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T-lymphocytes are key mediators of adaptive cellular immunity and knowledge about distinct subsets of these cells in healthy and infected mammary gland secretions remains limited. In this study, we used a multiplex cytometry panel to show that staphylococcal mastitis causes the activation of CD4+, CD8+and γδ T-cells found in bovine milk. We also highlight remarkable differences in the proportions of naïve and memory T-cells subsets found in blood and milk. These observations will contribute to a better understanding of cell-mediated immune mechanisms in the udder and to the development of new therapeutic and preventive strategies targeting mastitis.

Bovine mastitis are mainly caused by bacterial infection. They are responsible for economic losses and have an impact on the health and welfare of animals. The increase in the somatic cell count in milk during mastitis is mainly due to the influx of neutrophils which have a crucial role in the elimination of pathogens. For a long time, these first line defenders has been view as microbes’ killers with limited role in the orchestration of the immune response. However, their role is more complex and we recently characterized a MHC-II expressing neutrophil subset with regulatory capacities in cattle. In this study, we questioned the implication of different neutrophils subsets in the mammary gland immunity during clinical and subclinical mastitis. Here, we described for the first time that, in blood as in milk, neutrophils are a heterogeneous population and encompass at least two subsets distinguishable with their expression of MHC-II. We observed higher bactericidal capacities of milk MHC-IIpos neutrophils as compared to their classical counterparts, due to a higher production of ROS and phagocytosis ability. MHC-IIpos neutrophils are enriched in milk during a subclinical mastitis as compared to blood. Moreover, we observed a positive and highly significant correlation between MHC-IIpos neutrophils and T lymphocytes present in milk during subclinical mastitis. To conclude, our study could open the way to the discovery of new biomarkers of mastitis inflammation.

Neutrophils that reside in the bone marrow are switly recruited from circulating blood to fight infections. For a long time, these first line defenders were considered as microbe killers. However their role is far more complex as cross talk with T cells or dendritic cells have been described for human or mouse neutrophils. In cattle, these new roles are not documented yet. We identified a new subset of regulatory neutrophils that is present in the mouse bone marrow or circulate in cattle blood under steady state conditions. These regulatory neutrophils that display MHC-II on the surface are morphologically indistinguishable from classical MHC-IInegneutrophils. However MHC-IIposand MHC-IInegneutrophils display distinct transcriptomic profiles. While MHC-IInegand MHC-IIposneutrophils display similar bacterial phagocytosis or killing activity, MHC-IIposonly are able to suppress T cell proliferation under contact-dependent mechanisms. Regulatory neutrophils are highly enriched in lymphoid organs as compared to their MHC-IInegcounterparts and in the mouse they express PDL-1, an immune checkpoint involved in T-cell blockade. Our results emphasize neutrophils as true partners of the adaptive immune response, including in domestic species. They open the way for discovery of new biomarkers and therapeutic interventions to better control cattle diseases.

International audience; Flow cytometry and magnetic bead technology enable the separation of cell populations with the highest degree of purity. Here, we describe protocols to sort bovine neutrophils from blood, the labeling and sorting, including gating strategies. We also provide advice to preserve neutrophil viability and detail a protocol to measure phagocytosis and oxidative species production.

Flow cytometry and magnetic bead technology enable the separation of cell populations with the highest degree of purity. Here, we describe protocols to sort bovine neutrophils from blood, the labeling and sorting, including gating strategies. We also provide advice to preserve neutrophil viability and detail a protocol to measure phagocytosis and oxidative species production.