Neutrophils expressing major histocompatibility complex class II molecules circulate in blood and milk during mastitis and show high microbicidal activity.

Bovine mastitis is mainly caused by bacterial infection and is responsible for important economic losses as well as alterations of the health and welfare of animals. The increase in somatic cell count (SCC) 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 have been viewed as microbe killers, with a limited role in the orchestration of the immune response. However, their role is more complex: we recently characterized a bovine neutrophil subset expressing major histocompatibility complex class II (MHC-II) molecules (MHC-IIpos), usually distributed on antigen-presenting cells, as having regulatory capacities in cattle. In this study, our objective was to evaluate the implication of different neutrophils subsets in the mammary gland immunity during clinical and subclinical mastitis. Using flow cytometry, we analyzed the presence of MHC-IIpos neutrophils in blood and in milk during clinical mastitis at different time points of inflammation (n = 10 infected quarters) and during subclinical mastitis, defined as the presence of bacteria and an SCC >150,000 cells/mL (n = 27 infected quarters). Our results show, for the first time, that in blood and milk, neutrophils are a heterogeneous population and encompass at least 2 subsets distinguishable by their expression of MHC-II. In milk without mastitis, we observed higher production of reactive oxygen species and higher phagocytosis capacity of MHC-IIpos neutrophils compared with their MHC-IIneg counterparts, indicating the high bactericidal capacities of MHC-IIpos neutrophils. MHC-IIpos neutrophils are enriched in milk compared with blood during subclinical mastitis but not during clinical mastitis. Moreover, we observed a positive and highly significant correlation between MHC-IIpos neutrophils and T lymphocytes present in milk during subclinical mastitis. Our experiments involved a total of 47 cows (40 Holstein and 7 Normande cows). To conclude, our study opens the way to the discovery of new biomarkers of mastitis inflammation.

Bovine blood and milk T-cell subsets in distinct states of activation and differentiation during subclinical Staphylococcus aureus mastitis.

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.

Neutrophils Encompass a Regulatory Subset Suppressing T Cells in Apparently Healthy Cattle and Mice.

Neutrophils that reside in the bone marrow are swiftly 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-IIneg neutrophils. However MHC-IIpos and MHC-IIneg neutrophils display distinct transcriptomic profiles. While MHC-IIneg and MHC-IIpos neutrophils display similar bacterial phagocytosis or killing activity, MHC-IIpos only are able to suppress T cell proliferation under contact-dependent mechanisms. Regulatory neutrophils are highly enriched in lymphoid organs as compared to their MHC-IIneg counterparts 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.

Isolation of Bovine Neutrophils by Fluorescence- and Magnetic-Activated Cell Sorting.

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.