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

Adult female C57Bl/6 mice were intracranially inoculated with 2x10 5 PFU of TMEV. RNA from hippocampus was prepared at 3, 6, 12, and 24 hours postinfection (hpi) and analyzed by microarray to measure changes in transcripts. Hippocampal RNA was also prepared from uninfected mice and from mice 24 hours after sham inoculation with viral growth media. Fold changes in expression for the 16900 genes present on the array were calculated relative to uninoculated/uninfected controls. A heat map of log 2 fold change ( Figure 1A ) reveals that numerous genes were already upregulated at 3 hpi and the number of upregulated genes steadily grew through the first 24 hours after infection. Extraction of chemokine, cytokine, and adhesion factor genes ( Figure 1B ) indicates that these pathways were robustly impacted by TMEV infection. For example, the CCR2 ligand CCL2 was upregulated 8-fold by 3 hpi and continued to increase to almost 50-fold induction by 12 hpi, remaining above 30-fold at 24 hpi. The CCR5 ligand CCL5 increased steadily from 5-fold at 3 hpi to over 60-fold by 24 hpi and the CCR2 ligand CCL7 increased from 8-fold at 3 hpi to over 80-fold at 24 hpi. Likewise, the CXCR2 ligand CXCL1 was upregulated over 150-fold at 3 hpi, decreasing to 20-fold by 24 hpi, while CXCL2 was upregulated 10-fold at 3 hpi and decreased slightly to 6-fold by 24 hpi. The CX3CR1 ligand CX3CL1 was not increased at these timepoints and was, in fact, modestly downregulated by 1.4-fold at 3 hpi. In parallel with changes in chemokine expression, we observed marked upregulation of adhesion factors, with ICAM-1 upregulated 12-fold at 3 hpi, decreasing to 6-fold by 24 hpi, and VCAM-1 upregulated between 4-and 5-fold at each timepoint. Other adhesion factors involved in leukocyte trafficking were either unregulated (CD34), downregulated (Selectin-P ligand, 2-fold), or modestly upregulated (MAdCAM-1, 2-fold; GlyCAM-1, over 2-fold). Finally, a subset of immune genes were validated by RT-PCR ( Figure 1C ), confirming the general pattern of expression revealed by the microarray though with greater dynamic range. For example, by RT-PCR we measured a 260-fold induction of CCL2 at 3 hpi that continued to increase to over 2000-fold upregulation by 12 hpi. Overall, we conclude that inoculation of the brain with TMEV induces rapid and robust upregulation of proinflammatory chemokines, cytokines, and adhesion factors in the hippocampus that can be detected as early as 3 hours after injection.