Platelets Impair Natural Killer Cell Reactivity and Function in Endometriosis Through Multiple Mechanisms

Human Reproduction, Vol.32, No.4 pp. 794–810, 2017 Advanced Access publication on February 9, 2017 doi:10.1093/humrep/dex014 ORIGINAL ARTICLE Gynaecology Platelets impair natural killer cell reactivity and function in endometriosis through multiple mechanisms Yanbo Du, Xishi Liu, and Sun-Wei Guo* Shanghai Obstetrics and Gynecology Hospital, Fudan University Shanghai College of Medicine, 419 Fangxie Road, Shanghai 200011, China *Correspondence Address. Shanghai Obstetrics and Gynecology Hospital, Fudan University Shanghai College of Medicine, Shanghai 200011, China. Fax: 86-21-6345-5090; E-mail: [email protected] Submitted on October 28, 2016; resubmitted on December 27, 2016; accepted on January 13, 2017 STUDY QUESTION: Do platelets have any role in the reduced cytotoxicity of natural killer (NK) cells in endometriosis? SUMMARY ANSWER: Platelets impair NK cell reactivity and function in endometriosis through multiple mechanisms. WHAT IS KNOWN ALREADY: Platelets play an important role in the development of endometriosis, and platelet-derived transforming growth factor-β1 (TGF-β1) suppresses the expression of NK Group 2, Member D (NKG2D) on NK cells, resulting in reduced cytotoxicity in women with endometriosis. STUDY DESIGN SIZE, DURATION: Experiments on mice with induced endometriosis in which either platelets, NK cells or both were depleted and controls (none depleted). In vitro experiments with NK cells, platelets and, as target cells, endometriotic epithelial cell and endometrial stromal cell lines. PARTICIPANTS/MATERIALS SETTING METHODS: Immunohistochemistry analysis of ectopic endometrial tissues from mice with induced endometriosis receiving either platelet depletion (PD), NK cell depletion, or both or none. Immunofluorescence, flow cytometry and gene expression analysis for major histocompatibility complex class I (MHC-I) expression in target cells. Cytotoxicity and degranulation assays and the measurement of interferon (IFN)-γ secretion for the evaluation of NK cytotoxicity. Flow cytometry and gene expression for the expression of NK cell receptors. MAIN RESULTS AND THE ROLE OF CHANCE: PD resulted in significantly reduced lesion weight in mice with induced endometriosis, but NK cell depletion as well as concomitant platelet and NK cell depletion increased the weight, suggesting that the anti-endometriosis effect of PD is mediated, at least in part, by increased NK cell cytotoxicity against endometriotic cells. Co-incubation of target cells with platelets resulted in rapid platelet coating as well as increased MHC-I expression in these cells, effectively providing a cloak of ‘pseudo-self’ to these cells to shield against NK cell lysis. It also reduced the expression of NKG2D ligands MICA and MICB and reduced the NK cell cytotoxicity. In addition, co-incubation of NK cells with platelets impaired the NK cell cytotoxicity as well. This impaired NK cell cytotoxicity was not due to the increased NK cell apoptosis, but, rather, through reduced NK cell degranulation and IFN-γ production, and reduced expression of activating receptors NKG2D and NKp46 and increased expression of inhibitory receptor KIR2DL1 in NK cells. Inhibition of TGF-β1 signaling partially restored the aberrant expression of NKG2D, NKp46 and KIR2DL1, and partially restored the impaired NK cell cytotoxicity induced by activated platelets and their releasate. LARGE SCALE DATA: Not applicable. LIMITATIONS REASONS FOR CAUTION: This study is confined by the limitation of animal and in vitro experimentation and the lack of direct human data. WIDER IMPLICATIONS OF THE FINDINGS: Anti-platelet treatment holds promise in treating endometriosis. STUDY FUNDING/COMPETING INTERESTS: The National Natural Science Foundation of China (81471434 to S.W.G., 81270676 to S.W.G., 81370695 to X.S.L. and 81671436 to X.S.L). None of the authors has anything to disclose. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: [email protected] Platelets impair natural killer cell function 795 Key words: cytotoxicity / endometriosis / IFN-γ / MHC class I / NK cells / NKG2D / platelet / TGF-β1 also expresses higher MHC-I than that of healthy women (Vernet- Introduction Tomas Mdel et al., 2006). Thus, the aberrant expression of NK cell Characterized by lesions of endometrial-like tissue outside of the uter- KIRs and activating receptors may be responsible for decreased NK ine cavity, endometriosis is an estrogen-dependent and inflammatory cell cytotoxicity in women with endometriosis. Conceivably, the disease associated with pelvic pain and infertility (Johnson et al., 2013). reduced cytotoxicity would diminish the removal of ectopic endomet- Despite extensive research, our understanding of the pathogenesis rial tissues, allowing their survival, implantation and proliferation. As and pathophysiology of endometriosis is still fragmentary (Johnson such, impaired NK cell function may play an essential role in the patho- et al., 2013). It has long been suspected that a deficient immune sys- genesis of the disease (Lebovic et al., 2001). Despite all these findings, tem may be involved (Dmowski et al., 1981; 1989). however, the molecular mechanisms underlying the reported dysregu- As a component of innate immunity, natural killer (NK) cells are a lated NK cell surface receptors are poorly understood, and it is subset of lymphocytes that provide the first-line defense against unclear what the factors are in the peritoneal or sera of women with infected or transformed cells by release of cytotoxic granules and cyto- endometriosis that impair the NK cell cytotoxicity (Thiruchelvam kine secretion without prior sensitization (Smyth et al., 2002; Caligiuri, et al., 2015; Jeung et al., 2016). It is even unclear whether depletion of 2008). The function of NK cells is tightly regulated by a combination of NK cells would facilitate the development of endometriosis. functionally opposing cell surface receptors, inhibitory killer We have recently shown that platelets play important roles in the immunoglobulin-like receptors (KIRs) that bind major histocompatibil- development of endometriosis (Ding et al., 2015; Guo et al., 2015; ity complex class I (MHC-I) molecules and protect ‘self’ and activating Zhang et al., 2015, 2016a,b) and found that endometriotic stromal receptors that bind ligands on transformed cells and eliminate them cells release platelet-activating molecules (Guo et al., 2016b). We (Lanier, 2003). Cells with low or absent expression of MHC-I (‘missing have also reported that platelet-derived transforming growth factor-β1 self’), such as tumor cells, will be identified as ‘non-self’ and targeted (TGF-β1) suppresses the expression of NKG2D on NK cells, resulting by NK cells while harmful or transformed cells expressing stress- in reduced cytotoxicity in women with endometriosis, but neutraliza- induced activating ligands (‘induced self’ or ‘altered self’) will turn on tion of TGF-β1 reverses the reduction (Guo et al., 2016c). However, it and energize NK cell activating receptors, resulting in lysis by NK cells is unclear whether the three entities, i.e. endometriotic cells, NK cells (Vivier et al., 2008; Orr and Lanier, 2010). Both activating and inhibi- and platelets, engage in any cross-talk, and, if so, what the molecular tory receptors can transduce, respectively, positive (‘missing self’ or mechanisms and the consequences of the cross-talk between platelets ‘altered self’) or negative (‘self’) signals to regulate NK cell cytotoxicity and NK cells and between platelets and endometriotic cells might be. and cytokine release that kills the cells deemed to be harmful (Cheent Given the well-documented roles of platelets in modulation of NK cell and Khakoo, 2009). Two checkpoints control NK cell activation: the function in tumorigenesis and metastasis (Nieswandt et al., 1999; first checkpoint is represented by ligands for activating receptors that Palumbo et al., 2007; Kopp et al., 2009; Placke et al., 2012a,b,c), it is are expressed de novo by potentially dangerous cells while the second likely that platelets may also modulate NK cell functions in the devel- involves the MHC-I-specific KIRs that sense the loss of ‘self’ labels on opment of endometriosis. these cells (Moretta et al., 2002). We hypothesized that through interaction with either NK cells or In endometriosis, the impaired NK cell cytotoxicity as well as the target cells, platelets and their secreted soluble factors impair NK decreased NK cells in peritoneal fluid (PF) and sera were noted in early cell cytotoxicity by regulating NK cell surface receptors, reducing inter- 1990s (Oosterlynck et al., 1991, 1992; Tanaka et al., 1992; Garzetti feron (IFN)-γ production and attenuation of NK cell degranulation. In et al., 1993; Kikuchi et al., 1993; Somigliana et al., 1996a, b). Peritoneal addition, since activated platelets express large amount of MHC-I fluid (Oosterlynck et al., 1993) and sera (Kanzaki et al., 1992) from (Chapman et al., 2012), the close proximity of NK cells with endome- patients with endometriosis as well as endometriosis-secreted factors triotic cells following adhesion and aggregation of platelets to ectopic (Hirata et al., 1994; Somigliana et al., 1996a, b) have been reported to implants may transfer MHC-I to endometriotic cells, conferring a reduce NK cell cytotoxicity. In addition, the increased expression of ‘pseudo-self’ phenotype and thus subverting NK cell immunosurveil-

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