Sofie Notebaert

Regulation of the transcriptional activity of the nuclear factor-κB p65 subunit

Abstract Nuclear factor-κB (NF-κB) is well known for its role in inflammation, immune response, control of cell division and apoptosis. The function of NF-κB is primarily regulated by IκB family members, which ensure cytoplasmic localisation of the transcription factor in the resting state. Upon stimulus-induced IκB degradation, the NF-κB complexes move to the nucleus and activate NF-κB-dependent transcription. Over the years, a second regulatory mechanism, independent of IκB, has become generally accepted. Changes in NF-κB transcriptional activity have been assigned to phosphorylation of the p65 subunit by a large variety of kinases in response to different stimuli. Here, we give an overview of the kinases and signalling pathways mediating this process and comment on the players involved in tumour necrosis factor-induced regulation of NF-κB transcriptional activity. Additionally, we describe how other posttranslational modifications, such as acetylation and methylation of transcription factors or of the chromatin environment, may also affect NF-κB transcriptional activity.

Mouse models to study the pathogenesis and control of bovine mastitis. A review

Summary Mastitis is a major infectious disease affecting high yielding cows in dairy herds. Because of its economic impact and due to the animal welfare policy, the pathogenesis of this intramammary infection was studied extensively over the past 50 years. Still, the costs associated with the use of dairy cows for mastitis research constitute a major drawback. As an alternative, a mouse model of experimentally induced mastitis was developed some decades ago. This model has been increasingly used as it appears to be very suited for studying ruminant mastitis due to similarities between mice and cows. The various techniques for inducing mastitis in mice as well as the different pathogens and initial inoculum doses used are also compared in this review. Moreover, recent findings concerning the administration of antimicrobial and immunomodulatory agents are discussed. In addition, information is provided on the most novel approaches for the study of mastitis including the use of mutant pathogen strains and transgenic mice.

Inflammatory mediators in Escherichia coli-induced mastitis in mice.

Escherichia coli (E. coli) infections in mouse mammary glands are rarely described and poorly characterized. In order to investigate the host immune response during coliform mastitis, several inflammatory parameters were evaluated at 24 and 48h following inoculation of mouse mammary glands with E. coli. Successfully challenged mice showed high values of the acute phase protein serum amyloid A (SAA) in blood. Systemic concentrations of the major inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were also increased as compared to control mice, while interleukin-1 (IL-1) levels remained negligible. Infected mammary glands showed a significant increase of all cytokine levels as compared to control glands. In accordance, mammary expression of the biologically inactive proform of IL-1beta was strongly up-regulated. Remarkably, data obtained in wild type as well as caspase-1 knockout mice showed that IL-1beta maturation seemed to occur independently from caspase-1. Finally, E. coli infection also triggered activation of the nuclear transcription factor-kappaB (NF-kappaB) in the mammary gland. In conclusion, the current study provides novel insights on the contribution of major regulatory proteins to the acute inflammatory host response at the local and systemic level during E. coli mastitis in mice.

In vivo imaging of NF‐κB activity during Escherichia coli‐induced mammary gland infection

In mammary gland infections, the contribution of NF‐κB is not well defined, and was therefore investigated following intramammary inoculation of Escherichia coli. Non‐invasive real‐time in vivo imaging of the transcription factor activation was performed in mammary glands of transgenic mice expressing luciferase under the control of NF‐κB. Bacterial inoculation resulted in a major increase in luminescence as compared with control glands. This activation was confirmed by immunohistochemical nuclear staining of the NF‐κB p65 subunit in mammary epithelium of infected glands, while nuclear p50 was not detected. The systemic response to the intramammary inoculation of Escherichia coli was also studied. NF‐κB activation in the liver increased over time, and a relatively mild but longer‐lasting response was observed as compared with the acute hepatic response of mice receiving lipopolysaccharide. This systemic reaction was confirmed by increased circulating levels of the acute phase protein serum amyloid A, tumour necrosis factor‐α and interleukin‐6. In addition, high concentrations of both cytokines in the mammary gland inoculated with bacteria showed that the infection was also well established at the local level. These results indicate that in vivo monitoring of NF‐κB activation is an attractive novel approach to study mammary gland inflammation, and that this transcription factor is imperative in the early stages of the host immune response towards coliform intramammary infections, both at the local and systemic level.

Escherichia coli induces bovine neutrophil cell death independent from caspase-3/-7/-1, but with phosphatidylserine exposure prior to membrane rupture

Neutrophils are essential for the innate immune response against bacterial pathogens and play a key role during the early phases of infection, including mastitis and endometritis in cows. When directly challenged with bacteria, neutrophils undergo phagocytosis induced cell death (PICD). The molecular mechanisms of this cell death modality are poorly understood, especially for bovine neutrophils. Therefore, this study aimed to determine the mechanisms and hallmarks of PICD in bovine neutrophils after in vitro challenge with Escherichia coli (E. coli). Our data show that various apoptotic hallmarks such as blebbing, chromatin condensation and executioner caspase (C)-3/-7 activity are only observed during constitutive bovine neutrophil apoptosis. In contrast, bovine neutrophil PICD is characterized by production of reactive oxygen species (ROS), pro-inflammatory C-1 activation, nuclear factor (NF)-κB activation, and interleukin (IL)-1β and IL-6 secretion. Nevertheless, under both conditions these phagocytes undergo cell death with the exposure of phosphatidylserine (PS). Although PS exposure is generally attributed to the anti-inflammatory features of executioner caspase-dependent apoptosis, it surprisingly preceded plasma membrane rupture during bovine neutrophil PICD. Moreover, C-1 inhibition strongly affected IL-1β production but not the PICD kinetics. This indicates that the secretion of the latter pro-inflammatory cytokine is a bystander effect rather than a regulator of PICD in bovine neutrophils, in marked contrast to the IL-1β-dependent pyroptosis reported for macrophages.