Barbara A. Katzenback

Isolation and functional characterization of neutrophil-like cells, from goldfish (Carassius auratus L.) kidney

Neutrophils are one of the first cells to arrive at the site of inflammation and are central cells of innate immunity of vertebrates. Upon activation, neutrophils are capable of producing an intense respiratory burst response, they rapidly degranulate in the presence of pathogens, and deploy neutrophil extracellular traps that efficiently remove infections agents. We report on the isolation, cultivation and characterization of neutrophil-like cells isolated from the goldfish kidney. Neutrophil-like cells stained positive for acid phosphatase and Sudan black, expressed MPO, and produced a dose-dependent respiratory burst response after stimulation with phorbol esters (PMA) and calcium ionophore A23187 (CaI). Treatment of neutrophil-like cultures with PMA, CaI or zymosan induced a dose-dependent non-reversible degranulation of neutrophil-like cells. Goldfish neutrophil-like cells also exhibited a chemotactic response, degranulation, and respiratory burst response when exposed to the virulent fish pathogen, Aeromonas salmonicida A449.

Fish and Mammalian Phagocytes Differentially Regulate Pro-Inflammatory and Homeostatic Responses In Vivo

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 development or whether the capacity to balance between these two seemingly contradictory processes is a feature already displayed in lower vertebrates. In this study, we used murine (C57BL/6) and teleost fish (C. auratus) in vitro and in vivo models to assess the evolutionary conservation of this dichotomy at a site of inflammation. At the level of the macrophage, we found that teleost fish already displayed divergent pro-inflammatory and homeostatic responses following internalization of zymosan or apoptotic bodies, respectively, and that these were consistent with those of mice. However, fish and mice displayed significant differences in vivo with regards to the level of responsiveness to zymosan and apoptotic bodies, the identity of infiltrating leukocytes, their rate of infiltration, and the kinetics and strength of resulting antimicrobial responses. Unlike macrophages, significant differences were identified between teleost and murine neutrophilic responses. We report for the first time that activated murine, but not teleost neutrophils, possess the capacity to internalize apoptotic bodies. This internalization translates into reduction of neutrophil ROS production. This may play an important part in the recently identified anti-inflammatory activity that mammalian neutrophils display during the resolution phase of inflammation. Our observations are consistent with continued honing of inflammatory control mechanisms from fish to mammals, and provide added insights into the evolutionary path that has resulted in the integrated, multilayered responses that are characteristic of higher vertebrates.

Characterization of three Nod-like receptors and their role in antimicrobial responses of goldfish (Carassius auratus L.) macrophages to Aeromonas salmonicida and Mycobacterium marinum.

The nucleotide-binding oligomerization domain proteins Nod1, Nod2 and Nlrx1 are cytoplasmic pathogen recognition receptors (PRRs) of the Nod-like receptor (NLR) family. In this report, goldfish Nod1 (gfNod1), Nod2 (gfNod2) and Nlrx1 (gfNlrx1) genes were cloned and characterized. The full length of gfNod1, gfNod2 and gfNlrx1 were 3234bp, 3129bp and 4900bp, encoding 937, 982 and 1008 amino acids, respectively. The three Nod-like receptors have a NACHT domain and C-terminal leucine rich repeat (LRR) domains. In addition to these, gfNod1 and gfNod2 also had an N-terminal CARD domain (two in gfNod2). Phylogenetic analysis showed that the three NLRs are highly conserved. Quantitative gene expression analysis of the three receptors revealed the greatest mRNA levels in the spleen, and in isolated neutrophils and splenocytes. Furthermore, treatment of goldfish macrophages with LPS, Poly I:C, MDP, PGN, heat-killed Aeromonas salmonicida or Mycobacterium marinum differentially altered the expression of the Nod-like receptors. Our results indicate that Nod-like receptors are functionally highly conserved and that they play a pivotal role in recognition of fish pathogens such as A. salmonicida and M. marinum.

Antimicrobial Peptides as Mediators of Innate Immunity in Teleosts

Antimicrobial peptides (AMPs) have been identified throughout the metazoa suggesting their evolutionarily conserved nature and their presence in teleosts is no exception. AMPs are short (18–46 amino acids), usually cationic, amphipathic peptides. While AMPs are diverse in amino acid sequence, with no two AMPs being identical, they collectively appear to have conserved functions in the innate immunity of animals towards the pathogens they encounter in their environment. Fish AMPs are upregulated in response to pathogens and appear to have direct broad-spectrum antimicrobial activity towards both human and fish pathogens. However, an emerging role for AMPs as immunomodulatory molecules has become apparent—the ability of AMPs to activate the innate immune system sheds light onto the multifaceted capacity of these small peptides to combat pathogens through direct and indirect means. Herein, this review focuses on the role of teleost AMPs as modulators of the innate immune system and their regulation in response to pathogens or other exogenous molecules. The capacity to regulate AMP expression by exogenous factors may prove useful in modulating AMP expression in fish to prevent disease, particularly in aquaculture settings where crowded conditions and environmental stress pre-dispose these fish to infection.

Development of macrophages of cyprinid fish

The innate immune responses of early vertebrates, such as bony fishes, play a central role in host defence against infectious diseases and one of the most important effector cells of innate immunity are macrophages. In order for macrophages to be effective in host defence they must be present at all times in the tissues of their host and importantly, the host must be capable of rapidly increasing macrophage numbers during times of need. Hematopoiesis is a process of formation and development of mature blood cells, including macrophages. Hematopoiesis is controlled by soluble factors known as cytokines, that influence changes in transcription factors within the target cells, resulting in cell fate changes and the final development of specific effector cells. The processes involved in macrophage development have been largely derived from mammalian model organisms. However, recent advancements have been made in the understanding of macrophage development in bony fish, a group of organisms that rely heavily on their innate immune defences. Our understanding of the growth factors involved in teleost macrophage development, as well as the receptors and regulatory mechanisms in place to control them has increased substantially. Furthermore, model organisms such as the zebrafish have emerged as important instruments in furthering our understanding of the transcriptional control of cell development in fish as well as in mammals. This review highlights the recent advancements in our understanding of teleost macrophage development. We focused on the growth factors identified to be important in the regulation of macrophage development from a progenitor cell into a functional macrophage and discuss the important transcription factors that have been identified to function in teleost hematopoiesis. We also describe the findings of in vivo studies that have reinforced observations made in vitro and have greatly improved the relevance and importance of using teleost fish as model organisms for studying developmental processes.

The Analysis of Goldfish (Carassius auratus L.) Innate Immune Responses After Acute and Subchronic Exposures to Oil Sands Process-Affected Water

We examined the immunotoxic effects of acute and subchronic exposures of goldfish to aged, fresh, and ozonated oil sands process-affected water (OSPW) using a flow-through exposure apparatus. We measured the expression of proinflammatory cytokine genes, the antimicrobial responses of primary macrophages isolated from OSPW-exposed fish, and the ability of the goldfish to control infection with a protozoan parasite, Trypanosoma carassii. After acute (1 week) exposure to aged OSPW, we observed upregulation in the expression of interferon gamma (IFN-γ), tumor necrosis factor alpha-2 (TNF-α2) in the kidney and spleen but not in gills of the fish. After subchronic (12 weeks) exposure to aged OSPW, we observed significant increases in mRNA levels of proinflammatory genes in the gill (IFN-γ, interleukin-1 beta 1 [IL1-β1], TNF-α2), kidney (IL1-β1, TNF-α2), and spleen (IL1-β1). An upregulation of immune gene expression in the gill and kidney (IFN-γ, IL1-β1, TNF-α2) and spleen (IL1-β1, TNF-α2) was observed after acute exposure of fish to diluted fresh OSPW. Following subchronic exposure to diluted fresh OSPW, we observed high mRNA levels of IL1-β1 in all tissues examined. However, there were significant decreases in the mRNA levels of IFN-γ and TNF-α2 in the kidney and spleen and gill and spleen (IL-12p35 and IL-12p40) of exposed fish. There were no changes in the expression of anti-inflammatory cytokine IL-10 after both acute and subchronic exposures to diluted fresh OSPW. In fish exposed to ozonated fresh OSPW, immune gene expression was similar to nonexposed control fish in all organs examined, with exception of IL1-β1. The ability of primary kidney macrophages to generate reactive oxygen and nitrogen intermediates was significantly reduced in fish exposed to fresh OSPW. The enhanced proinflammatory response after acute exposure to diluted fresh OSPW was confirmed by the parasite challenge experiments, where OSPW-exposed fish controlled the infection better than nonexposed fish.

Characterization of granulocyte colony stimulating factor receptor of the goldfish (Carassius auratus L.)

Granulocyte colony stimulating factor receptor (GCSFR) is a member of the class I cytokine receptor superfamily and signaling through this receptor is important for the proliferation, differentiation and activation of neutrophils and their precursors. In this study we report on the cloning and molecular characterization of goldfish GCSFR. The identified goldfish GCSFR sequence possesses the conserved Ig-like domain, the cytokine receptor homology domain (CRH), three fibronectin domains as well as several intracellular signaling motifs characteristic of other vertebrate GCSFRs. Goldfish gcsfr mRNA was highly expressed in kidney and spleen, and in primary kidney neutrophils. The neutrophils have significantly higher mRNA levels of the transcription factors pu.1 and cebpα, and down-regulated levels of transcription factors important for macrophage development such as egr1 and cjun, compared to progenitor cells from the kidney. The gcsfr mRNA was present in the kidney progenitor cells, albeit at much lower levels compared to the neutrophils, and the expression of gcsfr in progenitor cells was not affected by duration of cultivation. Furthermore, gcsfr mRNA levels were up-regulated in neutrophils after treatment with heat-killed Aeromonas salmonicida A449 or with mitogens. Our results indicate that GCSFR may be a useful marker for fish neutrophils.

Molecular and functional characterization of erythropoietin receptor of the goldfish (Carassius auratus L.)

Erythropoietin receptor (EPOR) is a member of the class I cytokine receptor superfamily and signaling through this receptor is important for the proliferation, differentiation and survival of erythrocyte progenitor cells. This study reports on the molecular and functional characterization of goldfish EPOR. The identified goldfish EPOR sequence possesses the conserved EPOR ligand binding domain, the fibronectin domain, the class I cytokine receptor superfamily motif (WSXWS) as well as several intracellular signaling motifs characteristic of other vertebrate EPORs. The expression of epor mRNA in goldfish tissues, cell populations and cells treated with recombinant goldfish EPO (rgEPO) were evaluated by quantitative PCR revealing that goldfish epor mRNA is transcribed in both erythropoietic tissues (blood, kidney and spleen) and non-hematopoietic tissues (brain, heart and gill), as well as in immature erythrocytes. Recombinant goldfish EPOR (rgEPOR), consisting of its extracellular domain, dose-dependently inhibited proliferation of progenitor cells induced by rgEPO. In vitro binding studies indicated that rgEPO exists as monomer, dimer and/or trimmer and that rgEPOR exists as monomer and/or homodimer, and when incubated together, formed a ligand-receptor complex. Our results demonstrate that goldfish EPO/EPOR signaling has been highly conserved throughout vertebrate evolution as a required mechanism for erythrocyte development.

Colony-stimulating factor-1 receptor protein expression is a specific marker for goldfish (Carassius auratus L.) macrophage progenitors and their differentiated cell types.

Signaling through the colony-stimulating factor-1 receptor (CSF-1R) mediates the proliferation, differentiation, and activation of macrophages and their progenitors. In this study we report on the use of an anti-goldfish CSF-1R antibody to specifically recognize a population of CSF-1R positive cells from goldfish tissues. Furthermore, using our previously characterized primary kidney macrophage culture system, we show that CSF-1R positive cells include monocytes, macrophages, and their progenitor cells. Freshly isolated progenitor cells had a higher median florescent intensity ratio than those progenitor cells cultured for up to four days. The decrease in CSF-1R expression on the progenitor cells coincides with the appearance and development of monocytes and macrophages. Monocytes were consistently CSF-1R+ and maintained the high level of CSF-1R expression as they developed into macrophages. Like that of mammalian systems, CSF-1R is expressed on all macrophage sub-populations (progenitors, monocytes, macrophages), and CSF-1R expression increases with macrophage development in teleosts.

Molecular and functional characterization of kita and kitla of the goldfish (Carassius auratus L.)

Kit ligand and its type III tyrosine kinase receptor Kit promotes the survival, proliferation and differentiation of progenitor cells involved in mammalian myelopoiesis. In this study we report on the molecular and functional characterization of kit receptor A (kita) and kit ligand A (kitla) from the goldfish. Both kita and kitla were ubiquitously expressed in goldfish tissues, with higher mRNA levels observed in the kidney and spleen, the major hematopoietic organs of fish. Furthermore, both kita and kitla expressions decreased in a time-dependent manner in goldfish primary kidney macrophage (PKM) cultures, as progenitor to macrophage development progressed, and the highest expressions of both the receptor and ligand were observed in sorted progenitor cell populations. Activation of mature macrophage cultures increased both kita and kitla expressions. Kit ligand A induced chemotactic response, proliferation and survival of PKM cells in a dose-dependent manner, but did not induce differentiation of early PKM cells. These results are consistent with the role of kita and kitla during myelopoiesis of higher vertebrates and suggest a conserved mechanism of macrophage development throughout vertebrates.