Human Herpesvirus 6B Greatly Increases Risk of Depression by Activating Hypothalamic-Pituitary -Adrenal Axis during Latent Phase of Infection

Abstract

Little is known about the effect of latent-phase herpesviruses on their host. Human herpesvirus 6B (HHV-6B) is one of the most ubiquitous herpesviruses, and olfactory astrocytes are one of the most important sites of its latency. Here, we identified SITH-1, an HHV-6B latent protein specifically expressed in astrocytes. Mice induced to produce SITH-1 in their olfactory astrocytes exhibited olfactory bulb apoptosis, a hyper-activated hypothalamic-pituitary-adrenal (HPA) axis and depressive symptoms. The binding of SITH-1 to the host protein calcium-modulating ligand (CAML) to form an activated complex promoted the influx of extracellular calcium. The serum antibody titers for depressive patients with respect to this activated complex were significantly higher than for normal controls (p = 1.78 × 10^-15), when the antibody positive rates were 79.8% and 24.4%, respectively, and the odds ratio was 12.2. These results suggest that, in the latent phase, HHV-6B may be involved in the onset of depression.

Keywords: Behavioral Neuroscience; Molecular Biology; Virology.

Graphical abstract

Figure 1

Identification and Characterization of SITH-1 (A) Structures of HHV-6B DNA, H6LT, and SITH-1 mRNA. R1, R2, and R3 indicate repeat regions. (B) SITH-1 mRNA expression in macrophage (MΦ) cell lines (THP-1 and HL-60) and astrocyte cell lines (U373 and A172). Ratios of SITH-1 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are shown. (C) SITH-1 protein production in HHV-6B-infected U373 cells. Confocal imaging of HHV-6B infected and mock infected U373 cells. The upper six panels show staining with anti-SITH-1 antibodies and anti-gB antibodies and the lower six panels staining with anti-SITH-1 antibodies and anti-gH antibodies. Green, SITH-1; red, gB or gH; blue, DAPI. (D) Detection of apoptosis in OB using TUNEL staining. Green, TUNEL; red, propidium iodide (PI). (E) Numbers of TUNEL-positive apoptotic cells in each section. (F) Enhancement of OB apoptosis detected by increase in Bax/Bcl-2 ratio (G) Prolonged immobility in TST. One-way ANOVA with Fisher's post hoc test; ∗, p < 0.05; n.s., not significant. (E and F) Values are means ± SEM. Unpaired t test; ∗, p < 0.05; ∗∗, p < 0.01. Scale bars, 100 μm (C and D).

Figure 2

Depression-like Changes in SITH-1 Mice (A) Enhanced stress in SITH-1 mice. Vector control and SITH-1 mice were caged with (+) or without (−) tilt, and sucrose preference was calculated as % sucrose intake. (B–E) Up-regulation of indicated mRNAs related to HPA axis activation. (B–D) Up-regulation of indicated mRNAs related to HPA axis activation in the brain. (E) Up-regulation of StAR mRNA in the adrenal gland. (F) DCX staining of hippocampal dentate gyrus (DG). Green, DCX; blue, DAPI. Scale bar, 100 μm. (G) Numbers of DCX-positive cells in subgranular zone (SGZ) in each section. Comparison of vector control and SITH-1 mice. Unpaired t test; ∗∗∗, p < 0.001. (A, B, D, and E) Bars represent median values; Mann-Whitney U-test. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; n.s., not significant. (C) Values are means ± SEM. Unpaired t test; ∗, p < 0.05.

Figure 3

Characterization of SITH-1 (A) Binding activity of SITH-1 and CAML. (B) Colocalization of SITH-1 and CAML in astrocytes. U373 cells expressing SITH-1 (SITH-1[+]) and not expressing SITH-1 (SITH-1[−]) were immunostained. Confocal images are shown. Red, SITH-1; green, CAML. Scale bar, 100 μm. (C and D) Promotion of intracellular calcium influx by SITH-CAML complex with extracellular calcium (C) and without it (D). U373 cells expressing (SITH-1[+]) or not expressing SITH-1(SITH-1[−]) were stimulated with thapsigargin (TG) at time 0. Values are means ± SEM. (E) Binding activity of SITH-1 and mCAML determined by a mammalian two-hybrid assay. (F) Co-immunoprecipitation assay in cells co-expressing SITH-1 and CAML. After immunoprecipitation using anti-CAML antibodies, western blot analyses were performed using anti-CAML antibodies and anti-SITH-1 antibodies. The upper two panels show input cell lysate and the lower two panels show immunoprecipitated samples. hCAML, human CAML; mCAML, mouse CAML; M, molecular weight marker. (G) Increased calcium concentration in mouse 3T3 cells due to SITH-1 expression. Bars represent median values; Mann-Whitney U-test. ∗∗∗∗, p < 0.0001. (H) Enhancement of OB apoptosis by calcium ionophore. (A, E, and H) Unpaired t test; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. Values are means ± SEM (H).

Figure 4

Formation of SITH-CAML Complex in Depressive Patients (A) Intracellular calcium concentrations in various protein expressing cells. (B) Antibody response to various antigens in mice. Shows representative IFA images for SITH-1 mice and vector control mice for various antigens. Scale bar, 100 μm. (C) Comparison of antibody titers for N-SITH-CAML-C, N-CAML-SITH-C and SITH-1in SITH-1 mice. (D) Antibody reactions to various antigens in depressive patients. Shows representative IFA images for individual antigens in one patient and one normal control (NC). Scale bar, 100 μm. (E) Comparison of antibody titers for N-SITH-CAML-C, N-CAML-SITH-C, and SITH-1 in depressive patients. (F) Elevated anti-N-SITH-CAML-C titers in depressive patients. (G) Comparison of anti-N-SITH-CAML-C antibody titers in normal controls with BDI up to 3 and 4 or greater. Bars represent median values, and Kruskal-Wallis test with Steel's post hoc test (A, C, and E) and Mann-Whitney U-test (F and G). ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001.