The coating of microbiota by secretory immunoglobulins (sIgs) determines which bacteria colonize the gut and influences bacterial metabolism. Previous work has identified sIgA and sIgT as mediators of gut homeostasis. However, sIgM coats a large proportion of the gut microbiota in humans and teleost fish, thus suggesting a conserved role of sIgM in microbiota homeostasis. Here, to investigate this hypothesis, we used the teleost rainbow trout as a model system. Depletion of IgM from trout resulted in severe microbiota-dependent gut tissue damage, body weight loss, bacterial translocation and gut dysbiosis. IgM depletion led also to alterations in microbiota-derived metabolites, including short-chain fatty acids and essential amino acids. Supporting a protective role for sIgM in the gut, high mortality of IgM-depleted fish occurred in an experimental colitis model as a result of severe systemic bacteraemia and septic shock. Our findings uncover sIgM as a previously unrecognized regulator of microbiota homeostasis and metabolism.

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.

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.

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.

Ebola (EBOV) and Marburg viruses (MARV) cause severe hemorrhagic fever associated with high mortality rates in humans. A better understanding of filovirus-host interactions that regulate the EBOV and MARV lifecycles can provide biological and mechanistic insight critical for therapeutic development. EBOV glycoprotein (eGP) and MARV glycoprotein (mGP) mediate entry into host cells primarily by actin-dependent macropinocytosis. Here, we identified actin-binding cytoskeletal crosslinking proteins filamin A (FLNa) and B (FLNb) as important regulators of both EBOV and MARV entry. We found that entry of pseudotype psVSV-RFP-eGP, infectious recombinant rVSV-eGP-mCherry, and live authentic EBOV and MARV was inhibited in filamin A knockdown (FLNaKD) cells, but was surprisingly enhanced in filamin B knockdown (FLNbKD) cells. Mechanistically, our findings suggest that differential regulation of macropinocytosis by FLNa and FLNb likely contributes to their specific effects on EBOV and MARV entry. This study is the first to identify the filamin family of proteins as regulators of EBOV and MARV entry. These findings may provide insight into the development of new countermeasures to prevent EBOV and MARV infections.

The air-filled organs (AOs) of vertebrates (lungs and swim bladders) have evolved unique functions (air-breathing or buoyancy control in water) to adapt to different environments. Thus far, immune responses to microbes in AOs have been described exclusively in the lungs of tetrapods. Similar to lungs, swim bladders (SBs) represent a mucosal surface, a feature that leads us to hypothesize a role for SB in immunity. In this study, we demonstrate that secretory IgT (sIgT) is the key SB immunoglobulin (Ig) responding to the viral challenge, and the only Ig involved in viral neutralization in that organ. In support of these findings, we found that the viral load of the SB from fish devoid of sIgT was much higher than that of control fish. Interestingly, similar to the lungs in mammals, the SB represents the mucosal surface in fish with the lowest content of microbiota. Moreover, sIgT is the main Ig class found coating their surface, suggesting a key role of this Ig in the homeostasis of the SB microbiota. In addition to the well-established role of SB in buoyancy control, our findings reveal a previously unrecognized function of teleost SB in adaptive mucosal immune responses upon pathogenic challenge, as well as a previously unidentified role of sIgT in antiviral defense. Overall, our findings indicate that despite the phylogenetic distance and physiological roles of teleost SB and mammalian lungs, they both have evolved analogous mucosal immune responses against microbes which likely originated independently through a process of convergent evolution.

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.

Ray-finned fishes comprise more than half the *60,000 known vertebrate species, and are pivotal to the functionality of aquatic ecosystems and success of global multibillion dollar industries. Understanding ray-finned fish immune systems is essential to predicting how species will respond to known or emergent pathogens as well as to the development of effective vaccines for aquaculture. However, the diversity of species, including in aquaculture, necessitates that immunology and translational medicine research groups investigating the immune system in one or a number of species employ a common language for describing homologous immune components. Unfortunately for Immunoglobulin (Ig) genes that encode antibodies, this has not been the case. Ig genes are restricted to jawed vertebrates (gnathostomes) with all lineages encoding common heavy chains IgM and IgD (aka IgW in cartilaginous fish, lungfish, and coelacanths). Before 2005, it was believed that bony fish encoded only IgM and IgD. In 2005, Hansen et al. described a new Ig heavy chain encoded within the rainbow trout heavy chain locus and named it IgT for ‘‘teleost.’’ However, during the

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.

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.

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.

Abstract Induction of adaptive immune responses in higher vertebrate species occur within organized lymphoid structures (e.g. lymph nodes, Peyer’s patches). It has been proposed that such structures emerged throughout evolutionary time with the goal to maximize encounters between antigens, antigens-presenting cells and B-T lymphocytes. Fish lack such structures and thus, it remains unknown how and where antigen-specific immunoglobulin responses are induced in these species. To understand how systemic immune responses are induced in teleost lymphoid organs, Rainbow Trout were immunized with several soluble protein antigens. Overall, our results identified the spleen as the major site for CD4+ T and IgM+ B cell proliferation in systemic lymphoid organs upon immunization. The proliferating splenic IgM+ B cells were frequently observed as clusters in the vicinity of melano-macrophage centers. Moreover, in these areas we observed aggregates of B and T lymphocytes with a loose organized structure reminiscent of the cellular architecture frequently associated with tertiary lymphoid organs. Laser dissection microdissection of these areas enabled us to evaluating the immunoglobulin IgM repertoires within these structures upon immunization. Critically, repertoire analysis identified processes of antigen-specific B cell clonal expansion. In conclusion, these data points to the previously unrecognized existence of primordial semi-organized lymphoid tissue in the spleen of teleost fish in which adaptive IgM immune responses are induced. Our findings provide strong evidence that the induction of antigen-specific immune responses in all bony vertebrates requires the formation of organized or semi-organized lymphoid structures.

Anaerococcus spp. are Gram-positive anaerobic cocci involved in human skin and soft-tissue infections, among other diseases. We report the first known case of bacteremia due to A. octavius, detected in an elderly patient. A 71-year-old woman with a history of breast cancer presented with fever and cognitive impairment for more than three days. A. octavius was isolated in blood cultures, and only resistance to clindamycin was reported. Her general condition improved after treatment with metronidazole and she was discharged.