Mecanismos inmunológicos en Cnidaria: un sistema inmune basal de gran complejidad e interés bioprospectivo / Immunological mechanisms in Cnidaria: A highly complex basal immune system with bioprospective interest

Introducción: La protección ante agentes biológicos propios y externos de los cnidarios dependen de la inmunidad innata, la cual consta de tres procesos inmunológicos principales: 1) reconocimiento inmunológico, 2) señalización intracelular, y 3) respuesta efectora. Objetivo: Revisar críticamente el conocimiento actual del repertorio molecular involucrado en la respuesta inmune en cnidarios, así como, su papel en el establecimiento de la simbiosis, y las posibles aplicaciones biotecnológicas de las moléculas involucradas en el proceso de inmunidad. Métodos: Se realizó una revisión de artículos científicos encontrados a través de las bases de datos del NCBI, Google Scholar y Scielo, con palabras claves como inmunidad y/o reconocimiento inmunológico en cnidarios, en una ventana de tiempo de la última década, sin descartar literatura clásica más antigua. Resultados: El reconocimiento inmunológico consiste en receptores inmunológicos que reconocen patrones moleculares e inducen respuestas efectoras como la movilización de moléculas al sitio de la infección, la ingestión microbiana y la formación de moléculas que activan cascadas de señalización. La fase de señalización involucra mediadores de la traducción de señales que activan genes de trascripción, y cascadas de señalización intracelular que inician respuestas de defensa adecuadas. Las respuestas efectoras incluyen la capa superficial del mucus, péptidos antimicrobianos, especies reactivas de oxígeno y la respuesta celular mediada por fagocitosis. Por último, se presenta un esquema y una tabla integral de las vías de respuesta inmune en los cnidario. Conclusiones: La inmunidad en Cnidaria está mediada por mecanismos de defensa complejos integrados por receptores de reconocimiento de patógenos, vías de señalización intracelular, células y moléculas efectoras encargadas de la eliminación del patógeno, y reconocimiento-aceptación de simbiontes. El estudio de compuestos activos del sistema inmune en Cnidaria ha sido poco explorado, sin embargo, el trabajo realizado con otros compuestos presentes en las toxinas de este filo, los sitúa como una fuente importante de moléculas antimicrobianas dignas de un análisis de bioprospección.

Introduction: Cnidarians depend on innate immunity for protection against both their own and external biological agents. It consists of three main immunological processes: 1) immune recognition, 2) intracellular signaling, and 3) effector response. Objective: To critically review current knowledge of the molecular repertoire involved in the immune response in cnidarians, its role in symbiosis, and possible biotechnological applications. Methods: We used keywords such as immunity, and immunological recognition in cnidarians, in the NCBI, Scielo and Google Scholar databases, for the last decade. Results: Cnidarian immune recognition consists of molecular pattern receptors and responses such as the mobilization of molecules to the site of infection, microbial ingestion, and the formation of molecules that activate signaling cascades. The signaling phase involves translation mediators that activate transcriptional genes and intracellular signaling cascades that initiate defenses. Effector responses include surface layer mucus, antimicrobial peptides, reactive oxygen species, and the cellular response mediated by phagocytosis. Conclusions: Immunity in Cnidaria is mediated by complex defense mechanisms composed of pathogen recognition receptors, intracellular signaling pathways, effector cells and molecules responsible for pathogen elimination, and recognition of symbionts. There is a potential for toxin compounds useful as antimicrobial molecules.

Cnidarian Pattern Recognition Receptor Repertoires Reflect Both Phylogeny and Life History Traits.

Pattern recognition receptors (PRRs) are evolutionarily ancient and crucial components of innate immunity, recognizing danger-associated molecular patterns (DAMPs) and activating host defenses. Basal non-bilaterian animals such as cnidarians must rely solely on innate immunity to defend themselves from pathogens. By investigating cnidarian PRR repertoires we can gain insight into the evolution of innate immunity in these basal animals. Here we utilize the increasing amount of available genomic resources within Cnidaria to survey the PRR repertoires and downstream immune pathway completeness within 15 cnidarian species spanning two major cnidarian clades, Anthozoa and Medusozoa. Overall, we find that anthozoans possess prototypical PRRs, while medusozoans appear to lack these immune proteins. Additionally, anthozoans consistently had higher numbers of PRRs across all four classes relative to medusozoans, a trend largely driven by expansions in NOD-like receptors and C-type lectins. Symbiotic, sessile, and colonial cnidarians also have expanded PRR repertoires relative to their non-symbiotic, mobile, and solitary counterparts. Interestingly, cnidarians seem to lack key components of mammalian innate immune pathways, though similar to PRR numbers, anthozoans possess more complete immune pathways than medusozoans. Together, our data indicate that anthozoans have greater immune specificity than medusozoans, which we hypothesize to be due to life history traits common within Anthozoa. Overall, this investigation reveals important insights into the evolution of innate immune proteins within these basal animals.

AmAMP1 from Acropora millepora and damicornin define a family of coral-specific antimicrobial peptides related to the Shk toxins of sea anemones.

A candidate antimicrobial peptide (AmAMP1) was identified by searching the whole genome sequence of Acropora millepora for short (<125AA) cysteine-rich predicted proteins with an N-terminal signal peptide but lacking clear homologs in the SwissProt database. It resembled but was not closely related to damicornin, the only other known AMP from a coral, and was shown to be active against both Gram-negative and Gram-positive bacteria. These proteins define a family of AMPs present in corals and their close relatives, the Corallimorpharia, and are synthesised as preproproteins in which the C-terminal mature peptide contains a conserved arrangement of six cysteine residues. Consistent with the idea of a common origin for AMPs and toxins, this Cys motif is shared between the coral AMPs and the Shk neurotoxins of sea anemones. AmAMP1 is expressed at late stages of coral development, in ectodermal cells that resemble the "ganglion neurons" of Hydra, in which it has recently been demonstrated that a distinct AMP known as NDA-1 is expressed.

The Complicated Evolutionary Diversification of the Mpeg-1/Perforin-2 Family in Cnidarians.

The invertebrate innate immune system is surprisingly complex, yet our knowledge is limited to a few select model systems. One understudied group is the phylum Cnidaria (corals, sea anemones, etc.). Cnidarians are the sister group to Bilateria and by studying their innate immunity repertoire, a better understanding of the ancestral state can be gained. Corals in particular have evolved a highly diverse innate immune system that can uncover evolutionarily basal functions of conserved genes and proteins. One rudimentary function of the innate immune system is defense against harmful bacteria using pore forming proteins. Macrophage expressed gene 1/Perforin-2 protein (Mpeg-1/P2) is a particularly important pore forming molecule as demonstrated by previous studies in humans and mice, and limited studies in non-bilaterians. However, in cnidarians, little is known about Mpeg-1/P2. In this perspective article, we will summarize the current state of knowledge of Mpeg-1/P2 in invertebrates, analyze identified Mpeg-1/P2 homologs in cnidarians, and demonstrate the evolutionary diversity of this gene family using phylogenetic analysis. We will also show that Mpeg-1 is upregulated in one species of stony coral in response to lipopolysaccharides and downregulated in another species of stony coral in response to white band disease. This data presents evidence that Mpeg-1/P2 is conserved in cnidarians and we hypothesize that it plays an important role in cnidarian innate immunity. We propose that future research focus on the function of Mpeg-1/P2 family in cnidarians to identify its primary role in innate immunity and beyond.

Innate immunity and cnidarian-Symbiodiniaceae mutualism.

The phylum Cnidaria (sea anemones, corals, hydra, jellyfish) is one the most distantly related animal phyla to humans, and yet cnidarians harbor many of the same cellular pathways involved in innate immunity in mammals. In addition to its role in pathogen recognition, the innate immune system has a role in managing beneficial microbes and supporting mutualistic microbial symbioses. Some corals and sea anemones undergo mutualistic symbioses with photosynthetic algae in the family Symbiodiniaceae. These symbioses can be disrupted by anthropogenic disturbances of ocean environments, which can have devastating consequences for the health of coral reef ecosystems. Several studies of cnidarian-Symbiodiniaceae symbiosis have implicated proteins in the host immune system as playing a role in both symbiont tolerance and loss of symbiosis (i.e., bleaching). In this review, we critically evaluate current knowledge about the role of host immunity in the regulation of symbiosis in cnidarians.

Response of Cellular Innate Immunity to Cnidarian Pore-Forming Toxins.

A group of stable, water-soluble and membrane-bound proteins constitute the pore forming toxins (PFTs) in cnidarians. They interact with membranes to physically alter the membrane structure and permeability, resulting in the formation of pores. These lesions on the plasma membrane causes an imbalance of cellular ionic gradients, resulting in swelling of the cell and eventually its rupture. Of all cnidarian PFTs, actinoporins are by far the best studied subgroup with established knowledge of their molecular structure and their mode of pore-forming action. However, the current view of necrotic action by actinoporins may not be the only mechanism that induces cell death since there is increasing evidence showing that pore-forming toxins can induce either necrosis or apoptosis in a cell-type, receptor and dose-dependent manner. In this review, we focus on the response of the cellular immune system to the cnidarian pore-forming toxins and the signaling pathways that might be involved in these cellular responses. Since PFTs represent potential candidates for targeted toxin therapy for the treatment of numerous cancers, we also address the challenge to overcoming the immunogenicity of these toxins when used as therapeutics.

Metagenomic sequencing suggests a diversity of RNA interference-like responses to viruses across multicellular eukaryotes.

RNA interference (RNAi)-related pathways target viruses and transposable element (TE) transcripts in plants, fungi, and ecdysozoans (nematodes and arthropods), giving protection against infection and transmission. In each case, this produces abundant TE and virus-derived 20-30nt small RNAs, which provide a characteristic signature of RNAi-mediated defence. The broad phylogenetic distribution of the Argonaute and Dicer-family genes that mediate these pathways suggests that defensive RNAi is ancient, and probably shared by most animal (metazoan) phyla. Indeed, while vertebrates had been thought an exception, it has recently been argued that mammals also possess an antiviral RNAi pathway, although its immunological relevance is currently uncertain and the viral small RNAs (viRNAs) are not easily detectable. Here we use a metagenomic approach to test for the presence of viRNAs in five species from divergent animal phyla (Porifera, Cnidaria, Echinodermata, Mollusca, and Annelida), and in a brown alga-which represents an independent origin of multicellularity from plants, fungi, and animals. We use metagenomic RNA sequencing to identify around 80 virus-like contigs in these lineages, and small RNA sequencing to identify viRNAs derived from those viruses. We identified 21U small RNAs derived from an RNA virus in the brown alga, reminiscent of plant and fungal viRNAs, despite the deep divergence between these lineages. However, contrary to our expectations, we were unable to identify canonical (i.e. Drosophila- or nematode-like) viRNAs in any of the animals, despite the widespread presence of abundant micro-RNAs, and somatic transposon-derived piwi-interacting RNAs. We did identify a distinctive group of small RNAs derived from RNA viruses in the mollusc. However, unlike ecdysozoan viRNAs, these had a piRNA-like length distribution but lacked key signatures of piRNA biogenesis. We also identified primary piRNAs derived from putatively endogenous copies of DNA viruses in the cnidarian and the echinoderm, and an endogenous RNA virus in the mollusc. The absence of canonical virus-derived small RNAs from our samples may suggest that the majority of animal phyla lack an antiviral RNAi response. Alternatively, these phyla could possess an antiviral RNAi response resembling that reported for vertebrates, with cryptic viRNAs not detectable through simple metagenomic sequencing of wild-type individuals. In either case, our findings show that the antiviral RNAi responses of arthropods and nematodes, which are highly divergent from each other and from that of plants and fungi, are also highly diverged from the most likely ancestral metazoan state.

Immune-directed support of rich microbial communities in the gut has ancient roots.

The animal gut serves as a primary location for the complex host-microbe interplay that is essential for homeostasis and may also reflect the types of ancient selective pressures that spawned the emergence of immunity in metazoans. In this review, we present a phylogenetic survey of gut host-microbe interactions and suggest that host defense systems arose not only to protect tissue directly from pathogenic attack but also to actively support growth of specific communities of mutualists. This functional dichotomy resulted in the evolution of immune systems much more tuned for harmonious existence with microbes than previously thought, existing as dynamic but primarily cooperative entities in the present day. We further present the protochordate Ciona intestinalis as a promising model for studying gut host-bacterial dialogue. The taxonomic position, gut physiology and experimental tractability of Ciona offer unique advantages in dissecting host-microbe interplay and can complement studies in other model systems.

Cnidarian-microbe interactions and the origin of innate immunity in metazoans.

Most epithelia in animals are colonized by microbial communities. These resident microbes influence fitness and thus ecologically important traits of their hosts, ultimately forming a metaorganism consisting of a multicellular host and a community of associated microorganisms. Recent discoveries in the cnidarian Hydra show that components of the innate immune system as well as transcriptional regulators of stem cells are involved in maintaining homeostasis between animals and their resident microbiota. Here I argue that components of the innate immune system with its host-specific antimicrobial peptides and a rich repertoire of pattern recognition receptors evolved in early-branching metazoans because of the need to control the resident beneficial microbes, not because of invasive pathogens. I also propose a mutual intertwinement between the stem cell regulatory machinery of the host and the resident microbiota composition, such that disturbances in one trigger a restructuring and resetting of the other.

Allorecognition triggers autophagy and subsequent necrosis in the cnidarian Hydractinia symbiolongicarpus.

Transitory fusion is an allorecognition phenotype displayed by the colonial hydroid Hydractinia symbiolongicarpus when interacting colonies share some, but not all, loci within the allorecognition gene complex (ARC). The phenotype is characterized by an initial fusion followed by subsequent cell death resulting in separation of the two incompatible colonies. We here characterize this cell death process using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and continuous in vivo digital microscopy. These techniques reveal widespread autophagy and subsequent necrosis in both colony and grafted polyp assays. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays and ultrastructural observations revealed no evidence of apoptosis. Pharmacological inhibition of autophagy using 3-methyladenine (3-MA) completely suppressed transitory fusion in vivo in colony assays. Rapamycin did not have a significant effect in the same assays. These results establish the hydroid allorecognition system as a novel model for the study of cell death.

Neglected biological features in cnidarians self-nonself recognition.

Cnidarian taxa, currently of the most morphologically simplest extant metazoans, exhibit many salient properties of innate immunity that are shared by most Animalia. One hallmark constituent of immunity exhibit by most cnidarians is histocompatibility, marked by wide spectrum of allogeneic and xenogeneic effector arms, progressing into tissue fusions or inflammatory rejections. Scientific propensity on cnidarians immunity, while discussing historecognition as the ground for immunity in these organisms, concentrates on host-parasitic and disease oriented studies, or focuses on genome approaches that search for gene homologies with the vertebrates. Above tendency for mixing up between historecognition and host-parasitic/disease, highlights a serious obstacle for the progress in our understanding of cnidarian immunobiology. Here I critically overview four 'forgotten' cnidarian immune features, namely, specificity, immunological memory, allogeneic maturation and natural chimerism, presenting insights into perspectives that are prerequisite for any discussion on cnidarian evolution. It is evident that cnidarian historecognition embraces elements that the traditional field of vertebrate immunology has never encountered (i.e., variety of cytotoxic outcomes, different types of effector mechanisms, chimerism, etc.). Also, cnidarian immune features dictating that different individuals within the same species seem to respond differently to the same immunological challenge, is far from that recorded in the vertebrates' adaptive immunity. While above features may be connected to host-parasitic and disease phenomena and effector arms, they clearly attest to their unique critical roles in shaping cnidarians historecognition, calling for improved distinction between historecognition and host-response/ disease disciplines. The research on cnidarians immunity still suffers from the lack of accepted synthesis of what historecognition is or does. Mounting of an immune response against conspecifics or xenogeneic organisms should therefore be clearly demarcated from other paths of immunity, till cnidarian innate immunity as a whole is expounded.

Interactions of cnidarian toxins with the immune system.

Cnidarians comprise four classes of toxic marine animals: Anthozoa, Cubozoa, Scyphozoa and Hydrozoa. They are the largest and probably the oldest phylum of toxic marine animals. Any contact with a cnidarian, especially the box jellyfish (Chironex fleckeri), can be fatal, but most cnidarians do not possess sufficiently strong venomous apparatus to penetrate the human skin, whereas others rarely come into contact with human beings. Only a small, almost negligible percentage of the vast wealth of cnidarian toxins has been studied in detail. Many polypeptide cnidarian toxins are immunogenic, and cross-reactivity between several jellyfish venoms has been reported. Cnidarians also possess components of innate immunity, and some of those components have been preserved in evolution. On the other hand, cnidarian toxins have already been used for the design of immunotoxins to treat cancer, whereas other cnidarian toxins can modulate the immune system in mammals, including man. This review will focus on a short overview of cnidarian toxins, on the innate immunity of cnidarians, and on the mode of action of cnidarian toxins which can modulate the immune system in mammals. Emphasis is palced on those toxins which block voltage activated potassium channels in the cells of the immune system.

Shared antigenicity between the polar filaments of myxosporeans and other Cnidaria.

Nematocysts containing coiled polar filaments are a distinguishing feature of members of the phylum Cnidaria. As a first step to characterizing the molecular structure of polar filaments, a polyclonal antiserum was raised in rabbits against a cyanogen bromide-resistant protein extract of mature cysts containing spores of Myxobolus pendula. The antiserum reacted only with proteins associated with extruded polar filaments. Western blot and whole-mount immunohistochemical analyses indicated a conservation of polar filament epitopes between M. pendula and 2 related cnidarians, i.e., the anthozoan, Nematostella vectensis, and the hydrozoan, Hydra vulgaris. This conservation of polar filament epitopes lends further support to a shared affinity between Myxozoa and cnidarians.

Domain combination of the vertebrate-like TLR gene family: implications for their origin and evolution.

Domain shuffling, which is an important mechanism in the evolution of multi-domain proteins, has shaped the evolutionary development of the immune system in animals. Toll and Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate and adaptive immune systems. Draft genome sequences provide the opportunity to compare the Toll/TLR gene repertoire among representative metazoans. In this study, we investigated the combination of Toll/interleukin-1 receptor (TIR) and leucine-rich repeat (LRR) domains of metazoan Toll/TLRs. Before Toll with both domains occurred in Cnidaria (sea anemone, Nematostella vectensis), through domain combinations, TIR-only and LRR-only proteins had already appeared in sponges (Amphimedon queenslandica). Although vertebrate-like TIR (V-TIR) domain already appeared in Cnidaria, the vertebrate-like TLR (V-TLR) with both domains appeared much later. The first combination between V-TIR domain and vertebrate-like LRR (V-LRR) domain for V-TLR may have occurred after the divergence of Cnidaria and bilateria. Then, another combination for V-TLR, a recombination of both domains, possibly occurred before or during the evolution of primitive vertebrates. Taken together, two rounds of domain combinations may thus have co-shaped the vertebrate TLRs.

Cnidarian immunity: a tale of two barriers.

The phylum Cnidariais one of the earliest branches in the animal tree of life providing crucial insights into the early evolution of immunity. The diversity in cnidarian life histories and habitats raises several important issues relating to immunity. First, in the absence of specific immune cells, cnidarians must have effective mechanisms to defend against microbial pathogens. Second, to maintain tissue integrity, colonial forms have to rely on their capacity of self/nonself discrimination to rapidly detect approaching allogeneic cells as foreign and to eliminate them. And third, since cnidarians are colonized by complex bacterial communities and in many cases are home to algal symbionts, successful growth means for cnidarians to be able to distinguish between beneficial symbionts and pathogenic intruders. The aim of this chapter is to review the experimental evidence for innate immune reactions in Cnidaria. We show that in these diploblastic animals consisting of only two cell layers; the epithelial cells are able to mediate all innate immune responses. The endodermal epithelium appears as a chemical barrier employing antimicrobial peptides while the ectodermal epithelium is a physicochemical barrier supported by a glycocalix. Microbial recognition is mediated by pattern recognition receptors such as Toll- and Nod-like receptors. Together, the data support the hypothesis that the establishment of epithelial barriers represents an important step in evolution of host defense in eumetazoan animals more than 600 million years ago.

The diversity of C-type lectins in the genome of a basal metazoan, Nematostella vectensis.

C-type lectins (CTLs) are involved in cell-cell adhesion, recognition, and innate immunity in higher vertebrates, but little is known about CTLs in basal metazoans. The recent sequencing of the cnidarian Nematostella vectensis genome allowed us to explore the CTL-like gene family at the base of metazoan evolution. Sixty-seven predicted CTLs, with a total of 92 putative C-type lectin domains (CTLDs), were classified according to number of CTLDs present and their association with other protein domains in the CTL. Conserved residues in the glycan-binding pocket suggest that approximately half of the CTLDs retain glycan-binding function. Phylogenetic analysis of N. vectensis CTLDs with respect to other model invertebrates and humans indicates N. vectensis CTLD sequences more closely resemble vertebrate CTLDs. This study provides a N. vectensis CTL database that can be used for further research on the evolution of cnidarian CTLs and the role of CTLs in cnidarian innate immunity.

Skin prick test of Kudoa sp. antigens in patients with gastrointestinal and/or allergic symptoms related to fish ingestion.

A majority of Kudoa spp. infects the somatic muscle of fish establishing cysts. Previously, elevated humoral responses were detected in BALB/c mice immunised with Kudoa sp. pseudocyst extracts and in BALB/c mice orally inoculated with Kudoa sp. pseudocysts, as well as the presence of anti-Kudoa sp. antibodies in human sera by enzyme-linked immunosorbent assay. The objective of this work was to test Kudoa sp. pseudocyst extracts by the skin prick test. Fifteen patients with gastroallergic and/or allergic symptoms related to fish ingestion were examined. Kudoa sp. pseudocyst extracts were administered (1 mg/ml) on the volar forearm skin. Four of the 15 selected patients were positive to Kudoa sp. extracts. The saline solution negative control did not induce any reaction.

Anisakis simplex and Kudoa sp.: evaluation of specific antibodies in appendectomized patients.

High prevalence and intensity of infection with anisakid larvae has been reported in commercially important fish in Spain. Likewise, Kudoa-infected fish have lately been detected in both fresh and frozen fish. In the present study the possible relation between appendectomy and specific antibodies to these fish parasites was investigated. One hundred and sixty patients were enrolled in this study. They were divided into two groups of eighty patients each and matched for sex and age: Group 1 (appendectomized) and Group 2 (control group). Total immunoglobulins (Ig's), IgG, IgM, IgA and IgE against Anisakis simplex or Kudoa sp. antigens were analysed by ELISA. The mean values of the specific antibodies were lower in the appendectomy group, although significant differences were not observed in the case of IgG, IgA and IgE anti-A. simplex and IgE anti-Kudoa sp. In summary, appendectomy significantly decreased serum specific immunoglobulin levels against these food borne parasite antigens. This decrease was detectable from three months to three years post-appendectomy. It is necessary to study the influence of the surgical removal of other important parts of the GALT on these anti-parasite humoral immune responses.

From Darwin and Metchnikoff to Burnet and beyond.

Phagocytosis in unicellular animals represents the most ancient and ubiquitous form of defense against foreign material. Unicellular invertebrates can phagocytose for food and defense. Multicellular invertebrates and vertebrates possess phagocytic cells and have evolved more complex functions attributed to immunodefense cells that specialized into cellular and humoral immune responses. Thus all animals possess: innate, natural, nonspecific (no memory) nonanticipatory, nonclonal, germline (hard wired) host defense functions. In addition, all vertebrates possess: adaptive, induced, specific (memory), anticipatory, clonal, somatic (flexible) immune responses. A similar situation exists with respect to components of the signaling system, immunity and development. With multicellularity, clearly numerous immune response characteristics are not possible in unicellular forms or even those that straddle the divide between unicellularity and multicellularity, beginning with colonial/social protozoans. Still, it is instructive to elucidate a hierarchy of animals based upon immunologic characteristics and how they parallel other physiological traits. Evidence is presented that the most primitive of invertebrates prior to the evolution of multicellular organisms possess varying degrees of complexity at the molecular level of those hallmarks that now characterize the immune system.