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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. 

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. 

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. 

Abstract 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. 

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. 

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.