Genetic variations in the SPP1 promoter affect gene expression and the level of osteopontin secretion into bovine milk.

Osteopontin (OPN) is now recognized as an important cytokine and extracellular integrin-binding protein at the crossroads of inflammation and homeostasis. In a previous study, we found that OPN gene (SPP1) polymorphisms are associated with milk performance traits and somatic cell score (SCS), a parameter used to estimate the genetic value of udder health in dairy cattle. In this study, we assessed whether the genetic variations had an impact on SPP1 promoter activity, immune response and the level of OPN secreted into milk. The influence of DNA polymorphisms on the promoter activity of SPP1 was confirmed in vitro. To measure the impact of the genetic variations on OPN secretion into milk, we measured OPN levels in both plasma and milk throughout lactation. Cows were grouped by the OPN haplotypes associated with a high (H2 × H3) or low (H1 × H4) SCS. For both H2 × H3 and H1 × H4, the OPN level in plasma remained low throughout lactation, although the concentration in the milk of H1 × H4 cows increased more in late lactation. Moreover, the macrophages of H1 × H4 cows expressed a lower SPP1 and proinflammatory IL6 in response to infection. Regarding the immune cell response, cows with the genetic potential to secrete higher OPN levels during late lactation had macrophages expressing fewer proinflammatory cytokines, a situation that might explain the genetic association with low somatic cells. Although OPN's favorable roles during late lactation remain to be elucidated, the tissue remodeling properties associated with OPN may be beneficial for reducing the incidence of infection during the transition period in lactating cows.

Increased blood-circulating interferon-γ, interleukin-17, and osteopontin levels in bovine paratuberculosis.

Paratuberculosis-infected cattle initially develop an effective cell-mediated immune response that declines as the disease progresses. Blood is one of best sources for characterizing the inflammatory status of infected cows and for studying mediators related to chronic diseases. The aim of this study was to evaluate the cow-level association between blood cytokine concentration, the influence of serum on immune cell proliferation, and dairy cows naturally infected with Mycobacterium avium ssp. paratuberculosis (MAP). Positive animals (n=41) from 19 herds were selected on the basis of 2 positive fecal culture results and divided into 2 groups: single-positive, or serum ELISA-negative cows (n=32), and double-positive, or cows that gave positive results for both mycobacterial culture and serum ELISA (n=9). Negative animals (n=39) were selected from paratuberculosis-negative herds in which at least 80% of the animals had been diagnosed as negative by fecal culture and ELISA and that did not produce positive results during the 2-yr study. Analysis of plasma levels of the cytokines IL-4, IL-10, IL-17, IFN-γ, and osteopontin was performed, revealing distinct patterns. The ELISA-positive cows with MAP shedding had similar plasma concentrations of IL-4 and IL-10 but elevated levels of IFN-γ, IL-17, and osteopontin, which is indicative of inflammatory disease in these subclinical positive cows. In vitro MAP infection of bovine macrophages showed increased gene expression of tumor necrosis factor-α, IL-1ß, IL-6, IL-23, and transforming growth factor-ß as early as 6h postinfection for all of the cytokines involved in the establishment of a T-helper type-17 immune response. To determine the systemic influence of serum on immune cell functions, lymphoproliferation assays were also performed in presence of JD serum. The serum from shedding cows showed 15% less proliferation. These results indicate that infected cows have a lower systemic capacity to maintain a protective immune response and that, as the disease progresses, an emerging T-helper type-17 immune response is established.

Proteomic analysis and immunodetection of the bovine milk osteopontin isoforms.

The aim of this study was to characterize the osteopontin (OPN) secreted in bovine milk and to determine whether the different forms are the product of spliced variants. Spliced variants of the human gene and secreted osteopontin isoforms have been reported in human tumor tissue. In bovine milk, we identified 2 major forms: one corresponding to the full-length coding transcript and a truncated version of this form. No alternative spliced transcripts were detected in the lactating mammary gland tissue, in milk somatic cells, or in peripheral blood immune cells. The 60-kDa bovine osteopontin (bOPN) and a truncated 40-kDa protein isoform were confirmed by mass spectrometry and further characterized by immunoblotting using a panel of 6 antibodies targeting different domains of the protein. Of the 3 human anti-OPN antibodies targeting the N-terminal segment of the protein, only one detected all forms on sodium dodecyl sulfate-PAGE; one human anti-OPN antibody failed to detect bOPN, whereas the other detected only the 60-kDa protein, albeit barely in its phosphorylated form. Detection was generally more sensitive when the 60-kDa protein was dephosphorylated. Two polyclonal antibodies raised against bOPN were tested: one targeting the milk-purified bOPN (bOPN-121) and one targeting a bovine epitope (synthetic peptide) corresponding to a carboxy-terminal domain of the protein (bOPN-117). The bOPN-121 antibody detected all forms irrespective of the phosphorylation status of bOPN. The bOPN-117 and the mouse anti-human OPN (hOPN-4) antibodies, which recognized different domains of the carboxy-terminal segment of the protein, also preferentially detected the dephosphorylated 60-kDa protein. Whereas phosphorylation had a major effect on detection for several antibodies, deglycosylation slightly decreased immunodetection for the tested antibodies. In particular, phosphorylation is the major posttranslational modification that influenced the weak detection capacity of several antibodies. This fact needs to be taken into account for immunodetection of milk content. In conclusion, the OPN forms secreted in bovine milk are not the product of alternative splicing. The 40-kDa protein appears to be a truncated hypophosphorylated variant of the full-length 60-kDa form, which is highly phosphorylated. Together, the proteomic and immunoblotting analyses used to characterize bovine milk OPN revealed the complex nature of the bovine milk OPN forms.