Immune profiling of experimental murine mastitis reveals conserved response to mammary pathogenic Escherichia coli, Mycoplasma bovis, and Streptococcus uberis.

Mastitis is one of the most prevalent and economically important diseases of dairy animals. The disease is caused by ascending bacterial infection through the teat canal. Among the most common mastitis-causing bacteria are Gram-negative coliforms, Gram-positive streptococci and staphylococci, and mycoplasma. The most prominent cellular hallmark of acute mammary infection is a massive recruitment of blood neutrophils into the tubular and alveolar milk spaces. The complex biological processes of leukocyte recruitment, activation, adhesion, and migration in the mammary gland remain largely elusive to date. While field research of mastitis in dairy animals contributed a lot to the development of mitigation, control, and even eradication programs, little progress was made toward understanding the molecular mechanisms underlying the pathogenesis of the disease. We report here experimental mastitis model systems in lactating mice challenged with field strains of common udder pathogens in dairy cows. We used these model systems to apply recently developed multiplex gene expression technology (Nanostring nCounter), which enabled us to study the expression of over 700 immune genes. Our analysis revealed a core of 100 genes that are similarly regulated and functionally or physically interacting in E. coli, M. bovis, and Strep uberis murine mastitis. Common significantly enriched gene sets include TNFɑ signaling via NFkB, Interferon gamma and alpha response, and IL6-JAK-STAT3 signaling. In addition, we show a significantly enriched expression of genes associated with neutrophil extracellular traps (NET) in glands challenged by the three pathogens. Ligand-receptor analysis revealed interactions shared by the three pathogens, including the interaction of the cytokines IL1ß, IL1ɑ, and TNFɑ with their receptors, and proteins involved in immune cell recruitment such as complement C3 and ICAM1 (with CD11b), chemokines CCL3 and CCL4 (with CCR1), and CSF3 (with CSF3R). Taken together, our results show that mammary infection with E. coli, M. bovis, and Strep uberis culminated in the activation of a conserved core of immune genes and pathways including NET formation.

Mesenchymal stromal cells modulate infection and inflammation in the uterus and mammary gland.

The use of mesenchymal stromal cells (MSCs) is emerging as an efficacious and safe treatment for many infectious and non-infectious inflammatory diseases in human and veterinary medicine. Such use could be done to treat mastitis and metritis, which are the most common disease conditions affecting dairy cows leading to considerable economic losses and reduced animal welfare. Currently, both disease conditions are commonly treated using local and systemic administration of antibiotics. However, this strategy has many disadvantages including low cure rates and the public health hazards. Looking for alternative approaches, we investigated the properties of MSCs using in-vitro mammary and endometrial cell systems and in-vivo mastitis and metritis murine model systems. In-vitro, co-culture of mammary and uterus epithelial cells constructed with NF-kB reporter system, the master regulator of inflammation, demonstrated their anti-inflammatory effects in response to.LPS. In vivo, we challenge animals with field strains of mammary and utero pathogenic Escherichia coli and evaluated the effects of local and systemic application of MSC in the animal models. Disease outcome was evaluated using histological analysis, bacterial counts and gene expression of inflammatory markers. We show that MSC treatment reduced bacterial load in metritis and significantly modulated the inflammatory response of the uterus and mammary gland to bacterial infection. Most notably are the immune modulatory effects of remotely engrafted intravenous MSCs, which open new avenues to the development of MSC-based cell-free therapies.

Lipoproteins Are Potent Activators of Nuclear Factor Kappa B in Mammary Epithelial Cells and Virulence Factors in Mycoplasma bovis Mastitis.

Mastitis due to Mycoplasma bovis is a worldwide problem, which leads to significant economic losses and affects animal welfare. However, the mechanisms by which M. bovis establishes and maintains intra-mammary infections (IMI) in dairy cows are largely unknown. To study in further detail the pathogenesis of M. bovis IMI, time- and cost-effective experimental models are needed. To this end, we established and characterized an in vitro murine mammary alveolar epithelial (EpH4) cell-based model and an in vivo murine mastitis model. Our results showed that live and UV-treated M. bovis field strain 161791 and its lipid-associated membrane proteins (LAMP) activated nuclear factor kappa B (NF-kB) in EpH4 cells in a dose-dependent manner. In the murine mastitis model, temporal and spatial dynamics of inflammation in the mammary tissues were evident. Live M. bovis elicited diffuse inflammation affecting the whole challenged gland peaking at 48 h post infection (pi) in contrast to LAMP challenge, which elicited only focal inflammation peaking at 24 h and resolving at 48 h pi. Inflammation was characterized by massive neutrophil recruitment into the milk spaces and by elevated expression of the inflammatory mediators TNF-α, KC, iNOS and NF-kB dependent genes: A20 and IkBα. Moreover, the presence of intraepithelial bacterial communities in glands challenged with live M. bovis bacteria was shown. The developed models can be used efficiently for future characterization of M. bovis virulence factors and host immune response to IMI.