Distinctive Toll-like Receptors Gene Expression And Glial Response in Different Brain Regions Of Natural Scrapie Part 3

We investigated the possible correlations between the neuropathological hallmarks of prion disease and TLR gene expression in four different brain regions of sheep naturally infected with scrapie. In all four brain areas, we observed significant differences in PrPSc deposition, spongiosis, astrogliosis, and microgliosis in the naturally infected versus control sheep. 

Prion disease, also known as temple fair disease, wax-eating disease, etc., is an acute viral infection caused by prions. The disease is mainly transmitted by eating uncooked food containing prions. Once infected with prions, it may cause brain inflammation and nervous system damage, and symptoms such as headache, vomiting, and fever may occur.

However, when encountering such a situation, we cannot lose confidence in our memory because of the existence of prion disease. Most infected people can recover and restore their memory after treatment.

Of course, from a preventive perspective, we can try to eat more cooked food to avoid prion infection. At the same time, if you feel any symptoms, you should also see a doctor as soon as possible to prevent the disease from worsening.

In daily life, we can try some simple memory training, such as reading, compiling lists, organizing events and information, etc. to improve our memory. These training do not require much time and energy, but they are very important for maintaining a healthy brain and effective memory function.

In short, when encountering diseases such as prion disease, we should face it positively, seek professional medical treatment and preventive measures, and pay attention to daily memory exercises to ensure our physical health and efficient work. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because it can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche can also improve blood flow and promote oxygen delivery, which can ensure that the brain obtains sufficient nutrition and energy, thereby improving brain vitality and endurance.

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These differences were most evident in the medulla oblongata, the most caudal area. By contrast, the mildest lesions were observed in the frontal cortex, the most rostral area. This sequential pattern is in agreement with the route of prion neuroinvasion and dissemination throughout the CNS, beginning at entry sites in the spinal cord and obex and ultimately reaching the frontal cortex [1,5,51]. 

Unexpectedly, considering their anatomical proximity, we observed a drastic decrease in PrPSc deposition and vacuolization, and a significant decline in astrogliosis, when moving from the thalamus to the hippocampus, not following the gradual caudo–rostral progression of the pathology. 

A specific microglia morphology known as rod microglia was observed in the proximity of pyramidal cell neurons at CA3 but was absent from all of the other neuroanatomic regions studied. 

To our knowledge, this microglial profile has not been associated with neuroinflammation triggered by prions to date, although a recent study reported the presence of rod-shaped microglia in the cerebellum of human patients with CJD [52]. 

Although little studied since the first descriptions in the 1900s, rod microglia have been recently reported in neurological disorders such as epilepsy, Lewy body dementia, Huntington's disease, and AD, specifically in moderately damaged areas of the cerebral cortex and hippocampus [40,41,53]. The phenotypic expression and functions of rod microglia are not yet clear. 

However, the fact that this morphological feature typically coincides with the presence of neuronal elements that are damaged or vulnerable to damage suggests a neuroprotective role [42,54,55]. 

Rod microglia are not thought to be associated with severe lesions, as the progression toward ameboid morphology is expected in these conditions [56]. The presence of rod microglia in the hippocampus of scrapie-infected sheep, but not in other brain regions, may be indicative of a neuroprotective environment, in good agreement with the limited neuronal loss observed in this area. 

Our analysis of TLR expression reveals a direct correlation between lesion severity and TLR gene overexpression that coincides with the aforementioned caudo–rostral progression of prion neuropathology. 

Conversely, TLR4 was similarly overexpressed in all areas, regardless of the lesion severity. TLR4 overexpression in areas of milder neuronal damage (i.e., the hippocampus and frontal cortex) may reflect a neuroprotective role, as previously described for phagocytic cells such as macrophages and microglia [23,57]. The upregulation of TLR7 has been previously reported in mouse models of prion disease and human patients [22,26,31]. 

We only observed the upregulation of TLR7 in the medulla oblongata, the most damaged area in which the highest levels of spongiosis and PrPSc deposition were detected. 

Neuronal death via apoptosis has been previously associated with TLR7 stimulation [58,59], and its overexpression in the medulla oblongata may be related to this mechanism [60]. Although the role of TLR1 in neurodegenerative diseases is not yet clear, TLR2 involvement in microglial activation has been increasingly demonstrated in amyotrophic lateral sclerosis, MS, and AD [61,62]. 

While the role of TLR2 in prion diseases is not fully understood, beneficial effects have been proposed: survival time is reduced in mice that do not express TLR2 following intracerebral inoculation with scrapie [22]. 

TLR2 and MyD88 overexpression may be also indicative of a proinflammatory microglia phenotype, which could explain the increase in TNF-α and IL-6 that we observed in the thalamus [8,63]. 

Interestingly, experiments using EOC 13.31 cells, an immortalized microglia-like mouse cell line, have shown a dysregulation of the inflammatory response pathway in response to TLR2 activation and suggested a link between TLR2 expression and the accumulation of microglia in a state not optimal for phagocytosis [26]. 

Therefore, TLR2 overexpression in the medulla oblongata and the thalamus, the most damaged areas in which excessive levels of phagocytic microglia were also detected, suggests that the dysfunctional clearance of PrPSc may lead to sustained accumulation of PrPSc and neuronal damage [9,17]. 

Our findings in the hippocampus of scrapie-infected sheep reveal an opposite pattern compared with other analyzed brain areas, with the downregulation of TLR1, TLR2, and MyD88 and no cytokine alterations. 

This may indicate that microglia respond differently in the hippocampus; indeed, it has been shown that TLR2 deficiency in primary microglial cell cultures, from neonatal mice (0–3 days old) and stimulated with neurotoxic peptide PrP106-126, shifts microglial activation from a neurotoxic to a neuroprotective phenotype [63]. 

However, the relevance of the PrP106-126 peptide in prion pathology has been questioned [64]. CD36 is a different type of pattern recognition receptor, able to recognize endogenously derived ligands such as amyloid-forming peptides, that has established roles in the endocytic uptake of those components [65,66]. This receptor has been associated with a proinflammatory microglial status [67]. 

In vitro, stimulation of BV-2 cells, a type of immortalized microglial cell, with PrP106-126 results in CD36 upregulation, increasing proinflammatory cytokines and iNOS and NO production [68,69]. 

Additionally, the recognition of β-amyloid peptide by CD36 triggers the assembly of a novel heterotrimeric complex CD36-TLR4-TLR6 that activates the innate immune response [70,71]. 

In our study, all regions showed significant upregulation of CD36 except the hippocampus. The upregulation of CD36, TLR4, and TLR6 in the most damaged areas, the medulla oblongata and obex, suggests the involvement of this triad in triggering a pro-inflammatory microglial status in response to prion infection. 

Conversely, the absence of CD36 and TLR6 overexpression in the hippocampus suggests that this heterotrimer is not formed, indicative again of a neuroprotective environment in this brain region.

It remains to be explained why microglia respond differently in this brain region. While prion strain-specific cell tropism could determine the pattern of microgliosis and astrogliosis, recent findings suggest that both reactions are mainly influenced by the brain region [18,72]. 

Neither microglia nor astroglia respond uniformly across the CNS, and this region-specific response could result in the selective vulnerability of some brain regions in prion diseases [16,18]. 

In this regard, it has been postulated that the inflammatory response of microglia to prion infection is regulated by the sialylation of PrPSc [14,73–75] and that PrPSc sialylation is brain region-dependent [18]. Specifically, a higher level of sialylation of PrPSc is found in the hippocampus and cortex than in the thalamus and brainstem, suggesting a potential role in the selective vulnerability of these brain regions [18,73,75].

The high level of PrPSc sialylation in the hippocampus may be associated with decelerated prion replication, leading to the distinctive prion-induced microglial activation in this region, consistent with the reduced susceptibility of this region implied by our findings. 

Interestingly, previous findings suggest that the hippocampus may be protected from prion neurotoxicity in natural CJD infection [76,77], and detailed neuropathological studies of CJD cases have reported milder lesions in the hippocampus than in other brain regions [76]. 

Specifically, the archicortex, which primarily comprises the hippocampus, appears to be relatively spared compared with other cortical regions in CJD [77]. This mild hippocampal involvement described in natural CJD is in agreement with the present findings in sheep naturally infected with scrapie. 

Interestingly, this partial protection against at least two natural prion diseases occurs in the hippocampus, which is phylogenetically the oldest region of the cerebral cortex and consists of the most basic type of cortical tissue. 

Further studies will be required to explore the relevance of this correlation. In summary, our results reveal particularly mild neuropathology in the hippocampus of natural scrapie-infected sheep, characterized by lower levels of spongiosis, PrPSc deposition, and astrogliosis than expected given the caudal-to-rostral spread of scrapie lesions. 

Moreover, the presence in the hippocampus of an exclusive microglial morphology, rod microglia, which may play a neuroprotective role [54], together with a distinct pattern recognition receptor (TLR genes and CD36) gene expression profile, further differentiate this brain region from the other neuroanatomic regions in natural scrapie-infected sheep. 

These findings suggest a degree of neuroprotection against natural prion infection in the hippocampus that merits further investigation. Neurodegenerative diseases caused by protein misfolding involve a vicious cycle of inflammation consisting of misfolded protein accumulation, glial activation, and the release of glial inflammatory mediators, which exacerbate protein deposition and neuroinflammation. 

Interrupting this vicious cycle by targeting microglia activation using specific TLR inhibitors at a specific disease stage may constitute a promising approach to limit further neuroinflammation. Our findings highlight TLR2 downregulation as a potential target for such an approach, as this may induce a shift in microglia from a neurotoxic to a neuroprotective phenotype [63]. 

Finally, while great progress has been made in characterizing the diversity of glial phenotypes using mouse models of neurodegenerative diseases, whether mouse models faithfully reflect key aspects of prion diseases is a matter of some debate [78,79]. 

Our results in the tg338 transgenic model reproduced the common pathological signs of prion infection, including marked PrPSc deposition, neuropil spongiosis, astrogliosis, and microgliosis. 

However, infected mice displayed significant overexpression of TLR1 and TLR2 and a tendency towards TLR7 overexpression. While the upregulation of these genes has been previously described in other mouse models infected with scrapie [22,26], this pattern contrasts with our findings in the ovine brain. Remarkably, in the mouse brain, we observed no alterations in the expression of TLR4, the gene for which the greatest changes in expression were observed in the sheep samples. 

These conflicting findings may be a consequence of the different routes of infection and/or the prion protein expression levels in tg338 mice [80,81]. Nonetheless, our results ultimately indicate that the intracerebral inoculation of scrapie in oversized tg338 mice does not reproduce the immune response observed in natural scrapie infection.

4. Materials and Methods

4.1. Scrapie-Infected and Control Sheep

Twenty-one female Rasa Aragonesa sheep (aged from 2–6 years) were included in the present study. All were genotyped for PRNP polymorphisms, as previously reported [82], and were found to display an ARQ/ARQ genotype. Control animals (n = 8) were selected from a flock in which no scrapie cases had been reported. 

Scrapie-infected animals (n = 13) were obtained from scrapie-affected flocks and had been diagnosed by immunohistochemistry (IHC) of rectal mucosa biopsies. Infection was confirmed by post-mortem immunodetection of PrPSc in the obex following published criteria [83]. 

The animals were euthanized by an intravenous overdose of barbiturates and exsanguination. 

At the time of euthanasia, all scrapie-infected sheep showed clinical signs of diverse severity: some animals displayed incipient signs such as pruritus of the back and flanks after digital stimulation and mild reduction of the body condition, whereas others displayed advanced clinical signs such as spontaneous scratching of the tail root, lumbar area, and limbs, neurological signs including ataxia and head tremors, and teeth grinding, wool loss, and intense weight loss [84].

4.2. Infection of Tg338 Mouse

To evaluate TLR gene expressions in the brain of a murine model of scrapie, ten-week old tg338 mice (n = 8) (overexpressing the ovine VRQ/VRQ PrPC 8- to 10-fold [85]) were inoculated with a brain pool from a natural scrapie-infected Rasa Aragonesa sheep sacrificed at the clinical stage. 

The mice were inoculated with 20 uL of the scrapie inoculum (diluted 2% w/v in PBS) into the right cerebral hemisphere under isoflurane anesthesia. Intracerebral injections were performed using a 50 µL syringe and a 25G needle. After inoculation, the mice were administered a subcutaneous injection of buprenorphine (0.3 mg/kg) to induce analgesia. 

As controls, tg338 mice (n = 8) were inoculated with brain homogenate from a scrapie-negative sheep following the same procedure described above. The mice were monitored for the development of clinical signs and euthanized by cervical dislocation when terminal signs of disease such as severe ataxia and inability to feed appeared. 

Mice infected with the scrapie-positive inoculum displayed a mean survival time of 187 ± 26 dpi.

4.3. Tissue Collection

Samples from the CNS were collected and divided sagittally into two halves; one was fixed in 10% neutral-buffered formalin for histopathological and immunohistochemical analysis, and the other was directly frozen and maintained at −80 ◦C for protein analysis or stabilized in RNAlaterTM Solution (InvitrogenTM, Waltham, MA, USA) for RNA extraction and then frozen and stored at −80 ◦C.

4.4. PRNP Sequencing

DNA was extracted from blood samples with Speedtools Tissue DNA Extraction kit (Biotools, Madrid, Spain) according to the manufacturer's instructions. PCR amplification and sequencing were done as described previously [82].

4.5. Immunohistochemistry

Formalin-fixed tissues were processed according to standard histopathological procedures. Tissue sections were paraffin-embedded, cut into 4 µm thick sections, and stained with hematoxylin-eosin (HE) for the evaluation of vacuolation and neuropil spongiosis. 

IHC for PrPSc detection was performed using the mouse monoclonal primary antibody L42 in sheep (1:500 dilution at room temperature for 30 min) (R-Biopharm, Darmstadt, Germany) and rabbit polyclonal antibody R486 in mice (1:8000 dilution, overnight at 4 ◦C) (R. Jackman, unpublished) as previously described [86,87]. 

Sections were also subjected to conventional immunostaining for the astrocyte marker glial fibrillary acidic protein (GFAP) (1:500; Dako, Glostrup, Denmark), and the microglia marker ionized calcium-binding adaptor molecule 1 (Iba1) (1:1000; Wako, Richmond, VA, USA), according to published protocols [88]. All histological and IHC evaluations were performed by two veterinary pathologists blinded to the clinical data. 

Assessments of spongiosis and PrPSc staining intensity were semi-quantitatively performed and adapted following the criteria described in previous studies: vacuolation of the neuropil and the perikarya was scored from 0 (absent) to 5 (very numerous and confluent) [51], the PrPSc signal was quantified based on the extent of immunostaining from 0 (no labeling) to 5 (intense uniform labeling) as previously reported [89], and the extent of GFAP and Iba1 immunolabelling was scored on a scale ranging from 0 to 5 (0 = weak staining; 5 = substantial immunolabelling throughout the region) as described [88]. Four brain regions were evaluated: frontal cortex (Fc) thalamus (Th), hippocampal formation (Hc), and medulla oblongata (Mo), and each area was globally analyzed for the scoring and graphically represented as mean ± standard error.

4.6. Western Blot

100 mg of brain tissue of each brain area (Fc, Th, Hc, and Mo) from 13 scrapie-infected sheep, 8 with advanced clinical signs and 5 with incipient clinical signs, were homogenized in 1 mL of lysis buffer. 

Hemiencephalons of the 8 infected and 8 control mice were homogenized at 10% (w/v) in lysis buffer. Tissue samples were homogenized in grinding tubes (Bio-Rad, Hercules, CA, USA) using a TeSeEPrecess 48 TM homogenizer (Bio-Rad, Hercules, CA, USA), and the protein concentration was measured using the PierceTM BCA Protein Assay kit (ThermoScientificTM, Waltham, MA, USA) according to the manufacturer's instructions. 

For the PrPres analysis, equal protein amounts from tissue homogenates were incubated for 10 min at 37 ◦C with proteinase K solution, as previously described [90].

The resulting samples were subjected to electrophoresis in 12% CriterionTM XT Bis-Tris Protein Gel (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes that were blocked for 1 h with 2% non-fat dry milk in TBST (Tris-buffered saline with 0.1% Tween 20). 

For immunoblotting, the membranes were incubated overnight at 4 ◦C with Sha31 primary antibody (SPI-Bio, Montigny-le-Bretonneux, France) at a concentration of 1 µg/mL followed by 1 h incubation at room temperature (RT) with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1:5000) (Santa Cruz Biotechnology, Dallas, TX, USA). 

Immunoreactivity was detected using the chemiluminescent substrate Immobilon Crescendo Western HRP (Merck, Darmstadt, Germany). The TLR4 protein expression was analyzed from the tissue homogenates mixed with 2×Laemmli Sample buffer (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. 

Forty micrograms of total protein were loaded per well, run in 7.5% CriterionTM TGXTM Precast Midi Protein Gel (Bio-Rad, Hercules, CA, USA), and transferred to PVDF membranes, which were then blocked for 2 h with 4% bovine serum albumin (BSA) (Merck, Darmstadt, Germany) in TBST at RT. The membranes were incubated overnight at 4 ◦C with rabbit polyclonal anti-TLR4 antibody (Novus Biological, Minneapolis, MN, USA) at a concentration of 0.5 µg/mL, and then washed and incubated with goat antirabbit IgG (H + L) HRP secondary antibody (ThermoScientificTM, Waltham, MA, USA) at 1:20,000 for 1 h at RT. 

Blots were visualized as described above. Next, the membranes were stripped with RestoreTM Western Blot Stripping buffer (ThermoScientificTM, Waltham, MA, USA) for 15 min at 37 ◦C, washed, and blocked again. 

Then, the membranes were incubated overnight at 4 ◦C with mouse monoclonal β-actin primary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) at 1:1000, washed, and incubated with anti-mouse m-IgGк BP-HRP secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h. After washing, the blots were developed as described above. 

Densitometries were carried out with ImageJ software and the values were normalized using β-actin. The normalized values were represented with GraphPad Prism 6.0 (San Diego, CA, USA). 

The statistical analyses to compare the infected and control groups were performed with a Student's t-test, and the equality of variances was determined by Levene's test using the SPSS software (SPSS Statistics for Windows, Version 17.0, Chicago, IL, USA). Differences between groups were considered statistically significant at * p <0.05.

4.7. RNA Extraction, cDNA Synthesis, and Gene Expression

Sheep total RNA was extracted from 90 mg of tissue samples from the frontal cortex, thalamus, hippocampus, and medulla oblongata. Mouse brains were divided at the midline and total RNA was extracted from <90 mg obtained from the thalamic area. 

Tissues were homogenized using a TeSeEPrecess 48TM homogenizer (Bio-Rad) with RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) combined with TURBO DNase (Invitrogen TM, Waltham, MA, USA) to remove possible genomic DNA contamination. RNA concentration was determined spectrophotometrically with a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and for each sample, 260/280 and 260/230 ratios were analyzed to verify the sample purity. 

One microgram of complementary DNA (cDNA) was synthesized using qScriptTM cDNA SuperMix (Quantabio BiosciencesTM, Beverly, MA, USA) according to the manufacturer's instructions. In addition, the effectiveness of the DNase treatment was assessed in RT-negative samples. After reverse transcription, the same batch of diluted cDNA was subjected to qPCR to amplify TLRs. 

Two commonly used housekeeping (HK) genes were selected to normalize the expression of the target genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin-beta (ACTβ) [91]. The stability of this HK gene was verified under our experimental conditions. The messenger RNA (mRNA) expression was determined by qPCR for 1 to 10 ovine TLR genes, 1 to 9 murine TLR genes, and the MyD88 gene for both species. 

Two proinflammatory (TNF-α and IL-6) and two anti-inflammatory (IL-10 and TGF-β) cytokines were also studied in the sheep thalamus and hippocampus. Primer sequences and efficiencies had been previously published or were designed using the Primer3Plus tool [92] (Table 2). 

We verified the efficiency of each gene generating a standard curve by amplifying 1:2 serial dilutions of control cDNA and then checking for linearity between the initial template concentration and cycle threshold (Ct) values. All genes showed a correlation coefficient between 0.9 and 0.99, with a slope value of the standard curves in the ranges of −3.2 to −3.5 and a qPCR efficiency of 90–110%.

The qPCR reactions were run using Applied BiosystemsTM QuantStudioTM 5 RealTime PCR System, 96-well with universal amplification conditions: an initial activation and cDNA denaturation step of 10 min at 95 ◦C, followed by 40 cycles of 3 s at 95 ◦C and 30 s at 60 ◦C. 

To identify the presence of nonspecific PCR amplicons or high levels of primer dimers, we performed a dissociation curve protocol after each qPCR reaction. Each sample was analyzed in triplicate in a total reaction volume of 10 µL, consisting of 15 ng of cDNA, 5 µL Fast SYBR Green Master Mix (2X) (ThermoFisher Scientific, Waltham, MA, USA), and the required amount of forward and reverse primers (Table 2). 

Nuclease-free water was added to a final volume of 10 µL. The levels of gene expression were determined using the comparative Ct method. The results were represented as fold-change and the gene expression differences relative to the mean level of the control group scaled to 1.

4.8. Data Analysis and Statistics

All quantitative data collected were tested for normality with the Shapiro–Wilk W test. Histological and immunohistochemical differences between the infected and control groups were evaluated using a Student's t-test or Mann–Whitney U test depending on the parametric or nonparametric data distribution. 

Statistical differences between the four different brain regions in scrapie infected-sheep were determined using one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test or Kruskal–Wallis test, depending on the parametric or nonparametric data distribution. Statistical analyses of the qPCR data were conducted from the mean ∆Ct values for each gene. 

For the statistical comparison of infected and control groups, a Student's t-test or Mann–Whitney U test was performed depending on the normal distribution of each gene, and the equality of variances was determined by a Levene's test. Differences in expression were considered to be significant at p < 0.05. 

The following notations were used to denote p-values in the figures: * p < 0.05; ** p ≤ 0.01; # p < 0.1. SPSS (SPSS Statistics for Windows, Version 17.0, Chicago, IL, USA) software was used for the statistical analyses. Graphs were generated with GraphPad Prism 6.0 (San Diego, CA, USA) and the data shown in the figures represent the mean and the standard error of the mean (mean ± SEM).

5. Conclusions

To our knowledge, the present study is the first to describe the expression levels of TLR genes in different brain regions of natural scrapie-infected sheep and oversized tg338 mice experimentally infected with scrapie. 

Our study clearly shows that TLRs, and especially TLR4 in sheep and TLR1 and TLR2 in mice, are involved in the pathogenesis of scrapie. In addition, in contrast to all other regions studied, the distinctive profile of TLR gene expression, together with the unique microglial morphology and mild neuropathology observed in the hippocampus, suggests a brain region-specific immune response in natural scrapie infection. 

However, further studies will be necessary to characterize TLR expression in microglia, astroglia, and neurons in scrapie infection to understand the precise contribution of each cell type to neuroinflammation. Furthermore, it is yet to be determined whether TLR activation is a direct response to prion toxicity or occurs secondary to other inflammatory mechanisms. 

TLRs constitute a promising target for therapeutic approaches to prion diseases, and therefore a better understanding of TLR-regulated neuroinflammatory responses will be needed to ensure further advances in this area.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/ijms23073579/s1.

Author Contributions: Conceptualization, research, and data curation, M.C.G. and L.M.C.; writing- original draft preparation, M.G.-M., M.C.G., and L.M.C.; experimental methodology, M.G.-M.; writing-review and editing, M.G.-M., M.C.G., L.M.C., and A.O.; in vivo methodology, M.B.; funding acquisition, R.B., and J.J.B.; molecular methodology, B.S.-P.; supervision, M.C.G., and J.J.B. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by "Departamento de Ciencia, Universidad y Sociedad del Conocimiento" (Aragon Government) through the project "A05_20R: Enfermedades Priónicas, Vectoriales y Zoonosis Emergentes".

Institutional Review Board Statement: All procedures involving animals adhered to the guidelines included in the Spanish Law for Animal Protection RD53/2013 and the European Union Directive 2010/63 on the protection of animals used for experimental purposes. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Zaragoza (Permit Number: PI38/159 October 2015 and PI19/14 11 April 2014).

Informed Consent Statement: Not applicable.

Data Availability Statement: The data presented in this study are available within the article text, figures, and supplementary material.

Acknowledgments: We thank Olivier Andreoletti (UMR INRAEENVT 1225-IHAP) and Vincent Beringue (UMR VirologieImmunologieMoleculaires (VIM-UR892), INRAE, Universite Paris-Saclay) for kindly providing the tg338 mice used for these experiments.

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. The pictures and figures in this manuscript are original data.

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