Role of TOLL-like Receptors in Adjuvant-Augmented Immune Therapies by T. Seya

I have two approaches to CAM and therefore to eCAM. First as an experimental biologist, I would like to see CAM emerge not as a highly descriptive branch of biomedicine. CAM as a biomedical pursuit should be driven by imaginative probing—a search for evidence through endless questioning. Second, because of my interest in an evolutionary approach, especially to the mysteries of the immune system, I seek to search for origins as a way to understand the length and breadth or vertical and horizontal positions of biomedical phenomena. The extensiveness in either of the four directions provides important clues as to underlying explanations that often serve to strip away the mystique of what are more complex mechanisms in mammals, especially in humans, which after all is what CAM is all about—its essence. Taking the question of TOLL as one infinitesimal example of gene products, we can view signaling with respect to CAM but from the evolutionary position. It was this evolutionary connection that inspired me to pick up where Seya left off. Actually, Seya and co-workers (1) have considered evolutionary questions by examining TOLL in fish. ‘Rainbow trout Onchorhynchus mikiss possess two genes encoding putative leucine-rich repeat (LRR)-containing proteins similar to human TLR5. Molecular cloning of these two LRR proteins suggested the presence of a TLR5-like membrane form (rtTLR5M) and a soluble form (rtTLR5S). [They] elucidated the primary structures and the unique combinational functions of these fish versions of TLR5. Thus, two distinct gene encodes the TLR5 orthologs in fish, one of which has a consensus intracellular domain (TIR)’. According to Seya et al. (2), ‘the TLR system (TLRs, TOLLlike receptors) is essentially a foreign substance-recognition system. Thus, it is expected that many constituents of CAMs can be agonists of TLRs, thereby being possible candidates for oral vaccine adjuvants. Several reports have focused on the molecular mechanism by which CAMs are effective in association with TLRs. For example, Platycodon grandiflorum, a traditional oriental herbal medicine exerts immunostimulatory and antitumor effects. PG, a polysaccharide isolated from P. grandiflorum, activates macrophages and induces nitric acid production and mRNA expression of iNOS. PG-mediated nitric oxide production was sustained in macrophages by the TLR4 pathway involving NF-kB (3). An edible fungus used in traditional medicine, Agaricus blazei, induced expression of IL-12. IL-12 production was induced by a TLR4-dependent fashion in macrophages. Oral administration of Agaricus blazei further enhanced NK activity (4). The TLR signaling system is involved in the functional features of CAMs. If this is the case, functional genomics and proteomics and the comprehension of the cell signaling networks may substantially contribute to the development of molecular evidence-based CAM. The potential application of SAGE, DNA microarrays and the concept of signalosome to CAM systems may lead to the identification and characterization of genes and signaling pathways that are involved in CAM modalities (5)’.

Is There an Evidence-based Approach to TOLL and CAM?
I have two approaches to CAM and therefore to eCAM. First as an experimental biologist, I would like to see CAM emerge not as a highly descriptive branch of biomedicine. CAM as a biomedical pursuit should be driven by imaginative probing-a search for evidence through endless questioning. Second, because of my interest in an evolutionary approach, especially to the mysteries of the immune system, I seek to search for origins as a way to understand the length and breadth or vertical and horizontal positions of biomedical phenomena. The extensiveness in either of the four directions provides important clues as to underlying explanations that often serve to strip away the mystique of what are more complex mechanisms in mammals, especially in humans, which after all is what CAM is all about-its essence. Taking the question of TOLL as one infinitesimal example of gene products, we can view signaling with respect to CAM but from the evolutionary position. It was this evolutionary connection that inspired me to pick up where Seya left off. Actually, Seya and co-workers (1) have considered evolutionary questions by examining TOLL in fish. 'Rainbow trout Onchorhynchus mikiss possess two genes encoding putative leucine-rich repeat (LRR)-containing proteins similar to human TLR5. Molecular cloning of these two LRR proteins suggested the presence of a TLR5-like membrane form (rtTLR5M) and a soluble form (rtTLR5S). [They] elucidated the primary structures and the unique combinational functions of these fish versions of TLR5. Thus, two distinct gene encodes the TLR5 orthologs in fish, one of which has a consensus intracellular domain (TIR)'.
According to Seya et al. (2), 'the TLR system (TLRs, TOLLlike receptors) is essentially a foreign substance-recognition system. Thus, it is expected that many constituents of CAMs can be agonists of TLRs, thereby being possible candidates for oral vaccine adjuvants. Several reports have focused on the molecular mechanism by which CAMs are effective in association with TLRs. For example, Platycodon grandiflorum, a traditional oriental herbal medicine exerts immunostimulatory and antitumor effects. PG, a polysaccharide isolated from P. grandiflorum, activates macrophages and induces nitric acid production and mRNA expression of iNOS. PG-mediated nitric oxide production was sustained in macrophages by the TLR4 pathway involving NF-kB (3). An edible fungus used in traditional medicine, Agaricus blazei, induced expression of IL-12. IL-12 production was induced by a TLR4-dependent fashion in macrophages. Oral administration of Agaricus blazei further enhanced NK activity (4). The TLR signaling system is involved in the functional features of CAMs. If this is the case, functional genomics and proteomics and the comprehension of the cell signaling networks may substantially contribute to the development of molecular evidence-based CAM. The potential application of SAGE, DNA microarrays and the concept of signalosome to CAM systems may lead to the identification and characterization of genes and signaling pathways that are involved in CAM modalities (5)'.
follows: (i) to identify the characteristics of the cell population in peripheral blood mononuclear cells (PBMC) stimulated by A. blazei Murill fraction H (ABH); (ii) to determine the optimal concentration of ABH required to induce cytokine production in vitro; (iii) to identify the nature of TLR interacting with ABH on the cell surface; and (iv) to evaluate the influence of ABH on natural killer (NK) cell activity. In Brazil, this fungus is used as a traditional medicine for the prevention of cancer, diabetes, hyperlipidemia, arteriosclerosis and chronic hepatitis.
In a similar approach, Yoon et al. (18) looked for a TLR-4dependent activation of macrophages by polysaccharide isolated from the radix Platycodon grandiflorum. 'Platycodon grandiflorum is a traditional oriental herbal medicine known to have immunostimulatory and antitumor effects. PG, a polysaccharide isolated from P. grandiflorum activates macrophages and B cells.
[They] investigated the membrane receptor and intracellular signaling responsible for the activation of macrophages by PG. Taken together, their results suggest that PGmedicated induction of nitric oxide production and iNOS mRNA expression in macrophages is mediated, at least in part, by TLP4/NF-kappa B signaling pathway'.

TOLL in a Protostome Invertebrate: Mosquito
During the latter part of the 20th century, the discovery of mammalian TLRs, homologs of insect TOLL receptors, linked innate and adaptive immunity. This was the first instance that an immune pathway discovered outside of mammals could be superimposed onto the human immune system, thus unifying aspects of evolution of immune competence. In their study on mosquitoes, Christophides et al. (19) have identified 242 Anopheles gambiae genes from 18 gene families implicated in innate immunity and have detected marked diversification relative to Drosophila melanogaster. Immune-related gene families involved in recognition, signal modulation and effector systems show a marked deficit of orthologs and excessive gene expansions, possibly reflecting selection pressures from different pathogens encountered in these insects' very different lifestyles. In contrast, the multifunctional TOLL signal transduction pathway is substantially conserved, presumably because of counter selection for developmental stability. Representative expression profiles confirm that sequence diversification is accompanied by specific responses to different immune challenges. Alternative RNA splicing may also contribute to the expansion of the immune repertoire.

TOLL/IL-1 in Innate Immunity of Nematode Worm
There are entire sequences of genomes from several invertebrates (e.g. the fruitfly Drosophila, the nematode Caenorhabditis elegans, the tunicate Ciona, the mosquito Anopheles). In addition, there are established roles of JAK/ STAT signaling in Drosophila immune responses (20,21). Genetic and functional genomic approaches have begun to define the molecular determinants of pathogen resistance in C. elegans (22). For resistance to pathogens, conserved signal transduction components are required and these include a TOLL/IL-1 receptor (TIR) domain adaptor protein that functions upstream of a conserved p38 MAP kinase pathway. This pathway may be an ancestral innate immune signaling pathway found in a putative common ancestor of nematodes, arthropods and even vertebrates. Furthermore, it probably predated the involvement of the well known TOLL signaling pathways in innate immunity. Pathogen resistance in C. elegans offers fertile opportunities for continuous probing into evolutionary and mechanistic insights of signal transduction and function in innate immunity.

Invertebrates (Earthworms) Recognize Pathogens by Germ Line Receptors but there is Yet no Apparent TOLL
According to Engelmann et al. (23), 'Earthworm innate immunity depends upon small and large leukocytes (coelomocytes) that synthesize and secrete humoral antimicrobial molecules (e.g. lysenin, fetidin, eiseniapore, coelomic cytolytic factor [CCF]; Lumbricin I). Small coelomocytes (cytotoxic) are positive (CD11a, CD45RA, CD45RO, CDw49b, CD54, b 2 -m and Thy-1 [CD90]; CD24; TNF-a) but negative using other mammalian markers. Large coelomocytes (phagocytic) are uniformly negative. Specific earthworm anti-EFCC 1, 2, 3, 4 mAbs are negative for Drosophila melanogaster hemocytes and mammalian cells but positive those of earthworms. Coelomocytes contain several lysosomal enzymes involved in phagocytosis and a pattern recognition molecule (CCF) that may trigger the prophenoloxidase cascade a crucial innate immune response. Earthworms and other invertebrates possess natural, non-specific, non-clonal, and nonanticipatory immune response governed by germ line genes. TOLL and TOLL-like receptor signaling is essential for phagocytosis and antimicrobial peptide synthesis and secretion in insects and vertebrates but has not yet been shown to be essential in earthworm innate responses'.
In multicelluar organisms, the phagocytic response plays a major role in immune mechanisms against environmental pathogens and in clearance of apoptotic cells. The innate immune system is capable of recognizing conserved microbial structures or products of microbial metabolism [pathogenassociated molecular patterns (PAMPs)] through a set of germ line encoded receptors called pattern recognition receptors (PRRs). The PRRs of the innate immune system, particularly the family of TLRs is responsible for initiating inflammatory responses against invading pathogens. TOLL and TLRs signaling is essential for phagocytosis and antimicrobial peptide production in insects and vertebrates (24)(25)(26)(27)(28). TLR is also present in the nematode worm C. elegans; however, antimicrobial peptide expression is independent from TOLL pathway (29). The point of this review therefore is to present some of the recent characteristics of the innate cellular response in earthworms (annelid worms), a complement to humoral immune functions as summarized in the review by Cooper et al. (30). Earthworms possess a pattern recognition molecule (CCF), which may trigger the prophenoloxidase cascade (29,31), a crucial innate response, and an impressive array of other innate responses, but TOLL-like proteins have not yet been discovered.

Vertebrate Ancestors: The Tunicates Typical Deuterostomes
According to a multiauthored effort (32), the first chordates appear in the fossil records at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. Therefore, to explain the origins of chordates and vertebrates, Paramvir et al. (32) generated a draft of the protein-coding portion of the genome of the common ascidian, Ciona intestinalis. The Ciona genome contains 16 000 protein-coding genes, similar to that of other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development.
Although there is no evidence of adaptive immunity, a search of the Ciona genome reveals a variety of genes that are likely to mediate innate immunity. There are a large number of possible complement genes, including C1q-like and C6-like genes, three TLR genes and a variety of lectin genes. No interleukin or interleukin-receptor genes were identified except for an IL-1 receptor and an IL-17 receptor gene. It is possible that Ciona has evolved distinctive innate-immunity genes, because a search for the protein domains found in vertebrate innate-immunity genes identified a number of Ciona genes that contain these domains in previously unknown combinations. Despite this somewhat negative information, there is evidence of lytic activity in numerous invertebrates that destroy experimental targets (30).
Azumi et al. (33) proposed that the mammalian genome encodes several TLRs, with each TLR responsible for detecting corresponding pathogen-associated molecular patterns (34). The Ciona genome has only three TLR genes, characterized by the extracellular leucine-rich repeat (LRR) motif and the intracellular TIR domain. The genes involved in the TLR signaling pathway have been identified, including MyD88, characterized by the TIR and Death domains, IRAK (IL-1 receptor-associated kinase), TRAF (TNF receptor-associated factor), NFB and IB. Mouse RP105 protein is an atypical member of the mammalian TLR family as it possesses only multiple LRR motifs and no TIR domain. Ten gene models with domain architecture similar to that of RP105 have been identified. As LRR is a motif that also functions in protein-protein interactions and is involved in cell-cell communication, it is conceivable that some of the LRR-containing Ciona genes actually encode cell-adhesion molecules and not pathogen-recognizers.

Receptors and External Agents that can Affect the Immune System
Innate Sensing from the Perspective of NK Cells In a recent commentary by Cooper (35), CAM was viewed with respect to NK cells. According to Medzhitov and Janeway (36), the survival of multicellular organisms is dependent on their ability to recognize invading microbial pathogens and to induce several immune reactions. Recent evidence suggests that an evolutionally ancient family of TLRs plays a crucial role in the detection of microbial infection and the induction of immune and inflammatory responses. According to Beutler et al. (37), in humans, innate immune sensing usually proceeds through the activation of 10 TLRs, which in turn leads to the production of cytokine mediators that create the inflammatory milieu and abet the development of an adaptive immune response.
Each TLR senses a different molecular component of microbes that have invaded the host. TLR4 senses bacterial endotoxins (lipopolysaccharide); TLR9 senses unmethylated DNA, and TLR3 senses double-stranded RNA. Each receptor has a conserved signaling element called the TIR (TIR/resistance) motif that transduces a signal through five cytoplasmic adapter proteins, each of which has a homologous motif. The integration of signals that the receptors emit is a key mechanism that requires resolution with respect to TLRs. By creating random germ line mutations in mice and screening for individual animals with differences in signaling potential, the complex biochemical circuitry of the innate immune response can be unraveled. Currently, more than 35 000 germ line mutants have been produced, and 20 000 have been screened to predict innate immunodeficiency states (37).

How do CAM Agents Activate NK Cells?
Takeda and Okumura (38) have recently reviewed CAM and NK cells and asked the question are Toll-like receptors involved? According to them Toll-like receptors (TLRs) function as the pattern-recognition receptors in mammals and play an essential role in their recognition of microbial and fungal components. Ten members of the TLR family have been identified in humans. TLRs activate NK-kB and other signalling eCAM 2006;3 (1) pathways, which results in the secretion of various inflammatory cytokines. It has been reported that TLR-2/6 recognize some components of zymosan, but not ß-glucan, which result in production of cytokines and chemokines. Thus, TLRs might play important roles in the biological effects of some intaketype CAM agents. This is a very interesting subject for further studies.
TLRs function as the pattern-recognition receptors in mammals and play an essential role in their recognition of microbial and fungal components (39)(40)(41)(42). Ten members of the TLR family have been identified in humans. TLRs activate NK-kB and other signaling pathways, which results in the secretion of various inflammatory cytokines (39)(40)(41)(42)(43). There is also a report that TOLL-like receptors is recruited to macrophage phagosomes and discriminates between pathogens (44). It has been reported that TLR-2/6 recognize some components of zymosan, but not b-glucan, which result in production of cytokines and chemokines (45,46). Thus, TLRs might play important roles in the biological effects of some intaketype CAM agents. This is a very interesting subject for further studies.

Mechanisms by Which Foods Influence Immune Functions
According to Kaminogawa and Nanno (46), 'Probiotics ingested may be partially digested in the gut and incorporated into M cells present in FAE, and then captured by dendritic cells or macrophages in the interfollicular area of PPs. These professional phagocytic cells hold various receptors on their surface capable of binding common structures of microbes, the pathogen-associated molecular patterns (PAMPs). Among the receptors for PAMPs, molecular structure and functions of TLRs (TOLL-like receptors) have been recently unveiled. Ten TLR families (TLR1-TLR10) have been identified and ligands recognized by some TLRs have been determined. TLR2 recognizes peptidoglycans and lipopeptides as TLR4 does lipoteichoic acids and lipopolysaccharides. Moreover, the CpG oligonucleotides universally detected in bacterial DNA are recognized by TLR9. The signaling response to stimuli recognized by TLRs is mainly mediated by an intracellular adaptor molecule, MyD88 (myeloid differentiation factor 88). Thereafter, the nuclear transport of NF-B (nuclear factor-B) is stimulated and de novo synthesis of cytokines is induced (40). It has been proposed that stimuli through TLR2 activate both JNK (c-Jun N-terminal kinase) and ERK (extracellular signal regulated kinase) and induce production of IL-10, while stimuli through TLR4 activate JNK and induce production of IL-12 (47)'.

TOLL and CAM: Perspectives
TOLL is universal and has clear evolutionary counterparts as evidenced by its presence in invertebrates and at least in fish as a representative of lower vertebrate, thus there seems to be a dearth of information derived from many invertebrates and vertebrates. This in a sense is curious and that absence of evidence most probably indicates that no investigators have bothered to search. It is highly unlikely that the genes that control the expression of TOLL have hopscotched throughout the animal kingdom making erratic stops here and there. Another clear indication rings from this commentary concerns the modification of TOLL activities by products usually from plants (48)(49)(50)(51)(52) since there is very little information on modifications caused by natural animal products (48).