Leon Grayfer

• Professor (Assistant) at George Washington University

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 k...

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, macro...

Probiotic therapies have been suggested for amelioration efforts of wildlife disease such as chytridiomycosis caused by Batrachochytrium spp. in amphibians. However, there is a lack of information on how probiotic application affects resident microbial communities and immune responses. To better understand these interactions, we hypothesized that p...

Introduction The California purple sea urchin, Strongylocentrotus purpuratus, relies exclusively on an innate immune system to survive in its pathogen rich marine environment. Central to this defense is the SpTransformer (SpTrf) gene family that is unique to the euechinoid group of echinoderms. These genes were initially identified based on their s...

Ranaviruses (RV, family Iridoviridae ) infect fish, amphibians, and reptiles, raising considerable ecological and commercial concerns due to the escalating infection prevalence and the resulting die-offs of wild and aquacultural species. Notably, ranaviruses exhibit uncanny capacities to cross host species barriers, likely owing to their potent imm...

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...

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...

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 bot...

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 bo...

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 noth...

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 noth...

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 a...

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 n...

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...

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 cel...

Xenopus is a genus of African 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, inac...

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...

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...

Cross-species comparison of vertebrate genomes has unraveled previously unknown complexities of interferon (IFN) systems in amphibian species. Recent genomic curations 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-con...

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 coelomo...

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...

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 rap...

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...

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,...

Ranaviruses have been associated with rising numbers of mass die-offs in amphibian populations around the globe. However, most studies on ranaviruses to date focused on larval amphibians. To assess the role of postmetamorphic amphibians in the epidemiology of ranaviruses and to determine the role of viral immune-suppression genes, we performed a ba...

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 challen...

The ranavirus Frog Virus 3 (FV3) and the chytrid fungus Batrachochytrium dendrobatidis (Bd) are significant contributors to the global amphibian declines and both pathogens target the amphibian skin. We previously showed that tadpoles and adults of the anuran amphibian Xenopus laevis express notable levels of granulocyte chemokine genes (cxcl8a and...

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 fa...

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...

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 hematopoi...

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 cau...

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 ch...

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...

Ranaviruses have been associated with rising numbers of mass die-offs in amphibian populations globally. With life-stages occupying different environments and presenting distinct physiologies, amphibian of different ages are likely to play an important role in pathogen persistence. To assess the potential role of post-metamorphic amphibians as a Ra...

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 class...

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 se...

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 (...

Western blot of the produced, purified, and eluted rCXCL8a and rCXCL8b fractions. The eluted recombinant (r)CXCL8a and rCXCL8b were resolved by SDS PAGE, transferred onto nitrocellulose membranes and western blots were performed using an HRP-conjugated mouse anti-V5 (Sigma) to determine which elution fractions contained rCXCL8a (15 kDa) and rCXCL8b...

Analyses of the anti-G-CSFR polyclonal antibody and the rCXCL8a- and rCXCL8b-elicited granulocyte expression of the Cxcr1 and Cxcr2 genes. (A) Rabbits were immunized with rG-CSFR and the resulting serum was applied to a HiTrap Proetin A HP column (GE Health) to isolate the IgG fraction, and to a rG-CSFR-bound Sulfo-Link Protein column to purify the...

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 increa...

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 vertebra...

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 mortali...

Infections by ranaviruses such as Frog virus 3 (Fv3) are significantly contributing to the 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 re...

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 mononuc...

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...

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...

Amphibians represent a key stage in vertebrate evolution and exhibit incredible richness of physiological diversities across the species comprising this class. Unfortunately, the worldwide amphibian decline threatens amphibian communities, populations and even entire species. It is presently believed that compounding factors result in immune suppre...

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 t...

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 supbcapsular liver and inste...

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 specie...

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 funct...

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 resu...

We have identified and characterized two putative virulence and/or immune evasion genes in the ranavirus FV3 by engineering knockout (KO) defective mutants and using Xenopus tadpoles and Xenopus kidney cell lines.

The ITAM-bearing transmembrane signaling subunits (TSS) are indispensable components of activating leukocyte receptor complexes. The TSS-encoding genes map to paralogous chromosomal regions, which are thought to arise from ancient genome tetraploidization(s). To assess a possible role of tetraploidization in the TSS evolution, we studied TSS and ot...

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 d...

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 no...

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 reveale...

Ranaviruses (RV, Iridoviridae) are now known to infect fish, amphibians, and reptiles, raising considerable ecological and commercial concerns due to the escalating infection prevalence and the resulting die-offs of wild and aquacultural species. Notably, ranaviruses exhibit uncanny capacities to cross host species barriers of their poikilothermic...

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 imm...

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 respectiv...

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 the...

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...

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...

Human and murine MHC nonclassical class Ib-restricted invariant T (iT) cell subsets, such as invariant natural killer T cells (iNKT) and mucosal-associated invariant T cells, have specialized functions early in immune responses, especially in modulating subsequent adaptive immune responses. Here, we characterize a prominent iT population in the amp...

Macrophage precursors originate from and undergo lineage commitment within designated sites of hematopoiesis, such as the mammalian bone marrow. These cells subsequently differentiate in response to stimulation with macrophage colony-stimulating factor-1 (CSF-1). The amphibian bone marrow, unlike that of mammals, has been overlooked as a source of...

Phagocytosis is a cellular mechanism that is important to the early induction of antimicrobial responses and the regulation of adaptive immunity. At an inflammatory site, phagocytes serve as central regulators for both pro-inflammatory and homeostatic anti-inflammatory processes. However, it remains unclear if this is a recent evolutionary developm...

Cycloheximide primarily induces apoptosis in treated cells and can be effectively removed from apoptotic body preparations. (A) Representative experiments show apoptotic bodies generated for treatment of goldfish phagocytes. 3B11 catfish B cells were cultured for 24 h in the presence of 10 μg/mL cycloheximide. Cells were subsequently harvested and...

Characterization of goldfish peritoneal myeloid cells. (A) Total peritoneal exudates were analyzed by imaging flow cytometry and four distinct cellular subsets characterized based on area, internal complexity, and morphology. Unlike macrophage and lymphocyte subsets, monocytes and neutrophils could not be subdivided into two distinct populations so...

Mean fluorescence intensity of teleost and murine phagocytes. Goldfish and mice were injected intraperitoneally with saline, species-specific apoptotic bodies (5×106) or zymosan (2.5 mg). Apoptotic bodies were also pre-injected 4 h before zymosan injections. Cells from injected animals were harvested by peritoneal lavage and respiratory burst was a...

Two distinct sources of apoptotic bodies repress goldfish macrophage respiratory burst to equivalent levels. PKM cultures were incubated with apoptotic bodies derived from 3B11 B cells or goldfish kidney leukocytes. Cells were incubated for 2 h (5∶1, particle: cell ratio) and respiratory burst was analyzed by DHR. There was no significant differenc...

Gating strategy for cell subpopulations isolated from murine peritoneum. (A) Peritoneal cells from C57BL/6 mice were stained with combination of CD11b-FITC/Gr1-PE/F4/80-APC or CD11b-PE-Cy7/CD3-FITC/B220-PE/NK1.1-APC to determine the infiltration of granulocytes (F4/80−/Gr1+/CD11b+), macrophages (F4/80hi/Gr1+/−/CD11b+), monocytes (F4/80lo/Gr1+/−/CD1...

The rapid doubling time and genetic relatedness of the fish pathogen Mycobacterium marinum to Mycobacterium tuberculosis has rendered the former an attractive model for investigating mycobacterial host-pathogen interactions. We employed the M. marinum-goldfish infection model to investigate the in vivo immune responses to this pathogen in the conte...

Xenopus laevis adults mount effective immune responses to ranavirus Frog Virus 3 (FV3) infections and clear the pathogen within 2-3 weeks. In contrast, most tadpoles cannot clear FV3 and succumb to infections within a month. While larval susceptibility has been attributed to ineffective adaptive immunity, the contribution of innate immune component...

Ranaviruses (RV, Iridoviridae) are large double-stranded DNA viruses that infect fish, amphibians and reptiles. For ecological and commercial reasons, considerable attention has been drawn to the increasing prevalence of ranaviral infections of wild populations and in aquacultural settings. Importantly, RVs appear to be capable of crossing species...

The role of lipid rafts in non-mammalian leukocytes has been scarcely investigated. We performed biochemical and functional analysis of lipid rafts in fish leukocytes. Fish Flotillin-1 and a fish GM1-like molecule (fGM1-L) were found in low density detergent-resistant membranes (LD-DRM) in goldfish macrophages and catfish B lymphocytes, similarly t...

This is the first report of the identification and molecular characterization of an interleukin-10 receptor 1 in bony fish. By gene synteny analysis, we identified the zebrafish interleukin-10 receptor 1 (IL10R1) and using this IL10R1 sequence, we cloned the goldfish IL10R1 cDNA transcript. The identified fish IL10R1 protein sequences had a putativ...

This is the first report of comprehensive functional analysis of an interleukin-10 in bony fish. Quantitative expression analysis of goldfish IL-10 revealed the greatest mRNA levels in the spleen tissues, peripheral blood leukocytes and granulocytes. The stimulation of cells with recombinant goldfish (rg) TNFα2 significantly reduced IL-10 mRNA leve...

Unlike mammals, bony fish have two type II interferons, IFNgamma and IFNgammarel, whose pro-inflammatory functions have not been fully characterized. To elucidate the distinct roles of these type II interferons of bony fish, we examined the effects of recombinant goldfish (rg) IFNgamma and IFNgammarel on the macrophage antimicrobial responses, immu...

We previously reported that SHP-1 regulates IRAK-1 activity by binding to an ITIM-like motif found within its kinase domain, which we named kinase tyrosine-based inhibitory motif (KTIM). Herein, we further investigated the presence, number, location, and evolutionary time of emergence of potential KTIMs in many cytosolic kinases, all known to play...