Restraint Stress-Induced Neutrophil Inflammation Contributes to Concurrent Gastrointestinal Injury in Mice

Abstract

Psychological stress increases risk of gastrointestinal tract diseases. However, the mechanism behind stress-induced gastrointestinal injury is not well understood. The objective of our study is to elucidate the putative mechanism of stress-induced gastrointestinal injury and develop an intervention strategy. To achieve this, we employed the restraint stress mouse model, a well-established method to study the pathophysiological changes associated with psychological stress in mice. By orally administering gut-nonabsorbable Evans blue dye and monitoring its plasma levels, we were able to track the progression of gastrointestinal injury in live mice. Additionally, flow cytometry was utilized to assess the viability, death, and inflammatory status of splenic leukocytes, providing insights into the stress-induced impact on the innate immune system associated with stress-induced gastrointestinal injury. Our findings reveal that neutrophils represent the primary innate immune leukocyte lineage responsible for stress-induced inflammation. Splenic neutrophils exhibited elevated expression levels of the pro-inflammatory cytokine IL-1, cellular reactive oxygen species, mitochondrial burden, and cell death following stress challenge compared to other innate immune cells such as macrophages, monocytes, and dendritic cells. Regulated cell death analysis indicated that NETosis is the predominant stress-induced cell death response among other analyzed regulated cell death pathways. NETosis culminates in the formation and release of neutrophil extracellular traps, which play a crucial role in modulating inflammation by binding to pathogens. Treatment with the NETosis inhibitor GSK484 rescued stress-induced neutrophil extracellular trap release and gastrointestinal injury, highlighting the involvement of neutrophil extracellular traps in stress-induced gastrointestinal inflammation. Our results suggest that neutrophil NETosis could serve as a promising drug target for managing psychological stress-induced gastrointestinal injuries.

Keywords: NETosis; activating transcription factor 3; gastrointestinal injury; neutrophil extracellular trap; restraint stress.

Figure 1

Restraint stress-induced gastrointestinal (GI) leakage is associated with the infiltration of innate immune leukocytes in the spleen of mice. (A) Experiment outline. (B) Plasma Evans blue levels, indicating the stress-induced GI leakage and injury levels. ND: not detected. (C) An example of the flow cytometry gating of Ly6G⁺ leukocytes after 9 h stress; the same method is applied to the analyses in (D). In (C), the letters A and C represent the gated total live splenic leukocytes and the LY6G⁺ cell population, respectively. (D) Relative cell-number levels of different leukocyte lineages (Ly6G⁺ neutrophil, F4/80⁺ macrophage, CD11b⁺ monocyte, CD11c⁺ dendritic cell) without (no stress groups) and with (stress groups) stress in the spleen of mice. The levels of respective leukocyte lineages in the no stress groups were normalized to one-fold. (B,C) * p > 0.05, ** p > 0.01, vs. no stress groups. n = 6 (3 experiments with a total of 6 mice per group). The mouse graph was created with BioRender.com, accessed on 1 January 2024.

Figure 2

Restraint stress on the regulation of the cytokine expression of splenic innate immune leukocytes in mice. (A) Experiment outline. Blue: no stress groups; Red: stressed groups. (B) Relative levels of spleen splenocytes expressing pro-inflammatory cytokines IL-1β, TNF-α, IL-6, and anti-inflammatory cytokine IL-10, in mice with (stress groups) and without (no stress groups) 9 h stress. The levels of respective leukocyte lineages in the no stress groups were normalized to one-fold. (C) An example of flow cytometry gating for analyzing IL-1 expression in Ly6G⁺ leukocytes; the same method is applied to the analyses in (C–F). (C–F) Relative expression levels of IL-1β (D), TNF-α (E), IL-6 (F), IL-10 (G) in different leukocyte lineages (Ly6G⁺ neutrophil, F4/80⁺ macrophage, CD11b⁺ monocyte, CD11c⁺ dendritic cell) without (no stress groups) and with (stress groups) stress. The expression percentage of tested cytokines was normalized to a baseline value of 1 for the respective leukocyte lineage population in the no stress groups. (B,D–G) * p > 0.05, ** p > 0.01, vs. no stress groups; n = 6 (3 experiments with a total of 6 mice per group). The mouse graph was created with BioRender.com, accessed on 1 January 2024.

Figure 3

Relative levels of cell death, cellular ROS, and mitochondrial superoxide of splenic innate immune leukocytes. (A–D) Relative percentage of live and death cells of innate immune splenocytes, including (A) Ly6G⁺ neutrophil, (B) F4/80⁺ macrophage, (C) CD11b⁺ monocyte, (D) CD11c⁺ dendritic cell, in mice with (stress + groups) and without (stress − groups) stress. The levels of respective leukocyte lineages in the no stress groups were normalized to one-fold. (E–G) Relative levels of cellular ROS (E), and mitochondrial superoxide (F) without (no stress groups) and with (stress groups) stress in mice were analyzed. (E,F) The levels of respective leukocyte lineages in the no stress groups were normalized to one-fold. (A–G) * p > 0.05, ** p > 0.01, vs. respective no stress groups; n = 6 (3 experiments with a total of 6 mice per group). The mouse graph was created with BioRender.com, accessed on 1 January 2024.

Figure 4

Restraint stress-induced cell death of splenic neutrophil in wild-type (ATF3^+/+) and ATF3 deficient (ATF3^−/−) mice. (A) Experiment outline. (B) Live and death cell percentage of splenic neutrophil from ATF3^+/+ and ATF3^−/− mice without (stress − groups) and with (stress + groups) stress were analyzed by flow cytometry using Zombie NIR live/death analysis kit. Light blue represents the live cell population, while pink represents the dead cell population. (C–H) The regulated cell death (RCD) profiles, denoted by the alterations in the percentage of RCD-signal positive neutrophils in ATF3^+/+ and ATF3^−/− mice under unstressed (stress − groups) and stressed (stress + groups) conditions, were assessed. The percentages of neutrophil RCD-marker⁺ cells, including NETosis (C), apoptosis (D), autophagy (E), ferroptosis (F), necroptosis (G), and pyroptosis (H), were delineated. In each group, the signal from wild-type mice was normalized to one-fold; n = 6 (3 experiments with a total of 6 mice per group); * p < 0.05, ** p < 0.01, significantly higher vs. without stress control groups; # p < 0.05, significantly higher vs. wild-type (WT) control groups. The mouse graph was created with BioRender.com, accessed on 1 January 2024.

Figure 5

Restraint stress-induced changes in splenic neutrophil in wild-type (ATF3^+/+) and ATF3 deficient (ATF3^−/−) mice. (A) Relative cell abundance, (B) mitochondrial mass, (C) mitochondrial membrane potential, and (D) cellular ROS levels in the spleen neutrophils of ATF3^+/+ and ATF3^−/− mice, with (stress + groups) and without (stress − groups) stress, were analyzed by flow cytometry. Blue denotes the no-stress groups, while red indicates the stressed groups. n = 6 (3 experiments with a total of 6 mice per group); * p < 0.05, vs. vehicle control (0 g/mL) groups; # p < 0.05, significantly higher vs. wild-type (WT) control groups.

Figure 6

Treatments of NETosis inhibitor GSK484 rescued restraint stress-induced GI leakage and pro-inflammatory changes in splenic neutrophil in mice. (A) Experiment outline. (B) Plasma Evans blue levels, (C) neutrophil cell death levels, (D) neutrophil NETosis levels, in the spleen of mice, with (stress + groups) or without (stress − groups) stress, and with (GSK484 + groups) or without (GSK484 − groups) GSK484 treatments, were analyzed; n = 6 (3 experiments with a total of 6 mice per group); # p < 0.05, ## p < 0.01, exacerbated vs. no stress control groups; * p < 0.05, ** p < 0.01, rescued vs. vehicle control (0 g/mL, without GSK484 treatments) groups. (B) ND: not detected. (C) Light blue represents the live cell population, while pink represents the dead cell population. (D) The mouse graph was created with BioRender.com, accessed on 1 January 2024.