Treg-specific AMPKα1 deficiency alters immune cell compositions in immune organs of mice

Abstract

AIM: Regulatory T cells (Tregs) are a specialized subset of CD4+ T cells primarily involved in immunosuppressive functions. AMP-activated protein kinase (AMPK) serves as a metabolic sensor that governs the differentiation, maturation, and immune functions of Tregs through metabolic reprogramming. However, the impact of AMPKα1 (the catalytic subunit of AMPK) knockout specifically in Tregs on the host's immune microenvironment remains largely unexplored. METHODS: Histological changes in immune organs were assessed using HE staining. The types of immune cells and their relative population percentages in immune organs and blood were quantified through flow cytometry in both AMPKα1flox/flox (AMPKα1fl/fl) mice and Treg-specific AMPKα1 knockout mice (AMPKα1fl/flFoxp3cre mice). RESULTS: Compared to AMPKα1fl/fl mice, the percentage of eosinophils in the bone marrow of AMPKα1fl/flFoxp3cre mice was significantly reduced. Additionally, while the thymus of AMPKα1fl/flFoxp3cre mice exhibited normal structure, both its size and the ratio of thymus weight to body weight were significantly decreased. The knockout of AMPKα1 in Tregs led to a notable reduction in the total percentage of immature double-negative (DN) cells. Consequently, the percentage of CD4+ T cells derived from these DN cells also decreased, even though the percentages of DN1 and DN4 cells were higher in the thymus of AMPKα1fl/flFoxp3cre mice compared to AMPKα1fl/fl mice. Importantly, the proportion of Siglec-F+ CD11b+ eosinophils in the thymus was significantly lower in AMPKα1fl/flFoxp3cre mice. Knockout of AMPKα1 in Tregs resulted in a marked increase in the percentage of CD4+ T cells in peripheral blood, alongside a decrease in the proportion of mature CD8+ T cells. Similarly, the proportion of CD4+ T cells in the spleen of AMPKα1fl/flFoxp3cre mice was elevated compared to AMPKα1fl/fl mice. In contrast, the proportion of neutrophils significantly decreased, while mononuclear cell proportions increased in the spleen of AMPKα1fl/flFoxp3cre mice. In lymph nodes, the medullary boundaries in AMPKα1fl/flFoxp3cre mice were blurred, and the lymphoid follicles were missing, a feature not observed in AMPKα1fl/fl mice. Furthermore, the knockout of AMPKα1 in Tregs reduced the CD3+ T cell population, particularly the CD8+ T cell population, in lymph nodes. Although the mature Treg cell population was significantly lower in AMPKα1fl/flFoxp3cre mice, the percentage of CD4+ T cells was markedly increased. In contrast, there was no statistically significant difference in granulocyte populations between AMPKα1fl/flFoxp3cre and AMPKα1fl/fl mice. CONCLUSION: The populations of mature Tregs, CD8+ T cells and eosinophils in various immune organs were significantly altered in mice with Treg-specific AMPKα1 knockout, suggesting a potential remodeling of the host immune microenvironment in response to inflammatory stimuli.

1. Introduction

Regulatory T cells (Tregs) are an immunosuppressive subset of CD4+ helper T cells that are involved in the maintenance of self-tolerance, immune homeostasis, and repair functions [1-3]. The transcription factor forkhead box P3 (Foxp3) is required for the differentiation of naïve CD4+ T cells [4] and is essential for Treg function and development [5]. Tregs develop in the thymus and are exported throughout the body through blood circulation; consequently, they regulate local immune microenvironments by suppressing the activation and proliferation of potentially autoreactive T cells [6].

AMP-activated kinase (AMPK) is a highly conserved serine/threonine protein kinase that acts as an energy sensor and metabolic regulator to maintain cellular energy homeostasis [7-8]. Low bioenergetic resource levels or high intracellular AMP levels in cells activate AMPK to inhibit the mammalian target of rapamycin (mTOR) to block cell expansion and induce a shift toward catabolic metabolism and oxidative phosphorylation [9].

The metabolism of immune cells is fundamental to their survival and function. Naïve T cells rely on ATP produced via oxidative phosphorylation (OXPHOS) to maintain their survival and basic functionality [10-11] in the resting state, while activated naïve T cells initiate metabolic reprogramming that triggers rapid glucose uptake and promote a cellular metabolic transition from OXPHOS to aerobic glycolysis [12], and robust activation of initial CD4+ T cells lead to their rapid proliferation and differentiation into the effector T (Teff) and Treg subpopulations. Importantly, Tregs primarily use fatty acid and pyruvate oxidation (mitochondrial oxidative metabolism) for energy production [13], while effector T cells use mTORC1-AKT-driven glycolysis pathways [14]. AMPK activity is substantially greater in Tregs than in Teff cells, and the activation state of AMPK has been used to phenotypically characterize and distinguish Tregs.