West Nile virus (WNV) is a neurotropic flavivirus that can cause significant neurological disease. Mouse models of
WNV infection demonstrate that a proinflammatory environment is induced within the central nervous system (CNS) after
WNV infection, leading to entry of activated peripheral immune cells. We utilized ex vivo spinal cord slice cultures (SCSC) to demonstrate that anti-inflammatory mechanisms may also play a role in WNV-induced pathology and/or recovery. Microglia are a type of macrophage that function as resident CNS immune cells. Similar to mouse models,
infection of SCSC with WNV induces the upregulation of proinflammatory genes and
proteins that are associated with microglial activation, including the microglial activation marker Iba1 and CC motif
chemokines CCL2, CCL3, and CCL5. This suggests that microglia assume a proinflammatory phenotype in response to
WNV infection similar to the proinflammatory (M1) activation that can be displayed by other macrophages. We now show that the WNV-induced expression of these and other proinflammatory genes was significantly decreased in the presence of
minocycline, which has antineuroinflammatory properties, including the ability to inhibit proinflammatory microglial responses.
Minocycline also caused a significant increase in the expression of anti-inflammatory genes associated with alternative anti-inflammatory (M2) macrophage activation, including
interleukin 4 (IL-4),
IL-13, and FIZZ1.
Minocycline-dependent alterations to M1/M2 gene expression were associated with a significant increase in survival of neurons, microglia, and astrocytes in WNV-infected slices and markedly decreased levels of
inducible nitric oxide synthase (iNOS). These results demonstrate that an anti-inflammatory environment induced by
minocycline reduces viral cytotoxicity during
WNV infection in ex vivo CNS tissue.IMPORTANCE West Nile virus (WNV) causes substantial morbidity and mortality, with no specific therapeutic treatments available.
Antiviral inflammatory responses are a crucial component of WNV pathology, and understanding how they are regulated is important for tailoring effective treatments. Proinflammatory responses during
WNV infection have been extensively studied, but anti-inflammatory responses (and their potential protective and reparative capabilities) following
WNV infection have not been investigated.
Minocycline induced the expression of genes associated with the anti-inflammatory (M2) activation of CNS macrophages (microglia) in WNV-infected SCSC while inhibiting the expression of genes associated with proinflammatory (M1) macrophage activation and was protective for multiple CNS cell types, indicating its potential use as a therapeutic
reagent. This ex vivo culture system can uniquely address the ability of CNS parenchymal cells (neurons, astrocytes, and microglia) to respond to
minocycline and to modulate the inflammatory environment and cytotoxicity in response to
WNV infection without peripheral immune cell involvement.