Low serum vitamin D concentrations in Spring-born dairy calves are associated with elevated peripheral leukocytes.

A role for vitamin D in the immune system is emerging from human research but data in the bovine is limited. In the current study, 48 Holstein-Friesian calves were randomly assigned to one of 4 groups designed to expose calves to divergent vitamin D levels for a 7 month period and to determine its effects on circulating immunity in young calves. Concentrations of circulating 25-hydroxyvitamin D (25OHD) was measured in serum using a commercial ELISA with validated bovine standards. Results showed that mean circulating concentrations of 25OHD at birth was 7.64 ± 3.21 ng/ml indicating vitamin D deficiency. Neither the injection of Vit D3 at birth nor the elevated levels in milk replacer yield discernible changes to pre-weaning circulating concentration of 25OHD. No calf reached the recommended level of vitamin D immune sufficiencyof 30 ng/ml of 25OHD until at least 3 months of age (T4). Increasing dietary Vit D3 via ration in the post-weaning period significantly elevated 25OHD concentrations in serum in VitD-In calves. Maximal levels of circulating 25OHD were achieved in VitD-Out calves, reaching 60.86 ± 7.32 ng/ml at 5 months of age (T7). Greatest divergence in haematology profile was observed between Ctl-In vs VitD-In groups with Ctl-In calves showing an elevated count of neutrophils, eosinophils, and basophils associated with reduced 25OHD concentrations. Neither IL-8 expression nor ROS production in serum were significantly different between calves with high and low 25OHD, indicating that other vitamin D-dependent mechanisms may contribute to the divergent circulating cellular profiles observed. This novel data on the vitamin D status of neonatal calves identifies a significant window of vitamin D insufficiency which is associated with significant differences in circulating immune cell profiles. Vitamin D insufficiency may therefore exacerbate pre-weaning disease susceptibility, and further work in now warranted.

Effect of IL-8 haplotype on temporal profile in circulating concentrations of interleukin 8 and 25(OH) vitamin D in Holstein-Friesian calves.

Interleukin-8 (IL-8) is an inflammatory chemokine released during the primary innate immune response to recruit neutrophils to the site of infection. Two distinct gene promoter haplotypes have been previously reported to segregate in the Holstein-Friesian breed (IL8-h1 and IL8-h2). Our earlier work showed how these divergent IL8 haplotypes influence IL-8 concentration and other immune response parameters at a systemic level. While a close relationship has been established between vitamin D and IL-8 in other species, the role of genetic haplotype on temporal variation in vitamin D concentrations and its impact on immunity remains unexplored in cattle. Therefore this study had two objectives - 1: to establish the temporal variation in IL-8 concentration profile in healthy calves of each IL-8 haplotype (n = 5/6 per group) and 2: to identify the relationship between systemic 25(OH)D concentration and IL8 haplotype in blood at 10 time points across their first year of life. Elevated IL-8 protein concentration profiles were apparent in IL8-h2 calves at multiple time points throughout the year (P < 0.05). In contrast, circulating concentrations of 25(OH) vitamin D were negatively correlated (0.38) with IL-8, with elevated concentrations in calves of the IL8-h1 haplotype. Increased numbers of innate immune cells - specifically monocytes and basophils, were also detected in blood from IL8-h2 calves (P < 0.05). Importantly, circulating concentrations of vitamin D were substantially below recommended concentrations of 30 ng/mL serum for optimal immunity in the first five months of life, indicating a window of potentially heightened disease susceptibility - particularly in calves of the IL8-h1 haplotype. In conclusion, this study has established that IL8 haplotype confers divergent chemokine concentrations and which contrasts with circulating concentrations of vitamin D. Accounting for both IL8 haplotype and vitamin D concentration may be critical to provide dairy calves with optimal immune protection in early life.

The immune response in bovine primary dermal fibroblasts is influenced by Interleukin 8 promoter haplotype and vitamin D.

Interleukin-8 (IL-8) is a potent inflammatory chemokine, and two gene promoter haplotypes have been previously reported to segregate in cattle populations. Our earlier work showed how these divergent IL8 genotypes influence IL-8 expression and other immune response parameters at a systemic level. Here we extend that work to characterise the influence of haplotype on the local immune response - in primary bovine dermal fibroblasts. Furthermore, we also investigated how this response is modulated by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Significant induction of IL8 expression was observed in cells from both haplotypes at 3 and 24 h post-stimulation with the TLR1/2 ligand, Pam3CSK4 and with the TLR4 ligand, LPS. IL8 expression was elevated in response to both LPS and Pam3CSK4 in fibroblasts carrying the IL8-h1 haplotype and this result was supported by significantly enhanced IL-8 protein secretion. Gene expression profiles for other known fibroblast immune mediators (SAA3 and CCL20) did not show significant differences between haplotypes but NOS2 gene expression was significantly elevated in response to vitamin D, even above the level detected in response to both TLR ligands. In conclusion, this work has demonstrated that the IL-8 response of dermal fibroblasts is dependent on IL8 haplotype and that the immune response profile in these cells is significantly differentially regulated by 1,25(OH)2D3. Fibroblasts have important immune response capacity and their function in driving inflammatory responses (including iNOS) is underappreciated. Understanding the relationship between cattle genotype and immune function is critically important for uncovering sustainable solutions for animal disease.

Effect of IL8 haplotype on immunological traits in periparturient dairy cows.

Interleukin 8 (IL8) is a major mediator of the innate immune response. Polymorphisms in this gene are associated with susceptibility to inflammatory disease in humans. Two major promoter polymorphic haplotypes (IL8-h1 and IL8-h2) segregating in cattle populations have shown a significant effect on the immune response profile in calves but their implications for transition cow immunity have not been established. The aims of this study were to assess functional relevance of the IL8 haplotypes on the immunological traits of periparturient cows (n = 32) belonging to three genetic groups: Holstein (HO), Simmental (SI) and their crosses (CR) and to evaluate the frequency of IL8 haplotypes in the HO (dairy) and SI (dual purpose) pure breeds. IL8 haplotypes showed a significant effect on circulating number of both T helper lymphocytes (P = 0.0133) and T cytotoxic lymphocytes (P = 0.0024). Differences in percentage of CD14+ monocytes and T lymphocyte subsets were found between haplotype groups at different time points. Plasma concentrations of Serum Amyloid A (SAA) and Haptoglobin (Hp) were enhanced at calving in IL8-h2 (P = 0.0019, P = 0.0029) and IL8-het (P = 0.050 and P = 0.052) respectively, compared with IL8-h1 cows. In contrast, significantly lower levels of reactive oxygen metabolites (d-ROMs) activation were identified in IL8-h2 and IL8-het cows after calving compared with IL8-h1 cows. Furthermore, genotyping results showed that SI cows have a high frequency of the homozygous IL8-h2 haplotype compared to the HO cows (87.5 % vs 40 %) which reflects the different selective pressure between the two pure breeds. In conclusion, our preliminary data suggests that IL8 promoter haplotype is associated with significant and dynamic changes in immunological traits during peripartum and early lactation period. Future work will focus on a more comprehensive assessment of immune changes in additional cows.

1,25(OH)D vitamin D promotes NOS2 expression in response to bacterial and viral PAMPs in primary bovine salivary gland fibroblasts.

OBJECTIVES: The faecal-oral route is a predominant mode of infectious disease transmission and yet the immunology of the bovine oral cavity is poorly understood. The objectives of this study were to develop an in vitro cell model of bovine salivary gland cells and to characterize the role of vitamin D on the expression of innate immune genes induced by stimulation with bacterial and viral pathogen-associated molecular patterns (PAMPs). METHODS: Submandibular glandular tissue was excised post-mortem, processed, cells isolated and cultured until confluency after which cells were incubated with the active form of vitamin D (1,25(OH)D) for 18 h before stimulation with lipopolysaccharide (LPS µg/ml), lipoteichoic acid (LTA µg/ml) or polyinosinic:polycytidylic acid (poly I:C-20 µg/ml) PAMPs for 6 h and immune gene expression was assessed by Quantitative Real-Time PCR (RT-qPCR). RESULTS: RT-qPCR analysis of vimentin expression in cells derived from the bovine submandibular gland shows that cultured cells were fibroblast in origin. These cells significantly induce the pro-inflammatory cytokine IL1B, ß-defensin and cathelicidin genes but these were not significantly altered in response to 1,25(OH)D. In contrast, 1,25(OH)D significantly up-regulates the expression of the NOS2 gene encoding iNOS in bovine submandibular stromal cells compared to EtOH (vehicle) control and this is a maintained response to all three bacterial and viral ligands. We have developed a new in vitro model to allow detailed investigations of mechanisms to enhance oral immunity in cattle. We show that these cells are fibroblast in nature, immunologically competent and vitamin D responsive. Their vitamin D-mediated enhancement of NOS2 expression warrants further investigation in saliva as a potential mechanism to boost oral immunity against infectious agents.

Application of the TruCulture® whole blood stimulation system for immune response profiling in cattle.

Capturing the phenotypic variation in immune responses holds enormous promise for the development of targeted treatments for disease as well as tailored vaccination schedules. However, accurate detection of true biological variation can be obscured by the lack of standardised immune assays. The TruCulture® whole blood stimulation system has now been extensively used to detect basal and induced immune responses to a range of pathogen-associated molecular patterns (PAMPs) in human peripheral blood. This study demonstrates the optimisation of this commercially available assay for systemic immune phenotyping in cattle. The early immune response in Holstein-Friesian bull calves (n = 10) was assessed by haematology, flow cytometry and cytokine expression profiling after 24 h ex-vivo PAMP (LPS, poly (I:C) and zymosan) stimulation in TruCulture® tubes. A comparative analysis was also performed with a traditional whole blood stimulation assay and cell viability using both systems was also evaluated. Results: Supernatant collected from TruCulture® tubes showed a significant increase in IL-1ß and IL-8 expression compared to null stimulated tubes in response to both LPS and zymosan. In contrast, a detectable immune response was not apparent at the standard concentration of poly (I:C). Conventional whole blood cultures yielded similar response profiles, although the magnitude of the response was higher to both LPS and zymosan, which may be attributed to prokaryotic strain-specificity or batch of the stimulant used. Despite being a closed system, HIF1A expression - used as a measure of hypoxia was not increased, suggesting the TruCulture® assay did not negatively affect cell viability. This represents the first reported use of this novel standardised assay in cattle, and indicates that the concentration of poly (I:C) immunogenic in humans is insufficient to induce cytokine responses in cattle. We conclude that the low blood volume and minimally invasive TruCulture® assay system offers a practical and informative technique to assess basal and induced systemic immune responses in cattle.