Dissection of immunity controlling spread and growth of Listeria monocytogenes in neuronal cells

Sammanfattning: Listeria monocytogenes is a Grain-positive, facultative intracellular bacterium that causes severe infections in the nervous system of domestic animals and humans. Rhombencephalitis (brainstem encephalitis) due to L. monocytogenes, is a localized brainstem infection that is frequent in ruminants, and has been reported in humans as well. The asymmetric bacterial load, and neuropathological and ultrastructural findings indicate that axonal transport of the bacteria along one or more branches of cranial nerves to the brainstem may be the cause of L. monocytogenes rhombencephalitis. This thesis project aims to use L. monocytogenes as a model to analyze the interactions between a pathogen, a neuron and immune cells. More specifically the goal is to answer how peripheral sensory neurons take up L. monocytogenes to provide a pathway for entry into the brain and how innate and adaptive immune responses, especially the cytokine interferon gamma (IFN-ã), may control the replication and elimination of L. monocytogenes in neuronal cells. The initial experiments showed that L. monocytogenes spreads to the brain via a trigeminal nerve route after snout (where the trigeminal nerve innervates the vibrissae) infection. IFN-ã plays a protective role in such neuroinvasion; inducible nitric oxide synthase (iNOS) accounts partially for the protection. M< cells played the protective role in neuroinvasion by releasing IFN-ã but not through a perforin-dependent mechanism. By using mice lacking colony-stimulating factor 1 (CSF-1), and thereby deficient in macrophages and dendritic cells, we showed that these cells play a dual role: they constitute a major defense against systemic infection but they also facilitate L. monocytogenes brain invasion along the trigeminal nerves. In an in vitro system, L. monocytogenes could infect both axons and nerve cell bodies of rat dorsal root ganglia (DRG) neurons; they migrated in both retrograde and anterograde directions. Primary cultures of fetal mouse hippocampal neurons (PHN) and a neuronal cell line derived from mouse hypothalamus were infected by L. monocytogenes. Treatment with IFN-ã did not affect bacterial uptake, but resulted in increased killing of intracellular bacteria as measured by a plasmid segregation test. IFN-ã-mediated bacterial killing mapped to the infected neuronal cytosol, before listerial actin tail formation. Treatment with IFN-ã induced phosphorylation of the transcription factor STAT1 in neurons. In turn, IFN-ã-mediated listerial killing was not observed in STAT1-/- neurons or in neurons treated with inhibitory interferon regulatory factor (IRF-1) antisense oligonucleotides. IFN-ã-treated neurons showed higher levels of iNOS mRNA; iNOS antagonists and antisense iNOS oligonucleotides hampered the protective effect of IFN-ã-treatment, while neuronal NOS (nNOS) inhibitors had no effect. This novel neuronal function, i.e. that of a microbe killer, could play a crucial role in the control of infections in the immunoprivileged nervous system. Co-incubation of L. monocytogenes-infected PHN with spleen cells from immune (re-infected) but not uninfected or infected animals also reduced the intraneuronal bacteria. This is likely due to direct neuron-T cell interactions. CD4+ and CD8+ T cells were needed for spleen cell-mediated L. monocytogenes control, which was both perform- and IFN-ã-dependent and was partially inhibited in absence of MHC-class I antigen. In conclusion, this thesis further defines the role of immune responses in controlling dissemination to and replication of L. monocytogenes in the brain. The results showed that L. monocytogenes can spread to the brain via a neural route and that IFN-ã plays a protective role in bacterial neuroinvasion. CSF-1-dependent cells facilitate the neuroinvasion but also control systemic infections. Our data suggest that neurons can not only allow spread of bacteria into the brain, but they may also kill the bacteria upon stimulation with IFN-ã, which defines a new property of neurons that should be considered for understanding the interactions between a nerve cell and a pathogen.