Jonathan P. Rast

Inflammation is widely considered a hallmark of animal immune responses. In mammals, this process is mediated in part by pro-inflammatory cytokines of the interleukin 1 (IL-1) superfamily, which establish and support microenvironments that regulate immune cell activity. However, the evolutionary origins of IL-1 cytokines remain elusive, as homologous proteins have been difficult to confidently identify outside vertebrates using sequence-based approaches. Although IL-1 family members have been described in all vertebrate lineages, including cartilaginous and jawless fishes, their distribution and evolutionary relationships beyond these groups remain unclear. Here we applied protein structure prediction algorithms to identify genes encoding a previously unrecognized family of IL-1 proteins (termed ‘IL-1anc’ to reflect their ancestral evolutionary origin) with homologues throughout Eumetazoa. Here, using Petromyzon marinus (sea lamprey) and Strongylocentrotus purpuratus (purple sea urchin) as model systems, we demonstrate that IL-1anc proteins share key features with mammalian IL-1α/IL-1β, including expression patterns, subcellular localization and post-translational processing. Together with previous evidence identifying other IL-1 axis components in invertebrates, our data support a model in which the IL-1 superfamily and associated circuitry represent a foundational module of animal immunity that far predates the jawed vertebrates.

The evolutionary origin of vertebrate adaptive immunity has been a longstanding problem in evolutionary biology. The diversified proteins that orchestrate this complex system are present throughout the jawed vertebrates but are absent in extant jawless vertebrates and invertebrates. From work initially carried out in the sea lamprey ( Petromyzon marinus ) and subsequently extended to other lampreys and hagfishes, we now know that, instead of immunoglobulin domain-based receptors, jawless vertebrates have leucine-rich repeat-based variable lymphocyte receptors (VLRs). Like immunoglobulins (Igs) and T cell receptors (TCRs), these proteins are somatically diversified in lymphocyte-like cells. Of the six VLR loci described in the sea lamprey (VLRA-VLRF), five are expressed exclusively as transmembrane receptors on T-like lymphocytes (VLRA and VLRC-VLRF). Here we focus on VLRB which, like jawed vertebrate immunoglobulins, is expressed both as a cell surface receptor on B-like cells and as a secreted, polyvalent VLRB antibody. Although little is known about the mucosal functions of VLR antibodies, VLRB-expressing cells diversify in gut-associated lymphopoietic tissues and are present in the gut epithelium and other regions of the intestine. We discuss what is known about VLRB and some future directions for understanding its gut-associated functions in relation to what is known in the jawed vertebrates.

Interleukin-6 (IL-6) is a central regulator of vertebrate immunity, yet its existence in jawless vertebrates has remained obscure because of extreme sequence divergence. The extant jawless vertebrates (lampreys and hagfish), which deploy variable lymphocyte receptors (VLRs) instead of immunoglobulins and T cell receptors, provide a unique window into the earliest interface between cytokines and adaptive immunity. Here, we identify IL-6-like genes in jawless vertebrates by combining computational structural comparisons, phylogenetic reconstruction, and conserved local and long-range synteny analyses. The encoded proteins adopt the canonical four-helix bundle characteristic of immune-related IL-6 family cytokines and align topologically with mammalian IL-6. In sea lamprey, three IL-6 paralogs show distinct leukocyte and tissue expression patterns and are differentially induced by pathogen-associated molecular patterns and skin injury, indicating early functional diversification of IL-6-mediated inflammatory responses. Stimulation of myeloid peritoneal leukocytes with recombinant lamprey IL-6 induces STAT5 phosphorylation and rapid upregulation of SOCS1/3 genes, consistent with an IL-6/STAT5/SOCS regulatory axis. These findings extend the repertoire of immune-related four-helix bundle cytokines to jawless vertebrates and indicate that IL-6-dependent inflammatory programs were already in place before the divergence of VLR-based and Ig/TCR-based adaptive immune systems in vertebrates.

Summary: Inflammation is a hallmark of immune responses. Its mechanistic underpinnings in mammals are well-defined: pro-inflammatory cytokines of the interleukin 1 (IL-1) superfamily establish and support microenvironments that promote immune cell activities. Despite a growing number of reports on inflammatory processes and components of the IL-1 signaling axis in several invertebrate lineages, orthologs of these central cytokines have not been detected outside of the jawed vertebrates. Here, protein structure prediction algorithms were applied to identify genes encoding a family of IL-1 proteins with homologs throughout Eumetazoa (termed “IL-1anc” to reflect their ancestral evolutionary origin). Using Petromyzon marinus (sea lamprey) and Strongylocentrotus purpuratus (purple sea urchin) as model systems, we demonstrate that IL-1anc proteins share important features with mammalian IL-1α/IL-1β including expression patterns, protein localization, and processing. Together, our data indicate that the IL-1 superfamily and associated circuitry represent a foundational module of animal immunity that far predates the jawed vertebrates.

All extant jawless vertebrates (lampreys and hagfishes) possess a unique adaptive immune system characterized by highly variable lymphocyte receptors (VLR) that are assembled in developing lymphocytes using leucine-rich-repeat donor cassettes. Five VLR types have been identified in lampreys: VLRA, VLRB, VLRC, VLRD, and VLRE. VLRB-expressing lymphocytes are functional analogs to B cells, whereas VLRA, VLRC, VLRD, and VLRE-expressing lymphocytes are more akin to T cells of jawed vertebrates. Here we define an additional VLR, designated VLRF. VLRF is phylogenetically closest to VLRA, with which it likely shares a common ancestral gene of at least 250 million years in the past. VLR assembly analyses show that VLRA, VLRC, VLRD, VLRE, and VLRF share donor cassettes through long-range intra- and inter-chromosomal interactions, whereas VLRB utilizes a distinct, dedicated cassette set. The pattern of gene expression, donor cassette usage, and distinctive amino acid composition in the C-terminal stalk suggest that VLRF⁺ lymphocytes may represent an additional T-like sub-lineage, adding further complexity to the VLR-based adaptive immune system.

Exposure to and colonization by bacteria during development have wide-ranging beneficial effects on animal biology but can also inhibit growth or cause disease. The immune system is the prime mediator of these microbial interactions and is itself shaped by them. Studies using diverse animal taxa have begun to elucidate the mechanisms underlying the acquisition and transmission of bacterial symbionts and their interactions with developing immune systems. Moreover, the contexts of these associations are often confounded by stark differences between “wild type” microbiota and the bacterial communities associated with animals raised in conventional or germ-free laboratories. In this study, we investigate the spatio-temporal kinetics of bacterial colonization and associated effects on growth and immune function in larvae of the purple sea urchin (Strongylocentrotus purpuratus) as a model for host-microbe interactions and immune system development. We also compare the host-associated microbiota of developing embryos and larvae raised in natural seawater or exposed to adult-associated bacteria in the laboratory. Bacteria associated with zygotes, embryos, and early larvae are detectable with 16S amplicon sequencing, but 16S-FISH indicates that the vast majority of larval bacterial load is acquired after feeding begins and is localized to the gut lumen. The bacterial communities of laboratory-cultured embryos are significantly less diverse than the natural microbiota but recapitulate its major components (Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes), suggesting that biologically relevant host-microbe interactions can be studied in the laboratory. We also demonstrate that bacterial exposure in early development induces changes in morphology and in the immune system. In the absence of bacteria, larvae grow larger at the 4-arm stage. Additionally, bacteria-exposed larvae are significantly more resistant to lethal infection with the larva-associated pathogen Vibrio lentus suggesting that early exposure to high levels of microbes, as would be expected in natural conditions, affects the immune state in later larvae. These results expand our knowledge of microbial influences on early sea urchin development and establish a model in which to study the interactions between the developing larval immune system and the acquisition of larval microbiota.

The TNF superfamily ligands BAFF and APRIL interact with three receptors, BAFFR, BCMA, and TACI, to play discrete and crucial roles in regulating B cell selection and homeostasis in mammals. The interactions between these ligands and receptors are both specific and redundant: BAFFR binds BAFF, whereas BCMA and TACI bind to either BAFF or APRIL. In a previous phylogenetic inquiry, we identified and characterized a BAFF-like gene in lampreys, which, with hagfish, are the only extant jawless vertebrates, both of which have B-like and T-like lymphocytes. To gain insight into lymphocyte regulation in jawless vertebrates, in this study we identified two BCMA-like genes in lampreys, BCMAL1 and BCMAL2, which were found to be preferentially expressed by B-like lymphocytes. In vitro analyses indicated that the lamprey BAFF-like protein can bind to a BCMA-like receptor Ig fusion protein and to both BCMAL1- and BCMAL2-transfected cells. Discriminating regulatory roles for the two BCMA-like molecules are suggested by their differential expression before and after activation of the B-like lymphocytes in lampreys. Our composite results imply that BAFF-based mechanisms for B cell regulation evolved before the divergence of jawed and jawless vertebrates.

The embryo of the purple sea urchin has been a fruitful model for the study of developmental gene regulatory networks. For similar reasons, the feeding sea urchin larva provides a gene regulatory model to investigate immune interactions at the gut epithelium. Here we describe what is known of the gut structure and immune cells of the sea urchin larva, and the cellular and gene expression response of the larva to gut-associated immune challenge. As a focused example of how the sea urchin larva can be compared with vertebrate systems, we discuss the expression and function of the IL-17 signalling system in the course of the larval immune response.