Cold-blooded vertebrates evolved organized germinal center-like structures.

Germinal centers (GCs) or analogous secondary lymphoid microstructures (SLMs) are thought to have evolved in endothermic species. However, living representatives of their ectothermic ancestors can mount potent secondary antibody responses upon infection or immunization, despite the apparent lack of SLMs in these cold-blooded vertebrates. How and where adaptive immune responses are induced in ectothermic species in the absence of GCs or analogous SLMs remain poorly understood. Here, we infected a teleost fish (trout) with the parasite Ichthyophthirius multifiliis (Ich) and identified the formation of large aggregates of highly proliferating IgM+ B cells and CD4+ T cells, contiguous to splenic melanomacrophage centers (MMCs). Most of these MMC-associated lymphoid aggregates (M-LAs) contained numerous antigen (Ag)-specific B cells. Analysis of the IgM heavy chain CDR3 repertoire of microdissected splenic M-LAs and non-M-LA areas revealed that the most frequent B cell clones induced after Ich infection were highly shared only within the M-LAs of infected animals. These M-LAs represented highly polyclonal SLMs in which Ag-specific B cell clonal expansion occurred. M-LA-associated B cells expressed high levels of activation-induced cytidine deaminase and underwent significant apoptosis, and somatic hypermutation of Igµ genes occurred prevalently in these cells. Our findings demonstrate that ectotherms evolved organized SLMs with GC-like roles. Moreover, our results also point to primordially conserved mechanisms by which M-LAs and mammalian polyclonal GCs develop and function.

Secretory IgM (sIgM) is an ancient master regulator of microbiota homeostasis and metabolism.

The co-evolution between secretory immunoglobulins (sIgs) and microbiota began with the emergence of IgM over half a billion years ago. Yet, IgM function in vertebrates is mostly associated with systemic immunity against pathogens. sIgA and sIgT are the only sIgs known to be required in the control of microbiota homeostasis in warm- and cold-blooded vertebrates respectively. Recent studies have shown that sIgM coats a large proportion of the gut microbiota of humans and teleost fish, thus suggesting an ancient and conserved relationship between sIgM and microbiota early in vertebrate evolution. To test this hypothesis, we temporarily and selectively depleted IgM from rainbow trout, an old bony fish species. IgM depletion resulted in a drastic reduction in microbiota IgM coating levels and losses in gutassociated bacteria. These were accompanied by bacterial translocation, severe gut tissue damage, inflammation and dysbiosis predictive of metabolic shifts. Furthermore, depletion of IgM resulted in body weight loss and lethality in an experimental colitis model. Recovery of sIgM to physiological levels restores tissue barrier integrity, while microbiome homeostasis and their predictive metabolic capabilities are not fully restituted. Our findings uncover a previously unrecognized role of sIgM as an ancient master regulator of microbiota homeostasis and metabolism and challenge the current paradigm that sIgA and sIgT are the key vertebrate sIgs regulating microbiome homeostasis. One-Sentence Summary: IgM, the most ancient and conserved immunoglobulin in jawed vertebrates, is required for successful symbiosis with the gut microbiota.

CD4 and LAG-3 from sharks to humans: related molecules with motifs for opposing functions.

CD4 and LAG-3 are related molecules that are receptors for MHC class II molecules. Their major functional differences are situated in their cytoplasmic tails, in which CD4 has an activation motif and LAG-3 an inhibitory motif. Here, we identify shark LAG-3 and show that a previously identified shark CD4-like gene has a genomic location, expression pattern, and motifs similar to CD4 in other vertebrates. In nurse shark (Ginglymostoma cirratum) and cloudy catshark (Scyliorhinus torazame), the highest CD4 expression was consistently found in the thymus whereas such was not the case for LAG-3. Throughout jawed vertebrates, the CD4 cytoplasmic tail possesses a Cx(C/H) motif for binding kinase LCK, and the LAG-3 cytoplasmic tail possesses (F/Y)xxL(D/E) including the previously determined FxxL inhibitory motif resembling an immunoreceptor tyrosine-based inhibition motif (ITIM). On the other hand, the acidic end of the mammalian LAG-3 cytoplasmic tail, which is believed to have an inhibitory function as well, was acquired later in evolution. The present study also identified CD4-1, CD4-2, and LAG-3 in the primitive ray-finned fishes bichirs, sturgeons, and gars, and experimentally determined these sequences for sterlet sturgeon (Acipenser ruthenus). Therefore, with CD4-1 and CD4-2 already known in teleosts (modern ray-finned fish), these two CD4 lineages have now been found within all major clades of ray-finned fish. Although different from each other, the cytoplasmic tails of ray-finned fish CD4-1 and chondrichthyan CD4 not only contain the Cx(C/H) motif but also an additional highly conserved motif which we expect to confer a function. Thus, although restricted to some species and gene copies, in evolution both CD4 and LAG-3 molecules appear to have acquired functional motifs besides their canonical Cx(C/H) and ITIM-like motifs, respectively. The presence of CD4 and LAG-3 molecules with seemingly opposing functions from the level of sharks, the oldest living vertebrates with a human-like adaptive immune system, underlines their importance for the jawed vertebrate immune system. It also emphasizes the general need of the immune system to always find a balance, leading to trade-offs, between activating and inhibiting processes.

Cutting Edge: Neutralizing Public Antibody Responses Are an Ancient Form of Defense Conserved in Fish and Mammals.

The repertoire of Abs is generated by genomic rearrangements during B cell differentiation. Although V(D)J rearrangements lead to repertoires mostly different between individuals, recent studies have shown that they contain a substantial fraction of overrepresented and shared "public" clones. We previously reported a strong public IgHµ clonotypic response against the rhabdovirus viral hemorrhagic septicemia virus in a teleost fish. In this study, we identified an IgL chain associated with this public response that allowed us to characterize its functionality. We show that this public Ab response has a potent neutralizing capacity that is typically associated with host protection during rhabdovirus infections. We also demonstrate that the public response is not restricted to a particular trout isogenic line but expressed in multiple genetic backgrounds and may be used as a marker of successful vaccination. Our work reveals that public B cell responses producing generic Abs constitute a mechanism of protection against infection conserved across vertebrates.

Mucosal immunoglobulins of teleost fish: A decade of advances.

Immunoglobulins (Igs) are complex glycoproteins that play critical functions in innate and adaptive immunity of all jawed vertebrates. Given the unique characteristics of mucosal barriers, secretory Igs (sIgs) have specialized to maintain homeostasis and keep pathogens at bay at mucosal tissues from fish to mammals. In teleost fish, the three main IgH isotypes, IgM, IgD and IgT/Z can be found in different proportions at the mucosal secretions of the skin, gills, gut, nasal, buccal, and pharyngeal mucosae. Similar to the role of mammalian IgA, IgT plays a predominant role in fish mucosal immunity. Recent studies in IgT have illuminated the primordial role of sIgs in both microbiota homeostasis and pathogen control at mucosal sites. Ten years ago, IgT was discovered to be an immunoglobulin class specialized in mucosal immunity. Aiming at this 10-year anniversary, the goal of this review is to summarize the current status of the field of fish Igs since that discovery, while identifying knowledge gaps and future avenues that will move the field forward in both basic and applied science areas.

Prevailing Role of Mucosal Igs and B Cells in Teleost Skin Immune Responses to Bacterial Infection.

The skin of vertebrates is the outermost organ of the body and serves as the first line of defense against external aggressions. In contrast to mammalian skin, that of teleost fish lacks keratinization and has evolved to operate as a mucosal surface containing a skin-associated lymphoid tissue (SALT). Thus far, IgT representing the prevalent Ig in SALT have only been reported upon infection with a parasite. However, very little is known about the types of B cells and Igs responding to bacterial infection in the teleost skin mucosa, as well as the inductive or effector role of the SALT in such responses. To address these questions, in this study, we analyzed the immune response of trout skin upon infection with one of the most widespread fish skin bacterial pathogens, Flavobacterium columnare This pathogen induced strong skin innate immune and inflammatory responses at the initial phases of infection. More critically, we found that the skin mucus of fish having survived the infection contained significant IgT- but not IgM- or IgD-specific titers against the bacteria. Moreover, we demonstrate the local proliferation and production of IgT+ B cells and specific IgT titers, respectively, within the SALT upon bacterial infection. Thus, our findings represent the first demonstration that IgT is the main Ig isotype induced by the skin mucosa upon bacterial infection and that, because of the large surface of the skin, its SALT probably represents a prominent IgT-inductive site in fish.

Specialization of mucosal immunoglobulins in pathogen control and microbiota homeostasis occurred early in vertebrate evolution.

Although mammalian secretory immunoglobulin A (sIgA) targets mucosal pathogens for elimination, its interaction with the microbiota also enables commensal colonization and homeostasis. This paradoxical requirement in the control of pathogens versus microbiota raised the question of whether mucosal (secretory) Igs (sIgs) evolved primarily to protect mucosal surfaces from pathogens or to maintain microbiome homeostasis. To address this central question, we used a primitive vertebrate species (rainbow trout) in which we temporarily depleted its mucosal Ig (sIgT). Fish devoid of sIgT became highly susceptible to a mucosal parasite and failed to develop compensatory IgM responses against it. IgT depletion also induced a profound dysbiosis marked by the loss of sIgT-coated beneficial taxa, expansion of pathobionts, tissue damage, and inflammation. Restitution of sIgT levels in IgT-depleted fish led to a reversal of microbial translocation and tissue damage, as well as to restoration of microbiome homeostasis. Our findings indicate that specialization of sIgs in pathogen and microbiota control occurred concurrently early in evolution, thus revealing primordially conserved principles under which primitive and modern sIgs operate in the control of microbes at mucosal surfaces.

Convergent Evolution of Mucosal Immune Responses at the Buccal Cavity of Teleost Fish.

The buccal mucosa (BM) is a critical first line of defense in terrestrial animals. To gain further insights into the evolutionary origins and primordial roles of BM in teleosts here we show that rainbow trout, a teleost fish, contains a diffuse mucosal associated lymphoid tissue (MALT) within its buccal cavity. Upon parasite infection, a fish immunoglobulin specialized in mucosal immunity (sIgT) was induced to a high degree, and parasite-specific sIgT responses were mainly detected in the buccal mucus. Moreover, we show that the trout buccal microbiota is prevalently coated with sIgT. Overall our findings revealed that the MALT is present in the BM of a non-tetrapod species. As fish IgT and mucus-producing cells are evolutionarily unrelated to mammalian IgA and salivary glands, respectively, our findings indicate that mucosal immune responses in the BM of teleost fish and tetrapods evolved through a process of convergent evolution.

Pichia pastoris yeast as a vehicle for oral vaccination of larval and adult teleosts.

Oral vaccination is of major interest because it can be used for mass vaccination of fish of various size and age. Given that their administration is relatively easy and stress-free, oral vaccines have both economic and animal welfare benefits. Yet, mostly due to their limited efficacy, only very few oral vaccines are available to aquaculture industry. Here we present a method for oral vaccine delivery based on the yeast Pichia pastoris. We could express a model antigen, green fluorescent protein (GFP), in this yeast and subsequently show delivery of the GFP protein to the intestine of juvenile flounder or adult carp and trout. We tested this approach in several commercially-relevant fish species, from juvenile to adult stage. To test the oral delivery of antigen to larval fish, the GFP-expressing Pichia pastoris was first fed to planktonic crustacean Daphnia or rotifers that served as 'bioencapsulation vehicles' and afterwards, fed to flounder larvae. Again, we could show delivery of intact GFP protein to the intestine. In rainbow trout, the orally-administered GFP-expressing yeast elicited a rapid local innate immune response in the intestine and a subsequent systemic response in the spleen. Our results show that Pichia pastoris is a good vehicle for oral antigen delivery and that it can be used in non-encapsulated form for older fish or in bioencapsulated form for larval fish. We discuss the immunomodulatory properties of the yeast itself, and its potential to enhance local immune responses and act as an adjuvant.

Author Correction: Profiling Atlantic salmon B cell populations: CpG-mediated TLR-ligation enhances IgM secretion and modulates immune gene expression.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Profiling Atlantic salmon B cell populations: CpG-mediated TLR-ligation enhances IgM secretion and modulates immune gene expression.

While TLR-activated pathways are key regulators of B cell responses in mammals, their impact on teleost B cells are scarcely addressed. Here, the potential of Atlantic salmon B cells to respond to TLR ligands was shown by demonstrating a constitutive expression of nucleic-acid sensing TLRs in magnetic sorted IgM+ cells. Of the two receptors recognizing CpG in teleosts, tlr9 was the dominating receptor with over ten-fold higher expression than tlr21. Upon CpG-stimulation, IgM secretion increased for head kidney (HK) and splenic IgM+ cells, while blood B cells were marginally affected. The results suggest that CpG directly affects salmon B cells to differentiate into antibody secreting cells (ASCs). IgM secretion was also detected in the non-treated controls, again with the highest levels in the HK derived population, signifying that persisting ASCs are present in this tissue. In all tissues, the IgM+ cells expressed high MHCII levels, suggesting antigen-presenting functions. Upon CpG-treatment the co-stimulatory molecules cd83 and cd40 were upregulated, while cd86 was down-regulated under the same conditions. Finally, ifna1 was upregulated upon CpG-stimulation in all tissues, while a restricted upregulation was evident for ifnb, proposing that salmon IgM+ B cells exhibit a type I IFN-response.

Perspective on the Development and Validation of Ab Reagents to Fish Immune Proteins for the Correct Assessment of Immune Function.

Understanding of immune function in humans and model organisms, such as mice, has advanced in the last few decades because of technological breakthroughs and availability of reagents. While novel genomic technologies have helped to increase knowledge of many aspects of immunology, most developments in immunology have occurred because of the availability of antibodies to identify and sort different cell types, as well as to identify and quantify the protein products of cells. Unfortunately, many studies performed in fish make use of poorly characterized antibody reagents that may affect the conclusions of those studies. In light of this, we would like to offer some insight and discussion points based on our research experience on the strategies and techniques that are required for proper validation of antibody reagents to fish immune molecules. Our main goal is to encourage a much needed discussion in our field to foster the use of correctly validated reagents that enable the study of fish immune function.

Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress.

Ebola virus (EBOV) is a member of the Filoviridae family and the cause of hemorrhagic fever outbreaks. The EBOV VP40 (eVP40) matrix protein is the main driving force for virion assembly and budding. Indeed, expression of eVP40 alone in mammalian cells results in the formation and budding of virus-like particles (VLPs) which mimic the budding process and morphology of authentic, infectious EBOV. To complete the budding process, eVP40 utilizes its PPXY L-domain motif to recruit a specific subset of host proteins containing one or more modular WW domains that then function to facilitate efficient production and release of eVP40 VLPs. In this report, we identified additional host WW-domain interactors by screening for potential interactions between mammalian proteins possessing one or more WW domains and WT or PPXY mutant peptides of eVP40. We identified the HECT family E3 ubiquitin ligase WWP1 and all four of its WW domains as strong interactors with the PPXY motif of eVP40. The eVP40-WWP1 interaction was confirmed by both peptide pulldown and coimmunoprecipitation assays, which also demonstrated that modular WW domain 1 of WWP1 was most critical for binding to eVP40. Importantly, the eVP40-WWP1 interaction was found to be biologically relevant for VLP budding since (i) small interfering RNA (siRNA) knockdown of endogenous WWP1 resulted in inhibition of eVP40 VLP egress, (ii) coexpression of WWP1 and eVP40 resulted in ubiquitination of eVP40 and a subsequent increase in eVP40 VLP egress, and (iii) an enzymatically inactive mutant of WWP1 (C890A) did not ubiquitinate eVP40 or enhance eVP40 VLP egress. Last, our data show that ubiquitination of eVP40 by WWP1 enhances egress of VLPs and concomitantly decreases cellular levels of higher-molecular-weight oligomers of eVP40. In sum, these findings contribute to our fundamental understanding of the functional interplay between host E3 ligases, ubiquitination, and regulation of EBOV VP40-mediated egress.IMPORTANCE Ebola virus (EBOV) is a high-priority, emerging human pathogen that can cause severe outbreaks of hemorrhagic fever with high mortality rates. As there are currently no approved vaccines or treatments for EBOV, a better understanding of the biology and functions of EBOV-host interactions that promote or inhibit viral budding is warranted. Here, we describe a physical and functional interaction between EBOV VP40 (eVP40) and WWP1, a host E3 ubiquitin ligase that ubiquitinates VP40 and regulates VLP egress. This viral PPXY-host WW domain-mediated interaction represents a potential new target for host-oriented inhibitors of EBOV egress.

Rainbow trout (Oncorhynchus mykiss) secretory component binds to commensal bacteria and pathogens.

Commensal bacteria co-exist on the mucosal surfaces of all vertebrates. The host's mucosal immune system must tolerate commensals while fighting pathogens. One of the mechanisms used by the mucosal immune system to maintain homeostasis is the secretion of immunoglobulins (Igs) across epithelial barriers, which is achieved via the polymeric immunoglobulin receptor (pIgR). Rainbow trout pIgR is known to transport IgT and IgM across epithelia. However, other biological functions for trout pIgR or trout secretory component (tSC) remain unknown. This study investigates the interaction of tSC with commensal bacteria, pathogenic bacteria and a fungal pathogen. Our results show that the majority of trout skin and gut bacteria are coated in vivo by tSC. In vitro, tSC present in mucus coats trout commensal isolates such as Microbacterium sp., Staphylococcus warneri, Flectobacillus major, Arthrobacter stackebrantii, and Flavobacterium sp. and the pathogens Vibrio anguillarum and Edwardsiella ictaluri with coating levels ranging from 8% to 70%. Moreover, we found that the majority of tSC is in free form in trout mucus and free tSC is able to directly bind bacteria. We propose that binding of free SC to commensal bacteria is a key and conserved mechanism for maintenance of microbial communities in vertebrate mucosal surfaces.

Novel Teleost CD4-Bearing Cell Populations Provide Insights into the Evolutionary Origins and Primordial Roles of CD4+ Lymphocytes and CD4+ Macrophages.

Tetrapods contain a single CD4 coreceptor with four Ig domains that likely arose from a primordial two-domain ancestor. Notably, teleost fish contain two CD4 genes. Like tetrapod CD4, CD4-1 of rainbow trout includes four Ig domains, whereas CD4-2 contains only two. Because CD4-2 is reminiscent of the prototypic two-domain CD4 coreceptor, we hypothesized that by characterizing the cell types bearing CD4-1 and CD4-2, we would shed light into the evolution and primordial roles of CD4-bearing cells. Using newly established mAbs against CD4-1 and CD4-2, we identified two bona-fide CD4(+) T cell populations: a predominant lymphocyte population coexpressing surface CD4-1 and CD4-2 (CD4 double-positive [DP]), and a minor subset expressing only CD4-2 (CD4-2 single-positive [SP]). Although both subsets produced equivalent levels of Th1, Th17, and regulatory T cell cytokines upon bacterial infection, CD4-2 SP lymphocytes were less proliferative and displayed a more restricted TCRß repertoire. These data suggest that CD4-2 SP cells represent a functionally distinct population and may embody a vestigial CD4(+) T cell subset, the roles of which reflect those of primeval CD4(+) T cells. Importantly, we also describe the first CD4(+) monocyte/macrophage population in a nonmammalian species. Of all myeloid subsets, we found the CD4(+) population to be the most phagocytic, whereas CD4(+) lymphocytes lacked this capacity. This study fills in an important gap in the knowledge of teleost CD4-bearing leukocytes, thus revealing critical insights into the evolutionary origins and primordial roles of CD4(+) lymphocytes and CD4(+) monocytes/macrophages.

Preferential combination between the light and heavy chain isotypes of fish immunoglobulins.

Immunoglobulin light chain (IgL) is necessary for the assembly of an Ig molecule, which plays important roles in the immune response. IgL genes were identified in various teleost species, but the basic functions of different IgL isotypes and the preferential combination between IgL and IgH (Ig heavy chain) isotypes remain unclear. In the current study, by EST database searching and cDNA cloning in rainbow trout, 8 IgL sequences were obtained, which could be classified into the IgLκF, IgLκG, IgLσ and IgLλ isotypes, respectively. Trout IgL isotypes were highly expressed in the immune-related tissues, and participated in the immune responses in spleen and gut by stimulation with LPS and poly (I:C). The results of FACS and LC-MS/MS indicated that the IgLκG and IgLσ isotypes preferentially bonded with the heavy chains of IgM and IgT, respectively, in trout B cells and serum. In addition, the genomic organization of trout IgL isotypes and the utilization of recombination signal sequences were studied.

B cells and their role in the teleost gut.

Mucosal surfaces are the main route of entry for pathogens in all living organisms. In the case of teleost fish, mucosal surfaces cover the vast majority of the animal. As these surfaces are in constant contact with the environment, fish are perpetually exposed to a vast number of pathogens. Despite the potential prevalence and variety of pathogens, mucosal surfaces are primarily populated by commensal non-pathogenic bacteria. Indeed, a fine balance between these two populations of microorganisms is crucial for animal survival. This equilibrium, controlled by the mucosal immune system, maintains homeostasis at mucosal tissues. Teleost fish possess a diffuse mucosa-associated immune system in the intestine, with B cells being one of the main responders. Immunoglobulins produced by these lymphocytes are a critical line of defense against pathogens and also prevent the entrance of commensal bacteria into the epithelium. In this review we will summarize recent literature regarding the role of B-lymphocytes and immunoglobulins in gut immunity in teleost fish, with specific focus on immunoglobulin isotypes and the microorganisms, pathogenic and non-pathogenic that interact with the immune system.

Mucosal immunoglobulins at respiratory surfaces mark an ancient association that predates the emergence of tetrapods.

Gas-exchange structures are critical for acquiring oxygen, but they also represent portals for pathogen entry. Local mucosal immunoglobulin responses against pathogens in specialized respiratory organs have only been described in tetrapods. Since fish gills are considered a mucosal surface, we hypothesized that a dedicated mucosal immunoglobulin response would be generated within its mucosa on microbial exposure. Supporting this hypothesis, here we demonstrate that following pathogen exposure, IgT(+) B cells proliferate and generate pathogen-specific IgT within the gills of fish, thus providing the first example of locally induced immunoglobulin in the mucosa of a cold-blooded species. Moreover, we demonstrate that gill microbiota is predominantly coated with IgT, thus providing previously unappreciated evidence that the microbiota present at a respiratory surface of a vertebrate is recognized by a mucosal immunoglobulin. Our findings indicate that respiratory surfaces and mucosal immunoglobulins are part of an ancient association that predates the emergence of tetrapods.

Induction of anti-inflammatory cytokine expression by IPNV in persistent infection.

Infectious Pancreatic Necrosis Virus (IPNV) is the agent of a well-characterized acute disease that produces a systemic infection and high mortality in farmed fish species but also persistent infection in surviving fish after outbreaks. Because viral persistence of susceptible mammal hosts appears to be associated with the modulation of anti-inflammatory cytokine expression, in this study we examined the expression levels of key pro- and anti-inflammatory cytokines in kidney and spleen of trout, as well as humoral immune response (IgM and IgT) during experimental persistent viral infection and in the acute phase of infection as a comparison. IPNV infection in rainbow trout resulted in a distinct profile of cytokine expression depending on the type of infection, acute or persistent. Levels of early pro-inflammatory cytokines, IL-1ß and IL-8, did not increase in the head kidney of the fish with persistent asymptomatic infection but increased in some of the symptomatic infected fish. The antiviral cytokine IFNα was not significantly induced in any of the infected fish groups. The level of expression of the Th1-related cytokine IL-12 was significantly higher in trout with persistent asymptomatic infection than in symptomatic fish. This was also accompanied by an increase in IFNγ. The anti-inflammatory cytokines IL-10 and TGF-ß1 had distinct expression profiles. While IL-10 expression increased in all infected fish, TGF-ß1 was only up-regulated in fish with persistent infection. All infected fish had significantly lower total IgM levels than the non-infected fish whereas IgT levels did not change. Specific and neutralizing antibodies against IPNV were not observed in acute and persistent infection except in the group of fish with the lowest degree of clinical signs. Interestingly, the lack of humoral immune response could be associated with the high expression of anti-inflammatory cytokines, which might inhibit antibody production. The balance between pro-inflammatory Th1 type cytokines and the regulatory cytokines could explain the high percentage of survival and the resolution of the inflammatory response in the IPNV-infected fish but also the establishment of viral persistence.