Miodrag Belosevic

Antimicrobial mechanisms of fish phagocytes and their role in host defense

Phagocytosis is a primitive defense mechanism in all multicellular animals. Phagocytes such as macrophages and neutrophils play an important role in limiting the dissemination of infectious agents, and are responsible for the eventual destruction of phagocytosed pathogens. These cells have evolved elaborate killing mechanisms for destroying pathogens. In addition to their repertoire of degradative enzymes and antimicrobial peptides, macrophages and neutrophils can be activated to produce a number of highly toxic molecules. Production of reactive oxygen and nitrogen intermediates by these cells are potent cytotoxic mechanisms against bacteria and protozoan pathogens. Studies in fish suggest that the biological basis of these inducible killing mechanisms is similar to those described in mammals. More recent work suggest novel roles for regulating these killing responses in fish. In this review, we describe the biological basis of these killing mechanisms and how they are regulated in fish.

Inactivation of cryptosporidium parvum oocysts using medium- and low-pressure ultraviolet radiation

The effect of ultraviolet radiation from low- and medium-pressure mercury arc lamps on Cryptosporidium parvum oocysts was studied using a collimated beam apparatus. Experiments were conducted using parasites suspended in both filtered surface water and phosphate buffered laboratory water. Inactivation of oocysts was measured as reduction in infectivity using a CD-1 neonatal mouse model and was found to be a non-linear function of UV dose over the range of germicidal doses tested (0.8-119 mJ/cm2). Oocyst inactivation increased rapidly with UV dose at doses less than 25 mJ/cm2 with two and three log-units inactivation at approximately 10 and 25 mJ/cm2, respectively. The cause of significant leveling-off and tailing in the UV inactivation curve at higher doses was not determined. Maximum measured oocyst inactivation ranged from 3.4 to greater than 4.9 log-units and was dependent on different batches of parasites. Water type and temperature, the concentration of oocysts in the suspension, and the UV irradiance did not have significant impacts on oocyst inactivation. When compared on the basis of germicidal UV dose, the oocysts were equally sensitive to low- and medium-pressure UV radiation. With respect to Cryptosporidium, both low- and medium-pressure ultraviolet radiation are attractive alternatives to conventional chemical disinfection methods in drinking water treatment.

Macrophage activating factor(s) secreted by mitogen stimulated goldfish kidney leukocytes synergize with bacterial lipopolysaccharide to induce nitric oxide production in teleost macrophages

Recent studies in our laboratory demonstrated that fish macrophages produce nitric oxide. To elucidate the mechanisms which regulate nitric oxide production in teleosts, we examined whether macrophage activating factors (MAFs) secreted by mitogen stimulated leukocytes, induced nitric oxide production in a long-term cultured macrophage cell line and in primary cultures of kidney macrophages from the goldfish. The results indicate that both primary and long term cultured goldfish macrophages produce nitric oxide in response to MAF or bacterial lipopolysaccharide (LPS), and co-stimulation with both factors results in a synergistic induction of nitric oxide production. MAF that induced nitric oxide production were present in leukocyte supernatants as early as 24 h after addition of mitogens to cell cultures. The production of MAF was dependent upon the incubation temperature, presence of serum in the culture medium and duration of incubation: maximal MAF activity was detected in 72-96 h supernatants raised in media with serum at 30 degrees C. MAF-induced nitric oxide production by long term cultured macrophages was inhibited by 1000 microM NG-monomethyl-L-arginine or amino-guanidine, indicating an L-arginine-dependent metabolic pathway for the production of the reactive nitrogen intermediates in teleosts. The biochemical events of cytokine induced nitric oxide production by teleost macrophages appear to be similar to those of mammalian macrophages.

Transferrin and the innate immune response of fish: identification of a novel mechanism of macrophage activation

We have previously demonstrated that a non-cytokine serum protein called transferrin was a primary activating molecule of the goldfish (Carassius auratus) macrophage antimicrobial response. The ability of the enzymatically cleaved forms of this protein to modulate fish macrophage function is novel and may represent a primitive and evolutionary conserved mechanism for the induction of NO response of macrophages. In the present study we confirm our earlier findings using immunoaffinity purified goldfish transferrin from mitogen-stimulated leukocyte supernatants. In addition we demonstrate that: (1). products released by necrotic/damaged cells contain transferrin-cleaving activity; (2). the cleavage site is located within the bridge peptide connecting the two lobes of the transferrin molecule; (3). transferrin is expressed by activated goldfish macrophages but not mitogen-stimulated kidney leukocytes; and (4). addition of transferrin significantly enhanced the killing response of goldfish macrophages exposed to different pathogens or pathogen products (e.g. lipopolysaccharide, Mycobacterium chelonei, Trypanosoma danilewskyi, Aeromonas salmonicida, and Leishmania major). We propose a model of fish macrophage activation that is mediated by a non-cytokine host protein (i.e. transferrin) in combination with highly conserved innate immunity recognition receptors that are almost certain to exist in teleost.

Photodegradation of emerging micropollutants using the medium-pressure UV/H2O2 Advanced Oxidation Process.

A medium-pressure (MP) ultraviolet (UV) process has been applied to investigate the direct UV photolysis and UV/H2O2 oxidation of selected model micropollutants (naproxen, carbamazepine, diclofenac, gemfibrozil, ibuprofen, caffeine, 2,4-D, 2,4-DCP, and mecoprop). The quantum yields were found to be between 0.0010 and 0.13 at pH = 7. In the MP UV/H2O2 oxidation, the pseudo first-order rate constants for the selected compounds were found to be dependent on their initial concentrations (at mg/L levels) and on the H2O2 concentration. The UV doses required for 50% and 90% removal at various H2O2 levels varied widely among the compounds tested. Second-order rate constants (ranging from 4.1 × 10(9) to 1.4 × 10(10) M(-1) s(-1)) for the reaction between the selected compounds and hydroxyl radicals were determined using a competition-kinetics approach, where para-chlorobenzoic acid (pCBA) was chosen as the reference compound. Further, as an evaluation of electrical energy efficiency, the Figure-of-Merit, Electrical Energy per Order (EEO) was determined for the selected compounds using a batch reactor at 25 and 50 mg/L H2O2 concentrations. The electrical energy (in kWh) required to reduce a pollutant concentration by 90% ranged from 1.3 to 7.1 kWh m(-3).

Macrophage-mediated innate host defense against protozoan parasites.

Macrophages are immune cells that play a pivotal role in the detection and elimination of pathogenic microorganisms. Macrophages possess a variety of surface receptors devoted to the recognition of non-self by discriminating between host and pathogen-derived structures. Recognition of foreign microorganisms by the macrophage ultimately results in phagocytosis and the eventual destruction of microorganisms by lysosomal enzymes, toxic reactive oxygen and nitrogen intermediates, and/or nutrient deprivational mechanisms. However, protozoan parasites such as Toxoplasma gondii, Trypanosoma cruzi, and Leishmania spp., parasitize macrophages, utilizing them as a host cell for their growth, replication, and/or maintenance of their life cycles. The protozoan parasites of the genus Leishmania are unique in that their intracellular replication in the host is predominantly restricted to a single cell type, the macrophage. This review focuses on the cellular processes involved in macrophage-mediated host defense against protozoan parasites, from the initial host-parasite interactions that mediate recognition to the mechanisms employed by macrophages to destroy and eliminate the pathogen. As an example model system of experimental study, we describe in more more detail the cellular interactions between macrophages and the obligate intracellular parasite of mammalian macrophages, Leishmania spp.

A toll-like receptor (TLR) gene that is up-regulated in activated goldfish macrophages.

An expressed sequence tag screen of a macrophage activation factor and lipopolysaccharide (LPS) stimulated goldfish macrophage subtractive library generated several transcripts of a putative teleost homologue of the toll-like receptor (TLR) family. The full-length TLR cDNA was sequenced and is predicted to encode a type I transmembrane protein with an extracellular domain containing leucine rich repeats and a cytoplasmic tail encoding a toll/interleukin-1 receptor domain. These findings indicate that the gene identified is the first teleost homologue of the TLR family reported. Constitutive expression of TLR was observed in unstimulated macrophages and was also observed in goldfish spleen and kidney but not in heart and liver tissues. A significant up-regulation of the TLR mRNA in cultured macrophages following treatments with each of bacterial LPS, heat-killed Aeromonas salmonicida, and live Mycobacterium chelonei was observed after 3 and 6 h post-stimulation, though with different kinetics from each other. A relative decline in TLR expression was observed after 24 h, but expression levels were still higher than that of unstimulated cells. Thus pathogen-derived factors appear to differentially modulate the expression of TLR in goldfish macrophages, which undoubtedly contributes to the orchestration and/or induction of functional immune responses in fish.

Comparison of select innate immune mechanisms of fish and mammals.

Abstract:  The study of innate immunity has become increasingly popular since the discovery of homologs of many of the innate immune system components and pathways in lower organisms including invertebrates. As fish occupy a key position in the evolution of the innate and adaptive immune responses, there has been a great deal of interest regarding similarities and differences between their defense mechanisms and those of higher vertebrates. This review focuses on describing select mechanisms of the innate immune responses of fish and the implications for evolution of immunity in higher vertebrates.

Molecular characterization, expression and functional analysis of goldfish (Carassius aurutus L.) interferon gamma.

We report on the characterization and functional analysis of IFNgamma of the goldfish. Quantitative analysis indicated the highest expression of goldfish IFNgamma in the spleen, with lower expressions in brain, gill, kidney, heart, intestine and muscle. An increase in IFNgamma expression was observed in kidney leukocytes following stimulation with PHA, PolyI:C and during mixed leukocyte reaction. IFNgamma was expressed in different goldfish immune cell populations and this expression was upregulated in several of these populations following treatment of cells with recombinant goldfish TNFalpha-2. A recombinant form of goldfish IFNgamma (rgIFNgamma) was produced and functionally analyzed. The rgIFNgamma primed goldfish macrophages and neutrophils for enhanced respiratory burst responses and increased the phagocytic and nitric oxide responses of macrophages. Furthermore, treatment of goldfish macrophages with rgIFNgamma induced increased expression of several proinflammatory genes including TNFalpha-1, TNFalpha-2, IL-beta-1, IL-beta-2, IL-12-p35, IL-12-p40, RSAD-2 (=viparin), CXCL-8 (=IL-8), CCL-1, iNOS A and B and IFNgamma, and decreased the expression of TLR-3, while the expression of TGFbeta remained unchanged. Our results indicate that IFNgamma is a central proinflammatory cytokine of teleosts.

Two Macrophage Colony-Stimulating Factor Genes Exist in Fish That Differ in Gene Organization and Are Differentially Expressed

Mammalian macrophage CSF (MCSF; CSF-1) is the primary regulator of the mononuclear phagocyte lineage. We, for the first time, report the complete sequencing of five MCSF cDNAs from three fish species, rainbow trout, zebrafish, and goldfish. Despite the difference in the lengths of the MCSF transcripts, all of the fish MCSF molecules encode a signal peptide, a CSF-1 domain, a transmembrane domain, and an intracellular region. Each fish MCSF gene has a unique exon/intron structure. The primordial MCSF gene may have had a nine exon/eight intron structure. In this model, insertion of an intron in exon 6 in primitive fish created the fish type I MCSF, while the loss of this exon or part of the original exon 6 created the fish type II MCSF. Investigation of alternative splicing variants in trout suggests that no mammalian equivalent splice variants exist. The two trout MCSF genes are differentially expressed in vivo and contributed differently to the high-level expression of MCSF in spleen and head kidney. In contrast to the up-regulation of MCSF by PMA in mammals, in trout MCSF1 expression is down-regulated by PMA treatment. As in mammals, recombinant trout MCSF1 can promote the growth of head kidney leukocytes, and it up-regulates the expression of CXCR3 in head kidney macrophages, with the latter suggesting a role of MCSF in the trafficking of macrophages to sites of inflammation or injury where the CXCR3 ligands are expressed. Thus MCSF has an important role in the immune system of fish as in mammals.