Fluorosis is a prevalent disease worldwide and particularly endemic in China, which is one of the countries where F causes serious harm[11, 12]. In 2015, a review reported that F ion concentrations in the groundwater can reach at 48 mg/L[12]. This means that drinking water is the main source of excessive F ions, causing fluorosis in humans and animals. Although the exact link between fluorosis and infertility was established as early as 1925, the mechanisms linking them are poorly understood. Hence, it is very pivotal to evaluate the mechanisms of reproductive toxicity caused by F using an effective and reliable animal model.
Firstly, we exposed the WT C57B/6J mice to different concentrations of NaF, including 25, 50, and 100 mg/L NaF for 90 days. We observed that IL-17A was highly expressed in the testes of mice exposed to 50 mg/L NaF and IL-6, IL-23, IL-1β, TGF-β and TNF-α are closely related to the secretion and function of IL-17A induced by fluoride in testis. We also found that the expression of IL-17A was increased evidently in the interstitium of the testes using ELISA assay. A recent report by Hossam et al suggested that IL-17A mainly exists in the testicular interstitium of azoospermia patients. This is consistent with our results that IL-17A is abundant in the testicular interstitium of the fluorosis model. Although the previous studies has certificated that IL-17A is found mainly in the interstitium of the testes undergoing fluoride stimulation, the mechanisms behind fluoride-induced testicular damage and IL-17A production were not demonstrated. Hence, we selected IL-17A gene knockout fluorosis mice as an available model to gain more insights about fluorosis.
Although low doses of fluoride are beneficial to mammalian teeth and bones, its long-term exposure can cause dental fluorosis and may interfere with bone formation[13]. Therefore, the F content in the skeleton is an important index to determine whether the fluorosis model is successfully established or not. In this experiment, when the mice (WT and IL-17A-/-) were exposed to 50 mg/L NaF at 90 days, the F content was significantly abundant in the femur of mice, indicating that the establishment of the animal model of fluorosis was successful.
Earlier studies revealed that the height and weight of children in F-contaminated areas were significantly lower than those in normal areas[14]. Recent works using animal models reported that rats or mice exposed to superfluous F could experience a significant reduction in weight and organ coefficient of liver[15, 16]. This corroborates with our results, which shows that the weight gain of mice exposed to 50 mg/L NaF for 90 days significantly differs from the control group, indicating that excessive F exposure causes an adverse effect on the growth performance of mice. It should be noted that F did not significantly affect the weight gain of IL-17A deficient mice, possibly the absence of IL-17A antagonizes the damage caused by F in other tissues.
Studies have shown that excessive F consumption can take adverse effects on semen quality and damage the normal structure of testis[17, 18]. In our study, the use of different genotype animal models of fluorosis revealed that excessive NaF intake in the testes of WT mice could induce sperm count reduction and increase the rate of abnormal spermatozoa. In addition, it was found that the distance between seminiferous tubules increased and the number of cells in spermatozoa and stroma decreased in testis of fluorosis mice[19]. However, IL-17A deficient mice treated with 50 mg/L NaF for 90 days could effectively suppress these abnormal changes of semen quality and structure. Furthermore, the TEM were used to study the target of fluoride on testis more accurately, we found that excessive intake of fluoride can damage the normal structure of Leydig cells, and may decrease the ability of testosterone secretion. These results are not only consistent with the previous studies but also proved that functional alterations in IL-17A might play a key role in F-induced testicular damage.
IL-17A, mainly produced by Th17 cells, is an inflammatory cytokine with various functions, such as eliminating pathogenic microorganisms and inducing autoimmune inflammation[20, 21]. IL-17A can also recruit Act1 and TRAF6 by binding to IL-17RA or IL-17RC heterodimer receptor complexes in the process of inflammation[22]. On the one hand, self-ubiquitinated TRAF6 binds to TAK1 complexes and activates IKK complexes, leading to activation of NF-kappa B and MAPK pathways[23]. On the other hand, Act1 also participates in the activation of C/EBP. Activation of NF-kappa B, MAPK, and C/EBP up-regulates the expression of chemokines and cytokines, which both induce immune cell recruitment, inflammatory response, and tissue damage[24, 25]. These findings indicate that the balanced expression of the signaling molecules downstream of IL-17R decides the cellular response. In the WT NaF fluorosis group of our study, we found that the protein levels of IL-17A, IL-17R, Act1, NF-κB, C/EBP-α and TRAF6 presented a sharply upward trend. This means thatIL-17A and its signaling pathway might be involved in regulating the process of testicular damage in fluorosis. Although the mice in the IL-17A-/- group were also exposed to NaF, the related proteins in the IL-17A pathway were largely unaltered compared with the normal mice. Hence, these revealed that F-induced IL-17A production can cause damage to the testes. Our findings corroborate with the results from Wu, who revealed that excessive F can cause orchitis and damage the normal function of the testes[8].
Secreted by Leydig cells, testosterone can promote and maintain spermatogenesis in the testes[26]. There have many studies have confirmed that excessive fluoride intake can reduce the level of testosterone in vivo and in vitro[27, 28]. Based on our research in this experiment, we also detected the level of testosterone in IL-17A gene knock-out fluorosis mice, and we were surprised to find that the level of testosterone in the serum of this kind of mice was even equal to that of normal mice. We also detected the related proteins of testosterone secretion in mouse testes. The AKR1C3, also called HSD3β1, is responsible for the final step in the biosynthesis of testosterone from androstenedione[29]. Unlike HSD11β1, CYP11A1 exists in all stages of the Leydig cells and encodes the Cytochrome P450 Family 11 Subfamily A Member 1A[30]. Both AKR1C3 and CYP11A1 are the key rate-limiting enzyme in the process of steroid hormone synthesis. It has been confirmed that excess F feeding can reduce the activity of AKR1C3 and CYP11A1 in the testes and further inhibit the secretion of testosterone[31]. Our study confirms the previous findings that fluorine can cause a decrease in AKR1C3 and CYP11A1 levels. However, after the knockout of IL-17A in the testes of mice, the expression of AKR1C3 and CYP11A1 returned to the level similar to the normal mice.
To elucidate the effect of IL-17A deficiency on the testicular immune response of fluorosis mice, we detected the content of IL-17RA, IL-17RC, IL-6, IL-1β, IFN- γ, TNF- α, IL-23 and TGF-β by ELISA. Our current findings showed that in the testicular injury caused by fluorosis, IL-17A and its receptors occurs mainly in the interstitial tissue of testes. Interestingly, IL-17RC was significantly increased in the testis of WT fluorosis mice, but the content of IL-17RA was not significant and this needs further study. Consistented with previous results, the IL-6, IL-23, IL-1β and TGF-β were increased in the testis when the WT mice exposed 50 mg/L NaF[6]. There have a investigation also indicated that the mRNA levels of TGF-β, IL-1β, IL-6, and IL-21 up-regulated intestis after F exposure[32]. More importantly, after knockout of IL-17A, the expression of IL-6 and IFN-γin testis was still increased compared with the control group. This result suggests that the IL-17A deficiency can alleviate fluorosis by reducing the immune response in testis and damage of IL-17A to testis may not related to IL-6 and IFN -γ.
There are numerous innate immune cells in the interstitium of testes, such as T cells, macrophages, NK cells, etc[33]. Nevertheless, it is not clear which immune cells secrete IL-17A under the stimulation of F. By combining the PCR and ELISA results on IL-17A secretion related factors, we found that the secretions of IL-6, IL-23, and IL-1β were increased in testicular fluorosis WT in mice. However, after knockout of IL-17A, the expression of these inflammatory factors except IL-6 did not increase. This suggests that the expressions of IL-6, IL-23, and IL-1β depend on the increase of IL-17A in the process of fluoride-induced testicular injury. Previous findings showed that the increase of IL-17A can promote the secretion of IL-6 in some inflammation and cancer[34]. Also, γδ T cells can produce IL-17A upon stimulation by IL-1β and IL-23 in the testes[35, 36]. Featured by the expression of γδ TCRs and secreted extensive pro-inflammatory cytokines, γδ T cells are usually the main providers of IL-17A in various models of inflammatory diseases[37, 38]. Besides, in vitro studies have confirmed that the secretion of IL-17A will increase when γδ T cells bind to IL-23[39]. Therefore, we speculated that among the cytokines related to IL-17A, the expressions of IL-1β and IL-23, which were supposed to stimulate γδ T cells to secrete IL-17A, would be sharply up-regulated in the process of a testicular injury caused by F, indicating their role in IL-17A production. Testes have established effective self-protection through its immunosuppressive microenvironment, which is free from pathogens and inflammatory damage[40]. Once the microenvironment of the testes is imbalanced, it may lead to the occurrence of inflammation and lesion[41]. The overexpression of IL-17A, IL-6, IL-1β, and IL-23 may change the immunosuppressive microenvironment of the testes, causing testicular damage.
The changes of testicular environment caused by abnormal expression of cytokines can induce excessive apoptosis[42, 43]. Hence, we detected the expression of two key apoptosis proteins in testis, caspase-3 and caspase-9 acted an irreplaceable role in apoptosis[44]. Caspase-9 can be activated by self-cutting after being stimulated, followed by induction of the caspase cascade reaction to activate caspase-3[45]. As a key death protease downstream of the apoptosis pathway in mammalian cells, caspase-3 can shear multiple polymerases and lead to apoptosis[46]. Numerous studies have confirmed that excessive F intake can lead to apoptosis of multiple tissues and cells[47, 48]. Furthermore, Yan et al showed that F can induce the apoptosis of singular cells by accelerating the release of caspase-3 and caspase-9[49]. In our study, we found that excessive NaF exposure can indeed boost the expression of caspase-3 and caspase-9. Interestly, with the deletion of IL-17A, the expression of caspase-3 and caspase-9 in the testes have shifted as well. Notably, the fluoride-induced apoptosis in the testicular interstitium can be inhibited after knocking out the IL-17A. In addition, the observation from the immunohistochemical chart pointed out the apoptosis of testes mainly occurs in the interstitial tissue. We also demonstrated that NaF treatment in vivo can stimulate a significant decrease in testosterone secretion, however, IL-17A-depleted mice exposed to NaF can restore the ability to secrete testosterone. Therefore, we speculated that Leydig cells may be a target in the process of IL-17A-mediated fluoride-induced testicular damage.
We observed that fluoride-induced testicular damage occurs mainly in the Leydig cells. Therefore, we chose Leydig cells as our target to further elucidate the role of F in inducing reproductive toxicity. Leydig cells, referred to as interstitial cells, play a critical role in the testicular endocrine and immunologic systems[50]. The main function of Leydig cells is endocrine because they can secrete a high level of androgens[51]. Previous studies reported that excessive F exposure can lead to proliferation, autophagy, and apoptosis of the Leydig cells[52]. In the present study, fluoride and IL-17A can promote late apoptosis of the Leydig cells, and can significantly reduce the expression of genes involved in testosterone secretion. Therefore, we hypothesize that the increase of IL-17A after fluoride exposure can cause Leydig cell apoptosis leading to testosterone reduction, which will then negatively impact the semen quality. Our results do not only indicate that Leydig cells are susceptible to F-related changes in the testes, but also prove the antagonistic role of IL-17A during the process of testicular damage caused by F.