Introduction. Cell migration plays a central role in tissue homeostasis and mounting effective responses during infection and inflammation. This is particularly evident in the brain, where recruited immune cells can readily cause irreversible damage. Consequently, cell migration to and within the brain is carefully orchestrated and appears central to various neuropathologies, including encephalitis, stroke and multiple sclerosis.

Cell migration is predominantly controlled by a chemoattractant cytokines (chemokines) and their receptors. This process is poorly understood for migration to and within the brain, despite the therapeutic potential. Indeed, we have previously demonstrated that targeting cell migration improved the outcome in a mouse model of lethal viral encephalitis¹.

Here we have used viral encephalitis as a model for acute viral infection to investigate migration of monocytes and macrophages to and within the brain. Monocytes are circulating immune cells that contribute to host defence and tissue repair, and serve as macrophage precursors. CCR2 is the main chemokine receptor for monocyte bone marrow egress and migration in the periphery. CCR2 is expressed in a discrete locus with CCR1, CCR3 and CCR5. In humans, CCR5 deficiency is associated with increased morbidity during viral encephalitis.

Aim: To characterise chemokine-mediated cell migration of monocytes/macrophages to the brain.

Methods/ethics. Animal experiments were performed with approval by local ethics and UK home office. Our in vitro mixed glial/neuronal cell culture system uses embryonic day 17 mouse brains². Subcut injection with Semliki Forest Virus strain A7(74) was used to induce viral encephalitis.  

Results. Using FISH-imaging on myelinating rodent cultures, we previously observed that certain immune genes are expressed differentially by brain resident cells³. We now fully characterised the immune response to IFN-b, a key cytokine in the antiviral immune response in our mixed glial/neuronal cultures. RNAseq on cells separated using fluorescence-activated cell sorting after 24 hours of IFN-b or mock treatment demonstrated that each cell type initiated a unique immune profile. All cell types expressed multiple chemokines relevant for monocyte migration in response to IFN-b (fold change padj < 0.05, n=4 biological replicates).

We next investigated monocyte migration in our in vivo viral encephalitis model. Using our chemokine receptor reporter mice⁴, we found a reduction in CCR2 expression compared to bone marrow and blood (average 46% vs 85% and 97%, n=6 mice). Surprisingly, we observed an increase in CCR5 expression (average 28% monocytes CCR5+ in brain vs 2% in bone marrow and 5% in blood, n=6).

To determine the importance of CCR2 for monocyte migration to the brain, we compared monocyte migration in mice lacking the full cluster CCR1/2/3/5, or deficient for CCR1/3/5 but not CCR2. Full locus deletion reduced the presence of monocytes and their macrophage progeny in the encephalitic brain by 97%, and this phenotype could be fully restored by re-establishing only CCR2 expression (WT vs full KO p=0.0006; WT vs CCR2 restored p=0.2028; n=4-6).   

Conclusions. CCR2 expression is critical for monocyte migration to the inflamed brain. We hypothesise that CCR5 is important for movement within the brain, and further experiments to investigate this are ongoing.