Intrinsic Glutathione/Mitochondrial-IL-22 Axis in TH17 Cells and Its Protective Mechanism Against Intestinal Inflammation

Background Introduction

A large amount of reactive oxygen species (ROS) is generated in the intestine, and the role of T-cell antioxidant mechanisms in maintaining intestinal homeostasis remains unclear. This paper studies the impact of specifically deleting glutamate-cysteine ligase (GCLC) in T cells on glutathione (GSH) synthesis and further explores the importance of IL-22 produced by TH17 cells in intestinal protection. Under homeostatic conditions, GCLC deficiency does not alter cytokine secretion; however, after infecting mice with the pathogen Citrobacter rodentium, increased ROS disrupts mitochondrial function and mitochondrial gene expression driven by TFAM, leading to reduced cellular ATP, thereby inhibiting the PI3K/AKT/mTOR pathway and reducing IL-22 translation. Low levels of IL-22 lead to poor bacterial clearance, severe intestinal injury, and high mortality. These findings reveal the crucial role of intrinsic GSH in TH17 cells in promoting mitochondrial function and cellular signaling, vital for intestinal integrity and defense against intestinal infections.

Source Introduction

This paper is jointly completed by Lynn Bonetti, Veronika Horkova, Melanie Grusdat, and several other authors, affiliated with institutions such as the Department of Infection and Immunity at the Luxembourg Institute of Health, the Luxembourg Centre for Systems Biomedicine, the University of Zurich’s Institute of Experimental Immunology, and Yale School of Public Health. The paper was published in the August 6, 2024 issue of the journal “Cell Metabolism,” with an impact factor of 36.

Detailed Research Process

1. Research Process:

a) Experimental Model and Treatment: Using a mouse model with specific deletion of the GCLC gene (cd4cre-gclcfl/fl), the study examines the impact of glutathione on intestinal inflammation. Intestinal symptoms were induced by infection with Citrobacter rodentium, with mice grouped into infected and uninfected groups, and experiments conducted on day 7 and day 12 post-infection.

b) Cellular and Molecular Analysis: - Antioxidant Response: Flow cytometry analysis of thiol levels in CD4+ T cells from the lamina propria of infected mice, to detect increases in intracellular glutathione (GSH). - Mitochondrial Function: Assessment of mitochondrial ROS levels and their impact on ATP production, using oxygen consumption rate (OCR) to measure mitochondrial activity, and analyzing the impact of ROS on mitochondrial electron transport chain (ETC) related gene expression.

c) Cytokine Analysis: Detection of IL-17 and IL-22 expression in CD4+ T cells in the intestines of differently grouped mice, with particular attention to changes under infection conditions.

d) Antioxidant Treatment: Administering antioxidant NAC (N-acetylcysteine) in drinking water to evaluate its role in restoring mitochondrial function and IL-22 production in GCLC-deficient mice.

e) Cell Differentiation Experiment: In vitro culture for differentiation of TH17 cells to verify the mechanism by which IL-17 and IL-22 production is influenced by GSH.

f) Gene Expression Recovery Experiment: Using transgenic mouse models carrying promoters for IL-22 or IL-17 expression to further elucidate the role of GCLC loss in TH17 cells during intestinal infections.

2. Research Findings:

a) Relationship Between Antioxidant Response and Mortality: Flow cytometry data show increased GSH levels in T cells post-infection, with ROS maintained at low levels. GCLC deficiency leads to reduced ROS buffering capacity, increasing mortality and intestinal damage in infected mice.

b) Role of IL-22 in Bacterial Clearance: GCLC deficiency reduces IL-22 production and impairs bacterial clearance. NAC administration effectively restores ROS levels and mitochondrial function in GCLC-deficient mice, maintaining IL-22 production.

c) Intestinal Damage and Protective Mechanism: - Intestinal Structural Damage: GCLC-deficient mice show significant intestinal ulceration and shortening, severe crypt loss, and epithelial necrosis post-infection. - Recovery Mechanism: Treatment with antioxidant NAC and recombinant IL-22 effectively restores intestinal structure in GCLC-deficient mice, demonstrating the critical role of IL-22 in maintaining intestinal integrity.

d) Regulation of Gene Expression and Mechanism: RNA sequencing shows that GCLC deficiency leads to decreased activity of the PI3K/AKT/mTOR pathway, affecting mitochondrial gene TFAM expression, reducing ATP levels, and ultimately inhibiting IL-22 protein translation rather than its mRNA transcription.

e) Clinical Relevance Study: Analysis of samples from human IBD patients shows a positive correlation between GCLC expression and the expression of genes related to intestinal integrity, particularly IL-22 and Claudin (tight junction protein).

Research Conclusions and Significance

This research reveals the critical role of the intrinsic GSH/mitochondrial-IL-22 axis in TH17 cells in protecting the intestine from bacterial infection, uncovering the link between ROS regulation, mitochondrial function, and IL-22 protein translation. It provides new insights into T cell antioxidant mechanisms in intestinal inflammation and a theoretical basis for exploring new strategies for IBD treatment, such as improving TH17 cell function through the regulation of antioxidant use to protect intestinal integrity. Moreover, the study suggests that fine regulatory mechanisms on mitochondria may have broader significance in more cellular functions and diseases, warranting further exploration.

Research Highlights

1. Innovative Discovery: The study reveals for the first time how glutathione (GSH) within TH17 cells regulates mitochondrial ROS levels to influence IL-22 protein production, safeguarding intestinal integrity.
2. Systematic Experimental Design: From mouse models to in vitro cell experiments and clinical sample analysis, the research establishes a comprehensive scientific methodological framework.
3. Clinical Relevance: Human IBD sample genetic analysis confirms the correlation between GCLC and the expression of genes related to intestinal integrity, supporting the potential clinical application of the research findings.

This study highlights the complexity of antioxidant mechanisms in the immune system and their importance to disease and health states, providing a wealth of information and new perspectives for future IBD research and treatment.