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

The profound reduction in hippocampal CCL2 at 6 hpi in the neuron-specific CCL2-deficient mice suggested that monocyte infiltration would also be impaired in these animals. Brain-infiltrating leukocytes were collected from wildtype B6 mice ( Figure 6A ), CCL2-RFP fl/fl reporter mice (+CCL2; Figure 6B ), and CCL2-RFP fl/fl x Syn-Cre mice (-CCL2; Figure 6C ) at 18 hpi and analyzed by flow cytometry. Gates were established as described above on a CD45 hi parent gate. The total number of CD45 hi cells isolated from B6 mice and CCL2-RFP fl/fl reporters was not different (F=44.8489, P<0.0001 by one-way ANOVA; B6 vs +CCL2: P=1.000 by Dunnett's method) but the number of these cells in the neuron-specific CCL2-deficient mice was significantly reduced (-CCL2 vs B6: P=0.0001; -CCL2 vs +CCL2: P=0.0004) ( Figure 6D ). The trend toward more CD45 hi cells in the CCL2 reporter mice suggested by Figure 6D was exacerbated in the neutrophil counts ( Figure 6E ), though the difference was not significant (F=87.2340, P<0.0001 by one-way ANOVA; B6 vs +CCL2: P=0.1908 by Dunnett's method). In contrast, the number of neutrophils infiltrating the brain was reduced in the neuron-specific CCL2-deficient mice (-CCL2 vs B6: P=0.0003; -CCL2 vs +CCL2: P<0.0001) ( Figure 6E ). Finally, the number of inflammatory monocytes in the brain at 18 hpi did not differ between B6 and reporter mice (F=56.8592, P<0.0001 by one-way ANOVA; B6 vs +CCL2: P=1.000 by Dunnett's method) but was significantly reduced in the neuron-specific CCL2-deficient mice (-CCL2 vs B6: P<0.0001; -CCL2 vs +CCL2: P=0.0024) ( Figure 6F ). These observations support the conclusion that the limited CCL2 production and release measured in neuron-specific CCL2-deficient mice at 6 hpi results in a strong reduction in inflammatory monocyte infiltration into the brain at 18 hpi.