Bone marrow is the hematopoietic tissue of the human body. Hematopoietic stem cells in the bone marrow may proliferate and differentiate into erythroid, granuloid, lymphoid, mononuclear, and other blood cells and macrophages (22). Platelets exert hemostatic effects, and white blood cells may kill and inhibit various pathogens, including bacteria and viruses. Some lymphocytes specifically recognize antigens and produce antibodies (23). Therefore, bone marrow is a blood-forming and important immune organ. Owing to its strong affinity for bone, most fluoride accumulates in the calcified tissues of adults, resulting in skeletal fluorosis (24). Therefore, fluoride toxicity in human bones has been widely studied (25). However, the immunotoxic mechanism of fluoride exposure in bone injury remains unclear.

Bone marrow mesenchymal stem cells (BMSCs) are widely used as model cells to evaluate bone toxicity after exposure to environmental toxins. They may self-renew, undergo pluripotent differentiation and maintain immune regulation. Multi-omics analysis combining transcriptomics and metabolomics has shown that the influence of excessive fluoride on BMSCs is primarily caused by the alteration of lysosomes, which are severely damaged by fluoride exposure (26). Osteocytes have a strong tolerance to high fluoride concentrations, while osteoclasts are more sensitive to changes of fluoride dose (25). Fluoride can cause chromosome aberration and micronuclei increase in bone marrow cells, resulting in inhibited proliferation of bone marrow cells (27). In addition, fluoride can not only induce osteoblast differentiation, initiate endoplasmic reticulum stress and unfolded protein response, but also induce genotoxicity, oxidative stress and bone mineralization, thereby affecting cell proliferation and producing inflammatory factors (25, 28–30). However, the limited evidence available suggests that low level of fluoride have no immunosuppressive effect on bone marrow and do not cause immune damage. And low levels of NaF can promote soft tissue healing and hard tissue regeneration (31). Analysis at the transcriptomic level showed that human osteosarcoma (HOS) cells stimulated with sublethal concentration of sodium fluoride (8mg/L) for 30 days significantly changed the expression of genes associated with oxidative stress, inflammation, osteoblast differentiation, and bone development pathways. This suggests that chronic oxidative and inflammatory stress may occur in osteoblasts at sublethal fluoride concentrations, affecting the immune function of bone marrow (27, 32–34). These findings provide new insights to study the mechanism of bone marrow immune damage induced by fluoride.

The thymus is an important immune and secretory organ that releases hormones. It is the third line of defense in the human body, providing surveillance and protection and specifically recognizing a variety of pathogens, tumors, antigens, and tissue damage agents. The thymus is the main lymphoid organ involved in T lymphocyte development and regulates T cell development and function. Specific differentiation and generation of T lymphocytes occur in the cortical and medullary regions and are controlled by age (35–37).

Excessive fluoride intake in water may induce immunosuppression, decreasing the number of immune cells and damaging the immune function of the thymus, which can lead to damage via various apoptotic mechanisms and signaling pathways (38). In mature rats, the thymus medulla accounts for 1/3 of the entire lobule, and the ratio of the thymic cortex to the medulla is approximately 2:1 under normal conditions. Fluoride treatment may lead to a decreased ratio of the cortex to the medulla of the thymus, uneven distribution of the cortical layer, irregular ultrastructure and morphology of thymus lymphocytes, and enlargement or cavitation of the mitochondrial crista and apoptotic bodies (39). In addition, fluoride causes enlargement of the thymic nuclear space, condensation, and marginalization of chromatin (40). Moreover, high fluoride levels may decrease the thymus immune index and reduce the number of thymus cells by inhibiting lymphocyte division and promoting cell apoptosis, thereby inhibiting immune function. According to the literature on high fluoride-induced apoptosis, mitochondria-mediated, endoplasmic reticulum (ER)-mediated, and death receptor-mediated pathways are involved (41). However, current studies on the fluoride-induced apoptotic pathways in thymus cells have primarily focused on oxidative stress mediated by the endoplasmic reticulum. Excessive intake of fluoride leads to endoplasmic reticulum stress, which in turn leads to excessive accumulation of the unfolded protein response, activation of RNA activated the protein kinase-like endoplasmic reticulum kinase (PERK), inosin-required protein-1 (IRE1), and activated transcription factor (ATF)-6 signaling pathways. It also triggers eukaryotic initiation factor-2α and tumor necrosis factor (TNF) receptor-associated factor 2 expression. Under continuous external stimulation, ATF4 induces the expression of GADD153, which leads to apoptosis. The immunosuppressant effect of fluoride in the thymus primarily restores cell division reduced by fluoride treatment by inhibiting the PERK and IRE1 signaling pathways, thus inhibiting fluoride-induced apoptosis (42–44).

Fluoride induces the apoptosis of thymus cells and leads to immunosuppression; additionally, high concentrations of fluoride damages thymic epithelial cells (TECs) by altering the expression of T cell function–related genes and the production of immunomodulatory cytokines (45). In TECs, the expression of the Foxn1, Cbx4, DLL4, and IL-7 genes related to T cell development and differentiation are reduced (46). Thus, a decrease in CD4⁺ and CD8⁺ thymus T cells was induced. The mRNA levels of the immunoregulatory cytokines interleukin-2 (IL-2) and interleukin-10 (IL-10), which are involved in T cell proliferation, were also decreased, and the expression of the T cell function–related genes CD2, PTPRC, CD69, and CD101 decreased with the action of fluoride. This leads to dysfunction of the thymus gland (47–50). The ingestion of high fluoride levels can hence lead to decreased T cell numbers and T cell dysfunction by reducing the expression of these genes, which may become a new entry point for the study of fluoride-induced immune damage.

The spleen is the largest immune organ in the body, accounting for 25% of the total lymphoid tissue. It contains many lymphocytes and macrophages and is the center of cellular and humoral immunity. The spleen’s main function is to filter blood, which also plays an antitumor role via various mechanisms. Removal of the spleen leads to cellular and humoral immunity disorders (51–53).

The immune damage caused by high fluoride levels primarily manifests as a decline in humoral immune function and immune cell apoptosis, thereby reducing the immune response (50). Through the establishment of Wistar rat model of water-induced fluorosis, fluoride was shown to inhibits splenic cell division in rats, decreases the splenic immune index, promotes apoptosis, and leads to the expansion and vesiculation of the endoplasmic reticulum of splenic lymphocytes, with the presence of a large number of vacuoles between cells. Under fluoride treatment, the nuclear space was evident, the chromatin of lymphocytes in the spleen tissue was marginalized, and the nuclear chromatin coagulated (39, 54). In addition, fluoride has been shown to induce endoplasmic reticulum-mediated oxidative stress, activation of PERK and IRE1 signaling, and associated apoptosis. After PERK and IRE1 knockout, cell division ability was restored and apoptosis was reduced in fluoride-treated lymphocytes. This result correlated well with the expression of PERK and IRE1 signaling–related proteins, confirming the key role of these pathways in excessive fluoride immunosuppression. Therefore, the mechanism of fluoride immune damage in the spleen is related to the activation of the PERK and IRE1 signaling pathways. Inhibition of PERK and IRE1 signaling pathways may reduce cell division and apoptotic damage, and high fluoride levels can affect the development and differentiation of immune cells and induce immune cell apoptosis by activating PERK and IRE1 signaling pathways (42, 55–57).

In addition, fluoride may inhibit the activation of the p38/mitogen-activated protein kinase (MAPK) pathway in mouse splenic lymphocytes. p38/MAPK mRNA is expressed in macrophages, T lymphocytes, and B lymphocytes in mice. Fluoride may reduce the transcription of MLK3, MKK6, and p38/MAPK genes and thus reduce the expression of MKK6 and p38/MAPK proteins, thereby affecting the immune function of lymphocytes. With an increase in fluoride concentration, the expression of MKK6, p38/MAPK, and their protein products decreased, which may have been caused by the disruption of homeostasis by chronic fluorosis. These genes are not phosphorylated properly and exhibit differences in gene expression that directly alter protein expression, thereby reducing pathway activity. The p38/MAPK pathway is activated, and the c-Jun N-terminal kinase (JNK) and extracellular regulated protein kinases (ERK) pathways may also be activated, and high fluoride concentrations have a substantial inhibitory effect on mouse B lymphocytes, reducing the proliferative activity of lymphocytes, indicating that sodium fluoride (NaF) may inhibit the proliferation and differentiation of lymphocytes via the p38/MAPK pathway (58–61).

The damage of fluoride to the spleen is not only manifested in morphology and apoptosis but also leads to an increase in the percentage of G0/G1 phase cells and a decrease in the number of S phase cells in the spleen (62). Cell cycle arrest is the molecular basis for NaF toxicity caused by NaF during spleen development. Moreover, the protein expression levels of IL-2, transforming growth factor-β (TGF-β), TNF-α, interferon-γ (IFN-γ), and cyclin E/D and CDK2/4 were significantly decreased. In contrast, the IL-10 protein expression level was significantly increased. The expression levels of cyclin E/CDK2 and cyclin D/CDK4 proteins were significantly reduced, indicating that NaF treatment slowed the G1 cell cycle progression, blocked the G1/S transition, inhibited splenic lymphocyte proliferation, and damaged DNA synthesis, thereby resulting in cytotoxicity in mice (49, 63–66).

The intestine is an important digestive organ in the human body by playing key roles in food digestion and absorption, water and electrolyte balance, and immune regulation. The intestines can absorb the necessary nutrients in food, regulate the osmotic pressure of plasma crystals, and monitor and eliminate pathogenic bacteria via specific and non-specific immunity to protect the body’s internal environment (67). People living in fluoride-endemic areas often report stomach abnormalities with symptoms including loss of appetite, nausea, anorexia, gas, constipation, and intermittent diarrhea (68). Fluoride is absorbed in the gastrointestinal tract, and numerous studies in laboratory animals and humans have shown that ingestion of NaF may cause gastrointestinal damage (69–71). In addition, long-term excessive intake of fluoride has a certain damaging effect on the intestine, inhibiting the proliferation of epithelial cells (72), leading to increased intestinal permeability, and thus destroying the mechanical and immune barrier function of the intestine (73).

Dietary high fluoride–induced oxidative damage decreased the percentage of T cell subgroups in the cecal tonsil and decreased Immunoglobulin A (IgA), Immunoglobulin G (IgG), and Immunoglobulin M (IgM) levels in the cecal tonsil, which ultimately affected the local mucosal immune function of broilers (71, 74). Morever, ingested fluoride is absorbed from the stomach and intestinal epithelium. Excess fluoride may inhibit intestinal epithelial cell and mast cell proliferation, promote the release of bioactive molecules, stimulate inflammatory cells, activate lymphocytes, reduce the concentrations of immunoglobulins, reduce the ability to resist pathogen infection, and affect the immune function of the intestinal mucosal (69, 75–78). The intestinal barrier is considered to be the most important defense against microbial pathogens that enter the host through the gut (79). Excessive fluoride can stimulate the expression of pro-inflammatory factors, reduce the expression level of tight-junction related genes and proteins, activate inflammatory response, promote cell pyrogen, inhibit intestinal development, and thus cause intestinal inflammation and diarrhea (80). In addition, using high-throughput 16S rRNA gene sequencing technology, it has been found that fungi may be one of the important factors affecting intestinal function, and excessive fluorine can affect the species richness of intestinal fungi in mice, suggesting that fluorine may cause adverse intestinal symptoms by causing an imbalance in the intestinal fungal population, thus affecting the normal intestinal function (81, 82). Moreover, fluoride exposure not only disturbed the compositional balance of the gut microbiome, but also triggered metabolic disorders (83). Alterations in intestinal microbiota and metabolome play a critical role in regulating disease susceptibility and multi-organ damage after excessive fluoride exposure (73). More and more studies have shown that there is a significant difference in the abundance of bacteria in the gut after excessive fluoride intake, and excessive fluoride changes the composition of intestinal microbes in animals (77, 84, 85).

Macrophages are an important part of the innate immune system, and their activation is indispensable for several aspects of immune defense, inflammatory responses, tissue remodeling, and homeostasis. Macrophages are found in almost all body tissues and are highly heterogeneous. Macrophages function in different physiological states primarily via cell polarization (90).

High fluoride stimulation may affect macrophage polarization, resulting in decreased M2 differentiation and inhibition of osteogenic differentiation and formation (91). Moreover, macrophages exposed to fluoride concentrations ≥50 mg/L (2.6 mM NaF) produce active substances, significantly increasing lipid peroxidation. Even within a short period, it leads to macrophage redox imbalance, resulting in a decline in mitochondrial activity and phagocyte vitality (49). Additionally low fluoride levels can also damage the immune system. Even 1 and 3μM NaF can also affect the expression and activity of 15 lipoxygenases, the number and activity of cyclo-oxygenase, and the activation of phospholipase A2 in human peripheral blood mononuclear cell differentiation mononuclear cells, and promote the biosynthesis of inflammatory mediators, leading to inflammation (92, 93). Macrophages are the main source of reactive oxygen species (ROS) in the human body, participate in the body’s response to various pathogenic factors, and play a key role in inflammation. Under the effect of high fluoride concentrations, the formation of ROS in macrophages increases oxidative stress, thus affecting the expression of inflammatory factors (94).

Neutrophils are effective antibacterial cells, capable of attacking microorganisms within the limited intracellular compartment with powerful antibacterial properties (95). Inappropriate or dysregulated neutrophil activation may damage the host and lead to autoimmune and inflammatory diseases (96).

Due to the harmful effects of fluoride on the bone marrow and hematopoietic organs, fluoride may lower white blood cell, platelet, and neutrophil counts (97). Fluoride stimulation of neutrophils isolated from the tail venous blood of healthy carp showed that NaF significantly increased the release of neutrophil extracellular traps (NET) by regulating the Adenosine 5’-monophosphate (AMP)-activated protein kinase/p38 (AMPK/p38) signaling pathway, suggesting that the toxic effect of fluoride on the immune system of carp may be caused by the release of NET (98). In addition, NaF-induced NET formation in bovine neutrophils is accompanied by increased ROS production and decreased antioxidant enzyme activity, and oxidative stress and NET formation may cause immunotoxicity in neutrophils via the p38/MAPK and ERK pathways (99).

NK cells are lymphocytes of the innate immune system that play an important role in innate and adaptive immune responses to tumors and viral infections. They not only exert cell-mediated cytotoxicity toward tumor and infected cells but also play a regulatory role by secreting cytokines and chemokines to promote or inhibit the functions of other immune cells (100, 101).

NaF has immunosuppressive activity, and fluoride may enhance the in vitro reactivity of human lymphocytes to mitogen or specific antigens. Increased levels of IFN-γ and sIL-2R, which are products of activated T and NK cells, suggest that NaF may primarily act on T and/or NK cells (102). The levels of primary immune response cells, namely CD4 cells, IgG1 cells, and NK cells, may be reduced by different fluoride concentrations. Lipid peroxidation increases significantly, and the antioxidant defense system is damaged (103).

T lymphocytes are derived from pluripotent stem cells in the bone marrow. During the embryonic and neonatal stages, some pluripotent stem cells or pro-T cells in the bone marrow migrate to the thymus and mature into immunoactive T cells under the induction of thymus hormones (104–106). T lymphocytes are divided into two functionally distinct subsets: CD4⁺ T helper (Th) cells and CD8⁺ cytotoxic T lymphocytes (CTL). These cells are indirectly involved in clearing infections by regulating the activities of other immune cells. CTL cells induce apoptosis by secreting pro-inflammatory cytokines. Adequate CD4⁺ and CD8⁺ T cell activation, proliferation, clonal amplification, and effector functions are essential to clear the infection effectively (107, 108).

Exposure to excessive fluoride reduces the number of lymphocytes, resulting in a decrease in the percentage of CD4⁺ and CD8⁺ T cells in the thymus, leading to T cell dysfunction (109). Excessive fluoride intake can alter T-bet and GATA3 protein expression levels in the spleen. T-bet and GATA3 are transcription factors that effectively regulate the development, differentiation, and memory formation of Th1/Th2 cells, disrupting the balance of Th1/Th2 cells (110). Splenic injury is eventually induced by alterations in the expression of Th1/Th2 cell–related cytokines in the splenic microenvironment. In addition, excessive fluoride intake has been found to affect the number of Th17 cells and the expression of related cytokines, leading to changes in the immune system (111–113).

B lymphocytes are derived from the mammalian bone marrow or bird bursa lymphoid stem cells, also called bone marrow- or sac-dependent lymphocytes. They primarily settles in the superficial cortical area of the lymph nodes and lymph nodules of the white pulp of the spleen. In peripheral blood, B cells account for approximately 10%-15% of lymphocytes (113). B cells are the main cells involved in humoral immunity and produce immune effects primarily by secreting antibodies (114–117).

A study using flow cytometry to determine the percentages of CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺ T, and CD19⁺ B lymphocytes in the spleens of mice (118). The results showed that the percentage of B lymphocytes was significantly reduced under high fluoride conditions. In addition, a high fluoride diet reduced the number of tonsil B lymphocytes in the cecum and B lymphocytes in the intestinal mucosal of broilers (71, 74, 119). The decrease in the number and activity of B lymphocytes may be caused by the apoptosis of B lymphocytes induced by NaF or the inhibition of lymphocyte proliferation. Some studies have shown that NaF may induce apoptosis and inhibit the proliferation of splenic B lymphocytes both in vivo and in vitro, thus impairing humoral immunity (120).