Transmissible Spongiform Encephalopathies (TSEs) or prion diseases are a group of neurodegenerative diseases associated with misfolded prion protein deposits that affect both animals and humans, being along spongiosis, neuronal loss and gliosis, the neuropathological features-hallmarks of this group of diseases. Scrapie is an endemic TSE in many countries worldwide that naturally affects sheep, goats and mouflons. This prion disease has been widely studied and is used in animal models as a prototype of this group of diseases (1). Thus, there is renewed interest in studying this naturally occurring neurological disease in the host species (sheep) to provide reliable information.

Currently, the crucial role of the immune responses in the central nervous system (CNS) of individuals affected with a TSE and other neurodegenerative disorders is being increasingly acknowledged (2–5). In contrast to the theory that described the brain as an ‘immune privileged site’ (6), the neuroinflammation hypothesis postulates that neuroinflammatory mechanisms play a role in the neurodegenerative processes that are associated with brain pathologies such as prion diseases (7–10). Overall, the presence of cytokines has been assessed in several human neurodegenerative diseases, demonstrating that they are produced by different cellular types, including glial cells (11–14). Some studies have proposed that cytokines and other acute phase proteins may contribute to the development of chronic neuropathologies such as Alzheimer’s disease (AD) (15–20).

In TSEs, few studies have focused on the host immune response, probably because it has always been accepted that this group of diseases does not trigger the typical immune response that is observed in other infectious diseases (2). From those studies made in natural model of scrapie, which continue to be a small proportion of all the studies made in prion and prion-like diseases, innate immune responses mediated by dendritic cells, macrophages or microglia have been described during the course of the ovine infection (21, 22). Moreover, an altered profile of inflammatory intermediaries has been specifically shown in some experimental murine models of prion diseases (13, 23–30). However, the local inflammatory response has rarely been assessed (31, 32), and the local inflammatory response has not been previously studied in tissues from different natural models throughout the progression of neurodegeneration. A growing body of evidence indicates that prion protein deposits in a specific anatomical area may act as a stimulus that leads to glial activation and the subsequent cytokine production in vivo (31, 32). These signaling proteins can essentially function to activate neural components of the immune response, such as the hypothalamic–pituitary axis, thus serving as regulatory signals between the immune system and the CNS (33). Consequently, the cytokine-mediated host immune response is probably involved in the neuroinflammatory mechanisms that contribute to neurodegeneration (34–36). The actual role of prion protein-induced glial activation and the subsequent cytokine secretion during infection is still incompletely understood. Thus, the involvement of these cytokines in CNS pathology is a growing area of clinical research (34, 37).

Some previous studies about scrapie have focused on the cerebellum because it is a region of the brain that is particularly vulnerable to insults, and abnormalities in this area are usually easy to recognize (38). In addition, the cerebellum is an encephalic area that has been demonstrated to serve as a pseudo reference region to detect neuroinflammation (39). Specifically to scrapie in sheep, our previous studies (40, 41) have shown that Purkinje cells play a relevant role, being at the same time the most damaged and the most protected cellular type in this brain area. These cells are relevant to neurodegenerative progression because constitute the efferent pathway of the cerebellar cortex, playing a pivotal role in the cerebellar circuitry and, as a consequence, their death results in a functional lesion of the cerebellum leading to defects such as ataxia, dysmetria, dysphagia or tremor. Why these cells are the main target of neurodegenerative damage needs to be explained.

This study aimed to ascertain whether inflammatory cytokines could directly affect Purkinje neuron damage. Despite an alteration of these intermediaries had been evidenced in some experimental murine models (24, 27, 30), some authors showed that deficiencies in most of these molecules have little if any influence on CNS prion disease (42–44). As scarce studies have focused on in situ tisular expression of these proteins (32, 45) and none of them on a natural model, this is the main novelty and relevance of the present study.

Here, IHC was applied to determine the expression level of IL-1, IL-1R, IL-2R, IL-6, IL-10R, TNFαR and IFNγR in the Purkinje cell layer of the cerebellum from affected sheep throughout the progression of neurodegeneration (at the preclinical, clinical and terminal stages of the disease). Among all the cytokines and their receptors, we decided to focus on these markers because an exhaustive literature review revealed that most authors proposed that these cytokines are significant factors that regulate the pathogenesis of neurodegeneration in experimental models (7–10). To measure some other cytokines of potential interest (e.g., lL-2, IL-10 or IFNγ) was intended but technical problems prevented it.

The objective of this study is to assess the local expression of several cytokines and/or cytokine receptors in this specific affected encephalic area (cerebellum) by studying animals naturally affected by different clinical stages (preclinical, clinical and terminal) of scrapie. To the best of our knowledge, this is the first attempt to evaluate whether the local expression levels of these cytokines and/or receptors vary throughout the natural progression of the disease.

This study was approved by the Ethical Committee for Animal Welfare from the University of Zaragoza (Reference number: PI 41/16).

This study provides the inflammatory kinetics of a few innate-immune system related protein markers in the Purkinje cells of cerebellum from sheep with natural scrapie. Our results showed evident changes in the expression of most of the assessed markers for cytokines/receptors in the prion -affected compared to the healthy control sheep.

Previous reports did not observe an association between the upregulation of any pro-inflammatory cytokines and the course of disease by means of IHC (49), probably because, as introduced, natural models were not used nor were cytokines assessed in situ. However, the technique applied here shows differences in the expression of almost all of the assessed cytokines in the scrapie -affected sheep compared to the healthy control sheep, supporting the indisputable role of cytokines in the pathogenesis of TSE. Particularly, compared to the healthy control sheep, the clinical and terminal sheep expressed significantly increased levels of IL-1 and IL-6, while in contrast, the clinical and terminal sheep expressed decreased levels of IL-1R and IL-2R. Moreover, IL-10R and TNFαR expression was substantially increased from the preclinical stage onwards, and the expression of TNFαR in all the stages of scrapie was significantly higher compared to that in the healthy sheep.

As mentioned before, we considered it of interest to determine cytokine expression throughout the progression of neurodegeneration in a natural model. Previous studies of cytokines in models of prion diseases have not done so. Most of the knowledge about neuroinflammation in prion diseases has come from studies using transgenic mouse models. Our study contributes to the growing research on the complex relationship of natural prion diseases with cytokines. However, the upregulation of these mediators requires further research to confirm or exclude its possible detrimental role in chronic neurodegeneration. The fact that cytokines play a key role in neuroinflammation and neurodegeneration has opened new areas of scientific investigation.

There are several studies of prion diseases in which the expression of pro-inflammatory cytokines has been described (24, 25, 31), often in the context of the onset of different clinical stages. However, the profile of cytokines described here differs from previous results, as an increase in some cytokines was detected even at the preclinical stage of scrapie. The differences might be because none of these previous studies focused on the progression of neurodegeneration, and they did not use natural models.

Here, it is shown that IL-1 is highly expressed in ovine scrapie, mainly at the clinical and terminal stages. This interleukin is one of the main pro-inflammatory cytokines and, consequently, its overexpression its linked with neuroinflammation. Likewise, Cunningham et al. (26) demonstrated an overexpression of this cytokine in a mouse model of prion disease. In fact, the early overexpression of IL-1 has been suggested to be a candidate mechanism that contributes to the development of murine scrapie (27), given its relationship with neuroinflammation. This suggestion is consistent with our results in sheep. Actually, the presence of an impaired cytokine response in mice orally infected with scrapie, which mimics the natural route of entry (50), has been described. Similarly, IL-1 gene expression has been associated with susceptibility to scrapie in sheep, and differences in the expression of this cytokine have been shown in the cerebellum but not in the spleen (9). By IHC, we confirmed here the differences in IL-1 expression in ovine cerebellar tissue. Similar to studies of AD in humans (16, 51, 52), we observed that IL-1 is overexpressed in the preclinical animals with scrapie compared to the healthy controls.

To date, there are studies indicating that IL-6 can have both neurotrophic and neurotoxic effects on CNS neurons (53). In addition, IL-6 has been reported to exert trophic effects on glial cells, particularly oligodendrocytes (54). In this study, we found that the expression of this cytokine was significantly increased in the Purkinje cells in the clinical stage of scrapie and reached the maximum expression in the terminal stage. Therefore, this observation is in agreement with previous reports, in which the overexpression of IL-6 was demonstrated to produce neurological disease in mice, activating astrocytes and microglia (55). Moreover, IL-6 was shown to activate astrocytes in vitro (56). In our results, a basal level of this marker was observed in the healthy controls. Indeed, it has been previously described it is constitutively expressed in discrete regions of the CNS not only during illness but also under normal physiological conditions (57).

As explained above, natural models that provide reliable conclusions about neurodegenerative disorders are rarely available. Nevertheless, it has been reported that the anti-inflammatory cytokine IL-10 is neuroprotective concerning prion disease (29) and traumatic brain injury (58), respectively. It has been suggested that this cytokine not only plays a neuroprotective role by acting on IL-10R present on the surrounding neurons but also by increasing the secretion of TGF-1β by astrocytes, which is known to play an important role on the interaction between microglia and astroglia. That could explain the increase of the expression of the IL-10R throughout the scrapie progression observed in the present experiment, and it remarks the importance that the glial cells have in the development of prion and prion-like diseases.

TNFαR staining was consistently observed from the preclinical stage of disease, demonstrating that cytokine immunoreactivity was present before the appearance of clinical signs; this outcome agrees with the report by Williams et al. (48). However, from the preclinical stage onwards, a significant increase in the immunostained Purkinje cells was detected here, and it lasted until the terminal stage of the disease. The meaning of this overexpression, and whether it corresponds to either neuroprotective or neurotoxic roles as suggested by other authors (42, 59), is still unknown. However, since TNFα not only causes the production of cytokines but also plays an important role in the extrinsic apoptosis pathway, changes in either TNFα or TNFαR could be linked with changes in this process of cellular death. More scientific research on this topic should be done in order to clarify the link existing between apoptosis and neurodegeneration.

In the present study in sheep, several IL-1R immunopositive cells in the Purkinje cell layer of the preclinical animals exhibited a morphology consistent with that of astrocytes, which was consistent with previous studies using murine models (25, 27, 31, 45). Nevertheless, one remarkable finding was that neither IL-1R nor IL-2R were increased from the preclinical stage onwards. In fact, there was a progressive decrease in both of these markers in our study. Taken together, these data indicate that, similar to IL-1, a role for IL-2 receptors in TSE neuropathology cannot be excluded. Additionally, previous studies had reported that some cytokine receptors are localized in the granular layer but not in the Purkinje cell layer in rats (60). In contrast, other authors have found that immunostaining for such receptors in the granular layer was low and that this immunostaining was higher in the Purkinje cells in mice (61). This latter distribution is similar to that observed in our study; the cytokine receptors were more highly expressed by the Purkinje cells than by other cells in the sheep cerebellum.

On the basis of results provided in the present study regarding IFNγR, this cytokine is suggested not to play a significant role in natural ovine scrapie.

As cited, some problems arose regarding background in IHC applied here, as previously described by Parker and Smith (62). A possible explanation could be that the recommended PLP fixation and low-temperature paraffin embedding, described as ideal protocols for the IHC detection of cytokines in mouse tissues (63), were not followed in the present study. But most likely, the commercially available antibodies are not as specific for detecting the target epitopes in sheep. Another limitation of the study consisted of the range of age between control and affected animals. However, natural infection studies depend on sample availability.

This preliminary study shows an inflammatory profile in the cerebellum of sheep naturally affected by scrapie at different stages of the disease. Provided that our results suggest a relevant immunological component in the progression of natural neurodegeneration, in the future, this study will be extended to include other inflammatory proteins and brain regions to provide a global profile of the neuroinflammatory process in a natural model of this group of prion diseases, potentially extrapolable to other neurodegenerative disorders. Both quantitative and molecular studies to identify the specific cell types expressing these receptors have been initiated with the objective of elucidating the consistent differences among the distinct disease stages. The observed differences in terms of expression levels confirm that a complex network of cytokines is involved in the pathogenesis of natural scrapie. But moreover, these immunostaining patterns of cytokines would potentially serve as neuroinflammatory markers throughout natural scrapie progression and also as therapeutic target. For instance, Gabay (64) proposed blocking IL-6 as a possible treatment against chronic inflammatory diseases.

By identifying the cell type that expresses these inflammatory mediators, paying particular attention to astroglial and microglial populations as the main components of the host immune response in prion diseases, reliable conclusions about the neurodegeneration process can be also drawn. Undoubtedly, the characterization of cytokines in glial cells will enable us to gain information about the bidirectional communication between the immune and nervous systems. Understanding the actions of cytokines in scrapie and other prion and prion-like diseases could lead to novel therapeutic strategies.