Restraint Stress-Induced Immunosuppression Is Associated with Concurrent Macrophage Pyroptosis Cell Death in Mice

Abstract

Psychological stress is widely acknowledged as a major contributor to immunosuppression, rendering individuals more susceptible to various diseases. The complex interplay between the nervous, endocrine, and immune systems underlies stress-induced immunosuppression. However, the underlying mechanisms of psychological-stress-induced immunosuppression remain unclear. In this study, we utilized a restraint stress mouse model known for its suitability in investigating physiological regulations during psychological stress. Comparing it with cold exposure, we observed markedly elevated levels of stress hormones corticosterone and cortisol in the plasma of mice subjected to restraint stress. Furthermore, restraint-stress-induced immunosuppression differed from the intravenous immunoglobulin-like immunosuppression observed in cold exposure, with restraint stress leading to increased macrophage cell death in the spleen. Suppression of pyroptosis through treatments of inflammasome inhibitors markedly ameliorated restraint-stress-induced spleen infiltration and pyroptosis cell death of macrophages in mice. These findings suggest that the macrophage pyroptosis associated with restraint stress may contribute to its immunosuppressive effects. These insights have implications for the development of treatments targeting stress-induced immunosuppression, emphasizing the need for further investigation into the underlying mechanisms.

Keywords: ambient cold exposure; cell death; immunosuppression; intravenous immunoglobulin; macrophage; psychological stress; pyroptosis; restraint stress.

Figure 1

Restraint stress resulted in the development of anxiety-like behaviors in mice. During the 20 h stress procedure, both the no stress and stress groups were deprived of access to food and water. After the termination of restraint stress, both the unstressed and stressed groups of mice were given access to food and water for a period of 2 h to restore their resources. Subsequently, the OFT was conducted. Representative video tracking images captured during a 5-min OFT are presented (A). A comparison was made between the control group (no stress) and the restraint stress group (stress) for total distance traveled (B), distance traveled in the central zone (C), and time spent in the central area (D). The number of samples used for analysis was 3 (n = 3). * indicates statistical significance at p < 0.05, ** indicates statistical significance at p < 0.01, when compared to their respective no stress groups.

Figure 2

Treatments of IVIg, cold exposure, and restraint-stress-induced immunosuppression as indicated by the amelioration of anti-CD41 Ig (an antiplatelet Ig)-induced immune thrombocytopenia (ITP). (A–C) IVIg (+IVIg) vs. vehicle (−IVIg) treatments. (D–F) Cold exposure (4 °C) vs. room temperature (25 °C). (G–I) Restraint stress (+stress) vs. no stress (−stress). (A,D,G) Experiment outlines. (B,E,H) Experiments employed wild-type (WT) mice. (C,F,I) Experiments employed P-selectin deficient mice (Selp^−/−) mice. * p < 0.05, ** p < 0.01, vs. respective 0 h groups; # p < 0.05, vs. respective no treatment groups [e.g., vehicle (−IVIg) vs. IVIg; 25 °C vs. 4 °C; no stress (−stress) vs. stress]. n = 6 (three experiments with total six mice per group).

Figure 3

Treatments of IVIg, cold exposure, and restraint-stress-induced immunosuppression as indicated by the suppressed bacterial clearance. (A,B) IVIg (+IVIg) vs. vehicle (−IVIg) treatments. (C,D) Cold exposure (4 °C) vs. room temperature (25 °C). (E,F) Restraint stress (+stress) vs. no stress (−stress). (A,C,E) Experiment outlines. (B,D,F) Viable bacterial levels (CFU/g spleen). Wild-type (WT); P-selectin deficient mice (Selp^−/−). * p < 0.05, ** p < 0.01, vs. respective no treatment groups [e.g., vehicle (−IVIg) vs. IVIg; 25 °C vs. 4 °C; no stress (−stress) vs. stress]. n = 6 (three experiments with total six mice per group).

Figure 4

Time-dependent increase in GI leakage, splenic macrophage infiltration, and stress hormone levels in response to the stress in experimental mice. (A,D) Experimental outlines. (B) Plasma levels of Evans blue. (C) Levels of F4/80⁺ macrophages in the spleen. (E) Plasma corticosterone levels. (F) Plasma cortisol levels. * p < 0.05, compared to respective 0 h untreated groups; # p < 0.05, compared to respective no stress (−stress) groups. The study involved six mice per group, with three experiments conducted in total (n = 6).

Figure 5

Treatments of IVIg, cold exposure, and restraint stress suppressed the phagocyte function as indicated by the suppression of the engagement between splenic macrophage and fluorescent nanoparticles (NPs). (A) Experiment outline. (B) Percentage of double-labeled (cell fluorescence + NP fluorescence) cells. * p < 0.05, ** p < 0.01, vs. respective 0 h groups; # p < 0.05, ## p < 0.01, vs. respective no treatment groups [e.g., vehicle (−IVIg) vs. IVIg; 25 °C vs. 4 °C; no stress (−stress) vs. stress]. n = 6 (three experiments with total six mice per group).

Figure 6

Treatments of IVIg, cold exposure, and restraint stress on the induction of cell death in the population of total splenocytes and splenic macrophages. (A) Experiment outline. (B) Percentage cell death of total splenocytes. (C) Percentage cell death of F4/80⁺ splenic macrophages. * p < 0.05, vs. respective 0 h groups; # p < 0.05, vs. respective no treatment groups [e.g., 25 °C vs. 4 °C; no stress (−stress) vs. stress]. n = 6 (three experiments with total six mice per group).

Figure 7

Treatments with NLRP3 inflammasome inhibitors OLT1177 and Z-WEHD effectively rescued restraint-stress-induced spleen infiltration, IL-1β expression, and pyroptotic cell death of spleen macrophages. (A) Experiment outline, (B–D) OLT1177 treatments, and (E–G) Z-WEHD-FMK (Z-WEHD) treatments are indicated. (B,E) Relative IL-1β expression levels of F4/80+ spleen macrophages. (C,F) Relative pyroptosis levels of F4/80⁺ spleen macrophages. (D,G) Relative macrophage infiltration levels in the mouse spleen. * p < 0.05, vs. respective 0 h untreated groups; # p < 0.05, vs. ## p < 0.01, respective no stress (–stress) groups; + p < 0.05, vs. respective vehicle groups. n = 6 (three experiments with total six mice per group).