Bystander activation of microglia by brucella abortus-infected neurovascular cells induces neuronal death

Abstract

Central nervous system invasion by bacteria of the genus Brucellaresults in an inflammatory disorder called neurobrucellosis. We havepreviously demonstrated that B. abortus is able to elicit neuronaldeath by activating microglia (with release of nitric oxide and proinflammatorycytokines) and inducing primary phagocytosis. The aimof this work was to investigate the role of soluble mediators releasedby infected cells of the neurovascular unit in the modulation of thisphenomenon. For this, murine astrocytes and human brain microvasculatureendothelial cells (HBMECs) were infected or not with B.abortus for 24h. After that, cell free culture supernatants were usedto stimulate co-cultures of murine primary neuron/microglia during48h. Neuronal density was evaluated by fluorescence microscopy.Stimulation of neuron/microglia co-cultures with supernatants fromB. abortus-stimulated astrocytes or HBMECs induced a significantreduction in the density of neurons comparing with non-treatedco-cultured or treated with supernatants from non-infected cells(p<0.005 for astrocytes supernatants and p<0.05 for HBMECs supernatants).Furthermore, supernatants obtained from infected cellsincreased several microglia function as proliferation (determinedby microscopy, p<0.05), secretion of TNF-α (measured by ELISA;p<0.005) and phagocytic activity (evaluated by phagocytosis assaywith Escherichia coli; p<0.005). In order to determine possiblesoluble mediators involved in these phenomenon, we use monoclonal antibodies to neutralize TNF-α and IL-6 cytokines in culture supernatants of infected astrocytes. Neutralization of IL-6 prevented neuronal loss in microglia-neurons co-cultures (p<0.05), whereasneutralization of TNF-α did not (p>0.05). Overall, these results demonstrate that soluble mediators secreted by B. abortus-infected neurovascular cells activate resting microglia and this bystander activation could be involved in worsening the neuronal death induced by B. abortus.