Supplementary Material for: Effects of oral administration of the probiotic Lactobacillus rhamnosus GG on the proteomic profiles of cerebrospinal fluid and immunoregulatory signaling in the hippocampus of adult male rats

Introduction: The microbiome-gut-brain axis, by modulating bidirectional immune, metabolic, and neural signaling pathways in the host, has emerged as a target for the prevention and treatment of psychiatric and neurological disorders. Oral administration of the probiotic bacterium Lactobacillus rhamnosus GG (LGG; ATCC 53103) exhibits anti-inflammatory effects, although the precise mechanisms by which LGG benefits host physiology and behavior are not known. The goal of this study was to explore the general effects of LGG on the cerebrospinal fluid (CSF) proteome and a biological signature of anti-inflammatory signaling in the central nervous system (CNS) of undisturbed, adult male rats.

Methods: Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics were conducted using CSF samples collected after 21 days of oral treatment with live LGG (3.34 x 107 colony-forming units (CFU)/mL in the drinking water (resulting in an estimated delivery of ~1.17 x 109 CFU/day/rat) or water vehicle. Gene enrichment analysis (using DAVID, v. 6.8) and protein-protein interactions (using STRING, v. 11) were used to explore physiological network changes in CSF. Real time reverse transcription polymerase chain reaction (real time RT-PCR) was performed to assess gene expression changes of anti-inflammatory cytokines in the hippocampus. Genes associated with anti-inflammatory signaling that were analyzed included Il10, Tgfb1, Il4, and IL-4-responsive genes, Cd200, Cd200r1, and Mrc1 (Cd206).

Results: Oral LGG administration altered the abundance of CSF proteins, increasing the abundance of five proteins (cochlin, NPTXR, reelin, Sez6l, and VPS13C) and decreasing the abundance of two proteins (CPQ, IGFBP-7) in the CSF. Simultaneously, LGG increased the expression of Il10 mRNA, encoding the anti-inflammatory cytokine interleukin 10, in the hippocampus.

Conclusion: Oral LGG altered the abundance of CSF proteins associated with extracellular scaffolding, synaptic plasticity, and glutamatergic signaling. These data are consistent with the hypothesis that oral administration of LGG may improve memory and cognition, and may promote a physiological resilience to neurodegenerative disease, by increasing glutamatergic signaling and promoting an anti-inflammatory environment in the brain.