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The chytrid fungus, Batrachochytrium dendrobatidis (Bd), infects amphibian skin, causing chytridiomycosis, which is a contributing cause of worldwide declines and extinctions of amphibians. Relatively little is known about the roles of amphibian skin-resident immune cells, such as macrophages, in these antifungal defenses. Across vertebrates, macrophage differentiation is controlled through the activation of colony-stimulating factor-1 (CSF1) receptor by CSF1 and interleukin-34 (IL34) cytokines. While the precise roles of these respective cytokines in macrophage development remain to be fully explored, our ongoing studies indicate that frog (Xenopus laevis) macrophages differentiated by recombinant forms of CSF1 and IL34 are functionally distinct. Accordingly, we explored the roles of X. laevis CSF1- and IL34-macrophages in anti-Bd defenses. Enriching cutaneous IL34-macrophages, but not CSF1-macrophages, resulted in significant anti-Bd protection. In vitro analysis of frog macrophage-Bd interactions indicated that both macrophage subsets phagocytosed Bd. However, IL34-macrophages cocultured with Bd exhibited greater pro-inflammatory gene expression, whereas CSF1-macrophages cocultured with Bd showed greater immunosuppressive gene expression profiles. Concurrently, Bd-cocultured with CSF1-macrophages, but not IL34-macrophages, possessed elevated expression of genes associated with immune evasion. This work marks a step forward in our understanding of the roles of frog macrophage subsets in antifungal defenses. 

IntroductionThe 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 theSpTransformer(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 marineVibrio. A recombinant (r)SpTrf-E1-Ec protein produced byE. colialso bindsVibriobut does not augment phagocytosis. MethodsTo address the question of whether other rSpTrf isoforms function as opsonins and augment phagocytosis, six rSpTrf proteins were expressed in insect cells. ResultsThe 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. ConclusionThese 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. 

Amphibian metamorphosis represents a dramatic example of post-embryonic development. In the anuran Xenopus laevis frog, this process involves extensive changes to larval tissues, structures, and physiology to produce its adult form. As a long-standing model to study tissue remodeling, both amphibian metamorphosis and mammalian development are under the control of thyroid hormone. Successful remodeling though, also requires precise temporospatial regulation of immune activation. Yet there is much to learn about the immune components linked to metamorphosis. In turn, granulocytes are a class of innate immune cells recently touted for their participation in processes beyond classical immune defenses, including in pathological and non-pathological tissue remodeling. In this manuscript, we explore the roles of granulocytes in perhaps the most conspicuous anuran metamorphic event: tadpole tail reabsorption. We characterize granulocyte infiltration into the tail as metamorphosis progresses. Although some granulocyte subpopulations exist in both Xenopus and mammals, our previous work has identified additional Xenopus-specific populations. Thus, here we further explored subpopulation dynamics through distinct stages of natural metamorphosis, their likely roles during this process, and their relationship with thyroid hormone. As endocrine disruptors continue to threaten species across the animal kingdom, the work described here offers much-needed insight into immune contributions to endocrine-linked development. 

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 controllingBd, yet almost nothing is known about the roles of skin-resident immune cells in anti-Bddefenses. Mammalian mast cells reside within and serve as key immune sentinels in barrier tissues like skin. Accordingly, we investigated the roles ofXenopus laevisfrog mast cells duringBdinfections. Our findings indicate that enrichment ofX. laevisskin mast cells confers anti-Bdprotection and ameliorates the inflammation-associated skin damage caused byBdinfection. This includes a significant reduction in infiltration ofBd-infected skin by neutrophils, promoting mucin content within cutaneous mucus glands, and preventingBd-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 theX. laevisIL4 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-Bddefenses and illuminates a novel avenue for investigating amphibian host–chytrid pathogen interactions. 

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. 

Macrophage (Mϕ)-lineage cells are integral to the immune defences of all vertebrates, including amphibians. Across vertebrates, Mϕdifferentiation 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) Mϕs differentiated with CSF1 and IL34 are morphologically, transcriptionally and functionally distinct. Notably, mammalian Mϕs share common progenitor population(s) with dendritic cells (DCs), which rely on fms-like tyrosine kinase 3 ligand (FLT3L) for differentiation whileX. laevisIL34-Mϕs exhibit many features attributed to mammalian DCs. Presently, we comparedX. laevisCSF1- and IL34-Mϕs with FLT3L-derivedX. laevisDCs. Our transcriptional and functional analyses indicated that indeed the frog IL34-Mϕs and FLT3L-DCs possessed many commonalities over CSF1-Mϕs, including transcriptional profiles and functional capacities. Compared toX. laevisCSF1-Mϕs, the IL34-Mϕs and FLT3L-DCs possess greater surface major histocompatibility complex (MHC) class I, but not MHC class II expression, were better at eliciting mixed leucocyte responsesin vitroand generatingin vivore-exposure immune responses againstMycobacterium marinum. Further analyses of non-mammalian myelopoiesis akin to those described here, will grant unique perspectives into the evolutionarily retained and diverged pathways of Mϕand 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. 

Global amphibian declines are largely driven by deadly disease outbreaks caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd). In the time since these disease outbreaks were first discovered, much has been learned about the roles of amphibian skin-produced antimicrobial components and skin microbiomes in controlling Bd. Yet almost nothing is known about the roles of skin-resident immune cells in anti-Bd defenses. Notably, mammalian mast cells reside within and serve as key immune sentinels in barrier tissues like the skin. Thus, they are critical to immune recognition of pathogens and to orchestrating the ensuing immune responses. 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 significant anti-Bd protection and ameliorates the inflammation-associated skin damage caused by Bd infection. Moreover, enriching X. laevis mast cells promotes greater mucin content within cutaneous mucus glands and protects frogs from Bd-mediated changes to their skin microbiomes. Together, this work underlines the importance of amphibian skin-resident immune cells in anti-Bd defenses and introduces a novel approach for investigating amphibian host-chytrid pathogen interactions. 

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.