Introductory Chapter: Evolution of Toll-Like Receptors

1. Introduction

The story of TLRs started with the discovery of Toll protein in the common fruit fly or Drosophila melanogaster (D. melanogaster) controlling the dorsoventral body patterning during embryonic development [1, 2]. Later studies identified that the Toll protein in Drosophila also provides antifungal immunity [3]. Furthermore, serine protease network/cluster plays a critical role in the Drosophila Toll protein activation, like cysteine clusters present in the leucine-rich repeats (LRRs) comprising TLR ectodomains (ECDs) [4, 5]. Nineteen-to-Twenty-five tandem copies of LRR motifs form TLR ECDs [6]. The LRR motifs of TLRs form a horseshoe-shaped solenoid to directly interact with their corresponding ligands [5, 7, 8, 9]. The Drosophila Toll protein is like human IL-1 receptor (IL-1R), which, upon recognizing IL-1, activates NF-κB (a human homolog of Drosophila dorsal protein) and generates associated pro-inflammatory molecules [10, 11, 12]. Human TLR4, which recognizes LPS, was identified as a human homolog of the Drosophila Toll protein to generate signaling events activating adaptive immune response [13]. This discovery revolutionized immunology research. Now, we have identified thirteen TLRs in the laboratory mice and ten functional TLRs in humans (Figure 1), which are expressed on the outer cell surface (TLR1, 2, 4, 5, and 6) and intracellularly (TLR2, 7, 8, 9, and TLR13 (in mice only) recognize cytosolic nucleic acids) in cytosolic organelles, such as endosomes [14, 15, 16, 17]. These TLRs recognize different bacteria, viruses, fungi, parasite-derived microbe, or pathogen-associated molecular patterns (MAMPs or PAMPs) and host-derived damage or death-associate molecular patterns (DAMPs), which have been mentioned elsewhere [14, 18, 19, 20]. Thus, the discovery of TLR4 in humans strengthened Janeway’s PRR-PAMP theory [13, 21].

Figure 1.

Evolution of TLRs in the animal kingdom. Conventional TLRs are not present in placozoans, the smallest living animals. They first evolved in porifers or sponges more than 600 MYA. Thereafter, cnidarians, except Hydra and coral, express TLRs involved in host defense and development. Nematodes (C. elegans), arthropods (D. melanogaster), echinoderms (Purple Sea urchin), chordates, including amphioxus, sea squirts (Ciona intestinalis), and mammals, such as humans and mice, express different numbers of TLRs. Please see the text for details.

Evolution has played a critical role in the immune system’s development and function [22, 23]. TLRs are critical mediators of innate immune response against diverse pathogens and host-derived DAMPs. Their activation generates a pro-inflammatory immune response to central immune homeostasis and critically regulates adaptive immune response [18, 24, 25, 26, 27]. Hence, the current chapter introduces the evolution of TLRs, which is essential to understand as their dysregulation is associated with different inflammatory diseases and developmental defects.

2. Evolution of TLRs

Placozoans (Trichoplax sp.), the simplest animals on earth with no body part or organ, lack canonical TLRs (Figure 1) and have only TIR domain proteins with LRR motifs [28]. However, they have orthologs of downstream mediators of canonical TLR signaling, indicating the presence of components of primordial TLR signaling in placozoans. For example, they have complete activator protein-1 (AP-1) and mitogen-activated protein kinase (MAPK) pathways working downstream of TLR signaling in higher animals but lack IκBα of the NF-κB pathway. Their NF-κB called NF-κB-like protein (p1000-subunit-like) lacks death domain (DD), indicating the absence of canonical TLR signaling. Placozoan (Trichoplax sp.) also lacks TBK1 and TRIF, indicating the absence of myeloid differentiation factor 88 (MyD88)-independent TLR signaling [28]. Earlier studies have indicated that evolutionarily, TLRs existed during protostome (develop mouth first during embryogenesis) development [29]. However, a study has indicated the existence of TLRs as a part of innate immune defense in sponges (Demospongiae: Phylum Porifera) (Figure 1), which are lower to protostomes and deuterostomes (develop anus first and mouth develops later) [30]. Along with the TLR, demosponge Suberites homunculus (SD) also express IL-1receptor-associated kinase-4-like protein (IRAK-4 L) and a novel effector caspase, which shares significant sequence similarity with their homologs in higher metazoans. The SDTLR and SDIRAK-4 L are expressed constitutively [30]. However, SDCASPL (caspase) expression is highly artificial triacyl lipopeptide Pam(3)Cys-Ser-(Lys)(4) inducible. Additionally, sponges or porifers also express MyD88 with two protein interaction domains, TIR (Toll/IL-1 receptor) and DD (Death domain), like a higher animal, including human’s MyD88, an adaptor protein working downstream to TLRs [31, 32, 33]. Evolutionarily, sponges (Phylum Porifera) are among the most ancient of the metazoans, dating back to more than 600 million years ago (MYA) (Figure 1) or at least 60 million years before the Cambrian period (between 541 and 485 MYA) [34]. Thus, evolutionarily TLRs are more ancient PRRs than expected earlier.

Starlet Sea anemones or Nematostella vectensis have at least one TLR belonging to the Phylum Cnidaria, Class Anthozoa, and Order Actinaria (Figure 1) [5, 35, 36]. Cnidarians are early diverged metazoans descended from a common ancestor of protostomes and deuterostomes [37]. Another deuterostome invertebrate, the ascidian Ciona intestinalis, expresses only two TLR genes (Ci-TLR1 and Ci-TLR2), which possess a hybrid functionality of human TLRs, which cannot be predicted by the sequence comparison of vertebrate TLRs [38]. This indicates the confounding evolutionary lineages of deuterostome invertebrate TLRs. N. vectensis has separate NF-κB and IκB genes. The N. vectensis NF-κB lacks the C-terminal IκB-like sequences present in all other NF-κB proteins, and two IκB-like genes are present on the loci different from the Nv-NF-κB gene [35]. A gene fusion event has created the NF-κB gene in D. melanogaster and humans. N. vectensis also expresses genes involved in up (Toll- and tumor necrosis-like receptors and ligands, adaptor proteins (Trafs, Myd88), caspases, and a TBK-like kinase) and downstream (NF-κB coactivator protein Bcl-3 and several NF-κB target genes) signaling components of vertebrate NF-κB signaling. Thus, N. vectensis in their cnidocytes have a functional TLR to detect pathogens, such as Vibrio coralliilyticus, and activate NF-κB signaling to mount an effective immune response and their development [36, 39].

Notably, genomes of other cnidarians, such as Hydra (Hydra magnipapillata) or the coral species Acropora millepora, lack TLR genes and conventional TLRs (Figure 1) [35, 40, 41]. However, they (Hydra and Acropora millepora) have receptors with the TIR domain with ECDs lacking LRR motifs, which cluster with other higher animal TIRs, suggesting their relation as TLR-related molecules [5, 40]. They produce antimicrobial peptides (AMPs) by interacting with the LRRs domain-containing proteins with the TIR domain with ECDs lack LRR motifs [41]. Furthermore, they express MyD88 and NF-κβ to exhibit ancestral TLR signaling as MyD88 deficiency decreases their ancient TLR or TIR-dependent antibacterial immunity [42]. Caenorhabditis elegans (C. elegans, a Nematode) has only one TLR called TOL-1 that provides innate immunity by supporting the correct expression of antibacterial factor-2 (ABF-2), a defensin-like molecule, and development and function of chemosensory BAG neurons activated by carbon dioxide (CO₂) for their pathogen-avoidance behavior [43, 44].

The TOL-1 also regulates chemosensory Amphid Wing B (AWB) neurons sensing the cyclic lipodepsipentapeptide, Serrawettin W2, produced by Serratia marcescens [45]. Thus, TOL-1 in C. elegans is critical for the innate immune response against infection and behavioral response to avoid the pathogenic environment. The TOL-1 downstream signaling in C. elegans occurs in the absence of MyD88 and NF-κB as their homologs are absent but involve trf-1, pik-1, and ikb-1, which are homologs of Drosophila Traf, pelle, and cactus genes [44, 46, 47]. The purple sea urchins (Strongylocentrotus purpuratus) have maximum numbers of TLRs (222), which is followed by annelids expressing 105 TLR homologs in Capitella and 16 in Helobdella, Brachiostoma floridae (Amphioxus) expressing 42 TLRs, Xenopus tropicalis (Xenopus) having 19 TLRs, and Danio rerio (Zebrafish) expressing 17 TLRs [14, 48, 49, 50]. Humans have functional TLR10, which is inactive in laboratory mice. Most sea urchin and amphioxus TLR genes are paralogues, indicating these animals have expanded their TLR genes in a species-specific manner [38]. The details of TLRs in different animal phyla have been discussed elsewhere [38, 51, 52, 53].

TLRs have not evolved due to coincidental evolution; instead, they originated due to multigene evolution, except for TLR5 and TLRS5, which may have evolved due to coincidental evolution [49]. Furthermore, they have evolved at a constant and conserved rate. Significant TLR families have diverged during or before the Cambrian period, which lasted for 53.4 million years from the end of the preceding Ediacaran Period 538.8 MYA to the beginning of the Ordovician Period 485.4 MYA and produced the most intense burst of evolution ever known [49]. Synonymous/non-synonymous substitution ratio evaluation has further not supported the positive selection pressure in the vertebrate phylogeny. The coding sequence, function, and signaling pathways initiated by vertebrate TLRs are highly conserved upon recognizing their corresponding ligands [49, 54, 55]. Hence, the TLRs are an example of the evolutionary conservation of a biological system at multiple levels, such as genes, proteins, and networks.

Further studies have indicated that the rapid speciation and adaptation to freezing water temperatures are not critical for the evolution of TLR numbers in Nototheniidae (Perciformes order, Notothenioidei sub-order). This stenothermal monophyletic teleost clade evolved relatively recently in the cold-stable waters of Antarctica). However, it induces a shift in the LRR pathogen recognition domain common to all the Nototheniidae analyzed, and of the six subfamilies of TLR in Nototheniidae fishes, TLR22 was most affected [56].

Furthermore, MyD88-dependent and MyD88-independent downstream TLR signaling pathways have evolved separately with common ancestors for vertebrate and invertebrate orthologs of the MyD88 adaptor molecule [57]. Thus, the MyD88 signaling pathway is very ancient as it originated in sponges), like TLRs, and early duplication events generated different adaptor molecules and their corresponding TLRs.

3. Conclusion

TLRs exist in sponges (Phylum Porifera), which have evolved at least 600 MYA. TLRs protected sponges from infections and critically regulated cellular (cnidocytes) development in cnidarians. They have carried over this ancestry to the hierarchy of the animal kingdom (humans). Hence, TLRs evolved in lower animals (Poriferans) to protect them from invading microbes.