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

Not surprisingly, intracranial inoculation with TMEV induced a robust inflammatory program in the hippocampus, involving upregulation of dozens of chemokine, cytokine, and adhesion factor genes ( Figure 1 ). Somewhat surprising, however, was the speed with which this induction occurred. We reproducibly detected large changes in transcription by 3 hpi, and in some experiments measured variable, but sizable, induction of chemokines and cytokines by 1 hpi (not shown). Moreover, these factors were detected at the protein level over the same acute period, with CCL2 measurable in the serum and brain as early as 1 hpi (not shown). Not only are these responses fast with regard to biosynthesis, but it is unlikely that a significant number of cells have even been infected in the brain by 3 hpi. Mathematical modeling constrains the infection rate to only 0.01 to 0.1 cells per minute [26] , suggesting that fewer than 20 cells would be productively infected by 3 hpi. Even if this rate is off by a factor of 100, fewer than 2000 cells are infected by 3 hpi. Obviously, some cells can respond to virions and virus constituents via mechanisms that do not require active infection, such as pathogen (or pattern) recognition receptors [27] , but our findings indicate that inoculation with UV-inactivated virus did not elicit CCL2 production in the brain at 3 hpi. This finding also rules out induction via non-specific trauma-induced effects associated with intracranial inoculation. Moreover, even assuming a direct effect of each active virion on target cells, we only inoculated the animals with 200,000 plaque forming units and these were not introduced directly into the hippocampus [20] . Yet, at 6 hpi the hippocampus produced essentially all of the CCL2 measured in the brain and presumably contributed the majority of serum CCL2. This issue is further confounded by the pattern of CCL2 expression revealed by immunostaining, which shows that effectively every neuron in dorsal hippocampal CA1, CA3, and dentate gyrus expressed this factor at 6 hpi ( Figure 2 ). As we have previously reported, even by 3 dpi only a fraction of CA1 neurons are directly infected with TMEV and DG neurons are never positive for virus by immunostaining [22] . These observations and discrepancies suggest that a currently unidentified amplification event occurs almost immediately after inoculation with live virus that results in widespread, albeit tissue-specific, upregulation of CCL2 production. Furthermore, this induction occurs almost exclusively in neurons, as synapsin-promoter driven deletion of CCL2 results in nearly complete suppression of both hippocampal and serum CCL2 at 6 hpi.