Transmissible spongiform encephalopathies or prion diseases, such as Creutzfeldt-Jakob disease (CJD), are fatal neurodegenerative disorders that affect humans and many other mammals (Aguzzi and Calella, 2009). The infectious agent consists of PrP^Sc. PrP^Sc can aggregate, recruit and convert benign cellular prion protein (PrP^C) into abnormal pathological isoforms (Aguzzi and Calella, 2009). Thereby, prions act as “seeds” that trigger a chain reaction of PrP misfolding and aggregation (Jarrett and Lansbury, 1993). Prion diseases have a heterogeneous etiology, as they can be genetic, infectious or sporadic. Infectivity requires the transfer of prion seeds from affected individuals into healthy hosts, whereas in genetic and idiopathic cases prion protein undergoes a spontaneous misfolding of PrP molecules into self-propagating seeds. In humans, sporadic CJD (sCJD) is the most common prion disease, followed by genetic CJD (gCJD) and transmitted CJD (iatrogenic CJD and variant CJD; Aguzzi and Calella, 2009). CJD has been also shown to be transmitted through blood or blood derivatives (Llewelyn et al., 2004; Bishop et al., 2013).

The prion neuropathology is characterized by spongiform degeneration, synaptic and neuronal loss, gliosis and the accumulation of aggregated PrP^SC (DeArmond and Prusiner, 1995; Cunningham et al., 2003; Wadsworth and Collinge, 2011; Hilton et al., 2013). Prion disease typically has long incubation times and a rapid disease progression, and can be manifested in different ways, with behavioral and pathological differences within and across species (Prusiner, 1998; Tanaka et al., 2006; Collinge and Clarke, 2007; Colby and Prusiner, 2011). Interestingly, different strains of prion (e.g., ME7, 79A, 22L, 22A) preferentially affect specific regions of the brain in mice (Cunningham et al., 2005a). The simplest explanation for such regional selectivity would be a differential tropism of prion strains and thereby a regional aggregation and toxicity. However, recent experiments assessing prion misfolding using highly sensitive techniques showed that prion protein seeds accumulate in all brain regions irrespective of neurodegeneration (Alibhai et al., 2016).

Prions disease also provides an interesting experimental approach to model many aspects of neurodegenerative diseases associated with protein misfolding. In mouse models of prion disease, microglia become activated early in the disease process thereby representing a valuable tool for elucidating the impact of neuroinflammation in chronic neurodegenerative disorders.

Prion disease is characterized by an increase in the number of microglia, associated with an activated and phagocytic phenotype (Perry et al., 2002; Perry and O’Connor, 2010; Figure 1). The relative contribution of local proliferation of microglia vs. the infiltration of bone-marrow derived progenitors to this increase has been a source of debate during recent years (Gomez-Nicola and Perry, 2015). However, a recent study demonstrated that, in a murine model of prion disease, local proliferation of resident microglial cells is a major component in the evolution of chronic neurodegeneration (Gómez-Nicola et al., 2013). The increase in microglial density and proliferative activity varies across different regions such as the hippocampus (CA1) and the thalamus, the later showing the biggest increase in cell numbers (Gómez-Nicola et al., 2013). This increase in microglial numbers is independent of the recruitment of circulating monocytes, evidenced by comparing microglial density in prion diseased mice with a C-C chemokine receptor type 2 (CCR2)^−/− background with WT mice (Gómez-Nicola et al., 2014).

The proliferation of microglia in prion disease is regulated by the activation of CSF1R and the transcription factors PU.1 and CCAAT/enhancer-binding protein alpha (C/EBPα, being this system also active in human variant CJD and Alzheimer’s disease (AD; Gómez-Nicola et al., 2013; Olmos-Alonso et al., 2016). The inhibition of CSF1R blocks the proliferation of microglia, leading to a decrease in neuronal death in the hippocampus (Gómez-Nicola et al., 2013). A recent study showed that prolonged inhibition of CSF1R in APPswe/PSEN1dE9 (APP/PS1) mice, a model of AD-like pathology, blocks microglial proliferation and leads to the prevention of synaptic degeneration and to an improvement of performance in memory and exploratory tasks (Olmos-Alonso et al., 2016). CSF1R blockade also showed positive effect in mutant superoxide dismutase 1 (SOD1) models of Amyotrophic Lateral Sclerosis (ALS) by reducing microglial proliferation in the spinal cord and macrophage infiltration into peripheral nerves (Martínez-Muriana et al., 2016). The studies focused to targeting CSF1R suggest that microglial proliferation in prion disease, AD and ALS has a net detrimental contribution to the disease progression.

Targeting microglial proliferation by the specific inhibition of CSF1R renders a different experimental outcome than the unspecific removal of microglia. Some studies have aimed at eliminating microglial cells in prion disease, either by the transgenic expression of thymidine kinase (TK) and “suicide” of proliferating CD11b+ cells (Zhu et al., 2016), or by the non-specific blocker of mitosis cytosine arabinoside (Ara-C; Gómez-Nicola et al., 2013). These approaches indicated a neutral or beneficial role of microglia, as their elimination did not change the trajectory of the disease. However, the technical limitations of these targeting approaches difficult the interpretation. For example, the use of CD11b-TK mice leads to a prominent and uncontrolled death of microglia in the context of on-going neurodegeneration, not providing a physiologically silent way to address the contribution of the cells. Also, the TK transgene in CD11b-TK mice is activated by the administration of ganciclovir, an agent recently identified to have a potent anti-proliferative impact on microglia during brain pathology (Ding et al., 2014). Similarly, the use of Ara-C causes a shift in the microglial phenotype towards a detrimental pro-inflammatory profile, independent from its effects on cell proliferation, accelerating neuronal death (Gómez-Nicola et al., 2013). Together, these findings suggest that the specific and selective targeting of microglial proliferation, instead to their elimination, is an optimal approach to understand the contribution of these cells to the pathology.

Activation of microglia is detectable from early stages of prion disease pathogenesis (Betmouni et al., 1996) and becomes more widespread as the disease progresses, closely associated with the spread of neurodegeneration (Perry, 2016). Microglial activation appears simultaneously with first behavioral deficits (Guenther et al., 2001), at a time point when synapses start to degenerate in the stratum radiatum of the hippocampus, but no neuronal loss occurs yet (Cunningham et al., 2003; Gray et al., 2009). Whether microglia activation is directly caused by accumulating misfolded PrP^sc or as a response to synaptic damage cannot be reliably concluded. While studies in vitro have demonstrated that microglia can be activated directly by PrP and subsequently damage neurons (Brown et al., 1996; Giese et al., 1998), there is limited evidence of a direct response to PrP^Sc aggregates in vivo. In contrast, the mere presence of misfolded prion protein, as detected in various brain regions using high sensitivity techniques, might not be sufficient to induce a microglia-mediated immune response in all brain regions (Alibhai et al., 2016). However, it is widely accepted that microglia activation precedes neuronal degeneration and the onset of clinical disease (Williams et al., 1997; Giese et al., 1998).

The cytokine profile in the prion-diseased brain is associated with the expression of both pro- and anti-inflammatory molecules (Perry, 2016; Figure 1). While a number of studies demonstrated a profile shifted to the anti-inflammatory spectrum, dominated by the expression of transforming growth factor β (TGFβ, C-C Motif Chemokine Ligand 2 (CCL2) and prostaglandin E2 (PGE2) with a limited pro-inflammatory response characterized by IL-1β and tumor necrosis factor α (TNFa; Minghetti et al., 2000; Walsh et al., 2001; Cunningham et al., 2002, 2005b; Perry et al., 2002), other studies have reported that also pro-inflammatory factors are up-regulated in the prion brain (Campbell et al., 1994; Williams et al., 1994; Kordek et al., 1996), suggestive of a mixed inflammatory profile. The lack of consensus regarding the inflammatory profile in the prion brain may arise from the fact that different prion strains, stages of disease and techniques of detection were used. Recent studies using a broader panel of markers support the hypothesis that both pro-and anti-inflammatory factors contribute to the immune response in prion disease. Vincenti et al. (2015) re-analyzed a large transcriptomic database of brains from multiple mouse strains exposed to various prion strains and collected at different stages of disease progression (Hwang et al., 2009) and proposed that most of the differentially expressed genes in the prion brain were of microglial origin and associated with the inflammatory response. Microglia isolated from 79A-infected mice showed increased expression of IL-1β, TNFα and CSF1, but not IL-6, IL-10 or TGFβ, which correlates with disease progression and indicates a classical activation phenotype of microglia in this prion model (Vincenti et al., 2015). A recent longitudinal study reported new inflammatory genes upregulated early in the prion brain, including genes involved in inflammation, monocyte recruitment and growth regulation (Carroll et al., 2015). Concerning signal transduction pathways, an early activation predominantly of the Signal transducer and activator of transcription (STAT)- and Nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) pathways has been observed in prion disease models, determined by the up-regulation of STAT- and NFkB-responsive genes, including many cytokines and chemokines, as well as by the detection of increased phosphorylation of STAT1 and STAT3 specifically (Llorens et al., 2014; Carroll et al., 2015). The inflammatory response seems to be quite consistent between different murine prion strains. Although prion strains 22L, RML and ME7 revealed cellular and regional differences in PrP^Sc accumulation, they showed a similar up-regulation mainly of pro-inflammatory genes and chemokines which correlated with the deposition of PrP^Sc and the onset of glial activation (Carroll et al., 2016).

Targeting individual immune pathways which are dysregulated in prion disease have shown differential effects in modifying pathology. The absence of CCL2 does not drastically affect the disease course (Felton et al., 2005; O’Shea et al., 2008) and similarly, reducing PGE2 levels using dapsone or Nonsteroidal anti-inflammatory drugs (NSAIDs) did not impact disease progression (Guenther et al., 2001; Perry, 2010). On the contrary, inhibition of TGFβ enhanced neurodegeneration, indicating that this immune mediator is critically involved in regulation of the innate immune response in prion disease (Boche et al., 2006). TNFα as well as TNF receptor 1 knockout mice show normal prion disease progression after intracerebral injection of the prion protein (Klein et al., 1997; Mabbott et al., 2000), whereas deficiency of IL-1 receptor type 1 prolongs prion incubation time (Tamgüney et al., 2008) and delays disease onset and protein aggregation and increases survival of diseased mice (Schultz et al., 2004). PrP fibrils have been found to induce IL-1β secretion by microglia in vitro, dependent on components of the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, is sufficient to induce neuronal toxicity (Hafner-Bratkovič et al., 2012; Shi et al., 2012). However, an in vivo study using mice deficient in NLRP3 or the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and thereby lacking functional NLRP3 inflammasome, failed to demonstrate a significant impact on prion pathogenesis, indicating that inflammasomes do not contribute to prion progression (Nuvolone et al., 2015).

Numerous studies have reported signs of oxidative stress in brains of CJD patients as well as in murine models of prion disease (Guentchev et al., 2002; Van Everbroeck et al., 2004; Yun et al., 2006), which might play a role in disease progression by contributing to neurotoxicity and neurodegeneration. Stimulation of microglia with PrP fragments in vitro has been shown to induce growth arrest and the release of nitric oxide (NO) and PGE2 (Villa et al., 2016). Furthermore, in patients with CJD as well as in a mouse model of prion, production of NADPH oxidase 2 (NOX2) was up-regulated specifically by microglia in the affected brain regions (Sorce et al., 2014). Prion-induced mice deficient in NOX2 demonstrated decreased production of reactive oxygen species (ROS), a delayed onset of motor deficits and an increased survival time, indicating that microglia-specific NOX2 production leads to the release of ROS and affects prion pathology (Sorce et al., 2014).

It has been shown that microglia are able to engulf and clear apoptotic neurons (Hughes et al., 2010; Kranich et al., 2010). The phagocytic function of microglia is increased in the prion-diseased brain and associated with enhanced expression of scavenger receptors, cathepsins, and proteins of the respiratory burst, while phagocytic microglia were characterized by a lack of IL-1β expression (Hughes et al., 2010). While microglia were efficient in the uptake of injected latex beads and apoptotic cells, they were unable to remove prion protein aggregates, even upon additional stimulation with lipopolysaccharide (LPS; Hughes et al., 2010). Phagocytosis of apoptotic neurons by microglia has been shown to be dependent on milk fat globule epidermal growth factor 8 (MFGE8). Ablation of mfge8 resulted in accelerated prion pathology, with reduced clearance of apoptotic bodies and increased prion protein accumulation, indicating that microglia phagocytosis via MFGE8 is a protective mechanism in prion disease (Kranich et al., 2010). While in vitro studies using microglia cells or organotypic brain slices proposed that microglia can clear PrP and thereby decrease prion titers (McHattie et al., 1999; Falsig et al., 2009; Kranich et al., 2010; Zhu et al., 2016), evidence that they do so in vivo is still missing. It is possible that the removal of misfolded prion protein is simply inefficient or that PrP^sc is not a sufficient trigger to induce phagocytosis, a similar concept to that proposed for phagocytosis of amyloid β (Aβ in AD (Guillot-Sestier and Town, 2013; Prokop et al., 2013). Furthermore, while the phagocytic function of microglia in prion disease is mostly considered to be beneficial and protective, it is also conceivable that microglia, by taking up cell debris from prion infected cells or possibly PrP aggregates, might even contribute to the spreading of the pathogenic protein (Baker et al., 2002).

TREM2 has been implicated in several neurodegenerative diseases such as AD (Jonsson et al., 2012; Guerreiro R. J. et al., 2013), frontotemporal dementia (Guerreiro R. et al., 2013) and ALS (Cady et al., 2014). TREM2 is an innate immune cell receptor expressed on microglia and other myeloid cells is thought to be involved in phagocytosis of apoptotic neurons and promoting an anti-inflammatory phenotype (Takahashi et al., 2005, 2007; Hsieh et al., 2009). In the context of prion disease, TREM2 has been demonstrated to be up-regulated after prion infection, but the depletion of TREM2 did neither change incubation time and survival, nor microglia immune phenotype during prion disease (Zhu et al., 2015).

Recently, there has been evidence of the involvement of non-coding microRNAs (miR) in regulating the microglia inflammatory response in prion disease. A number of miRs implicated in the regulation of gliosis, glial cell proliferation, the innate-immune response, inflammatory signaling, deficits in neurotrophic signaling and synaptogenesis have been found to be upregulated in human prion disease cases (Zhao et al., 2016). Among them, miR-146a was observed to influence immune response and activation state of microglia (Saba et al., 2012).

While increasing evidence indicates that microglia activation seems to contribute to prion pathogenesis, not much is known about the mechanistic underpinnings. We have shown recently that microglial expansion negatively affects prion disease pathology (Gómez-Nicola et al., 2013), pointing towards a net detrimental role of microglia during chronic neurodegeneration. Another recent study emphasized a protective role of microglia in prion disease, demonstrating accelerated disease pathology upon microglia ablation in the brain (Zhu et al., 2016). This dual role of microglia during prion disease provides further proof that microglia function is highly dynamic and versatile, by promoting neurotoxic effects through facilitating a potentially aberrant and harmful inflammation in prion disease, but also by provoking a protective response through tissue maintenance and repair.

The study of immune to brain communication is gaining interest, with the immune system as a transducer of both endogenous and exogenous challenges to the host. It is known that systemic infection and inflammation are able to produce behavioral changes, known as “sickness behaviors”, that include fever, malaise, anorexia, lethargy and depression (Dantzer et al., 1998), demonstrating an important influence of the immune system on CNS processes. LPS challenge in healthy humans has been shown to produce sickness behavior with fever and neuropsychological symptoms, including reduced declarative memory performance (Krabbe et al., 2005) and increased symptoms of depression (DellaGioia et al., 2012). Systemic inflammation communicates with the brain by several neural and humoral pathways. Receptors for inflammatory mediators present on vagus nerve fibers can respond to inflammatory signals and inform the nucleus of the solitary tract and other regions of the brain (Wang et al., 2003). Macrophages in the circumventricular organs (CVOs), which are regions of the CNS without a tight blood-brain barrier (BBB), are also able to communicate with systemic inflammatory mediators. Signals generated in the CVOs subsequently travel to other regions of the brain (Lacroix et al., 1998). A third route of communication involves cerebral endothelial cells of the BBB, which can transduce signals from blood to the CNS (Laflamme and Rivest, 1999). The molecular pathways by which systemic inflammation alters brain function leading to sickness behavior are not completely elucidated. It has been suggested that the link between the systemic immune system and the CNS is located at the level of the hypothalamus, the main brain region controlling the neuroendocrine system. Systemic inflammation or ageing can modify the hypothalamic function though NFkB activation and microglial-neuronal crosstalk (Li et al., 2012; Zhang et al., 2013). Further studies are needed to elucidate the exact mechanism underpinning the alteration of neuronal function by systemic inflammation.

It is known that microglia play an important role in immune-to-brain communication (Perry and Teeling, 2013). Within this context, the concept of microglial priming has been proposed. Microglial priming occurs as a result of damage associated with chronic neurodegenerative conditions (e.g., misfolded protein, neuronal debris or vascular changes), or several other stressors affecting the nervous system, such as maternal separation, acute injury or aging. It consists of an exaggerated or heightened microglial response—much stronger than that observed in stimulus-naïve microglia—to a second inflammatory stimulus. Most of the evidence supporting the hypothesis of microglial priming in chronic neurodegenerative conditions arises from the study of prion models. In fact, microglial priming was first described in prion brains subjected to a systemic challenge mimicking systemic infection (Combrinck et al., 2002). Cunningham et al. (2005b) showed how intracerebral LPS injection in prion mice results in exacerbated IL-1β expression, neutrophil infiltration and NO expression, accompanied with increased cell death. Systemic injection of LPS (Cunningham et al., 2009), Polyinosinic-polycytidylic acid (poly I:C; Cunningham et al., 2002, 2005b, 2007; Field et al., 2010) or TNFα (Hennessy et al., 2017) in prion mice also leads to exacerbated in the brain, with increased expression of pro-inflammatory cytokines and chemokines including IL-1β TNFα and CCL2. Interestingly, the increased inflammation triggered by LPS in ME7 prion brain is independent on circulating IL-1β and IL-6 (Murray et al., 2011; Hennessy et al., 2017). Although the exact mechanism by which microglia is primed is still unknown, it has been suggested that cyclooxygenase 1 (COX-1) expression in microglia mediates the systemic effects of LPS in the prion brain (Griffin et al., 2013). More recently, Hennessy et al. (2017) reported that astrocytes could be also primed in the prion brain, and generate exaggerated levels of cytokines and chemokines after systemic LPS challenge. Although these findings suggest that microglia in the prion diseased brain are primed by the ongoing pathology and that a secondary stimulus switches these cells to an aggressive phenotype, there is still no defining criteria to classify this state, and it remains unclear whether different patterns of priming result from different forms of neurodegeneration or different systemic inflammatory stimuli.

Interestingly, priming observations and the impact of the immune system on the CNS are not restricted to prion disease (Perry, 2010; Cunningham, 2013). Systemic LPS injection can induce degeneration of cells in the substantia nigra (Gao et al., 2002; Qin et al., 2007) and systemic challenge with IL-1β in an animal model of PD leads to enhanced degeneration of substantia nigra neurons and pro-inflammatory cytokine production (Pott-Godoy et al., 2008). Systemic challenge with LPS or other bacterial toxins in an experimental model of multiple sclerosis, Experimental autoimmune encephalomyelitis (EAE), can also exacerbate neurological symptoms (Schiffenbauer et al., 1993). Systemic LPS challenge caused increased synthesis of pro-inflammatory cytokines in the CNS of transgenic mouse models of AD-like pathology (Sly et al., 2001), and tau phosphorylation (Roe et al., 2011). Chronic systemic inflammation in the form of osteoarthritis results in accelerated neuroinflammation and Aβ pathology in APP/PS1/Col1-IL1βXAT mice (Kyrkanides et al., 2011). There is also evidence about the impact of the immune system upon human neurodegenerative diseases. Systemic infection and increased systemic inflammation have been associated to an enhanced cognitive decline in AD patients (Holmes et al., 2003, 2009, 2011), and systemic infections are associated with relapses in multiple sclerosis in patients (Buljevac et al., 2002).

Overall, evidence suggests that systemic inflammation contributes to the pathology during chronic neurodegenerative conditions, with microglia acting as a hub of communication between the systemic and CNS compartments. According to this view, it is likely that a correct treatment and management of systemic inflammation could potentially delay the progression of neurodegenerative disorders.

Evidence from the literature supports a role of microglia-mediated inflammation in human prion diseases. Activated microglia are found in the brains of CJD patients (Sasaki et al., 1993; Szpak et al., 2006) where they appear to be closely associated with PrP^Sc deposits (Miyazono et al., 1991; Guiroy et al., 1994; Muhleisen et al., 1995). However the degree of microglial reactivity seems to depend on the subtype of prion disease and the type of biochemical PrP^Sc (Puoti et al., 2005; Shi et al., 2013). Increased levels of inflammatory cytokines such as IL-8, CCL2, TGFβ, TNFα and IL-1β have been found in the cerebrospinal fluid (CSF) of sporadic CJD cases (Sharief et al., 1999; Stoeck et al., 2006, 2014) and the inflammatory response seems to correlate with the severity of lesions (Van Everbroeck et al., 2002). A recent study demonstrated subtype-specific and region-specific changes in glia activation and the expression of inflammatory mediators in CJD, with inflammation being pre-dominant in the cerebral cortex in the CJD subtype PRNP codon 129 Met/Met type 1 (MM1) and in the cerebellum in PRNP codon 129 Val/Val type 2 (VV2) cases (Llorens et al., 2014). Microglia markers such as CD11b, Iba-1 and CD68 were found to be up-regulated in a region- and subtype-specific manner which correlated with the up-regulation of pro- and anti-inflammatory cytokines, members of the complement system, the integrin family and C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily, toll-like receptors, colony-stimulating factors and cathepsins (Llorens et al., 2014). Additionally, regulatory proteins IL-34, PU.1 and C/EBPα involved in microglial proliferation are increased in variant CJD (Gómez-Nicola et al., 2013). The increased inflammatory response observed in the two subtypes of CJD was further associated with an activation of NFkB and STAT1, 3 signaling pathways (Llorens et al., 2014). The finding that key regulators of inflammationCOX-1/2 and PGE2, are elevated in brains of CJD (Minghetti et al., 2000; Deininger et al., 2003; Llorens et al., 2014) further speaks for a crucial role of inflammatory processes in human prion disease, however the exact role of microglia-mediated inflammation during the course of the fatal disease remains to be elucidated.

Increasing evidence highlights the major contribution of the expansion and activation of microglia to the pathogenesis of prion disease. Activated microglia adopts a variety of functionally diverse phenotypes depending on the disease stage and systemic influences. While their response is manifold during prion disease, targeting the microglia-mediated immune response appears to be a useful approach to modify the disease course. Determining the exact mechanistic underpinnings of the neuroinflammatory processes in prion disease is an informative step in order to develop novel treatment strategies targeting neurodegenerative disease.

JO, ES and RM jointly wrote the manuscript and contributed to the drafting. DG-N designed the contents of the revision and drafted the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.