CD8⁺ T cells contribute to diet-induced memory deficits in aged male rats

Abstract

We have previously shown that short-term (3-day) high fat diet (HFD) consumption induces a neuroinflammatory response and subsequent impairment of long-term memory in aged, but not young adult, male rats. However, the immune cell phenotypes driving this proinflammatory response are not well understood. Previously, we showed that microglia isolated from young and aged rats fed a HFD express similar levels of priming and proinflammatory transcripts, suggesting that additional factors may drive the exaggerated neuroinflammatory response selectively observed in aged HFD-fed rats. It is established that T cells infiltrate both the young and especially the aged central nervous system (CNS) and contribute to immune surveillance of the parenchyma. Thus, we investigated the modulating role of short-term HFD on T cell presence in the CNS in aged rats using bulk RNA sequencing and flow cytometry. RNA sequencing results indicate that aging and HFD altered the expression of genes and signaling pathways associated with T cell signaling, immune cell trafficking, and neuroinflammation. Moreover, flow cytometry data showed that aging alone increased CD4⁺ and CD8⁺ T cell presence in the brain and that CD8⁺, but not CD4⁺, T cells were further increased in aged rats fed a HFD. Based on these data, we selectively depleted circulating CD8⁺ T cells via an intravenous injection of an anti-CD8 antibody in aged rats prior to 3 days of HFD to infer the functional role these cells may be playing in long-term memory and neuroinflammation. Results indicate that peripheral depletion of CD8⁺ T cells lowered hippocampal cytokine levels and prevented the HFD-induced i) increase in brain CD8⁺ T cells, ii) memory impairment, and iii) alterations in pre- and post-synaptic structures in the hippocampus and amygdala. Together, these data indicate a substantial role for CD8⁺ T cells in mediating diet-induced memory impairments in aged male rats.

1. Introduction

It is well-accepted that overnutrition and consumption of a Western diet, typically enriched with a combination of saturated fats, refined carbohydrates, and low fiber is associated with impaired cognitive function, especially with aging. This has been demonstrated in rodents, non-human primates, and humans (Bernier et al., 2016; Miller and Spencer, 2014; Sridharan et al., 2013; Taylor et al., 2021). In line with this, our lab has shown that even just short-term (3 days) high fat diet (HFD) consumption, a time course that precedes perturbations in peripheral glucose or insulin homeostasis, leads to an impairment in long-term hippocampal- and amygdalar-dependent memory in aged, but not young adult, rats (Spencer et al., 2017). This age-specific memory impairment is associated with an exaggerated HFD-induced proinflammatory response in the hippocampus and amygdala that, if blocked, prevents the memory impairment (Spencer et al., 2017). However, the underlying immune mechanisms driving this exaggerated proinflammatory response in aged rats are not well understood.

We have previously published that HFD has no impact on microglial morphology in the hippocampus, and has minimal impact on amygdalar microglial morphology (Spencer et al., 2019). Moreover, we have shown that microglia isolated from young and aged rats fed a HFD express similar levels of proinflammatory transcripts (Butler et al., 2020), suggesting that additional factors or cell types may drive the exaggerated neuroinflammatory response in aged HFD-fed rats. For the various reasons outlined below, we turned our attention to T cells. T cells infiltrate both the young and aged CNS and contribute to immune surveillance of the brain parenchyma (Chen and Holtzman, 2022; Filiano et al., 2016; Herz et al., 2021; Pasciuto et al., 2020; Rustenhoven et al., 2021). T cells belong to the adaptive arm of the immune system and play integral roles in host defense and regulation of innate immune cells by providing antigen-specific immunity. Briefly, upon activation via peptides presented on major histocompatibility complex (MHC) class II by antigen-presenting cells, naïve CD4⁺ T helper cells will differentiate into either proinflammatory (Th1 or Th17 cells) or anti-inflammatory (Th2 cells) subtypes and migrate to non-lymphoid tissues and either promote or resolve inflammation, respectively (Ruterbusch et al., 2020). CD8⁺ T cells, or cytotoxic T cells, are proinflammatory and mainly react to antigens presented via MHC class I (Coder et al., 2015). In addition to antigen-specific immune functions, T cells have recently been found to play roles in promoting tissue homeostasis (Muñoz-Rojas and Mathis, 2021) and, as aging occurs, contribute to the chronic low-grade inflammation observed with aging, likely due to increased infiltration of T cells into non-lymphoid tissues (Coder et al., 2015). In the brain, T cells can impact the neuroimmune environment directly, through the release of cytokines, or indirectly via interactions with resident microglia (Batterman et al., 2021; Schetters et al., 2018). While increased T cell trafficking to the aged brain has been observed in rodents, non-human primates, and humans (Batterman et al., 2021; Gate et al., 2020; Ritzel et al., 2016), the impact of diet on this process has not been investigated. However, the impact of HFD on T cell function has been investigated to some extent in the periphery, as multiple studies have demonstrated HFD and saturated fatty acids can increase T cell-driven inflammation and increase trafficking of T cells to non-lymphoid tissues (de Jong et al., 2014; McCambridge et al., 2019; McLaughlin et al., 2014; Stentz and Kitabchi, 2006; Strissel et al., 2010).

In the current study, we hypothesized that short-term HFD consumption increases T cell presence in the aged brain, which contributes to the diet-induced proinflammatory response and subsequent hippocampal- and amygdalar-dependent memory impairment. To test this hypothesis, we utilized bulk RNA sequencing of the hippocampus and amygdala to investigate the extent to which T cell signaling pathways may be differentially expressed between young adult and aged HFD-fed rats, and flow cytometry to identify the extent to which both CD4⁺ and CD8⁺ T cells exist in the brain and are altered by diet. To delineate the functional role of brain T cells, we depleted peripheral CD8⁺ T cells in vivo and used contextual fear conditioning to test long-term memory, as well as protein assays to measure inflammatory and synaptic changes. Taken together, our data implicate a major role for CD8⁺ T cells in mediating diet-induced alterations to memory, cytokines, and synaptic elements in aged male rats.

2. Materials and methods

2.1. Subjects

Three- and 24-month old male F344×BN F1 rats (N=98; n = 3–8 per group; and two per cage) were utilized. Rats were obtained from the National Institute on Aging (NIA) Rodent Colony maintained by Charles River (Indianapolis, IN), where they are exclusively available. Due to aged female rats of this strain not being available at the time these experiments were conducted, only male rats were used in this study. Future work will include the use of females as they become more readily available through the NIA. The animal colony was maintained at 22°C on a 12-h light/dark cycle (lights on at 07:00 h). Rats were co-housed two per cage and allowed free access to food and water and were given at least 1 week to acclimate to colony conditions before experimentation began. All experiments were conducted in accordance with protocols approved by the Ohio State University Institutional Animal Care and Use Committee.

2.2. Diet

At study onset, cages of young and aged rats were randomly assigned to either continue consuming regular chow (Teklad Diets, TD.8640; energy density of 3.0 kcal/g; 29% calories from protein, 54% from carbohydrates [no sweetener added], and 17% from fat [0.9% saturated, 1.2% monounsaturated, 2.7% polyunsaturated]), or an adjusted calorie 60% HFD (TD.06414, Envigo, energy density of 5.1 kcal/g; 18.4% calories from protein, 21.3% from carbohydrates [90 g/kg sucrose, 160 g/kg maltodextrin], and 60.3% from fat [37% saturated, 47% monounsaturated, 16% polyunsaturated]). Rats consumed chow or HFD ad libitum for 3 days for all experiments.

2.3. Tissue collection

For all experiments, rats were given a lethal dose of sodium pentobarbital and transcardially perfused with ice-cold saline (0.9%) for 3 min to remove peripheral immune leukocytes from the CNS vasculature. Complete necropsies of each subject were conducted for all experiments to insure there were no tumors, cysts, or tissue abnormalities. For RNA sequencing experiments, brains were rapidly extracted, placed on ice, and hippocampus and amygdala were dissected and frozen in liquid nitrogen and stored at −80°C until processing. For other experiments, one whole hemisphere of the cerebrum (excluding cerebellum and olfactory bulbs) was collected and processed for flow cytometry, while from the other hemisphere, the hippocampus and amygdala were dissected and stored at −80°C until further processing.

2.4. RNA extraction and bulk RNA-sequencing

RNA was extracted from the hippocampus and amygdala of young and aged rats fed either a chow or 3-day HFD (n = 3/group), as previously described (Butler et al., 2021). RNA was reverse transcribed to complementary DNA, and 20 million 75-bp reads were sequenced on an Illumina NovaSeq 6000 SP/S1. Library preparation and sequencing was conducted by the Genomics Shared Resource at The Ohio State University College of Medicine and the Genomics Services Laboratory at Nationwide Children’s Hospital, respectively. FASTQ files were aligned to the rat Rnor 7.1 reference genome using STAR (Spliced Transcripts Alignment to a Reference) Aligner. Data were normalized and differential expression testing was determined using DESeq2 in R. Genes with p < 0.05 were considered differentially expressed. Volcano plots and heat maps were generated in R. Canonical pathway analysis was performed using Ingenuity Pathway Analysis (IPA, Qiagen). The full RNAseq dataset was deposited in NCBI’s Gene Expression Omnibus database and can be accessed using the accession number GSE223802.

2.5. Flow cytometry

Following 3 days of either chow or HFD, young and aged rats (n = 6/group) were euthanized and perfused as described above. A single-cell suspension of brain mononuclear cells were generated using a 70/30% Percoll density gradient following tissue homogenization and filtering through a 70 μM cell strainer, as previously described (Pino and Cardona, 2011). Total isolated cells were counted using a Countess III automated cell counter and incubated in Fc block + 1% bovine serum albumin (Biolegend 101320, TruStain FcX) for 10 min at 4°C and then primary antibodies for 15 min at 4°C. Primary antibodies included: CD3 (BioLegend 201412, clone 1F4), CD4 (BioLegend 201511, clone W3/25), and CD8 (BioLegend 200610, clone G28). Samples were run on either an LSR II or a Cytek Northern Lights flow cytometer (Cytek Biosciences, Fremont, CA) and analyzed using FlowJo software (FlowJo, Ashland, OR, USA). The percentage of brain CD3⁺CD4⁺ and CD3⁺CD8⁺ cells were quantified by gating on live (determined by live/dead staining) lymphocytes following doublet exclusion. The total number of CD4⁺ and CD8⁺ T cells in a cerebral hemisphere was calculated by multiplying the total starting number of isolated mononuclear cells by the percentage of each subsequent gate. Staining specificity was verified with the use of appropriate isotype controls. Splenocytes were used as a positive control for CD3, CD4, and CD8 staining. Each flow cytometry experiment was conducted across two cohorts of rats ran one week apart with the same antibody lots and flow cytometer.

2.6. Evans blue dye injection and quantification in tissue

Young and aged rats were fed either a standard chow or HFD (n=4/group) for 3 days. On the morning of the 4^th day, all rats were injected intraperitoneally (i.p.) with 1mL of 4% Evans blue dye (Sigma-Aldrich, St. Louis, MO, USA) diluted in pharmaceutical grade saline. Within minutes, the dye was circulating throughout the rat as evidenced by the feet and nose turning blue. At 90 minutes post Evans blue dye injection, rats were saline-perfused and peritoneal tissue and the pituitary gland were dissected from the periphery. In the brain, the hippocampus and amygdala were dissected. All tissues were weighed and sonicated in 50% trichloroacetic acid (Sigma-Aldrich) and then spun at 10,000 g for 10 min at 4°C. The supernatant was collected and plated in a 96-well plate and fluorescence intensity was read at 620/680nm on a plate reader. Absorbance values were converted to ng of dye based on a standard curve and expressed per mg of tissue as in (Goldim et al., 2019).

2.7. CD8⁺ T cell depletion

Only aged rats were used in depletion experiments, as HFD had no impact on the number of brain CD8⁺ T cells in young rats. To deplete CD8⁺ cells in vivo, aged rats were injected intravenously with 0.5 mg anti-rat CD8α (OX-8 clone) depleting antibody or IgG1 isotype control antibody (BioXcell) 3 days before starting HFD or remaining on a standard chow diet. Dose and timing was determined based on previous work (Hartlage et al., 2019) and a small pilot study. Depletion of cells in blood were tracked via flow cytometry using the G28 antibody listed above, which binds a separate CD8 epitope than the OX-8 clone. In brief, heparinized blood collected from aged rats fed either a chow or 3-day HFD and treated with either the anti-CD8 antibody or isotype control (n = 6/group) was stained for 20 min with anti-rat CD3, CD4, and CD8 antibodies, followed by direct RBC lysis and fixation (BD lyse/fix buffer). Validation of in vivo CD8⁺ T cell depletion at critical experimental time points is included in figure 4. Body weights were recorded prior to, and during, T cell depletion experiments to evaluate any possible impact of antibody injection on food intake and sickness behavior.

2.8. Contextual Fear Conditioning

After 3 days of consuming either chow or HFD, and 6 days after receiving either an anti-CD8 antibody injection or IgG isotype control, aged rats (n = 7–8/group) were taken two at a time from their home cage and each placed separately in a conditioning chamber (26 L x 21 W x 24 H, cm) made of stainless steel with a transparent fiberglass door. Rats were allowed to explore the chamber for 2 minutes before the onset of a 15-second tone (76 dB), followed immediately by a 2-second footshock (1.5 mA) delivered through a removable floor of stainless steel rods that were wired to a shock generator (Coulbourn Instruments, Allentown, PA, USA). To assess lethargy or sickness, activity was scored during conditioning. Immediately after the termination of the shock, rats were removed from the chamber and returned to their home cage. At this point, previously HFD-fed rats were switched back to a chow diet. After 4 days, all rats were tested for fear of the conditioning context, a hippocampal-dependent task, and then for fear of the tone, an amygdala-dependent task. Chambers were cleaned with water before each rat was conditioned or tested. For the contextual fear test, rats were placed in the exact context in which they were conditioned (in the absence of the tone) and were observed for 6 minutes and manually scored for freezing behavior. For the auditory-cued fear test, rats were placed in a completely novel context (e.g., differently shaped and sized chamber, with bedding, toys, dim lighting), observed, and manually scored for freezing behavior for 3 minutes. Following the 3 minutes, the tone was activated and freezing behavior was scored for an additional 3 minutes. A single tone-shock pairing in our rats consistently yields excellent conditioning (~65% freezing) in a young adult control rat (Baartman et al., 2017; Barrientos et al., 2006a; Bilbo et al., 2008; Frank et al., 2010a; Kwilasz et al., 2021; Spencer et al., 2017). Freezing is the rat’s dominant fear response and is a common measure of conditioned fear. In this case, freezing was defined as the absence of all visible movement, except for respiration. Using a time-sampling procedure, every 10 seconds each rat was judged in real time and scored as either freezing or active at the instant the sample was taken by two observers who were blind to experimental conditions. Inter-rater reliability exceeded 95% for all experiments. An experimental timeline and body weights are included in Figure 6.

2.9. Tissue processing for protein analysis

In preparation for protein assays, one hemisphere of the hippocampus or amygdala was manually sonicated in 0.3 mL or 0.2 mL buffer, respectively, for 20 sec using an ultrasonic cell disrupter (ThermoFisher Scientific). Sonication buffer contained 50 mM Tris base and an enzyme inhibitor cocktail that included 100 mM amino-n-caproic acid, 1 mM EDTA, 5 mM benzamidine HCl, 0.2 mM phenylmethyl sulfonyl fluoride, and a cOmplete^™ Mini protease inhibitor cocktail tablet (Millipore Sigma). Following sonication, samples were centrifuged at 10,000 rpm at 4°C for 10 min and supernatants were transferred into a clean tube. Bradford protein assays were performed on all samples (prior to the first freeze) to determine total protein concentrations. Samples were divided into 10 μL aliquots and frozen at −80°C until assays were performed.

2.10. Multiplex enzyme-linked immunosorbent assays

Protein levels of interferon gamma (IFNγ), interleukin (IL)-1β, and IL-4 were determined using a commercially available rat-specific multiplex ELISA (Meso Scale Discovery, MD, USA). The assays were performed according to the manufacturer instructions. Values of all cytokines were determined and normalized to total protein (n = 6/group).

2.10. Western Blot

An equal amount of total protein (25ug) from each sample was loaded into each lane. The NuPAGE Bis-Tris (10 well, 4–12%, 1.5 mm) gel electrophoresis system was used under reducing conditions (Life Technologies). The iBlot dry-blotting system (Life Technologies) was used to electrophoretically transfer gels to nitrocellulose membranes. Odyssey TBS blocking solution (Li-Cor) was used to prevent nonspecific protein binding (1 hr at RT). Primary antibodies, in Odyssey blocking solution containing 0.2% Tween20 (overnight at 4°C), were: Synaptophysin (SYP) (1:1,000; sc-17750; Santa Cruz) and PSD95 (1:1,000; 2507S; Cell Signaling). Anti-GAPDH (1:50,000; 8245; Abcam) was used as an internal loading control. Blots were washed 4 × 5 min with TBS+0.1% Tween20 and then probed with the appropriate fluorescent secondary antibody (1:20,000; Invitrogen) for 1 hr at RT. The Odyssey Infrared Imaging System (LI-COR Biosciences) was used to image membranes and Empiria Studio v1.3 software was used for quantification of bands. Each protein band was normalized to its respective loading control and then analyzed as percentage of control (n = 6/group).

2.11. Statistical analysis

For all experiments, n = 4–8 rats/group were utilized, based on statistical power established by our previous work (Barrientos et al., 2012a; Muscat et al., 2021; Spencer et al., 2017). Statistical analyses were performed using Prism v.9 software. Outliers, as determined by Grubb’s test, were removed prior to statistical tests. Data met the requirements for parametric analysis, based on Shapiro-Wilk tests for normality and Q-Q plots. Two-way ANOVAs were run for the experiments that had a 2 × 2 factorial design. In the case of significant interactions, Tukey’s multiple comparisons posthoc tests were run. The threshold for significance was set as α = 0.05. Only significant F-values were reported in the results section.

3. Results

3.1. Aging and HFD regulate thousands of genes in the hippocampus and amygdala, including genes associated with T cell signaling and synaptic function

Due to our previous reports of diet impacting both hippocampal- and amygdalar-dependent memory function and inflammation (Butler et al., 2021; Spencer et al., 2017), we evaluated the impact of these variables on transcriptomic changes in these regions. In the hippocampus, 822 (471 up; 351 down) genes were differentially expressed between young and aged chow-fed rats (Fig. 1A), 544 (297 up; 247 down) were differentially expressed between aged HFD-fed and aged chow-fed rats (Fig. 1B), 1346 (675 up; 671 down) between aged HFD-fed and young HFD-fed rats (Fig. 1C), and 607 (345 up; 242 down) between young HFD-fed and young chow-fed rats (Fig. 1D). Specific genes of interest, along with other top genes implicated in the dataset, are displayed on volcano plots, Venn diagrams, and heatmaps (Fig. 1A–G). Ingenuity Pathway analysis revealed that for all key group comparisons, pathways associated with T cell receptor signaling, leukocyte extravasation, neuroinflammatory signaling, and synaptic transmission and plasticity were enriched and/or downregulated (Fig. 1H–K).

Figure 1.

Bulk RNA sequencing of the hippocampus (n=3/group). (A-D) Volcano plots depicting differentially expressed genes between all relevant pairwise comparisons. The red triangles represent genes that were significantly increased (log2FoldChange > 1.5, p < 0.05). The blue triangles represent genes that were significantly decreased with (log2FoldChange > 1.5, p < 0.05) (E) Venn diagram of significantly differentially expressed genes in the Young-HFD vs. Young-chow and the Aged-HFD vs. Aged-chow comparison. (F) Venn diagram of significantly differentially expressed genes in the Aged-chow vs. Young-chow and the Aged-HFD vs. Young-HFD comparison. (G) Heat map of genes that were differentially expressed by either age or HFD. (H-K) Ingenuity Pathway Analysis of differentially expressed genes (z-score). The same canonical pathways associated with neuroinflammation, T cell signaling, and synaptic function are displayed across all pairwise comparisons.

In the amygdala, 2,798 (1,210 up; 1,588 down) genes were differentially expressed between young and aged chow-fed rats (Fig. 2A), 2,395 (1,380 up; 1,015 down) differentially expressed genes between aged HFD-fed and aged chow-fed rats (Fig. 2B), 779 (316 up; 463 down) between aged HFD-fed and young HFD-fed rats (Fig. 2C), and 532 (353 up; 179 down) between young HFD-fed rats and young chow-fed rats (Fig. 2D). Specific genes of interest, along with other top genes implicated in the dataset, are displayed on volcano plots, Venn diagrams, and heatmaps (Fig. 2A–G). Similar to the hippocampus, Ingenuity Pathway analysis implicated T cell receptor signaling, leukocyte extravasation, neuroinflammatory signaling, and synaptic transmission and plasticity as canonical pathways that were enriched or downregulated in the amygdala in response to aging and HFD consumption (Fig. 2H–K).

Figure 2.

Bulk RNA sequencing of the amygdala (n=3/group). (A-D) Volcano plots depicting differentially expressed genes between all relevant pairwise comparisons. The red triangles represent genes that were significantly increased (log2FoldChange > 1.5, p < 0.05). The blue triangles represent genes that were significantly decreased with (log2FoldChange > 1.5, p < 0.05). (E) Venn diagram of significantly differentially expressed genes in the Young-HFD vs. Young-chow and the Aged-HFD vs. Aged-chow comparison. (F) Venn diagram of significantly differentially expressed genes in the Aged-chow vs. Young-chow and the Aged-HFD vs. Young-HFD comparison. (G) Heat map of genes that were differentially expressed by either age or HFD. (H-K) Ingenuity Pathway Analysis of differentially expressed genes (z-score). The same canonical pathways associated with neuroinflammation, T cell signaling, and synaptic function are displayed across all pairwise comparisons.

3.2. Short-term HFD consumption increases CD8+ T cell presence in the brain of aged rats

Because T cell-related pathways were enriched in our RNAseq analyses, we performed flow cytometry to quantify the presence of CD4⁺ and CD8⁺ T cells in the brains of young and aged rats following 3 days of either chow or HFD consumption. A 2-way ANOVA demonstrated a main effect of age, indicating an increase in the percentage and number of CD4⁺ T helper cells in the aged brain compared to younger counterparts (percentage: F_(1,20) = 90.82, p < 0.0001; number: F_(1,20) = 25.46, p < 0.0001; Fig. 3B–C). There was no effect of diet on the percentage or number of CD4⁺ T cells in the brain (p > 0.05). There was a similar main effect of age on brain CD8⁺ T cell percentage and number (percentage: F_(1,20) = 95.26, p < 0.0001; number: F_(1,20) = 33.11, p < 0.0001). Furthermore, there was a significant age x diet interaction for the percentage and number of brain CD8⁺ T cells (percentage: F_(1,20) = 6.265, P < 0.05); number: F_(1,20) = 4.485, p < 0.05; Fig. 3D–E). A Tukey’s posthoc analysis revealed a significant increase in the percentage and number of CD8⁺ T cells in the brain of aged HFD-fed rats relative to both young HFD-fed and aged chow-fed rats. To test whether or not the diet-induced increase in brain CD8⁺ T cells was due to increases in blood brain barrier permeability, we injected (i.p.) a separate cohort of rats with Evans blue dye and measured dye leakage into the peritoneal tissue, pituitary gland, hippocampus, and amygdala following saline perfusion. Results indicated significant uptake of dye in peripheral tissues (peritoneum and pituitary gland), but not in the hippocampus or amygdala (Fig. 3F). There was no impact of age or diet on Evans blue dye leakage in any of the tissues, including brain tissues. Lastly, we measured the impact of HFD on weight gain in young and aged rats. We report an age x diet interaction on the percent of body weight gained across the 3-day diet (F_(1,20) = 29.420, P < 0.0001; Fig. 3G). Posthoc analysis revealed young and aged rats fed a HFD had increased weight gain relative to their chow-fed counterparts, with HFD-fed aged rats showing even greater weight gain than HFD-fed young rats.

Figure 3.

Brain T cell populations identified via flow cytometry. (A) Representative plots demonstrating our gating strategy for identification of CD4+ and CD8+ T cell populations. Isotype controls and splenocytes were included as a negative and positive control, respectively. (B-C) The percentage and number of brain CD4+ T cells. (D-E) The percentage and number of brain CD8+ T cells. (F) Quantification of Evans blue dye absorbance into peripheral and central tissues. *p < 0.05, ** p < 0.01, *** p < 0.001, ****p < 0.0001.

3.3. CD8 antibody depletes peripheral CD8⁺ T cells and prevents the HFD-induced increase of brain CD8⁺ T cells in aged rats

In a small pilot study using chow-fed aged rats injected with either a CD8 antibody or IgG control, we determined that a single dose of peripherally-administered CD8 antibody produced > 99% depletion of CD8⁺ T cells from blood and spleen by 3 days post injection (Fig. 4A–C), and this depletion was maintained for at least 10 days post injection (Fig. 4A–C). However, CD8⁺ T cells in the brain were unaltered at any time point (Fig. 4A,D).

Figure 4.

Validation of peripheral CD8+ T cell depletion via flow cytometry. (A) Representative plots of CD8+ T cells following either an isotype control or anti-CD8 (OX-8) injection at day 3 and day 10 post-injection. Percentages of (B) blood, (C) spleen, and (D) brain are also included.

To determine the effects of depleting peripheral CD8⁺ cells on their expression in blood and brain in the context of HFD, we injected aged rats with either CD8 antibody or IgG control 3 days prior to being fed HFD or chow. First, a 2-way ANOVA demonstrated that starting body weights did not differ between groups (Fig 5A) and although, as expected, there was a main effect of diet on body weight change, with HFD increasing the percentage of body weight change (F_(1,20) = 44.72, p < 0.0001), there was no effect of CD8⁺ T cell depletion on weight gain (Fig. 5B). There was a main effect of the CD8 antibody on CD8⁺ T cell depletion in the blood, regardless of diet condition (F_(1,20) = 145.8, p < 0.0001; Fig. 5C–D). Diet condition did not impact the ratio of CD8⁺ to CD4⁺ T cells in the blood.

Figure 5.

Validation of peripheral CD8+ T cell depletion in chow and HFD-fed aged rats via flow cytometry. (A) Starting body weight of aged rats showing no differences across groups. (B) Weight change across the 3-day HFD exposure showing no impact of CD8+ T cell depletion on weight change. (C) The percentages of blood CD8+ T cells. (D) Representative plots from each group showing nearly full depletion of blood CD8+ T cells. **** p < 0.0001.

In the brain, a 2-way ANOVA indicated there was a significant diet x treatment interaction for the percentage and number of CD8⁺ T cells (percentage: F_(1,20) = 9.66, p < 0.01; number: F_(1,20) = 7.69, p < 0.05; Fig 6A–C). A Tukey’s posthoc analysis confirmed that aged HFD-fed rats injected with the IgG control exhibited increased CD8⁺ T cells relative to all other groups (p < 0.01). In contrast, depleting these cells peripherally significantly decreased their diet-induced increase in the brain (p < 0.01) such that they did not differ from chow-fed controls (p > 0.05). Neither diet nor antibody treatment altered the percentage or number of brain CD4⁺ T cells (percentage: F_(1,20) = 0.25, p = 0.41; number: F_(1,20) = 0.58 p = 0.97; Fig 6D–E)

Figure 6.

Brain T cell responses to peripheral CD8+ T cell depletion and HFD consumption in aged rats. (A) Representative plots of brain CD8+ and CD4+ T cells following HFD consumption and control or anti-CD8 injections. Isotype controls and splenocytes were included as a negative and positive control, respectively. (B-C) Percentages and number of brain CD8+ T cells. (D-E) Percentage and number of brain CD4+ T cells. ** p < 0.01.

3.4. Depletion of peripheral CD8⁺ T cells prevents HFD-induced memory deficits in aged rats

To investigate the extent to which depletion of peripheral CD8⁺ T cells may protect against HFD-induced memory deficits, aged rats were injected (i.v.) with a CD8 antibody 3 days prior to either remaining on standard chow or receiving a HFD for 3 days, followed by behavior testing (see Fig 7A for schematic of experimental design). Starting body weights did not differ (Fig 7B), and CD8⁺ T cell depletion had no impact on body weight (Fig. 7C). There was no effect of diet or treatment on freezing behavior during the conditioning phase (Fig. 7D) or during the pre-tone phase/novel context test (Fig. 7F). A 2-way ANOVA revealed a significant diet x treatment interaction for hippocampal-dependent contextual memory (F_(1,27) = 6.51, p < 0.05; Fig. 7E). A Tukey’s posthoc analysis indicated that IgG-injected HFD-fed rats froze for significantly less time than all other groups, indicating impaired memory. In contrast, HFD-fed rats with depleted peripheral CD8⁺ T cells froze for significantly more time than did the IgG-treated controls and were indistinguishable from chow-fed controls, indicating that memory function had been rescued.

Figure 7.

Long-term memory function of aged rats assessed via contextual fear conditioning following HFD and peripheral CD8+ T cell depletion. (A) Experimental timeline. (B) Starting body weight of aged rats showing no differences across groups (C) Weight change across the 3-day HFD exposure showing no impact of CD8+ T cell depletion on weight change. (D) Freezing behavior scored during conditioning phase. (E) Freezing behavior during testing phase for hippocampal-dependent contextual memory. (F) Freezing behavior in the pre-tone phase of the cued-fear test. (G) Freezing behavior during the test phase for amygdala-dependent cued-fear memory. * p < 0.05, ** p < 0.01, **** p < 0.0001.

For amygdala-dependent cued-fear memory, there was also a significant diet x treatment interaction (F_(1,25) = 14.66, p < 0.001; Fig. 7G). A Tukey’s posthoc comparisons showed that HFD-fed rats with depleted peripheral CD8⁺ T cells froze at a higher rate than IgG-injected HFD-fed rats and chow-fed rats with depleted peripheral CD8⁺ T cells, indicating that this memory was also rescued by depleting peripheral CD8⁺ T cells.

3.5. Depletion of peripheral CD8⁺ T cells decreases cytokine protein concentration in the hippocampus of aged rats

To further understand the impact of increased CD8⁺ T cell presence in the brain, we depleted peripheral CD8⁺ T cells prior to 3 days of either chow or HFD consumption and then measured the concentration of multiple pro- and anti-inflammatory cytokines in the hippocampus and amygdala of aged male rats. There was a main effect of CD8⁺ T cell depletion on the concentration of IFNγ (F_(1,20) = 31.33, p < 0.0001, Fig. 8A), IL-1β (F_(1,20) = 10.81, p < 0.005, Fig. 8C), and IL-4 (F_(1,20) = 15.66, p < 0.005, Fig. 8E) in the hippocampus, in that CD8⁺ T cell depletion decreased the concentration of these cytokines. In the amygdala, there was no impact of diet or CD8⁺ T cell depletion on the measured cytokines (Fig. 8B,D,F).

Figure 8.

Protein concentration of hippocampal and amygdalar cytokines following HFD and peripheral CD8+ T cell depletion. Cytokine values were normalized to total protein. (A) Hippocampal and (B) amygdalar levels of IFNγ. (C) Hippocampal and (D) amygdalar levels of IL-1β. (E) Hippocampal and (F) amygdalar levels of IL-4. ** p < 0.01, **** p < 0.0001.

3.6. Depletion of peripheral CD8⁺ T cells prevents HFD-induced decreases in synaptic elements in the hippocampus and amygdala of aged rats

Because memory impairments are often associated with changes in pre- and post-synaptic elements, we evaluated whether 3 days of HFD results in such changes and investigated the impact of peripheral CD8⁺ T cell depletion on this process. Our data show there were no changes in hippocampal SYP in any condition (Fig. 9A). However, in the amygdala, there was a significant diet x treatment interaction for SYP protein (F_(1,19) = 12.82, p < 0.01). A Tukey’s posthoc analysis revealed decreased SYP in IgG-injected HFD rats and in chow-fed rats with depleted CD8⁺ T cells relative to IgG-injected chow-fed controls (Fig 9B). HFD-fed rats with depleted CD8⁺ T cells did not differ from any other groups. Furthermore, our data showed there was a significant diet x treatment interaction for PSD95 protein concentration in the hippocampus (F_(1,18) = 4.79, p < 0.05). A Tukey’s posthoc analysis revealed IgG-injected HFD-fed rats had significantly less PSD95 than IgG-injected chow-fed controls. Chow-and HFD-fed rats with depleted peripheral CD8⁺ T cells did not differ from IgG-chow controls (Fig. 9C). There was also a significant diet x treatment interaction for amygdalar PSD95 (F_(1,19) = 7.90, p < 0.05), with IgG-injected HFD-fed rats having significantly less PSD95 than IgG-injected chow-fed controls. Chow-and HFD-fed rats with depleted peripheral CD8⁺ T cells did not differ from IgG-chow controls (Fig. 9D).

Figure 9.

Protein concentration of hippocampal and amygdalar pre- and post-synaptic elements following HFD and peripheral CD8+ T cell depletion. Representative Western blots are included at the bottom of the figure. (A) Hippocampal and (B) amygdalar levels of synaptophysin. (C) Hippocampal and (D) amygdalar levels of PSD-95. * p < 0.05.

4. Discussion

The current study investigated the role of T cells on HFD-induced memory deficits and neuroinflammation in aged rats. RNA sequencing and pathway analysis of the hippocampus and amygdala revealed several pathways related to neuroinflammation, immune cell trafficking, and T cell signaling that were enriched by aging and/or HFD. We also showed, via flow cytometry, that HFD significantly increases the number of CD8⁺, but not CD4⁺, T cells in the brains of aged rats, with no impact on young rats. To understand the functional impact of increased CD8⁺ T cell numbers in the aged brain, we depleted peripheral CD8⁺ T cells in aged rats prior to HFD consumption. These data show that peripheral depletion of CD8⁺ T cells prevents the diet-induced increase in brain CD8⁺ T cells and prevents diet-induced hippocampal- and amygdalar-dependent memory deficits. Peripheral CD8⁺ T cell depletion also decreased the concentration of several pro and anti-inflammatory cytokines in the hippocampus, but not the amygdala. Finally, we showed that HFD consumption decreased synaptic elements in the hippocampus and amygdala of aged rats and that CD8⁺ T cell depletion prevented that decrease. Taken together, these data demonstrate, for the first time, that increased presence of CD8⁺ T cells in the brain contribute to HFD-induced memory impairments, owing perhaps to their effects on the neuroinflammatory milieu and synaptic structure in the aged brain.

RNA sequencing of the hippocampus and amygdala revealed multiple genes and canonical signaling pathways associated with neuroinflammation, immune cell trafficking, T cell signaling, and synaptic plasticity that were altered in response to HFD and/or aging. Some of these key genes include complement-associated genes, integrins, cytokines and cytokine receptors, chemokines, glutamate receptor subunits, and neurotrophic factors. Of note, chemokines associated with T cell recruitment and migration across endothelia (Carr et al., 1994; Kollikowski et al., 2020; Zhang et al., 2021) were upregulated as a function of HFD in aged rats in both the hippocampus (Ccl2) and amygdala (Cxcl11). Furthermore, in the aged hippocampus, HFD increased the expression of MHC class I-associated genes, Lilrb1 and Lilrb2, which function to modulate MHC class I signaling (Barkal et al., 2018; Zhao et al., 2019).

In addition to T cell signaling and immune cell trafficking, genes associated with synaptic function, such as glutamate receptor subunits (Grin2a, Grin2c, Gria2, Gna15, Grm8), postsynaptic elements (Synpo2), and neurotrophic factors (Igf1, Igf2, Igfbp1) were dysregulated by aging and HFD consumption in both the hippocampus and amygdala, which is in line with synaptogenesis and synaptic plasticity pathways being implicated in the IPA results. While further validation with functional LTP or spine density analysis is required, these data suggest a combination of aging and diet might impair synaptic function, which is consistent with the observed memory deficits in aged HFD-fed rats. In this dataset, in terms of gene expression, the amygdala had more changes associated with both aging and/or HFD than the hippocampus. Interestingly, amygdala-dependent memory deficits in aged rats are not observed in the context of other insults we have studied, such as peripheral bacterial infection or post-operative cognitive dysfunction (Barrientos et al., 2012b, 2006b). The origin of this apparent sensitivity to HFD is unclear, but it could potentially be tied to the fact that the amygdala is a critical mediator of gut-brain signaling (Cowan et al., 2018).

To validate the transcriptional changes implicating a role for immune cell trafficking and T cells in diet-induced neuroinflammation, we measured T cell presence in the brain of young and aged rats fed either a standard chow or 3-day HFD. Indeed, our data show, for the first time, that HFD increased CD8⁺, but not CD4⁺, T cells in aged brain. While this is the first evidence that diet can impact brain CD8⁺ T cell presence, our findings add to the growing literature implicating T cells in secondary challenges in the aged brain (i.e. traumatic brain injury, stroke, and neurodegenerative disease) (Ritzel et al., 2016; Schindowski et al., 2007; Unger et al., 2020). Therefore, it is conceivable that this CD8⁺ T cell observation could be a general mechanism through which inflammatory challenges impair memory in aged brains, but this hypothesis requires further testing in other models. While the amount of brain CD4⁺ T cells were not altered by diet, this does not rule out potential changes in T helper cell subtype in the brain as a function of diet. For example, previous work has shown that HFD can increase the polarization of naïve CD4⁺ T cells into the Th1 phenotype and increase proinflammatory cytokine production in those cells (Mauro et al., 2017; Strissel et al., 2010). Interestingly, our RNAseq dataset revealed that the anti-inflammatory Th2 transcription factor Gata3 is decreased in the amygdala of aged-HFD rats, but increased in young-HFD rats. These transcript data further raise the possibility of a nuanced dynamic between diet and T helper cells in the aged brain. Due to limited antibodies available for rat flow cytometry, we did not further investigate the T helper cell subtype; however, this will be the focus of future experiments.

Of note, the HFD-induced increase in CD8⁺ T cells does not appear to be due to deficits in blood brain barrier (BBB) function. Previous reports have shown that HFD consumption and obesity are associated with increased BBB permeability (Chang et al., 2014; Hargrave et al., 2016; Popescu et al., 2009). Here, we evaluated BBB function by measuring Evans blue dye leakage into brain tissues in young and aged rats fed either a chow or HFD. Our findings show no impairment in BBB function, as aging or diet did not alter the low-level leakage of Evans blue dye into the hippocampus and amygdala. Importantly, the pituitary gland, which is proximal to the brain, but not protected by the BBB, had significant uptake of the Evans blue dye. Thus, it could not be argued that there was insufficient time for the dye to travel from the periphery to the brain. The lack of effect of HFD on BBB permeability in the current study is likely due to the short-term nature of the diet, as the majority of previous reports have used a longer diet regimen (Chang et al., 2014; Hargrave et al., 2016; Popescu et al., 2009). Nonetheless, these data are useful in highlighting that, in the context of HFD consumption, BBB breakdown is not necessary for CD8⁺ T cells to populate the brain.

To understand the functional impact of increased CD8⁺ T cell presence in the brain, we used a well-validated anti-CD8 depleting antibody to eliminate circulating CD8⁺ T cells (Hartlage et al., 2019). Importantly, this antibody did not deplete basal levels of brain CD8⁺ T cells, likely due to not being able to cross the BBB. Nevertheless, depletion of all peripheral CD8⁺ T cells allowed us to understand the contribution of peripheral cells to the diet-induced increase in brain CD8⁺ T cells. A single injection of the anti-CD8 antibody depleted > 99% of blood and spleen CD8⁺ T cells for at least 10 days in aged rats. Indeed, this depletion of peripheral CD8⁺ T cells prior to 3-day HFD consumption completely prevented their HFD-induced increase in aged rat brains. This finding strongly suggests the diet-induced increase in brain CD8⁺ T cells is due to peripheral T cell trafficking to the brain, though further studies are needed to confirm this. Importantly, this depletion had no impact on the number of brain CD4⁺ T cells, highlighting its specificity to CD8⁺ T cells.

In addition to understanding if peripheral CD8⁺ T cell depletion would prevent their increased presence in the brain after HFD consumption, we also wanted to understand what impact this increase has on long-term memory function. Thus, CD8⁺ T cells were depleted from the blood prior to 3-day HFD consumption and contextual fear conditioning. As we have shown previously (Spencer et al., 2017), HFD significantly impaired hippocampal- and amygdalar-dependent memory function. Importantly, peripheral CD8⁺ T cell depletion completely prevented these diet-induced memory impairments. These data establish, for the first time, a role for elevated CD8⁺ T cells in diet-induced memory deficits. These data also extend previous work implicating CD8⁺ T cells in cognition with healthy aging and neurodegenerative disease models (Garber et al., 2019; Laurent et al., 2017; Schindowski et al., 2007; Stojić-Vukanić et al., 2020; Unger et al., 2020). Interestingly, depletion had no impact on hippocampal memory function in the absence of a HFD challenge; however, amygdala-dependent memory was moderately impacted. While these data were not statistically significant, they might suggest that peripheral CD8⁺ T cells play a role in basal amygdala function, though future studies are needed to confirm this hypothesis and understand the mechanism through which this occurs.

There is a well-established role for elevated proinflammatory cytokines to disrupt long-term potentiation and impair memory function, and previous work from our lab indicates IL-1β as a key cytokine that mediates diet-induced memory impairments (Frank et al., 2010b; Muscat and Barrientos, 2021; Sobesky et al., 2014a; Spencer et al., 2017; Tanaka et al., 2018). Thus, we investigated the role of CD8⁺ T cells in regulating hippocampal and amygdalar cytokines in aged rats. Our findings showed that peripheral CD8⁺ T cell depletion decreased the protein concentration of IFNγ, IL-1β, and IL-4 in the hippocampus, but not the amygdala. While IFNγ and IL-4 are traditionally T cell-associated cytokines, it is noteworthy that IL-1β in the brain is predominantly produced by microglia (Smith et al., 2012). Given that CD8⁺ T cell depletion decreased IL-1β concentration, it suggests a potential role of CD8⁺ T cells to regulate myeloid cell-derived cytokines in the brain. Unexpectedly, HFD did not alter the levels of the measured cytokines. This is in contrast to our previous work showing HFD increased hippocampal and amygdalar IL-1β in aged rats (Spencer et al., 2017). However, in those experiments, brain tissues were collected 2 h following a learning experience that included an electric shock, which might have potentiated a heightened inflammatory response, as has been demonstrated elsewhere (Sobesky et al., 2014b), which was lacking in the current study. Notably, our depletion protocol does not deplete basal levels of brain CD8⁺ T cells, suggesting this reduction in hippocampal cytokine signaling is due to signals, or lack thereof, initiated in the periphery.

In addition to inflammatory signaling, structural changes to synapses can also contribute to deficits in synaptic plasticity and long-term memory (Fitzgerald et al., 2014; Nithianantharajah and Murphy, 2008; Tampellini et al., 2010). To gain a general understanding of diet- and T cell-mediated changes in synaptic structure, we measured changes in pre and postsynaptic elements, SYP and PSD95, respectively. Our data demonstrated that HFD decreased hippocampal PSD95 in aged rats and that CD8⁺ T cell depletion prevented this deficit. Similarly, in the amygdala, HFD decreased both SYP and PSD95 and CD8⁺ T cell depletion attenuated these deficits. These data extend prior work showing CD8⁺ T cells regulate synaptic genes in a mouse model of Alzheimer’s disease by showing synaptic protein changes within the context of HFD consumption (Unger et al., 2020). Given that microglia play a large role in regulating synaptic structure and integrity (Wang and Li, 2021), it is plausible that brain CD8⁺ T cells interact with resident microglia to alter synaptic elements. Our data demonstrating CD8⁺ T cells regulate a microglia-associated cytokine (IL-1β) strengthens this possibility and brain T cell interactions with resident cells will be a focus of future experiments. Overall, these data pair nicely with our transcriptomic data indicating diet-induced alterations in genes associated with synaptic function and strongly implicate either a direct or indirect role of CD8⁺ T cells in regulating synaptic structure.

An important caveat to the current data regarding increased CD8⁺ T cell presence in the brain is that it lacks regional specificity due to needing a whole hemisphere of brain tissue to generate enough cells for reliable flow cytometry data. Thus, it is unclear where in the cerebrum this increase in CD8⁺ T cells is occurring. However, our behavior, protein, and transcriptomic data strongly implicate both the hippocampus and amygdala. On a related note, while the meninges were removed upon brain dissection, we did not remove the choroid plexus, which has been shown to be a major hub for a variety of different immune cells, including T cells (Baruch and Schwartz, 2013; Meeker et al., 2012; Strominger et al., 2018). Thus, it is possible this increase in brain CD8⁺ T cells is localized to the choroid plexus. Future studies will investigate the spatial-anatomical characteristics of diet-induced T cell changes in the brain. Moreover, while our depletion data demonstrate a causal role of T cells in diet-induced memory deficits and neuroinflammation, we cannot rule out the contribution of other immune cells that may infiltrate the aged brain following HFD consumption. Another important note concerns the mechanism through which HFD signals to CD8⁺ T cells. While lipids can be presented as antigens to T cells (mostly natural killer T cells) via CD1-mediated antigen presentation, it is unclear whether excess dietary lipids can alter this process (De Libero and Mori, 2005). While more experiments are needed to determine the exact mechanisms, a more likely explanation is that HFD is rapidly altering T cell signaling, independent of antigen presentation, to promote a hyperinflammatory state. This hypothesis is based on the precedent for fatty acids alone to induce a proinflammatory response in T cells independent of canonical activation (de Jong et al., 2014; McCambridge et al., 2019; Stentz and Kitabchi, 2006). Lastly, another important limitation of the current study is these data were all collected in male rats for reasons stated above, so results should not be generalized to females as there are significant sex differences in response to HFD, memory, and T cell signaling (Ahnstedt et al., 2018; Chowen et al., 2017; Hwang et al., 2010).

To conclude, we have presented, using a variety of techniques, the first evidence for CD8⁺ T cell involvement in HFD-induced memory deficits. We have also shown that peripheral CD8⁺ T cells can regulate pro and anti-inflammatory cytokines in the hippocampus, as well as impact synaptic elements in both the hippocampus and amygdala. Future studies will investigate the direct and indirect mechanisms through which CD8⁺ T cells impact resident cells in the brain parenchyma.

Highlights.

• 3-day high fat diet (HFD) increased the number of brain CD8⁺ T cells in aged rats.

• CD8⁺ T cell depletion prevented HFD-induced memory deficits in aged rats.

• CD8⁺ T cell depletion decreased hippocampal cytokine levels in aged rats.

• 3-day HFD decreased synaptic elements in the aged hippocampus and amygdala.

• CD8⁺ T cell depletion attenuated the diet-induced decrease in synaptic elements.