Low-dose fluoride exposure disrupts CD4+ T cell balance in humans and rats.

Abstract

Introduction

Fluorine, a widely distributed natural element, exerts various biological effects on organisms. The primary human exposure routes include drinking water, food, and air, with groundwater being the predominant source of fluoride intake (Taher et al., 2024; Vasisth et al., 2024). Excessive fluoride intake has been widely confirmed to cause damage to both skeletal and non-skeletal tissues in the human body (Pal et al., 2023; Solanki et al., 2022). At present, the impact of fluoride on non-skeletal tissues has received extensive attention from researchers. Long-term or excessive fluoride exposure can cause various acute and chronic diseases, including organ damage (e.g., to the heart and liver) and neurological impairments (e.g., brain injury) (Wang et al., 2023a, 2023b; Hou et al., 2024). Many countries worldwide have implemented measures to reduce fluoride concentrations in water (Arab et al., 2024). However, emerging evidence indicates that systemic damage can occur even when water fluoride levels meet the World Health Organization (WHO)-recommended limit of <1.5 mg/L (Wang et al., 2021). Low-fluoride exposure [humans: <1.5 mg/L, rats: <20.9 mg/L sodium fluoride (NaF), mice: <30.2 mg/L NaF, cells: <3.9 uM NaF] similarly induces skeletal tissue damage, such as dental fluorosis. It also adversely affects multiple systems, including cardiovascular, nervous, hepatic/renal, reproductive, thyroid, and immune functions (Zhou et al., 2023). However, current population-based and animal studies on low-fluoride exposure remain limited, and the underlying mechanisms are inadequately explored.

During the aforementioned tissue damage processes, the immune system plays a crucial role. As the core defense system of the human body, the immune system undertakes important functions such as immune surveillance, defense, and regulation (De Martino et al., 2024). T cells, as critical immune cells, recognize and eliminate pathogens and aberrant cells while regulating other immune components through cytokine secretion, thereby orchestrating immune system modulation (Dong, 2021). Epidemiological and animal studies consistently show reduced CD3⁺/CD4⁺/CD8⁺ T cell counts and altered CD4⁺/CD8⁺ ratios in both endemic fluorosis populations (drinking water fluoride: 0.89–2.66 mg/L) and animal models with elevated urinary fluoride, confirming fluoride’s immunotoxicity (Reddy et al., 2013; Yin et al., 2016; Zhu et al., 2024). Concurrently, with the increase in urinary fluoride concentration, fluoride exposure may affect the function of CD4⁺ T cells in the blood or thymus of humans and experimental animals, leading to an increase in the proportion of CD4⁺ regulatory T cell (CD4⁺Treg), which in turn triggers an immune imbalance between Treg and effector T cells (Zhu et al., 2024). Such imbalance may critically impact the homeostasis of the CD4⁺ T cells by inhibiting the activation and differentiation of effector CD4⁺ T cell subsets such as T helper (Th) 1, Th2, and Th17 cells, as well as by regulating their metabolism and cytokine environment (Gocher-Demske et al., 2023; So et al., 2023). Given the central role of CD4⁺ T cells in adaptive immune responses and immune balance (Kruse et al., 2023), these findings highlight CD4⁺ T cell dysregulation as a primary pathway for fluoride-induced immune system damage. Previous animal studies have also shown that excessive fluoride exposure (>50 mg/L or 24 mg/kg NaF) can impact cellular immune function, reducing the number of T cells in the thymus, spleen, and blood of mice, and causing changes in cytokine levels [transforming growth factor-B (TGF-B), tumor necrosis factor-a (TNF-a), interferon-y (IFN-y), interleukin (IL)-2, IL-10, etc.] (Guo et al., 2017; Kuang et al., 2017; Yin et al., 2016). The above studies suggest that high-fluoride exposure can lead to changes in T cell subsets and the expression levels of related cytokines. However, there are few reports on the impact of low-fluoride exposure (humans: water fluoride concentration <1.5 mg/L, experimental animals exposed to fluoride concentrations converted accordingly) on these indicators.

The Th subset of CD4⁺ T cells plays a pivotal role in orchestrating immune responses (Cenerenti et al., 2022; Mousset et al., 2019; Sun et al., 2023). Under the combined action of IL-12 and IFN-y, Th1 cells may further promote IFN-y secretion by activating specific signaling pathways and initiating a positive feedback loop, thereby participating in and enhancing cell-mediated immune responses (Elsner et al., 2024; Gao et al., 2023). Th2 cells, under the positive feedback loop of IL-4, further secrete IL-4, IL-5, and IL-13, contributing to humoral immune responses (Wang et al., 2017; Zhang et al., 2014). Th17 cells, induced jointly by TGF-B and IL-6, produce IL-17A and IL-17F, participating in cell-mediated immunity against extracellular bacteria and fungi, as well as pathological processes in certain inflammatory and autoimmune diseases (Harbour et al., 2020; Mills, 2023). Meanwhile, changes in the Th1/Th2 and Th1/Th17 ratios can reflect the body’s immune balance status (Yu et al., 2022). The balance of Th1/Th2 and Th1/Th17 plays a crucial role in maintaining the homeostasis of the human immune system (Leung et al., 2010; Li et al., 2021; Luo et al., 2022), regulating distinct immune responses to ensure the stability and balance of the immune microenvironment, thereby preserving the appropriateness of immune responses and preventing excessive inflammation or immune dysregulation (Basu et al., 2021; Wang et al., 2020).

In studies on the relationship between fluoride and the immune system, there remains a lack of population-based and experimental evidence establishing a definitive link between low-fluoride exposure and immune damage in the body. To investigate the role of specific CD4⁺ T cell subsets in immune damage caused by low-fluoride exposure, this study employed cytometry by time-of-flight (CyTOF) technology for the first time to conduct cluster analysis of CD4⁺ T cell subsets in populations with varying water fluoride exposure concentrations, identifying key CD4⁺ T cell subsets through the establishment of population immune profiles. Concurrently, we developed rat models with low-, medium-, and high-fluoride exposure under different durations (short-term and long-term) and water improvement intervention. To clarify the impact of low-fluoride exposure on the composition of CD4⁺ T cell subsets and the balance of Th1/Th2 and Th1/Th17, and to explore the role of related cytokines, we analyzed changes in the proportions of Th1, Th2, and Th17 cells as well as the expression levels of related cytokines. These findings will provide both population-based and experimental evidence for studying immune injury caused by low-fluoride exposure.

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