Antimicrobial Immunity Macrophage and granulocyte lineage immune cells are integral to all vertebrate antimicrobial defenses. These cells are the first to recognize infiltrating pathogens and orchestrate the ensuing immune responses. Perhaps for these reasons, many pathogens have evolved to evade, thrive and disseminate within these cell types.
While the best-characterized animal model of amphibian immunity is the Xenopus laevis African clawed frog, the immune mechanisms governing frog susceptibility and resistance to pathogens (such as those contributing to their declines) remain largely undefined.
We are currently exploring the roles of distinct tadpole and adult frog macrophage and granulocyte populations in the susceptibility and immunological resistance of these animals to viral, bacterial and fungal pathogens. Our research is presently focused on defining the molecular drivers of those antimicrobial mechanisms that confer cellular resistance against these respective pathogens and the molecular determinants that render distinct immune cell populations more susceptible to distinct pathogens.
Cells of the Innate Immune System To understand the facets of amphibian susceptibility and resistance to different pathogens, we are exploring the cellular differentiation pathways that culminate in distinct macrophage and granulocyte subsets. To this end, we are generating in recombinant form, key frog immune cell differential factors and exploring the roles of these moieties in innate immune cell development, both in vitro and in vivo.
For example, we found that macrophages differentiated with the macrophage colony-stimulating factor (M-CSF) and interleukin-34 (IL-34) growth factors are morphologically and functionally distinct. In particular, we are seeing that while the IL-34-macropahges are highly resistant to intracellular pathogens and protect animals from infections, the M-CSF-macrophages are significantly more susceptible to infiltrating pathogens and compromise infected animals.
Viruses, Bacteria and Fungi Macrophages and granulocytes represent indispensable barriers to invading pathogens, but may also be usurped by infectious agents in order to evade the immune system and thrive within their hosts. To better understand the facts of these cells' susceptibility and resistance to different types of pathogens, we have established several approaches for differentiating distinct frog macrophage and granulocyte populations, both in vitro and in vivo. We are now using these methods to define the roles of distinct macrophage and granulocyte subsets in host susceptibility and resistance to important viral, bacterial and fungal pathogens.
The Frog Virus 3 Ranavirus and antiviral immunity: Amphibian infections by Frog Virus 3 (FV3) and other ranavirus genus members are causing large-scale population die-offs, thus significantly contributing to the worldwide amphibian declines. Ranaviruses are large, icosahedral, dsDNA viruses that manifest in systemic diseases, hemorrhaging and necrotic cell death within multiple afflicted organs of their amphibian hosts. Our recent work indicates that the frog IL-34-macrophages and certain granulocyte subsets are crucial to the clearance of this virus. By contrast, the frog M-CSF-macrophages are highly susceptible to this pathogen and exacerbate FV3 infections. It is particularly notable that amphibian tadpoles are significantly more susceptible to, and die from ranavirus infections, whereas adult frogs also suffer from these infections but more readily clear this pathogens. Notably, our research indicates that this tadpole susceptibility to ranaviruses stems at least in part from their failure to generate appropriate antiviral macrophage and granulocyte responses.
Antiviral interferon cytokines: The antiviral immune responses of all vertebrate species depend on antiviral interferon (IFN) cytokines (soluble mediators), and primarily rely on type I and type III IFNs. Mammals, birds and reptiles possess intronless type I IFN cytokines and type III IFNs with a five exon/four intron gene organization. By contrast, bony fish possess only type I IFNs that are phylogenetically related to the mammalian type I IFNs but exhibit five exon/four intron gene organization. Strikingly, amphibians encode highly expanded repertoires of intron-containing and intronless type I and type III IFNs (IFNs, IFNLs, IFNXs, IFNLXs, respectively). Although the antiviral efficacies of these frog cytokines remain largely undefined, it is intuitive that greater insights into the roles of these respective type I and type III IFNs during ranavirus infections will significantly contribute to the understanding of amphibian immune susceptibility and resistance to these pathogens. We recently showed that tadpoles and adult frogs mount drastically different antiviral IFN responses to FV3, which presumably also reflects the differences in the capacities of tadpoles and adult frogs to control these viral infections. This suggests that the different tadpole- and adult frog-expressed type I and type III IFNs possess distinct antiviral capacities; a hypothesis that we are now actively pursuing. Mycobacteria and antimicrobial immunity: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains the leading global cause of death resulting from an infectious agent. Presently, there is no single animal model of this disease that permits both in vitro and in vivo study of mycobacteria-host immune interactions. This has stalled the development of more effective preventative and therapeutic measures against TB. Notably, Mycobacterium marinum, the causative agent of fish and frog tuberculosis has become an auspicious model for TB research, owing to its close genetic relatedness to TB and the availability of alternative, natural host animal models such as zebrafish, and as proposed here, frogs. Both M. tuberculosis and M. marinum rely on macrophages to infiltrate their hosts and establish latent infections through the formation of granulomas, which are characteristic of TB disease. We have established the X. laevis frog as model of vertebrate macrophage immunity to mycobacteria. Notably, akin to their FV3-susceptibility, the frog M-CSF-macrophages are much more susceptible to M. marinum infection, transmission and establishment of disease. By contrast, our work indicates that the IL-34-macrophages are highly resistant to mycobacteria, transmit significantly less of this pathogen and restrict the progression of mycobacterial infection. Using these two macrophage populations as archetypes of cellular susceptibility and resistance to this pathogen; we are exploring the molecular determinants of macrophage susceptibility and resistance to mycobacteria.
The chytrid fungus and amphibian anti-fungal defenses: Infections of amphibians with the Batrachochytrium dendrobatidis (Bd) chytrid fungus has culminated in devastating population declines and even extinction, with the global amphibian declines significantly compounded by this pathogen. While the host-Bd immune interactions remain poorly understood, this pathogen is now know to possess numerous mechanisms by which it modulates and evades the amphibian immune system. Because the amphibian macrophages and granulocytes are the first immune cells to encounter Bd, we are focusing our research on defining the immune outcomes to Bd detection/contact with distinct frog macrophage and granulocyte subsets. In particular, we are interested in determining which of these frog immune cell subsets are important to fighting off this invading fungus and which of the subsets fall victim to the immuno-modulatory effects of this pathogen.
Integrating Physiology and Immunity Many 'immune' cells such as macrophages and granulocytes play important 'non-immune' roles in maintaining the physiological integrity of respective vertebrate species. In turn and as you may imagine, different animals posses very distinct physiologies, depending on the environmental, developmental and reproductive pressures that have shaped them. These differences also reflect in the pathways by which immune cells such as macrophages and granulocytes may develop as well as the routes of infection and the tissues targeted by the pathogens that plague the respective organisms. Thus, to better understand amphibian immunity in the context of their physiology, we have adopted comprehensive approaches to discern the tissue sources of distinct innate immune cell subsets, how these differentiation pathways may differ between tadpoles and adult frogs and what consequences these differences may have on the pathogens that target the distinct organs of the tadpole and adult frog stages.
Hepatic and bone marrow blood cell development: Most vertebrates possess designated sites for blood cell development such as the mammalian bone marrow or the bony fish head kidney. Until recently, the anuran (frog, toad) peripheral liver was thought to be the principal site of all blood cell development. We recently demonstrated that while most frog blood cell precursors, including those of red blood cells, reside in this sub-capsular liver area, the adult frog bone marrow (but not peripheral liver) host macrophage and granulocyte precursors. This suggests that by contrast to other animals, frog segregate their blood cell development to (at least) two distinct organs. Considering that frogs do not develop their bone marrow until some time into metamorphosis; we are interested in determining where the tadpole granulocyte and macrophage precursors arise and the consequences of these differences in the tadpole and adult frog cell ontogeny on their physiology and immunity.