Peripheral adaptive immunity of the triple transgenic mouse model of Alzheimer’s disease

The 3xTg-AD mouse model of AD used in this study was developed by Oddo and colleagues [15] and bred in our animal facility. These mice harbor three mutant genes, namely genes coding for the human beta-amyloid precursor protein (APP), tau (in Thy1.2 expression cassettes), and presenilin-1 (PS1, knockin) with human mutations from familial AD (APP_swe, PS1_M146V) and frontotemporal dementia (tau_P301L). The 3xTg-AD replicates many features of AD including Aβ and tau pathologies as well as cognitive deficits. Previous studies showed that 3xTg-AD mice fully develop neuropathological and behavior changes around 12 months of age [6, 7]. The non-transgenic (NTg) controls used in this study were generated from the backcross of our 3xTg-AD colony with B6126SF1/J animals, maintained on a mixed B6129 background and bred in our animal facility. The Laval University Animal Research Committee (Québec, QC, Canada) approved all procedures. The animals used for post-mortem analyses of adaptive immunity are the control group (vehicle) from preclinical evaluation of human intravenous immunoglobulin (IVIg) efficacy reported earlier and included 8 NTg and 12 3xTg-AD animals, 50% females in each group [4]. These animals received intraperitoneal administrations of sterile 0.2 M glycine pH 4.25, endotoxin free, twice a week for 3 months (27 injections). Mice were killed when overt AD-like neuropathologic changes are observed (12 ± 0.1 months) under deep anesthesia (100 mg/kg ketamine, 10 mg/kg xylazine) via terminal intracardiac perfusion of PBS containing protease and phosphatase inhibitors. Serum was prepared from intracardiac blood, and splenocytes and bone marrow (both tibia and femur) cells were isolated and stored in liquid nitrogen until used. Cytokines and chemokines were quantified before and after the development of overt AD-like neuropathological processes: blood from untreated 9- (9.0 months ± 0.1) and 13- (12.9 months ± 0.2) month-old mice (N = 7–8 mice per group, 4 females in each group) was drawn from the saphenous vein, clotted on ice, and centrifuged at 5000 g for 5 min for serum recovery. Serum was frozen at − 80 °C until used.

Unless otherwise specified, all biochemical reagents were purchased from JT Baker (Phillipsburg, NJ, USA).

To study cell marker expression, the following fluorochrome-conjugated antibodies were used as described [4, 16]: anti-CD45 (leukocyte marker; clone 30-F11; eBioscience, San Diego, CA, USA), anti-CD3 (T lymphocyte marker; clone 17A2; BD Biosciences, Mississauga, ON, Canada), anti-B220 (B lymphocyte marker; clone RM4-5; BD Biosciences), anti-CD4 (helper T lymphocyte marker; clone GK1.5; eBioscience), anti-CD8b (cytotoxic T lymphocyte marker; clone eBioH35-17.2; eBioscience), anti-CD25 (regulatory T lymphocyte marker; clone PC61.5; eBioscience), anti-Foxp3 (regulatory T lymphocyte marker; clone FJK-16 s; eBioscience), anti-F4/80 (macrophage marker; clone BM8; eBioscience), anti-CD11b (macrophage and granulocyte marker; clone M1/70; eBioscience), anti-Gr1 (granulocyte marker; Ly-6G, Clone RB6-8C5; eBioscience), or relevant isotypic controls in PBS-1%BSA. We used 7-amino-actinomycin D to assess cell viability (eBioscience). Cells were acquired and analyzed using a CyFlow ML cytometer (Partec North America, Inc., Swedesboro, NJ, USA) and FCS Express software (De Novo Software, Los Angeles, CA, USA).

Hematopoietic stem cells were quantified in bone marrow with the Mouse Hematopoietic Stem Cell Isolation Kit (BD Biosciences). It includes a lineage cocktail (lin), which contains antibodies against CD3, CD11b, B220, Ly6C, Gr1, and TER-119 to exclude differentiated cells. Both long-term reconstituting (LTR) and STR hematopoietic stem cells express the cell markers Sca-1 and c-Kit, whereas only STR expresses CD34. Therefore, lin⁻/Sca-1⁺/cKit⁺/CD34⁻ cells are LTR and lin⁻/Sca-1⁺/cKit⁺/CD34⁺ cells are designated STR.

A mouse IgG-specific enzyme-linked immunosorbent assay (ELISA) was performed to assess IgG concentration in serum and cortex using goat anti-mouse IgG Fc-specific antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, USA) [17]. Cytokine and chemokine concentrations were determined using a multiplex ELISA (Q-plex Mouse Cytokine—Screen (16-plex); Quansys Bioscience, Logan, UT, USA). The following cytokines and chemokines were analyzed: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17, tumor necrosis factor (TNF)α, granulocyte-macrophage colony-stimulating factor (GM-CSF), regulated on activated, normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1, also called CCL2), and macrophage inflammatory protein 1α (MIP-1α).

Splenocytes isolated using dissociation were thawed, washed, counted, and plated on mouse anti-IgG-coated wells (Multiscreen®HTS filter plate; Millipore Corporation, Billerica, MA, USA) and left immobile for antibody secretion overnight at 37 °C. After washing the cells, wells were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG Fc-specific antibody and mouse immunoglobulins were detected with TrueBlue peroxidase substrate (VWR, Ville Mont-Royal, QC, Canada). Each spot was counted under a dissection microscope and considered as a single IgG-secreting B cell.

At sacrifice, a separate set of 12-month-old mice (N = 3 NTg and 6 3xTg-AD, treated with glycine as described above) were perfused with PBS, brain hemispheres were recovered, fixed in 4% paraformaldehyde for 48 h then cryoprotected in 20% sucrose at 4 °C for > 72 h. Then, 25-μm-thick sections were cut and stained as previously described [4, 17]. For detection of cerebral IgG and amyloid plaques, free-floating sections were blocked with 5% normal horse serum, 0.2% Triton X-100, and 2.4G2 antibody (to block mouse Fc receptors) in PBS. Sections were incubated overnight at 4 °C, with an Alexa-Fluor 568 conjugated goat anti-mouse IgG antibody. After the overnight incubation, sections were washed in PBS and incubated for 2 h with an Alexa-Fluor 568 donkey anti-goat antibody (Life Technologies, Burlington, ON, Canada) at room temperature. The sections were further washed and placed in a 4′,6-diamino-2-phenylindole (DAPI) for 7 min to stain the nuclei, then mounted on slides, incubated for 5 min with a 0.1% thioflavin-S solution to stain amyloid plaques, and treated with 0.5% Sudan Black to minimize the autofluorescence. After drying, the slides were coverslipped in Fluoromount mounting media. Images were acquired using a Zeiss AxioImager M2 microscope. Analysis was performed with ZEN 2012 SP2 and ImageJ 1.51 s Software in the hippocampus [4].

Statistical analyses were performed using Prism 7.0a (GraphPad Software Inc., San Diego, CA, USA) and JMP 14 (SAS, Marlow, Buckinghamshire, UK). The threshold for statistical significance was set to P < 0.05. Homogeneity of variance and normality was determined for all data sets using D’Agostino & Pearson’s normality test. When normality was verified, unpaired t test was performed. When needed, logarithmic transformation was performed to reduce variances and provide more normally distributed data. Otherwise, Mann-Whitney was applied when Gaussian distribution was not confirmed. For cytokine quantification, we consulted the Statistical Consulting Service from the Université Laval. To compare the effect of the transgenes in two age groups, we used two-way ANOVA when normality of the residuals was confirmed. When normality was not confirmed, data were analyzed first using Kruskal-Wallis test followed by Dunn’s multiple comparison. Then to identify the global effect of genotype, results from the two ages were grouped and analyzed using Mann-Whitney test. All statistical analyses are described in Table 1.

To determine if the decrease in lymphocytes previously observed in 3xTg-AD blood [4] was generalized to primary and secondary lymphoid organs, we evaluated the cell populations of hematopoietic cells extracted from bone marrow and spleen. Comparable numbers of T and B lymphocytes as well as T subpopulations (helper, cytotoxic, and regulatory T lymphocyte) were detected in the spleen of 3xTg-AD mice compared to age-matched NTg controls (Fig. 1a). Similarly, in the bone marrow of 3xTg-AD animals, the levels of lymphocytes but also monocytes and granulocytes were unchanged (Fig. 1b). In humans, reduction in hematopoietic stem cell number is observed in early AD and correlates negatively with age and cerebrospinal fluid Aβ42/40 ratio [18]. To determine whether hematopoietic stem cell population could explain the decreased number of lymphocytes observed in the blood of 3xTg-AD mice, we analyzed the hematopoietic stem cell progenitor populations in the bone marrow using Sca-1, c-kit, and CD34 expression on Lin− cells. Lin− cells do not express markers normally present on lineage committed hematopoietic cells. We quantified LTR and STR hematopoietic stem cells using Sca-1⁺/cKit⁺/CD34⁻/lin⁻ and Sca-1⁺/cKit⁺/CD34⁺/lin⁻ cells, respectively [19, 20] (Fig. 1b and Fig. 2). Although the proportions of LTR hematopoietic stem cells were similar between groups, we observed a significant decrease of STR in 3xTg-AD animals (0.58 fold, P = 0.0116, Fig. 1b). These cells are precursors of common myeloid progenitors and common lymphoid progenitors, which migrate to secondary lymphoid organs and produce lymphoid differentiated cells [21]. The diminution in STR observed here, likely the lymphoid-committed progenitors, could explain decreasing levels of circulating B and T lymphocytes observed in the blood of 3xTg-AD mice.

Activation and maturation of immune cells are tightly regulated to ensure adequate immune response while protecting from autoimmune response. To evaluate lymphocyte activation, we first determined the serum IgG concentration. Interestingly, we observed a 5.4-fold increase in IgG concentration in 3xTg-AD mice (P < 0.0001, Fig. 3a). We also detected a 2.3-fold rise of IgG levels in the cortex of 3xTg-AD mice (P = 0.0002) but no co-localization with amyloid plaques was observed (Fig. 3a). To confirm the increase B lymphocyte activation, we next assessed the proportion of IgG-secreting B lymphocytes among total splenocytes using enzyme-linked immunosorbent spot (ELISPOT) quantification. We detected a trend toward an increase in IgG-secreting cells in the spleen (3.8-fold, P = 0.059, Fig. 3a). Because mature B lymphocytes (plasma cells) are responsible for the production of large amounts of immunoglobulins, we next measured the number of plasma cells (CD138⁺ lymphocytes) in bone marrow and observed a 1.3-fold increase of CD138⁺ cells in 3xTg-AD mice compared to controls (P = 0.0405, Fig. 3). Because sex differences in behavior and Aβ load have been observed in the 3xTg-AD model [6, 22‐24], we further analyzed males and females separately (data not shown). Albeit low statistical power, we did not observe major sex differences. Indeed, although a trend toward more numerous plasma CD138+ cells was observed in female mice only, higher IgG levels were noted in the serum and cortex of both males and females, suggesting that the overall activation of B lymphocytes is not sex dependent. Taken together, these results provide evidence of B lymphocytes activation in the 3xTg-AD mouse model.

Serum cytokine quantification using multiplex ELISA probing in 9- and 13-month-old 3xTg-AD animals revealed increased levels of IL-2, TNF-α, IL-17, and GM-CSF compared with controls but no changes in other cytokine/chemokine investigated (Fig. 4 and data not shown, detailed statistics: Table 1). Interestingly, from the panel of cytokine/chemokine tested, IL-2 is secreted almost exclusively by activated CD4⁺ T helper lymphocyte [25]. Moreover, IFN-γ, TNF-α, and IL-2 are secreted by Th1 T lymphocyte; IL-4, IL-5, and IL-10 are the signature of Th2 lymphocytes, whereas IL-2, IL-6, IL-17, and GM-CSF relates to Th17 activation [26‐28]. Therefore, our results suggest increased activation of T helper lymphocytes along with Th17 polarization in the 3xTg-AD model, particularly in 9-month-old mice.