation of metallothioneins in astrocytes could be part of a compensatory mechanism in goats devoid of PrP
C.
Systemic administration of LPS has been shown to activate microglia in the hypothalamus, thalamus, and brainstem as early as 8–24 h after LPS challenge [
58], but murine hippocampal microglia were not activated until 48 h post challenge [
59]. The latter study is consistent with our results as we did not observe increased AIF1 expression or altered Iba1 immunohistochemical labeling in hippocampus 29 h after the first LPS injection. Altogether, the transcriptional and morphological findings indicate that only a modest inflammation, with a predominance of astrocytes, was present in the hippocampus. This might not be sufficient to manifest clearly potential phenotypes related to the loss of PrP
C and further suggests that this brain region is relatively protected from circulating endotoxins. Still, the clinical signs of sickness behavior, and difference between the
PRNP genotypes in this respect, demonstrate the sensitivity of the CNS towards inflammatory insult and that this sensitivity is increased in the absence of PrP
C.
Although not statistically significant, LPS upregulated
PRNP transcripts in both the hippocampus and ChP of
PRNP
+/+ goats, indicating a role for PrP
C in acute inflammation. Similarly, systemic LPS upregulated PrP
C in circulating neutrophils [
60], whereas LPS incubation increased
PRNP expression in neuronal cell cultures [
61]. As expected,
PRNP expression was low in PrP
C-deficient goats, regardless of treatment, which probably reflects nonsense-mediated mRNA decay [
62].
Acknowledgements
The authors are grateful to Siri Bjerkreim Hamre and Wenche Okstad for skillful laboratory work. The authors acknowledge Lucy Robertson for proofreading the manuscript.