Repeated Low-Dose Lipopolysaccharide Pretreatment Improves Behavioral Abnormalities and Aberrant Immune Responses Induced by Repeated Social Defeat Stress

Background

Stress significantly impacts human health, with chronic stress particularly inducing various physiological and psychological disorders. The hypothalamic-pituitary-adrenal (HPA) axis is the main neuroendocrine system, secreting glucocorticoids such as corticosterone and cortisol to regulate stress responses. However, prolonged chronic stress exposure disrupts HPA axis regulatory function, affecting glucocorticoid negative feedback mechanisms and leading to physiological dysfunction. Concurrently, glucocorticoids regulate the immune system, including immunomodulation of inflammatory responses. These stress hormones stimulate immune cells in vitro and enter the central nervous system through the blood-brain barrier or central nervous system second messenger signaling pathways, activating resident immune cells, especially microglia. Numerous studies have shown that chronic inflammation is closely related to brain dysfunction, including emotions, cognitive function, and behavioral patterns.

Recent research has found that low-dose lipopolysaccharide (LPS) pretreatment can induce a tolerant state, reconfiguring the inflammatory response to reduce the production of inflammatory factors. LPS tolerance has shown neuroprotective effects in some neurological disease models, but its preventive role in affective disorders has not been fully elucidated.

Research Source

This paper titled “Repetitive Administration of Low-Dose Lipopolysaccharide Improves Repeated Social Defeat Stress-Induced Behavioral Abnormalities and Aberrant Immune Response” was jointly completed by scholars Vichuda Charoensaensuk, Wei-Lan Yeh, et al., and published in the Journal of Neuroimmune Pharmacology 2024, Volume 19, Page 38. The paper aims to explore the effects of LPS pretreatment on behavioral abnormalities and aberrant immune responses in mice subjected to repeated social defeat stress (RSDS) and its potential mechanisms.

Research Process

This study used eight-week-old male C57BL/6 mice and twenty-five-week-old male CD-1 (ICR) mice. C57BL/6 mice were divided into experimental and control groups and raised under standard laboratory conditions. All animal experiments followed the animal care and use guidelines of China Medical University.

Animal Treatment and RSDS Procedure

C57BL/6 mice were pretreated with intraperitoneal injections of 0.5 mg/kg LPS once daily for 4 consecutive days to induce endotoxin tolerance. Subsequently, mice underwent a 6-day RSDS procedure. The RSDS procedure was conducted according to standard operating procedures, placing several C57BL/6 mice in different ICR aggressor cages for 2 hours of physical contact daily, returning to their own cages for 22 hours after contact.

Behavioral Tests and Sample Collection

On day 11 after the RSDS procedure, social interaction tests, elevated plus maze tests, and light-dark box tests were conducted, and serum and brain tissue samples were collected for further analysis. In another experiment, brain, blood, spleen, and bone marrow samples were collected within 12-13 hours after the RSDS procedure for leukocyte analysis.

Cell Culture and Endotoxin Tolerance Induction

Adult mouse brain microglia (IMG) and neonatal mouse brain microglia (BV2) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. To induce cellular endotoxin tolerance, cells were treated with 10 ng/ml LPS for 20 hours, then treated with 500 ng/ml recombinant mouse lipocalin 2 (LCN2) protein for 12 hours before mRNA extraction.

Research Results

LPS Tolerance Alleviates RSDS-Induced Social Avoidance Behavior

LPS pretreatment significantly prolonged the time RSDS mice spent in the interaction zone when the target was present, while reducing time spent in the corner zone. This indicates that LPS tolerance can alleviate social avoidance behavior in RSDS mice, with no significant effect on social interaction in normal mice.

LPS Tolerance Improves RSDS-Induced Anxiety Behavior

Anxiety behavior was tested through elevated plus maze and light-dark box experiments. Results showed that LPS pretreatment significantly increased the time RSDS mice spent in open arms and light areas, and reduced time in closed arms and dark areas, indicating that LPS tolerance can alleviate RSDS-induced anxiety behavior.

LPS Tolerance Affects Stress-Related Proteins in the Hippocampus of RSDS Mice

Expression levels of monoamine oxidase (MAO-A and MAO-B) and glucocorticoid receptor (GR) in the hippocampus of RSDS mice changed significantly. LPS pretreatment significantly reduced the expression of MAO-A and MAO-B, and increased GR expression, indicating that LPS tolerance improves social avoidance and anxiety behaviors by regulating stress-related proteins.

LPS Pretreatment Regulates Peripheral Inflammatory Monocyte Infiltration into the Brain via the CCL2-CCR2 Axis

Flow cytometry analysis revealed a significant increase in CD11b+CD45hi peripheral leukocytes and CD11b+CD45hily6chiccr2+ inflammatory monocytes in the brains of RSDS mice, while LPS pretreatment significantly reduced the number of these cells. Additionally, LPS pretreatment reduced the role of the CCL2/CCR2 axis in peripheral inflammatory monocyte infiltration into the brain, suggesting that LPS tolerance can alleviate RSDS-induced inflammation and behavioral abnormalities through this axis.

LPS Tolerance Inhibits Lipocalin 2 (LCN2)-Induced Microglial Activation and CCL2 Expression

LPS pretreatment significantly reduced the activation state of microglia and LCN2 expression in the brains of RSDS mice. In vitro experiments also showed that LPS pretreatment effectively inhibited LCN2-induced overexpression of CCL2, IL-6, IL-1β, and TNF-α in microglia, indicating that LCN2 plays an important regulatory role in microglial activation, and LPS tolerance can improve RSDS-induced immune abnormalities by regulating LCN2 expression.

LPS Tolerance Promotes Endogenous Antioxidant Levels in the Hippocampus of RSDS Mice

The study further found that the expression of endogenous antioxidants such as GCLM, GCLC, NQO1, and anti-inflammatory phenotype marker ARG1 in the hippocampus of RSDS mice was significantly reduced, while LPS pretreatment significantly increased the levels of these antioxidant proteins. This indicates that LPS tolerance improves social avoidance and anxiety behaviors by promoting the production of endogenous antioxidants and inhibiting inflammatory responses.

Research Conclusion

This study demonstrates that repeated low-dose LPS pretreatment can protect mice from RSDS-induced behavioral abnormalities and immune response aberrations through multiple mechanisms. The results support that LPS tolerance protects mice from RSDS-induced abnormal immune responses and behavioral changes by regulating the interaction between the immune system and the central nervous system, reducing peripheral inflammatory cell infiltration into the brain, and maintaining the balance between inflammation and anti-inflammation in the brain and immune system.