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

Circulating levels of CCL2 were detected at 300 pg/mL as early as 3 hpi, with serum levels falling by 6 and 12 hours (2A). To measure the production of this chemokine in the CNS, separate animals were perfused with PBS to remove circulating factors and whole brain was homogenized for ELISA ( Figure 2B ). As with the serum, CCL2 was robustly detected at 3 hpi, at levels in excess of 4000 pg per brain, and decreased sharply at 6 hpi ( Figure 2B ). However, in contrast to serum, the brain levels rose again at 12 hpi and reached levels comparable to the 3 hpi values by 24 hpi. Based on our previous findings regarding the locus of neuronal injury during acute TMEV infection [7, 22] , we also measured CCL2 in microdissected hippocampus at the same timepoints in separate animals ( Figure 2C ). CCL2 levels peaked at 6 hpi and were measured at 1500 pg per mouse (hippocampi pooled for each individual animal). Notably, this amount of CCL2 represents approximately the same amount of CCL2 measured in the whole brain at this timepoint ( Figure 2B ), suggesting that the hippocampus is the primary site for production at 6 hpi. Finally, to separate non-infectious pathogen recognition receptor-mediated effects from chemokine induction triggered by infectious virus, especially within the context of the very rapid response following inoculation, we compared the levels of CCL2 in the whole brain at 3 hr after inoculation with UV-inactivated TMEV or live TMEV. In this experiment we measured 3010 ± 425 pg/brain with live virus, 176 ± 65 pg/brain with dead virus, and 88 ± 16 pg/brain in sham mice (live vs dead: P<0.001; live vs sham: P<0.001; dead vs sham: P=0.703; by one-way ANOVA with Dunnett's pairwise comparison). We conclude that inoculation with infectious TMEV induces the rapid production of CCL2 in the brain and particularly in the hippocampus.