Dušan Palić

Chicken heterophil extracellular traps (HETs): Novel defense mechanism of chicken heterophils

Recent findings in mammals and fish have revealed that neutrophil nuclear material associated with cytoplasmic granular content is released in the form of neutrophil extracellular traps (NETs) that can trap and kill invading microorganisms in vitro and in vivo. To determine if a similar mechanism is present in chicken heterophils, hydrogen peroxide (H(2)O(2)) and phorbol myristate acetate (PMA) were used for stimulation of blood-derived heterophils. Stimulated heterophils produced structures that were characterized using immunocytochemistry and confocal microscopy as heterophil extracellular traps (HETs). Released HETs contained DNA, histone-DNA complex and elastase from heterophil cytoplasmic granules. HETs released from chicken heterophils are structurally similar to NETs found in mammalian and fish neutrophils. Extracellular DNA released from heterophils was quantified by Picogreen assay. Stimulation with PMA or H(2)O(2) significantly increased the HET-DNA release index in vitro compared to non-stimulated heterophils (1.11+/-0.04 and 1.55+/-0.10, respectively), and H(2)O(2) stimulation induced significantly higher HET-DNA release than PMA (P<0.001). Thus, HETs are now characterized as an important heterophil-mediated defense mechanism in chickens.

Immunotoxicology of non-functionalized engineered nanoparticles in aquatic organisms with special emphasis on fish—Review of current knowledge, gap identification, and call for further research

The rapid increase in use of nanotechnology products is increasing the presence of metal, metal-oxide and carbon-based nanoparticles in the aquatic environment. These non-functionalized engineered nanoparticles can interact with the immune system of fish and invertebrates, and tip the ecological balance of population sustainability. Most nanoparticle types present in the aquatic environment, such as titanium dioxide, do not exhibit or have very low direct toxicity, but instead display silent or concealed sub-lethal effects on the immune system with serious implications. There is a gap in current available information regarding the immunotoxic potential of engineered nanoparticles toward aquatic organisms. Therefore, there is a critical need to provide the first comprehensive review of the effects of engineered non-functionalized nanoparticles on the immune system of aquatic animals, address the major gaps in current existing information, and recommend the future focus of research. This manuscript identifies cell mediated immunity and the phagocytic cells as the primary target of nanoparticle immunotoxicity. The immunotoxicity is primarily govern by lysosomal destabilization, frustrated phagocytosis, and change in function of the phagocytic cells, which decrease the ability of animals to defend themselves against pathogens and infectious diseases. Humoral immune system is a lesser target of direct immunotoxicity, but plays a critical role in dissemination of the nanoparticles through the body and their presentation to the phagocytic cells. The external innate immunity and the acquired immunity have not been connected with overly important and direct immunotoxic effects, but instead a big gap in current targeted research has been acknowledged.

Effects of nanosized titanium dioxide on innate immune system of fathead minnow (Pimephales promelas Rafinesque, 1820).

Effects of nanosized (<100 nm) titanium dioxide (TiO(2)) particles on fish neutrophils and immune gene expression was investigated using the fathead minnow (Pimpehales promelas). Expanded use of TiO(2) in the cosmetic industry has increased the potential exposure risk to aquatic ecosystems and human health. Effects of nano-TiO(2) on neutrophil function of the fathead minnow was investigated using oxidative burst, neutrophil extracellular traps (NETs) release and degranulation of primary granules. The innate immune gene expression was determined with quantitative PCR (qPCR). Application of 0.1 μg mL(-1) of nano-TiO(2) in vitro stimulated oxidative burst and NET release. Intraperitoneal injection of 10 μg g(-1) of nano-TiO(2) caused a significant decrease in oxidative burst, NETs release and degranulation (21%; 11%; and 30%, decrease, respectively). Fish exposed to nano-TiO(2) for 48 h in vivo had significantly increased expression of interleukin 11, macrophage stimulating factor 1, and neutrophil cytosolic factor 2 (4; 2.5; and 2 fold increase, respectively). Nano-TiO(2) has potential to interfere with the evolutionary conserved innate immune system responses, as evidenced with observed changes in gene expression and neutrophil function. This finding encourages the use of fish models in the studies of nanoparticle immunotoxicity. The lowest significant response concentration studied in vitro is four times greater than the estimated environmental concentration for TiO(2) (0.025 μg mL(-1)) causing concern about potential impact of nano-TiO(2) on aquatic animals and ecosystems.

Chicken heterophils from commercially selected and non-selected genetic lines express cytokines differently after in vitro exposure to Salmonella enteritidis.

Resistance to pathogens such as Salmonella enteritidis (SE) is a heritable trait important in maintaining the health of chickens and reducing bacterial contamination of poultry products. In chickens, heterophils act as the first responders to bacterial infections and are, therefore, responsible for initiating the immune response against SE challenge. This study measured mRNA expression of several immune response genes [interleukin-6 (IL-6), IL-10, transforming growth factor-beta4 (TGF-beta4), granulocyte macrophage-colony stimulating factor (GM-CSF), and Toll-like receptor-4 (TLR-4)] by heterophils from broiler, Leghorn, and Fayoumi chickens, either non-stimulated or stimulated in vitro with SE using quantitative reverse-transcriptase PCR. We found that heterophils of commercially selected broiler and Leghorn birds had differing early heterophil responses to SE in comparison with the native Fayoumi line. Heterophil stimulation with SE in vitro increased expression of pro- (IL-6 and GM-CSF) and anti-inflammatory cytokine mRNA (IL-10 and TGF-beta4) in the Fayoumi line, while the broiler and Leghorn line heterophils had decreased or no changes in the cytokine gene expression levels. The unique response of the Fayoumi line is in contrast to the lines with a history of genetic selection to increase growth or reproduction, a process which may favor reduced or suppressed inflammatory responses. The findings illustrate the potential value of native lines to provide biodiversity to enhance innate health in commercially selected poultry.

Gene expression of zebrafish embryos exposed to titanium dioxide nanoparticles and hydroxylated fullerenes

Increased release of engineered nanoparticles to the environment suggests a rising need for the monitoring and evaluation of potential toxicity. Zebrafish frequently have been used as a model species in human and aquatic toxicology studies. In this study, zebrafish embryos were microinjected in the otic vesicle with a sublethal dose of engineered nanoparticles (titanium dioxide/TiO(2) and hydroxylated fullerenes/C(60)(OH)(24)). A gene microarray analysis was performed on injected and control embryos to determine the potential for nanoparticles to change the expression of genes involved in cross talk of the nervous and immune systems. The exposure to TiO(2) and hydroxylated fullerenes caused shifts in gene regulation response patterns that were similar for downregulated genes but different for upregulated genes. Significant effects on gene regulation were observed on genes involved in circadian rhythm, kinase activity, vesicular transport and immune response. This is the first report of circadian rhythm gene deregulation by nanoparticles in aquatic animals, indicating the potential for broad physiological and behavioral effects controlled by the circadian system.

Hydroxylated fullerenes inhibit neutrophil function in fathead minnow (Pimephales promelas Rafinesque, 1820).

Hydroxylated fullerenes act as potent inhibitors of cytochrome P450-dependent monooxygenases, and are reported to be very strong antioxidants quenching reactive oxygen species (ROS) production. Effects of nanosized hydroxylated fullerenes on fish neutrophil function and immune gene transcription was investigated using fathead minnow (Pimephales promelas). Neutrophil function assays were used to determine the effects of fullerene exposure in vitro and in vivo on oxidative burst, degranulation and extracellular trap (NETs) release, and the innate immune gene transcription was determined with quantitative PCR (qPCR). Application of fullerenes (0.2-200 microgmL(-1)in vitro) caused concentration dependent inhibition of oxidative burst and suppressed the release of NETs and degranulation of primary granules (up to 70, 40, and 50% reduction in activity compared to non-treated control, respectively). Transcription of interleukin 11 and myeloperoxidase genes was significantly increased and transcription of elastase 2 gene was significantly decreased in fish exposed to hydroxylated fullerenes for 48h in vivo (12 and 3 fold increase, and 5 fold decrease, respectively). Observed changes in gene transcription and neutrophil function indicate potential for hydroxylated fullerenes to interfere with the evolutionary conserved innate immune system responses and encourages the use of fish models in studies of nanoparticle immunotoxicity.

Differential splenic cytokine responses to dietary immune modulation by diverse chicken lines

Nutritional modulation of the immune system is an often exploited but poorly characterized process. In chickens and other food production animals, dietary enhancement of the immune response is an attractive alternative to antimicrobial use. A yeast cell wall component, beta-1,3/1,6-glucan, augments the response to disease in poultry and other species; however, the mechanism of action is not clear. Ascorbic acid and corticosterone are better characterized immunomodulators. In chickens, the spleen acts both as reservoir and activation site for leukocytes and, therefore, splenic gene expression reflects systemic immune function. To determine effects of genetic line and dietary immunomodulators, chickens of outbred broiler and inbred Leghorn and Fayoumi lines were fed either a basal diet or an experimental diet containing beta-glucans, ascorbic acid, or corticosterone from 56 to 77 d of age. Spleens were harvested, mRNA was isolated, and expression of interleukin (IL)-4, IL-6, IL-18, macrophage inflammatory protein-1beta, interferon-gamma, and phosphoinositide 3-kinase p110gamma transcripts was measured by quantitative reverse transcription PCR. Effects of diet, genetic line, sex, and diet x genetic line interaction on weight gain and gene expression were analyzed. At 1, 2, and 3 wk after starting the diet treatments, birds fed the corticosterone diet had gained less weight compared with birds fed the other diets (P < 0.001). Sex affected expression of IL-18 (P = 0.010), with higher levels in males. There was a significant interaction between genetic line and diet on expression of IL-4, IL-6, and IL-18 (P = 0.021, 0.006, and 0.026, respectively). Broiler line gene expression did not change in response to the experimental diet. Splenic expression of IL-6 was higher in Leghorns fed the basal or ascorbic acid diets, rather than the beta-glucan or corticosterone diets, whereas the opposite relationship was observed in the Fayoumi line. Expression of IL-4 and IL-18 responded to diet only within the Fayoumi line. The differential splenic expression of birds from diverse genetic lines in response to nutritional immunomodulation emphasizes the need for further study of this process.

Genetic control of chicken heterophil function in advanced intercross lines: associations with novel and with known Salmonella resistance loci and a likely mechanism for cell death in extracellular trap production

Heterophils, the avian polymorphonuclear leukocyte and the counterpart of mammalian neutrophils, generate the primary innate response to pathogens in chickens. Heterophil performance against pathogens is associated with host disease resistance, and heterophil gene expression and function are under genetic control. To characterize the genomic basis of heterophil function, heterophils from F13 advanced intercross chicken lines (broiler × Leghorn and broiler × Fayoumi) were assayed for phagocytosis and killing of Salmonella enteritidis, oxidative burst, and extracellular trap production. A whole-genome association analysis of single nucleotide polymorphisms at 57,636 loci identified genomic locations controlling these functional phenotypes. Genomic analysis revealed a significant association of extracellular trap production with the SAL1 locus and the SLC11A1 gene, which have both been previously associated with resistance to S. enteritidis. Fine mapping supports SIVA1 as a candidate gene controlling SAL1-mediated resistance and indicates that the proposed cell-death mechanism associated with extracellular trap production, ETosis, likely functions through the CD27/Siva-1-mediated apoptotic pathway. The SLC11A1 gene was also associated with phagocytosis of S. enteritidis, suggesting that the Slc11a1 protein may play an additional role in immune response beyond depleting metal ions to inhibit intracellular bacterial growth. A region of chromosome 6 with no characterized genes was also associated with extracellular trap production. Further characterization of these novel genes in chickens and other species is needed to understand their role in polymorphonuclear leukocyte function and host resistance to disease.

Heterophil functional responses to dietary immunomodulators vary in genetically distinct chicken lines

The effect of dietary supplementation of immunomodulators on in vitro chicken heterophil function was investigated using three diverse genetic lines of chickens (broiler, Fayoumi, and Leghorn). Dietary supplementation with β-glucan, ascorbic acid, and corticosterone was fed from 8 to 11 weeks of age. Heterophil function was evaluated weekly during supplementation using phagocytosis, bacterial killing, and heterophil extracellular traps (HETs)-DNA release. Fayoumis fed the basal diet had significantly higher HETs-DNA release (P=0.002) than Leghorns and broilers. Both genetic line and immunomodulator diet supplementation had significant effects on bacterial killing (line and diet effect: P<0.001) and HETs-DNA release (line: P<0.001; diet: P=0.043). Dietary supplementation with immunomodulators, therefore, shows potential to affect and augment heterophil function in chickens. The current results also suggest the important role of genetics in innate immune responses.

Proportion of circulating chicken heterophils and CXCLi2 expression in response to Salmonella enteritidis are affected by genetic line and immune modulating diet.

Genetic line and diet affect chicken heterophil activity and gene expression, and the combination of these factors can enhance disease resistance. This study evaluated the effects of immune modulating diets on heterophil/lymphocyte (H/L) ratio and heterophil chemokine expression in distinct genetic lines. Fayoumi and Leghorn chickens were fed a basal diet or immune modulating diets enhanced with β-glucans, ascorbic acid, or corticosterone. H/L ratios and heterophil gene expression in response to in vitro stimulation with Salmonella enteritidis (SE) were evaluated on days 1, 3, 7, and 21 of diet treatment. The stress-mimicking corticosterone diet influenced H/L ratio in the Leghorn line, but not the Fayoumi line, suggesting resistance to stress-induced immunosuppression in the Fayoumi line. Leghorn line H/L ratios were increased on days 1 and 3 of corticosterone diet treatment, but not days 7 or 21. Expression of CXCLi2 by SE stimulated heterophils was higher in the Leghorn line, suggesting that Leghorns rely more heavily on inflammatory response than do Fayoumis. Corticosterone diet was associated with reduced CXCLi2 expression in heterophils from both lines. Dietary β-glucan or ascorbic acid did not affect H/L ratio or CXCLi2 expression, suggesting that benefits of these immunomodulators may not be evident in healthy birds.