Differential phenotype of immune cells in blood and milk following pegylated granulocyte colony stimulating factor (PEG-gCSF) therapy during a chronic Staphylococcus aureus infection in lactating Holsteins

Authors: POWELL, ELLIS - Oak Ridge Institute For Science And Education (ORISE); EDER, JORDEN - Iowa State University; Reinhardt, Timothy; Sacco, Randy; Casas, Eduardo; Lippolis, John

Submitted to: Journal of Dairy Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/10/2019
Publication Date: 10/1/2019
Citation: Powell, E.J., Eder, J.M., Reinhardt, T.A., Sacco, R.E., Casas, E., Lippolis, J.D. 2019. Differential phenotype of immune cells in blood and milk following pegylated granulocyte colony stimulating factor (PEG-gCSF) therapy during a chronic Staphylococcus aureus infection in lactating Holsteins. Journal of Dairy Science. 102(10):9268-9284. https://doi.org/10.3168/jds.2019-16448.
DOI: https://doi.org/10.3168/jds.2019-16448

Interpretive Summary: Mastitis disease is defined as the inflammation of the mammary gland and can affect all lactating animals. Mastitis can result from various bacterial infections including Staph. aureus. In particular, mastitis is of great health and economic concern to dairy cattle, where an infection can result in discomfort and damage to the animal, as well as a loss of safe, consumable milk. Preventing and treating mastitis is thus critically important to the dairy cow industry. One developing therapy is to treat the cows with a cytokine (cell signaling protein) designed to activate the immune cells most important in fighting mastitis causing bacteria. In this study we analyzed eight cows with a chronic Staph. aureus infection. We treated four cows with two injections of a cytokine, and compared their immune responses to the four untreated controls. We examined data such as milk production, blood cell profile, and the presence of bacteria and immune cells in the milk. We knew from a previous study that immune cells in the blood stream respond to the cytokine treatment we used. Our primary goals were to further examine the effects of the cytokine therapy and learn more about the mechanism of immune system response to mastitis specifically in the immune cells in the mammary gland itself. We found that the cows that had been treated with the cytokine produced the same amount of milk, but surprisingly had more bacteria in their milk compared to untreated cows. The treated cows also had biological changes on immune cells in blood and milk. Overall, we show that this cytokine is not a treatment for Staph. aureus mastitis infections. These findings are important in the development of mastitis prevention and treatment therapies.

Technical Abstract: Neutrophils are principal host innate immune cell responders to mastitis infections. Thus, therapies have been developed that target neutrophil expansion. This includes the neutrophil-stimulating cytokine granulocyte colony-stimulating factor (G-CSF). Pegylated G-CSF (PEG-gCSF) (Imrestor, Elanco Animal Health) has been shown to reduce the natural incidence of mastitis in periparturient cows in commercial settings and reduce severity of disease against experimental mastitis challenge. PEG-gCSF stimulates neutrophil expansion, but also induces changes in monocyte and lymphocyte circulating numbers and/or surface protein expression changes. The aim of this study is to better understand the how PEG-gCSF therapy modulates immune cell populations in the mammary gland during a chronic mastitis infection. We challenged eight mid-lactation Holsteins with ~150 CFU of Staphylococcus aureus (Newbould) in a single quarter via intra mammary infusion. All animals developed chronic infections as assessed by bacteria counts and SCCs. Ten to sixteen weeks post challenge four of the animals were treated with two SC injections of PEG-gCSF, seven days apart. CBCs, SCCs, bacterial counts, milk yield, feed intake, NET analysis, and flow cytometric analyses of milk and blood samples were performed at indicated time points for 14 days after the first PEG-gCSF injection. PEG-gCSF treated cows had significantly increased numbers of blood neutrophils and lymphocytes compared to control cows. Flow cytometric analyses revealed increased surface expression of myeloperoxidase (MPO) on neutrophils and macrophages in milk, but not in blood of treated cows. Neutrophils isolated from blood of PEG-gCSF treated cows had decreased surface expression of CD62L (L-selectin) in blood, consistent with cell activation. Surprisingly, CD62L cell surface expression was increased on neutrophils and macrophagses sourced from milk from treated animals compared to cells isolated from controls. PEG-gCSF treated cows did not clear the S. aureus infection, nor did they significantly differ from controls in SCCs. These findings provide evidence that PEG-gCSF therapy modifies cell surface expression of neutrophils and monocytes. However, while surface MPO+ cells accumulate in the mammary gland, the lack of bacterial control from these milk-derived cells suggest an incomplete role for PEG-gCSF treatment against chronic S. aureus infection and maybe chronic mammary infections in general.