The Total Bile Acid Assay was purchased from Diazyme Laboratories (Poway, CA, USA). Antibodies against IBA1 were purchased from Wako Chemicals USA (Richmond, VA, USA). The real-time PCR (RT-PCR) primers for CCL2, IL-6, S1PR2 and TNFα were purchased from SABiosciences (Frederick, MD, USA). CCL2, IL-6 and TNFα ELISAs were purchased from R&D systems (Minneapolis, MN, USA). JTE-013 was purchased from Tocris (Minneapolis, MN, USA). The cholestyramine and AIN-93G control diets were formulated and supplied by Dyets Inc. (Bethlehem, PA, USA). Cell culture media and supplements were purchased from Thermofisher Scientific (Waltham, MA, USA). All other assays and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted, and were of the highest grade available.

Mouse in vivo experiments were performed using male C57Bl/6 mice (25–30 g; Charles River Laboratories, Wilmington, MA, USA). All animal experiments were approved by the Baylor Scott & White Health IACUC committee and were performed in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals.

Acute liver failure and HE was induced via a single intraperitoneal injection of 100 μg/g of azoxymethane (AOM) into mice. After injection, mice were placed on heating pads set to 37°C to ensure they remained normothermic. Hydrogel and rodent chow were placed on cage floors to ensure access to food and hydration. After 12 h and every 4 h thereafter, mice were injected subcutaneously with 5% dextrose in 250 μL saline to ensure euglycemia and hydration. Following injection, mice were monitored at least every 2 h (starting at 12 h post AOM injection) for body temperature, weight and neurological score using previous published methodology (McMillin et al., 2014a,b; McMillin M. et al., 2015). Once neurological impairment was evident, mice were continuously monitored with formal assessments of temperature, body weight and neurological score performed each hour. The neurological score was assessed by an investigator blind to the treatments by assigning a score between 0 (absent) and 2 (intact) to each of the following parameters: the pinna reflex, corneal reflex, tail flexion, escape response, righting reflex and ataxia. The summation of these six reflexes gives a neurological score between 0 and 12. Tissue was collected prior to neurological symptoms (pre neurological), when minor ataxia and weakened reflexes were present (minor neurological), when major ataxia and deficits in reflexes were evident (major neurological) and at coma defined by a loss of righting and corneal reflexes.

In a subset of experiments mice were fed a diet containing cholestyramine (2%) or the control diet AIN-93G for 3 days prior to the injection of AOM. Specific inhibition of S1PR2 in the brain was achieved by infusing JTE-013 reconstituted in 50% DMSO directly into the lateral ventricle of the brain using Alzet brain infusion kits coupled to subcutaneous implanted minipumps (Cupertino, CA, USA) at 2 μg/kg/day for 3 days at coordinates AP -0.34, ML -1.0, DV -2.0 prior to AOM injection.

Primary neurons were isolated from P1 mouse pups from FVB/N mice or S1PR2 knockout mice (S1PR2^−/−) using methodology previously described (McMillin et al., 2014b). Dr. Kazuaki Takabe from Virginia Commonwealth University School of Medicine provided the S1PR2^−/− mice used to generate pups for neuron cell isolations. Mouse pups were decapitated and whole brains were removed. Cortex was isolated and meninges and dura were removed. Cortical tissue was mechanically disrupted and filtered through a 100-μm filter. Neurons were pelleted by centrifugation at 1400 g. Neurons were resuspended in DMEM/F12 media containing 10% heat inactivated fetal bovine serum, 1% penicillin/streptomycin and 1% gentamycin and were plated on 12 well plates at 750,000 cells per well. After 24 h, the neurons were washed and media was replaced with Neurobasal^® media containing 2% B27 growth supplement, 1% penicillin/streptomycin and 1% gentamycin. After 10–12 days, neurons were treated with 10 μM TCA or 50 μM JTE-013. Cells were lysed and RNA was isolated for RT-PCR analyses.

Whole brains from adult C57Bl/6 mice were homogenized using the Miltenyi Biotec gentleMACS™ Dissociator (San Diego, CA, USA). Solutions used to ensure viability of cells were part of the Neural Tissue Dissociation Kit supplied from Miltenyi Biotec. Following dissociation into a single cell suspension, cells were passed through LS columns (Miltenyi Biotec) containing beads coated with CD11b antibodies (to isolate microglia) or GLAST antibodies (to isolate astrocytes) localized to the columns. The remaining cells not bound to the columns were kept as the neuron enriched fraction. LS columns were washed to remove the CD11b-bound cells or the GLAST-bound cells. The enriched cell fractions were then used for subsequent assays.

Determination of total bile acid concentrations in the brain was performed as previously described (McMillin et al., 2016a). Vehicle and AOM-treated mice were euthanized and cortex tissue was dissected. Cortex homogenates were prepared by calculating the wet weight of brain tissue with subsequent homogenization in 100 mg/ml of ultrapure water using the Miltenyi Biotec gentleMACS™ Dissociator. Homogenates were spun down for 5 min at 16,100 g and supernatants were collected. Total bile acid content was assessed in these homogenates using the manufacturer’s instructions from Diazyme Laboratories. This kit was previously validated to effectively measure mouse total bile acids in cortex homogenates using this methodology (McMillin et al., 2016a).

Serum and cortex tissue were isolated from vehicle and AOM-treated mice at the pre-neurological and minor neurological states of decline and were sent to Metabolon (Morrisville, NC, USA) to assess certain bile acid levels as part of a larger metabolomics panel. Metabolon performed the assay and statistical analyses of these samples with n = 5 or greater per group.

Free-floating immunofluorescence staining was performed on 30 μm brain sections using anti-IBA1 immunoreactivity to detect morphology and relative staining of microglia. Immunoreactivity was visualized using fluorescent secondary antibodies labeled with Cy3 and counterstained with ProLong^© Gold Antifade Reagent containing 4′,6-diamidino-2-phenylindole (DAPI). Slides were viewed and imaged using a Leica TCS SP5-X inverted confocal microscope (Leica Microsystems, Buffalo Grove, IL, USA). Field fluorescence area of photomicrographs was determined by converting images to grayscale, inverting their color and quantifying field staining intensity with ImageJ software.

RNA was extracted from tissue, primary neurons or isolated cell fractions using an RNeasy Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. Synthesis of cDNA was accomplished using a Bio-Rad iScript™ cDNA Synthesis Kit (Hercules, CA, USA). RT-PCR was performed as previously described (Frampton et al., 2012) using commercially available primers designed against mouse IL-6, CCL2, TNFα, S1PR2 and glyceraldehyde 3-phosphate dehydrogenase (SABioscience, Frederick, MD, USA). A ΔΔCT analysis was performed using vehicle-treated tissue or untreated primary neurons as controls for subsequent experiments (Livak and Schmittgen, 2001; DeMorrow et al., 2008).

Cortex tissue from all treatment groups was homogenized using a Miltenyi Biotec gentleMACS™ Dissociator and total protein was quantified using a ThermoFisher Pierce™ BCA Protein Assay kit. Capture antibodies against CCL2, IL-6, or TNFα were incubated overnight in 96-well plates. Each ELISA was performed according to the instructions provided from R&D Systems and the total input protein for each sample was 100 μg or serum diluted 1:10. Absorbance was read using a SpectraMax^® M5 plate reader from Molecular Devices (Sunnyvale, CA, USA). Data were reported as CCL2, IL-6 or TNFα concentration per mg of total lysate protein or per ml of serum.

Paraffin-embedded livers were cut into 4 μm sections and mounted onto positively charged slides (VWR, Radnor, PA, USA). Slides were deparaffinized and stained with Hematoxylin QS (Vector Laboratories, Burlingame, CA, USA) for 1 min followed by staining for 1 min with eosin Y (Amresco, Solon, OH, USA) and rinsed in 95% ethanol. The slides were then dipped into 100% ethanol and subsequently through two xylene washes. Coverslips were mounted onto the slides using CytoSeal XYL mounting media (ThermoFisher). The slides were viewed and imaged using an Olympus BX40 microscope with an Olympus DP25 imaging system (Olympus, Center Valley, PA, USA). Serum alanine aminotransferase (ALT) concentrations were measured using a commercially available kit from Sigma-Aldrich with all assays and subsequent analyses performed according to the instructions provided by the manufacturer.

All statistical analyses were performed using Graphpad Prism software (Graphpad Software, La Jolla, CA, USA). Results were expressed as mean ± SEM. For data that passed normality tests, significance was established using the Student’s t-test when differences between two groups were analyzed, and analysis of variance when differences between three or more groups were compared followed by an appropriate post hoc test. If tests for normality failed, two groups were compared with a Mann-Whitney U test or a Kruskal-Wallis ranked analysis when more than two groups were analyzed. For the neurological score analyses, a two-way analysis of variance was performed followed by a Bonferroni multiple comparison post hoc test. Differences were considered significant for p values less than 0.05.

As bile acids have been shown to be present in brain homogenates from patients with fulminant hepatic failure (Bron et al., 1977), bile acid concentrations were assessed throughout the time course of AOM-induced liver failure and HE. Levels of total cortical bile acids are increased as an early event during AOM-induced HE, with a significant increase beginning at a stage of minor neurological decline until coma when compared to vehicle-treated controls (Figure 1A). Due to this increase of bile acids occurring when minor neurological decline was present, a metabolic screen was performed in serum and cortex to identify metabolites that may contribute to the development of neurological complications in this model. While a variety of metabolites were dysregulated at stages of minor neurological decline compared to control, TCA was significantly elevated in both the serum (Figure 1B) and cortex (Figure 1C). Concentrations of the bile acids cholic acid, deoxycholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, tauroursodeoxycholic acid, α-muricholic acid and tauromuricholic acid were not significantly altered in the serum and tauromuricholic acid was not significantly increased in the cortex during minor neurological decline (data not shown). Together this identifies that total bile acids and the composition of the bile acid pool are dysregulated in the brain during AOM-induced HE.

Previously we have identified that cholestyramine feeding reduces serum and cortical bile acid concentrations and subsequent neurological decline after AOM injection compared to control diet-fed mice (McMillin et al., 2016a). It is conceivable that the mechanism that led to the reduction of AOM-induced neurological decline after cholestyramine feeding was due to its ability to modulate systemic inflammation. While the levels of all cytokines remained significantly elevated in the serum of AOM-treated mice fed cholestyramine-enriched chow compared to vehicle-treated mice, levels of CCL2 and TNFα were significantly reduced while IL-6 was significantly increased in cholestyramine-fed AOM-treated mice compared to AOM-treated mice on a control diet (Figures 2A–C). Therefore cholestyramine does appear to have an effect on modulating systemic inflammation, though this appears to be dependent upon the cytokine measured.

In regards to bile-acid mediated signaling, FXR signaling contributed in part to the neurological decline observed during AOM-induced HE, though FXR signaling does not account for the exacerbated neuroinflammatory response observed during this disease state (data not shown). Neuroinflammation is a known pathological contributor to HE and significant microglia proliferation, as assessed by IBA1 staining, was observed in control diet-fed AOM-treated mice compared to cholestyramine diet-fed mice administered AOM or vehicle-treated mice on either diet (Figures 3A,B). Microglia activation and proliferation can occur in this model due to increased CCL2 secretion from neurons that acts on microglia (McMillin et al., 2014a). There was a significant increase of CCL2 expression in control diet-fed AOM-treated mice but this increase was significantly reduced in cholestyramine-fed AOM-treated mice as shown by mRNA (Figure 3C) and protein expression (Figure 3D). In addition to this, expression of IL-6 was significantly increased in control diet-fed AOM-treated mice but was significantly lessened in cholestyramine-fed AOM-treated mice as shown by mRNA (Figure 3E) and protein expression analyses (Figure 3F). TNFα mRNA (Figure 3G) and protein (Figure 3H) expression were significantly increased in control diet-fed AOM-treated mice but was significantly reduced in cholestyramine-fed AOM-treated mice. Cholestyramine-fed AOM-treated mice had reduced levels of S1PR2 mRNA in the cortex compared to control diet-fed AOM-treated mice, supporting a role for total bile acids regulating S1PR2 expression in the brain (Figure 3I).

In order to determine the cellular localization and expression of S1PR2, neurons, astrocytes and microglia were isolated from vehicle and AOM-treated mice and were assessed for S1PR2 mRNA expression. There was a significant increase of S1PR2 mRNA in neurons isolated from AOM-treated mice compared to neurons isolated from vehicle-treated controls, while mRNA levels were static in astrocytes and decreased in microglia following AOM administration (Figure 4A). As neurons had a significant increase in S1PR2 expression during AOM-induced HE, and we have previously demonstrated that neuron-derived CCL2 expression contributes to the neuroinflammation observed during HE (McMillin et al., 2014a), the effects of TCA on neuronal CCL2 expression were assessed. Treatment of primary mouse neurons with 10 μM TCA was found to significantly increase CCL2 expression which was attenuated if the cells were co-treated with the S1PR2 antagonist JTE-013 (Figure 4B). In contrast, TCA treatment had no effect on CCL2 mRNA expression in primary neurons isolated from S1PR2^−/− mice (Figure 4C).

In order to see the effects of S1PR2 signaling during AOM-induced HE, mice were implanted with osmotic minipumps to infuse JTE-013 directly into the lateral ventricle of the brain prior to vehicle or AOM injection. JTE-013 infusion was found to significantly lengthen the time taken to reach coma in AOM-treated mice compared to 50% DMSO-infused controls (Figure 5A). Neurological decline of AOM-treated mice began earlier and at a greater rate than AOM-treated mice infused with JTE-013 with a significant difference between the two groups (p = 0.0381; Figure 5B). As AOM-induced HE is a model of liver failure, overall liver histology and function were assessed to determine if the effect of JTE-013 could be due to an improvement in liver pathology rather than a direct protective action on the brain. No difference in liver damage was observed between vehicle and JTE-013-infused mice after saline or AOM injection as observed by H&E histochemistry (Figure 5C) or serum ALT concentrations (Figure 5D).

Due to the protective effect of JTE-013 on AOM-induced HE without a concurrent improvement in liver pathology suggests that the protective mechanism of JTE-013 infusion is primarily in the brain. As inflammation is a core component of HE, neuroinflammation was assessed in JTE-013-infused mice. The significant increase in number of microglia observed during AOM-induced HE was not observed in AOM-treated mice infused with JTE-013 (Figures 6A,B). CCL2 mRNA expression (Figure 6C) and protein expression (Figure 6D) in the cortex were significantly increased in AOM-treated mice infused with 50% DMSO and this increase in expression was not present in AOM-treated mice infused with JTE-013. IL-6 mRNA (Figure 6E) and protein (Figure 6F) expression were significantly increased in the cortex of AOM-treated mice infused with 50% DMSO and were significantly reduced in vehicle-treated mice or AOM-treated mice infused with JTE-013. The mRNA and protein expression of TNFα was elevated in AOM-treated mice infused with 50% DMSO compared to vehicle-treated mice or AOM-treated mice infused with JTE-013 (Figures 6G,H). Taken together, these data support that the inhibition of S1PR2 by JTE-013 infusion reduces microglia proliferation and neuroinflammation during AOM-induced HE.