Colony Specificity in the Colonial Tunicate Botryllus and the Origins of Vertebrate Immunity

SYNOPSIS. Colonies of the compound tunicate Botryllus show thecapacity for self—nonself discrimination by fusion betweenseparated pieces of the same colony and rejection between piecesof unrelated colonies. We have found that genes controllingthis colony specificity are similar to those which cause transplantrejection in the vertebrates. Like the loci within the vertebratemajor histocompatibility complex (MHC), Botryllus fusibility(or histocompatibility) genes are highly polymorphic. In Botryllus,the histocompatibility complex also controls self—sterility,and limits cross—fertilization between colonies sharinghistocompatibility alleles. The mouse MHC, the H-2 region, islinked to loci which also affect the frequencies of allelesat H-2 loci in mouse populations. Thus both systems containcharacters which could act to promote the heterozygous conditionat the linked histocompatibility loci. We suggest that suchlinked characters are responsible for the evolution of allogeneicpolymorphism in vertebrates (however currently maintained),and that tunicate fusibility loci may be the evolutionary precursorsof vertebrate MHC genes.     

Distinct Innate Immunity Pathways to Activation and Tolerance in Subgroups of Chronic Lymphocytic Leukemia with Distinct Immunoglobulin Receptors

Subgroups of patients with chronic lymphocytic leukemia (CLL) have distinct expression profiles of Toll-like receptor (TLR) pathway–associated genes. To test the hypothesis that signaling through innate immunity receptors may influence the behavior of the malignant clone, we investigated the functional response triggered by the stimulation of TLRs and NOD2 in 67 CLL cases assigned to different subgroups on the basis of immunoglobulin heavy variable (IGHV ) gene usage, IGHV gene mutational status or B-cell receptor (BcR) stereotypy. Differences in the induction of costimulatory molecules and/or apoptosis were observed in mutated versus unmutated CLL. Different responses were also identified in subsets with stereotyped BcRs, underscoring the idea that “subset-biased” innate immunity responses may occur independently of mutational status. Additionally, differential modulation of kinase activities was induced by TLR stimulation of different CLL subgroups, revealing a TLR7-tolerant state for cases belonging to stereotyped subset #4. The distinct patterns of TLR/NOD2 functional activity in cells from CLL subgroups defined by the molecular features of the clonotypic BcRs might prove relevant for elucidating the immune mechanisms underlying CLL natural history and for defining subgroups of patients who might benefit from treatment with specific TLR ligands.     

Development of Panel of Monoclonal Antibodies Specific to Urochordate Cell Surface Antigens

Monoclonal antibodies are an important tool in the study of botryllid ascidians’ immunology and developmental biology. Here we describe the development of a panel of 38 monoclonal antibodies that are specific to Botryllus schlosseri (Ascidiacea; subfamily Botryllinae) cell surface antigens. Many of these hybridomas recognize (by enzyme-linked immunosorbent assay and immunohistochemistry) epitopes of Botrylloides subpopulations (SP) II and III from the Mediterranean coast of Israel and show, on blood cell smear assays, reactions with subsets of Botryllus circulating blood cells. Fluorescence-activated cell sorting analyses using antibodies positive for botryllid tissues revealed up to 3.6% positive cells. ELISA screenings were performed with 64 new monoclonal antibodies on 5 different individual botryllid ascidian colonies (B. schlosseri, Botrylloides). The positive antibodies in this panel identified a large number of different antigenic determinants, some of which distinguish Botryllus versus Botrylloides colonies, and other, different colonies within these two species, or different cell types within tissues, embryos, and buds of individual colonies. Only 21 monoclonal antibodies tested positive with all colonies. Cross-reactivity with at least one Botrylloides colony was recorded in 49 hybridomas that identified Botryllus cells. This wide panel of monoclonal antibodies is the first such detailed set of monoclonals available for studies on botryllid ascidians.     

A Shift from Cellular to Humoral Responses Contributes to Innate Immune Memory in the Vector Snail Biomphalaria glabrata

Discoveries made over the past ten years have provided evidence that invertebrate antiparasitic responses may be primed in a sustainable manner, leading to the failure of a secondary encounter with the same pathogen. This phenomenon called “immune priming” or "innate immune memory" was mainly phenomenological. The demonstration of this process remains to be obtained and the underlying mechanisms remain to be discovered and exhaustively tested with rigorous functional and molecular methods, to eliminate all alternative explanations. In order to achieve this ambitious aim, the present study focuses on the Lophotrochozoan snail, Biomphalaria glabrata, in which innate immune memory was recently reported. We provide herein the first evidence that a shift from a cellular immune response (encapsulation) to a humoral immune response (biomphalysin) occurs during the development of innate memory. The molecular characterisation of this process in Biomphalaria/Schistosoma system was undertaken to reconcile mechanisms with phenomena, opening the way to a better comprehension of innate immune memory in invertebrates. This prompted us to revisit the artificial dichotomy between innate and memory immunity in invertebrate systems.     

A trade-off switch of two immunological memories in Caenorhabditis elegans reinfected by bacterial pathogens

Recent studies have suggested that innate immune responses exhibit characteristics associated with memory linked to modulations in both vertebrates and invertebrates. However, the diverse evolutionary paths taken, particularly within the invertebrate taxa, should lead to similarly diverse innate immunity memory processes. Our understanding of innate immune memory in invertebrates primarily comes from studies of the fruit fly Drosophila melanogaster, the generality of which is unclear. Caenorhabditis elegans typically inhabits soil harboring a variety of fatal microbial pathogens; for this invertebrate, the innate immune system and aversive behavior are the major defensive strategies against microbial infection. However, their characteristics of immunological memory remains infantile. Here we discovered an immunological memory that promoted avoidance and suppressed innate immunity during reinfection with bacteria, which we revealed to be specific to the previously exposed pathogens. During this trade-off switch of avoidance and innate immunity, the chemosensory neurons AWB and ADF modulated production of serotonin and dopamine, which in turn decreased expression of the innate immunity-associated genes and led to enhanced avoidance via the downstream insulin-like pathway. Therefore, our current study profiles the immune memories during C. elegans reinfected by pathogenic bacteria and further reveals that the chemosensory neurons, the neurotransmitter(s), and their associated molecular signaling pathways are responsible for a trade-off switch between the two immunological memories.     

Sequencing,Annotation, and Characterization of the Influenza Ferret Infectome

Ferrets have become an indispensable tool in the understanding of influenza virus virulence and pathogenesis. Furthermore, ferrets are the preferred preclinical model for influenza vaccine and therapeutic testing. Here we characterized the influenza infectome during the different stages of the infectious process in ferrets with and without prior specific immunity to influenza. RNA from lung tissue and lymph nodes from infected and naïve animals was subjected to next-generation sequencing, followed by de novo data assembly and annotation of the resulting sequences; this process generated a library comprising 13,202 ferret mRNAs. Gene expression profiles during pandemic H1N1 (pdmH1N1) influenza virus infection were analyzed by digital gene expression and solid support microarrays. As expected during primary infection, innate immune responses were triggered in the lung tissue; meanwhile, in the lymphoid tissue, genes encoding antigen presentation and maturation of effector cells of adaptive immunity increased dramatically. After 5 days postinfection, the innate immune gene expression was replaced by the adaptive immune response, which correlates with viral clearance. Reinfection with homologous pandemic influenza virus resulted in a diminished innate immune response, early adaptive immune gene regulation, and a reduction in clinical severity. The fully annotated ferret infectome will be a critical aid to the understanding of the molecular events that regulate disease severity and host-influenza virus interactions among seasonal, pandemic, and highly pathogenic avian influenzas.     

The immune responses in Chinese mitten crab Eriocheir sinensis challenged with double-stranded RNA

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis.     

Not like Botryllus: indiscriminate post-metamorphic fusion in a compound ascidian

Fusion trials between metamorphs of the aplousobranch compound ascidian Diplosoma listerianum indicated that chimera formation was not dependent on relatedness. Similar, high rates of union were observed between full siblings, half siblings, unrelated individuals from the same population, and individuals from two geographically distant localities. This is in contrast to the well-studied ascidian genus Botryllus, in which a highly polymorphic allorecognition system governing the fusion–non-fusion reaction (colony specificity) largely limits fusion to close relatives. Fusion in Botryllus establishes a vascular chimera throughout which stem cells may circulate, promoting cell lineage competition between the fusion partners. The restriction of fusion to close kin in Botryllus is thought to reduce the inclusive fitness costs of competitive interactions between cell lineages within the chimera. In contrast to Botryllus, modules (zooids) of a D. listerianum colony are not interlinked by blood vessels, seemingly precluding the exchange of stem cells. The apparent absence of strict colony specificity in D. listerianum is thus in keeping with the predictions of the Botryllus model for the maintenance of allorecognition polymorphism. However, colony specificity has been reported in other species of aplousobranch ascidian that also lack a common vascular system. In these, the threat of migrating blood-borne stem cells cannot be responsible for the presence of colony specificity. One possibility, requiring experimental investigation, is that stem cells could perhaps migrate between zooids by another route, such as through the matrix of the colonial tunic. Even in the absence of stem cell exchange, cheating on the costs of colony maintenance and defence could also produce selective forces favouring colony specificity. In compound ascidians, this could involve unequal contribution to extrazooidal structures, principally the tunic and related tissues. This consideration seems potentially relevant to the lack of discrimination during fusion in D. listerianum, since extrazooidal somatic investment in this species appears minimal, severely limiting the scope for this other form of cheating. The various possible modes of exploitative interaction between fused colonies are not mutually exclusive, and offer fundamentally similar explanations for colony specificity. If none of them can be shown to occur in non-botryllid species possessing colony specificity, the generality of the Botryllus model may require re-evaluation.     

Gene expression associated with white syndromes in a reef building coral,Acropora hyacinthus

Background

Corals are capable of launching diverse immune defenses at the site of direct contact with pathogens, but the molecular mechanisms of this activity and the colony-wide effects of such stressors remain poorly understood. Here we compared gene expression profiles in eight healthy Acropora hyacinthus colonies against eight colonies exhibiting tissue loss commonly associated with white syndromes, all collected from a natural reef environment near Palau. Two types of tissues were sampled from diseased corals: visibly affected and apparently healthy.

Results

Tag-based RNA-Seq followed by weighted gene co-expression network analysis identified groups of co-regulated differentially expressed genes between all health states (disease lesion, apparently healthy tissues of diseased colonies, and fully healthy). Differences between healthy and diseased tissues indicate activation of several innate immunity and tissue repair pathways accompanied by reduced calcification and the switch towards metabolic reliance on stored lipids. Unaffected parts of diseased colonies, although displaying a trend towards these changes, were not significantly different from fully healthy samples. Still, network analysis identified a group of genes, suggestive of altered immunity state, that were specifically up-regulated in unaffected parts of diseased colonies.

Conclusions

Similarity of fully healthy samples to apparently healthy parts of diseased colonies indicates that systemic effects of white syndromes on A. hyacinthus are weak, which implies that the coral colony is largely able to sustain its physiological performance despite disease. The genes specifically up-regulated in unaffected parts of diseased colonies, instead of being the consequence of disease, might be related to the originally higher susceptibility of these colonies to naturally occurring white syndromes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1540-2) contains supplementary material, which is available to authorized users.     

Specific Gene Expression Responses to Parasite Genotypes Reveal Redundancy of Innate Immunity in Vertebrates

Vertebrate innate immunity is the first line of defense against an invading pathogen and has long been assumed to be largely unspecific with respect to parasite/pathogen species. However, recent phenotypic evidence suggests that immunogenetic variation, i.e. allelic variability in genes associated with the immune system, results in host-parasite genotype-by-genotype interactions and thus specific innate immune responses. Immunogenetic variation is common in all vertebrate taxa and this reflects an effective immunological function in complex environments. However, the underlying variability in host gene expression patterns as response of innate immunity to within-species genetic diversity of macroparasites in vertebrates is unknown. We hypothesized that intra-specific variation among parasite genotypes must be reflected in host gene expression patterns. Here we used high-throughput RNA-sequencing to examine the effect of parasite genotypes on gene expression patterns of a vertebrate host, the three-spined stickleback (Gasterosteus aculeatus). By infecting naïve fish with distinct trematode genotypes of the species Diplostomum pseudospathaceum we show that gene activity of innate immunity in three-spined sticklebacks depended on the identity of an infecting macroparasite genotype. In addition to a suite of genes indicative for a general response against the trematode we also find parasite-strain specific gene expression, in particular in the complement system genes, despite similar infection rates of single clone treatments. The observed discrepancy between infection rates and gene expression indicates the presence of alternative pathways which execute similar functions. This suggests that the innate immune system can induce redundant responses specific to parasite genotypes.     

Expression of putative immune response genes during early ontogeny in the coral Acropora millepora

Background

Corals, like many other marine invertebrates, lack a mature allorecognition system in early life history stages. Indeed, in early ontogeny, when corals acquire and establish associations with various surface microbiota and dinoflagellate endosymbionts, they do not efficiently distinguish between closely and distantly related individuals from the same population. However, very little is known about the molecular components that underpin allorecognition and immunity responses or how they change through early ontogeny in corals.

Methodology/Principal Findings

Patterns in the expression of four putative immune response genes (apextrin, complement C3, and two CELIII type lectin genes) were examined in juvenile colonies of Acropora millepora throughout a six-month post-settlement period using quantitative real-time PCR (qPCR). Expression of a CELIII type lectin gene peaked in the fourth month for most of the coral juveniles sampled and was significantly higher at this time than at any other sampling time during the six months following settlement. The timing of this increase in expression levels of putative immune response genes may be linked to allorecognition maturation which occurs around this time in A.millepora. Alternatively, the increase may represent a response to immune challenges, such as would be involved in the recognition of symbionts (such as Symbiodinium spp. or bacteria) during winnowing processes as symbioses are fine-tuned.

Conclusions/Significance

Our data, although preliminary, are consistent with the hypothesis that lectins may play an important role in the maturation of allorecognition responses in corals. The co-expression of lectins with apextrin during development of coral juveniles also raises the possibility that these proteins, which are components of innate immunity in other invertebrates, may influence the innate immune systems of corals through a common pathway or system. However, further studies investigating the expression of these genes in alloimmune-challenged corals are needed to further clarify emerging evidence of a complex innate immunity system in corals.     

Proteolysis of Human Thrombin Generates Novel Host Defense Peptides

The coagulation system is characterized by the sequential and highly localized activation of a series of serine proteases, culminating in the conversion of fibrinogen into fibrin, and formation of a fibrin clot. Here we show that C-terminal peptides of thrombin, a key enzyme in the coagulation cascade, constitute a novel class of host defense peptides, released upon proteolysis of thrombin in vitro, and detected in human wounds in vivo. Under physiological conditions, these peptides exert antimicrobial effects against Gram-positive and Gram-negative bacteria, mediated by membrane lysis, as well as immunomodulatory functions, by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, they are protective against P. aeruginosa sepsis, as well as lipopolysaccharide-induced shock. Moreover, the thrombin-derived peptides exhibit helical structures upon binding to lipopolysaccharide and can also permeabilize liposomes, features typical of “classical” helical antimicrobial peptides. These findings provide a novel link between the coagulation system and host-defense peptides, two fundamental biological systems activated in response to injury and microbial invasion.     

Insect Innate Immunity Database (IIID): An Annotation Tool for Identifying Immune Genes in Insect Genomes

The innate immune system is an ancient component of host defense. Since innate immunity pathways are well conserved throughout many eukaryotes, immune genes in model animals can be used to putatively identify homologous genes in newly sequenced genomes of non-model organisms. With the initiation of the “i5k” project, which aims to sequence 5,000 insect genomes by 2016, many novel insect genomes will soon become publicly available, yet few annotation resources are currently available for insects. Thus, we developed an online tool called the Insect Innate Immunity Database (IIID) to provide an open access resource for insect immunity and comparative biology research (http://www.vanderbilt.edu/IIID). The database provides users with simple exploratory tools to search the immune repertoires of five insect models (including Nasonia), spanning three orders, for specific immunity genes or genes within a particular immunity pathway. As a proof of principle, we used an initial database with only four insect models to annotate potential immune genes in the parasitoid wasp genus Nasonia. Results specify 306 putative immune genes in the genomes of N. vitripennis and its two sister species N. giraulti and N. longicornis. Of these genes, 146 were not found in previous annotations of Nasonia immunity genes. Combining these newly identified immune genes with those in previous annotations, Nasonia possess 489 putative immunity genes, the largest immune repertoire found in insects to date. While these computational predictions need to be complemented with functional studies, the IIID database can help initiate and augment annotations of the immune system in the plethora of insect genomes that will soon become available.     

No Genetic Tradeoffs between Hygienic Behaviour and Individual Innate Immunity in the Honey Bee,Apis mellifera

Many animals have individual and social mechanisms for combating pathogens. Animals may exhibit short-term physiological tradeoffs between social and individual immunity because the latter is often energetically costly. Genetic tradeoffs between these two traits can also occur if mutations that enhance social immunity diminish individual immunity, or vice versa. Physiological tradeoffs between individual and social immunity have been previously documented in insects, but there has been no study of genetic tradeoffs involving these traits. There is strong evidence that some genes influence both innate immunity and behaviour in social insects – a prerequisite for genetic tradeoffs. Quantifying genetic tradeoffs is critical for understanding the evolution of immunity in social insects and for devising effective strategies for breeding disease-resistant pollinator populations. We conducted two experiments to test the hypothesis of a genetic tradeoff between social and individual immunity in the honey bee, Apis mellifera. First, we estimated the relative contribution of genetics to individual variation in innate immunity of honey bee workers, as only heritable traits can experience genetic tradeoffs. Second, we examined if worker bees with hygienic sisters have reduced individual innate immune response. We genotyped several hundred workers from two colonies and found that patriline genotype does not significantly influence the antimicrobial activity of a worker’s hemolymph. Further, we did not find a negative correlation between hygienic behaviour and the average antimicrobial activity of a worker’s hemolymph across 30 honey bee colonies. Taken together, our work indicates no genetic tradeoffs between hygienic behaviour and innate immunity in honey bees. Our work suggests that using artificial selection to increase hygienic behaviour of honey bee colonies is not expected to concurrently compromise individual innate immunity of worker bees.     

Sociogenomics of Cooperation and Conflict during Colony Founding in the Fire Ant Solenopsis invicta

One of the fundamental questions in biology is how cooperative and altruistic behaviors evolved. The majority of studies seeking to identify the genes regulating these behaviors have been performed in systems where behavioral and physiological differences are relatively fixed, such as in the honey bee. During colony founding in the monogyne (one queen per colony) social form of the fire ant Solenopsis invicta, newly-mated queens may start new colonies either individually (haplometrosis) or in groups (pleometrosis). However, only one queen (the “winner”) in pleometrotic associations survives and takes the lead of the young colony while the others (the “losers”) are executed. Thus, colony founding in fire ants provides an excellent system in which to examine the genes underpinning cooperative behavior and how the social environment shapes the expression of these genes. We developed a new whole genome microarray platform for S. invicta to characterize the gene expression patterns associated with colony founding behavior. First, we compared haplometrotic queens, pleometrotic winners and pleometrotic losers. Second, we manipulated pleometrotic couples in order to switch or maintain the social ranks of the two cofoundresses. Haplometrotic and pleometrotic queens differed in the expression of genes involved in stress response, aging, immunity, reproduction and lipid biosynthesis. Smaller sets of genes were differentially expressed between winners and losers. In the second experiment, switching social rank had a much greater impact on gene expression patterns than the initial/final rank. Expression differences for several candidate genes involved in key biological processes were confirmed using qRT-PCR. Our findings indicate that, in S. invicta, social environment plays a major role in the determination of the patterns of gene expression, while the queen''s physiological state is secondary. These results highlight the powerful influence of social environment on regulation of the genomic state, physiology and ultimately, social behavior of animals.     

Binding of Glycoprotein Srr1 of Streptococcus agalactiae to Fibrinogen Promotes Attachment to Brain Endothelium and the Development of Meningitis

The serine-rich repeat glycoprotein Srr1 of Streptococcus agalactiae (GBS) is thought to be an important adhesin for the pathogenesis of meningitis. Although expression of Srr1 is associated with increased binding to human brain microvascular endothelial cells (hBMEC), the molecular basis for this interaction is not well defined. We now demonstrate that Srr1 contributes to GBS attachment to hBMEC via the direct interaction of its binding region (BR) with human fibrinogen. When assessed by Far Western blotting, Srr1 was the only protein in GBS extracts that bound fibrinogen. Studies using recombinant Srr1-BR and purified fibrinogen in vitro confirmed a direct protein-protein interaction. Srr1-BR binding was localized to amino acids 283–410 of the fibrinogen Aα chain. Structural predictions indicated that the conformation of Srr1-BR is likely to resemble that of SdrG and other related staphylococcal proteins that bind to fibrinogen through a “dock, lock, and latch” mechanism (DLL). Deletion of the predicted latch domain of Srr1-BR abolished the interaction of the BR with fibrinogen. In addition, a mutant GBS strain lacking the latch domain exhibited reduced binding to hBMEC, and was significantly attenuated in an in vivo model of meningitis. These results indicate that Srr1 can bind fibrinogen directly likely through a DLL mechanism, which has not been described for other streptococcal adhesins. This interaction was important for the pathogenesis of GBS central nervous system invasion and subsequent disease progression.     

Expression of maize prolamins in Escherichia coli

We have constructed a cDNA expression library of developing corn (Zea mays L.) endosperm using plasmid pUC8 as vector and Escherichia coli strain DH1 as host. The expression library was screened with non-radioactive immunological probes to detect the expression of gamma-zein and alpha-zein. When anti-gamma-zein antibody was used as the probe, 23 colonies gave positive reactions. The lengths of cDNA inserts of the 23 colonies were found to be 250–900 base pairs. When anti-alpha zein antibody was used, however, fewer colonies gave positive reactions. The library was also screened by colony-hybridization with ³²P-labeled DNA probes. Based on immunological and hybridization screening of the library and other evidence, we conclude that alpha-zein was either toxic to E. coli cells or rapidly degraded whereas gamma-zein and its fragments were readily expressed.