In vivo inflammatory challenge to elucidate the role of the toll-like receptor 4 pathway in depression

Project Summary Approximately 1/3 of individuals with major depressive disorder (MDD) display inflammation that is believed to play a causal role in the disorder, but the precise mechanisms are not understood. Experimental endotoxemia studies in healthy individuals have shown behavioral, immunological, and physiological changes leading to a transient depressive-like state that resolves 4-5 hours after administration of lipopolysaccharide (LPS). While informative, these studies cannot reveal putative, aberrant inflammatory and regulatory mechanisms in MDD. Hence, a mechanistic approach is required to pinpoint which immunoregulatory mechanisms are defective in MDD. This proposal will leverage an experimental medicine study involving acute administration of LPS or saline to individuals with MDD and healthy controls (HC) to identify depression-relevant changes in the TLR4 pathway and its regulation by microribonucleic acids (miRNAs) in both the blood and the brain. The central hypothesis is that TLR4 signaling is sensitized in MDD by a history of exposure to exogenous (e.g. pathogens, LPS from leaky gut) and/or endogenous (e.g. chronic stress) ligands, and thus, TLR4-mediated inflammatory and regulatory mechanisms are impaired in MDD subjects compared to HC. TLR4 detects pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) for production of inflammatory cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor (TNF), which are elevated in some individuals with MDD. Whilst PAMPs, like LPS play a role in the pathogenesis of MDD, stress-associated production of DAMPs may also activate TLR4. Despite the theoretical importance of the TLR4 pathway, the evidence base is currently small, the extant clinical data are cross-sectionally derived, and TLR4 downstream signaling has not been assessed despite aberrant expression of TLR4-responsive miRNAs in MDD. These miRNAs can be isolated from extracellular vesicles (EVs) that cross the blood brain barrier, thus, isolating EVs of glial origin will provide a readout of CNS-associated TLR4 regulation in MDD. Our preliminary data suggest that the inflammatory cytokines, IL-6 and TNF, as well as TLR4 are increased in MDD compared to HC in response to LPS. The K99 phase will identify peripheral and central TLR4-mediated immune signaling mechanisms in MDD with the use of high-sensitivity, immunoassay detection to identify changes in cytokine expression and TLR4 pathway proteins and support the PI’s training in flow cytometry, miRNA extraction, astrocyte-enriched EV (AEEV) isolation, and RNA-seq. The R00 phase will identify peripheral and central TLR4-mediated regulatory mechanisms in MDD with the use of Next Generation Sequencing to identify miRNAs dysregulated in MDD and will provide support for transition to an independent investigator specializing in CNS-dependent TLR4- inflammatory mechanisms in depression. The ability to delineate TLR4-mediated peripheral and CNS immunoregulatory responses with cutting edge techniques, still novel to the field of psychiatry, will move us one step closer to understanding the mechanisms underlying inflammation-associated depression.