The addition of melatonin improves the degranulation and IFN-γ secretion abilities of RMA-S co-cultured NK cells

Our previous studies have revealed that disrupting the circadian rhythm of mice impairs the immune surveillance capacity of NK cells, emphasizing the importance of maintaining a normal circadian rhythm for NK cell function [36]. Melatonin, a hormone secreted by the pineal gland, is known to regulate circadian rhythms. Tian et al. [27] demonstrated NK cell recovery activity in aged mice after melatonin supplementation. Currier et al. (2000) showed an increase in the absolute number of NK cells in the spleen and bone marrow of mice fed melatonin compared to control mice. In this study, we conducted in vitro experiments by adding melatonin to explore its effect on NK cell function. Our results demonstrate that the additional addition of melatonin increases the expression of CD107a in NK cells co-incubated with RMA-S cells (Fig. 1A, B). Furthermore, melatonin supplementation also enhances the ability of NK cells co-incubated with RMA-S cells to secrete IFN-γ (Fig. 1C, D). These findings lead us to speculate that melatonin treatment can improve the function of NK cells.

Fig. 8

Melatonin promotes NK cell function by enhancing IL-2 secretion from CD4 + T cells rather than directly acting on melatonin receptors. (A) Splenic NK cells were isolated from aged mice (1 × 10^6), pre-treated with S26131 (MT1 antagonist, 10µM) or 4-P-PDOT (MT2 antagonist, 10µM) for 1 h, followed by treatment with 100µM melatonin for 24 h, NK cell CD107a expression was detected using flow cytometry. (B) The statistical graph of NK cell CD107a expression. (C) Splenic NK cells were isolated from aged mice (1 × 10^6), pre-treated with S26131 (MT1 antagonist, 10µM) or 4-P-PDOT (MT2 antagonist, 10µM) for 1 h, followed by treatment with 100µM melatonin for 24 h, NK cell IFN-γexpression was detected using flow cytometry. (D) The statistical graph of NK cell IFN-γ expression. (E) Flow cytometry was employed to assess the expression of IL-2 in CD4 + T cells from the spleens of aged mice treated with melatonin or control. (F) The statistical graph of IL-2 expression in CD4 + T cells. (G) We used a CD4 + T cell isolation kit to deplete CD4 + T cells from splenic PBMCs of aged mice, followed by treatment of the remaining PBMCs with 100 µM melatonin for 24 h, and assessed NK cell IFN-γ expression using flow cytometry. (H) The statistical graph of NK cell IFN-γ expression. The data is presented as means ± SD. n = 6. Unpaired two-tailed Student’s t-tests or one-way ANOVA. were conducted using Prism software. A p-value of less than 0.05 was considered statistically significant. *P < 0.05, **P < 0.01

In vivo Melatonin treatment increases the number of NK cells in the spleen, bone marrow, and liver of aged mice

Given the positive effects of melatonin on NK cell function observed in vitro, we were curious to investigate whether melatonin treatment also impacts the number of NK cells in the immune organs of aged mice. To explore this, we administered melatonin to aging wildtype mice and isolated lymphocytes from the spleen, bone marrow, lymph nodes, liver, and lungs. By employing flow cytometry, we analyzed the proportion and number of NK cells in each lymphatic organ or tissue. Our results revealed a significant increase in the proportion and number of NK cells in the spleen, bone marrow, and liver of mice following melatonin treatment (Fig. 2A-C). This finding suggests that melatonin treatment contributes to maintaining a higher number of NK cells in the body, which may, in turn, enhance the immune function of elderly mice. However, when we treated young mice (8–12 weeks) with melatonin, we found that melatonin did not increase the number of NK cells in the spleen, bone marrow, and liver (Fig. S1A-C) nor did it enhance the function of NK cells (Fig. S1D-G).

Melatonin treatment promotes the development and maturation of NK cells in aged mice

The proper development of NK cells is pivotal for determining their functionality. In light of this, we investigated the impact of melatonin treatment on the development of NK cells in the spleen and bone marrow of aged mice using flow cytometry. We utilized CD27 and CD11b markers to classify NK cells into four developmental stages: CD27 single positive (CD27SP, pre-stage), double positive (DP, immature), and CD11b single positive (CD11bSP, mature). Our results demonstrated a notable increase in mature NK cells, specifically in the CD11bSP subset, and a reduction in the CD27SP subset following melatonin treatment (Fig. 3A-C). Additionally, we employed another gating method using NK1.1 and CD11b markers to distinguish different developmental stages, including NK cell precursors (DN), immature NK cells (NK1.1SP), and mature NK cells (DP). Similar results were observed, with a significant increase in mature NK cells and a decrease in immature NK cells in both the spleen and bone marrow after melatonin treatment (Fig. 3D-F). These findings indicate that melatonin promotes the maturation of NK cells.

Melatonin treatment improves NK cell degranulation and IFN-γ secretion in aging mice

To further validate the beneficial effects of melatonin on NK cell function in aged mice, we isolated splenocytes from aging mice treated with melatonin and stimulated them with RMA-S or YAC-1 tumor cells. Flow cytometry analysis was conducted to assess the expression of CD107a and IFN-γ in NK cells. The results showed an increase in CD107a expression in NK cells stimulated by RMA-S or YAC-1 after melatonin treatment, indicating an enhancement in NK cell degranulation (Fig. 4A, B). Additionally, melatonin treatment also augmented the ability of NK cells to secrete IFN-γ (Fig. 4C, D). These findings provide further evidence of melatonin’s positive impact on NK cell function in aging mice.

Melatonin treatment increases the proliferation of CD27−CD11b+ NK cells in the spleen of aged mice

To understand the underlying reason behind the increase in NK cell numbers after melatonin treatment in aged mice, we analyzed the proliferation and apoptosis of NK cells at various developmental stages in the spleen of melatonin-treated aging mice. Interestingly, we observed a significant increase in Ki67+ cells among the total NK cell population, particularly in the CD27−CD11b+ subset (Fig. 5A, B). This finding suggests that melatonin treatment may increase the number of NK cells in aging mice by promoting the proliferation of CD27−CD11b+ mature NK cells. Furthermore, we evaluated NK cell apoptosis in melatonin-treated aged mice and found no significant changes in the total NK cell population or the number of Annexin V+ cells in each developmental stage (Fig. 5C, D). These results suggest that melatonin’s effects on NK cell numbers in aging mice are likely attributed to increased proliferation rather than reduced apoptosis.

Melatonin treatment activates NK cells in aged mice

To gain insights into the mechanism underlying melatonin’s positive influence on NK cell function in aged mice, we investigated the expression of activation markers CD71 and CD98 in melatonin-treated aged mice. CD71 and CD98 are nutrient receptors, and their expression levels reflect cell metabolism levels. Our results demonstrated significantly higher expression levels of CD71 on NK cells in melatonin-treated mice compared to aging mice. Specifically, the CD11bSP mature NK cell subset showed a notable increase in CD71 expression, while other developmental stages did not exhibit significant differences (Fig. 6A, B). Furthermore, we observed increased expression of CD98 in NK cells from melatonin-treated mice compared to aging mice (Fig. 6C, D). Notably, the expression of CD98 was significantly increased in most developmental stages, except for the early developmental stage of CD27SP. These findings indicate that melatonin may improve NK cell function by activating NK cells.

Melatonin enhances T-bet expression through the JAK3/STAT5 signaling pathway to enhance NK cell function

To further elucidate the molecular mechanism underlying melatonin’s positive effects on NK cell function, we focused on the signaling pathways involved in NK cell development and function, particularly the JAK3/STAT5 pathway. We assessed the changes in JAK3/STAT5 in NK cells of aged mice treated with melatonin and found a significant increase in JAK3 expression, particularly in the CD11bSP mature NK cell subset (Fig. 7A, B). Additionally, we observed a notable increase in the phosphorylation level of STAT5, a downstream molecule of JAK3, in the spleen NK cells of melatonin-treated mice, indicating the activation of STAT5 signaling (Fig. 7C, D). As T-bet is a crucial transcription factor involved in NK cell development and function, we investigated its expression in spleen NK cells of melatonin-treated mice. Remarkably, the expression of T-bet was significantly increased in melatonin-treated mice, including in NK cells at various developmental stages (Fig. 7E-F). However, the expression of another important transcription factor regulating NK cell function, Eomes, did not change (Fig. 7G-H). We used the inhibitor BD750 to inhibit STAT5 expression in NK cells and examined the expression of T-bet. The results showed that after inhibiting STAT5, there was a significant decrease in T-bet expression in NK cells (Fig. 7I-L). From these findings, we speculate that melatonin enhances the expression of T-bet through the JAK3/STAT5 signaling pathway, ultimately augmenting the immune surveillance function of NK cells.

Melatonin enhances T-bet expression through the JAK3/STAT5 signaling pathway to enhance NK cell function

We hypothesize that melatonin may enhance NK cell function either by directly activating intracellular signals through surface melatonin receptors on NK cells or indirectly by increasing IL-2 secretion from CD4 + T cells. We treated aged mice NK cells with either the melatonin receptor 1 (MT1) antagonist S26131 or the melatonin receptor 1 (MT2) antagonist 4-P-PDOT, followed by melatonin treatment, and assessed NK cell expression of CD107a and IFN-γ.The results indicate that inhibiting melatonin receptors does not affect melatonin’s effects on NK cells (Fig. 8A-D), suggesting that melatonin does not exert its effects directly through it’s receptors expression on NK cells. We found that melatonin treatment significantly increases IL-2 expression in CD4 + T cells of aged mice (Fig. 8E-F). Using a CD4 + T cell sorting kit, we depleted CD4 + T cells from mouse splenic mononuclear cells (PBMCs). Results showed that PBMCs depleted of CD4 + T cells and treated with melatonin significantly reduced NK cell IFN-γ production (Fig. 8G-H), indicating that melatonin enhances NK cell function in aged mice by increasing IL-2 expression in CD4 + T cells.