Neuronal CCL2 expression drives inflammatory monocyte infiltration into the brain during acute virus infection

Charles L Howe; Reghann G. LaFrance-Corey; Emma N Goddery; Kanish Mirchia

Overall, we conclude that infection of the brain with TMEV rapidly induces CCL2 expression in neurons and this cellular source is central to driving CCR2-dependent infiltration of inflammatory monocytes into the brain during the most acute stage of encephalitis. While the relevance of our current findings to the neuropathological and electrophysiological sequelae of TMEV encephalitis remains to be determined, our observations lend further support to the therapeutic relevance of targeting the CCL2:CCR2 axis to confer neuroprotection. Our findings also highlight a unique role for neuronal production of chemokines in the initiation of leukocytic infiltration into the infected central nervous system. Hippocampal tissue was collected for RNA purification at early timepoints following intracranial inoculation of adult female C57Bl/6 mice with 2x10 5 PFU of the Daniel's strain of TMEV. Transcriptional changes were analyzed using Illumina MouseWG-6 v2.0 beadchips. Foldchange relative to uninfected control for 16900 genes present on the array was calculated, converted to log 2 , and heatmapped using Gitools. Sham controls were injected intracranially with culture media used for the virus preparation and RNA was collected at 24 h postinfection (hpi). Expression data for all conditions except sham are the means from 6 individual mice analyzed in two separate experiments (3 mice per experiment). Three sham mice were analyzed in the second experiment. (A) Heatmap showing pattern of transcriptional changes at 3, 6, 12, and 24 hpi relative to uninfected controls. The discontinuity in expression coding is the result of removing from analysis all genes with changes between -1.2-fold and +1.2-fold at 24 hpi, leaving 6764 genes in the heatmap. Color-coded heatmaps range from >8-fold downregulated (log 2 <-3.0; blue) to >8-fold upregulated (log 2 >+3.0; red). (B) The same expression data remapped to highlight chemokines, cytokines, and adhesion factors. In contrast to (A), values are continuous between -8-fold and +8-fold. Note that the color range was selected to reveal weaker changes in some genes, resulting in saturation at all timepoints for highly upregulated genes such as CCL2. (C) A further subset of highly upregulated and notable genes was validated by RT-PCR and the results heatmapped on a scale from 0 to 1000-fold (log 2 =+10) to reveal large expression changes over the first 12 hours.