Fig 2: Behavioral tests and NKG2D expression in the spleen of CSDS-susceptible mice and CSDS-resilient mice. a The schedule of CSDS model, social interaction test, behavioral tests, and collection of spleen. CSDS was performed for 10 days (day 1–day 10). On day 11, the social interaction test was performed to separate CSDS-susceptible mice and CSDS-resilient mice. On day 12, locomotion and TST were performed. On day 13, FST was performed. On day 14, SPT was performed. On day 15, spleen from the three groups was collected. Control (no CSDS) mice were used as control group. b Locomotion (one-way ANOVA, F2,21 = 0.118, P = 0.889). c TST (one-way ANOVA, F2,21 = 8.494, P = 0.002). d FST (one-way ANOVA, F2,21 = 13.317, P < 0.001). e SPT (one-way ANOVA, F2,21 = 5.395, P = 0.013). f NKG2D expression in the spleen (one-way ANOVA, F2,21 = 4.278, P = 0.028). g A positive correlation (r = 0.767, P < 0.001) between TST immobility time and NKG2D expression in the spleen. h A positive correlation (r = 0.929, P < 0.001) between FST immobility time and NKG2D expression in the spleen. Data are shown as mean ± SEM. (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001. ANOVA analysis of variance, FST forced swimming test, N.S. not significant, SPT sucrose preference test, TST tail suspension test

Fig 3: Effects of (R)-ketamine on depression-like phenotypes, abnormal splenic functions in the CSDS-susceptible mice. a The schedule of CSDS model, social interaction test, treatment, behavioral tests, and collection of spleen. CSDS was performed for 10 days (day 1–day 10). On day 11, the social interaction test was performed to select CSDS-susceptible mice. Control (no CSDS) mice were used as control group. On day 12, saline (10 ml/kg) or (R)-ketamine (10 mg/kg) was administered i.p. into CSDS-susceptible mice. Saline was administered i.p. into control (no CSDS) mice. Locomotion and TST were performed 1 and 3 h after injection, respectively. On day 13, FST was performed. On day 14, SPT was performed. On day 15, spleen from the three groups was collected. b Locomotion (one-way ANOVA, F2,15 = 0.270, P = 0.767). c TST (one-way ANOVA, F2,15 = 5.188, P = 0.019). d FST (one-way ANOVA, F2,15 = 4.446, P = 0.030). e SPT (one-way ANOVA, F2,15 = 6.376, P = 0.010). f Representative picture of spleen from saline-treated control (no CSDS) mice, saline-treated CSDS-susceptible mice, and (R)-ketamine-treated CSDS-susceptible mice. Spleen weight of (R)-ketamine-treated CSDS-susceptible mice was significantly lower higher than that of saline-treated CSDS-resilient mice (one-way ANOVA, F2,15 = 12.052, P = 0.001). g Western blot analysis of NKG2D expression in the spleen (one-way ANOVA, F2,15 = 7.591, P = 0.005). The representative bands of Western blot analysis were shown. Data are shown as mean ± SEM. (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001. ANOVA analysis of variance, ANOVA analysis of variance, FST forced swimming test, NKG2D natural-killer receptor group 2, member D, N.S. not significant, SPT sucrose preference test, TST tail suspension test

Fig 4: Proposed mechanisms of the role of brain–spleen axis in the stress-induced psychiatric disorders. Stress caused increase of spleen size and weight, resulting in abnormalities in spleen functions. Brain inflammation by stress might be mediated by brain–spleen axis (i.e., vagus nerve). NKG2D in spleen may play a role in the stress-related psychiatric disorders such as depression. Interestingly, (R)-ketamine ameliorates abnormalities of spleen functions and depressive symptoms by stress

Fig 5: FACS analysis of spleen samples of three groups. a Total cell number of spleen (one-way ANOVA, F2,15 = 4.659, P = 0.027). b The number of NKG2D+ cells in the spleen (one-way ANOVA, F2,15 = 5.207, P = 0.019). Data are shown as mean ± SEM. (n = 6). *P < 0.05, **P < 0.01. c FACS analysis of cells stained with antibodies against NKG2D and DX5 in spleen samples from saline-treated control mice, saline-treated CSDS-susceptible mice, and (R)-ketamine-treated CSDS-susceptible mice. (R)-ketamine ameliorated the increased percentage of NKG2D+ cells in the spleen of CSDS-susceptible mice. ANOVA analysis of variance, DX5 CD49b, NKG2D natural-killer receptor group 2, member D