This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Lyme neuroborreliosis is likely caused by inflammatory effects of the spirochete B. burgdorferi on the central and/or peripheral nervous systems. Microglia, the resident macrophages within the central nervous system (CNS), are important in initiating immune responses to microbial products; these responses result in the production of chemokines and inflammatory cytokines. Astrocytes, the major glial cell type in the CNS, are known to contribute to the inflammatory activity in the brain. TLRs may be used by cells of the innate immune system, eg microglia and astrocytes, to recognize pathogen-associated molecular patterns (PAMP) and initiate acquired immune responses. Previous studies have detected levels of mRNA for TLRs 1-10 in murine microglia and astrocytes. Because of their PAMP specificities, TLR 1, 2, 5, 6, and 9 may be involved in the immunobiology of B. burgdorferi. Primary cultures of rhesus monkey astrocytes or microglia were used to determine the involvement of TLRs in mediating cytokine production in response to B. burgdorferi in the CNS. Cells were incubated with lipidated outer surface protein A, live B. burgdorferi (1:10 cell-to-spirochete ratio), and sonicated B. burgdorferi (1:10), and the results were analyzed by qRT-PCR (TLRs 2, 5, and 9), Western Blot (WB), and ELISA. All stimulants led to up-regulation of TLR2 mRNA, but only sonicated B. burgdorferi caused up-regulation of TLR9 mRNA. The expression of TLR2 and TLR9 protein was detected through WB and/or immunofluorescence. TLR5 expression was constitutive in both cell types, and no increased expression was observed after stimulation. Astrocytes and microglia produced IL6, IL8, and TNF-a in response to all of the stimulants tested. Our results indicate that microglia and astrocytes respond to B. burgdorferi by inducing inflammatory cytokine secretion through TLR2 and 9. Next we plan to analyze TLR1 and 6, which may also respond to B. burgdorferi in the CNS.
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