Effect of Copper, Zinc, and Selenium on the Migration of Bovine Neutrophils

Neutrophils represent the first line of mammary gland defense against invading pathogens by transmigration across the mammary epithelial cell barrier. The effect of trace elements on the migration of bovine neutrophils is not clear. In this study, we investigated the effect of copper (Cu; 0.5, 1.0 and 1.5 mg/L), zinc (Zn; 1.0, 5.0 and 10 mg/L) and selenium (Se; 0.1, 1.0 and 2.0 mg/L) on the migration of bovine neutrophils by using a Transwell assay. The results showed that Cu, Zn and Se promoted the number of neutrophils in the trans-mammary epithelium. With the increased concentration of Cu at 1.5 mg/L, the number of neutrophils in the trans-mammary epithelium was increased significantly (p < 0.05). Zn (5.0 mg/L) and Se (0.1 mg/L) increased the migrated number of neutrophils (p < 0.01) to an extremely significant degree. These findings provided a theoretical and experimental basis for mammary gland immunity in dairy cows. Thus, we suggest that adding moderate amounts of different trace elements can improve the immune function of dairy cows.

Engineering and characterization of human β-defensin-3 and its analogues and microcin J25 peptides against Mannheimia haemolytica and bovine neutrophils

Mannheimia haemolytica-induced bovine respiratory disease causes loss of millions of dollars to Canadian cattle industry. Current antimicrobials are proving to be ineffective and leave residues in meat. Antimicrobial peptides (AMPs) may be effective against M. haemolytica while minimizing the risk of drug residues. Cationic AMPs can kill bacteria through interactions with the anionic bacterial membrane. Human β-Defensin 3 (HBD3) and microcin J25 (MccJ25) are AMPs with potent activity against many Gram-negative bacteria. We tested the microbicidal activity of wild-type HBD3, three HBD3 peptide analogues (28 amino acid, 20AA, and 10AA) derived from the sequence of natural HBD3, and MccJ25 in vitro against M. haemolytica. Three C-terminal analogues of HBD3 with all cysteines replaced with valines were manually synthesized using solid phase peptide synthesis. Since AMPs can act as chemoattractant we tested the chemotactic effect of HBD3, 28AA, 20AA, and 10AA peptides on bovine neutrophils in Boyden chamber. Minimum bactericidal concentration (MBC) assay showed that M. haemolytica was intermediately sensitive to HBD3, 28AA and 20AA analogues with an MBC of 50 µg/mL. The 10AA analogue had MBC 6.3 µg/mL which is likely a result of lower final inoculum size. MccJ25 didn’t have significant bactericidal effect below an MBC < 100 µg/mL. Bovine neutrophils showed chemotaxis towards HBD3 and 20AA peptides (P < 0.05) but not towards 28AA analogue. Co-incubation of neutrophils with any of the peptides did not affect their chemotaxis towards N-formyl-l-methionyl-l-leucyl-phenylalanine (fMLP). The data show that these peptides are effective against M. haemolytica and are chemotactic for neutrophils in vitro.

Synergistic Activation of Bovine CD4+ T Cells by Neutrophils and IL-12

CD4+ T cell activation requires inflammatory cytokines to provide a third signal (3SI), such as interleukin-12 (IL-12). We recently reported that bovine neutrophils can enhance the activation of bovine CD4+ T cells. To explore the interactions between neutrophils and third signal cytokines in bovine CD4+ T cell activation, naïve CD4+ T cells were isolated from cattle lymph nodes and stimulated for 3.5 days with anti-bovine CD3 (first signal; 1SI), anti-bovine CD28 (second signal; 2SI), and recombinant human IL-12 (3SI) in the presence or absence of neutrophils harvested from the same animals. Indeed, the strongest activation was achieved in the presence of all three signals, as demonstrated by CD25 upregulation, IFNγ production in CD4+ T cells, and secretion of IFNγ and IL-2 in cell supernatants. More importantly, 1SI plus neutrophils led to enhanced CD25 expression that was further increased by IL-12, suggesting synergistic action by IL-12 and neutrophils. Consistently, neutrophils significantly increased IFNγ production in 1SI plus IL-12-stimulated CD4+ T cells. Our data suggest the synergy of neutrophils and IL-12 as a novel regulator on bovine CD4+ T cell activation in addition to three signals. This knowledge could assist the development of immune interventions for the control of infectious diseases in cattle.

Innate immune response in bovine neutrophils stimulated with Mycoplasma bovis

Mycoplasma bovis (M. bovis) is a significant worldwide pathogen of cattle. Neutrophils have an important role in the innate immune response during infection with M. bovis. However, even though neutrophils accumulate in M. bovis infection, the interaction of M. bovis and neutrophils has not been fully elucidated. We attempted to elucidate the innate immune response of neutrophils stimulated with M. bovis and evaluate the transcriptome and functional analysis of bovine neutrophils stimulated with M. bovis. Proinflammatory cytokines, such as inducible nitric oxide (iNOS), which was the most increased gene in transcriptome analysis, were increased in quantitative polymerase chain reaction analysis of bovine neutrophils stimulated with live or heat-killed M. bovis. Nitric oxide and intracellular reactive oxygen species production of neutrophils stimulated with M. bovis was significantly increased. Neutrophils stimulated with M. bovis showed an increased ratio of nonapoptotic cell death compared to unstimulated controls. We demonstrated that neutrophil extracellular traps (NETs) formation was not recognized in neutrophils stimulated with live M. bovis. However, heat-killed M. bovis induced NETs formation. We also showed the interaction with M. bovis and bovine neutrophils regarding proinflammatory cytokine gene expression and functional expression related to NETs formation. Live and killed M. bovis induced innate immune responses in neutrophils and had the potential to induce NETs formation, but live M. bovis escaped NETs.

Cellular Response of Neutrophils to Bismuth Subnitrate and Micronized Keratin Products In Vitro

The aim of this study was to assess the effect of bismuth subnitrate and micronized keratin on bovine neutrophils in vitro. We hypothesized that recruitment and activation of neutrophils into the teat canal and sinus are the mechanisms of action of bismuth subnitrate and keratin-based teat sealant formulations. To test this, a chemotaxis assay (Experiment 1) and a myeloperoxidase (MPO) assay (Experiment 2) were conducted in vitro. Blood was sampled from 12 mid-lactation dairy cows of variable ages. Neutrophils were extracted and diluted to obtain cell suspensions of approximately 106 cells/mL. In Experiment 1, test substances were placed in a 96-well plate, separated from the cell suspension by a 3 µm pore membrane and incubated for 3 h to allow neutrophils to migrate through the membrane. In Experiment 2, neutrophils were exposed to the test products and the amount of MPO released was measured by optical density. Results showed that neutrophils were not activated by bismuth or keratin products (p < 0.05) in all of the tests performed. These results suggest that the mechanisms of action of bismuth subnitrate and keratin-based teat sealants do not rely on neutrophil recruitment and activation in the teat canal and sinus after treatment.

Polysialic Acid Modulates the Binding of External Lactoferrin in Neutrophil Extracellular Traps

Neutrophil extracellular traps (NETs) are formed by neutrophils during inflammation. Among other things, these DNA constructs consist of antimicrobial proteins such as lactoferrin and histones. With these properties, NETs capture and destroy invading microorganisms. The carbohydrate polysialic acid (polySia) interacts with both lactoferrin and histones. Previous experiments demonstrated that, in humans, lactoferrin inhibits the release of NET and that this effect is supported by polySia. In this study, we examined the interplay of lactoferrin and polySia in already-formed NETs from bovine neutrophils. The binding of polySia was considered to occur at the lactoferricin (LFcin)-containing domain of lactoferrin. The interaction with the peptide LFcin was studied in more detail using groups of defined polySia chain lengths, which suggested a chain-length-dependent interaction mechanism with LFcin. The LFcin domain of lactoferrin was found to interact with DNA. Therefore, the possibility that polySia influences the integration of lactoferrin into the DNA-structures of NETs was tested by isolating bovine neutrophils and inducing NETosis. Experiments with NET fibers saturated with lactoferrin demonstrated that polySia initiates the incorporation of external lactoferrin in already-loaded NETs. Thus, polySia may modulate the constituents of NET.

Galectin-8 Modulates Innate and Adaptive Immune Response Genes in Bovine Neutrophils

Galectins (Gals) are a family of animal lectins that bind &beta;-galactosides through a carbohydrate recognition domain. Galectin-8 is a tandem-repeat galectin, secreted intracellularly and extracellularly. It is associated with neutrophil migration and has been studied as a possible therapeutic to combat inflammation. The objective of this study was to evaluate the translational and the transcriptional effects of recombinant Galectin-8 (rGal-8) on cow neutrophils. Blood was collected aseptically from Holstein-Friesian cows (n=10) from the North Carolina A&amp;T State University Dairy Unit. Neutrophils isolated were treated with rGal-8 (2&mu;g), or PBS (control) and were incubated at 37&deg;C, 5% CO2 for 1 hour. Supernatant from treated neutrophils was evaluated for total protein concentration, and galectin-8 secretion using bovine Galectin-8 Enzyme Linked-Immuno-Sorbent Assay (ELISA) kit. Total RNA was extracted, reverse transcribed, and RT-qPCR was performed using the RT&sup2; Profiler Cow Innate &amp; Adaptive Immune Responses Array with 84 genes. The Livak method was used to calculate transcript abundance and fold change (FC&gt;2 considered significant). Total protein concentration increased (P=0.0361) after rGal-8 treatment compared to the untreated control. Galectin-8 secretion was not significantly different in control compared to treated group (P=0.5819). Out of the 84 genes, 81 genes were differentially expressed in response to rGal-8; 14 up-regulated, 5 down-regulated, 61 genes remained unchanged. Treatment with rGal8 induced the expression of IRF7. The top five up-regulated genes include FAS, CD40, CD86, IFNGR1, STAT1; down-regulated genes were TLR9, CD14, CCR6, TICAM1, and TLR1. Selected genes were probed to validate fold change; the levels of gene expression were comparable to data from RT2 array. Exposure of bovine neutrophils to rGal-8 modified expression of immune response genes. The functional significance of the change needs further studies.