Leon Grayfer

Comparison of macrophage antimicrobial responses induced by type II interferons of the goldfish (Carassius auratus L.).

Unlike mammals, bony fish have two type II interferons, IFNγ and IFNγrel, 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) IFNγ and IFNγrel on the macrophage antimicrobial responses, immune gene expression, and their signaling pathways. Our findings indicate that rgIFNγ and rgIFNγrel possess unique capacities to mediate each of the above processes. Q-PCR analysis revealed similar expression of both cytokines in tissues and immune cell populations of the goldfish, although IFNγ mRNA levels were generally higher in most tissues and cell types. Whereas rgIFNγ had long-lasting effects on the priming of goldfish monocyte ROI production, the rgIFNγrel had relatively short-lived ROI priming potential and eventually down-regulated the priming of ROI production induced by rgIFNγ or rgTNFα2. Whereas rgIFNγ induced relatively modest phagocytic and nitric oxide responses of goldfish macrophages, rgIFNγrel induced significantly higher phagocytosis, iNOSA and iNOSB gene expression and nitric oxide production compared with rgIFNγ. The rgIFNγ and rgIFNγrel induced different gene expression profiles in goldfish monocytes. These differences included significantly higher induction of TNFα2, CXCL8, ceruloplasmin, and interferon regulatory factor (IRFs) expression after activation of monocytes with rgIFNγrel. The rgIFNγrel was more abundant in whole cell lysates compared with rgIFNγ. Both cytokines induced the phosphorylation of Stat1, while the nuclear localization of Stat1 was only observed following treatment of monocytes with rgIFNγ. Our findings suggest the presence of functional segregation of the induction of macrophage antimicrobial functions by type II interferons of bony fish.

Immune Evasion Strategies of Ranaviruses and Innate Immune Responses to These Emerging Pathogens

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 barriers of numerous poikilotherms, suggesting that these pathogens possess a broad host range and potent immune evasion mechanisms. Indeed, while some of the 95–100 predicted ranavirus genes encode putative evasion proteins (e.g., vIFα, vCARD), roughly two-thirds of them do not share significant sequence identity with known viral or eukaryotic genes. Accordingly, the investigation of ranaviral virulence and immune evasion strategies is promising for elucidating potential antiviral targets. In this regard, recombination-based technologies are being employed to knock out gene candidates in the best-characterized RV member, Frog Virus (FV3). Concurrently, by using animal infection models with extensively characterized immune systems, such as the African clawed frog, Xenopus laevis, it is becoming evident that components of innate immunity are at the forefront of virus-host interactions. For example, cells of the macrophage lineage represent important combatants of RV infections while themselves serving as targets for viral infection, maintenance and possibly dissemination. This review focuses on the recent advances in the understanding of the RV immune evasion strategies with emphasis on the roles of the innate immune system in ranaviral infections.

Colony-Stimulating Factor-1-Responsive Macrophage Precursors Reside in the Amphibian (Xenopus laevis) Bone Marrow rather than the Hematopoietic Subcapsular Liver

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 leukocyte precursors in favor of the liver subcapsular region, where hematopoiesis occurs in anurans. Here we report that the bone marrow rather than the liver periphery provides macrophage progenitors to the amphibian Xenopus laevis. We identified the amphibian CSF-1, examined its gene expression in developing and virally infected X. lae vis and produced it in recombinant form (rXlCSF-1). This rXlCSF-1 did not bind or elicit proliferation/differentiation of subcapsular liver cells. Surprisingly, a subpopulation of bone marrow cells engaged this growth factor and formed rXlCSF-1 concentration-dependent colonies in semisolid medium. Furthermore, rXlCSF-1-treated bone marrow (but not liver) cultures comprised of cells with characteristic macrophage morphology and high gene expression of the macrophage marker CSF-1 receptor. Together, our findings indicate that in contrast to all other vertebrates studied to date, committed Xenopus macrophage precursor populations are not present at the central site of hematopoiesis, but reside in the bone marrow.

Biology of Bony Fish Macrophages

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.

Amphibian macrophage development and antiviral defenses.

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.

Inflammation-Induced Reactivation of the Ranavirus Frog Virus 3 in Asymptomatic Xenopus laevis

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.

Water Temperature Affects Susceptibility to Ranavirus.

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.

Immune roles of amphibian (Xenopus laevis) tadpole granulocytes during Frog Virus 3 ranavirus infections

Abstract 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 TNF&agr; 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. HighlightsG‐CSF differentiates X. laevis tadpole granulocytes, which upregulated TNF gene expression in response to FV3.G‐CSF extends FV3‐infected tadpole survival and reduced their kidney viral loads.G‐CSF granulocytes are less susceptible to viral entry but support greater FV3 replication compared to M‐CSF macrophages.Tadpole susceptibility to FV3 may reflect their inability to recruit granulocytes into their kidney sites of FV3 infection.

Amphibian (Xenopus laevis) tadpoles and adult frogs mount distinct interferon responses to the Frog Virus 3 ranavirus.

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.

Mechanisms of amphibian macrophage development: characterization of the Xenopus laevis colony-stimulating factor-1 receptor.

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.