TLR4 inhibitor TAK-242 attenuates the adverse neural effects of diet-induced obesity

Ten-week-old male C57BL6/J mice were purchased from Jackson Labs (Bar Harbor, ME, USA) and allowed to acclimate to our vivarium facility at the University of Southern California for 2 weeks. Animals were housed under a 12-h light/dark cycle with lights on at 6 AM and ad libitum access to food and water. At 12 weeks of age, mice were randomized to a total of four dietary and drug treatments groups (N = 10–14/group). Dietary treatments were either control (CTL; 10% fat; #D12450J, Research Diets, New Brunswick, NJ, USA) or high-fat diet (HFD; 60% fat; #D12492, Research Diets). Drug treatments were either vehicle (0.09% sterile saline) or the TLR4 inhibitor TAK-242 (3 mg/kg in saline; #614316, EMD Millipore, Billerica, MA, USA). Drugs were administered via intraperitoneal (IP) injection 6 days/week. Dosage was based upon a previous study in which TAK-242 delivered at 3 mg/kg via IP injection yielded significant brain levels of the drug that were sufficiently maintained for at least 24 h after administration [60]. Treatments were maintained over a 12-week experimental period, during which body weights were recorded daily and food consumption was measured weekly.

At the conclusion of the experimental period, mice were euthanized with inhalant carbon dioxide and the brains were rapidly removed. One hemi-brain was immersion fixed for 48 h in 4% paraformaldehyde/0.1 M PBS, then stored at 4 °C in 0.1 M PBS/0.03% NaN₃ until processed for immunohistochemistry. Hippocampus was dissected and snap frozen for subsequent use in RNA extraction, while the remainder of the hemi-brain was snap frozen for subsequent use in protein extraction to examine soluble β-amyloid (Aβ) levels. Blood was collected via cardiac puncture into EDTA-coated tubes and centrifuged to separate plasma, which was stored in aliquots at − 80 °C. Gonadal and retroperitoneal (RP) fat pads were dissected and weighed as measures of adiposity. Both fat pads were snap frozen for subsequent RNA extraction. All animal procedures were conducted under protocols approved by the University of Southern California Institutional Animal Care and Use Committee and in accordance with National Institute of Health standards.

Body composition was determined 1 day prior to euthanization using the Bruker LF90 Minispec (Bruker Optics, Billerica, MA, USA). Mice were placed and loosely restrained inside an acrylic cylinder. The cylinder was placed inside the bore of the magnet, and measurements of fat, lean, and fluid mass percentages were recorded. Animals were returned to their home cages in less than 2 min.

At weeks 0, 4, 8, and 11, blood glucose readings were measured after overnight fasting (16 h). Blood was collected from the lateral tail vein and immediately assessed for glucose levels using the Precision Xtra Blood Glucose and Ketone Monitoring System (Abbott Diabetes Care, Alameda, CA, USA).

At week 11, glucose tolerance testing (GTT) was performed. First, baseline fasting glucose levels were taken. Mice were then administered a glucose bolus (2 g/kg body weight) via IP injection. Blood glucose levels were recorded from lateral tail vein 15, 30, 60, and 120 min after the glucose bolus. Area under the curve (AUC) was calculated.

Plasma cholesterol and triglyceride levels were measured enzymatically at the conclusion of the experimental period. Commercially available kits for both cholesterol (Total Cholesterol Colorimetric Assay kit, #K603, BioVision, Milpitas, CA, USA) and triglycerides (LabAssay Triglycerides, #290-63701, Wako Chemicals, Richmond, VA, USA) were used following the manufacturers’ protocols.

All behavioral testing was conducted between the hours of 6 AM and 1 PM. For all behavioral assays, mice were brought into the behavior room and allowed to acclimate for 30 min prior to testing. After each trial, animals were returned to their home cages and the testing arenas were disinfected with 70% ethanol.

Open field and forced swim testing were video recorded and analyzed by a rater blind to experimental treatment groups. Elevated plus maze and spontaneous alternation behavior were scored live. Fear conditioning was recorded using Noldus Ethovision XT software (Leesburg, VA, USA) and the Ugo Basille Fear Conditioning System NG (Gemonia, Varese, Italy).

Fixed hemi-brains were completely sectioned at 40 μm in the horizontal plane, using a vibratome (Leica Biosystems, Buffalo Grove, IL, USA). A standard avidin/biotin peroxidase approach using ABC Vector Elite kits (Vector Laboratories, Burlingame, CA, USA) was used to perform immunohistochemistry, as previously described [63]. Every eighth section was processed for ionized calcium binding adaptor molecule 1 (IBA-1), doublecortin (DCX), and bromodeoxyuridine (BrdU). A different initial antigen retrieval step was performed for each antibody, after which the same protocol was followed. For IBA-1 staining, sections were boiled in 10 mM EDTA, pH 6.0 for 10 min, then rinsed in water three times for 5 min each. For DCX staining, tissue was pretreated with 95% formic acid for 5 min, followed by rinsing in TBS. Finally, for BrdU staining, sections were placed in 1% NP40 detergent for 20 min, rinsed in TBS, then incubated in 2 N HCl at 37 °C for 30 min, followed by 10 min in 0.1 M boric acid and rinsing in TBS. Following the various antigen retrieval steps, sections were treated with an endogenous blocking solution for 10 min, then rinsed with 0.2% Triton-X in TBS, 3 times for 10 min each. Tissue was then incubated for 1 h in a blocking solution consisting of 2% bovine serum albumin and 0.2% Triton-X in TBS for IBA-1, plus 2% normal goat serum for BrdU. For DCX, the blocking solution was made up of 3% normal horse serum and 0.2% Triton-X in TBS. Blocked sections were incubated overnight at 4 °C in primary antibody directed against IBA-1 (#019-19741, 1:500 dilution, Wako Chemicals); DCX (#sc-271390, 1:1000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA); or BrdU (#MCA2483, 1:200 dilution, Bio-Rad, Hercules, CA, USA). All primary antibodies were diluted in the respective blocking solution used. On the following day, sections were rinsed and incubated in biotinylated secondary antibody diluted in blocking solution. Finally, immunoreactivity was visualized using 3,3′-diaminobenzidine (Vector Laboratories).

Density and activation states of microglia were determined using live imaging under bright-field microscopy with a × 40 objective (Olympus, BX50, CASTGrid software, Olympus, Tokyo, Japan). As previously described [63, 64], each cell was scored as having either a resting or reactive phenotype. Specifically, resting or type 1 microglia were defined as having spherical cell bodies with numerous thin, branched processes. Both type 2 and 3 microglia were considered reactive: type 2 cells had enlarged, rod-shaped cell bodies with fewer and thicker processes, while type 3 cells were enlarged and had either very few or no processes, or several filopodia. Microglia were quantified in the entorhinal cortex (4 fields/section), subiculum (4 fields/section), CA1 (5 fields/section), and CA2/3 (3 fields/section) across 4 tissue sections for a total of 64 fields and an average of ~ 450 cells per brain. Because increased soma size is a robust indicator of microglial activation [65], we also examined microglial soma size. Images of IBA-1 immunostaining in the CA1 subregion of the hippocampus were digitally captured using an Olympus BX50 microscope and DP74 camera paired with a computer running CellSens software (Olympus). Microglial cell bodies were outlined, and their area was determined using NIH ImageJ 1.50i (US National Institutes of Health, Bethesda, MD, USA).

DCX- and BrdU-immunoreactive cells were also quantified using live imaging under bright-field microscopy with a × 100 oil immersion lens (Olympus). Cells were counted in non-overlapping fields of the subgranular zone and granule cell layer of the dentate gyrus, across 8 sections per animal. Additionally, to examine the relative maturity of DCX-expressing cells, the morphology of their dendritic processes was assessed as previously described [66‐68]. Briefly, immature or type 1 cells were defined as having very short or no processes, intermediate or type 2 cells as having processes that extended only within the granule cell layer and do not extend into the molecular layer, and post-mitotic or type 3 cells as having dendrites that extend and branch into the molecular layer or having multiple branches within the granule cell layer. Morphology of DCX-positive cells was assayed across 4 sections per animal, and the relative proportions of type 1, 2, and 3 cells were calculated.

RNA was extracted from the gonadal fat pads and the hippocampus using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA), following the manufacturer’s protocol. To remove any remaining DNA contamination, the RNA pellet was treated with RNase-free DNase I (Epicentre, Madison, WI, USA) for 30 min at 37 °C after which a phenol-chloroform extraction was performed to isolate RNA. cDNA was reverse transcribed from 1 μg of purified RNA using the iScript cDNA synthesis system (Bio-Rad). The resulting cDNA was used to run real-time quantitative PCR using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and a Bio-Rad CFX Connect Thermocycler, as previously described [63]. Both hippocampus and adipose tissue were analyzed for expression levels of cluster of differentiation 68 (CD68), EGF-like module-containing mucin-like hormone receptor-like 1 (F4/80), major histocompatibility complex class II (MHC II), cluster of differentiation 74 (CD74) transcript variant 1, interleukin-6 (IL-6), and interleukin-1β (IL-1β). Additionally, hippocampal tissue was assessed for lipoprotein lipase (LPL) and CD36, as well as for the Aβ clearance and production factors neprilysin, insulin-degrading enzyme (IDE), and β-site APP cleaving enzyme (BACE1). Finally, levels of the cytokine tumor necrosis factor α (TNFα) transcript variant 1 were examined in adipose tissue. Primer pair sequences for target genes are shown in Table 1. All samples were run in duplicate, and PCR products were normalized with corresponding expression levels of β-actin and/or phosphoglycerate kinase 1 (Pgk1) in the brain and succinate dehydrogenase complex, subunit A, flavoprotein (SDHA) in the adipose tissue. The ΔΔ-CT method was used to determine relative mRNA levels. For hippocampal samples, the Ct value of each reference gene (β-actin and/or Pgk1) was subtracted separately from the target genes and the resulting values were averaged and used to calculate fold changes relative to the control-diet, vehicle-treated group. CD68, F4/80, MHCII, CD74, IL6, IL1β, LPL, and CD36 were run with both reference genes and neprilysin, IDE, and BACE1 only with β-actin.

Levels of soluble Aβ42 peptides were determined by enzyme-linked immunosorbent assay (ELISA) as described previously [69], with noted modifications. Briefly, the remaining hemi-brain portions were homogenized in buffer (0.2% diethylamine, 50 mM NaCl, 1 mL/200 mg tissue) using a polytron on ice. Resulting homogenates were centrifuged at 4 °C for 1 h at 15,000 g. Supernatants were collected and neutralized with 1/10th volume of 0.5 M Tris-HCl, pH 6.8. Samples were then analyzed using a commercially available Aβ42 ELISA (Human/Rat β Amyloid 42 ELISA Kit High Sensitive; 292-64501; Wako Chemicals) according to manufacturer’s directions.

We first examined measures of DIO in vehicle-treated and TAK-242-treated animals to assess whether drug treatment altered the obesogenic effects of HFD. The control diet was associated with a ~ 5% weight gain in both vehicle-treated and TAK-242-treated mice, whereas HFD was associated with a 39.6 ± 4.2% increase in body weight in vehicle-treated mice and a 34.4 ± 3.0% increase in TAK-242-treated mice (Fig. 1a). Two-way repeated measures ANOVA showed that HFD significantly increased body weight (F = 38.9, p < 0.0001). There was no significant effect of drug treatment on body weight. Between-group comparisons revealed that mice fed HFD weighed more than those fed CTL diets at the 4-, 8-, and 12-week time points (p < 0.05); this was true for both vehicle-treated and TAK-242-treated groups. When examining final body weight, we found a main effect of diet (F = 63.88, p < 0.0001; Fig. 1b), which was significant across both drug treatments (p < 0.0001). There were no significant interactions between diet and drug on measures of body weight.

Next, we examined adiposity by both body composition analysis and weights of gonadal and RP fat pads. We found that HFD was associated with a significant decrease in percent lean body mass (F = 103.0, p < 0.0001; Fig. 1c) and a corresponding significant increase in percent body fat (F = 98.7, p < 0.0001; Fig. 1d) in both vehicle-treated and TAK-242-treated mice (p < 0.0001). There was no interaction effect between diet and drug, nor were there significant main effects of drug on lean mass or body fat. The same pattern was found with fat depot weight, such that HFD significantly increased weights of both RP (F = 117.1, p < 0.0001; Fig. 1e) and gonadal (F = 108.4, p < 0.0001; Fig. 1f) fat pads. There were neither significant main effects of drug nor interaction effects between diet and drug.

We also examined food intake and found that HFD feeding was associated with a significant increase in the average daily kilocalorie consumption (F = 52.5, p < 0.0001; Fig. 1g), as would be expected given the higher caloric density of HFD relative to control diet. Importantly, drug treatment did not significantly affect caloric intake, and there were no significant interactions between diet and drug.

Another established outcome of DIO is dysregulation of glucose homeostasis [70, 71]. We first examined changes in fasting glucose levels over the treatment period. We found that HFD was associated with a significant increase in glucose levels (F = 23.78, p < 0.0001; Fig. 2a) at the 4-, 8-, and 11-week time points (p < 0.05). Additionally, there was a significant main effect of diet on percent change in glucose levels from baseline to the end of the treatment period (F = 13.7, p < 0.001; Fig. 2b). However, between-group comparisons revealed that the effect of HFD on increasing fasting glucose was only significant in vehicle-treated (p < 0.01), not in TAK-242-treated animals. There were neither significant interaction effects nor main effects of drug treatment on these measures of glucose homeostasis.

In addition to fasting glucose levels, we examined responses to a glucose bolus. There was a main effect of diet on glucose clearance in GTT (F = 49.95, p < 0.0001; Fig. 2c) that was significant in both vehicle-treated and TAK-242-treated mice (p < 0.05). We also calculated the AUC for GTT and again found a significant effect of diet (F = 55.11, p < 0.0001; Fig. 2d) such that HFD increased AUC regardless of drug treatment. There were no significant interactions or main effects of drug treatment on GTT measures.

Finally, we examined the effects of diet and drug treatments on levels of plasma triglycerides and cholesterol. HFD was associated with significantly increased triglyceride levels (F = 15.64, p < 0.001; Fig. 2e) in both drug treatment groups (p < 0.05). There was a non-significant trend towards increased cholesterol levels by HFD (F = 3.41, p = 0.07; Fig. 2f). There were no main effects of drug nor were there interactions between diet and drug on levels of either triglycerides or cholesterol.

DIO is known to increase inflammation in a number of organs, including adipose tissue [72]. To assess effects of HFD on peripheral tissue inflammation, we examined gene expression of markers of macrophage activation and inflammatory cytokines in gonadal fat (Fig. 3). We found a significant effect of diet on adipose tissue levels of CD68 (F = 17.14, p < 0.001; Fig. 3a), F4/80 (F = 10.06, p < 0.01; Fig. 3b), MHCII (F = 16.42, p < 0.001; Fig. 3c), CD74 (F = 7.42, p < 0.05; Fig. 3d), IL-6 (F = 6.52, p < 0.05; Fig. 3e), IL-1β (F = 9.99, p < 0.01; Fig. 3f), and TNFα (F = 10.85, p < 0.01; Fig. 3g). However, for the markers of CD68, F4/80, MHC II, IL6, and TNFα, this effect was only statistically significant in vehicle-treated HFD-fed mice and did not reach statistical significance in TAK-242-treated HFD-fed mice. For CD74 and IL-1β the main effect of diet failed to reach statistical significance in either of the HFD-fed groups. There was neither a main effect of drug, nor an interaction effect between diet and drug on expression of any probed genes.

In addition to causing macrophage activation and inflammation in peripheral tissues, HFD is associated with increased glial activation and inflammation in the brain [73, 74]. Because microglia express high levels of TLR4 [57, 58] and have been shown to adopt activated phenotypes in response to HFD [40, 75, 76], we examined microgliosis in brain sections. We analyzed both cell density and morphology of labeled cells following immunostaining for IBA-1 in entorhinal cortex and in the subiculum, CA1, and CA2/3 regions of the hippocampus. Figure 4a–c illustrates morphological phenotype characteristic of resting (type 1; Fig. 4a) microglia, with multiple, thin processes, and activated microglia with fewer, thicker processes (type 2; Fig. 4b), or amoeboid appearance (type 3; Fig. 4c). When examining microglial density, we found neither significant effects of diet or drug treatment, nor an interaction between these factors in entorhinal cortex (Fig. 4d), subiculum (Fig. 4f), CA1 (Fig. 4h), or CA2/3 (Fig. 4j). However, we found significant interactions between diet and drug treatment on microglial activation in entorhinal cortex (F = 36.27, p < 0.0001; Fig. 4e), subiculum (F = 38.93, p < 0.0001; Fig. 4g), CA1 (F = 47.16, p < 0.0001; Fig. 4i), and CA2/3 (F = 31.7, p < 0.0001; Fig. 4k). Between-group comparisons revealed that across all brain regions, HFD increased microglial reactivity exclusively in vehicle-treated animals, and TAK-242 was associated with significantly reduced microglial reactivity specifically in HFD-fed animals.

Activation phenotypes of microglia are also associated with increased soma size [65]. We measured microglial soma size as a complementary measure of microgliosis, specifically in the CA1 region of the hippocampus. Our data show similar results to findings on microglial morphology. That is, there is a significant interaction effect between diet and drug treatment (F = 8.62, p < 0.01; Fig. 4l), such that diet significantly increased soma size only in vehicle-treated animals (p < 0.0001), and TAK-242 treatment was associated with significantly decreased soma size compared to the vehicle group only in HFD-fed animals (p < 0.0001).

One frequent consequence of microgliosis is the increased expression of various microglia/macrophage markers and pro-inflammatory cytokines [77, 78]. We examined gene expression levels of several such factors in hippocampus (Fig. 5), including CD68 and F4/80 as general microglia/macrophage markers [79], with CD68 being characteristic of a more activated cell phenotype [80‐82]. We found a significant interaction between diet and drug (F = 7.06, p < 0.05; Fig. 5a) on expression levels of CD68. Between-group comparisons revealed that HFD increased CD68 expression only in vehicle-treated but not in TAK-242-treated animals (p < 0.01), and TAK-242 significantly decreased CD68 only in HFD-fed mice (p < 0.05). There were neither significant effects of diet or drug treatment, nor an interaction between these factors on hippocampal gene expression of F4/80 (Fig. 5b).

We next examined levels of MHC II, which is expressed specifically in microglia in the brain [83, 84], enhances TLR4 signaling [85], and is increased in microglia by HFD [10], and of CD74, which is also expressed specifically by microglia and macrophages [86], and is involved in the formation and transport of MHC II [87]. Additionally, CD74 expression has been shown to correlate with obesity-induced increases in body weight and metabolic changes in adipose tissue [88], and is increased in hippocampus of HFD-fed mice [89]. There was a statistically non-significant trend of increased MHC II in the HFD-vehicle group (Fig. 5c), and no significant diet or drug effects on CD74 expression (Fig. 5d).

Increased production of pro-inflammatory cytokines is a one potential outcome of increased microglial activation [78] as well as of obesity [21]. Thus, we probed for two pro-inflammatory cytokines: IL-6 (Fig. 5e) and IL-1β (Fig. 5f). Though diet did not significantly affect gene expression of either cytokine, we found a significant main effect of drug on levels of IL-6 (F = 4.73, p < 0.05); however, this did not reach statistical significance in either CTL-fed or HFD-fed mice.

Finally, we examined mRNA levels of two factors involved in fatty acid transport and uptake as well as regulation of activated microglial phenotypes: LPL [90, 91] and CD36 [92, 93]. Results demonstrated a statistically non-significant trend of a main effect of diet on LPL levels (F = 3.03, p = 0.08; Fig. 5g), as well as a statistically significant effect of diet on CD36 expression (F = 10.99; p < 0.01; Fig. 5h). Between-group comparisons showed that HFD significantly increased CD36 only in vehicle-treated but not in TAK-242-treated mice (p < 0.05).

An established negative consequence of DIO is impaired neurogenesis [8, 9]. We examined neurogenesis across groups using techniques to quantify both neural stem cells committed to a neuronal phenotype (DCX-labeling) and proliferation of neural stem cells (BrdU labeling) in the dentate gyrus region of the hippocampus. Figure 6 shows representative images of DCX immunohistochemistry, which qualitatively show a decrease in labeled cells with HFD that is prevented by TAK-242 treatment (Fig. 6a‐d). Quantification of DCX-labeled cell density showed a significant main effect of drug (F = 5.01, p < 0.05; Fig. 6e). Between-group comparisons revealed that this effect of drug treatment was significant only in HFD-fed mice (p < 0.05). In addition, there was a non-significant trend towards an interaction between diet and drug (F = 3.05, p = 0.08; Fig. 6e). There was no significant effect of diet on DCX-labeled cells. We also determined the relative maturity of DCX-positive cells by examining their dendritic morphology and arborization [66, 67]. Diet and drug treatments did not significantly affect maturation states of new neurons, as the proportion of subtypes was roughly equivalent between treatment groups (Fig. 6f). Parallel assessment of BrdU-labeled cells revealed neither significant main effects of diet or drug, nor an interaction between these factors (Fig. 6g).

DIO has been shown to promote Alzheimer-related amyloidogenic pathways in rodent models, in part by regulating neural expression of factors involved in Aβ production and clearance [94‐98]. We measured hippocampal expression levels of three key genes to investigate if HFD and TAK-242 treatments affect Aβ homeostasis pathways. We found no significant effects of diet or drug treatment, nor an interaction between these factors, on expression levels of the Aβ-degrading enzymes neprilysin (Fig. 7a) and IDE (Fig. 7b). However, we found a significant interaction between diet and drug treatment (F = 4.90, p < 0.05; Fig. 7c) on levels of the pro-amyloidogenic Aβ enzyme BACE1. Between-group comparisons revealed that HFD significantly increased BACE1 in vehicle-treated animals (p < 0.05) but not in TAK-242-treated mice. Finally, we determined levels of soluble Aβ42 peptides by ELISA. There were no statistically significant effects of diet or drug treatment and no interaction between these factors, though there were non-significant trends consistent with the BACE1 data (Fig. 7d).

To determine whether the drug treatment affected general behavioral performance, we compared animals on measures of activity, anxiety, and depression, using the behavioral assays of open field, elevated plus maze, and forced swim test, respectively. We found no statistically significant main effects of drug treatment on any of these three behavioral tasks (Additional file 1: Figure S1). There were only two statistically significant effects on any of the outcome measures in these assays, and both of these were in exploratory activity in the open field. First, there was a significant interaction between diet and drug treatment on the number of times the animals crossed into the center field (F = 4.91, p < 0.05; Additional file 1: Figure S1A), such that HFD increased center crossings only in TAK-242-treated mice (p < 0.05). Second, there was a significant main effect of diet on time spent in the center field (F = 4.23, p < 0.05; Additional file 1: Figure S1B), such that HFD again increased this measure specifically in TAK-242-treated animals (p < 0.05). There were no statistically significant effects on any measures of anxiety-like or depressive-like behaviors.

Finally, we examined cognitive performance in two behavioral assays. The spontaneous alternation behavior (SAB) task assays short-term working memory and visual attention. Total arm entries did not vary by either diet or drug treatment (Fig. 8a). However, there was a main effect of diet on alternation behavior (F = 7.86, p < 0.01; Fig. 8b), which was significantly only in TAK-242-treated mice (p < 0.05), though performance of the TAK-242-treated HFD mice (49%) is not significantly different from that observed in vehicle-treated HFD mice (50%).

As a measure of hippocampal-dependent and hippocampal-independent memory, we tested animals using the fear-conditioning paradigm. When examining freezing during the day 1 training trials, we found no significant differences between groups in initial freezing before the tone/shock pairing or in freezing during the tone/shock pairings and inter-trial periods. Figure 8c shows time spent freezing during the trace period between the tone and shock during the final presentation of the tone/shock pairing on day 1.

Cued memory was assessed on day 2 by changing the appearance and odor of the chamber and examining freezing to the tone, without presenting the shock. There were no group differences in freezing during the baseline period before presentation of the tone (data not shown). There was a main effect of diet on freezing during the trace period immediately after the first presentation of the tone (F = 4.76, p < 0.05; Fig. 8d), but this did not reach statistical significance across the different drug treatments. There were no significant group differences on freezing during the following 2 tone presentations (data not shown).

Contextual memory was examined on day 3 by placing animals back into the chamber with the same appearance and odor as during day 1 and examining freezing to this context. There were no significant effects of either diet or drug treatment, nor was there an interaction between these factors, on freezing in response to the context (Fig. 8e).