Studies on microglia in tumor biology and neurobiology

Abstract: Microglia are innate immune cells that reside in the central nervous system (CNS). Their activities are critical for ensuring the correct microenvironment for brain development and for maintaining homeostasis in the brain after birth. However, during the lifetime, the neuronal and the glial cells in the brain suffer from multiple challenges that require microglia to react and to execute different functions. Under diverse pathological conditions, polarized microglia have detrimental effects by either promoting neuronal death in neurodegenerative diseases, or shaping the microenvironment to enhance brain tumor growth and invasiveness. Although microglia contribute to the maintenance of brain homeostasis and the pathogenesis of brain tumors, the molecular mechanisms behind their polarization towards selective phenotypes remains elusive. In the first study, we describe a novel molecular mechanism employed by glioma cells to polarize microglia towards a tumor-supportive phenotype. We demonstrate that decreased basal caspase-3 activity in microglia is a necessary condition for their polarization into a tumor-supportive phenotype. We reveal that this process relies on the inhibition of microglial thioredoxin-2 denitrosylation activity, which in turn leads to increased S-nitrosylation of caspase-3. Furthermore, we demonstrate that microglial thioredoxin-2 becomes inactive due to nitric oxide (NO) originating from the glioma nitric oxide synthase-2 (NOS2) activity. Using a syngeneic glioma tumor model in immunocompetent mice, and through different strategies including the generation of a Casp3flox/floxCx3cr1CreERT2 mouse model, we validated that interfering with this glioma-microglia signaling pathway impacted on the recruitment of microglia towards the tumor and also the tumor growth. Our current findings, together with previous report from our lab, uncover a central role for distinct caspase-3-dependent signaling pathways in the regulation of different microglia phenotypes. Previously it was established that the sequential activation of caspase-8 and caspase-3/7 is of importance for the pro-inflammatory polarization of microglia. In the present study, we demonstrate a role for thioredoxin-2 mediated repression of caspase-3 in promoting a tumor-supportive phenotype in microglia. Mechanisms promoting a tumor-supportive phenotype in immune cells are of extraordinary importance, given the strong correlation between this phenotype and tumor malignancy. Our research work suggests that caspase-3 may function as a rheostat which modulates microglial polarization states in response to various stimuli. More specifically, we show that highly elevated activity of caspase-3 causes cell death, while moderate induced activity and reduced basal activity of caspase-3 regulates the pro-inflammatory and the tumor-supportive microglial polarization states, respectively. Brain injury is commonly followed by neuroinflammation, and microglia are critical cellular elements of the brain mediating this process. In the second study, neural stem cells (NSCs) were exposed to conditioned medium originating from non-stimulated microglia, or stimulated microglia exhibiting pro- or anti-inflammatory phenotype. We found that NSCs grown in conditioned medium collected from anti-inflammatory microglia had better survival, enhanced migration and lower astrocytic differentiation compared to NSCs kept in conditioned medium deriving from pro-inflammatory microglia. This study demonstrates that pro- and anti-inflammatory microglia regulate NSCs functions differentially, and they induce chemokine CCL2 expression in differentiated NSCs.