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

Brain-infiltrating leukocytes were collected from C57Bl/6 mice (WT; A, C) and CCR2 -/mice (B,D) at 18 hpi. Flow plots in (A-D) show cells in a CD45 + parent gate. The number of inflammatory monocytes (IM; CD45 hi CD11b + Gr1 ++ 1A8 -), neutrophils (N; CD45 hi CD11b ++ Gr1 + 1A8 + ), and microglia (M; CD45 mid CD11b mid Gr1 -1A8 -) was measured by flow cytometry. The percent of inflammatory monocytes, neutrophils, and microglia in the CD45 + population is shown as mean ± 95%CI calculated from 9 mice per genotype in 3 separate experiments (3x3) (E). Data were analyzed by Dunnett's method. No difference in microglia was observed, but neutrophils were significantly increased in CCR2 -/mice (P=0.0012 vs wt). Notably, the absence of CCR2 robustly inhibited inflammatory monocyte infiltration (P=0.0004 vs wt). **: P<0.001. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/208587 doi: bioRxiv preprint Figure 5 . Neurons are the primary source of CCL2 at 6 hpi. Ccl2-RFP fl/fl reporter mice (A, B) and Syn-Cre x Ccl2-RFP fl/fl neuron-specific CCL2-deficient mice (C, D) were intracranially inoculated with TMEV and brain sections were collected at 6 hpi for analysis of RFP expression. Single-channel RFP (A, C) and two-channel RFP (red) and DAPI (blue) (B, D) microscopy revealed that the reporter signal present in neurons at 6 hpi was specifically deleted in the Syn-Cre x Ccl2-RFP fl/fl mice. Serum (E) and hippocampal (F) CCL2 levels were measured by ELISA at 0, 6, and 24 hpi in the CCL2 reporter mice (+CCL2) and the neuron-specific CCL2-deficient mice (-CCL2). Each dot represents one animal; bar graphs represent mean ± 95%CI calculated from at least 3 mice per group per timepoint. Data were analyzed by two-way ANOVA with Tukey-Kramer pairwise analysis; statistical significance is only shown between genotypes at each timepoint; **: P<0.001; ***: P<0.0001. Figure 6 . Loss of acute neuronal CCL2 production results in reduced inflammatory monocyte infiltration into the brain. Brain-infiltrating leukocytes were analyzed by flow cytometry in wildtype B6 mice (A), Ccl2-RFP fl/fl reporter mice (+CCL2; B), and Syn-Cre x Ccl2-RFP fl/fl neuron-specific CCL2-deficient mice (-CCL2; C) at 18 hpi. The flow plots in (A-C) show Gr1-and CD11b-labeled cells in a CD45 hi parent gate. The number of CD45 hi cells (D), neutrophils (E), and inflammatory monocytes (F) were counted in the three groups (blue circles = B6; black circles = +CCL2; red circles = -CCL2). Each dot represents one animal; the line graph represents mean ± 95%CI calculated from at least two separate experiments. All cell types were significantly reduced in the neuron-specific CCL2-deficient mice but not in the parent reporter line. Data were analyzed by one-way ANOVA with Dunnett's pairwise comparison; *: P£0.0001.