Phosphoinositid 3-Kinase (PI3K) als Mediator inflammatorischer Funktionen in Mikrogliazellen
Microglial cells represent the macrophages of the brain. During fetal development precursor cells invade into the brain and differentiate to adult microglia cells. Microglial cells differ from circulating macrophages, but they exhibit similar surface receptors and reaction patterns. Disturbances of the homeostasis in the brain lead to the activation of microglia and induction of various cellular key functions. Activated microglial cells are able to migrate into damaged regions, eliminate pathological microorganisms and necrotic neurons by phagocytosis and secrete pro- and anti-inflammatory cytokines. Activation of microglial cells could be a part of regenerative and degenerative processes in the brain. An important mediator of immunological and inflammatory reactions is Phosphoinositide 3-kinase γ (PI3Kγ). The signaling protein generates via its lipid kinase activity PtdIns(3,4,5)P3 as a second messenger which controls many immunological key functions. Independent of its enzymatic activity PI3Kγ acts as a scaffold protein which forms complexes with other proteins. Investigations in the last years occupy more and more a regulatory function of PI3Kγ outside of the immune system. Detection of the signaling protein in neuronal cells and microglial cells (Jin et al., 2010, König et al., 2010) assumes that it might be involved in many neurologial disorders. Until now the regulatory function of PI3Kγ in these cells is largely unknown. The present thesis is a first contribution to understand the signaling function of PI3Kγ in microglial cell in vitro and in vivo. Functional pattern of PI3Kγ was investigated on isolated microglia cells and mouse models of septic encephalopathy and stroke. Pharmacological and genetic inhibition of PI3Kγ was used to analyze the specific function of the signaling protein in these models. For in vitro investigations the activation of the cells was induced by Lipopolysaccharide (LPS) and Adenosintriphosphate (ATP). These compounds mimic bacterial infection or trauma. To analyze the regulatory function of PI3Kγ primary microglia from wild type and PI3Kγ-deficient (knockout) mice were isolated. In addition microglial cells from transgenic mice, which lack the lipidkinase activity (knockin) of PI3Kγ were involved. Treatment of microglia with inflammatory mediators LPS or ATP leads to a distinct morphology change and the induction of an activated state. Depletion or pharmacological inhibition of PI3Kγ affects phagocytosis and proliferation of the cells. The role of lipidkinase activity of the signaling protein was investigated by using the PI3Kγ-knockin mutant. We could show that the phagocytotic activity of microglial cells is controlled in a lipidkinase independent way via the scaffold protein function of PI3Kγ. Interaction of PI3Kγ with Phosphodiesterase 3B (PDE3B) promotes the hydrolysis of cAMP and the abolishment of a known inhibitory effect of this second messenger on the phagocytotic activity. Additional unpublished investigations on primary microglia stimulated with LPS or ATP revealed a lipidkinase dependent regulation of proliferative capacities. Induction of ROS production after LPS/ATP describes its important function as second messenger. Low doses of LPS and ATP show a beneficial effect on proliferation of microglia, whereas high concentrations inhibit this cell function. The hormetic effect of both inflammatory mediators could be shown in wild type microglia controlled by PI3Kγ. The second part of the thesis focuses on specific functions of PI3Kγ in mouse models of septic encephalopathy (SE) and stroke. Different investigations identified PI3Kγ as protective mediator in the pathogenesis of these diseases. The in vivo model of septic encephalopathy showed that inhibition of cAMP controls activation of matrixmetalloprotease 9 (MMP9) in a PI3Kγ lipidkinase independent function. MMP are proteolytic enzymes which can lead to blood brain barrier damage. In stroke model we could also show protective effects of the signaling protein. PI3Kγ deficient mice exhibit an increased infarct volume. Our in vitro and in vivo investigations occupy a key function of PI3Kγ in the control of microglia functions. Because of the importance of microglia during inflammatory reactions within the nervous system our results seems to be of distinct medical relevance.
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