S. J. Williams-McLeod1, B. Eke1, C. Sodhi1, D. Hackam1 1The Johns Hopkins University School Of Medicine, Pediatric Surgery, Baltimore, MD, USA
Introduction: Lipopolysaccharide (LPS) is an endotoxin found in the cell wall of gram-negative bacteria that triggers the activation of inflammatory mediators of the innate immune system. Short-chain fatty acids (SCFAs) are metabolites that contain fewer than six carbon atoms. SCFAs have been shown to influence gut-brain communication, especially under conditions of gut dysbiosis such as sepsis. Despite this, the mechanisms underlying this crosstalk are not fully understood. β-hydroxybutyrate (BHB), a four-carbon short chain fatty acid, can exert anti-inflammatory and antioxidant effects in cells treated with LPS. Here, we investigated the effects of a BHB pre-treatment in a brain organoid model of inflammation as well as a mouse model of endotoxemia.
Methods: In-Vitro LPS Exposure: Pregnant C57/Bl6 mice were anesthetized with isoflurane and cervically dislocated prior to harvest of embryonic tissue. Brains from C57/Bl6 e15 mice were harvested for organoid development. Brain organoids are a novel model system that allow for the study of early brain development at the structural, cellular, and molecular level. Here, organoids were cultured for 21 days in Complete Brain Media (Neurobasal Media Plus – 2% B-27, 1% Glutamax, 0.01 ug/mL BDNF, 0.01 ug/mL GDNF, 1X primocin/gentamycin/penn/strep) before harvest (Fig. 1). Brain organoids were re-suspended in six-well plates at 1.5e6 M cells/well. Organoids were treated with BHB (5 Mm) for a 15-minute pre-treatment prior to treatment with LPS (50 μg). Samples were collected and stored at -80°C. A Qiagen RNeasy kit was used to isolate RNA and generate cDNA for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. In-Vivo Endotoxemia: P10 C57/Bl6 mice were administered BHB (80 mg/kg i.p.) for a 15-minute pre-treatment prior to treatment with LPS (2.5 mg/kg i.p.). All animals were sacrificed six hours after treatment. Tissue samples were collected in liquid nitrogen and stored at -80°C for analysis or were fixed in 4% paraformaldehyde and stored at 4°C for immunohistochemistry. Experiments were performed with six animals per group. Statistical significance was evaluated using student’s t-test and ordinary one-way ANOVA.
Results: Statistically significant differences were found between LPS and LPS+BHB groups in TLR4 (p= 0.0424) and IL-6 (p= 0.0011) genes. Surprisingly, BHB resulted in elevated expression levels of IL-6, TNF-a, LCN-2, GFAP, and Tubb3 genes as assessed by qRT-PCR, revealing LPS-induced feedback inhibition and the sustained activation of pro-inflammatory cytokines.
Conclusion: The short chain fatty acid BHB leads to increased LPS signaling in mouse brain, providing insights into the mechanisms by which a dysbiotic microbiome may lead to impaired gut-brain signaling during sepsis.