Stress transmission in social groups of mice: unveiling physiological responses, behavioral patterns, and immune dynamics

Abstract

In modern societies, stress is pervasive, requiring sophisticated physiological mechanisms for stability and survival, primarily through the sympatho-adrenal medullary (SAM) and hypothalamo-pituitary adrenal (HPA) axes. Chronic stress is linked to a range of mental and physical health problems and has been shown to affect immune function. In this study, a paradigm for social stress transmission in groups of mice was established, based on a restraint stress model to study how stress spreads among individuals. Mice exposed to indirect stress exhibited HPA-axis activation, elevated corticosterone (CORT) levels, enlarged adrenal glands, and anxiety-like behaviors in light-dark-box tests. Notably, female mice were more susceptible to stress transmission. While stress transmission enhanced innate immune responses, it did not affect adaptive immunity following vaccination with a poly(lactic-co-glycolic acid) (PLGA)-based vaccine. In contrast, direct stress impaired both immune responses and the effectiveness of immunotherapy in a melanoma model.

Keywords: Rodent behavior; Rodent immunology; Rodent physiology.

Graphical abstract

Figure 1

Effects of acute stress transmission on physiology, anxiety-like behavior, and immune parameters (A and B) Female C57BL/6 mice (n = 12–16 per group) were subjected to acute restraint stress (ARS, red squares), stress transmission (TS, blue triangles), or control treatment (CTRL, black dots). 1 day after the stress procedure, thymuses (A) and adrenal glands (B) were harvested, and the organ mass was determined relative to the body weight. (C) Corticosterone levels were analyzed in the blood plasma via ELISA. (D) 4 h after the stress procedure, mice were tested for exploratory behavior in a light-dark box using an automated tracking device. Cumulative duration in light box is depicted. (E and F) 1 day after the stress procedure, splenocytes were harvested and incubated for 72 h with 1 μg/mL LPS. Supernatants were analyzed for secreted IL-6 (E) and TNF (F) via ELISA. Pooled data from three independent experiments are presented as means ± SD. Statistics: one-way ANOVA followed by Tukey’s multiple comparison test ∗p < 0.05 and ∗∗p < 0.01.

Figure 2

Impacts of chronic stress transmission on physiology (A and C) Male (n = 10 per group) (A) or female (n = 10 per group) (C) C57BL/6 mice were subjected to chronic restraint stress (CRS, red squares), stress transmission (TS, blue triangles), or control treatment (CTRL, black dots) and the change in body weight was monitored from day 1 to day 10 of the stress procedure. Pooled data from five independent experiments are presented as means ± SD. Statistical significance was analyzed by ANOVA followed by Tukey’s multiple comparison test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant. (B and D–H) Male or female C57BL/6 mice (n = 10) were subjected to chronic restraint stress (CRS, red squares), stress transmission (TS, blue triangles), or control treatment (CTRL, black dots). One day after the last stress procedure, adrenal glands (B and D) and thymuses (F and H) were harvested, and the organ mass was determined relative to the body weight. (E and G) Corticosterone levels were analyzed in the blood plasma via ELISA. (B, D, E, F, and G) Pooled data from three independent experiments are presented as means ± SD. Statistics: ANOVA followed by Tukey’s multiple comparison test; ∗p < 0.05, ∗∗p < 0.01 ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant. (I) Thymocytes were analyzed by flow cytometry for CD4 and CD8 populations (n = 8 mice per group (4 female, 4 male)). Absolute cell numbers pooled from two independent experiments are presented as means ± SD. Statistics: two-way ANOVA, followed by Tukey’s multiple comparison test; ∗∗∗∗p < 0.0001; ns, not significant. (J) Representative flow cytometry plot showing thymocyte compositions of CRS and TS mice.

Figure 3

Chronic stress transmission reduces exploratory behavior in mice (A and B) Male (n = 10 per group) and female (n = 10 per group) C57BL/6 mice were subjected to chronic restraint stress (CRS, red squares), stress transmission (TS, blue triangles), or control treatment (CTRL, black dots) for 10 consecutive days. On day 10 of the stress procedure, mice were placed in a light-dark box arena and tested for exploratory behavior for 5 min. The cumulative duration spent in the light box (A) or dark box (B) was quantified by automated video tracking. (C–E) Merged trials per group of one representative experiment are shown as heatmaps. On day 8 of the stress procedure, mice were placed in an open field arena and tested for exploratory behavior for 5 min. The cumulative duration spent in the inner zone (D) or outer area (E) of the maze was quantified by automated video tracking. (F) Merged trials per group of one representative experiment are shown as heatmaps. (A, B, D, and E) Pooled data of five independent experiments are presented as means ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001; ns, not significant. (G) On day 9 of the stress procedure, mice were tested for cognitive performance in a novel object recognition test. Mice were trained for the familiar objects for 10 min. After 5 min, mice were tested for the novel object recognition for 5 min. Data of five independent experiments are presented as ratio of the cumulative durations exploring the novel object and the familiar object is presented. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test; ns, not significant.

Figure 4

Chronic stress transmission induces elevated cytokine secretion upon LPS stimulation Male (n = 5 per group) and female (n = 5 per group) C57BL/6 mice were subjected to chronic restraint stress, stress transmission, or control treatment for 10 consecutive days. On day 11, splenocytes were harvested and stimulated in vitro for 75 h with LPS. Supernatants were screened for cytokine secretion using a flow cytometry based multiplex assay (A). Pooled data of two independent experiments are presented as heatmap plot of fold change relative to controls. TNF (B) or IL-6 (C) levels in the supernatants of chronically restraint stressed (CRS, red squares, n = 10) mice or mice subjected to stress transmission (TS, blue triangles, n = 10) or control treated mice (CTRL, black dots, n = 10) were quantified by ELISA. Data of three independent experiments are presented as means ± SD. Statistical significance was analyzed by one-way ANOVA followed by Tukey’s multiple comparison test; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns, not significant.

Figure 5

Adaptive immune response is not affected by stress transmission (A) C57BL/6 mice (n = 8 per group [4 female, 4 male]) were subjected to chronic restraint stress (CRS, red squares), stress transmission (TS, blue triangles), or control treatment (CTRL, black dots) for 10 consecutive days. On day 5, mice were immunized subcutaneously with 5 mg PLGA MP Ova/Riboxxim. On day 21, bone marrow was harvested and the amount of Ova-specific IgG secreting cells was analyzed by FLUOROSPOT assay (A). Representative wells are shown per group. Pooled data of two independent experiments are presented as means ± SD. (B) Blood serum was analyzed for Ova-specific antibody titers by ELISA. Pooled data of two independent experiments are presented as geometric means ± geometric SD. (C) C57BL/6 mice (n = 10 per group [5 female, 5 male]) were subjected to chronic restraint stress (CRS, red squares), stress transmission (TS, blue triangles), or control treatment (CTRL, black dots) for 10 consecutive days. On day 5, mice were immunized subcutaneously with 5 mg PLGA MP Ova/Riboxxim. On day 11, splenocytes were harvested and Ova-specific CTL responses were analyzed by intracellular cytokine staining of CD8⁺ for IFNγ followed by flow cytometry. (D) Male C57BL/6 mice (n = 5 per group) were subjected to chronic restraint stress (CRS, red squares), stress transmission (TS, blue triangles), or control treatment (CTRL, black dots) for 10 consecutive days. On day 5, mice were double-route immunized subcutaneously with 4 mg and intranasally with 1 mg PLGA MP Ova/Riboxxim or empty PLGA MP (empty, gray diamonds). On day 11, mice were inoculated with 1 × 10⁵ B16-Ova tumor cells intravenously. 21 days post-tumor cell administration, lungs were harvested and formed metastasis were counted. Pooled data of two independent experiments are presented as means ± SD. (E) Representative H&E-stained tissue slides of lungs. Scale bars represent 100 μm. Statistical significance was analyzed by one-way ANOVA followed by Tukey’s multiple comparison test; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns, not significant.