LPS‐induced endotoxemia promotes blood‐brain barrier permeability via TSP1‐dependent TGFβ1 activation

During disease states, the blood-brain barrier (BBB) can become permeable due to endothelial injury or dysregulation of tight junction proteins. Endotoxemia, which induces systemic inflammation, causes BBB dysfunction and neuroinflammation. We have shown that transforming growth factor beta 1 (TGFβ1) is upregulated during systemic inflammation and promotes BBB permeability by disrupting claudin-5 and occludin. Therefore, we hypothesize that TGFβ1 is upregulated during endotoxemia and induces BBB permeability by disrupting tight junction proteins.C57Bl/6 mice were administered 3 mg/kg of LPS and monitored for neurological decline. In a second set of mice 6 hours after LPS administration, an antagonist to TGFβ1 activation, LSKL, or control, SLLK, were injected (1 mg/kg/ip). Mice were euthanized 24 hours after LPS injection and tissue was collected. In a subset of animals, 3 hours prior to euthanasia, Evans Blue was injected (8%/ip) to assess BBB function. TGFβ1, claudin-5, occludin, IL-1β, IL6 and TNFα expression was assessed by RTPCR, immunoblotting and immunofluorescence. In vitro studies using mouse brain endothelial cells (bEnd.3 cells) were treated with LPS (10 μg/ml) or rTSP1 (1 μg/ml) to activate TGFβ1. TGFβ1, TSP1, claudin-5 and occludin expression was assessed by immunofluorescence or RTPCR. Barrier function was determined in bEnd.3 cell monolayers using TEER or 10-kDa FITC-dextran diffusion. Data were analyzed using GraphPad Prism with results presented as mean +/- SEM. Differences between two groups were compared using Student's t-test and differences between three groups were determined using ANOVA.TGFβ1 expression was increased in cortex and cerebellum of LPS-treated mice. This was associated with decreased claudin-5 and occludin mRNA expression and immunofluorescence staining. Treatment of bEnd.3 cells with LPS increased TGFβ1 and TSP1 expression, decreased TEER, and increased FITC-dextran diffusion. Supplementation of bEnd.3 cells with rTSP1 led to decreased claudin-5 and occludin mRNA expression, decreased TEER, and increased FITC-dextran diffusion across the transwell. LSKL treatment in LPS-treated mice reduced sickness behavior, reduced Evan's blue penetrance into the brain, restored tight junction expression and alleviated neuroinflammation when compared to SLLK-treated mice.Strategies to target TGFβ1 signaling may prove effective at alleviating endotoxin-induced BBB dysfunction.