Jordan W. Hodgkinson

Characterization and functional analysis of goldfish (Carassius auratus L.) interleukin-10

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 levels in granulocytes and monocytes of the goldfish. To functionally assess the goldfish IL-10, we generated a recombinant form of the molecule (rgIL-10). The rgIL-10 substantially reduced the expression of TNFα1, TNFα2, IL-1β1, IL-10, CXCL-8, and NADPH oxidase component, p47(phox) in monocytes activated with heat-killed Aeromonas salmonicida and reduced the expression of IFNγ in A. salmonicida-activated splenocytes. Pre-treatment of monocytes with rgIL-10 resulted in substantial reduction of the ROI response of the A. salmonicida or rgIFNγ-primed monocytes. The rgIL-10 bound to goldfish monocytes and induced phosphorylation and nuclear translocation of Stat3. The rgIL-10 also induced rapid and robust increase in the mRNA levels of the goldfish monocyte SOCS-3. Our results indicate that the function of IL-10 is highly conserved through evolution.

Antimicrobial responses of teleost phagocytes and innate immune evasion strategies of intracellular bacteria

During infection, macrophage lineage cells eliminate infiltrating pathogens through a battery of antimicrobial responses, where the efficacy of these innate immune responses is pivotal to immunological outcomes. Not surprisingly, many intracellular pathogens have evolved mechanisms to overcome macrophage defenses, using these immune cells as residences and dissemination strategies. With pathogenic infections causing increasing detriments to both aquacultural and wild fish populations, it is imperative to garner greater understanding of fish phagocyte antimicrobial responses and the mechanisms by which aquatic pathogens are able to overcome these teleost macrophage barriers. Insights into the regulation of macrophage immunity of bony fish species will lend to the development of more effective aquacultural prophylaxis as well as broadening our understanding of the evolution of these immune processes. Accordingly, this review focuses on recent advances in the understanding of teleost macrophage antimicrobial responses and the strategies by which intracellular fish pathogens are able to avoid being killed by phagocytes, with a focus on Mycobacterium marinum.

Commercial naphthenic acids and the organic fraction of oil sands process water induce different effects on pro‐inflammatory gene expression and macrophage phagocytosis in mice

Naphthenic acids (NAs) are believed to be the major toxic component of oil sands process water (OSPW). Different OSPW preparations have distinct NA compositions, and additional organics, that differ from the commercial NAs (C‐NAs) often used for toxicology studies. To evaluate whether C‐NAs are an adequate model to study OSPW toxicity in complex organisms, we compared the effects of C‐NAs and the extractable organic fraction of OSPW (OSPW‐OF) on mice immune mechanisms. Mice were orally exposed to different C‐NA doses, or OSPW‐OF at the same NA dose, for up to 8 weeks, and the expression of pro‐inflammatory genes in different organs was determined using quantitative PCR. C‐NAs and OSPW‐OF altered the expression of pro‐inflammatory genes, inducing either expression down‐regulation or up‐regulation, depending on the organ examined and time after exposure. The time at which gene expression alterations occurred, and the specific sets of genes whose expression was altered, were very different between animals exposed to C‐NAs or to OSPW‐OF. We evaluated the ability of mouse peritoneal macrophages to phagocytose yeast cell wall, as a measure of the ability of mice to mount a central function of the innate immune response. Phagocytosis was significantly reduced in animals exposed to C‐NAs, but enhanced in mice exposed to OSPW‐OF. Our results indicate that studies using C‐NAs may not necessarily reflect the possible effects induced in animals by process water from tailing ponds. Copyright

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.

Neutrophil contributions to the induction and regulation of the acute inflammatory response in teleost fish

Neutrophils are essential to the acute inflammatory response, where they serve as the first line of defense against infiltrating pathogens. We report that, on receiving the necessary signals, teleost (Carassius auratus) neutrophils leave the hematopoietic kidney, enter into the circulation, and dominate the initial influx of cells into a site of inflammation. Unlike mammals, teleost neutrophils represent <5% of circulating leukocytes during periods of homeostasis. However, this increases to nearly 50% immediately after intraperitoneal challenge with zymosan, identifying a period of neutrophilia that precedes the peak influx of neutrophils into the challenge site at 18 h after injection). We demonstrate that neutrophils at the site of inflammation alter their phenotype throughout the acute inflammatory response, and contribute to both the induction and the resolution of inflammation. However, neutrophils isolated during the proinflammatory phase (18 h after injection) produced robust respiratory burst responses, released inflammation‐associated leukotriene B4, and induced macrophages to increase reactive oxygen species production. In contrast, neutrophils isolated at 48 h after infection (proresolving phase) displayed low levels of reactive oxygen species, released the proresolving lipid mediator lipoxin A4, and downregulated reactive oxygen species production in macrophages before the initiation of apoptosis. Lipoxin A4 was a significant contributor to the uptake of apoptotic cells by teleost macrophages and also played a role, at least in part, in the downregulation of macrophage reactive oxygen species production. Our results highlight the contributions of neutrophils to both the promotion and the regulation of teleost fish inflammation and provide added context for the evolution of this hematopoietic lineage.

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.

Analysis of the antimicrobial responses of primary phagocytes of the goldfish (Carassius auratus L.) against Mycobacterium marinum

The slow growth rate of Mycobacterium spp. that infect humans coupled with a lack of reliable in vitro infection model systems has hindered the progress of research in host cell-mycobacteria interactions. Recent studies have utilized the relatively fast growing Mycobacterium marinum to examine the host-pathogen interface in natural fish hosts. Here we describe the use of primary goldfish monocyte and mature macrophage cultures to investigate the immune cell-M. marinum interactions. Live and heat-killed M. marinum abrogated the recombinant goldfish (rg)TNFα2 and rgIFNγ-induced monocyte reactive oxygen production. Live but not heat-killed M. marinum also ablated rgIFNγrel and rg-TNFα2 induced macrophage nitric oxide production. M. marinum induced significant changes in gene expression of select NADPH oxidase components and inflammatory cytokine receptors and up-regulated the expression of immunosuppressive genes IL-10, TGFβ1 and SOCS-3. The exposure of monocytes and mature macrophages to M. marinum caused an increase in the mRNA levels of several pro-inflammatory genes. Stimulation of monocytes and macrophages with rgTNFα2, rgIFNγ, or rgIFNγrel reduced the survival of intracellular mycobacteria. The characterization of the interaction between M. marinum and natural host-derived primary phagocyte cultures will enable future studies on the host-pathogen interactions in mycobacterial infections.

Analysis of the immune response in infections of the goldfish (Carassius auratus L.) with Mycobacterium marinum.

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 context of a natural host. Histological analysis revealed mycobacterial infiltrates in goldfish kidney and spleen tissues, peaking 28 days post infections (dpi). Quantitative gene expression analysis showed significant increases of mRNA levels of pro-inflammatory cytokines (IFNγ, IL-12p40, IL-1β1) and cytokine receptors (IFNGR1-1, TNFR2) at 7 dpi. Conversely, the gene expression levels of key anti-inflammatory cytokines TGFβ and IL-10 were elevated at 14 dpi. Furthermore, M. marinum infections markedly increased the cytokine-primed oxidative burst responses of isolated kidney phagocytes at 7 but not 56 dpi. We believe that the M. marinum-goldfish infection model will be invaluable in furthering the understanding of the mycobacterium host-pathogen interface.

Identification and functional characterization of the goldfish (Carassius auratus L.) high mobility group box 1 (HMGB1) chromatin-binding protein.

We report on the identification and functional characterization of HMGB1 of the goldfish. Quantitative analysis indicated the highest expression of goldfish HMGB1 in the brain, with lower mRNA levels in spleen, intestine, kidney, gill and heart. HMGB1 was also differentially expressed in goldfish immune cell populations with highest mRNA levels present in splenocytes and neutrophils. We generated and functionally characterized the recombinant HMGB1 (rgHMGB1). The rgHMGB1 primed the respiratory burst response in monocytes and induced nitric oxide production of primary goldfish macrophages. Treatment of goldfish macrophages with heat-killed Mycobacterium marinum and Aeromonas salmonicida elevated the expression of HMGB1 and resulted in higher HMGB1 protein levels. The rgHMGB1 induced a dose-dependent production of TNFα-2 and IL-1β1 of goldfish macrophages. Furthermore, the dual luciferase reporter assay revealed that goldfish HMGB1 induced the activation of the NF-κB signaling pathway. Our results indicate that goldfish HMGB1 is a critical regulatory cytokine of inflammatory and antimicrobial response of the goldfish.

Recombinant IL-4/13A and IL-4/13B induce arginase activity and down-regulate nitric oxide response of primary goldfish (Carassius auratus L.) macrophages.

ABSTRACT We report on the expression analysis and functional characterization of IL‐4/13A and IL‐4/13B in goldfish. Quantitative analysis indicated the highest expression in the heart, spleen, brain, and kidney, with comparable expression patterns for both IL‐4/13A and IL‐4/13B. The mRNA levels of IL‐4/13A and IL‐4/13B in the immune cells examined were highest in macrophage and monocytes. Assessment of spleen mRNA following infection with Trypanosoma carassii, a prominent protozoan pathogen of fish, revealed decrease in IL‐4/13B and arginase expression 14 days post infection, followed by an increase in IL‐4/13B and arginase‐2 at 28 days post infection. Recombinant forms of IL‐4/13A and IL‐4/13B induced an increase in arginase activity in macrophages in a dose‐dependent manner. Recombinant IL‐4/13A and IL‐4/13B also induced significant increase in mRNA levels of arginase −2 in macrophages at 6, 12, 18 and 24 h after treatment. Furthermore, treatment with both IL‐4/13 recombinants interfered with the IFN&ggr;‐induced nitric oxide response of macrophages. Our results suggest a conserved role of IL‐4/IL‐13 in induction of alternative activation phenotype in teleost macrophages. HighlightsIL‐4/13A and IL‐4/13B increases mRNA levels of arginase‐2 and arginase activity in macrophages.Both IL4/13A and IL‐4/13B down‐regulate IFN&ggr;‐induced nitric oxide response of macrophages.Expression of IL‐4/13 and arginase‐1 and ‐2 lower on day 14 in T. carassi infected goldfish.Expression of IL‐4/13 and arginase‐2 up‐regulated on day 28 in T. carassii infected goldfish.