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Global amphibian declines are compounded by deadly disease outbreaks caused by the chytrid fungus, Batrachochytrium dendrobatidis ( Bd ). Much has been learned about the roles of amphibian skin-produced antimicrobial components and microbiomes in controlling Bd , yet almost nothing is known about the roles of skin-resident immune cells in anti- Bd defenses. Mammalian mast cells reside within and serve as key immune sentinels in barrier tissues like skin. Accordingly, we investigated the roles of Xenopus laevis frog mast cells during Bd infections. Our findings indicate that enrichment of X. laevis skin mast cells confers anti- Bd protection and ameliorates the inflammation-associated skin damage caused by Bd infection. This includes a significant reduction in infiltration of Bd -infected skin by neutrophils, promoting mucin content within cutaneous mucus glands, and preventing Bd -mediated changes to skin microbiomes. Mammalian mast cells are known for their production of the pleiotropic interleukin-4 (IL4) cytokine and our findings suggest that the X. laevis IL4 plays a key role in manifesting the effects seen following cutaneous mast cell enrichment. Together, this work underscores the importance of amphibian skin-resident immune cells in anti- Bd defenses and illuminates a novel avenue for investigating amphibian host-chytrid pathogen interactions., Competing Interests: KH, CG, RC, MH, DH, NR, LG, AY, NK, MZ, EJ, AD, LR, CM, LG No competing interests declared, (© 2023, Hauser et al.)

Introduction: The California purple sea urchin, Strongylocentrotus purpuratus , relies solely on an innate immune system to combat the many pathogens in the marine environment. One aspect of their molecular defenses is the SpTransformer ( SpTrf ) gene family that is upregulated in response to immune challenge. The gene sequences are highly variable both within and among animals and likely encode thousands of SpTrf isoforms within the sea urchin population. The native SpTrf proteins bind foreign targets and augment phagocytosis of a marine Vibrio . A recombinant (r)SpTrf-E1-Ec protein produced by E. coli also binds Vibrio but does not augment phagocytosis., Methods: To address the question of whether other rSpTrf isoforms function as opsonins and augment phagocytosis, six rSpTrf proteins were expressed in insect cells., Results: The rSpTrf proteins are larger than expected, are glycosylated, and one dimerized irreversibly. Each rSpTrf protein cross-linked to inert magnetic beads (rSpTrf::beads) results in different levels of surface binding and phagocytosis by phagocytes. Initial analysis shows that significantly more rSpTrf::beads associate with cells compared to control BSA::beads. Binding specificity was verified by pre-incubating the rSpTrf::beads with antibodies, which reduces the association with phagocytes. The different rSpTrf::beads show significant differences for cell surface binding and phagocytosis by phagocytes. Furthermore, there are differences among the three distinct types of phagocytes that show specific vs. constitutive binding and phagocytosis., Conclusion: These findings illustrate the complexity and effectiveness of the sea urchin innate immune system driven by the natSpTrf proteins and the phagocyte cell populations that act to neutralize a wide range of foreign pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Crow, Shaw, Grayfer and Smith.)

The world's reptiles and amphibians are experiencing dramatic and ongoing losses in biodiversity, changes that can have substantial effects on ecosystems and human health. In 2022, the first Global Amphibian and Reptile Disease Conference was held, using One Health as a guiding principle. The conference showcased knowledge on numerous reptile and amphibian pathogens from several standpoints, including epidemiology, host immune defenses, wild population effects, and mitigation. The conference also provided field experts the opportunity to discuss and identify the most urgent herpetofaunal disease research directions necessary to address current and future threats to reptile and amphibian biodiversity.

Macrophage-lineage cells are indispensable to immunity and physiology of all vertebrates. Amongst these, amphibians represent a key stage in vertebrate evolution and are facing decimating population declines and extinctions, in large part due to emerging infectious agents. While recent studies indicate that macrophages and related innate immune cells are critically involved during these infections, much remains unknown regarding the ontogeny and functional differentiation of these cell types in amphibians. Accordingly, in this review we coalesce what has been established to date about amphibian blood cell development (hematopoiesis), the development of key amphibian innate immune cells (myelopoiesis) and the differentiation of amphibian macrophage subsets (monopoiesis). We explore the current understanding of designated sites of larval and adult hematopoiesis across distinct amphibian species and consider what mechanisms may lend to these species-specific adaptations. We discern the identified molecular mechanisms governing the functional differentiation of disparate amphibian (chiefly Xenopus laevis) macrophage subsets and describe what is known about the roles of these subsets during amphibian infections with intracellular pathogens. Macrophage lineage cells are at the heart of so many vertebrate physiological processes. Thus, garnering greater understanding of the mechanisms responsible for the ontogeny and functionality of these cells in amphibians will lend to a more comprehensive view of vertebrate evolution., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Xenopus is a genus of African clawed frogs including two species, X. tropicalis and X. laevis that are extensively used in experimental biology, immunology, and biomedical studies. The availability of fully sequenced and annotated Xenopus genomes is strengthening genome-wide analyses of gene families and transgenesis to model human diseases. However, inaccuracies in genome annotation for genes involved in the immune system (i.e., immunome) hamper immunogenetic studies. Furthermore, advanced genome technologies (e.g., single-cell and RNA-Seq) rely on well-annotated genomes. The annotation problems of Xenopus immunome include a lack of established orthology across taxa, merged gene models, poor representation in gene pages on Xenbase, misannotated genes and missing gene IDs. The Xenopus Research Resource for Immunobiology in collaboration with Xenbase and a group of investigators are working to resolve these issues in the latest versions of genome browsers. In this review, we summarize the current problems of previously misannotated gene families that we have recently resolved. We also highlight the expansion, contraction, and diversification of previously misannotated gene families., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Macrophage (M ϕ )-lineage cells are integral to the immune defences of all vertebrates, including amphibians. Across vertebrates, M ϕ differentiation and functionality depend on activation of the colony stimulating factor-1 (CSF1) receptor by CSF1 and interluekin-34 (IL34) cytokines. Our findings to date indicate that amphibian ( Xenopus laevis ) M ϕ s differentiated with CSF1 and IL34 are morphologically, transcriptionally and functionally distinct. Notably, mammalian M ϕ s share common progenitor population(s) with dendritic cells (DCs), which rely on fms-like tyrosine kinase 3 ligand (FLT3L) for differentiation while X. laevis IL34-M ϕ s exhibit many features attributed to mammalian DCs. Presently, we compared X. laevis CSF1- and IL34-M ϕ s with FLT3L-derived X. laevis DCs. Our transcriptional and functional analyses indicated that indeed the frog IL34-M ϕ s and FLT3L-DCs possessed many commonalities over CSF1-M ϕ s, including transcriptional profiles and functional capacities. Compared to X. laevis CSF1-M ϕ s, the IL34-M ϕ s and FLT3L-DCs possess greater surface major histocompatibility complex (MHC) class I, but not MHC class II expression, were better at eliciting mixed leucocyte responses in vitro and generating in vivo re-exposure immune responses against Mycobacterium marinum . Further analyses of non-mammalian myelopoiesis akin to those described here, will grant unique perspectives into the evolutionarily retained and diverged pathways of M ϕ and DC functional differentiation. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

The amphibian declines are compounded by emerging pathogens that often preferentially target distinct amphibian developmental stages. While amphibian immune responses remain relatively unexplored, macrophage (Mφ)-lineage cells are believed to be important to both amphibian host defenses and to their pathogen infection strategies. As such, a greater understanding of tadpole and adult amphibian Mφ functionality is warranted. Mφ biology is interdependent of interleukin-34 (IL-34) and colony-stimulating factor-1 (CSF-1) cytokines and we previously showed that CSF-1- and IL-34-derived Mφs of the Xenopus laevis frog are morphologically, transcriptionally, and functionally distinct. Presently, we directly compared the cytology and transcriptomes of X. laevis tadpole and frog CSF-1- and IL-34-Mφs. Our results indicate that tadpole and frog CSF-1-Mφs possess greater non-specific esterase activity, typically associated with Mφ-lineage cells. By contrast, both tadpole and frog IL-34-Mφs have greater specific esterase activity, which is typically attributed to granulocyte-lineage cells. Our comparisons of tadpole CSF-1-Mφ transcriptomes with those of tadpole IL-34-Mφs indicate that the two tadpole populations possess significantly different transcriptional profiles of immune and non-immune genes. The frog CSF-1-Mφ gene expression profiles are likewise significantly disparate from those of frog IL-34-Mφs. Compared to their respective tadpole Mφ subtypes, frog CSF-1- and IL-34-Mφs exhibited greater expression of genes associated with antigen presentation. Conversely, compared to their frog Mφ counterparts, tadpole CSF-1- and IL-34-Mφs possessed greater levels of select Fc-like receptor genes. Presumably, these cytological and transcriptional differences manifest in distinct biological roles for these respective tadpole and frog Mφ subtypes., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cross-species comparison of vertebrate genomes has unraveled previously unknown complexities of interferon (IFN) systems in amphibian species. Recent genomic curation revealed that amphibian species have evolved expanded repertoires of four types of intron-containing IFN genes akin to those seen in jawed fish, intronless type I IFNs and intron-containing type III IFNs akin to those seen in amniotes, as well as uniquely intronless type III IFNs. This appears to be the case with at least ten analyzed amphibian species; with distinct species encoding diverse repertoires of these respective IFN gene subsets. Amphibians represent a key stage in vertebrate evolution, and in this context offer a unique perspective into the divergent and converged pathways leading to the emergence of distinct IFN families and groups. Recent studies have begun to unravel the roles of amphibian IFNs during these animals' immune responses in general and during their antiviral responses, in particular. However, the pleiotropic potentials of these highly expanded amphibian IFN repertoires warrant further studies. Based on recent reports and our omics analyses using Xenopus models, we posit that amphibian IFN complex may have evolved novel functions, as indicated by their extensive molecular diversity. Here, we provide an overview and an update of the present understanding of the amphibian IFN complex in the context of the evolution of vertebrate immune systems. A greater understanding of the amphibian IFN complex will grant new perspectives on the evolution of vertebrate immunity and may yield new measures by which to counteract the global amphibian declines., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Granulocyte-lineage cells are important innate immune effectors across all vertebrates. Named for conspicuous secretory granules, granulocytes have historically been studied for their antimicrobial roles. Although versions of these cells are found in all vertebrate species examined to date, disparate environmental and physiological pressures acting on distinct vertebrate classes have shaped many of the facets dictating granulocyte biology. Immune pressures further determine granulopoietic constraints, ultimately governing granulocyte functions. For amphibians that inhabit pathogen-rich aquatic environments for some or all their lives, their unique granulocyte biologies satisfy many of their antimicrobial needs. Amphibians also occupy an intermediate position in the evolution of vertebrate immune systems, using combinations of primitive (e.g., subcapsular liver) and more recently evolved (e.g., bone marrow) tissue sites for hematopoiesis and specifically, granulopoiesis. The last decade of research has revealed vertebrate granulocytes in general, and amphibian granulocytes in particular, are more complex than originally assumed. With dynamic leukocyte phenotypes, granulocyte-lineage cells are being acknowledged for their multifaceted roles beyond immunity in other physiological processes. Here we provide an overview of granulopoiesis in amphibians, highlight key differences in these processes compared to higher vertebrates, and identify open questions., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Ltd.)

The sea urchin, Strongylocentrotus purpuratus has seven described populations of distinct coelomocytes in the coelomic fluid that are defined by morphology, size, and for some types, by known functions. Of these subtypes, the large phagocytes are thought to be key to the sea urchin cellular innate immune response. The concentration of total coelomocytes in the coelomic fluid increases in response to pathogen challenge. However, there is no quantitative analysis of how the respective coelomocyte populations change over time in response to immune challenge. Accordingly, coelomocytes collected from immunoquiescent, healthy sea urchins were evaluated by flow cytometry for responses to injury and to challenge with either heat-killed Vibrio diazotrophicus , zymosan A, or artificial coelomic fluid, which served as the vehicle control. Responses to the initial injury of coelomic fluid collection or to injection of V. diazotrophicus show significant increases in the concentration of large phagocytes, small phagocytes, and red spherule cells after one day. Responses to zymosan A show decreases in the concentration of large phagocytes and increases in the concentration of small phagocytes. In contrast, responses to injections of vehicle result in decreased concentration of large phagocytes. When these changes in coelomocytes are evaluated based on proportions rather than concentration, the respective coelomocyte proportions are generally maintained in response to injection with V. diazotrophicus and vehicle. However, this is not observed in response to zymosan A and this lack of correspondence between proportions and concentrations may be an outcome of clearing these large particles by the large phagocytes. Variations in coelomocyte populations are also noted for individual sea urchins evaluated at different times for their responses to immune challenge compared to the vehicle. Together, these results demonstrate that the cell populations in sea urchin immune cell populations undergo dynamic changes in vivo in response to distinct immune stimuli and to injury and that these changes are driven by the responses of the large phagocyte populations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Barela Hudgell, Grayfer and Smith.)

The global amphibian declines are compounded by infections with members of the Ranavirus genus such as Frog Virus 3 (FV3). Premetamorphic anuran amphibians are believed to be significantly more susceptible to FV3 while this pathogen targets the kidneys of both pre- and postmetamorphic animals. Paradoxically, FV3-challenged Xenopus laevis tadpoles exhibit lower kidney viral loads than adult frogs. Presently, we demonstrate that X. laevis tadpoles are intrinsically more resistant to FV3 kidney infections than cohort-matched metamorphic and postmetamorphic froglets and that this resistance appears to be epigenetically conferred by endogenous retroviruses (ERVs). Using a X. laevis kidney-derived cell line, we show that enhancing ERV gene expression activates cellular double-stranded RNA-sensing pathways, resulting in elevated mRNA levels of antiviral interferon (IFN) cytokines and thus greater anti-FV3 protection. Finally, our results indicate that large esterase-positive myeloid-lineage cells, rather than renal cells, are responsible for the elevated ERV/IFN axis seen in the tadpole kidneys. This conclusion is supported by our observation that CRISPR-Cas9 ablation of colony-stimulating factor-3 results in abolished homing of these myeloid cells to tadpole kidneys, concurrent with significantly abolished tadpole kidney expression of both ERVs and IFNs. We believe that the manuscript marks an important step forward in understanding the mechanisms controlling amphibian antiviral defenses and thus susceptibility and resistance to pathogens like FV3. IMPORTANCE Global amphibian biodiversity is being challenged by pathogens like the Frog Virus 3 (FV3) ranavirus, underlining the need to gain a greater understanding of amphibian antiviral defenses. While it was previously believed that anuran (frog/toad) amphibian tadpoles are more susceptible to FV3, we demonstrated that tadpoles are in fact more resistant to this virus than metamorphic and postmetamorphic froglets. We showed that this resistance is conferred by large myeloid cells within the tadpole kidneys (central FV3 target), which possess an elevated expression of endogenous retroviruses (ERVs). In turn, these ERVs activate cellular double-stranded RNA-sensing pathways, resulting in a greater expression of antiviral interferon cytokines, thereby offering the observed anti-FV3 protection.

The sea urchin, Strongylocentrotus purpuratus, possesses at least seven distinguishable cell populations in the coelomic fluid, which vary in morphology, size, and function. Of these, the large phagocytes, small phagocytes, and red spherule cells are thought to be key to the echinoid immune response. Because there are currently no effective and rapid means of evaluating sea urchin coelomocytes, we developed a flow cytometry based approach to identify these subsets from unseparated, unstained, live cells. In particular our gating strategy distinguishes between the large phagocytes, small phagocytes, red spherule cells, and a mixed population of vibratile cells and colorless spherule cells. This flow cytometry based analysis increases the speed and improves the reliability of coelomocyte analysis compared to differential cell counts by microscopy., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Infections by Frog Virus 3 (FV3) and other ranavirus genus members are significantly contributing to global amphibian decline. The Xenopus laevis frog is an ideal research platform upon which to study the roles of distinct frog leukocyte populations during FV3 infections. Frog macrophages (MΦs) are integrally involved during FV3 infection, as they facilitate viral dissemination and persistence but also participate in immune defense against this pathogen. In turn, MΦ differentiation and functionality depend on the colony-stimulating factor-1 receptor (CSF-1R), which is ligated by CSF-1 and iterleukin-34 (IL-34) cytokines. Our past work indicated that X. laevis CSF-1 and IL-34 give rise to morphologically and functionally distinct frog MΦ subsets, and that these CSF-1- and IL-34-MΦs respectively confer susceptibility and antiviral resistance to FV3. Because FV3 targets the frog kidneys and establishes chronic infections therein, presently we examined the roles of the frog CSF-1- and IL-34-MΦs in seeding and maintaining these chronic kidney infections. Our findings indicate that the frog CSF-1-MΦs result in more prominent kidney FV3 infections, which develop into greater reservoirs of lingering FV3 marked by infiltrating leukocytes, fibrosis, and overall immunosuppressive states. Moreover, the antiviral effects of IL-34-MΦs are short-lived and are lost as FV3 infections progress.

The global amphibian declines are compounded by ranavirus infections such as Frog Virus 3 (FV3), and amphibian tadpoles more frequently succumb to these pathogens than adult animals. Amphibian gastrointestinal tracts represent a major route of ranavirus entry, and viral pathogenesis often leads to hemorrhaging and necrosis within this tissue. Alas, the differences between tadpole and adult amphibian immune responses to intestinal ranavirus infections remain poorly defined. As interferon (IFN) cytokine responses represent a cornerstone of vertebrate antiviral immunity, it is pertinent that the tadpoles and adults of the anuran Xenopus laevis frog mount disparate IFN responses to FV3 infections. Presently, we compared the tadpole and adult X. laevis responses to intestinal FV3 infections. Our results indicate that FV3-challenged tadpoles mount more robust intestinal type I and III IFN responses than adult frogs. These tadpole antiviral responses appear to be mediated by myeloid cells, which are recruited into tadpole intestines in response to FV3 infections. Conversely, myeloid cells bearing similar cytology already reside within the intestines of healthy (uninfected) adult frogs, possibly accounting for some of the anti-FV3 resistance of these animals. Further insight into the differences between tadpole and adult frog responses to ranaviral infections is critical to understanding the facets of susceptibility and resistance to these pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hauser, Singer, Hossainey, Moore, Wendel, Yaparla, Kalia and Grayfer.)

Nematode virulence factors are of interest for a variety of applications including biocontrol against insect pests and the alleviation of autoimmune diseases with nematode-derived factors. In silico "omics" techniques have generated a wealth of candidate factors that may be important in the establishment of nematode infections, although the challenge of characterizing these individual factors in vivo remains. Here we provide a fundamental characterization of a putative lysozyme and serine carboxypeptidase from the host-induced transcriptome of Heterorhabditis bacteriophora. Both factors accelerated the mortality rate following Drosophila melanogaster infections with Photorhabdus luminescens, and both factors suppressed phenoloxidase activity in D. melanogaster hemolymph. Furthermore, the serine carboxypeptidase was lethal to a subpopulation of flies and suppressed the upregulation of antimicrobial peptides as well as phagocytosis. Together, our findings suggest that this serine carboxypeptidase possess both toxic and immunomodulatory properties while the lysozyme is likely to confer immunomodulatory, but not toxic effects., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The differentiation of distinct leukocyte subsets is governed by lineage-specific growth factors that elicit disparate expression of transcription factors and markers by the developing cell populations. For example, macrophages (Mφs) and granulocytes (Grns) arise from common granulocyte-macrophage progenitors in response to distinct myeloid growth factors. In turn, myelopoiesis of the Xenopus laevis anuran amphibian appears to be unique to other studied vertebrates in several respects while the functional differentiation of amphibian Mφs and Grns from their progenitor cells remains poorly understood. Notably, the expression of colony stimulating factor-1 receptor (CSF-1R) or CSF-3R on granulocyte-macrophage progenitors marks their commitment to Mφ- or Grn-lineages, respectively. CSF-1R is activated by the colony stimulating factor-1 (CSF-1) and interleukin (IL-34) cytokines, resulting in morphologically and functionally distinct Mφ cell types. Conversely, CSF-3R is ligated by CSF-3 in a process indispensable for granulopoiesis. Presently, we explore the relationships between X. laevis CSF-1-Mφs, IL-34-Mφs and CSF-3-Grns by examining their expression of key lineage-specific transcription factor and myeloid marker genes as well as their enzymology. Our findings suggest that while the CSF-1- and IL-34-Mφs share some commonalities, the IL-34-Mφs possess transcriptional patterns more akin to the CSF-3-Grns. IL-34-Mφs also possess robust expression of dendritic cell-associated transcription factors and surface marker genes, further underlining the difference between this cell type and the CSF-1-derived frog Mφ subset. Moreover, the three myeloid populations differ in their respective tartrate-resistant acid phosphatase, specific- and non-specific esterase activity. Together, this work grants new insights into the developmental relatedness of these three frog myeloid subsets., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Insect pathogens have adopted an array of mechanisms to subvert the immune pathways of their respective hosts. Suppression may occur directly at the level of host-pathogen interactions, for instance phagocytic capacity or phenoloxidase activation, or at the upstream signaling pathways that regulate these immune effectors. Insect pathogens of the family Baculoviridae, for example, are known to produce a UDP-glycosyltransferase (UGT) that negatively regulates ecdysone signaling. Normally, ecdysone positively regulates both molting and antimicrobial peptide production, so the inactivation of ecdysone by glycosylation results in a failure of host larvae to molt, and probably a reduced antimicrobial response. Here, we examine a putative ecdysteroid glycosyltransferase, Hba_07292 (Hb-ugt-1), which was previously identified in the hemolymph-activated transcriptome of the entomopathogenic nematode Heterorhabditis bacteriophora. Injection of recombinant Hb-ugt-1 (rHb-ugt-1) into Drosophila melanogaster flies resulted in diminished upregulation of antimicrobial peptides associated with both the Toll and Immune deficiency pathways. Ecdysone was implicated in this suppression by a reduction in Broad Complex expression and reduced pupation rates in r Hb-ugt-1-injected larvae. In addition to the finding that H. bacteriophora excreted-secreted products contain glycosyltransferase activity, these results demonstrate that Hb-ugt-1 is an immunosuppressive factor and that its activity likely involves the inactivation of ecdysone.

Across vertebrates, hematopoiesis takes place within designated tissues, wherein committed myeloid progenitors further differentiate toward cells with megakaryocyte/erythroid potential (MEP) or those with granulocyte/macrophage potential (GMP). While the liver periphery (LP) of the Xenopus laevis amphibian functions as a principal site of hematopoiesis and contains MEPs, cells with GMP potential are instead segregated to the bone marrow (BM) of this animal. Presently, using gene expression and western blot analyses of blood cell lineage-specific transcription factors, we confirmed that while the X. laevis LP hosts hematopoietic stem cells and MEPs, their BM contains GMPs. In support of our hypothesis that cells bearing GMP potential originate from the frog LP and migrate through blood circulation to the BM in response to chemical cues; we demonstrated that medium conditioned by the X. laevis BM chemoattracts LP and peripheral blood cells. Compared to LP and by examining a comprehensive panel of chemokine genes, we showed that the X. laevis BM possessed greater expression of a single chemokine, CXCL12, the recombinant form of which was chemotactic to LP and peripheral blood cells and appeared to be a major chemotactic component within BM-conditioned medium. In confirmation of the hepatic origin of the cells that give rise to these frogs' GMPs, we also demonstrated that the X. laevis BM supported the growth of their LP-derived cells., (Copyright © 2020 Yaparla, Reeves and Grayfer.)

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains the leading global cause of death from an infectious agent. Mycobacteria thrive within their host Mϕs and presently, there is no animal model that permits combined in vitro and in vivo study of mycobacteria-host Mϕ interactions. Mycobacterium marinum (Mm), which causes TB in aquatic vertebrates, has become a promising model for TB research, owing to its close genetic relatedness to Mtb and the availability of alternative, natural host aquatic animal models. Here, we adopted the Xenopus laevis frog-Mm surrogate infection model to study host Mϕ susceptibility and resistance to mycobacteria. Mϕ differentiation is regulated though the CSF-1 receptor (CSF-1R), which is activated by CSF-1 and the unrelated IL-34 cytokines. Using combined in vitro and in vivo approaches, we demonstrated that CSF-1-Mϕs exacerbate Mm infections, are more susceptible to mycobacterial entry and are less effective at killing this pathogen. By contrast, IL-34-Mϕs confer anti-Mm resistance in vivo, are less susceptible to Mm entry and more effectively eliminate internalized mycobacteria. Moreover, we showed that the human CSF-1- and IL-34-Mϕs are likewise, respectively, susceptible and resistant to mycobacteria, and that both frog and human CSF-1-Mϕs are more prone to the spread of mycobacteria and to being infected by Mm-laden Mϕs than the respective IL-34-Mϕ subsets. This work marks the first report describing the roles of these Mϕ subsets in mycobacterial disease and may well lead to the development of more targeted anti-Mtb approaches., (©2019 Society for Leukocyte Biology.)

Pathogens such as the Frog Virus 3 (FV3) ranavirus are contributing to the worldwide amphibian declines. While amphibian macrophages (Mϕs) are central to the immune defenses against these viruses, the pathogen recognition capacities of disparate amphibian Mϕ subsets remain unexplored. In turn, Mϕ differentiation and functionality are interdependent on the colony-stimulating factor-1 receptor (CSF-1R), which is ligated by colony-stimulating factor-1 (CSF-1) and the unrelated interleukin-34 (IL-34) cytokines. Notably, the Xenopus laevis frog CSF-1- and IL-34-derived Mϕs are functionally distinct, and while the CSF-1-Mϕs are more susceptible to FV3, the IL-34-Mϕs are highly resistant to this pathogen. Here, we elucidate the pathogen recognition capacities of CSF-1- and IL-34-differentiated Mϕs by evaluating their baseline transcript levels of key pathogen pattern recognition receptors (PRRs). Compared to the frog CSF-1-Mϕs, their IL-34-Mϕs exhibited greater expression of PRR genes associated with viral recognition as well as PRR genes known for recognizing bacterial pathogen-associated molecular patterns (PAMPs). By contrast, the CSF-1-Mϕs displayed greater expression of toll-like receptors (TLRs) that are absent in humans. Moreover, although the two Mϕ types possessed similar expression of most downstream PRR signaling components, they exhibited distinct outcomes upon stimulation with hallmark PAMPs, as measured by their tumor necrosis factor-alpha and interferon-7 gene expression. Remarkably, stimulation with a TLR2/6 agonist conferred FV3 resistance to the otherwise susceptible CSF-1-Mϕs while treatment with a TLR9 agonist significantly ablated the IL-34-Mϕ resistance to FV3. These changes in Mϕ-FV3 susceptibility and resistance appeared to be linked to changes in their expression of key immune genes. Greater understanding of the amphibian macrophage pathogen-recognition capacities will lend to further insights into the pathogen-associated causes of the amphibian declines., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The adaptive immune response in jawed vertebrates is marked by the ability to diversify somatically specific immune receptor genes. Somatic recombination and hypermutation of gene segments are used to generate extensive repertoires of T and B cell receptors. In contrast, jawless vertebrates utilize a distinct diversification system based on copy choice to assemble their variable lymphocyte receptors. To date, very little evidence for somatic immune gene diversification has been reported in invertebrate species. Here we show that the SpTransformer ( SpTrf ; formerly Sp185/333 ) immune effector gene family members from individual coelomocytes from purple sea urchins undergo somatic diversification by means of gene deletions, duplications, and acquisitions of single nucleotide polymorphisms. While sperm cells from an individual sea urchin have identical SpTrf gene repertoires, single cells from two distinct coelomocyte subpopulations from the same sea urchin exhibit significant variation in the SpTrf gene repertoires. Moreover, the highly diverse gene sequences derived from single coelomocytes are all in-frame, suggesting that an unknown mechanism(s) driving these somatic changes involve stringent selection or correction processes for expression of productive SpTrf transcripts. Together, our findings infer somatic immune gene diversification strategy in an invertebrate.

Frog virus 3 (FV3) is the type species of the genus Ranavirus (family Iridoviridae). FV3 and FV3-like viruses are globally distributed infectious agents with the capacity to replicate in three vertebrate classes (teleosts, amphibians, and reptiles). At the cellular level, FV3 and FV3-like viruses can infect cells from virtually all vertebrate classes. To date, the cellular receptors that are involved in the FV3 entry process are unknown. Class A scavenger receptors (SR-As) are a family of evolutionarily conserved cell-surface receptors that bind a wide range of chemically distinct polyanionic ligands and can function as cellular receptors for other DNA viruses, including vaccinia virus and herpes simplex virus. The present study aimed to determine whether SR-As are involved in FV3 cellular entry. By using well-defined SR-A competitive and non-competitive ligand-blocking assays and absolute qPCR, we demonstrated that the SR-A competitive ligands drastically reduced the quantities of cell-associated viral loads in frog cells. Moreover, inducing the expression of a human SR-AI in an SR-A null cell line significantly increased FV3⁻cell association. Together, our results indicate that SR-As are utilized by FV3 during the cellular entry process.

The establishment of latent HIV-1 reservoirs in terminally differentiated cells represents a major impediment to the success of antiretroviral therapies. Notably, macrophages (Mϕs) are susceptible to HIV-1 infection and recent evidence suggests that they may be involved in long-term HIV-1 persistence. While the extensive functional heterogeneity seen across the Mϕ cell lineage parallels the spectrum of HIV-1 susceptibility reported across these cell subsets, the facets of Mϕ HIV-1 resistance and susceptibility remain to be fully defined. Notably, the differentiation of most Mϕ subsets depends on signaling through the macrophage colony-stimulating factor receptor (M-CSFR), which in addition to M-CSF, is now known to bind the unrelated interleukin-34 (IL-34) cytokine. The biological need for two M-CSFR ligands awaits full elucidation. Here, we report that Mϕs differentiated from human peripheral blood monocytes with IL-34 are substantially more resistant to HIV-1 infection than M-CSF-derived Mϕs. Moreover, while both Mϕ subsets express comparable surface protein levels of the HIV-1 receptor and co-receptor, CD4 and CCR5 respectively, the IL-34-Mϕs express significantly greater levels of pertinent restriction factor genes, potentially accounting for their greater resistance to HIV-1 infection than that observed in M-CSF-Mϕs. Together, our findings underline previously unexplored differentiation pathways resulting in HIV-1-susceptible and resistant Mϕ subsets and pave the way for further research that may overcome one of the last major hurdles in developing more successful antiretroviral therapy., (Copyright © 2018. Published by Elsevier Ltd.)

The glutamic acid-leucine-arginine (ELR) motif is a hallmark feature shared by mammalian inflammatory CXC chemokines such the granulocyte chemo-attractant CXCL8 (interleukin-8, IL-8). By contrast, most teleost fish inflammatory chemokines lack this motif. Interestingly, the amphibian Xenopus laevis encodes multiple isoforms of CXCL8, one of which (CXCL8a) possesses an ELR motif, while another (CXCL8b) does not. These CXCL8 isoforms exhibit distinct expression patterns during frog development and following immune challenge of animals and primary myeloid cultures. To define potential functional differences between these X. laevis CXCL8 chemokines, we produced them in recombinant form (rCXCL8a and rCXCL8b) and performed dose-response chemotaxis assays. Our results indicate that compared to rCXCL8b, rCXCL8a is a significantly more potent chemo-attractant of in vivo -derived tadpole granulocytes and of in vitro -differentiated frog bone marrow granulocytes. The mammalian CXCL8 mediates its effects through two distinct chemokine receptors, CXCR1 and CXCR2 and our pharmacological inhibition of these receptors in frog granulocytes indicates that the X. laevis CXCL8a and CXCL8b both chemoattract tadpole and adult frog granulocytes by engaging CXCR1 and CXCR2. To delineate which frog cells are recruited by CXCL8a and CXCL8b in vivo , we injected tadpoles and adult frogs intraperitoneally with rCXCL8a or rCXCL8b and recovered the accumulated cells by lavage. Our transcriptional and cytological analyses of these tadpole and adult frog peritoneal exudates indicate that they are comprised predominantly of granulocytes. Interestingly, the granulocytes recruited into the tadpole, but not adult frog peritonea by rCXCL8b, express significantly greater levels of several pan immunosuppressive genes.

While amphibians around the globe are facing catastrophic declines, in part because of infections with pathogens such as the Frog Virus 3 (FV3) ranavirus; the mechanisms governing amphibian susceptibility and resistance to such pathogens remain poorly understood. The type I and type III interferon (IFN) cytokines represent a cornerstone of vertebrate antiviral immunity, while our recent work indicates that tadpoles and adult frogs of the amphibian Xenopus laevis may differ in their IFN responses to FV3. In this respect, it is notable that anuran (frogs and toads) tadpoles are significantly more susceptible to FV3 than adult frogs, and thus, gaining greater insight into the differences in the tadpole and adult frog antiviral immunity would be invaluable. Accordingly, we examined the FV3-elicited expression of a panel of type I and type III IFN genes in the skin (site of FV3 infection) and kidney (principal FV3 target) tissues and isolated cells of X. laevis tadpoles and adult frogs. We also examined the consequence of tadpole and adult frog skin and kidney cell stimulation with hallmark pathogen-associated molecular patterns (PAMPs) on the IFN responses of these cells. Together, our findings indicate that tadpoles and adult frogs mount drastically distinct IFN responses to FV3 as well as to viral and non-viral PAMPs, while these expression differences do not appear to be the result of a distinct pattern recognition receptor expression by tadpoles and adults.

Peritoneal lavage of Xenopus laevis tadpoles and adult frogs is a reliable way of isolating resident and/or recruited innate immune populations. This protocol details the isolation of tadpole and adult amphibian ( Xenopus laevis ) peritoneal leukocytes. The isolated cells are comprised predominantly of innate immune populations and chiefly of mononuclear and polymorphonuclear granulocytes. As described here, these cells are typically elicited by peritoneal injections of animals with heat-killed Escherichia coli, causing peritoneal accumulation of inflammatory cell populations, which are then isolated from the stimulated animals by lavage. E. coli -mediated elicitation of tadpole and adult peritoneal leukocytes greatly enhances the total numbers of recovered cells, at the cost of their inflammatory activation. Conversely, lavage may be performed on naïve, unstimulated animals to isolate nonactivated cells with much lower yield. This protocol represents a reliable means of deriving tadpole and adult frog innate immune cell populations, and the conditions of the stimulation may be amended to suit the specifics of a given experimental design., (© 2018 Cold Spring Harbor Laboratory Press.)

Overcrowding conditions and temperatures shifts regularly manifest in large-scale infections of farmed fish, resulting in economic losses for the global aquaculture industries. Increased understanding of the functional mechanisms of fish antimicrobial host defenses is an important step forward in prevention of pathogen-induced morbidity and mortality in aquaculture setting. Like other vertebrates, macrophage-lineage cells are integral to fish immune responses and for this reason, much of the recent fish immunology research has focused on fish macrophage biology. These studies have revealed notable similarities as well as striking differences in the molecular strategies by which fish and higher vertebrates control their respective macrophage polarization and functionality. In this review, we address the current understanding of the biological mechanisms of teleost macrophage functional heterogeneity and immunity, focusing on the key cytokine regulators that control fish macrophage development and their antimicrobial armamentarium.

Infections by ranaviruses such as Frog virus 3 (Fv3), are significantly contributing to worldwide amphibian population declines. Notably, amphibian macrophages (Mφs) are important to both the Fv3 infection strategies and the immune defense against this pathogen. However, the mechanisms underlying amphibian Mφ Fv3 susceptibility and resistance remain unknown. Mφ differentiation is mediated by signaling through the colony-stimulating factor-1 receptor (CSF-1R) which is now known to be bound not only by CSF-1, but also by the unrelated interleukin-34 (IL-34) cytokine. Pertinently, amphibian ( Xenopus laevis ) Mφs differentiated by CSF-1 and IL-34 are highly susceptible and resistant to Fv3, respectively. Accordingly, in the present work, we elucidate the facets of this Mφ Fv3 susceptibility and resistance. Because cellular resistance to viral replication is marked by expression of antiviral restriction factors, it was intuitive to find that IL-34-Mφs possess significantly greater mRNA levels of select restriction factor genes than CSF-1-Mφs. Xenopodinae amphibians have highly expanded repertoires of antiviral interferon (IFN) cytokine gene families, and our results indicated that in comparison with the X. laevis CSF-1-Mφs, the IL-34-Mφs express substantially greater transcripts of representative IFN genes, belonging to distinct gene family clades, as well as their cognate receptor genes. Finally, we demonstrate that IL-34-Mφ-conditioned supernatants confer IFN-mediated anti-Fv3 protection to the virally susceptible X. laevis kidney (A6) cell line. Together, this work underlines the differentiation pathways leading to Fv3-susceptible and -resistant amphibian Mφ populations and defines the molecular mechanisms responsible for these differences., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The X. laevis sub-capsular liver is thought to be the principal hematopoietic site of Xenopodinae species from early development and, in case of certain species, into adulthood. The Xenopus bone marrow appears to be comprised of precursor cells committed to myeloid lineages, such as macrophage- and granulocyte-progenitor cells. With alarming increases in the contribution of pathogenic infections to the global amphibian declines, now more than ever a better understanding of the mechanisms controlling amphibian immune cell ontogeny and functionality is warranted. Accordingly, here we detail the isolation and culture of the X. laevis hematopoietic cells from the sub-capsular liver and bone marrow. Considering the immunological roles attributed to these amphibian organs, the respective cell isolation protocols described here will be pertinent to garnering further insights into the coordinated regulation of amphibian hematopoiesis and immune defense mechanisms.

Infections by Frog Virus 3 (FV3) and other ranaviruses (RVs) are contributing to the amphibian declines, while the mechanisms controlling anuran tadpole susceptibility and adult frog resistance to RVs, including the roles of polymorphonuclear granulocytes (PMNs) during anti-FV3 responses, remain largely unknown. Since amphibian kidneys represent an important FV3 target, the inability of amphibian (Xenopus laevis) tadpoles to mount effective kidney inflammatory responses to FV3 is thought to contribute to their susceptibility. Here we demonstrate that a recombinant X. laevis granulocyte colony-stimulating factor (G-CSF) generates PMNs with hallmark granulocyte morphology. Tadpole pretreatment with G-CSF prior to FV3 infection reduces animal kidney FV3 loads and extends their survival. Moreover, G-CSF-derived PMNs are resistant to FV3 infection and express high levels of TNFα in response to this virus. Notably, FV3-infected tadpoles fail to recruit G-CSFR expressing granulocytes into their kidneys, suggesting that they lack an integral inflammatory effector population at this site., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Infections of amphibians by Frog Virus 3 (FV3) and other ranavirus genus members are significantly contributing to the amphibian declines, yet much remains unknown regarding amphibian antiviral immunity. Notably, amphibians represent an important step in the evolution of antiviral interferon (IFN) cytokines as they are amongst the first vertebrates to possess both type I and type III IFNs. Accordingly, we examined the roles of type I and III IFNs in the skin of FV3-challenged amphibian Xenopus laevis) tadpoles and adult frogs. Interestingly, FV3-infected tadpoles mounted type III IFN responses, whereas adult frogs relied on type I IFN immunity. Subcutaneous administration of type I or type III IFNs offered short-term protection of tadpoles against FV3 and these type I and type III IFNs induced the expression of distinct antiviral genes in the tadpole skin. Moreover, subcutaneous injection of tadpoles with type III IFN significantly extended their survival and reduced FV3 dissemination., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Myeloid progenitors reside within specific hematopoietic organs and commit to progenitor lineages bearing megakaryocyte/erythrocyte (MEP) or granulocyte/macrophage potentials (GMP) within these sites. Unlike other vertebrates, the amphibian Xenopus laevis committed macrophage precursors are absent from the hematopoietic subcapsular liver and instead reside within their bone marrow. Presently, we demonstrate that while these frogs' liver-derived cells are unresponsive to recombinant forms of principal X. laevis macrophage (colony-stimulating factor-1; CSF-1) and granulocyte (CSF-3) growth factors, bone marrow cells cultured with CSF-1 and CSF-3 exhibit respectively archetypal macrophage and granulocyte morphology, gene expression and functionalities. Moreover, we demonstrate that liver, but not bone marrow cells possess erythropoietic capacities when stimulated with a X. laevis erythropoietin. Together, our findings indicate that X. laevis retain their MEP within the hematopoietic liver while sequestering their GMP to the bone marrow, thus marking a very novel myelopoietic strategy as compared to those seen in other jawed vertebrate species., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The occurrence of emerging infectious diseases in wildlife populations is increasing, and changes in environmental conditions have been hypothesized as a potential driver. For example, warmer ambient temperatures might favor pathogens by providing more ideal conditions for propagation or by stressing hosts. Our objective was to determine if water temperature played a role in the pathogenicity of an emerging pathogen (ranavirus) that infects ectothermic vertebrate species. We exposed larvae of four amphibian species to a Frog Virus 3 (FV3)-like ranavirus at two temperatures (10 and 25°C). We found that FV3 copies in tissues and mortality due to ranaviral disease were greater at 25°C than at 10°C for all species. In a second experiment with wood frogs (Lithobates sylvaticus), we found that a 2°C change (10 vs. 12°C) affected ranaviral disease outcomes, with greater infection and mortality at 12°C. There was evidence that 10°C stressed Cope's gray tree frog (Hyla chrysoscelis) larvae, which is a species that breeds during summer-all individuals died at this temperature, but only 10% tested positive for FV3 infection. The greater pathogenicity of FV3 at 25°C might be related to faster viral replication, which in vitro studies have reported previously. Colder temperatures also may decrease systemic infection by reducing blood circulation and the proportion of phagocytes, which are known to disseminate FV3 through the body. Collectively, our results indicate that water temperature during larval development may play a role in the emergence of ranaviruses.

Macrophage lineage cells represent the cornerstone of vertebrate physiology and immune defenses. In turn, comparative studies using non-mammalian animal models have revealed that evolutionarily distinct species have adopted diverse molecular and physiological strategies for controlling macrophage development and functions. Notably, amphibian species present a rich array of physiological and environmental adaptations, not to mention the peculiarity of metamorphosis from larval to adult stages of development, involving drastic transformation and differentiation of multiple new tissues. Thus it is not surprising that different amphibian species and their respective tadpole and adult stages have adopted unique hematopoietic strategies. Accordingly and in order to establish a more comprehensive view of these processes, here we review the hematopoietic and monopoietic strategies observed across amphibians, describe the present understanding of the molecular mechanisms driving amphibian, an in particular Xenopus laevis macrophage development and functional polarization, and discuss the roles of macrophage-lineage cells during ranavirus infections., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Macrophages are found across all vertebrate species, reside in virtually all animal tissues, and play critical roles in host protection and homeostasis. Various mechanisms determine and regulate the highly plastic functional phenotypes of macrophages, including antimicrobial host defenses (pro-inflammatory, M1-type), and resolution and repair functions (anti-inflammatory/regulatory, M2-type). The study of inflammatory macrophages in immune defense of teleosts has garnered much attention, and antimicrobial mechanisms of these cells have been extensively studied in various fish models. Intriguingly, both similarities and differences have been documented for the regulation of lower vertebrate macrophage antimicrobial defenses, as compared to what has been described in mammals. Advances in our understanding of the teleost macrophage M2 phenotypes likewise suggest functional conservation through similar and distinct regulatory strategies, compared to their mammalian counterparts. In this review, we discuss the current understanding of the molecular mechanisms governing teleost macrophage functional heterogeneity, including monopoetic development, classical macrophage inflammatory and antimicrobial responses as well as alternative macrophage polarization towards tissues repair and resolution of inflammation.

To identify ranavirus virulence genes, we engineered Frog Virus 3 (FV3) knockout (KO) mutants defective for a putative viral caspase activation and recruitment domain-containing (CARD) protein (Δ64R-FV3) and a β-hydroxysteroid dehydrogenase homolog (Δ52L-FV3). Compared to wild type (WT) FV3, infection of Xenopus tadpoles with Δ64R- or Δ52L-FV3 resulted in significantly lower levels of mortality and viral replication. We further characterized these and two earlier KO mutants lacking the immediate-early18kDa protein (FV3-Δ18K) or the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α). All KO mutants replicated as well as WT-FV3 in non-amphibian cell lines, whereas in Xenopus A6 kidney cells replication of ΔvCARD-, ΔvβHSD- and ΔvIF-2α-FV3 was markedly reduced. Furthermore, Δ64R- and ΔvIF-2α-FV3 were more sensitive to interferon than WT and Δ18-FV3. Notably, Δ64R-, Δ18K- and ΔvIF-2α- but not Δ52L-FV3 triggered more apoptosis than WT FV3. These data suggest that vCARD (64R) and vβ-HSD (52L) genes contribute to viral pathogenesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Although Mϕ represent the most primordial immune cell subsets, the mechanisms governing their functional heterogeneity remain poorly defined. However, it is well established that the CSF-1 cytokine contributes to monopoiesis and to this heterogeneity, whereas the unrelated IL-34 also binds the CSF-1R toward poorly understood immunologic roles. To delineate the molecular and evolutionary basis behind vertebrate Mϕ functional heterogeneity, we performed comprehensive transcriptional and functional studies of amphibian (Xenopus laevis) BM (in vitro) and PER (in vivo) Mϕ derived by rXlCSF-1 and rXlIL-34. Our findings indicate that these amphibian cytokines promote morphologically and functionally distinct Mϕ populations. Mϕ induced by rXlCSF-1 possess more robust iNOS gene expression, are substantially more phagocytic, display greater NO responses, and exhibit enhanced bactericidal capacities. By contrast, rXlIL-34-derived Mϕ express greater levels of Arg-1 and NADPH oxidase components and possess greater respiratory burst responses. Most notably, whereas CSF-1 Mϕ are highly susceptible to the emerging FV3 ranavirus, rXlIL-34 Mϕ exhibit potent antiviral activity against this pathogen, which is dependent on reactive oxygen production. This work marks an advance in our understanding of the possible mechanisms governing vertebrate Mϕ functional heterogeneity., (© Society for Leukocyte Biology.)

Nonclassical MHC class Ib-restricted invariant T (iT) cell subsets are attracting interest because of their potential to regulate immune responses against various pathogens. The biological relevance and evolutionary conservation of iT cells have recently been strengthened by the identification of iT cells (invariant Vα6 [iVα6]) restricted by the nonclassical MHC class Ib molecule XNC10 in the amphibian Xenopus laevis. These iVα6 T cells are functionally similar to mammalian CD1d-restricted invariant NKT cells. Using the amphibian pathogen frog virus 3 (FV3) in combination with XNC10 tetramers and RNA interference loss of function by transgenesis, we show that XNC10-restricted iVα6 T cells are critical for early antiviral immunity in adult X. laevis. Within hours following i.p. FV3 infection, iVα6 T cells were specifically recruited from the spleen into the peritoneum. XNC10 deficiency and concomitant lack of iVα6 T cells resulted in less effective antiviral and macrophage antimicrobial responses, which led to impaired viral clearance, increased viral dissemination, and more pronounced FV3-induced kidney damage. Together, these findings imply that X. laevis XNC10-restricted iVα6 T cells play important roles in the early anti-FV3 response and that, as has been suggested for mammalian invariant NKT cells, they may serve as immune regulators polarizing macrophage effector functions toward more effective antiviral states., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Unlabelled: Ranaviruses (Iridoviridae) are posing an increasing threat to amphibian populations, with anuran tadpoles being particularly susceptible to these viral infections. Moreover, amphibians are the most basal phylogenetic class of vertebrates known to possess both type I and type III interferon (IFN)-mediated immunity. Moreover, little is known regarding the respective roles of the IFN mediators in amphibian antiviral defenses. Accordingly, we transcriptionally and functionally compared the amphibian Xenopus laevis type I (IFN) and III (IFN-λ) IFNs in the context of infections by the ranavirus frog virus 3 (FV3). X. laevis IFN and IFN-λ displayed distinct tissue expression profiles. In contrast to our previous findings that X. laevis tadpoles exhibit delayed and modest type I IFN responses to FV3 infections compared to the responses of adults, here we report that tadpoles mount timely and robust type III IFN gene responses. Recombinant forms of these cytokines (recombinant X. laevis IFN [rXlIFN] and rXlIFN-λ) elicited antiviral gene expression in the kidney-derived A6 cell line as well as in tadpole leukocytes and tissues. However, rXlIFN-λ was less effective than rXlIFN in preventing FV3 replication in A6 cells and tadpoles and inferior at promoting tadpole survival. Intriguingly, FV3 impaired A6 cell and tadpole kidney type III IFN receptor gene expression. Furthermore, in A6 cultures rXlIFN-λ conferred equal or greater protection than rXlIFN against recombinant viruses deficient for the putative immune evasion genes, the viral caspase activation and recruitment domain (vCARD) or a truncated vIF-2α gene. Thus, in contrast to previous assumptions, tadpoles possess intact antiviral defenses reliant on type III IFNs, which are overcome by FV3 pathogens., Importance: Anuran tadpoles, including those of Xenopus laevis, are particularly susceptible to infection by ranavirus such as FV3. We investigated the respective roles of X. laevis type I and type III interferons (IFN and IFN-λ, respectively) during FV3 infections. Notably, tadpoles mounted timely and more robust IFN-λ gene expression responses to FV3 than adults, contrasting with the poorer tadpole type I IFN responses. However, a recombinant X. laevis IFN-λ (rXlIFN-λ) conferred less protection to tadpoles and the A6 cell line than rXlIFN, which may be explained by the FV3 impairment of IFN-λ receptor gene expression. The importance of IFN-λ in tadpole anti-FV3 defenses is underlined by the critical involvement of two putative immune evasion genes in FV3 resistance to IFN- and IFN-λ-mediated responses. These findings challenge the view that tadpoles have defective antiviral immunity and suggest, rather, that their antiviral responses are predominated by IFN-λ responses, which are overcome by FV3., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Macrophages are integral to amphibian immunity against RVs, as well as to the infection strategies of these pathogens. Although CSF-1 was considered to be the principal mediator of macrophage development, the IL-34 cytokine, which shares no sequence identity with CSF-1, is now believed to contribute to vertebrate monopoiesis. However, the respective roles of CSF-1- and IL-34-derived macrophages are still poorly understood. To delineate the contribution of these macrophage populations to amphibian immunity against the RV FV3, we identified the Xenopus laevis IL-34 and transcriptionally and functionally compared this cytokine with the previously identified X. laevis CSF-1. The X. laevis CSF-1 and IL-34 displayed strikingly nonoverlapping developmental and tissue-specific gene-expression patterns. Furthermore, only CSF-1 but not IL-34 was up-regulated in the kidneys of FV3-challenged tadpoles. Intriguingly, recombinant forms of these cytokines (rXlCSF-1, rXlIL-34) elicited morphologically distinct tadpole macrophages, and whereas rXlCSF-1 pretreatment decreased the survival of FV3-infected tadpoles, rXlIL-34 administration significantly prolonged FV3-challenged animal survival. Compared with rXlIL-34-elicited macrophages, macrophages derived by rXlCSF-1 were more phagocytic but also significantly more susceptible to in vitro FV3 infections. By contrast, rXlIL-34-derived macrophages exhibited significantly greater in vitro antiranaviral activity and displayed substantially more robust gene expression of the NADPH oxidase components (p67(phox), gp91(phox)) and type I IFN. Moreover, FV3-challenged, rXlIL-34-derived macrophages exhibited several orders of magnitude greater up-regulation of the type I IFN gene expression. This marks the first report of the disparate roles of CSF-1 and IL-34 in vertebrate antiviral immunity., (© 2014 Society for Leukocyte Biology.)

TCR-mediated specific recognition of antigenic peptides in the context of classical MHC molecules is a cornerstone of adaptive immunity of jawed vertebrate. Ancillary to these interactions, the T cell repertoire also includes unconventional T cells that recognize endogenous and/or exogenous antigens in a classical MHC-unrestricted manner. Among these, the mammalian nonclassical MHC class I-restricted invariant T cell (iT) subsets, such as iNKT and MAIT cells, are now believed to be integral to immune response initiation as well as in orchestrating subsequent adaptive immunity. Until recently the evolutionary origins of these cells were unknown. Here we review our current understanding of a nonclassical MHC class I-restricted iT cell population in the amphibian Xenopus laevis. Parallels with the mammalian iNKT and MAIT cells underline the crucial biological roles of these evolutionarily ancient immune subsets.

Natural infections of ectothermic vertebrates by ranaviruses (RV, family Iridoviridae) are rapidly increasing, with an alarming expansion of RV tropism and resulting die-offs of numerous animal populations. Notably, infection studies of the amphibian Xenopus laevis with the ranavirus Frog Virus 3 (FV3) have revealed that although the adult frog immune system is efficient at controlling RV infections, residual quiescent virus can be detected in mononuclear phagocytes of otherwise asymptomatic animals following the resolution of RV infections. It is noteworthy that macrophage-lineage cells are now believed to be a critical element in the RV infection strategy. In the present work, we report that inflammation induced by peritoneal injection of heat-killed bacteria in asymptomatic frogs one month after infection with FV3 resulted in viral reactivation including detectable viral DNA and viral gene expression in otherwise asymptomatic frogs. FV3 reactivation was most prominently detected in kidneys and in peritoneal HAM56+ mononuclear phagocytes. Notably, unlike adult frogs that typically clear primary FV3 infections, a proportion of the animals succumbed to the reactivated FV3 infection, indicating that previous exposure does not provide protection against subsequent reactivation in these animals.

The recent dramatic increase of the prevalence and range of amphibian host species and populations infected by ranaviruses such as Frog Virus 3 (FV3) raises concerns about the efficacies of amphibian antiviral immunity. In this context, the potential negative effects of water contaminants such as the herbicide atrazine, at environmentally relevant levels, on host antiviral immunity remains unclear. Here we describe the use of the amphibian Xenopus laevis as an ecotoxicology platform to elucidate the consequences of exposure to ecologically relevant doses of atrazine on amphibian antiviral immunity. X. laevis were exposed at tadpole and adult stages as well as during metamorphosis to atrazine (range from 0.1 to 10.0 ppb) prior to infection with FV3. Quantitative analysis of gene expression revealed significant changes in the pro-inflammatory cytokine, TNF-α and the antiviral type I IFN gene in response to FV3 infection. This was most marked in tadpoles that were exposed to atrazine at doses as low 0.1 ppb. Furthermore, atrazine exposure significantly compromised tadpole survival following FV3 infections. In contrast, acute atrazine exposure of mature adult frogs did not induce detectable effects on anti-FV3 immunity, but adults that were exposed to atrazine during metamorphosis exhibited pronounced defects in FV3-induced TNF-α gene expression responses and slight diminution in type I IFN gene induction. Thus, even at low doses, atrazine exposure culminates in impaired development of amphibian antiviral defenses., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Unlabelled: The increasing prevalence of ranavirus (RV; Iridoviridae) infections of wild and commercially maintained aquatic species is raising considerable concerns. While Xenopus laevis is the leading model for studies of immunity to RV, amphibian antiviral interferon (IFN) responses remain largely uncharacterized. Accordingly, an X. laevis type I interferon was identified, the expression of the gene for this IFN was examined in RV (frog virus 3 [FV3])-infected tadpoles and adult frogs by quantitative PCR, and a recombinant form of this molecule (recombinant X. laevis interferon [rXlIFN]) was produced for the purpose of functional studies. This rXlIFN protected the kidney-derived A6 cell line and tadpoles against FV3 infection, decreasing the infectious viral burdens in both cases. Adult frogs are naturally resistant to FV3 and clear the infection within a few weeks, whereas tadpoles typically succumb to this virus. Hence, as predicted, virus-infected adult X. laevis frogs exhibited significantly more robust FV3-elicited IFN gene expression than tadpoles; nevertheless, they also tolerated substantially greater viral burdens following infection. Although tadpole stimulation with rXlIFN prior to FV3 challenge markedly impaired viral replication and viral burdens, it only transiently extended tadpole survival and did not prevent the eventual mortality of these animals. Furthermore, histological analysis revealed that despite rXlIFN treatment, infected tadpoles had considerable organ damage, including disrupted tissue architecture and extensive necrosis and apoptosis. Conjointly, these findings indicate a critical protective role for the amphibian type I IFN response during ranaviral infections and suggest that these viruses are more pathogenic to tadpole hosts than was previously believed, causing extensive and fatal damage to multiple organs, even at very low titers., Importance: Ranavirus infections are threatening wild and commercially maintained aquatic species. The amphibian Xenopus laevis is extensively utilized as an infection model for studying ranavirus-host immune interactions. However, little is known about amphibian antiviral immunity and, specifically, type I interferons (IFNs), which are central to the antiviral defenses of other vertebrates. Accordingly, we identified and characterized an X. laevis type I interferon in the context of infection with the ranavirus frog virus 3 (FV3). FV3-infected adult frogs displayed more robust IFN gene expression than tadpoles, possibly explaining why they typically clear FV3 infections, whereas tadpoles succumb to them. Pretreatment with a recombinant X. laevis IFN (rXlIFN) substantially reduced viral replication and infectious viral burdens in a frog kidney cell line and in tadpoles. Despite reducing FV3 loads and extending the mean survival time, rXlIFN treatments failed to prevent tadpole tissue damage and mortality. Thus, FV3 is more pathogenic than was previously believed, even at very low titers.

During infection, macrophage lineage cells eliminate infiltrating pathogens through a battery of antimicrobial responses, where the efficacy of these innate immune responses is pivotal to immunological outcomes. Not surprisingly, many intracellular pathogens have evolved mechanisms to overcome macrophage defenses, using these immune cells as residences and dissemination strategies. With pathogenic infections causing increasing detriments to both aquacultural and wild fish populations, it is imperative to garner greater understanding of fish phagocyte antimicrobial responses and the mechanisms by which aquatic pathogens are able to overcome these teleost macrophage barriers. Insights into the regulation of macrophage immunity of bony fish species will lend to the development of more effective aquacultural prophylaxis as well as broadening our understanding of the evolution of these immune processes. Accordingly, this review focuses on recent advances in the understanding of teleost macrophage antimicrobial responses and the strategies by which intracellular fish pathogens are able to avoid being killed by phagocytes, with a focus on Mycobacterium marinum., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Macrophage-lineage cells are indispensable to vertebrate homeostasis and immunity. In turn, macrophage development is largely regulated through colony-stimulating factor-1 (CSF1) binding to its cognate receptor (CSF1R). To study amphibian monopoiesis, we identified and characterized the X. laevis CSF1R cDNA transcript. Quantitative analysis revealed that CSF1R tissue gene expression increased with X. laevis development, with greatest transcript levels detected in the adult lung, spleen and liver tissues. Notably, considerable levels of CSF1R mRNA were also detected in the regressing tails of metamorphosing animals, suggesting macrophage involvement in this process, and in the adult bone marrow; corroborating the roles for this organ in Xenopus monopoiesis. Following animal infections with the ranavirus Frog Virus 3 (FV3), both tadpole and adult X. laevis exhibited increased kidney CSF1R gene expression. Conversely, while FV3-infected tadpoles increased their spleen and liver CSF1R mRNA levels, the FV3-challenged adults did not. Notably, FV3 induced elevated bone marrow CSF1R expression, and while stimulation of tadpoles with heat-killed E. coli had no transcriptional effects, bacterial stimulation of adult frogs resulted in significantly increased spleen, liver and bone marrow CSF1R expression. We produced the X. laevis CSF1R in recombinant form (rXlCSF1R) and determined, via in vitro cross-linking studies, that two molecules of rXlCSF1R bound the dimeric rXlCSF1. Finally, administration of rXlCSF1R abrogated the rXlCSF1-induced tadpole macrophage recruitment and differentiation as well as bacterial and FV3-elicited peritoneal leukocyte accumulation. This work marks a step towards garnering greater understanding of the unique mechanisms governing amphibian macrophage biology.