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Evolutionary responses of innate immunity to adaptive immunity
Infection, Genetics and Evolution 21 (2014) 492–496
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Discussion
Evolutionary responses of innate immunity to adaptive immunity Amanda E. Ward a,⇑, Benjamin M. Rosenthal b,1 a b
University of Maryland-College Park, College Park, MD 20740, United States United States Department of Agriculture, Beltsville Agricultural Research Center, Building 1040, Room 103, 10300 Baltimore Avenue, Beltsville, MD 20705-2350, United States
a r t i c l e
i n f o
Article history: Received 25 August 2013 Received in revised form 18 December 2013 Accepted 20 December 2013 Available online 8 January 2014 Keywords: Toll-like receptors Pattern recognition receptors JAK/STAT signaling Agnathostome immunity Innate immunity VLR immunity
a b s t r a c t Innate immunity is present in all metazoans, whereas the evolutionarily more novel adaptive immunity is limited to jawed fishes and their descendants (gnathostomes). We observe that the organisms that possess adaptive immunity lack diversity in their innate pattern recognition receptors (PRRs), raising the question: did gnathostomes lose the diversity of their ancestors? Or might innate receptors have diversified in the lineage lacking adaptive immunity? We address this question by contextualizing PRRs in their distinct functional roles in organisms possessing or lacking adaptive immunity. In particular, limited PRR diversity in gnathostomes is accompanied by an expansion of the JAK/STAT signaling pathway, which would suggest that the development of adaptive immunity shifted the role of PRRs from the entirety of pathogen recognition to regulators of subsequent immune responses. As PRRs became essential upstream components of the increasingly complex JAK/STAT signaling cascade in organisms possessing adaptive immunity, it may have limited their freedom to diversify. By contrast, PRR diversity continues to confer an advantage for organisms lacking the means to generate non-self recognition receptors via somatic mutation. Extensive deuterostome PRR diversity may have been driven by gnathostome adaptive immunity inducing diversification of shared pathogens, which exerted strong diversifying selection on deuterostome PRRs. Thus, the development of adaptive immunity changed the role of PRRs in immunity as well as the selective forces on host receptors, deuterostomes, and pathogens. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Immunity has evolved in all metazoans to be protective against pathogens. The two major strategies employed by metazoans for the first step of this process, recognition, are the use of a large set of diverse, germ-line encoded pattern recognition receptors (PRRs) and a system for random generation and clonal selection of antigen specific receptors. While oversimplified, the first strategy describes the typical innate immune system and the second is characteristic of an adaptive immune system. Innate immunity is the evolutionarily more ancient system that is present, in some form, in almost all metazoans. In contrast, adaptive immunity has evolved in complement to innate immunity in jawed vertebrates (McCurley et al., 2011). Adaptive immunity, as elaborated in mammals and other jawed vertebrates (gnathostomes), is often viewed as the definitive solution to pathogen recognition. Recent evidence has complicated this classical picture with the discovery of alternatives to the prototypical model of adaptive immunity (Hedrick, 2004). The purpose of
⇑ Corresponding author. E-mail addresses: [email protected] (A.E. Ward), [email protected] (B.M. Rosenthal). 1 Tel.: +1 (301) 504 5408. 1567-1348/$ - see front matter Ó 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2013.12.021
this paper is to review several new lines of evidence concerning the advent of distinct (but functionally analogous) systems of adaptive immunity in vertebrates and the subsequent impact that such innovation had on the diversity and function of innate pathogen recognition receptors in organisms possessing, and in organisms lacking, the ability to synthesize nearly limitless repertoires of molecules capable of recognizing pathogens.
1.1. The evolution of adaptive immunity The cellular component of the adaptive immune system in jawed vertebrates consists of T lymphocytes and B lymphocytes, which are activated by antigen presenting cells akin to those also found in animals lacking such specific effector cells. As they sample the environment and phagocytose pathogens and damaged tissue, dendritic cells and other antigen presenting cells may be viewed as ‘‘surveillance’’ and a link between the innate and adaptive immune systems (Palucka and Banchereau, 1999). Until recently, no homologues for T and B lymphocytes were known in jawless fishes (agnathostomes). This apparent absence of adaptive immune cells sparked an immunological conundrum because sharks, hagfish, and lampreys exhibit graft rejection, a hallmark of a cytotoxic Tcell mediated immune response (Wang et al., 2008). More recently, it has been shown that hagfish and lampreys do have populations
A.E. Ward, B.M. Rosenthal / Infection, Genetics and Evolution 21 (2014) 492–496
of cells that have similar function to T and B cells but that lack the characteristic markers of T cell receptors and B cell receptors (TCRs, BCRs). In gnathostome immunity, immature T cells are shown selfantigens in the specialized lymphoid organ, the thymus. It is in the thymus that self-reactive T cells are negatively selected. A homologous organ could not be found in agnathostomes until it was discovered that the tips of hagfish and lamprey gills served a similar function to the gnathostome thymus (Boehm et al., 2012). In classical adaptive immunity, immature lymphocytes must undergo V(D)J recombination to specify the binding characteristics of each resulting TCR and BCR. This process of somatic recombination is accomplished by the rearrangement of germline encoded gene fragments to produce structurally and functionally new receptors. This process is engendered by two principal enzymes: recombination activating genes 1 and 2 (RAG1 and RAG2); which are members of a family of enzymes known as ‘‘recombinases’’ (Nishana and Raghavan, 2012). Although agnathostomes lack VDJ genes and RAG1/2, they nonetheless produce cell lines functionally analogous to gnathostome T and B cells (Boehm et al., 2012). Recent work by Pancer et al. (2004) showed that agnathostomes do have functionally analogous receptors, called Variable Lymphocyte Receptors (VLRs); these are produced by gene conversion of germ line encoded fragments, in a similar but not identical process to TCR/BCR development. However, VLR structure is drastically different from that of TCR/BCRs; VLRs feature leucine rich repeats which produce a concave binding region, similar to the structure of the highly evolutionarily conserved innate pattern recognition receptors, the Toll-like receptors (TLRs) (Pancer et al., 2004). Diversity in VLRs is produced through a process of gene rearrangement entirely distinct from the process by which RAG1 and RAG2 initiate V(D)J recombination. Instead, VLRs are produced by gene conversion, mediated by a different class of enzymes, the cytidine deaminases. Despite differences in molecular mechanisms, VLR-based immunity follows a similar functional pattern as classical adaptive immunity, where VLRA is analogous to the T cell receptor and VLRB is analogous to the secreted B cell receptor (Boehm et al., 2012). The adaptive immune systems of gnathostomes and agnathostomes achieve strikingly similar outcomes by entirely distinct molecular mechanisms. This creates an interesting puzzle about how the two systems could have evolved. The results of comparative genome analyses suggest that the ancestor of gnathostomes and agnathostomes possessed the precursors of both VLRs and TCR/BCRs, each of which was likely ‘‘repurposed’’ from other functions. The gnathostome lineage developed RAGs, which act on specific DNA sequences to produce diverse TCRs and BCRs through the V(D)J system of somatic recombination. Agnathostomes instead relied on cytidine deaminases to produce unique antigen recognition receptors. These enzymes each form double stranded DNA breaks, but at different sequence specificities. Thus, antigen recognition receptors with unique structures but similar function are mediated by these two systems of somatic diversification (Boehm et al., 2012), a fascinating example by which independent evolutionary lineages have elaborated functionally analogous (convergent, but not evolutionarily homologous) attributes. However, these systems of adaptive immunity did not develop in isolation from pathogens or existing innate immunity. Even though somatic recombination provides the adaptive immune system with the ability to generate receptors for the recognition of over 1014 different antigens (McCurley et al., 2011), adaptive immunity still requires activation signals from the innate immune system. Considering this functional link, it is reasonable to hypothesize that innate immunity shaped the evolution of adaptive immunity in all its forms. Less evident though, is whether the development of adaptive immunity shaped the evolution of innate immunity.
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1.2. The effect of adaptive immunity on the evolution of innate immunity We can observe the effect of the adaptive immune system on the innate immune system by comparing the diversity of innate immune components in organisms that possess or lack an adaptive immune system. The most easily studied are the pattern recognition receptors, which are present in some form in all metazoans. A comparison of PRRs and innate immune signaling adaptor molecules in humans (Homo sapiens), lampreys (Petromyzon marinus), sea urchins (Stronglyocentrotus purpuratus), amphioxus (Branchiostoma floridae), and fruit fly (Drosophila melanogaster), demonstrates far more PRRs in the species that lack adaptive immune systems (Huang et al., 2008). The Toll-like receptor family, a group of PRRs that are highly conserved across all metazoans and play integral roles in the innate recognition of bacterial and viral motifs (Janeway and Medzhitov, 2002) provide an instructive contrast. 10 Toll-like receptors have been identified in humans, 21 in lamprey, 72 in amphioxus, 222 in sea urchins, and 9 in Drosophila (Fig. 1) (Huang et al., 2008; Satake and Sekiguchi, 2012). Even though lamprey have VLR-based adaptive immunity, they still have twice the diversity in Toll-like receptors that humans have. Interestingly, the greatest diversity is found in amphioxus and sea urchins, which do not possess an adaptive immune system. Clearly, organisms possessing adaptive immunity harbor fewer innate immune receptors. Either of two hypotheses could conceivably account for this difference, however, the two hypotheses are not mutually exclusive and could conceivably occur simultaneously. The ‘‘gnathostome loss’’ hypothesis would posit that abundant ancestral innate immune receptor diversity was lost in the evolutionary lineage that inherited adaptive immunity. Reduced need for such heritable variation would, according to this view, have resulted in waning selective pressure to maintain and further diversify germline-encoded pathogen recognition. Adaptive immunity, powered by somatic recombination, would have ended such organisms’ reliance on a diverse set of innate immune receptors. In its simplest formulation, this hypothesis would predict selection against a number of pathogen recognition receptors or the lack of maintenance of those genes. The second explanation, which may be termed ‘‘deuterostome gain,’’ posits that ancestral innate immune receptors underwent marked diversification in those organisms lacking adaptive immunity. Why such organisms, and only such organisms, would have undergone diversification in their heritable pathogen recognition receptors remains a matter of some interest; could adaptive immunity in vertebrates have engendered PRR diversity in deuterostomes, indirectly, by promoting the antigenic diversification of shared pathogens? This possibility focuses attention on the difference in magnitude of the selective pressures faced by vertebrates and deuterostomes. An examination of the phylogeny and evolution of Toll-like receptors, cytokine receptors, and the JAK/STAT signaling pathway components, provides a means to evaluate the relative merits of ‘‘gnathosome loss’’ versus ‘‘deuterostome gain.’’ 1.3. Selective pressures on Toll-like receptors Toll-like receptors (TLRs) are a class of Type I transmembrane receptors featuring leucine-rich repeats which produce a concave ligand-binding site. They are highly evolutionarily conserved across metazoans (Wiens et al., 2007) however, recent evidence provided by Maximum Likelihood analysis by Areal et al. (2011) has shown that all mammalian TLRs are still undergoing positive selection and a similar analysis of primate TLRs by Wlasiuk and Nachman (2010) showed six of ten TLRs have recently experienced positive selection. It should be noted that human TLRs may have
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Toll-like Receptors Humans
STAT Family Members 7
10
Mammalia
Gnathostoma
Cytokine Receptor Chains
Reptilia Adaptive Immunity
Amphibia Osteichythes Bony Fishes
36
8
2
2
1
1
Chondrichthyes
Sharks
Petromyzondia
Lamprey
21
Myxini Urochordata
Sea Squirt
Deuterostomia
Cephalochordata
43
Echinodermata
222
Lancelet
Sea Urchin
Molluska Annelida Nematoda Arthropoda
Fruit Fly
9
Cnidaria Porifera
Sponges
1
Fig. 1. Toll-like receptor (TLR) diversity is greatest in echinoderms but smaller in mammals. The pattern of TLR diversity is mirrored by an expansion of cytokine receptors and the STAT family of adaptor signaling molecules (the JAK family experienced a similar expansion but for simplicity only the number of STAT family members are shown). Taken together, these patterns support the notion that with the advent of adaptive immunity, the role of pattern recognition receptors shifted to initiators of downstream immune signaling and appropriate responses.
experienced unusual selective pressures due to human migration patterns and more recently, health practices, but since both studies consider humans as part of larger groups, mammals and primates, the results do not merely reflect the selective pressures of humans. In both studies, the majority of positively selected codons were in the leucine rich repeats which comprise the ligand binding domain, so it is reasonable to conclude this evolution is driven by host pathogen interactions. Evidence for ongoing positive selection in gnathostome TLRs renders the ‘‘gnathostome loss’’ hypothesis less plausible, at least in its simplest formulation. If ancestral PRR diversity were selected against (owing to its metabolic costs and redundancy with the antigen recognition provided by adaptive immunity), this would be in contrast to extant data that substantiate the persistence of selection maintaining PRR function. However, the ‘‘deuterostome gain’’ hypothesis does not exclude the possibility that PRRs in vertebrates with adaptive immune systems also underwent positive selection, but rather that this selection was not as strong as the selective pressures experienced by deuterostome invertebrates. If the ancestor had similar TLR diversity to current protostomes (drosophila have 9 members) then it is reasonable to assume that vertebrates, after acquiring adaptive immunity, underwent a modest diversification in their TLR repertoire (mammals have between 10 and 13 TLRs) (Satake and Sekiguchi, 2012). Meanwhile, deuterostomes underwent a much larger diversification. It is interesting to consider how the advent of adaptive immunity may have created conditions necessitating such an expansion of PRRs in deuterostomes. This idea seems plausible (though by no means yet established) given the fact that certain deuterostomes and gnathostomes share a common pool of microbial and parasitic threats. If the advent of vertebrate adaptive immunity engendered antigenic diversifications among such pathogens, diversification of
PRRs in deuterostomes might have been a necessary response. In an evolutionary sense, immunity does not require perfect defense against all pathogens; to proliferate, an organism needs only comparatively more effective defense (Hedrick, 2004). A sudden increase in infection would exert a strong selective pressure on invertebrate deuterostomes, which might explain the extreme diversity of TLRs in basal chordates and echinoderms (48 and 222 members respectively). Marine ecosystems, where lower chordates and vertebrates frequently interact, would seem to magnify the potential of competition mediated by a shared pool of pathogens. For example, the parasitic worm Echinorhychnus coregoni Linkins is known to infect both sea lamprey (McLain, 1952; Van Cleave, 1921), which lack classical adaptive immunity and teleost ‘‘whitefish’’ (Van Cleave, 1919), which have adaptive immunity. Also, more distantly, the nematode Anisakis simplex is known to affect both mollusks such as squid (Pascual et al., 1995), which lack any form of adaptive immunity, and marine mammals such as dolphins (Aznar et al., 2003), which possess adaptive immunity. Both of these examples are parasitic worms which induce an IgE/CD4 based immune response in vertebrates (MacDonald et al., 2002) and both examples serve to illustrate the point that, despite large evolutionary distances, host species can still share common pathogens. If host–pathogen interactions of shared pathogens were sufficient to affect the evolution of immunity in gnathostomes and deuterostomes, perhaps the reverse is also true. It is reasonable to hypothesize that as the types of immunity increased, there was a mirrored increase in microbial diversity; more complex defensive systems may have engendered more ways to circumvent such defenses. It is well documented that microbes modulate immune responses in their favor by mimicking host signaling proteins (Epperson et al., 2012). If there were an increase in the complexity
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of signaling pathways, pathogens would adapt to new niches. Additionally, perhaps if pathogen recognition pathways were able to process signals with more subtlety, hosts would be able to differentiate between symbiotic and pathogenic microbes more easily and therefore develop more diverse microbiomes. However, all of this relies on the expansion of host signaling capabilities. 1.4. Expansion of host signaling capabilities Cytokines are a class of proteins that can be produced by all nucleated cells and generally have immuno-modulatory or hematopoietic functions. Cytokines binding to specific cell surface cytokine receptors initiate downstream signaling events that regulate the host immune system. Given their central role in shaping immune responses, especially adaptive immune responses, it is feasible that cytokine receptors could have been influenced by the development of adaptive immunity. Analysis by Liongue and Ward (2007) of the sea squirt, mosquito, fruit fly, zebrafish, and human genomes revealed only one cytokine receptor chain in mosquitos, two in sea squirts and fruit flies, and 36 cytokine receptor chains in the zebrafish genome. They reason that the large expansion of cytokine receptor chain diversity between urochordates and vertebrates occurred via a combination of whole genome, tandem, and en bloc duplication events. Downstream of cytokine receptors, the components of the JAK/ STAT pathway experienced a similar expansion. This is reasonable considering the pathway is used in many biological systems (including hematopoiesis, immune cell development, stem cell maintenance, organismal growth, and mammary gland development), but is heavily involved in the signaling pathways of interferons and cytokines (Rawlings et al., 2004). This expansion is evident in that Drosophila possess only one member of the JAK family, while jawed fishes (specifically teleosts) possess five and humans possess four. In a similar manner, Drosophila possess only one member of the STAT family, sea squirts possess two members, teleosts possess eight members and humans possess seven members. Clearly, there has been an expansion of the number of components and complexity of this signaling pathway, mostly in telosts and their descendants. In-depth bioinformatics has revealed that there was very little diversification of the JAK and STAT families before the divergence of urochordates. However, a major expansion coincided with the development of adaptive immunity in jawed fish, a pattern that is parallel to that seen in cytokine receptor chains. There is also evidence that between the divergence of urochordates and fish there were two whole genome duplication events (Liongue et al., 2012). Perhaps with the redundancy provided by a duplicated genome, changes in cytokine receptor chains and JAK/STAT components were more tolerated and could be positively selected until they became new members of the signaling pathway. Then, even as many paralagous genes that were created by the whole genome duplication were lost, the functionally distinct and positively selected new cytokine receptor chains and JAK/STAT members were retained in the genome of vertebrates. These changes took place at the same time that key lymphoid cytokines were evolving, so together they allowed more subtlety in regulation of the complex emerging adaptive immune system (Liongue et al., 2012). This pattern of diversified cellular communication seems to be indicative of a larger pattern wherein vertebrates, rather than evolving new PRRs, developed new ways to regulate the power of their newfound adaptive immune systems. With more signals and more ways to process and interpret those signals, adaptive and innate immune cells could respond to infection with more subtlety and nuance. Perhaps because the evolution of adaptive immunity required an expansion of cellular and intracellular signaling networks, there was reduced selective pressure on innate
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immune PRRs. Rather than developing new innate immune receptors to initiate immunological signals, immune cells in organisms that posses adaptive immune systems could process their current set of inflammatory signals in more ways with increased signaling network diversity. The expansion of cytokine and JAK/STAT signaling, in conjunction with the development of adaptive immunity, seems more consistent with the ‘‘deuterostome gain’’ hypothesis. If the evolution of adaptive immunity caused the expansion of cellular communication between the innate and adaptive immune systems, increased molecular cooperation would have ensued. The result may have been an increase in the efficacy of immune responses, which would thereby reduce the need for expansion of innate PRRs. Perhaps chordates, which did not experience the JAK/STAT expansion, had to compensate by diversifying their PRR repertoire. The expansion of the JAK/STAT signaling pathway would not rule out the reduction in PRR effectors envisioned by the ‘‘gnathostome loss’’ hypothesis, but rather a combination of the two mechanisms may have created the current pattern of PRR and signaling network diversity. It is reasonable that in the wake of two whole-genome duplication events, signaling network components faced positive selection while PRRs faced strong purifying selection.
2. Conclusion Through examination of both TLR and JAK/STAT diversity, it has become apparent that the two mechanisms for the evolution of innate immunity, as influenced by adaptive immunity, are not equally plausible but still compatible. The ‘‘gnathostome loss’’ explanation relies on the negative or purifying selection of innate PRRs, which is in contrast to extant evidence that PRRs maintain function in vertebrates. It is apparent that the major class of PRRs, the TLRs, had limited diversity in ancestral protostomes but have retained structure and function. Moreover, the adaptive immune system requires activating signals from the innate immune system to become effective at clearing infections. Those organisms that developed a costly and complex system for fighting infection maintained the core PRRs with the ability to activate it effectively. The ‘‘deuterostome gain’’ hypothesis posits, more plausibly, that organisms lacking adaptive immunity experienced increased pressure to diversify their PRRs, although notably in the absence of whole genome duplications making the massive expansion all the more impressive. Since vertebrate adaptive immunity can generate specific receptors for virtually any epitope, vertebrates rely less on their innate immune system to recognize all PAMPs when neutralizing infectious agents. In vertebrates, positive selection on the TLRs and the expansion of JAK/STAT signaling components substantiate the notion that increased complexity of their cellular signaling network reduced the pressure on their PRRs to further diversify. Indeed, situating each PRR in a complex signaling network may have created conditions where further changes in PRR structure would have disrupted their new signaling functions. Vertebrates still rely on innate immunity to help initiate adaptive immune responses, but the value of further diversification in PRRs has been reduced or even reversed. By contrast, organisms lacking adaptive immunity rely solely on their PRRs for immune surveillance; since their pathogens rapidly evolve, the diversity of deuterostome PRRs must quickly evolve as well. Situated as effectors of simpler signaling cascades, these PRRs would seem amenable to the continuous elaboration of new diversity. Gnathostome PRRs must have experienced negative selection after whole genome duplications (hence why mammals do not have four times as many TLRs as invertebrates), although it seems unlikely that purifying selection acting alone could cause the large
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observed difference in the number of deuterostome TLRs and gnathostome TLRs when already selecting against duplicated alleles. However, it does illustrate the point that the present pattern of PRR diversity was likely caused by a combination of ‘‘deuterostome gain’’ and ‘‘gnathostome loss’’ in varying strengths. ‘‘Deuterostome gain’’ acting alone is still plausible but ‘‘gnathostome loss’’ would require extraordinary negative selection on gnathostome PRRs in the wake of whole genome duplications; so if it played a role in shaping PRR diversity, it most likely acted in conjunction with ‘‘deuterostome gain.’’ The precise reasons that deuterostomes posses more diverse PRRs than do gnathostomes may never be known with certainty. Inferring the events impelling either loss or gain in extinct ancestors is, admittedly, fraught with difficulty and is not amenable to either direct observation or testing. Nonetheless, comparing various lines of evidence in extant descendants of each evolutionary lineage sheds light on the relative plausibility of possible hypotheses. Ultimately, the evidence to date favors the notion that deuterostomes underwent diversification in PRRs that was not also experienced in those of our ancestors who passed on to us adaptive immunity. Even though gnathostomes evolved an entirely new system of host defense, we will still be plagued by pathogens and the red queen must keep running her eternal race. Conflicts of interest None. Acknowledgement The University Honors Program at The University of Maryland. References Aznar, J., Herreras, V., Balbuena, J., Raga, J., 2003. Population structure and habitat selection by Anisakis simplex in 4 odontocete species from northern Argentina. Comp. Parasitol. 70 (1), 66–71. Areal, Helena, Abrantes, Joana, Esteves, Pedro, 2011. Signatures of positive selection in Toll-like receptor (TLR) genes in mammals. Evol. Biol. 11 (368), 1471–2148.
Boehm, Thomas, McCurley, Nathaniel, Sutoh, Yuichu, Schorpp, Michael, Kasahara, Masanori, Cooper, Max D., 2012. VLR-based adaptive immunity. Annu. Rev. Immunol. 30, 203–220. Epperson, M.L., Lee, C.A., Fremont, D.H., 2012. Subversion of cytokine networks by virally encoded decoy receptors. Immunol. Rev. 250, 199–215. Hedrick, Stephan, 2004. The acquired immune system: a vantage from beneath. Immunity 21, 607–615. Huang, Shengfeng, Yung, Shaochun, Guo, Lei, 2008. Genomic analysis of the immune gene repertoire of amphioxus reveals extraordinary innate complexity and diversity. Genome Res. 18, 1112–1126. Janeway, Charles A., Medzhitov, Ruslan, 2002. Innate immune recognition. Annu. Rev. Immunol. 20 (1), 197–216. Liongue, Clifford, Ward, Alister, 2007. Evolution of class I cytokine receptors. BMC Evol. Biol. 7 (120). Liongue, Clifford, O’Sullivan, Lydia, Trengrove, Monique, Ward, Alister C., 2012. Evolution of JAK–STAT pathway components: mechanisms and role in immune system development. PLoS One 7 (3), 1–16. MacDonald, Andrew S., Araujo, Maria Ilma, Pearce, Edward J., 2002. Immunology of parasitic helminth infections. Infect. Immun. 70, 427–433. McCurley, Nathanael, Hirano, Masayuki, Das, Sabyasachi, Cooper, Max, 2011. Immune related genes underpin the evolution of adaptive immunity in jawless vertebrates. Curr. Genomics 13, 86–94. McLain, A., 1952. Diseases and parasites of the sea lamprey, Petromyzon marinus, in the Lake Huron basin. Trans. Am. Fish. Soc. 81, 94–100. Nishana, Mayilaadumveettil, Raghavan, Sathees C., 2012. Role of recombination activating genes in the generation of antigen receptor diversity and beyond. Immunology 137, 271–281. Palucka, Karolina, Banchereau, Jacques, 1999. Dendritic cells: a link between innate and adaptive immunity. J. Clin. Immunol. 19, 12–25. Pancer, Z., Amemiya, C., Ehrhardt, G., Ceitlan, J., Gartland, L., Cooper, M., 2004. Somatic diversification of variable lymphocyte receptors in the agnathan sea lamprey. Nature 430, 174–180. Pascual, S., Gonzalez, A., Arias, C., Guerra, A., 1995. Helminth infection in the shortfinned squid Illex coindetii (Cephalopoda, Ommastrephidae) off NW Spain. Dis. Aquat. Organ. 23, 71–75. Rawlings, J., Rosler, K., Harrison, D., 2004. The JAK/STAT signaling pathway. J. Cell Sci. 117, 1281–1283. Satake, H., Sekiguchi, T., 2012. Toll-like receptors of deuterostome invertebrates. Front. Immunol. 3 (34), 1–7. Van Cleave, H.J., 1921. Acanthocephala from the Eel. Trans. Am. Microsc. Soc. 40 (1), 1–13. Van Cleave, H.J., 1919. Notes on the life cycle of two species of acanthocephala from freshwater fishes. J. Parasitol. 56 (4), 167–172. Wang, E., Worschech, A., Marincola, F., 2008. The immunologic constant of rejection. Trends Immunol. 29 (6), 256–262. Wiens, Matthias, Korzhev, Michael, Perovic-Ottstadt, Sanja, 2007. Toll-like receptors are part of the innate immune defense system of sponges. Mol. Biol. Evol. 24 (3), 792–804. Wlasiuk, G., Nachman, M.W., 2010. Adaptation and constraint at Toll-like receptors in primates. Mol. Biol. Evol. 27 (9), 2172–2186.
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Discussion
Evolutionary responses of innate immunity to adaptive immunity Amanda E. Ward a,⇑, Benjamin M. Rosenthal b,1 a b
University of Maryland-College Park, College Park, MD 20740, United States United States Department of Agriculture, Beltsville Agricultural Research Center, Building 1040, Room 103, 10300 Baltimore Avenue, Beltsville, MD 20705-2350, United States
a r t i c l e
i n f o
Article history: Received 25 August 2013 Received in revised form 18 December 2013 Accepted 20 December 2013 Available online 8 January 2014 Keywords: Toll-like receptors Pattern recognition receptors JAK/STAT signaling Agnathostome immunity Innate immunity VLR immunity
a b s t r a c t Innate immunity is present in all metazoans, whereas the evolutionarily more novel adaptive immunity is limited to jawed fishes and their descendants (gnathostomes). We observe that the organisms that possess adaptive immunity lack diversity in their innate pattern recognition receptors (PRRs), raising the question: did gnathostomes lose the diversity of their ancestors? Or might innate receptors have diversified in the lineage lacking adaptive immunity? We address this question by contextualizing PRRs in their distinct functional roles in organisms possessing or lacking adaptive immunity. In particular, limited PRR diversity in gnathostomes is accompanied by an expansion of the JAK/STAT signaling pathway, which would suggest that the development of adaptive immunity shifted the role of PRRs from the entirety of pathogen recognition to regulators of subsequent immune responses. As PRRs became essential upstream components of the increasingly complex JAK/STAT signaling cascade in organisms possessing adaptive immunity, it may have limited their freedom to diversify. By contrast, PRR diversity continues to confer an advantage for organisms lacking the means to generate non-self recognition receptors via somatic mutation. Extensive deuterostome PRR diversity may have been driven by gnathostome adaptive immunity inducing diversification of shared pathogens, which exerted strong diversifying selection on deuterostome PRRs. Thus, the development of adaptive immunity changed the role of PRRs in immunity as well as the selective forces on host receptors, deuterostomes, and pathogens. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Immunity has evolved in all metazoans to be protective against pathogens. The two major strategies employed by metazoans for the first step of this process, recognition, are the use of a large set of diverse, germ-line encoded pattern recognition receptors (PRRs) and a system for random generation and clonal selection of antigen specific receptors. While oversimplified, the first strategy describes the typical innate immune system and the second is characteristic of an adaptive immune system. Innate immunity is the evolutionarily more ancient system that is present, in some form, in almost all metazoans. In contrast, adaptive immunity has evolved in complement to innate immunity in jawed vertebrates (McCurley et al., 2011). Adaptive immunity, as elaborated in mammals and other jawed vertebrates (gnathostomes), is often viewed as the definitive solution to pathogen recognition. Recent evidence has complicated this classical picture with the discovery of alternatives to the prototypical model of adaptive immunity (Hedrick, 2004). The purpose of
⇑ Corresponding author. E-mail addresses: [email protected] (A.E. Ward), [email protected] (B.M. Rosenthal). 1 Tel.: +1 (301) 504 5408. 1567-1348/$ - see front matter Ó 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2013.12.021
this paper is to review several new lines of evidence concerning the advent of distinct (but functionally analogous) systems of adaptive immunity in vertebrates and the subsequent impact that such innovation had on the diversity and function of innate pathogen recognition receptors in organisms possessing, and in organisms lacking, the ability to synthesize nearly limitless repertoires of molecules capable of recognizing pathogens.
1.1. The evolution of adaptive immunity The cellular component of the adaptive immune system in jawed vertebrates consists of T lymphocytes and B lymphocytes, which are activated by antigen presenting cells akin to those also found in animals lacking such specific effector cells. As they sample the environment and phagocytose pathogens and damaged tissue, dendritic cells and other antigen presenting cells may be viewed as ‘‘surveillance’’ and a link between the innate and adaptive immune systems (Palucka and Banchereau, 1999). Until recently, no homologues for T and B lymphocytes were known in jawless fishes (agnathostomes). This apparent absence of adaptive immune cells sparked an immunological conundrum because sharks, hagfish, and lampreys exhibit graft rejection, a hallmark of a cytotoxic Tcell mediated immune response (Wang et al., 2008). More recently, it has been shown that hagfish and lampreys do have populations
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of cells that have similar function to T and B cells but that lack the characteristic markers of T cell receptors and B cell receptors (TCRs, BCRs). In gnathostome immunity, immature T cells are shown selfantigens in the specialized lymphoid organ, the thymus. It is in the thymus that self-reactive T cells are negatively selected. A homologous organ could not be found in agnathostomes until it was discovered that the tips of hagfish and lamprey gills served a similar function to the gnathostome thymus (Boehm et al., 2012). In classical adaptive immunity, immature lymphocytes must undergo V(D)J recombination to specify the binding characteristics of each resulting TCR and BCR. This process of somatic recombination is accomplished by the rearrangement of germline encoded gene fragments to produce structurally and functionally new receptors. This process is engendered by two principal enzymes: recombination activating genes 1 and 2 (RAG1 and RAG2); which are members of a family of enzymes known as ‘‘recombinases’’ (Nishana and Raghavan, 2012). Although agnathostomes lack VDJ genes and RAG1/2, they nonetheless produce cell lines functionally analogous to gnathostome T and B cells (Boehm et al., 2012). Recent work by Pancer et al. (2004) showed that agnathostomes do have functionally analogous receptors, called Variable Lymphocyte Receptors (VLRs); these are produced by gene conversion of germ line encoded fragments, in a similar but not identical process to TCR/BCR development. However, VLR structure is drastically different from that of TCR/BCRs; VLRs feature leucine rich repeats which produce a concave binding region, similar to the structure of the highly evolutionarily conserved innate pattern recognition receptors, the Toll-like receptors (TLRs) (Pancer et al., 2004). Diversity in VLRs is produced through a process of gene rearrangement entirely distinct from the process by which RAG1 and RAG2 initiate V(D)J recombination. Instead, VLRs are produced by gene conversion, mediated by a different class of enzymes, the cytidine deaminases. Despite differences in molecular mechanisms, VLR-based immunity follows a similar functional pattern as classical adaptive immunity, where VLRA is analogous to the T cell receptor and VLRB is analogous to the secreted B cell receptor (Boehm et al., 2012). The adaptive immune systems of gnathostomes and agnathostomes achieve strikingly similar outcomes by entirely distinct molecular mechanisms. This creates an interesting puzzle about how the two systems could have evolved. The results of comparative genome analyses suggest that the ancestor of gnathostomes and agnathostomes possessed the precursors of both VLRs and TCR/BCRs, each of which was likely ‘‘repurposed’’ from other functions. The gnathostome lineage developed RAGs, which act on specific DNA sequences to produce diverse TCRs and BCRs through the V(D)J system of somatic recombination. Agnathostomes instead relied on cytidine deaminases to produce unique antigen recognition receptors. These enzymes each form double stranded DNA breaks, but at different sequence specificities. Thus, antigen recognition receptors with unique structures but similar function are mediated by these two systems of somatic diversification (Boehm et al., 2012), a fascinating example by which independent evolutionary lineages have elaborated functionally analogous (convergent, but not evolutionarily homologous) attributes. However, these systems of adaptive immunity did not develop in isolation from pathogens or existing innate immunity. Even though somatic recombination provides the adaptive immune system with the ability to generate receptors for the recognition of over 1014 different antigens (McCurley et al., 2011), adaptive immunity still requires activation signals from the innate immune system. Considering this functional link, it is reasonable to hypothesize that innate immunity shaped the evolution of adaptive immunity in all its forms. Less evident though, is whether the development of adaptive immunity shaped the evolution of innate immunity.
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1.2. The effect of adaptive immunity on the evolution of innate immunity We can observe the effect of the adaptive immune system on the innate immune system by comparing the diversity of innate immune components in organisms that possess or lack an adaptive immune system. The most easily studied are the pattern recognition receptors, which are present in some form in all metazoans. A comparison of PRRs and innate immune signaling adaptor molecules in humans (Homo sapiens), lampreys (Petromyzon marinus), sea urchins (Stronglyocentrotus purpuratus), amphioxus (Branchiostoma floridae), and fruit fly (Drosophila melanogaster), demonstrates far more PRRs in the species that lack adaptive immune systems (Huang et al., 2008). The Toll-like receptor family, a group of PRRs that are highly conserved across all metazoans and play integral roles in the innate recognition of bacterial and viral motifs (Janeway and Medzhitov, 2002) provide an instructive contrast. 10 Toll-like receptors have been identified in humans, 21 in lamprey, 72 in amphioxus, 222 in sea urchins, and 9 in Drosophila (Fig. 1) (Huang et al., 2008; Satake and Sekiguchi, 2012). Even though lamprey have VLR-based adaptive immunity, they still have twice the diversity in Toll-like receptors that humans have. Interestingly, the greatest diversity is found in amphioxus and sea urchins, which do not possess an adaptive immune system. Clearly, organisms possessing adaptive immunity harbor fewer innate immune receptors. Either of two hypotheses could conceivably account for this difference, however, the two hypotheses are not mutually exclusive and could conceivably occur simultaneously. The ‘‘gnathostome loss’’ hypothesis would posit that abundant ancestral innate immune receptor diversity was lost in the evolutionary lineage that inherited adaptive immunity. Reduced need for such heritable variation would, according to this view, have resulted in waning selective pressure to maintain and further diversify germline-encoded pathogen recognition. Adaptive immunity, powered by somatic recombination, would have ended such organisms’ reliance on a diverse set of innate immune receptors. In its simplest formulation, this hypothesis would predict selection against a number of pathogen recognition receptors or the lack of maintenance of those genes. The second explanation, which may be termed ‘‘deuterostome gain,’’ posits that ancestral innate immune receptors underwent marked diversification in those organisms lacking adaptive immunity. Why such organisms, and only such organisms, would have undergone diversification in their heritable pathogen recognition receptors remains a matter of some interest; could adaptive immunity in vertebrates have engendered PRR diversity in deuterostomes, indirectly, by promoting the antigenic diversification of shared pathogens? This possibility focuses attention on the difference in magnitude of the selective pressures faced by vertebrates and deuterostomes. An examination of the phylogeny and evolution of Toll-like receptors, cytokine receptors, and the JAK/STAT signaling pathway components, provides a means to evaluate the relative merits of ‘‘gnathosome loss’’ versus ‘‘deuterostome gain.’’ 1.3. Selective pressures on Toll-like receptors Toll-like receptors (TLRs) are a class of Type I transmembrane receptors featuring leucine-rich repeats which produce a concave ligand-binding site. They are highly evolutionarily conserved across metazoans (Wiens et al., 2007) however, recent evidence provided by Maximum Likelihood analysis by Areal et al. (2011) has shown that all mammalian TLRs are still undergoing positive selection and a similar analysis of primate TLRs by Wlasiuk and Nachman (2010) showed six of ten TLRs have recently experienced positive selection. It should be noted that human TLRs may have
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Toll-like Receptors Humans
STAT Family Members 7
10
Mammalia
Gnathostoma
Cytokine Receptor Chains
Reptilia Adaptive Immunity
Amphibia Osteichythes Bony Fishes
36
8
2
2
1
1
Chondrichthyes
Sharks
Petromyzondia
Lamprey
21
Myxini Urochordata
Sea Squirt
Deuterostomia
Cephalochordata
43
Echinodermata
222
Lancelet
Sea Urchin
Molluska Annelida Nematoda Arthropoda
Fruit Fly
9
Cnidaria Porifera
Sponges
1
Fig. 1. Toll-like receptor (TLR) diversity is greatest in echinoderms but smaller in mammals. The pattern of TLR diversity is mirrored by an expansion of cytokine receptors and the STAT family of adaptor signaling molecules (the JAK family experienced a similar expansion but for simplicity only the number of STAT family members are shown). Taken together, these patterns support the notion that with the advent of adaptive immunity, the role of pattern recognition receptors shifted to initiators of downstream immune signaling and appropriate responses.
experienced unusual selective pressures due to human migration patterns and more recently, health practices, but since both studies consider humans as part of larger groups, mammals and primates, the results do not merely reflect the selective pressures of humans. In both studies, the majority of positively selected codons were in the leucine rich repeats which comprise the ligand binding domain, so it is reasonable to conclude this evolution is driven by host pathogen interactions. Evidence for ongoing positive selection in gnathostome TLRs renders the ‘‘gnathostome loss’’ hypothesis less plausible, at least in its simplest formulation. If ancestral PRR diversity were selected against (owing to its metabolic costs and redundancy with the antigen recognition provided by adaptive immunity), this would be in contrast to extant data that substantiate the persistence of selection maintaining PRR function. However, the ‘‘deuterostome gain’’ hypothesis does not exclude the possibility that PRRs in vertebrates with adaptive immune systems also underwent positive selection, but rather that this selection was not as strong as the selective pressures experienced by deuterostome invertebrates. If the ancestor had similar TLR diversity to current protostomes (drosophila have 9 members) then it is reasonable to assume that vertebrates, after acquiring adaptive immunity, underwent a modest diversification in their TLR repertoire (mammals have between 10 and 13 TLRs) (Satake and Sekiguchi, 2012). Meanwhile, deuterostomes underwent a much larger diversification. It is interesting to consider how the advent of adaptive immunity may have created conditions necessitating such an expansion of PRRs in deuterostomes. This idea seems plausible (though by no means yet established) given the fact that certain deuterostomes and gnathostomes share a common pool of microbial and parasitic threats. If the advent of vertebrate adaptive immunity engendered antigenic diversifications among such pathogens, diversification of
PRRs in deuterostomes might have been a necessary response. In an evolutionary sense, immunity does not require perfect defense against all pathogens; to proliferate, an organism needs only comparatively more effective defense (Hedrick, 2004). A sudden increase in infection would exert a strong selective pressure on invertebrate deuterostomes, which might explain the extreme diversity of TLRs in basal chordates and echinoderms (48 and 222 members respectively). Marine ecosystems, where lower chordates and vertebrates frequently interact, would seem to magnify the potential of competition mediated by a shared pool of pathogens. For example, the parasitic worm Echinorhychnus coregoni Linkins is known to infect both sea lamprey (McLain, 1952; Van Cleave, 1921), which lack classical adaptive immunity and teleost ‘‘whitefish’’ (Van Cleave, 1919), which have adaptive immunity. Also, more distantly, the nematode Anisakis simplex is known to affect both mollusks such as squid (Pascual et al., 1995), which lack any form of adaptive immunity, and marine mammals such as dolphins (Aznar et al., 2003), which possess adaptive immunity. Both of these examples are parasitic worms which induce an IgE/CD4 based immune response in vertebrates (MacDonald et al., 2002) and both examples serve to illustrate the point that, despite large evolutionary distances, host species can still share common pathogens. If host–pathogen interactions of shared pathogens were sufficient to affect the evolution of immunity in gnathostomes and deuterostomes, perhaps the reverse is also true. It is reasonable to hypothesize that as the types of immunity increased, there was a mirrored increase in microbial diversity; more complex defensive systems may have engendered more ways to circumvent such defenses. It is well documented that microbes modulate immune responses in their favor by mimicking host signaling proteins (Epperson et al., 2012). If there were an increase in the complexity
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of signaling pathways, pathogens would adapt to new niches. Additionally, perhaps if pathogen recognition pathways were able to process signals with more subtlety, hosts would be able to differentiate between symbiotic and pathogenic microbes more easily and therefore develop more diverse microbiomes. However, all of this relies on the expansion of host signaling capabilities. 1.4. Expansion of host signaling capabilities Cytokines are a class of proteins that can be produced by all nucleated cells and generally have immuno-modulatory or hematopoietic functions. Cytokines binding to specific cell surface cytokine receptors initiate downstream signaling events that regulate the host immune system. Given their central role in shaping immune responses, especially adaptive immune responses, it is feasible that cytokine receptors could have been influenced by the development of adaptive immunity. Analysis by Liongue and Ward (2007) of the sea squirt, mosquito, fruit fly, zebrafish, and human genomes revealed only one cytokine receptor chain in mosquitos, two in sea squirts and fruit flies, and 36 cytokine receptor chains in the zebrafish genome. They reason that the large expansion of cytokine receptor chain diversity between urochordates and vertebrates occurred via a combination of whole genome, tandem, and en bloc duplication events. Downstream of cytokine receptors, the components of the JAK/ STAT pathway experienced a similar expansion. This is reasonable considering the pathway is used in many biological systems (including hematopoiesis, immune cell development, stem cell maintenance, organismal growth, and mammary gland development), but is heavily involved in the signaling pathways of interferons and cytokines (Rawlings et al., 2004). This expansion is evident in that Drosophila possess only one member of the JAK family, while jawed fishes (specifically teleosts) possess five and humans possess four. In a similar manner, Drosophila possess only one member of the STAT family, sea squirts possess two members, teleosts possess eight members and humans possess seven members. Clearly, there has been an expansion of the number of components and complexity of this signaling pathway, mostly in telosts and their descendants. In-depth bioinformatics has revealed that there was very little diversification of the JAK and STAT families before the divergence of urochordates. However, a major expansion coincided with the development of adaptive immunity in jawed fish, a pattern that is parallel to that seen in cytokine receptor chains. There is also evidence that between the divergence of urochordates and fish there were two whole genome duplication events (Liongue et al., 2012). Perhaps with the redundancy provided by a duplicated genome, changes in cytokine receptor chains and JAK/STAT components were more tolerated and could be positively selected until they became new members of the signaling pathway. Then, even as many paralagous genes that were created by the whole genome duplication were lost, the functionally distinct and positively selected new cytokine receptor chains and JAK/STAT members were retained in the genome of vertebrates. These changes took place at the same time that key lymphoid cytokines were evolving, so together they allowed more subtlety in regulation of the complex emerging adaptive immune system (Liongue et al., 2012). This pattern of diversified cellular communication seems to be indicative of a larger pattern wherein vertebrates, rather than evolving new PRRs, developed new ways to regulate the power of their newfound adaptive immune systems. With more signals and more ways to process and interpret those signals, adaptive and innate immune cells could respond to infection with more subtlety and nuance. Perhaps because the evolution of adaptive immunity required an expansion of cellular and intracellular signaling networks, there was reduced selective pressure on innate
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immune PRRs. Rather than developing new innate immune receptors to initiate immunological signals, immune cells in organisms that posses adaptive immune systems could process their current set of inflammatory signals in more ways with increased signaling network diversity. The expansion of cytokine and JAK/STAT signaling, in conjunction with the development of adaptive immunity, seems more consistent with the ‘‘deuterostome gain’’ hypothesis. If the evolution of adaptive immunity caused the expansion of cellular communication between the innate and adaptive immune systems, increased molecular cooperation would have ensued. The result may have been an increase in the efficacy of immune responses, which would thereby reduce the need for expansion of innate PRRs. Perhaps chordates, which did not experience the JAK/STAT expansion, had to compensate by diversifying their PRR repertoire. The expansion of the JAK/STAT signaling pathway would not rule out the reduction in PRR effectors envisioned by the ‘‘gnathostome loss’’ hypothesis, but rather a combination of the two mechanisms may have created the current pattern of PRR and signaling network diversity. It is reasonable that in the wake of two whole-genome duplication events, signaling network components faced positive selection while PRRs faced strong purifying selection.
2. Conclusion Through examination of both TLR and JAK/STAT diversity, it has become apparent that the two mechanisms for the evolution of innate immunity, as influenced by adaptive immunity, are not equally plausible but still compatible. The ‘‘gnathostome loss’’ explanation relies on the negative or purifying selection of innate PRRs, which is in contrast to extant evidence that PRRs maintain function in vertebrates. It is apparent that the major class of PRRs, the TLRs, had limited diversity in ancestral protostomes but have retained structure and function. Moreover, the adaptive immune system requires activating signals from the innate immune system to become effective at clearing infections. Those organisms that developed a costly and complex system for fighting infection maintained the core PRRs with the ability to activate it effectively. The ‘‘deuterostome gain’’ hypothesis posits, more plausibly, that organisms lacking adaptive immunity experienced increased pressure to diversify their PRRs, although notably in the absence of whole genome duplications making the massive expansion all the more impressive. Since vertebrate adaptive immunity can generate specific receptors for virtually any epitope, vertebrates rely less on their innate immune system to recognize all PAMPs when neutralizing infectious agents. In vertebrates, positive selection on the TLRs and the expansion of JAK/STAT signaling components substantiate the notion that increased complexity of their cellular signaling network reduced the pressure on their PRRs to further diversify. Indeed, situating each PRR in a complex signaling network may have created conditions where further changes in PRR structure would have disrupted their new signaling functions. Vertebrates still rely on innate immunity to help initiate adaptive immune responses, but the value of further diversification in PRRs has been reduced or even reversed. By contrast, organisms lacking adaptive immunity rely solely on their PRRs for immune surveillance; since their pathogens rapidly evolve, the diversity of deuterostome PRRs must quickly evolve as well. Situated as effectors of simpler signaling cascades, these PRRs would seem amenable to the continuous elaboration of new diversity. Gnathostome PRRs must have experienced negative selection after whole genome duplications (hence why mammals do not have four times as many TLRs as invertebrates), although it seems unlikely that purifying selection acting alone could cause the large
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observed difference in the number of deuterostome TLRs and gnathostome TLRs when already selecting against duplicated alleles. However, it does illustrate the point that the present pattern of PRR diversity was likely caused by a combination of ‘‘deuterostome gain’’ and ‘‘gnathostome loss’’ in varying strengths. ‘‘Deuterostome gain’’ acting alone is still plausible but ‘‘gnathostome loss’’ would require extraordinary negative selection on gnathostome PRRs in the wake of whole genome duplications; so if it played a role in shaping PRR diversity, it most likely acted in conjunction with ‘‘deuterostome gain.’’ The precise reasons that deuterostomes posses more diverse PRRs than do gnathostomes may never be known with certainty. Inferring the events impelling either loss or gain in extinct ancestors is, admittedly, fraught with difficulty and is not amenable to either direct observation or testing. Nonetheless, comparing various lines of evidence in extant descendants of each evolutionary lineage sheds light on the relative plausibility of possible hypotheses. Ultimately, the evidence to date favors the notion that deuterostomes underwent diversification in PRRs that was not also experienced in those of our ancestors who passed on to us adaptive immunity. Even though gnathostomes evolved an entirely new system of host defense, we will still be plagued by pathogens and the red queen must keep running her eternal race. Conflicts of interest None. Acknowledgement The University Honors Program at The University of Maryland. References Aznar, J., Herreras, V., Balbuena, J., Raga, J., 2003. Population structure and habitat selection by Anisakis simplex in 4 odontocete species from northern Argentina. Comp. Parasitol. 70 (1), 66–71. Areal, Helena, Abrantes, Joana, Esteves, Pedro, 2011. Signatures of positive selection in Toll-like receptor (TLR) genes in mammals. Evol. Biol. 11 (368), 1471–2148.
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