Neutrophils Disrupt B-1a Cell Homeostasis by Targeting Siglec-G to Exacerbate Sepsis

Medical Laboratory Science Infectious Diseases Rheumatology and Immunology Medicine Life Sciences Immunology
Neutrophils B-1a Cells Siglec-G Sepsis Immune Regulation

Research Report: Neutrophils Disrupt B-1a Cell Homeostasis by Targeting Siglec-G, Exacerbating Sepsis

Background

Sepsis is a life-threatening organ dysfunction caused by a dysregulated immune response to infection. The immune system dysfunction accompanying sepsis is primarily triggered by pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), leading to an excessive inflammatory state in the body. B lymphocytes are mainly divided into two subgroups: B-1 cells and B-2 cells. Mouse B-1 cells are further divided into CD5+ B-1a cells and CD5- B-1b cells. B-1a cells are important innate immune cells that play crucial roles in tissue homeostasis and host defense by secreting natural IgM antibodies and IL-10.

In sepsis, the number of B-1a cells in the peritoneal cavity decreases significantly, partly due to their migration to the spleen and transformation into plasma cells, thus altering their phenotype and function. Currently, the regulatory mechanisms of this migration process are not fully understood. Research has indicated that the chemokine CXCL12 and its receptor CXCR4 play a key role in the migration of B-1a cells. Siglec-G (sialic acid-binding immunoglobulin-like lectin) is an immunoregulatory receptor mainly expressed on B-1a cells, which negatively regulates B cell receptor (BCR)-mediated B-1a cell proliferation and activation, and regulates B-1a cell migration through interaction with CXCR4 and CXCL12.

Studies have also emphasized the dual role of neutrophils in sepsis: they rapidly migrate to infection sites to eliminate invading pathogens, but also cause inflammation and organ damage by releasing neutrophil extracellular traps (NETs). However, it remains unclear whether neutrophils promote B-1a cell migration through excessive accumulation.

Research Source

The paper was written by Chuyi Tan, Bridgette Reilly, Gaifeng Ma, Atsushi Murao, Alok Jha, Monowar Aziz, and Ping Wang, from institutions including the Feinstein Institutes for Medical Research in New York, USA, Xiangya School of Medicine, Central South University, and Hunan, China, published in the journal “Cellular & Molecular Immunology” in 2024.

Research Process

Research Process Overview

This study primarily revealed the roles of neutrophils, neutrophil elastase (NE), and Siglec-G in B-1a cell migration and homeostasis maintenance through the following steps:

  1. Sepsis Model Experiments: Establishing a mouse cecal ligation and puncture (CLP) model to evaluate changes in B-1a cell numbers in the peritoneal cavity and spleen of septic mice.
  2. Cell Migration Experiments: Assessing B-1a cell migration and the regulatory role of Siglec-G through in vivo and in vitro experiments.
  3. Biochemical and Computational Biology Analyses: Studying the interactions between Siglec-G and CXCL12/CXCR4 using immunofluorescence and surface plasmon resonance techniques. Additionally, using bioinformatics approaches to predict and identify NE cleavage sites and designing and validating a small decoy peptide to protect Siglec-G from NE cleavage.
  4. Human Experimental Analysis: Evaluating the expression of Siglec-10 (human homolog of Siglec-G) and NE in B-1 cells of sepsis patients and healthy individuals.

Detailed Process

a) Specific Experimental Procedures:

  • Sepsis Model: Inducing sepsis in mice through cecal ligation and puncture. Assessing B-1a cell numbers and Siglec-G expression in the peritoneal cavity and spleen 20 hours post-surgery.
  • Cell Migration and Phenotype Analysis: Injecting dye-labeled B-1a cells into the peritoneal cavity and observing cell migration after 20 hours. Using flow cytometry to detect phenotypic changes of B-1a cells migrated to the spleen.
  • In Vitro Migration Experiments: Using transmembrane chambers to evaluate the migration ability of Siglec-G-deficient and wild-type B-1a cells induced by CXCL12.
  • Biochemical Analysis: Exploring interactions between Siglec-G, CXCL12, and CXCR4 using computational modeling and surface plasmon resonance technology.
  • NE Effects: Studying the cleavage effect of NE on Siglec-G and its impact on B-1a cell migration using neutralizing antibodies and specific inhibitors of neutrophil elastase. Further validating NE’s cleavage of Siglec-G using protein docking and immunoblotting.
  • Small Decoy Peptide Design and Validation: Designing a small protein peptide based on computational models, validating its protective effect on Siglec-G against NE cleavage through in vivo and in vitro experiments, and evaluating its protective effect on septic mice.
  • Human Data Analysis: Analyzing the expression of Siglec-10 and NE in B-1 cells of healthy individuals and sepsis patients using single-cell RNA sequencing data.

b) Results Analysis:

  • Septic Mouse Experiments: Found that under septic conditions, the number of B-1a cells in the mouse peritoneal cavity significantly decreased, while the number in the spleen significantly increased. This change was more pronounced in Siglec-G-deficient mice.
  • In Vitro Migration Experiments: Showed that Siglec-G deficiency significantly enhanced B-1a cell migration ability, supporting the negative regulatory role of Siglec-G in B-1a cell migration.
  • Interaction Studies: Both surface plasmon resonance and computational modeling results showed strong interactions between Siglec-G and CXCL12/CXCR4, indicating that Siglec-G inhibits B-1a cell migration by interfering with the CXCL12-CXCR4 signaling pathway.
  • NE Cleavage Effect: Using computational prediction, biochemical experiments, and protein docking simulations, the cleavage sites of NE on Siglec-G were revealed, and the mechanism by which NE promotes B-1a cell migration by cleaving Siglec-G was verified.
  • Small Decoy Peptide Efficacy: Experimental results showed that the small decoy peptide effectively prevented Siglec-G cleavage by NE, significantly reducing inflammatory responses in septic mice and improving survival rates.
  • Human Data Analysis: In sepsis patients, Siglec-10 expression on B-1 cells was significantly reduced, while NE in neutrophils was significantly increased, supporting the applicability of the experimental findings in humans.

Research Significance and Value

This study reveals a key mechanism by which neutrophils regulate B-1a cell migration through NE-mediated Siglec-G cleavage, providing a new perspective for understanding B-1a cell behavior in sepsis. Additionally, by designing a small decoy peptide to prevent NE cleavage of Siglec-G, the study successfully reduced inflammatory responses and improved survival rates in septic mice, demonstrating a potential therapeutic strategy.

Research Highlights:

  • Important Findings: First revelation of the mechanism by which Siglec-G regulates B-1a cell migration; discovery that NE cleavage is a key factor leading to B-1a cell migration dysregulation in sepsis.
  • Novel Methods: Design of a small decoy peptide that can prevent NE cleavage of Siglec-G, protecting B-1a cell homeostasis and reducing inflammatory responses, demonstrating an entirely new therapeutic approach.

Through this series of experiments, the regulatory role of Siglec-G in B-1a cell migration was preliminarily established, providing potential for new targets and therapies, and offering new directions for the treatment of sepsis and other inflammatory diseases.