8:30am - 8:45am
Macrophages Regulate Outcome of Sepsis via Connexin 43 Dependent ATP Release
Department for BioMedical Research, Visceral Surgery Research Laboratory, University of Bern, Switzerland
Sepsis is a critical clinical condition with no specific therapy options available so far. We investigate here the potential of purinergic signaling and ATP release mechanisms as new therapeutic targets for sepsis. During sepsis, ATP is released extracellulary to modulate immune responses via specific purinergic receptors present on immune cells. Screening ATP release mechanisms by using specific blockers revealed active ATP release from primary macrophages via hemichannel connexin 43 (CX43). The aim of the study is to identify mechanisms of endotoxemia elicited ATP release on innate immune responses during sepsis.
ATP release was assessed in vitro and in vivo using a luciferin-luciferase assay. CX43 was blocked using Gap27, a specific CX43 inhibitor. To model sepsis in rodents, caecal ligation and puncture (CLP) was performed. Conditional KO cx43fl/fllyz2cre/cre mice were developed to assess the impact of CX43 in macrophages. Inflammatory cells expressing CX43 were characterized using IHC and FACS. Cytokine levels (ELISA), gene expression (qPCR) and protein expression (immunoblot) were assessed.
We identified CX43 expression in primary macrophages upon LPS stimulation in vitro and in hepatic, pulmonary and peritoneal fluid macrophages following CLP in vivo. Macrophages were identified to release ATP in response to TLR 4 and 2 agonists, which was abrogated by CX43 blocking using Gap27. In addition, blocking of CX43 in vitro or specific deletion of CX43 on macrophages reduced activation of LPS stimulated macrophages and decreased levels of inflammatory cytokines (TNFA, IL1B, IL6) secreted.
Cell specific deletion of CX43 on macrophages during CLP prolonged survival and was associated with decreased systemic and local levels of inflammatory cytokines (TNFA, IL1B, IL6) and chemokines (CCL2).
Systemic pharmacological inhibition as well as macrophage specific deletion of CX43 improves sepsis outcome. This effect was associated with decreased systemic inflammation.
8:45am - 9:00am
Unravelling the Molecular Response to Dose-Rate and Delivery Time of Ionizing Radiation
1Department of Radiation Oncology, Inselspital, Bern University Hospital, and University of Bern, Switzerland; 2Division of Medical Radiation Physics and Department of Radiation Oncology, Inselspital, Bern University Hospital, and University of Bern, Switzerland
Aim: There is increasing evidence that modulation of dose-rate and delivery time can effect tumor cell survival. The clinical significance of this observation and the underlying molecular mechanisms are still unclear. In this study we hypothesize that decreasing delivery time or increasing dose-rate in radiation therapy leads to a significant change of the molecular response using the multicellular tumor spheroid (3D) model or monolayer (2D) leading to a more pronounced activation of cell death signaling pathways.
Methods: Experiments were performed using HCT116 p53+/+cells growing as spheroids in agarose-coated flat-bottomed 96-well plates, and monolayer. To compare cell death in both cell culture systems we used flow cytometry analysis and Annexin V and propidium iodide staining. In addition, we examined the DNA double strand break (gammaH2AX) and DNA damage (phosphor p53) pathway in response to ionizing radiation (IR) treatment. Furthermore the effects of IR on cell cycle in both models was deterimined.
Results: Preliminary results showed a decrease of cell viability in 2D culture (46.9%) compared to 3D culture (67.7%) when treated with 10Gy. An increase in gamma-H2AX expression was observed in two different dose rates/delivery times, however we noticed no significant change between treatment schedules. Using spheroids we showed no significant change in activation of p-p53 between the two different dose rates of radiation, neither after an early time point nor at a later time point. However, in the 2D cell model we could observe a tendency towards an increased activation of p-p53 in response to the irradiation applied within a shorter delivery time. We observed different radiation sensitivity of 2D and 3D cell culture in respect to cell cycle redistribution.
Conclusions: 3D cell culture is more resistant to IR treatment when compared to 2D cell culture. Further investigations are needed to clarify the molecular mechanisms of response to different delivery time.
9:00am - 9:15am
The Role of Mitochondrial Uncoupling for T Lymphocyte Function
Institute of Pathology, University of Bern, Switzerland
The mitochondrial membrane potential influences the cellular energy metabolism and the generation of reactive oxygen species (ROS) – two essential factors that control T cell function. The uncoupling proteins (UCPs) act as ionophores across the inner mitochondrial membrane that regulate the mitochondrial membrane potential, ROS production and metabolic fluxes. While UCP1 and UCP3 are specific for adipose and muscle tissue, respectively, UCP2 is expressed by a variety of cell types including immune cells. UCP2 is strongly induced in activated and memory T cells, but its physiological relevance remains unclear. We investigate the role of UCP2 for T cell function and maintenance using mice with a conditional deletion of UCP2 in T cells (UCP2∆T/∆T). Absence of UCP2 revealed no effect on the naïve CD4 and CD8 T cell compartment, neither in UCP2∆T/∆T mice nor in competitive bone marrow chimeras (BMC). However, the frequency of Foxp3+CD4+ regulatory T cells (Treg) was significantly increased in UCP2∆T/∆T mice; and UCP2-deficient Treg cells showed a marked competitive advantage over wild type Treg in mixed BMC. Yet, the expansion of the Treg cell population was not affected by UCP2 in a model of cytokine-driven expansion or during chronic viral infection. Deletion of UCP2 in P14-TCR transgenic CD8+ T cells massively increased the proliferation of effector T cells during viral infection. We detected no effects of UCP2 on the mitochondrial membrane potential regulation or the production of signalling ROS in effector T cells. Still, naïve and activated UCP2-deficient CD4+ and CD8+ T cells exhibited an increased spare respiratory capacity, indicating that UCP2 may influence T cell metabolism. Together, our results suggest a role of UCP2 for Treg cells and effector CD8+ T cells. We are currently investigating the impact of UCP2 on the effector functions and cellular metabolic programs of these T cell subsets in order to identify the underlying mechanism.
9:15am - 9:30am
Dietary Influences on the Microbiota: New Insights Thanks to Gnotobiology and Next-Generation Sequencing Techniques
1Department for BioMedical Research, Mucosal Immunology, University of Bern, Switzerland; 2Interfaculty Bioinformatics Unit and Department for BioMedical Research, University of Bern, Switzerland; 3Department of Physiology and Pharmacology and Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, AB, Canada.
The microbiota has been shown to be an important factor in the pathogenesis of metabolic and immune disorders, though the underlying molecular mechanisms remain elusive. The complexity and the lack of standardization of conventional animal models represents a major challenge. We created a gnotobiotic mouse model colonized with a medium-diversity microbiota that remains stable over years, the sDMDMm2 mouse. This allows us to study in a very detailed fashion the impact of controlled environmental pressure on the host and its microbiota.
As diet represents a major factor shaping microbiota and host metabolism, we investigated the impact of two purified diets, a starch- and a fat-enriched diet. Thanks to next-generation sequencing techniques, this approach gives us unprecedented insights into the interplay between the 12 bacteria species of our model and the host in different dietary conditions.
We show that metabolic syndrome develops regardless of macronutrient-specific changes in the microbiota composition. However, fat-diet fed animals harbor specific changes in the bacterial metatranscriptome. Moreover, we show that micronutrients and plant-cell wall materials heavily impact the gene expression profile and the replication rate of defined species, consistently with observed changes in their relative abundance.