Review articles

By Dr. Okom Ofodile
Corresponding Author Dr. Okom Ofodile
Center for Cardiovascular Research (CCR ), Institute for Phamacology and Toxicology, AG : THEURING, , Hessische Strasse 3-4, 10115 Berlin, Germany - Germany 10115
Submitting Author Dr. Okom Ofodile

Innate Immune system, Pathogenic Microorganisms, Inflammation, Immunotherapy, TNF1-TLR Inhibitor

Ofodile O. Toll-like Receptors (TLRs) and Prion Disease: Relevance to Pathology and Novel Therapy. WebmedCentral IMMUNOLOGY 2011;2(11):WMC002410
doi: 10.9754/journal.wmc.2011.002410
Submitted on: 03 Nov 2011 05:48:43 PM GMT
Published on: 04 Nov 2011 07:17:25 AM GMT


Transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of chronic, fatal neurodegenerative disorders of humans and animals, which have the unique property of being infectious, sporadic or genetic in origin. Creutzfeldt-Jakob disease (CJD) in humans, scrapie (Sc) in sheep and goats, and bovine spongiform encephalopathy (BSE) in cattle are typical prion diseases. Classical CJD can be considered as sporadic, infectious or familial, whereas the new variant of CJD (nvCJD) is considered a BSE derived human disease. The exact pathogenic mechanisms and the exact nature of the infectious agent of this disorder remain uncertain, however, it is largely believed that an abnormal form ( PrPSc) of a host cellular prion protein (PrPc) may compose the substantial parts of the infectious agent and that various factors such as oxidative stress, inflammation, perturbation of glutamatergic homeostasis, over-reactivity of the localized innate immune system of the brain, and microbial attack are implicated in the pathogenesis of PrD. Until very recently the signal-transducing receptors that trigger the acute inflammatory cascade have been elusive. However, this enigma has been recently elucidated with the discovery of a transmembrane receptor protein family, designated, Toll-like receptors (TLRs). TLRs are a family of highly conserved, germline-encoded transmembrane receptors that recognize conserved products of a variety of pathogen-derived molecular patterns (PAMPs), such as lipoteichoic acids(LTA), lipopolysaccharide (LPS), peptodoglycan (PGN), flagellin, unmethylated DNA with CpG motifs, viral double- stranded (ds) RNA and other components of microbial cell walls. In the last decade, advances in understanding mammalian host immune responses to microbial invasion suggest that the first line of defense against microbes is the recognition of pathogen-associated molecular patterns (PAMPs) by the Toll-like receptors (TLRs). TLRs mediate the recognition of PAMPs and inflammatory responses to a wide range of microbial products and they are crucial for effective host defense. Recent discoveries revealed that TLRs also have important role in recognizing and regulating responses to endogenous stimuli, such as heat shock proteins, necrotic cells, extracellular matrix breakdown products, and small synthetic molecules. Importantly, rapidly accumulating data have implicated the TLRs in the development and resolution of pathology in a wide range of neurological conditions. With regard to the aforementioned observations, and coupled, with the margins of what we now understand about the biology and activities of Toll-like receptors, it is conceivable to suggest that these receptor proteins, the TLRs, may play important role to play in the pathology of prion disease. Hence, elucidation and understanding the cellular and molecular basis responsible for both the biochemical and molecular alterations associated with the interactions between the TLRs and pathogenic agent(s), and the key processes of the pathogenic pathways in TSE pathology, and interactions between, and the interdependence of, the innate and adaptive immune responses may open a new dimension to understanding prion diseases.


Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative diseases in mammalian species that are sporadic, but also have been traced to mutations and to infectious transmission, including iatrogenic transfer. TSEs include kuru, Creutzfeldt-Jakob disease (CJD), Gerstemann-Sträussler syndrome (GSS), and fatal familial insomnia (FFI) in human beings, as well as scrapie in sheeps and goats, bovine spongiform encephalopathy (BSE) in cattle, and encephalopathies in mink, cats, mule, deer, elk, and several exotic ungulates [1,2]. Neuron loss, spongiform degeneration and glial proliferation are the main pathological consequences of TSEs [3, 4, 5]. Amyloid plaques are abundant in Kuru, Gerstermann-Sträussler-Schenker and certain forms of sporadic CJD. Fluoride plaques, particularly common in new-variant CJD (nvCJD) are composed of a central core of amyloid surrounded by prominent microvasculation [6]. Abnormal PrPSc accumulation occurs in the majority of, but not all, prion diseases [7, 8]. The pathogenic mechanism underlying these pathological conditions is largely believed to be a conformational conversion of the cellular prion proteinPrPc into disease-specific, beta-sheet-rich forms (PrPSc or Prp-res) that possess abnormal physiological properties such as detergent insolubility and protease resistance, PrPSc or PrP-res [9].Evidence indicates that PrPSc is substantially involved in the pathogenesis of brain changes, and in propagation of the transmissibility of the disease process, by converting PrPc into a likeness of itself [3, 9, 10]. The “protein only” hypothesis, which was later refined into “prion hypothesis” holds that TSEs are distinct from infectious diseases caused by bacteria, viruses, fungi , or viroids in that the origin of the disease is related to conformational alterations of an ubiquitous protein, the prion protein, PrP, and that nucleic acids are not essential for the propagation of the infectious agent [9,11,12,]. Thus, according to prion hypothesis: 1) the normal cellular form of PrP(PrPc) is transformed into disease-related, and possibly in itself infectious, scrapie form, 2) therefore PrPSc is solely responsible for the infectivity and transmissibility of prion diseases, for it it is the sole component of the infectious particle, which is termed prion, [12, 13] and the variations in the tertiary structures of PrPSc would account for the existence of prion strains with distinct biological properties [14]. However, despite the above data, the” protein only” hypothesis and the associated protein-only model cannot explain all the existing data. The prion hypothesis has not yet been experimentally proved [5, 15]. There are, at least, four hypotheses regarding the identity of the infectious agent in prion disease: virus [16], virino [17], Bacterium [18] and prion [19]. Indeed, it has been shown in vivo that PrPSc deposition in neuronal tissue not expressing PrPc has no pathological consequence [20]. In addition, in both infectious and genetic models of TSEs typical symptoms of disease outcome and neurodegeneration have been detected in the absence of observable PrPSc [21, 22]. Additionally, in commensurate to this, indeed, in vitro conversion experiments of PrPc to PrPSc, in which protease-resistance was, achieved by a denaturation/renaturation procedure, resulting in protease-resistant beta-sheet rich PrPSc but not infectious PrPSc [23, 24]. Second, the “prion hypothesis cannot explain the presence of many prion strains that retain and inherit unique incubation period [15, 25].
The cellular prion protein, PrPc, is a normal cellular glycophosphatidylinositol (GPI) -anchored sialoglycoprotein encoded by the PRNP gene [26]. PrPc is found predominantly in CNS, but a lower amount of it is also found in the other tissues [27, 28]. Despite a wide range of postulations, the physiological functions of PrPc have not yet been fully understood. Nevertheless, strong compelling evidence exist suggesting that PrPc is essential for the development of prion disease serving as a template for conformational change [29].This notion is evidenced by Bueler and associates [30]. These authors demonstrated that PrPc knockout (PRNP-/-) mice are resistant to scrapie infection. The finding that PrPc is multiply glycosidated indicates that PrPc may be linked to signal tranduction pathway [31]. Because PrPc has been found to be localized in the synapse, some investigators suggested that the absence of PrPc may alter synapse formation suggesting an involvement of PrPc in neurotransmitter system of CNS [32, 33]. There is also increasing evidence that suggests functional roles for PrPs in the copper metabolism [34, 35, 36]. Recent evidence indicates that PrPc controls the survival of the challenged cells by governing the induction of pro-and anti-apoptotic signaling pathways [37].
Together, the above data suggest that disturbing fundamental uncertainties remains in the pathology of TSEs. For instance, microbial pathogen agent(s), whose exact nature have yet to be fully resolved, should be substantially involved in driving prion disease. Hence the etiology of prion disease is unknown. This presents a substantial obstacle to the development of effective diagnosis, therefore, to date; neither preventive strategies nor long-term effective treatment modalities are available for these diseases. Nevertheless, abundant evidence now exists that neuroinflammation, and oxidative stress-damage contributes to the pathogenesis of several neurodegenerative disorders including Alzheimer’s disease (AD) [ 38, 39] and prion diseases [ 40,41, 42, 43, 44, 45], and all these processes have been shown to be engaged and mediated by Toll-like Receptor signaling actions. Therefore, at the margins of what we now understand, a major event in elucidating a plethora of fundamental uncertainties in TSE pathogenesis may largely rely on our increased understanding of both the nature of the exact infectious agent(s), and the nature of the receptors intimately involved in mediating the inflammatory activities in the pathogenesis of prion diseases.
Until very recently, the signal-transducing receptors that trigger the inflammatory cascade have remained elusive. However, this uncertainty has been a few years ago elucidated with the discovery of a transmembrane receptor protein family, designated, toll-like receptors. These advances in understanding the molecular basis for mammalian host immune responses to microbial invasion suggest that the first line of defense against microbes is the recognition of pathogen-associated molecular patterns (PAMPs) by the TLRs (reviewed in [46, 47]. TLRs have been identified as being part of a large family of pathogen-recognition receptors that play crucial role for the induction of both innate and adaptive immunity. Most recent studies have implicated TLRs in recognition of proteinaceous molecules such as heat shock proteins, and other endogenous ligands including extracellular matrix breakdown products, chromatin-IgG, pulmonary surficants, necrotic cells [ 48,49,50] and endogenous mRNA [51]. Heat shock proteins have been implicated in a plethora of central events in prion disease pathogenesis [52, 53]. Johannes van Noort and co-workers [54] have recently demonstrated the presence and activities of TLRs in the central nervous system, thereby indicating the implication of these receptors in executing physiological and pathophysiological processes in the CNS. Furthermore, Schluesener and colleagues [55] have reported that that astrocytes and microglia are activated upon intracranial injection of CpG ODN (synthetic oligodesoxynucleotides containing the unmethylated 5’-CpG-3’ dinucleotides motif). In addition to this, Hemmi and associates [56] disclosed earlier that Toll-like receptor 9 (TLR9) was responsible for the transduction of inflammatory intracellular signals upon stimulation with bacterial DNA and CpG ODN. Supports for the aforementioned notion have been evidenced by further studies [57, 59, 59]. Together, these data indicate that TLR9 is expressed on astrocytes and microglia, and points to an important role for TLR9-activated glial cells in CpGODN-induced neuropathological conditions. In this respect, I am tempted to suggest that TLRs may have an important role in the development and resolution of TSE pathology because of their critical role in initiation of inflammatory responses, and mediating a wide range of signal transduction pathways intimately associated in the development and resolution of pathology in a wide range of neuropathological conditions. Here I summarize the current state of knowledge on TLR-associated signaling, and interactions, alongside discuss the broad implications of these interactions for the pathology of prion diseases, and concluding that advances in understanding of the biology and TLR pathways may allow us to tackle a wide range of challenges in immunology and medicine.

The finding that the CNS is not immunologically privileged as was previously believed represents a major breakthrough and a milestone in the history of immunological sciences [60]. This recent discovery of the immune response in the brain revives the idea that immunological challenges might well be etiological factors in sporadic cases of neurodegeneration, and also indicates that primary causes of such degeneration could originate outside the central nervous system.
Immunity to infectious agents is mediated by two general systems, innate and acquired immunity (reviewed in ref: [61, 62]. In contrast to adaptive immunity , the innate immunity appears early during evolution and has the ability to recognize pathogenic microorganisms by germ-like encoded receptors, equipped with defined specificities for highly conserved structures present on most pathogenic organisms [63]. The innate immunity, in contrary to adaptive immunity, is activated immediately after infection and rapidly controls microorganism replication, until adaptive immunity takes over.
The innate immune response is activated by a few highly conserved structures present in most pathogenic microorganisms, instead of by recognition by a wide range of various antigens.These structures are defined as pathogen-associated molecular patterns (PAMP) sensed by pattern-recognition receptors (PRP).Among the well-defined PAMPs are bacterial lipopolysaccharide (LPS), peptidoglycan, mannans, bacterial DNA and double-stranded RNA.
PAMPs are believed to be produced almost solely by bacterial pathogens and are are essential for the survival or pathogenicity of microorganisms[64, 65, 66]Patter-recognition receptors are mainly expressed on effector , such as dendritic cells (DCs), macrophages and B cells. All PRRs are displayed by a given cell type and have identical specificities.When a PAMP is recognized, all cells are immediately triggered to perform their effector functions, thereby leading to a rapid innate immune response [67, 68].


Toll-like Receptors
Now, research over the last few years has greatly advanced our understanding of the mechanisms by which the immune system functions, and especially, the innate immune system [69, 70, 71]. In keeping with this, recent advances in understanding the molecular basis for mammalian host immune responses to microbial invasion suggest that the first line of defense against microbes is the recognition of pathogen-associated molecular patterns (PAMPs) by the Toll-like Receptors (TLRs). In addition, the findings of Lemaitre B and colleagues [72] and Poltorak A and colleagues [73] led to the realization that the proximal innate innate immune sensing tool of insects and mammals are related by descent, and thus further indicates a key pivotal role for TLRs in the primary recognition of infectious pathogens by mammals. The mammalian Toll-like receptors, major integral component of the innate immune system, are a family of highly conserved, germline-encoded transmembrane receptors that are critically involved in mammalian host defense. It is now known that there are 13 mammalian TLRs, which can sense molecular patterns that are common constituents of a wide variety of pathogens but are rarely found in the host; 10 in humans and 12 in mice [74,75]. Structurally, TLRs are characterized by the presence of a leucine-rich repeats domain in their extracellular regions and a Toll/IL-1R (TIR) domain in the intracellular regions. In respect of the amino acid sequence and genomic structure, TLRs can be divided into five subfamilies: TLR2, TLR3, TLR4, TLR5, and TLR9. The TLR2 subfamily is composed of TLR1, TLR2, TLR6, and TLR10, and TLR9 subfamily is composed of TLR7, TLR8, and TLR9. TLR1 and TLR6 form heterodimers with TLR2 [67].
TLRs serve to identify conserved products of microbial metabolism (PAMPs), such as lipoteichoic acids (LTA), lipopolysaccharide (LPS), peptidoglycan (PGN), other components of microbial cell walls [76, 77, 67], which enables the innate immune system to recognize invading microorganisms and to induce a protective immune response.
Recent discoveries disclosed that TLRs do not only mediate recognition and inflammatory responses to a wide range of microbial products but also to non-microbial endogenous proteinaceous molecules, heparan sulfate and RNA, DNA and small molecular synthetic products [50, 78,79,80, 51]. The TLRs, hence, play a key central role in innate immunity by recognizing conserved molecular patterns and generating signals leading to the initiation of an adaptive immune response, thereby serving as an important link between the innate- and the adaptive arms of the immune system. Recent discoveries disclosed the identification of a human TLR11. This was originally isolated from murine. Murine TLR 11 appears to be closely related to TLR5 and it is expressed abundantly in kidney and bladder. TLR11-deficient mice are reported to be highly susceptible to infection of the kidney by uropathogenic bacteria [81], suggesting that TLR11 play important role in urinary tract infection. The function of the human TLR11 is not known because of the presence of a stop codon in the gene [81]. Important updates suggested that a microbial profilin-like molecule isolated from the protozoan parasite Toxoplasma gondii (T. gondii) functions as a ligand for TLR11 [ 82]. This profilin-like molecule (a protein) was shown to trigger IL-12 through TLR11.

TLR signaling pathways
TLRs activate distinct signaling cascades via four different Toll/IL-1 receptor (TIR) domain-containing adapter proteins. The four adapter proteins are MyD88 (myeloid differentiation factor 88), MAL/TIRAP (MyD88-adapter-like/TIR-associated protein), TRIF or TICAM-1/ Toll-receptor-associated activator of interferon) and TRAM (Toll-receptor-associated molecule). The aforementioned four adapter proteins transduce signals from all of the TIR domains, activating protein kinases and then the transcription factors that lead to inflammatory effects. Recent discoveries disclosed the identification of a possibly fifth TIR adapter protein, designated, Sarm [83]. However, the function of Sarm is presently completely unknown.
Despite divergent PAMP ligands, all TLRs with the exception of TLR3 activate MyD88-dependent pathways to induce a core of stereotyped responses such as inflammatory events. The pathways that transduce TLR signals in mammals appear to have both similar and dissimilar characteristics from those in Drosophila [84]. In Drosophila the Toll-IMD-pathways are crucial for antifungal and anti-Gram negative bacterial responses, respectively. In mammals the host defense against microorganisms mainly relies on pathways that originate from the common TIR domain of TLRs. The TLR family signaling pathway is highly homologous to that of IL-1R family. Both TLR and IL-1R interact with an adapter protein MyD88, which has a TIR domain in its C-terminal segment but a death domain (DD) in its N-terminal segment instead of the transmembrane domain found in TLRs. MyD88 associates with both the TLRs and IL-1R via interaction between the respective TIR domains. Upon stimulation by a ligand, MyD88 recruits a death domain-containing serine kinase, the IL-1R –associated kinase (IRAK (IRAK 1 and IRAK4). IRAK is activated by phosphorylation through its N-terminal death domain and then activates TNFR associated factor 6 (TRAF6) to stimulate IKappaB Kinase (IKK) complex and MAP kinase. Phosphorylation of IkappaB by IKK complex induces the degradation of Ikappa B through the ubiquitin-proteasome pathway, and subsequent nuclear translocation of liberated NF-kappaB mediates transcription of pro-inflammatory cytokine gerne [85, 86].
The TLR3 ligand, double-stranded RNA has been reported to induce the activation of NF-kappaB in MyD88 knockout (KO) mice, thereby buttressing the notion that TLR3 signaling is independent of MyD88 [87] Furthermore, signaling can occur independently of MyD88 for TLR4, which also activates NF-kappaB through the adapter protein TIRAP and for TLR3, as indicated above, which induces an antiviral interferon (IFN)-? response through TICAM-1[67; 88]. Recently, a germline-induced mutation in TRIF led to identification of another adapter molecule with a TIR domain; TRIF-related adopter molecule (TRAM), shown to be required by TLR4, but not TLR3-mediated IFN response. In Tram-deficient mice, LPS induced persistent NF-kappaB activation, whereas the expression of IFN-inducible genes was defective, thereby strongly pointing to a crucially pivotal role for TRAM in the Toll-like receptor 4-mediated MyD88-independent signaling pathway[ 89, 90] .These compellingly indicate that studies designed to determine the activities of TLRs in any specific pathological condition, whereby strictly MyD88-deficient model was tested (solely putting MyD88-dependent pathway into consideration), could almost impossibly lead to findings, which could be enough to categorically rule out the involvement of TLRs in the development and resolution of the pathology in the concerned model, strongly suggesting that the earlier reports by Prinz et al [ 91 ] that Toll-like receptors were not involved in the pathology of TSE disease should not be considered as very accurate. Thus, individual TLR signaling pathways are divergent, although MyD88 is common to almost all TLRs. Nevertheless, it has become increasingly clear that there are MyD88-dependent and MyD88-independent pathways. Further support for this notion is evidenced by a huge body of important updates [92, 93, 94, 95,]. Judging from the enormous interest in research on these transmembrane receptor proteins, the TLRs, it is very likely that more Toll-like receptor ligands and signaling pathways will be identified in the future. It is also of note that signaling pathways mediated by Toll-like Receptors have also been revealed to be cell type-specific [96, 97].This indicate that , under certain conditions, some cells and tissues might be prone to favor this pathway to the other. The molecular mechanisms underlying this have yet to be fully resolved.

The realization that a ubiquitous protein called, prion protein, plays an important role in the pathology of transmissible encephalopathies is a milestone, and represents one of the most gratifying aspects of research carried out on transmissible encephalopathies during the last five decades. This discovery, concomitantly led to the birth of the “protein only” hypothesis [11], which was later refined into prion hypothesis [12, 9]. Albeit, recent intensive researches and significant advances in the province of prion diseases in the last ten years, fundamental uncertainties remain; the true nature of the causative agent (unconventional virus, virino, bacterium, fungus or “prion”?), mechanisms underlying the conversion of PrPc to PrPSc, the preference of PrPSc accumulation in glia and neurons or both, accurate pathogenic mechanisms of neurodegeneration, precise modes of infection, transmission, definite physiological functions of PrPc and exhaustive structural characterization of PrPc and its pathogenic isoform, PrPSc [98, 99] Hence, a plethora of fundamental uncertainties still remains in prion disease biology and pathology, as previous described. Among all these, the most pressing and conflicting question is that of the true nature of the causative agent in prion diseases. Support for this notion is evidenced by the following data:
Earlier biophysical studies carried out by Eigen M. [100] conclude that the infectious unit in vivo does not considerably correspond with an in vitro form of aggregated prion proteins, thus strongly indicative of the involvement of a non-prion protein identical pathogenic agent in the orchestrated network of events intimately associated with the pathogenesis of prion diseases

Bacterial Involvement
In spirited and thorough studies, coupled with enormous patience and determination, Bastian F.O. and associates [101, 102, 103, 104, 18, 105] followed up progressively the involvement of a bacterium, named, Spiroplasma, which belongs to a large family of mycobacterium, in the pathogenesis of Creutzfeldt-Jakob disease. Support for this notion has also been evidenced by studies carried out by others [106, 107, 108]. Spiroplasmas are helical mycoplasmas that play a significant role in insects and plants diseases [109] and they are also found in arthropods that are likely to bit animals and humans, such as such as ticks and mosquitoes [110]. This subject (Spiroplasmas and their characteristics) has been recently reviewed by Gasparich G. and associates [111, 112] and comprehensive and detailed disclosure of the morphological and the ultrastructural aspects of spiroplasmas have been earlier demonstrated by Cole and associates (109). The earlier report of Bastian FO [101] demonstrating the inclusion of Spiroplasma-like molecules in CJD, was followed by a plethora of data from both Bastian and associates,and other various laboratories substantiating and strengthening the notion, that Spiroplasma-associated inclusions have a role in TSEs pathogenesis [101, 102, 103, 18, 104, 105, 106, 107, 108] The most recent work of Bastian and associate on this subject, overwhelmingly and most compellingly implicated Spiroplasma sp (a Mycobacterium) in the pathology of TSEs [18]. Sequencing of the amplified PCR products of Spiroplasma 16S rDNA robustly confirmed the presence of Spiroplasma –like DNA (Spiroplasma 16S rDNA) in all the tested (5 in number) of TSE brains. This result presents a very clear and reproducible evidence for the involvement of a bacterium in TSE pathology. Consistent with this notion, Ebringer and co-workers earlier suggested the involvement of another bacterium, Acinrtobacter calcoaceiticus, in the brain of a BSE-affacted animal ([113]. Additionally, in commensurate to this, several lines of data have demonstrated the ability of various anti-bacterial drugs ( antibiotics) to suppress the progression of TSEs and in some cases , also to significantly hinder the conversion of PrPc (PrPsen) into PrPSc (PrPres) [ 114, 115]. Now, as we have been thought, the cell wall is responsible for many of the characteristic properties of bacteria (e.g. acid fastness, slow growth, resistance to detergents, resistance to common antibacterial antibiotics, antigenicity). In agreement with this, the mycoplasma, a bacterial family to whom Spiroplasma sp belongs, do not have cell wall, and as a result, the mycoplasma are resistance to penicillins, cephalosporins, vancomycin, and other antibiotics that predominantly interfere with the synthesis of the cell wall [116]. Theses findings are consistent with earlier report showing that spiroplasmas were highly sensitive to antibiotics such as tetracycline, and other antibiotics [117]. Furthermore, neither penicillins, nor very closely related antibacterial antibiotics, as previous described, have been reported to confer proctection to TSE [116,117]. Hence, essentially, the above data strongly suggest involvement of microbial pathogenic agent(s) in the pathology of TSEs [101, 102, 103, 18, 104, 105, 106, 107, and 108]. In addition to this, Seya and Matsumoto [118] have recently disclosed that the functions of three forms of a mycoplasma lipopedtide/protein, designated, macrophage-activating lipopeptide 2(MALP-2), P48, and M161Ag, which were isolated from a mycoplasma, M fermentans ( which thus belongs to the same family as its close relative, Spiroplasma sp), were largely mediated by Toll-like receptor 2. These protein molecules exert similar immunomodulatory effects on macrophages and dentritic cells. Immunomodulatory effects such as cytokine induction, NO production and maturation of antigen-presenting cells. M161Ag has also been associated with the capability to induce in vitro apoptotic cell death and to induce complement activation by binding macrophages via complement elements C3b/C3bi and their receptors [119]. Together, essentially, the above experimental evidence strongly suggests that; 1) microbial pathogen agent(s) should be implicated in driving prion disease, 2) that the TLRs may have critically important role in mediating the pathology of prion disease, and finally 3) the TLRs, indeed, represent attractive target for pharmacological interventions in the development of novel approaches for the management of TSE diseases and a variety of other pathological conditions.

C-reactive Protein
C-reactive protein (CRP) is a serum protein that is massively induced as a part of innate immune response to infection or tissue injury. The ability of CRP to recognize pathogens and to mediate their elimination by recruiting the complement system and phogocytic cells makes CRP and important component of the innate arm of the immune system, whict is solely responsible for the first line of host defense. According to the prion hypothesis, the main proteinaceous component in TSEs is the prion protein, PrPc, whose misfolded isoform, PrPSc, is suggested to be solely responsible for the infectivity and transmissibility of TSE diseases. Aside from prion protein, however, a large number of other proteins have been localized to the TSE-affected brain. These include, for instance, serum amyloid component (SAP) in mice Coe J. and associates [120] and C-reactive protein (CRP) and IL-6 in CJD (121). Equally, in other related neurodegenerative disease such as AD, acute phase reactants including CRP have been located in the diseased brain tissues [122, 123, 124, 125, 126]. Mouse serum amyloid component (SAP), a major acute phase protein, is a physiologic and functional counterpart of C-reactive protein (CRP) in humans. These proteins belong to the member of pentraxin family of proteins and they are potent acute phase reactants, characterized by by the cyclic pentameric structure; these proteins interact with their ligands in a calcium-dependent manner [127, 128, 129]. They are mainly serum constituents and are not formed in the brain under normal circumstances. On the other hand, under pathological conditions, associated with microbial attack, the concentrations of C-reactive protein, for instance, rises dramatically in host defense against the microbial attack. CRP has Ca2+-dependent binding specificity for phosphocholine (PCh), a constiruent of many bacterial and fungal polysaccharides and most biological cell membranes [130, 131]. Consistent with the primary function of CRP, CRP was discovered and named because of its reactivity with the PCh residues of C-polysaccharide (PNC) , the teichoic acid of Streptococcus pneumonia [132], pointing to an important role of TLR in mediating the actions of CRP. This notion is recently evidenced by Greenhalgh and co-workers [133]. These investigators provided evidence indicating the two components of the LPS receptor complex, CD14 and TLR4 should participate in the cellular and molecular events controlling the induction of acute phase proteins, SAA and SAP, in the lever after burn injury.. CRP is also bind other constituents, which do not contain phosphocholine (PCh), such as small ribonucleaoprotein particuiles [134]. Ligand-complexed CRP is recognized by C1q and efficiently activates the classical pathway of human complement system [135, 136]. It has also been proposed that CRP working in concert with the complement component plays a role in the clearance of apoptotic and necrotic cells, thereby contributing to restoration of normal structure and function of injured tissues [137]. However, as with most other elements of immunity, CRP has the lineage, the appropriate receptors, and the capacity to participate in both potentially destructive inflammatory responses and potentially protective responses. Thus recent discoveries have implicated C-reactive in atherogenesis [138, 139], mediation of tissue damage in acute myocardial infarction [140] and in the process of “autotoxicity” in neurodegenerative disorders [141, 142]. Despite the above mentioned observations, however, the functions of CRP are yet fully to be elucidated: Nevertheless, it is now well established that CRP, in host defense against pathogenic agents, exerts three major functions: activation the complement system; opsonization and the induction of phagocytosis [143]. Therefore, a very important function of CRP that could hardly be overlooked is its ability to offer protection against microbial agents. This is believed to be the primary function of CRP, whose blood levels increases from almost zero to several hundred micrograms per milliliter during inflammatory events in a variety of pathological conditions [144, 145]. In addition to this, recently, Szalai AJ [143] lent further support to this notion and extended on it by reporting that the CRP primarily functions in host defense against microbial pathogen agents of largely bacterial, in the first instance, and fungal origins. This notion was consistent with earlier studies carried out by Du Clos and associate [146, 147]. .Xia et al. reported the ability of CRP to offer protection against heightened inflammatory response mediated by toxins such as lipopolysaccharide, which are released by the infectious agents [147]. This finding may be considered consistent with the reports of Coe and colleagues [120], Peyrin and colleagues [40] and Sharief and associates [42]. These investigators demonstrated, totally, independent from one another that systemic inflammatory responses have pivotal role in the pathophysiology/pathogenesis of TSEs. Conclusively, 1) CRP is usually over-expressed as a result of microbial attack largely of bacterial and fungal origins and this represents the fundamental function of CRP, 2) the expression of pentraxin in TSE strongly indicates the involvement of systematic inflammatory events in the pathophysiology and /or pathogenesis of TSEs, 3) the generally believed “Marker” function of CRP is , indeed, purely a secondary function, which is of mere diagnostic importance, 4) the reported expression- and up-regulation of CRP in TSEs [121], could , therefore, very conceivably, be indicative of the presence of exogenous microbial agents, largely , if not, solely , of bacterial and fungal origins, consistent with the reports previously described [143, 145, 147, 146]. Finally, the upregulation of CRP in the context of fulfilling host defense function againt pathogenic microorganisms has been reported to be also implicated in Aspergillus fumigatus conidia-induced attack [148] and in malaria-parasite-related attack , as well [149]. The above observations, indeed, do not reconcile with the “prion hypothesis”. Taken together with the evidence that Toll-like receptors mediate the biological effects of CRP [133,150], coupled with the margins of our present understandings of the biology and actions of TLRs, the above observations strongly strengthens the notion that Toll-like receptors should have important role in the pathphysiology and pathogenesis of prion disease,

Cross-Talk with Bacterial CpG-DNA and TLR9 links Innate and Adaptive Immunity
It has been well established for decades that bacterial DNA is immunogenic in vertebrates. Introduction of bacterial DNA in mice induces anti-DNA antibody and NK cell activation. . In 1995, Krieg et al. [151] using a B cell proliferation essay discovered that 5´-CpG-3´dinuclöeotides with selective flanking bases are important for the immunogenicity of bacterial DNA, thereby disclosing the sequence –specific immunogenic characteristics of bacterial DNA. In addirtion to this, in 1998, Jakob and associates [152] demonstrated that bacterial DNA induces strong Th 1-like inflammatory responses .The usage of this sequence is suppressed in mammals and that eukaryotic CpG motifs are preferentially methylated, which then abolishes their immunogenic potential. Following the work of [153], in 2001, Hemmi et al. [56] disclosed that a member of the Toll-like receptor family, TLR9, is the true receptor for the CpG motif-containing oligodeoxynucleotide (CpG-ODN). Accordingly, the ability of the TLR9 to satimulate the vertebrate innate immune system , and subsequently that of the adaptive immune system, presents TLR9 agonists as attractivetarget for the development of highly effective vaccines for infectious diseases, and as well as a stand-alone therapies or in combination with other therapies in other diseases such as cancer. Advances in this field of research have led to the discovery of different types of CpG -ODNs, and disclosed that the biological activity of CpG - ODNs is not restricted to stimulatory cells of the primary. Recent advances revealed that CpG- ODNs also activate cells in the cental nervous system. For instance, particularly microglia and astrocytes present in the brain were induced to upregulate their expression of cytokine and chemokines following exposure to CpG -ODNs [57]. These observations strongly suggest that CpG- ODNs can directly activate immunologically relevant cells in the central nervous system. Now, in the relentless efforts to eventually work out effective therapeutic approaches for TSE disease management, Hans Kretzschmar and co-workers [154] recently revealed that appropriate employment of CpG deoxyoligonucleotides may have a role in significantly delaying the progression of TSE disease and may, at least, be beneficial as a potent post-exposure prophylactic measure. Thus, essentially, the above data widely imply that pathogenic agent of bacterial origin consistent with the compelling findings of Bastian et al [101, 102, 103, 18, 104, 105, 106, 107, 108] may have a role in driving the prion disease, and that endosomal –lysosomal compartmen, earlier disclosed as the major location for PrPSc synthesis [155], may represent a potential compartmental target for TLR-associated therapeutic interventions. Furthermore, the above data are consistent with the concept of protective role of innate immune system. Therfore, on the basis of the aforementioned observations , it is tempting to suggest that innate immune system is an important player in TSE pathology, and that TLRs may have important role in driving the pathology of TSE disease. Hence, increased understanding of the activities, and biology of TLRs may bear relevance to pathology and management of TSE diseases, and other neurodegenerative diseases .

PrP106-126 (a peptide mimetic of PrPSc) can mature and activate Dendritic Cells (DCs)
The activation of monocyte-derived cells is largely believed to play a pivotal role in the inflammatory process leading to the pathogenesis of many neurodegenerative diseases such as Alzheimer’s disease and prion diseases [156, 157, 158, 159, 160]. Denditic cells have been shown to mature and become highly activated in response to several different mediators, including inflammatory cytokines [161] oligo CpG motifs, LPS and other bacterial products, and dsRNA, and also by endogenous mRNA, as previously described. Support for this notion is evidenced by recent studies carried out by Bacot and associates [162], for instance. These investigators demonstrated, in a set of series of thorough and elegant experiments, the activation of NF-?B signaling pathway in monocyte-derived DCs, which led to the production of inflammatory cytokines and further differentiation of DCs by PrP106-126. These finding indicates that PrP106-126 (a peptide mimetic of the pathogenic PrPSc) has the ability to mature and activate DCs as determined by the increased expression of MHC classII (HLA-DR), costimulatory molecules CD40 and CD80, and the maturation marker, CD 83. This study also lends support to the notion that a proteinaceous molecule, in this case, pathogenic prion protein, PrPSc, (its mimetic) is in a full position to mature and activate DCs. However, despite the elegancy and the convincing strength of the study, essential questions were left open. These include the following points: 1) did PrP106-126 bind directly to the DCs or was the binding activity, as it should be expected, mediated by appropriate cell surface receptors(s). In this context, it is reasonable to assume that the activities of PrP106-126 could be best carried out with the help of appropriate cell surface receptors, which could then effectively mediate, and possibly regulate the activities of the PrP106-126 peptide fragments. In the face of this problem, and based on our present understanding, it is reasonable to contemplate that coordinated association of PrP106-126 with surface receptor proteins should be required to enable a successful interaction between the PrP106-120 and the DCs. Now, at the margins of what we now understand about the biology and the actions of TLRs, albeit that, there are also other available surface receptor proteins [48], it is reasonable to contemplate these family of transmembrane receptors may participate directly or indirectly in this event. As earlier reported by Lee et al. [163, 164], a G protein-coupled receptor formyl peptide receptor-like 1 (FPRL1) has been suggested to mediate the binding activities of PrP106-126, but with low interacting affinity to PrP106-126. This strongly indicates that other receptor proteins should be required in the mediation of PrP106-126 binding-activities, to enable the protein PrP106-126 achieve its objective effectively. Furthermore, Lee et al [163, 164] also concluded that FPRL1 also mediate the activities of amyloid beta peptide (Abeta 42) and that of almost all amyloidogenic molecules. Now, George Perry and associates [165] recently disclosed that TLR4 mediates the microglial activation-associated interaction between amyloid beta peptide and HSP70, thereby , pointing that a member of the Toll-like receptor family of proteins, could also work in concert with FPRL1 in mediating the biological effects of an amyloidogenic molecule( peptides). In this respect, coupled with the margins of what we presently now about the biology and activities of TLRs, and the findings concluding that neurodegenerative disease such as AD share various pathogenic factors with CJD [166, 167, 168, 169] , I am tempted to suggest that TLRs should have a role in mediating this process. The TLRs might be or might not be the cell surface receptors primarily and/or largely responsible for the mediation of PrP106-126 peptide fragments activities in the previously described process [162]. However, at least, TLRs, working in concert, with other receptor proteins including FPRL1 should be implicated, in one way or the other, in this event. It may also be noteworthy, that under certain circumstances the TLR signaling might not be required for particle binding or internalization. However, for mediation of inflammatory and immune responses TLR activation and signaling are indispensable [170] The TLRs should be directly or indirectly involved in the complex cascade of events that finally terminate in inflammatory responses, such as production of TNF-? and activation of NF-?B [171, 172, 173]. Therefore, the above observations lend further support to the notion that the TLRs may have important role in the development and resolution of TSE pathology. Hence, therapeutic strategies designed to inhibit the appropriate TLRs, and FPRL1 receptors and their corresponding intracellular signaling pathways may hold great promise for the management of TSE diseases and related cases. However, to this end, appropriate measures should be taken to see that the positive biological effects of the concerned receptor proteins are not excessively compromised.

The Variability of TLR-signaling Pathways
It is now generally accepted that all TLRs can utilize the adapter protein MyD88 to propagate signals to gene targets to and generate a relatively rapid protective response, by activating NF kappaB or through other routes. However, at least two Toll-like receptors (TLR3 and TLR4), as previously described, can use alternative adapters such as Toll/IL-1receptor resistance (TIR) domain –containing adapter inducing IFN-beta /TRIF) and TRIF-related adapter molecule (TRAM); used by TLR4 than can activate responses different from those elicited by MyD88. Therefore, they (TLR3 and TLR4) utilize the adapter protein MyD88-independent pathway to propagate signals to gene targets. Now, previous report that prion pathogenesis is unhampered in MyD88-/- mice [91] simply told us indirectly that the TLRs can utilize both the MyD88 adapter protein dependent-pathway and also (under certain conditions) the MyD88-independent pathway to propagate signals to gene targets [174, 175 ]. Aside from previously mentioned data, further support for this notion has been evidenced by very recent discovery, which revealed that the TLR3 efficiently mediated the entry of a microbial pathogen agent from the peripheral blood system into the brain [176].TLR3, notably, does not utilize the MyD88 dependent pathway for its activities. In addition to this, at the margins of what we now know about the biology and activities of TLRs in health and disease states, studies designed to investigate the involvement of Toll-like receptor signaling in the pathogenesis of a particular or any specific pathological condition, using a model, whereby only MyD88-dependent pathway, for instance, is strictly put into consideration, could hardly lead to results, which could generate enough data to categorically rule out the involvement of TLR signaling in the development and resolution of the concerned pathologic condition. In the face of the above considerations, the previous report that Toll-like receptors are not involved in prion disease pathogenesis [Prinz et al., 2003] should not be considered as very accurate. It is now generally recognized, supported by a rich vein of data [85, 93, 95, 174 ], that TLRs, working alone or in concert, can effectively carry out their biological functions through MyD88 –dependent, and/or MyD88-independent pathways, as well. Therefore, based on a plethora of data previously described, and, coupled with a huge body of compelling data persuasively arguing in favor of the involvement of pathogenic microorganisms in driving prion diseases, [18, 101, 102, 104, 105, 106, 107,108 113, 177, 178 ], and strong implication of the activated microglia -and activated complement system (both represent integral components of the innate immune system) in TSE pathogenesis [41, 179, 180, 181, 182,183], I am led to strongly contend that Toll-like receptors should have important role in the development and resolution of TSE pathology.

It is now largely recognized that the accumulation of reactive microglia in the degenerative areas represents the major cellular evidence pointing unequivocably to the presence of neuroinflammation in TSE disease and other neurodegenerative disorders, as well. Microglia are macrophage-like cells resident within the CNS, which can perform APC and proinflammatory effector functions following activation. These cells are derived from bone marrow stem cells and populate the CNS early during development and remain in the CNS as resident macrophage population. Microglial cells are quiescent in the CNS unable to perform effector and APC functions until activated by injury or infection, and have been suggested to represent the first line of defense for the CNS, which normally lacks professional APCs until they are recruited to the CNS by inflammatory stimuli [184,185]. The neuropathology of Alzheimer`s disease and prion disease has many common features and it has been suggested that microgial cells play a causative role in the pathogenic cascade of neuroidegeneration in Alzheimer`s disease and TSE disease [ 180, 186]. Support for this notion has been evidenced by histological studies demonstrating that microgial cells are associated with the accumulation of abnormal , disease-associated isoforms of prion protein, PrPSc, in in the central nervous system in prion disease [187]. The findings that fibrillar forms of amyloid –beta , or PrP peptides , stimulate microgial cells in vitro [ 188,189] buttress the notion that the deposition of amyloidogenic peptides induces microgial cell-mediated inflammatory responses that largely contribute to neurodegeneration and the concomitant cognitive decline observed in TSE disease and Alzheimer’s disease.Furthermore, Microglial cells may be activated to produce directly neurotoxic substances and inflammatory mediators in experimental prion disease and are very likely involved in phagocytosing PrPSc and/ or apoptotic neurons [157, 179, 190, 191]. The toxic effect of prion peptide PrP106-126 has been reported to require the presence of microglia, which are activated to release reactive oxygen species. Therefore, PrP106-126 reduces neuronal reistance to oxidative stress [179, 192]. The aforementioned observations points to a critical role for activated microglia as one of the mechanisms of neurodegeneration in prion disease. Support of this notion is evidenced by the pioneering studies of Meyermann and associates [193].These authors concluded that the actions of activated microglia contribute largely to the cascade of events that finally culminate in neurodegeneration in CJD. Now, as previously described, a wealth of scientific evidence has shown that TLRs engage and mediate both the activation and actions of microglia in CNS [54, 55, 57] .In addition to this, TAK 1 ( TGFbeta-activated kinase 1), is a member of the MKKK family that is increasingly being accepted to play a pivotal role in TLR signaling [ 194]. TAK 1 ( TGFbeta-activated kinase 1) is a common upstream kinase that mediates the signal transduction for inflammatory cells via mitogen-activation protein kinase(MAPK) and NF-kappaB pathways , after being activated by inflammatory cytokines and engagement of TLRs by bacterial and viral pathogens. Recent works that LPS/IFN-gamma induction of gene expression utilizes TAK1 as the major signal molecule in glial cells [195] and Bhat and associates [196] have seriously implicated TAK 1 in the induction of nitric oxide synthase gene expression in glia cells. These findings strongly implicate Toll-like receptor protein family activities in modulating the activities of glial cells. In line with this, the discovery that TLR 2 and TLR4 signal through TAK1, and that these two Toll-like receptors are expressed in astrocytes and microglia in vivo and potentially very strongly participate in mediating neuroinflammatory responses to infection and disease processes [ 54, 197], strongly reinforces the notion that TLR-signaling participates in mediating the activities of microglia in CNS ,and this suggests that TAK1 may represent a potent target for anti-inflammatory strategies against neuroinflammatory diseases including TSE disease. Futhermore, microglia, which express TLR9, release TNF and IL-12 when stimulated with non-methylated CpG DNA [198]. Chronic glial activation, neurodegeneration and IL-1beta induction in rat brain has been reported after administration of dsRNA, the ligand of TLR3 [199]. Now, in addition to this, interestingly, the important updates from Olson and Miller [200], most persuasively buttress this notion. These investigators, based on a set of elegant series of experiments, demonstrated a critical role for microglia in the innate immune response to CNS pathogens, leading to the activation of adaptive immune functions, in particular, antigene presenting capacity. The microglia hence represent important component of both the innate and adaptive immune response. Recent work by Ebert et al. [201] buttresses this notion. Together and coupled with the fact that the activation of microglia plays a pivotal in the pathogenesis of TSE disease, I am led to propose that Toll-like receptors should play important role in the pathology of TSE disease.

Toll-like Receptors mediate and regulate Oxidative Stress and Apoptotic Cascades
A wealth of scientific data has implicated TLRs in mediating and regulating the pathogenic cascades of oxidative stress damage [ 202, 203, 204, 205, 206, 207] , and apoptotic cell death [95, 208, 209, 210, 211 ]. Apoptosis is an active type of cell death. It differs from necrosis in its programmed manner, complex regulatory mechanisms, distintive morphological changes and lack of inflammation [212, 213]. Oxidative stress represents the imbalance between biochemical processes leading to production of reactive oxygen species (ROS) and the cellular antioxidant cascade. The consequence of this imbalance causes molecular damage that can lead to a critical failure of biological functions and ultimately cell death. Hence, in line with the aforementioned, oxidative stress, although largely a secondary event, emerges as an important driving force in the pathogenic cascade of events responsible for sustaining and exacerbation of the disease process in a plethora of chronic degenerative diseases. These findings broadly implicate that toll-like receptor activities-based therapeutic interventions directed at the key processes of these two pathogenic factors may positively influence the clinical outcome in the management of a plethora of pathological condiutions including TSE diseases. To this end, importantly, there is now strong evidence that oxidative stress [180, 214, 215] and apoptotic cascades [180, 216, 217, 218,219] are key pathogenic factors involved in the development and progressiong of TSE disease, and that these features contributes strongly in facilitating the processes of neurodegeneration in prion disease brain. Furthermore, the aforementioned obcervations, indeed, do not seem to argue against the involvement of Toll-like receptors in the pathophysiology and pathogenesis of prion disease.

PrPc Conversion to PrPSc and Lipid Rafts
A key feature of prion diseases is the conversion of the normal, cellular prion protein, PrPc, into the beta-sheet –rich disease-associated isoforms ,PrPSc, and the deposition of PrPSc is thought to lead to neurodegeneration [ 9]. The molecular mechanisms underlying the aforemention processes are incompletely understood. However, a convergence of scientific evidence strongly suggest that lipid rafts are intimately involved in the process of PrPc conversion to PrPSc [220, 221]. In this light, an important finding has reported that both PrPc and PrPSc have been found in detergent –resistant microdomains (DRMs), also named lipd rafts [222]. Both PrPc and PrPSc are thus associastzed with DRMs in a cholesterol-dependent. Cholesterol depletion of cells leads to decreased formation of PrPSc from PrPc. As reported by Baron et al. [ 220] the conversion of PrPs to PrPSc occurs when microsomes containing are fused with DRMs containing PrP. Lipid rafts have been defined as liquid-ordered cholesterol and spingolipid rich microdamains within the plasma membrane that are thought to function as platforms for signaling, internalization, and intracellular trafficking [ 223, 224] . They have been described to play a role in multiple prototypical cascades, such as the lipopolysaccharide pathways , and to host multiple signal proteins, including kinases and low molecular weight G-proteins. Little data currently exist regarding the role of lipid rafts in LPS involved-signaling within the macrophages. To this end , important updates by Olsson and Sundler reveal that lipid rafts may play important role in TLR signaling [225]. These authors investigated the significance of lipid rafts in LPS signaling in machrophages. They found that CD14, and MAP kinases (ERK2 and p38) are involved in lipid rafts-associated LPS-mediated signaling in macrophages and that they become translocated to lipid rafts after stimulation with LPS, thereby, pointing to an important role for lipid rafts in LPS-induced TLR4-driven signaling. The findings by Olsson and Sundler[225], buttress the earlier report that within the immune system , lipid rafts play important roles in coordinating and mediating the signaling cascades emanating from multichain receptor complexes such as Fc? receptor, T cell and B cell receptors, and pattern recognition receptors such as the TLRs [226, 227, 228]. Additionally, emerging evidence indicates that Toll-like receptor proteins may represent potential factor in regulating cholesterol metabolism [229]. This suggests a functional role for these transmembrane receptor proteins in influencing the pathogenesis of prion disease, for cholesterol depletion has been demonstrated to influence the conversion of PrPc into PrPSc [ 230]. Furthermore, PrPc is a glycosylphosphatidylinositol (GPI) –anchored protein located in lipid rafts or DRMS [231, 232]. Like other members of this class, many engulfing receptors including CD36 (a sensor of diacylglycerides:TLR-2 ligands [233]),CD44, and CD14 (a prominent co-receptor with TLR4, and a glycosylphosphatidylinositol (GPI) –anchored protein) are present in lipid rafts [234], and ( GPI)-anchor is notably a prominent TLR ligand [ 235], coupled with the fact that GPI-anchors of vasrious parasites have been reported to be associated with a role similar to that of bacterial LPS in the innate immune response, and that despite some structural differences noted among GPI-anchored proteins, all GPI anchors have a common core structure [236], I am tempted to suggest that Toll-like Receptors might have an important role in the process of PrPc to PrPSc conversion in prion disease pathogenesis. Moreover, the ability of cholesterol to mudulate the conversion of PrPc to PrPSc combined with ability of TLRs to regulate cholesterol metabolism suggest that Toll-like receptor protein family may be a fertile avenue of research for the management of TSE diseases. This hypothesis underscores the importance and urgency to proactively address this subject..
Collectively, the aforementioned considerations are, by all means, in agreement with the accepted value of scientific evidence, which generally revolves around the probability and chance. Nevertheless, additionally, in agreement with the elementary lessons in statistics, the probability or chance that all these findings are due to an indirect pathological effects or due to coincidental circumstances related to the conundrums and/or controversies, which prevail in the pathology of TSEs, seems extremely far-fetched. The sheer magnitude of the insidious nature of TSEs, coupled with the fact that neither preventive measures nor long-term treatment modalities are available for the management of this lesion, underscore the urgent need to proactively address the above-mentioned notions by researchers, clinical investigators, and industry.


The recent advances in our understanding of Toll-like receptor functions in the innate immune response shed new light on how immunoinflammatory responses are initiated and mediated within the central nervous system.. It has been increasingly recognized that Toll-like receptors (TLRs) play a major role in innate immunity to recognize specific molecular patterns derived from pathogens including lipids, protein, DNA, RNA, and also recognize endogenous ligands and small molecular synthetic compounds. These observations that TLRs are also able to sense endogenous ligands such as heat shock proteins [49], surfacted protein A [240] and hyaluronan [80] buttresses the “danger theory” of immune activation [241, 242], which holds that the induction of immunity requires stimulation of cells by not only non-self derived ligands but also the so-called “endogenous danger signals” constituting factors released by dead or dying cells as result of tissue damage, for instance. Now; quite irrespective of the evidence compellingly implicating pathogenic microorganisms in driving TSE disease. For instance, microglial activation and inflammatory mediators are detected in plaques in TSE patients as previously described [179, 180, 182, 183], and in Alzheimer’s disease patients [ 243], as well, in CNS inflammation. It is thus conceivable that “endogenous danger signal” exposed in the damage incurred in the brain during the progression of the disease, stimulates viable glia cells through TLRs, thereby initiating inflammatory responses.These events could well be extrapolated to the orchestrated network of events intimately involved in the pathogenesis of TSE diseases. For instance, heparan sulfate, a products of the degradation of heparan sulfate proteoglycan, suggested by a plethora of data to be intimately implicated in TSE pathogenesis [ 244, 245,246], can induce the maturation of DCs through the TLR4, as well, [79, 80,]. The maturation of DCs is an immunologically important process by which the dendritic cells acquire an ability to present antigens and to induce cellular response efficiently. Furthermore, putting into consideration that Drosophila Toll was originally identified as essential molecule for embryonic patterning in Drosophila [247] subsequently shown to be of key importance in antifungal immunity by Lemaitre B. et al. [72], and the Toll-like receptors are the mammalian homologue of the Drosophila Toll receptor [ 248], it is conceivable that certain members of the TLR family expressed on glia cells may be implicated in both the physiological and pathophysiological functions in the brain. To this end, importantly, a huge body of scientific data has compellingly prion protein in embyronic development [ 249; 250; 251]. Therefore, it seems very difficult, if not almost impossible, to exclude the involvement of TLR receptors in the development and resolution of pathology in neuro- pathological conditions including that of TSE diseases.
The ability of TLR-activated APC to activate CD4+ T cells and shape a TH 1-associated immune response has been well described elsewhere [252; 253]. In this respect, as previously described, TLRs activate multiple steps in the inflammatory reactions to eliminate the invading pathogens and subsequently coordinate the systemic defenses, which involves the modulation of multiple dendritic cell functions and the activation of signals that are of critical importance in initiation of adaptive immune responses, unequivocally pointing to a protective function for the innate immune system. On the other hands, when the innate immune system, or a component of the innate immune system, is inappropriately stimulated or activated, the immune responses driven by this inappropriate activation may lead to severe consequences. In this respect, for instance, excessive TLR signaling caused by microbial infection and/or other challenges can lead to detrimental inflammation, tissue /cell damage, and occasionally to death. Now, CpG-containing oligodeoxynucleotides (CpG-ODN) are known as strong stimulators of innate immunity by mimicking the effects of bacterial DNA. Thus, CpG-ODNs, as previously described, are recognized by Toll-like receptor 9 (TLR9). They can act as immune adjuvants, accelerating and boosting antigen-specific antibody responses by up to 500-fold. CpG motifs promote the production of T-helper 1 and pro-inflammatory cytokines and induce the maturation/activation of professional antigen-presenting cells including macrophage and dendritic cells. The therapeutic importance of these products are now being studied, and tested in a wide range of pathological conditions [254; 255; 256; 257; 258] including prion disease. Importantly, employloyment of this agent in the management of TSE disease by Hans Kretzschmar and co-workers [154] led to a protective effect on mice infected with scrapie agent, consistent with the well-established ability of the innate immune system to confer protection to cells. Interestingly, further studies carried out to replicate the efficacy CpG-ODN on the prion disease [259] in a different laboratory by Heikenwalder and associates surprisingly yielded disastrous results. These observations [ 259], however, may not be easy to explain, for very few studies are presently available on this subject with regard to prion diseases. Nevertheless, CpG-ODN employment as was documented by Heikenwalder and associates [259] apparently led to negative clinical outcome, because, very probably, the immune system in the brain of the concerned TSE-infected models must have been already over- stimulated. Therefore, judging from the results obtained by concerned colleagues [ 259], it might be reasonable to conclude that repeated application of the concerned agent to the infected models, whereby the infectious dosis, apparently, must not have been necessarily low enough, could almost impossibly lead to a positive result. Prion protein has now been realized to have the ability to induce the production of antibodies [98, 113] and autoantibodies [260], and the presence of PrPSc (or PrPres), can lead to activation of the complement system as well. In this respect, PrPres can activate the complement system, leading to production of membrane attack complex (MAC) among other complement proteins [41]. Now, as it can be the case under pathological condition, the complement system could be inappropriately stimulated in the process of prion disease progression, and over-stimulation of the complement leading to sustained inappropriate production of the membrane attack complex (MAC) in TSE pathogenesis. This, in turn, can initiate and sustain the process of autodestruction of neuronal cells and viable tissues, by host defense system, as previously described in the cases of Alzheimer`s disease and Amyotrophic lateral sclerosis [141, 142], and, very recently, in the case of CJD by Budka and associates [41]. Furthermore, the mere fact that the presence of PrPres could result to production of autoantibodies; that the structure of microglia (the potent professional APCs in the CNS) could be impaired in TSEs pathogenesis, leading to dysfunction of APCs; and that the homeostasis of the glutamatergic system could be significantly perturbed in TSE pathogenesis [261]; all these events possess the ability to elicit production of autoantibodies in the brain, which could invariably lead to a situation, where “Horror autotoxicus” becomes commonplace. These notions, therefore, strongly indicate that administration of CpG-ODN, under certain conditions, could also result or lead to premature hyperimmunity of autoimmunity, Horror autotoxicus = autoimmune reaction.”Horror autotoxicus,” a Latin expression, is a term coined by Paul Ehrlich at the turn of the last century to describe autoimmunity to self, or the attack of “self” by immune system, which ultimately results to autoimmune condition. For instance, dysfunction of glutamatergic system could lead to over-expression of glutamate receptors, which can in turn result to production of physiologically active autoantibodies [262]. These autoantibodies could be directed towards excitatory ionotropic glutamate receptors in the brain. Hence, it is well-established that appropriate employment of an agent (pharmaceutical agent), in most cases, result to amelioration of the concerned disease state or condition with minimal side-effect. The aforementioned consideration seems to be consistent with appropriate employment of CpG-ODN agents. Support for this notion is evidenced by the work of Zimmermann et al. [263] and that of Schetter and associaste [198]. Additionally, and importantly, this notion has been very recently stengthened in the case of prion disease by Kretzschmar and co-workers [154], who demonstrated positive clinical outcome as a result of CpG-ODN agent employment. The above observations further strengthen the concept that innate immunity may be an important player in TSE pathology, and that TLRs are crucial receptors for the activation of innate immune mechanisms, and therefore; points to a critical role for TLRs in the pathology and therapy of a wide range of diseases including prion diseases. Therefore, essentially the above notions strongly argue that the previous report [259] might not necessarily represent a negation of the well-established protective properties of the innate immune system, nor the efficacy of CpG-ODN in the management of diseases including TSE diseases. Nevertheless, it is of importance to point out that standard conditions for effective administration of CpG- ODN as therapeutic agent have yet to be fully determined. The aforementioned conflicting observations underscore urgent need for increased studies in this direction. To this end as recently suggested in a closely -related case by McGeer and McGeer [264], such studies could lead to less severe consequences, if two major conditions are put into consideration,:1) if the problems of autoimmune reaction were substantially and generally put under control, 2) if appropriate measures are taken to substantially or almost completely prevent specifically the over-reactivity of the complement. Moreover, importantly, the recent discovery disclosing that concerted actions of tumor necrosis factor receptor 1 (TNFR1)-and TLR are may imply that antagonists of TNFR1-and TLR , as well as inhibitors of their intracellular signaling pathways might be effective anti-neuroinvasion agents for the management of a plethora of neurological diseases, where the process of neuroinvasion plays a central role in the disease process. Whilst neuroinvasion is undoubtedly a complex process that may include other mechanisms, these findings by Wang et al. [174] provide important insights into the signaling pathways and processes that result in neuroinvasion. It should, however, not be ruled out, that these observations [174] may bear enormous relevance to TSE pathology, and may hold good promise for novel therapies for a plethora of devastating neurodegenerative diseases including TSE diseases. It is reasonable to speculate that more TLR proteins other than TLR3 may be involved or may have the capacity to mediate this particular process. Therefore, the mere fact that there are currently no preventive measures nor long-term treatment modalities for TSEs, underscores the urgent need for further investigations into this particular issue.
Human Toll-like receptors are transmembrane signal-transducing receptor proteins with an extracellular leucine-rich repeat domain and intracellular domain homologous to the IL-receptor. TLRs act as key receptors responsible not only for the detection of a variety of micobial cell-wall components and bacterial DNA, ds-RNA, endogenous m-RNA, endogeneous proteinaceous molecules, extracellular matrix breakdown particules, but also for the initiation and mediation of signal transduction events eventually leading to the production of various pro- inflammatory mediators. There is no longer any doubt that TLRs are capable of sensing microbial organisms ranging from protozoa [235] to bacteria, fungus and viruses. Therefore; the above observations strongly strengthen the notion that Toll-like receptors may play an important role in the development and resolution of TSE disease pathology. To our knowledge, this is the first work compellingly arguing in favor of the involvement of TLR activities in the development and resolution of TSE pathology. The message is simple; the innate immune system is a complex body. What started originally as the study of fruit flies and caterpillars has become the basis of our hopes for new cures for diseases as lethal as cerebral malaria, sepsis, and systemic lupus erythematosus, and possibly, Creutzfeldt-Jakob diseases, as well. Hence, enhanced understanding of the Toll-like receptor biology , the nature of their various ligands, and the molecular underpinnings responsible for their biochemical effects will allow a model to be constructed in which the type of immune response to any type of infection associated with prion disease, for instance, could be viewed as a function of many different determinants, including the transmembrane protein, TLRs, the form and the nature of the microbe and the cytokine microenvironments. Knowledge gained from these studies, could be employed in targeting appropriately the pathways associated with TLR signal-transducing activities for either inhibition or augmentation, thereby opening novel avenues in future approaches to TSE disease therapy and other various pathological conditions.


The author; Dr. Ofodile, is exceedingly grateful to Obiamaka, Ekene and Ifeanyi for their love, support, and understanding, and for the immense strength, which these brave and wonderful Girls gave to him (their Father) in the last nine years, without which it could not have been possible for him to successfully complete his investigations and bring forward this manuscript.
The author, also, greatly acknowledged immense collegial assistance given to him by Franz Theuring, PhD. during the preparation of this manuscript.

Authors Contribution(s)



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