We reported previously that sheep affected with FR have lower WB-Se concentrations and that parenteral Se-supplementation in conjunction with routine control practices accelerates recovery from FR [12
]. To determine whether Se acts as an immunonutrient and improves immune function in FR-affected sheep, we examined the effect of FR, Se treatment, and WB-Se status on measures of CMI, humoral immunity, and innate immunity. Our primary finding is that immune responses to a novel protein (KLH) are attenuated in FR-affected sheep with lower WB-Se status. Furthermore, the DTH and antibody titer responses to a novel protein were improved, in part, by Se treatment and high WB-Se status, supporting our hypothesis that Se acts as an immunonutrient in FR-affected sheep. Neutrophil function was suppressed by FR, but was not changed by Se supplementation or WB-Se status.
The purpose of this follow-up study was to investigate the mechanisms by which Se facilitates recovery from FR. The immune system has two functional divisions: innate and adaptive immunity. Both divisions involve various blood-borne factors (e.g., complement, antibodies, and cytokines) and cells (e.g., neutrophils, lymphocytes, and macrophages). There is large individual variation in immune function even among healthy animals. For example, differences in genetics, age, gender, levels of exercise, diet, stress, infectious disease history, vaccination status, and early life experiences are important contributors to this observed variation [17
]. Thus, demonstrating an improvement in immune function with Se supplementation is challenging.
Tests used to assess adaptive immunity include the DTH test, which is also known as a type IV hypersensitivity reaction. This test provides a general measure of CMI. Professional antigen presenting cells, e.g., dendritic cells, present antigen to T lymphocytes. This results in antigen-specific activation of T lymphocytes in local tissues. Inflammatory cytokines produced by these stimulated T lymphocytes cause other mononuclear cells (lymphocytes and macrophages) to migrate to the area and proliferate. To perform this test, foreign antigen is injected under the epidermis of the skin. The immune system responds to this antigen by producing a small raised wheal that can be measured 24 to 96 h after injection. The larger the wheal, the greater is the CMI response.
In our study, healthy control sheep demonstrated an enhanced CMI response to a novel protein (KLH) compared with FR-affected sheep based on the DTH test. Results were more definitive for the ear tip compared with two wool-free sites on the ventro-lateral abdomen, likely because higher tissue compliance allowed a more diffuse DTH reaction on the abdomen. For this reason, the latter may not be as useful of a test location as the ear tip for this assay in sheep. Both ear thickness and ear wheal diameter were similarly affected, although the DTH response resolved faster over time for ear wheal diameter. The DTH response in healthy sheep receded from 24 to 96 h. Although ear thickness was suppressed at 24 h in FR ewes compared to healthy controls, there was no change across time in FR-Se treated sheep. Thus, by 96 h FR-Se sheep had a greater ear thickness than FR-Sal sheep and a similar ear thickness compared with healthy control sheep. Similarly, FR-affected sheep with higher WB-Se concentrations had a more intense DTH response than FR-affected sheep with lower WB-Se concentrations. Our results suggest an attenuated T-lymphocyte response in FR-affected sheep, which could be the result of decreased activation, migration, proliferation, or a combination of these, and which may be improved, in part, by Se treatment. An enhanced DTH response after Se supplementation was also shown by Lacetera et al. [18
]. In their study, ewes given a single 5 mg Se injection 30 d prior to lambing had a greater DTH response to intradermal phytohaemagglutinin (PHA) injection at 6 h than ewes not treated with Se. Lambs born to Se-treated ewes had a greater DTH response to PHA 24 h after injection. In this study as well as ours, Se was supplemented to Se-adequate sheep. Methodology differences with injection site (ear vs. neck), antigen (KLH vs. PHA), response time (days vs. hours), and measurement technique (wheal reaction vs. skin thickness) likely account for differences between the studies.
Measuring an antibody titer in response to sensitization/immunization is another test to assess the adaptive (humoral) immune response. The animal is injected with a novel protein (e.g., KLH) that elicits an immune response. Following sensitization, antibody titers to KLH can be measured. Our results showed that FR disease was associated with lower KLH antibody titers 2 and 4 weeks after KLH immunization compared with healthy sheep, suggesting a lower antibody titer to a novel protein in FR-affected sheep. Stratification by WB-Se status showed that sheep with FR-High Se had KLH antibody titers more similar to healthy control sheep 14 days after the second immunization. There was no difference in antibody titers between FR-Se and FR-Sal treated ewes at 28 days, suggesting that titers may rise more rapidly in FR-Se treated ewes, similar to what we have shown in cattle [19
]. Our results suggest that Se may improve antibody production in response to a novel protein in FR-affected sheep. It would be interesting to look at earlier titers in future studies, for example at 7 days after the second immunization.
Sheep affected with FR, regardless of Se treatment had smaller ear wheal diameters compared with healthy sheep 30 min after KLH challenge. This is consistent with a reduction in the type I hypersensitivity reaction normally induced by histamine and inflammatory cytokines. The KLH stimulates inflammatory cytokine production. Bacterial infection may cause immunosuppression of Type I hypersensitivity by affecting the release of histamine, or virulence factors such as leukotoxin, endotoxin, haemolysin, haemagglutinin and adhesin that are used to overcome the host's defense mechanisms when infection is established may also suppresses the immune response.
Selenium treatment decreased the 30 min skin-test response to histamine in FR-affected sheep. Histamine normally increases capillary permeability and relaxes vascular smooth muscle, allowing edema fluid accumulation. Influx of proinflammatory cytokines triggers production of reactive oxygen species (ROS). When produced in excess, ROS are important mediators of cell and tissue injury. Because Se is involved in redox reactions, and immune activation is usually associated with increased production of ROS by cells of the immune system, higher Se may help suppress tissue damage caused by ROS (reviewed in Murr et al. [20
]). As a component of the glutathione peroxidase family of enzymes, Se contributes to the reduction of hydroperoxides in cells. Glutathione peroxidase reduces ROS to less reactive metabolites, decreasing oxidant stress. We suggest that Se suppressed oxidative stress and attenuated the early skin-test reaction at 30 min. Thus, Se may protect from type I hypersensitivity reactions, involving IgE antibody triggering of mast cells and oxidative insult, by preventing lipid hydroperoxide accumulation. On the other hand, Se enhanced the type IV hypersensitivity reaction at 96 h.
Neutrophils are the most numerous and important cellular component of innate immunity. Their primary functions are phagocytosis and destruction of microorganisms. They serve as the body's first line of defense against invading microorganisms. Phagocytosed bacteria are rapidly killed by proteolytic enzymes (e.g., myeloperoxidase), antimicrobial proteins, and ROS when membrane-bound granules fuse with phagocytic vesicles. In addition to phagocytosis, bacterial killing occurs via neutrophil extracellular traps (NETs) [21
]. Upon activation, neutrophils release extracellular fibers called NETS, which form a meshwork that kills bacteria extracellularly without the need for phagocytosis. NETs are made up of DNA, histones, and granule proteins such as elastase. Both Gram-positive and Gram-negative bacteria are killed, which suggests that NETs can kill a wide range of pathogens. These NETs serve as a physical barrier, which prevents the spread of bacteria.
To assess innate immunity of neutrophils, a biologic assay was performed using Lactococcus lactis
and measuring percent bacterial killing. Neutrophils from healthy control sheep demonstrated higher percent bacterial killing compared with neutrophils from FR-affected sheep, regardless of 15 months of Se supplementation. Even when FR-affected sheep were stratified by WB-Se status, neutrophil bacterial killing ability was not altered. Our results suggest that FR-affected sheep have reduced neutrophil bacterial killing ability, which cannot be improved by Se supplementation or high WB-Se status. It has been reported previously that neutrophils of Se-deficient cattle have reduced ability to kill phagocytosed bacteria [24
]. Although acute infections have been shown to decrease serum-Se levels [26
], sheep in the current study were Se-adequate. It is possible that supranutritional Se does not further improve neutrophil bacterial killing ability or that the disease state inhibits or prevents modulation of neutrophil bacterial killing ability.
Measuring the relative abundance of mRNA specific for neutrophil migration activity (L-selectin and IL-8R) by RT-qPCR is another method for assessing innate immunity. To protect against invading pathogens, neutrophils migrate to infected sites. Neutrophils roll along walls of blood vessels by the coordinated formation and breakage of selectin-carbohydrate chemical bonds. L-selectin is a cell surface glycoprotein that is constitutively expressed on the surface of most leucocytes [27
]. L-selectin is important for the binding and subsequent rolling of leucocytes along endothelial walls, facilitating migration into secondary lymphoid organs (e.g., naive T cells) and into sites of inflammation (e.g., neutrophils) [27
]. Chemokines such as IL-8 and its receptor control the interaction of neutrophils with the epithelial cell barrier. IL-8 is recognized mainly for its ability to induce neutrophil migration, but it also increases cytokine production, enhances phagocytosis and ROS generation, and regulates cell survival [28
]. In our study, we found that sheep affected with FR had lower concentrations of IL-8R mRNA compared with healthy sheep, and Se treatment had no effect. Our results suggest that attenuated IL-8R gene expression may play a role in reducing bacterial killing ability in FR-affected sheep; IL-8R gene expression, however, may not be responsive to Se treatment. The relative abundance of L-selectin mRNA was not affected by FR-status, however, mRNA concentrations tended to be higher in FR-High Se sheep, suggesting that L-selectin is a potential molecular target of Se treatment.
In conclusion, our goal was to determine if supplementing Se at concentrations above those currently recommended for sheep (supranutritional) can modulate the immune response in a way that reduces severity and/or improves recovery from a disease process. We reported previously that parenteral Se supplementation in conjunction with routine control practices resulted in higher WB-Se concentrations and more rapid improvement of foot lesions [12
]. In this study, we found that neutrophil function and the DTH and antibody titer responses to a novel protein (KLH) were attenuated in FR-affected sheep. The DTH and antibody titer responses to a novel protein were improved, in part, by Se treatment and WB-Se status, suggesting that FR in sheep is associated with reduced lymphocyte function, which may be partly restored by improving WB-Se status (≥ 250 ng/mL). Additional experiments are needed to determine the best source, route of administration, and dose of Se to optimize immune function in sheep with FR.