CD137L Inhibition Ameliorates Hippocampal Neuroinflammation and Behavioral Deficits in a Mouse Model of Sepsis-Associated Encephalopathy

Neurology Infectious Diseases Medicine Cell Biology Life Sciences Immunology
CD137L microglia inflammation anxiety cognition TKS-1

Background Introduction

Sepsis is a life-threatening multiple organ dysfunction syndrome caused by uncontrolled infection in the host, and is one of the leading causes of death in intensive care units. It is reported that there are approximately 189 adult sepsis hospitalization cases per 100,000 person-years, with a mortality rate of 26.7%. Sepsis-Associated Encephalopathy (SAE) is a central brain dysfunction caused by peripheral or systemic inflammatory infection. About 70% of severe sepsis patients develop SAE, which increases mortality, prolongs hospital stay, and leads to excessive medical resource consumption. Its symptoms range from delirium to coma, and early diagnosis and intervention are crucial for treating sepsis patients. However, the pathological mechanism of SAE has not been fully elucidated.

Microglia are resident macrophages in the central nervous system (CNS) and are closely related to the pathogenesis of SAE. Uncontrolled neuroinflammation is the main feature of SAE and the main cause of brain dysfunction and neuronal death in this condition. Studies have shown that microglial activation during SAE is closely associated with emotional distress, anxiety, depression, and cognitive impairment. In sepsis animal models, microglia are typically activated by lipopolysaccharide (LPS) stimulation and play a key role in mediating SAE-related behavioral changes. The activation state of microglia can be divided into two main phenotypes: M1 and M2. M1 microglia produce pro-inflammatory factors associated with tissue and neuronal damage, while M2 microglia produce anti-inflammatory factors that promote tissue and neuronal repair.

CD137L (TNFSF9, 4-1BBL) is a transmembrane glycoprotein belonging to the tumor necrosis factor (TNF) ligand family and plays an important role in adaptive immune responses. Studies have shown that blocking CD137L signaling can reduce the release of neuroinflammatory mediators, and neurodegenerative diseases hardly occur in CD137L-deficient mice. In this context, the authors hypothesized that the CD137 receptor/ligand system affects SAE-induced neuroinflammation and cognitive behavioral disorders by regulating the polarization state of microglia.

Paper Source

This research paper was written by Fang Qiu et al., with authors from multiple Chinese research institutions, including Shenzhen Longhua District Central Hospital, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong Medical University, and others. The paper was published in the journal “Neuromolecular Medicine” on October 5, 2023.

Research Process

Establishment of Sepsis-Associated Encephalopathy Mouse Model

The research strictly followed the ethical guidelines for animal research and was approved by the Animal Care and Use Committee of Guangdong Medical University. Adult male C57BL/6 mice were used, and the SAE model was established by intraperitoneal injection of LPS (2 mg/kg). LPS injection was performed 24 hours before the start of behavioral tests. Mice were divided into three groups: control group (Ctrl), LPS-treated group (LPS), and LPS plus TKS-1 treated group (LPS+TKS-1). Mice in the LPS+TKS-1 group were treated with CD137L neutralizing antibody TKS-1 (200 µg).

Behavioral Tests

All behavioral tests were conducted in a constant temperature room during the light cycle, with experimenters and analysts blinded to the experimental grouping. Behavioral tests included open field test (OFT), elevated plus maze test (EPM), and Y-maze spontaneous alternation behavior test to assess anxiety-like behavior and spatial memory ability in mice.

Cell Culture and Treatment

The BV2 microglial cell line used in the experiment was from Shenzhen University. To verify the role of the CD137L/CD137 signaling axis in microglia, recombinant CD137-Fc fusion protein and CD137L neutralizing antibody TKS-1 were used in cell experiments.

Flow Cytometry

The effect of LPS on CD137L expression in BV2 cells was detected by flow cytometry. Cells were grouped as follows: control group (Ctrl), LPS-treated group (LPS), and TKS-1+LPS treated group (TKS-1+LPS). Subsequently, detection was performed using secondary antibodies labeled with appropriate Alexa Fluor dyes.

Enzyme-Linked Immunosorbent Assay (ELISA)

The culture supernatant of BV2 cells and mouse hippocampal tissue were stored at -80°C for processing. ELISA kits were used to detect cytokine and protein expression levels.

Immunofluorescence Staining

Immunofluorescence staining was used to detect the expression and co-localization of Iba1, CD137L, TNF-α, and IL-6 in the hippocampus of experimental group mice.

Real-Time Quantitative PCR (RT-qPCR)

Total RNA was extracted from mouse hippocampus or BV2 cells and subjected to RT-qPCR detection. RT-qPCR was used to determine the relative expression levels of microglial activation marker genes and inflammatory factors.

Data Analysis

All data were analyzed using GraphPad Prism 7 software. Comparisons between two groups were made using two-tailed t-tests, while comparisons among multiple groups were made using one-way analysis of variance (ANOVA).

Main Results

The study found that CD137L expression was significantly upregulated in the hippocampus of mice after LPS treatment and was notably expressed in microglia. Meanwhile, pretreatment with CD137L neutralizing antibody TKS-1 significantly reduced CD137L levels, inhibited the expression of pro-inflammatory factors (such as TNF-α, IL-1β, and IL-6), and suppressed M1 polarization of microglia. Additionally, CD137-Fc fusion protein activated the synthesis and release of pro-inflammatory factors in BV2 microglia. Behaviorally, administration of TKS-1 significantly alleviated LPS-induced anxiety-like behavior and spatial memory decline.

These results suggest that the upregulation of CD137L in microglia is involved in LPS-induced neuroinflammation, anxiety-like behavior, and cognitive dysfunction. Therefore, modulating the CD137L/CD137 signaling pathway may be an effective approach to reduce sepsis-associated brain injury and prevent cognitive and emotional disorders.

Research Significance

This study reveals the key role of CD137L in LPS-induced sepsis-associated encephalopathy by regulating microglial polarization state, affecting neuroinflammation and cognitive behavioral disorders. This finding provides a new perspective for the treatment of SAE, especially by targeting the CD137L signaling pathway, which may provide an important theoretical basis and clinical application prospect for the clinical treatment of SI and SAE.

Research Highlights

  1. Important Discovery: First demonstration of the crucial role of CD137L in sepsis-associated encephalopathy.
  2. High Impact: The research results have significant implications for understanding and treating sepsis-associated encephalopathy.
  3. Innovative Approach: The CD137L neutralizing antibody TKS-1 used in the experiment showed effectiveness in targeting neuroinflammation, proposing a new therapeutic approach.

Through this study, scientists have further understood the mechanism of microglial action in sepsis-associated encephalopathy, providing important experimental data and theoretical support for developing effective methods to treat SAE in the future.