In this study, we observed that serum granulysin levels were higher in women positive for aPLs, especially anti-PE antibodies, and that heparin treatment significantly decreased serum granulysin levels. The miscarriage rate was significantly lower in women whose serum granulysin levels were reduced by heparin treatment, indicating that heparin reduces granulysin levels to prevent miscarriage. In this study, the mechanism by which heparin regulated granulysin levels remained unknown.
Although heparin primarily exerts antithrombotic effects [32, 33], it is also reported to exert an apoptotic effect on the placenta and villi [34, 35] and a suppressive effect on complement activation [9]. In recent years, heparin has also been shown to alter the configuration of high mobility group box 1 (HMGB1) by inhibiting its binding to the receptor of advanced glycation end products [36, 37], suppressing binding to toll-like receptors (TLR4), and suppressing inflammatory signals from TLR4 [38–40] on APCs. HMGB1 is an alarmin that is secreted by necrotic cells or passively secreted by immune cells into the extracellular matrix. It activates APCs, including DCs, as well as granulysin [41]. It acts as a damage-associated molecular pattern and induces pre-inflammatory responses [17]. HMGB1 expression is increased in patients with APS [42] as well as in individuals with RPL of unknown etiology [43]. These findings and our results indicate that heparin may block the binding of HMGB1 to its receptor and suppress the production of granulysin from effector cells. Furthermore, recent studies have revealed that granulysin also acts as an alarmin leading to the activation of TLR4 on APCs [20]. Therefore, we suspected that heparin may suppress the immunostimulatory effects of HMGB1 and granulysin.
It is unclear why granulysin levels were not correlated with NK cell activity in the present study. In general, granulysin is produced by CTLs as well as NK and NKT cells. Indeed, we observed that the production of cytotoxic factors (perforin and granzyme) by uterine NKT cells was significantly higher than that by uterine NK cells observed in our previous study [44]. Therefore, NK cell activation may not have decreased in response to heparin treatment because granulysin was produced by NK cells as well as CTLs and NKT cells. It is also unclear why granulysin levels were not correlated with the Th1/Th2 ratio in the present study. The Th1/Th2 ratio is used to evaluate interferon gamma (IFN-γ) levels (corresponding to Th1) and interleukin (IL)-4 levels (corresponding to Th2). However, it is not only IFN-γ that is involved in Th1 but also other cytokines such as IL-12, tumor necrosis factor-α, and IL-2 [45]. Since Th2 cytokines are also associated with IL-10 and IL-13 expression in addition to IL4 expression [46], their levels cannot be simply evaluated by the Th1/Th2 ratio. In addition, since Th17 and regulatory T cells also share a complex association [47], it is necessary to investigate the correlation between various cytokines and granulysin in future studies. In this study, we analyzed the NK cell activity and Th1/Th2 ratio in the peripheral blood of patients. In our previous study, we observed the elevation in the levels of cytotoxic granules and inflammatory cytokines, such as IL-2 and IL-12, as well as in the levels of perforin and granzyme B, at the implantation site rather than systemically in murine miscarriage induced by α-galactosylceramide (α-GalCer) [48]. Nakashima et al. also reported that granulysin produced by uterine NK cells induces the apoptosis of extravillous trophoblasts [25], and 85% of NK cells in the decidua of the uterus contain granulysin, which is twice the proportion observed in the periphery [49]. In future, it will be necessary to investigate local changes in these cells in aPL-positive patients.
We previously reported that activated DCs take up α-GalCer and activate NKT cells via CD1d and IL-12, which results in a miscarriage [48]. In this study, the abundance of uterine NKT cells was observed to increase after miscarriage. These cells express multiple cytokines, such as IL-2 and IFN-γ, along with perforin and granzyme, at high levels. However, the actual effector has not been identified. NKT cells produce granulysin [11], which has been reported to induce miscarriage [26]. Therefore, α-GalCer-induced sterile inflammatory miscarriage may be associated with granulysin expression. The alarmin activity of granulysin [15] may also play a role in this. Alarmins stimulate APCs, and granulysin, which is present at high levels in patients with APS (especially those who test positive for anti-PE antibodies), activates PRR-expressing DCs, which may further enhance granulysin secretion by NKT cells, NK cells, and CTLs, and eventually induce miscarriage via its direct effect on apoptosis.
In an earlier study, patients with APS who tested positive for anti-PE antibody showed high granulysin levels, and when this persisted for more than 12 weeks, the miscarriage rate was found to be high [50]. PE is an anti-inflammatory lipid [51]; therefore, anti-PE antibody-positive patients may show higher levels of inflammation, increased immune activity (including granulysin production), and a higher risk of miscarriage. Further research will be required to investigate this.
Serum granulysin concentration may be useful as a clinical marker for RPL. In our study, if heparin treatment reduced serum granulysin levels, the miscarriage rate was found to be lower than if the levels increased. PE antibody-positive patients had high granulysin levels; therefore, it is possible that granulysin is high in “seronegative” APS. In future studies, we intend to evaluate the effects of granulysin on seronegative APS, for which no treatment has been established, and test the effectiveness of heparin.
Preimplantation genetic testing for aneuploidy is being increasingly performed on patients with RPL, which has helped reduce the proportion of miscarriages of unknown etiologies. However, cases of recurrent miscarriage of unknown etiology continue to be reported, including some cases caused by immunological abnormalities. We have already reported the involvement of anti-C9 antibodies in patients of unknown etiology of recurrent miscarriage [52]. In future, we intend to increase the number of granulysin measurements recorded, establish key clinical thresholds, appropriately identify patients who require heparin, and treat them to reduce the risk of miscarriage. A limitation of this study is its relatively small sample size, which precluded the investigation of anti-β2GPI antibodies or other APS-related antibodies. We intend to increase the sample size in future studies and examine the associations between serum granulysin levels and multiple antibodies.