Elucidating the role of interferon gamma production and signaling in the generation of neuropathic pain in human dorsal root ganglia - Project Summary Neuropathic pain is a major healthcare concern in the US due to its prevalence and high-cost through healthcare treatment and lost wages. Molecular techniques have been utilized on human dorsal root ganglia (hDRG) to better understand mechanisms underlying neuropathic pain to lead to the development of novel therapeutics. This work has demonstrated a sex-specific neuroimmune mechanism in which females show increased expression of interferon gamma (IFNγ) induced genes. IFNγ is a pro-inflammatory cytokine known for its anti-viral effects which recently has been shown to produce neuro-inflammation and pain. In animal models, intrathecal delivery of IFNγ produces paw hypersensitivity, and animals lacking the IFNγ receptor (IFNGR) fail to produce hyperalgesia following induction of neuropathic pain. Thus, this combined data suggests a potential female specific role of IFNγ in the hDRG to produce neuropathic pain. Elucidation of IFNγ mechanisms can lead to the production of sex-specific therapeutics for the treatment of neuropathic pain. I propose to use hDRG neurons to examine the role of IFNγ in the production of changes consistent with the development of neuropathic pain. Specific Aim 1 will explore which immune cells present in hDRGs are capable of producing IFNγ. I will do this through fluorescently activated cell sorting of hDRG neurons of both sexes to isolate CD4 and CD 8 T cells and monocytes. Then, I will perform RNA sequencing and proteomic assessments to determine which immune cells possess genes and proteins responsible for IFNγ production and if any sex differences exist. Then, I will perform a ligand-receptor interactome analysis with the RNA sequencing and proteomic data sets of each immune cell subtype. This will lead to the generation of testable hypotheses through which each immune cell can interact with hDRG neurons to lead to the production of neuropathic pain and if differences exist between the sexes. In Specific Aim 2, I will perform a comprehensive assessment of the impact of IFNγ signaling on hDRG neurons. In these experiments, I will culture hDRG neurons of each sex and expose them to IFNγ. First, I will look at the functional impact of IFNγ on hDRG neurons by using calcium imaging to see if IFNγ can excite and/or sensitize hDRG neurons to later prostaglandin exposure. Then, I will again expose cultured hDRG neurons to IFNγ and isolate RNA and protein to perform bulk and single cell RNA sequencing and phosphoproteomic assessment. This will give unprecedented insight into how hDRG neurons respond functionally, transcriptionally, and proteomically to IFNγ exposure and if any differences exist between the sexes. In sum, this work can lead to the production of novel, sex-specific therapeutics for the treatment of neuropathic pain. This work will also provide substantial scientific and bioinformatic training vital to me reaching my long-term career goal of becoming an independent, translational researcher as a PI at a large academic institution conducting next generation sequencing and other omic experiments on human tissue.
