Brain-and spinal cord-infiltrating inflammatory monocytes and neutrophils contribute to pathogenesis, injury, and repair/regeneration in a wide array of neurologic diseases, including stroke, epilepsy, demyelinating disease, Alzheimer disease, ALS, cancer, pain, TBI, spinal cord injury, and infection. While a number of surface markers exist that provide variable and overlapping resolution of different myelomonocytic populations, these tools are suboptimal for identifying, tracking, and quantitating monocytes and neutrophils in target tissues such as the brain. The development of the LysM-eGFP mouse by Graf, in which only cells of myelomonocytic lineage express GFP, has provided a far more sophisticated tool for following monocytes and neutrophils. At the same time, a burgeoning but conflicted literature indicates that neutrophil and monocyte recruitment to the CNS is quite complex and dependent upon a variety of chemokines and chemokine receptor interactions. For example, in general, neutrophil trafficking may depend upon signaling through the CXCR2 and CXCR3 axis, while monocyte trafficking may depend upon CCR2 and CCR5 receptors. Based on this concept, and in an effort to dissect the role of specific chemokine receptors in the trafficking of myelomonocytic cells to the CNS, our first objective in this small grant proposal is to cross LysM-eGFP mice with mice that are homozygously deficient in CCR2, CCR5, CXCR2, or CXCR3. Our second objective is to characterize the kinetics and spatial distribution of myelomonocytic cells infiltrating the brain in mice infected with the Theiler's murine encephalomyelitis virus. To accomplish this objective, we intend to use a fiber optic-based fluorescence endoscope to acquire deep tissue images of GFP-positive neutrophil and inflammatory monocyte trafficking in live animals and determine whether chemokine receptor deficiency alters the trafficking of the cells. Our long- term goals are to use these four lines to identify the factors responsible for leukocyte infiltration into the CNS and to assess the temporal inter-relation between myelomonocytic cells by thwarting infiltration of one population (for example neutrophils via CXCR2 deficiency) and quantifying the infiltration of other populations (for example monocytes). We intend to use these mouse models to determine basic aspects of leukocyte trafficking into the brain in our specific virus model and to make these lines available to other investigators studying stroke, TBI, spinal cord injury, etc. This project is innovative because it will generate new mouse models for more carefully studying neutrophil and inflammatory monocyte trafficking into the CNS and because it employs a fiber optic microscope to observe the trafficking of these cells within deep brain structures in living animals. Our proposed project is significant because it is expected to provide tools that will resolve a number of conflicting concepts regarding the mechanisms of leukocyte trafficking to the CNS. By extending our knowledge of neutrophil and monocyte trafficking mechanisms, these tools have the potential to greatly impact the development of therapeutic strategies for ameliorating human disease.
The proposed research is relevant to public health because it will provide new models for the analysis of leukocyte trafficking into target tissues such as the central nervous system. The ability to better characterize neutrophil and monocyte entry into the infected brain, for example, may lead to the discovery of novel therapeutic strategies that will impact a wide array of human diseases. Thus, the proposed research is relevant to the mission of the NIH because it will provide tools for the extension of fundamental knowledge that will alleviate or reduce the burden of human disease.