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The NF-κB/leukemia inhibitory factor/STAT3 signaling pathway in antibody-mediated suppression of Sindbis virus replication in neurons.

ABSTRACT: Alphaviruses are positive-sense, enveloped RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV) is the prototype alphavirus and preferentially infects neurons in rodents to induce an encephalomyelitis similar to the human disease. Using a mouse model of SINV infection of the nervous system, many of the immune processes involved in recovery from viral encephalomyelitis have been identified. Antibody specific to the SINV E2 glycoprotein plays an important role in recovery and is sufficient for noncytolytic suppression of virus replication in vivo and in vitro. To investigate the mechanism of anti-E2 antibody-mediated viral suppression, a reverse-phase protein array was used to broadly survey cellular signaling pathway activation following antibody treatment of SINV-infected differentiated AP-7 neuronal cells. Anti-E2 antibody induced rapid transient NF-κB and later sustained Y705 STAT3 phosphorylation, outlining an intracellular signaling cascade activated by antiviral antibody. Because NF-κB target genes include the STAT3-activating IL-6 family cytokines, expression of these messenger RNAS (mRNAs) was assessed. Expression of leukemia inhibitory factor (LIF) cytokine mRNA, but not other IL-6 family member mRNAs, was up-regulated by anti-E2 antibody. LIF induced STAT3 Y705 phosphorylation in infected differentiated AP-7 cells but did not inhibit virus replication. However, anti-E2 antibody localized the LIF receptor to areas of E2 expression on the infected cell surface, and LIF enhanced the antiviral effects of antibody. These findings identify activation of the NF-κB/LIF/STAT3 signaling cascade as involved in inducing antibody-mediated viral suppression and highlight the importance of nonneutralizing antibody functions in viral clearance from neurons.

SUBMITTER: Yeh JX

PROVIDER: S-EPMC7682347 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Nuclear factor-kappa B (NF-κB) is a ubiquitous transcription factor that regulates immune and cell-survival signaling pathways. NF-κB has been reported to be present in neurons wherein it reportedly responds to immune and toxic stimuli, glutamate, and synaptic activity. However, because the brain contains many cell types, assays specifically measuring neuronal NF-κB activity are difficult to perform and interpret. To address this, we compared NF-κB activity in cultures of primary neocortical neurons, mixed brain cells, and liver cells, employing Western blot of NF-κB subunits, electrophoretic mobility shift assay (EMSA) of nuclear κB DNA binding, reporter assay of κB DNA binding, immunofluorescence of the NF-κB subunit protein p65, quantitative real-time polymerase chain reaction (PCR) of NF-κB-regulated gene expression, and enzyme-linked immunosorbent assay (ELISA) of produced proteins. Assay of p65 showed its constitutive presence in cytoplasm and nucleus of neurons at levels significantly lower than in mixed brain or liver cells. EMSA and reporter assays showed that constitutive NF-κB activity was nearly absent in neurons. Induced activity was minimal--many fold lower than in other cell types, as measured by phosphorylation and degradation of the inhibitor IκBα, nuclear accumulation of p65, binding to κB DNA consensus sites, NF-κB reporting, or induction of NF-κB-responsive genes. The most efficacious activating stimuli for neurons were the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin-beta (IL-β). Neuronal NF-κB was not responsive to glutamate in most assays, and it was also unresponsive to hydrogen peroxide, lipopolysaccharide, norepinephrine, ATP, phorbol ester, and nerve growth factor. The chemokine gene transcripts CCL2, CXCL1, and CXCL10 were strongly induced via NF-κB activation by TNFα in neurons, but many candidate responsive genes were not, including the neuroprotective genes SOD2 and Bcl-xL. Importantly, the level of induced neuronal NF-κB activity in response to TNFα or any other stimulus was lower than the level of constitutive activity in non-neuronal cells, calling into question the functional significance of neuronal NF-κB activity.

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The NF-κB/leukemia inhibitory factor/STAT3 signaling pathway in antibody-mediated suppression of Sindbis virus replication in neurons.

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