A small molecule screen identifies novel inhibitors of mechanosensory nematocyst discharge in Hydra.

Cnidarians are characterized by the possession of stinging organelles, called nematocysts, which they use for prey capture and defense. Nematocyst discharge is controlled by a mechanosensory apparatus with analogies to vertebrate hair cells. Members of the transient receptor potential (TRPN) ion channel family are supposed to be involved in the transduction of the mechanical stimulus. A small molecule screen was performed to identify compounds that affect nematocyst discharge in Hydra. We identified several [2.2]paracyclophanes that cause inhibition of nematocyst discharge in the low micro-molar range. Further structure-activity analyses within the compound class of [2.2]paracyclophanes showed common features that are required for the inhibitory activity of the [2.2]paracyclophane core motif. This study demonstrates that Hydra can serve as a model for small molecule screens targeting the mechanosensory apparatus in native tissues.

Evaluation of Hydra HALT-1 as a toxin moiety for recombinant immunotoxin.

BACKGROUND: Immunotoxin is a hybrid protein consisting of a toxin moiety that is linked to a targeting moiety for the purpose of specific elimination of target cells. Toxins used in traditional immunotoxins are practically difficult to be produced in large amount, have poor tissue penetration and a complex internalization process. We hypothesized that the smaller HALT-1, a cytolysin derived from Hydra magnipapillata, can be used as the toxin moiety in construction of a recombinant immunotoxin. RESULTS: In this study, pro-inflammatory macrophage was selected as the target cell due to its major roles in numerous inflammatory and autoimmune disorders. We aimed to construct macrophage-targeted recombinant immunotoxins by combining HALT-1 with anti-CD64-scFv in two orientations, and to assess whether their cytotoxic activity and binding capability could be preserved upon molecular fusion. The recombinant immunotoxins, HALT-1-scFv and scFv-HALT-1, were successfully constructed and expressed in Escherichia coli (E. coli). Our data showed that HALT-1 still exhibited significant cytotoxicity against CD64+ and CD64- cell lines upon fusion with anti-CD64 scFv, although it had half cytotoxic activity as compared to HALT-1 alone. As positioning HALT-1 at N- or C-terminus did not affect its potency, the two constructs demonstrated comparable cytotoxic activities with IC50 lower in CD64+ cell line than in CD64- cell line. In contrast, the location of targeting moieties anti-CD64 scFv at C-terminal end was crucial in maintaining the scFv binding capability. CONCLUSIONS: HALT-1 could be fused with anti-CD64-scFv via a fsexible polypeptide linker. Upon the successful production of this recombinant HALT-1 scFv fusion protein, HALT-1 was proven effective for killing two human cell lines. Hence, this preliminary study strongly suggested that HALT-1 holds potential as the toxin moiety in therapeutic cell targeting.

The Origin of Mucosal Immunity: Lessons from the Holobiont Hydra.

Historically, mucosal immunity-i.e., the portion of the immune system that protects an organism's various mucous membranes from invasion by potentially pathogenic microbes-has been studied in single-cell epithelia in the gastrointestinal and upper respiratory tracts of vertebrates. Phylogenetically, mucosal surfaces appeared for the first time about 560 million years ago in members of the phylum Cnidaria. There are remarkable similarities and shared functions of mucosal immunity in vertebrates and innate immunity in cnidarians, such as Hydra species. Here, we propose a common origin for both systems and review observations that indicate that the ultimately simple holobiont Hydra provides both a new perspective on the relationship between bacteria and animal cells and a new prism for viewing the emergence and evolution of epithelial tissue-based innate immunity. In addition, recent breakthroughs in our understanding of immune responses in Hydra polyps reared under defined short-term gnotobiotic conditions open up the potential of Hydra as an animal research model for the study of common mucosal disorders.

Rethinking the role of immunity: lessons from Hydra.

The ability of multicellular organisms to detect and respond to microorganisms is fundamental and has ancient evolutionary origins. In this review, I evaluate our current understanding of the evolution of epithelial-based innate immunity in Hydra, an apparently simple animal that shares deep evolutionary connections with all animals, including humans. I highlight growing evidence that the innate immune system with its host-specific antimicrobial peptides and rich repertoire of pattern recognition receptors has evolved in response to the need for controlling resident beneficial microbes rather than to defend against invasive pathogens. These findings provide new insight into how developmental pathways beyond those associated with the immune system, such as stem cell transcriptional programs, interact with environmental cues such as microbes.

Injury-induced immune responses in Hydra.

The impact of injury-induced immune responses on animal regenerative processes is highly variable, positive or negative depending on the context. This likely reflects the complexity of the innate immune system that behaves as a sentinel in the transition from injury to regeneration. Early-branching invertebrates with high regenerative potential as Hydra provide a unique framework to dissect how injury-induced immune responses impact regeneration. A series of early cellular events likely require an efficient immune response after amputation, as antimicrobial defence, epithelial cell stretching for wound closure, migration of interstitial progenitors toward the wound, cell death, phagocytosis of cell debris, or reconstruction of the extracellular matrix. The analysis of the injury-induced transcriptomic modulations of 2636 genes annotated as immune genes in Hydra identified 43 genes showing an immediate/early pulse regulation in all regenerative contexts examined. These regulations point to an enhanced cytoprotection via ROS signaling (Nrf, C/EBP, p62/SQSMT1-l2), TNFR and TLR signaling (TNFR16-like, TRAF2l, TRAF5l, jun, fos-related, SIK2, ATF1/CREB, LRRC28, LRRC40, LRRK2), proteasomal activity (p62/SQSMT1-l1, Ced6/Gulf, NEDD8-conjugating enzyme Ubc12), stress proteins (CRYAB1, CRYAB2, HSP16.2, DnaJB9, HSP90a1), all potentially regulating NF-κB activity. Other genes encoding immune-annotated proteins such as NPYR4, GTPases, Swap70, the antiproliferative BTG1, enzymes involved in lipid metabolism (5-lipoxygenase, ACSF4), secreted clotting factors, secreted peptidases are also pulse regulated upon bisection. By contrast, metalloproteinases and antimicrobial peptide genes largely follow a context-dependent regulation, whereas the protease inhibitor α2macroglobulin gene exhibits a sustained up-regulation. Hence a complex immune response to injury is linked to wound healing and regeneration in Hydra.

MyD88-deficient Hydra reveal an ancient function of TLR signaling in sensing bacterial colonizers.

Toll-like receptor (TLR) signaling is one of the most important signaling cascades of the innate immune system of vertebrates. Studies in invertebrates have focused on the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, and there is little information regarding the evolutionary origin and ancestral function of TLR signaling. In Drosophila, members of the Toll-like receptor family are involved in both embryonic development and innate immunity. In C. elegans, a clear immune function of the TLR homolog TOL-1 is controversial and central components of vertebrate TLR signaling including the key adapter protein myeloid differentiation primary response gene 88 (MyD88) and the transcription factor NF-κB are not present. In basal metazoans such as the cnidarians Hydra magnipapillata and Nematostella vectensis, all components of the vertebrate TLR signaling cascade are present, but their role in immunity is unknown. Here, we use a MyD88 loss-of-function approach in Hydra to demonstrate that recognition of bacteria is an ancestral function of TLR signaling and that this process contributes to both host-mediated recolonization by commensal bacteria as well as to defense against bacterial pathogens.

FoxO is a critical regulator of stem cell maintenance in immortal Hydra.

Hydra's unlimited life span has long attracted attention from natural scientists. The reason for that phenomenon is the indefinite self-renewal capacity of its stem cells. The underlying molecular mechanisms have yet to be explored. Here, by comparing the transcriptomes of Hydra's stem cells followed by functional analysis using transgenic polyps, we identified the transcription factor forkhead box O (FoxO) as one of the critical drivers of this continuous self-renewal. foxO overexpression increased interstitial stem cell and progenitor cell proliferation and activated stem cell genes in terminally differentiated somatic cells. foxO down-regulation led to an increase in the number of terminally differentiated cells, resulting in a drastically reduced population growth rate. In addition, it caused down-regulation of stem cell genes and antimicrobial peptide (AMP) expression. These findings contribute to a molecular understanding of Hydra's immortality, indicate an evolutionarily conserved role of FoxO in controlling longevity from Hydra to humans, and have implications for understanding cellular aging.

Where simplicity meets complexity: hydra, a model for host-microbe interactions.

For a long time, the main purpose of microbiology and immunology was to study pathogenic bacteria and infectious disease; the potential benefit of commensal bacteria remained unrecognised. Discovering that individuals from Hydra to man are not solitary, homogenous entities but consist of complex communities of many species that likely evolved during a billion years of coexistence (Fraune and Bosch 2010) led to the hologenome theory of evolution (Zilber-Rosenberg and Rosenberg 2008) which considers the holobiont with its hologenome as the unit of selection in evolution. Defining the individual microbe-host conversations in these consortia is a challenging but necessary step on the path to understanding the function of the associations as a whole. Untangling the complex interactions requires simple animal models with only a few specific bacterial species. Such models can function as living test tubes and may be key to dissecting the fundamental principles that underlie all host-microbe interactions. Here we introduce Hydra (Bosch et al. 2009) as such a model with one of the simplest epithelia in the animal kingdom (only two cell layers), with few cell types derived from only three distinct stem cell lineages, and with the availability of a fully sequenced genome and numerous genomic tools including transgenesis. Recognizing the entire system with its inputs, outputs and the interconnections (Fraune and Bosch 2010; Bosch et al. 2009; Fraune and Bosch 2007; Fraune et al. 2009a) we here present observations which may have profound impact on understanding a strictly microbe-dependent life style and its evolutionary consequences.

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.

Defining the origins of the NOD-like receptor system at the base of animal evolution.

Distinguishing self from nonself and the onset of defense effector mechanisms upon recognition of pathogens are essential for the survival of all life forms in the animal kingdom. The family of nucleotide -binding and oligomeriszation domain-like receptors (NLRs) was first identified in vertebrates and comprises a group of pivotal sensor protein of the innate immune system for microbial cell wall components or danger signals. Here, we provide first evidence that early diverging metazoans have large and complex NLR repertoires. The cnidarian NACHT/NB-ARC genes include novel combinations of domains, and the number of one specific type (NB-ARC and tetratricopeptide repeat containing) in Hydra is particularly large. We characterize the transcript structure and expression patterns of a selected HyNLR, HyNLR type 1 and describe putative interaction partners. In a heterologous expression system, we show induced proximity recruitment of an effector caspase (HyDD-Caspase) to the HyNLR type 1 protein upon oligomerization indicating a potential role of caspase activation downstream of NLR activation in Hydra. These results add substantially to our understanding of the ancestral innate immune repertoire as well as providing the first insights into putative cytoplasmic defense mechanisms at the base of animal evolution.

How Hydra senses and destroys microbes.

Molecular genetic evidence has revealed that the basic templates of innate immune sensors were laid down in ancient animals such as the cnidarian Hydra. Important functions of Hydra's innate immune sensors and effectors include not only protection against pathogens but also controlling tissue-microbiota homeostasis. The deep evolutionary connections imply that invertebrate and mammalian immune pathways have evolved from a reduced number of common ancestral building blocks to their present configurations.

Identification of a kazal-type serine protease inhibitor with potent anti-staphylococcal activity as part of Hydra's innate immune system.

In the absence of migratory phagocytic cells the basal metazoan Hydra has developed a very effective immune system. Previous work has shown that epithelial cells, both in the ectoderm and endoderm, recognize PAMPs by TLR and produce a number of antimicrobial peptides. In this study we demonstrate that not only epithelial cells but also gland cells are critically involved in Hydra's innate host defense by producing a kazal-type serine protease inhibitor, kazal2, that has potent in vitro bactericidal activity against Staphylococcus aureus. The discovery of an antimicrobial serine protease inhibitor in Hydra may shed new light on the mechanisms of host defense early in metazoan evolution, and promises to open new avenues for the development of potent anti-staphylococcal compounds.

[The antigen of the differentiated basal disc cells of Hydra].

A new antigenic marker of the differentiated basal disk cells of Hydra was characterized. An antigen named 3G11 was revealed by monoclonal antibody in granules of the basal disk gland cells of the ectoderm. The antigen appearance during budding, regeneration and ectopic foot formation evidences for the differentiation of the body column epithelial cells into basal disk gland cells. Antigen 3G11 is species-specific: among six hydra species investigated, the antigen was observed exclusively in polyps of vulgaris group which is a special taxon of the filum Hydra. Cell and tissue localization of the antigen 3G11 was similar to that of the well-established biochemical hydra marker, foot specific peroxidase, reported formerly. However, ELISA data suggest that the molecule bearing antigen 3G11 does not possess any peroxidase activity. Thus the new hydra antigenic marker 3G11 extends the number of previously used markers of differentiation and allows to improve the technique of the basal disk differentiated tissue identification.

Uncovering the evolutionary history of innate immunity: the simple metazoan Hydra uses epithelial cells for host defence.

Although many properties of the innate immune system are shared among multicellular animals, the evolutionary origin remains poorly understood. Here we characterize the innate immune system in Hydra, one of the simplest multicellular animals known. In the complete absence of both protective mechanical barriers and mobile phagocytes, Hydra's epithelium is remarkably well equipped with potent antimicrobial peptides to prevent pathogen infection. Induction of antimicrobial peptide production is mediated by the interaction of a leucine-rich repeats (LRRs) domain containing protein with a TIR-domain containing protein lacking LRRs. Conventional Toll-like receptors (TLRs) are absent in the Hydra genome. Our findings support the hypothesis that the epithelium represents the ancient system of host defence.

The innate immune repertoire in cnidaria--ancestral complexity and stochastic gene loss.

BACKGROUND: Characterization of the innate immune repertoire of extant cnidarians is of both fundamental and applied interest--it not only provides insights into the basic immunological 'tool kit' of the common ancestor of all animals, but is also likely to be important in understanding the global decline of coral reefs that is presently occurring. Recently, whole genome sequences became available for two cnidarians, Hydra magnipapillata and Nematostella vectensis, and large expressed sequence tag (EST) datasets are available for these and for the coral Acropora millepora. RESULTS: To better understand the basis of innate immunity in cnidarians, we scanned the available EST and genomic resources for some of the key components of the vertebrate innate immune repertoire, focusing on the Toll/Toll-like receptor (TLR) and complement pathways. A canonical Toll/TLR pathway is present in representatives of the basal cnidarian class Anthozoa, but neither a classic Toll/TLR receptor nor a conventional nuclear factor (NF)-kappaB could be identified in the anthozoan Hydra. Moreover, the detection of complement C3 and several membrane attack complex/perforin domain (MAC/PF) proteins suggests that a prototypic complement effector pathway may exist in anthozoans, but not in hydrozoans. Together with data for several other gene families, this implies that Hydra may have undergone substantial secondary gene loss during evolution. Such losses are not confined to Hydra, however, and at least one MAC/PF gene appears to have been lost from Nematostella. CONCLUSION: Consideration of these patterns of gene distribution underscores the likely significance of gene loss during animal evolution whilst indicating ancient origins for many components of the vertebrate innate immune system.

Enhanced antibacterial activity in Hydra polyps lacking nerve cells.

The nervous system evolved within cnidarians. When assessing antibacterial activity in the freshwater polyp Hydra, we observed a strong correlation between the number of neurons present and the antibacterial activity. Tissue lacking neurons had a drastically enhanced antibacterial activity against Gram-positive (Bacillus subtilis) and Gram-negative (E. coli) bacteria compared to control tissue. The results indicate direct and strong neural influences on immunity in the phylogenetically oldest animals having a nervous system.

Foot formation in hydra: commitment of the basal disk cells in the lower peduncle.

Foot regeneration in the freshwater hydra Pelmatohydra robusta was examined using a monoclonal antibody AE03 as a marker. This antibody specifically recognizes mucous-producing ectodermal epithelial cells in the basal disk, but not cells in the peduncle region located just above the basal disk in the foot. When the basal disk was removed by amputation at the upper or lower part of the peduncle, AE03-positive (basal disk) cells always appeared at the regenerating tip of the footless polyp approximately 12-16 h later. When a small piece of tissue was cut out from the upper or lower peduncle region, the tissue invariably turned into a smooth spherical or oblong shape within a few hours. AE03 signal appeared in these spheres variably depending on their origin: when tissue pieces were derived from the lower peduncle, the signal appeared in nearly all pieces and often covered the entire surface of the pieces within 24 h. In contrast, the signal appeared in less than 10% of pieces derived from the upper peduncle. Furthermore, the signal seldom covered more than half of the surface of these pieces. When maintained for many days, pieces derived from the upper peduncle often regenerated tentacles, whereas those from the lower peduncle seldom did. These and other observations suggest that epithelial cells in the peduncle can rapidly differentiate into basal disk cells when the basal tissue is removed. However, cells in the upper peduncle are not irreversibly committed to differentiate into basal disk cells because, when cut out as small tissue pieces, they could remain AE03 negative and become tentacle cells. In contrast, the cells in the lower peduncle apparently are irreversibly committed to differentiate into basal disk cells, as they always turned rapidly into AE03-positive cells once they were physically separated from (and freed from the influence of) the basal disk itself, regardless of the separation methods used.

Ontogeny recapitulates phylogeny: comparative immunology in Germany.

The innate immune system is in the spot light of modern immunology. Whenever protists, invertebrates and vertebrates are threatened by pathogens, they rapidly activate highly effective antimicrobial defense reactions. Because this young field develops very dynamically, it is important to ask what we really know about the mechanisms governing the innate immune defense system. This was the topic of a recent meeting entitled 'The Evolution of the Immune System', held at the Friedrich Schiller University in Jena, Germany. Leading scientists in the field of innate immunity presented their latest data in a historical and friendly setting.