Neutrophil extracellular traps in diseases of the female reproductive organs

Preventing NETs from forming or accelerating NETs degradation may be a potential therapeutic strategy ( 96 ). Several potential strategies can be considered, for example, treatment with deoxyribonuclease 1 (DNase I), which dissolves NETs or inhibition of PAD4 ( 214 ). Attempts to use NETs for treatment have been described in numerous studies and reviews ( 116 , 215 ). Given that NETs formation stimulated invasion and migration of breast cancer cells, it seems a logical conclusion that inhibition of NETs formation or NETs digestion by DNase I blocks these processes ( 123 ). Treatment with DNase I-coated nanoparticles markedly reduced breast cancer metastasis to the lungs in mice ( 123 ). In a study by Park et al. ( 123 ), the PAD4 inhibitor Cl-amidine reduced NETs formation and blocked the ability of neutrophils to promote tumor invasion. In a study by Qi et al. ( 155 ), degradation of NETs by DNase I also significantly inhibited the formation of breast cancer metastases in the lungs. To increase the efficacy of DNase-related therapy, since DNase is degraded quite rapidly under physiological conditions, Herre et al. ( 216 ) developed an adeno-associated virus (AAV) vector system to deliver mouse DNase I and tried it on a mouse model of metastatic breast cancer. The use of the AAV vector was aimed at prolonging DNAase viability ( 216 ). In addition to reduced breast cancer metastasis to the lungs in mice given the AAV-mDNase I (adeno-associated virus vector system for delivery of murine DNase I), they also observed a lower value of the renal hypoperfusion biomarker, neutrophil gelatinase-associated lipocalin (NGAL), in these mice compared to mice that received DNase without the vector ( 216 ). Sivelestat, an NE inhibitor, has been investigated for use in the treatment of breast cancer, specifically epithelial growth factor receptor 2 (HER2) positive breast cancer ( 217 ). NE interacts with tumor growth factor-α (TGF-α), which is present in breast cancer cells, and inhibiting this interaction would adversely affect tumor cell proliferation ( 217 ). Nawa et al. ( 217 ) showed that the combined use of sivelestat and trastuzumab inhibited cell proliferation more intensively than with either drug alone. Zhu et al. ( 152 ) found that the NF-κB essential modifier-binding domain (NBD) peptide reduced IL-8 levels and NETs formation, resulting in inhibition of primary tumor growth, inhibition of lung metastasis in mouse models of human breast cancer and in a mouse model of spontaneous breast cancer. Also, inhibition of NETs production by the PAD4 inhibitor reduced NF-κB activation, resulting in reduced metastasis ( 152 ). Tang et al. ( 166 ) showed that affecting cholesterol biosynthesis could be a therapeutic strategy for breast cancer. This is because cholesterol biosynthesis induced by ASPP2 depletion in mouse breast cancer cells and human breast cancer cell cultures promoted the formation of NETs in vitro , as well as in breast cancer metastasis to the lungs in ASPP2-deficient mice ( 166 )Cholesterol biosynthesis is also a positive regulator of CCDC25 expression, and increased CCDC25 expression is associated with breast cancer metastasis ( 166 ). Simvastatin and berberine, inhibitors of cholesterol synthesis, effectively blocked NETs formation induced by ASPP2 depletion, which may have therapeutic effects on breast cancer metastasis ( 166 ). Yu et al. ( 218 ) examined resveratrol (RES), a polyphenolic natural phytoalexin and silent information regulator-1 (SIRT1) agonist, which inhibited NETs formation after CTSC treatment. In in vivo studies, RES impeded the formation of breast cancer metastases in a mouse model of breast cancer ( 218 ). Also, serum levels of NETs markers, MPO-DNA and NE-DNA in the mouse model of breast cancer were significantly lower after treatment ( 218 ). RES, among other things, inhibits histone H3 citrullination, which is essential for NETs formation ( 218 ). The researchers also found that NETs were suppressed by RES in bone marrow neutrophils after CTSC treatment, while specific SIRT1 deficiency in neutrophils promoted their formation, and thus breast cancer metastasis to the lungs ( 218 ). Zeng et al. ( 219 ) studied kaempferol, a flavonoid, and found that it had an inhibitory effect on primary tumor growth and lung metastasis in a mouse model of breast tumor. After treating lung metastases with the compound, they also observed reduced expression of citH3, a biomarker of NETs ( 219 ). The researchers also found that kemferol is specific for NETs, with no effect on neutrophils ( 219 ). Lu et al. ( 220 ) developed a micellar nanoparticle of low-molecular-weight heparin and astaxanthin (LMWH-AST/DOX, LA/DOX NP) loaded with doxorubicin, which has the ability, among other things, to reduce the recruitment of neutrophils in the liver and myeloid-derived suppressor cells (MDSCs) in the lung and tumor by blocking P-selectin. The nanoparticle has the ability to inhibit the formation of NETs, thereby inhibiting breast cancer metastasis to the lung and liver ( 220 ). Zhao et al. ( 221 ) examined the effects of dihydrotanshinone I (DHT), a compound derived from Salvia miltiorrhiza Bunge ( S. miltiorrhiza ) on breast cancer. In their study, DHT inhibited the formation of NETs and attenuated breast cancer metastasis to the lungs induced by NETs ( 221 ). Metastasis to the omentum, a common occurrence in ovarian cancer, was reduced in mice deficient in neutrophil-specific PAD4 ( 174 ). Blocking NETs formation with a pharmacological PAD4 inhibitor also reduced omentum colonization ( 174 ). Doxorubicin (DOX), used in the treatment of ovarian cancer, is captured by NETs, preventing the substance’s therapeutic effect of inducing apoptosis of tumor cells ( 222 ). Tamura et al. ( 222 ) demonstrated that the reduced diffusion of the drug was restored after degradation of NETs by DNAase I. Ning et al. ( 42 ) demonstrated that digestion of NETs with DNAase 1 or inhibition of TLR2 with chloroquine eliminated the metastatic potential of cervical cancer, as observed by reduced metastasis to inguinal lymph nodes. No studies combining the inhibition of NETs formation and endometrial cancer have been conducted to date.

Neutrophils are the main, physiologically most abundant leukocyte population in peripheral blood in adults (50-70%), where they are present for about 12 hours ( 1 – 3 ). The neutrophil population can be divided into three main groups: bone marrow reserve, circulating and located in peripheral tissues ( 4 ). They are produced in the bone marrow within hematopoietic cords surrounded by venous sinusoids, while they arise from stem cells that proliferate and differentiate into mature neutrophils equipped with granules ( 1 , 5 ). Granules can be divided into primary (azurophils), secondary (specific) and tertiary (gelatinase) ( 6 ). Primary granules consist mainly of myeloperoxidase (MPO) and neutrophil serine proteases (NSPs) ( 6 ). NSPs include neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G and neutrophil serine protease-4 (NSP4) ( 7 ). Secondary granules contain lactoferrin, lysozymes, and pentraxin 3, while tertiary granules consist of matrix metalloproteinase-9 (MMP-9) and antimicrobial substances, including cathelicidin ( 6 , 8 ), which have been shown on the Figure 1 . Neutrophil granules The figure shows a neutrophil and the division of its granules, along with examples of substances that belong to them. The activity of neutrophils is the basis for the operation of the innate immune response, as they are among the first cells of the immune system to respond to pathogens (including bacteria, fungi and protozoa) ( 9 , 10 ). The life cycle of neutrophils and their maturation is associated with their acquisition of functions, and as the main effector cells of the immune system, they have numerous capabilities to combat pathogens: phagocytosis, migration, production of reactive oxygen species (ROS), degranulation and, consequently, release of cytotoxic granule components and recruitment of other immune cells ( 1 ). Neutrophils can shape the inflammatory and immune response through production of cytokines and chemokines, including, among others: tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), interleukin-1 receptor antagonist (IL-1Ra), interleukin 6 (IL-6), and interleukin 8 (IL-8) ( 11 – 13 ). They also have the ability to form neutrophil extracellular traps (NETs) ( 13 ). The neutrophil functions are shown in Figure 2 . Neutrophils’ functions. The figure shows neutrophil functions, which include degranulation, phagocytosis, activation, formation of NETs, production of chemokines and cytokines, and crawling. In recent years, attention has been drawn to the significant role of neutrophils, not only in fighting pathogens but also in the pathomechanism of cancer. It has been shown that in addition to macrophages, subpopulations of T lymphocytes, B lymphocytes, dendritic cells or Natural Killer cells (NK cells), tumor-associated neutrophils (TAN) are an important component of the tumor microenvironment (TME) ( 14 – 16 ). A study by Fridlender et al. ( 17 ) showed the existence of at least two different populations of tumor-associated neutrophils: pro-tumor and anti-tumor. The dichotomous role of neutrophils depends on cytokine signaling and epigenetic modifications and is enabled by signals from tumor cells or cells within the tumor microenvironment ( 2 , 18 ). Tumor-derived factors and the tumor microenvironment have been shown to have the ability to reprogram neutrophils from an anti-tumor phenotype to a pro-tumor phenotype ( 19 , 20 ). Tumor-derived cytokines: transforming growth factor-beta (TGF-β), granulocyte colony-stimulating factor (G-CSF), and interferon-beta (IFN-β) are involved in neutrophil polarization ( 21 ). G-CSF secreted by tumor cells can alter the hematopoietic function of the bone marrow and promote neutrophil differentiation toward the N2 phenotype ( 20 ). TGF-β activates the tumor-promoting neutrophil program, i.e., pro-tumor polarization, while IFN-β promotes the opposite process, i.e., anti-tumor polarization ( 21 ). The N1, “anti-tumor” neutrophil phenotype promotes tumor suppression ( 20 ). Studies indicate that in the pre-metastatic niche, factors such as TGF-β, for instance, hinder the emergence of the N1 phenotype, thereby preventing extensive killing of tumor cells ( 20 ). Antitumor neutrophils can directly kill tumor cells by releasing ROS and reactive nitrogen species (RNS) ( 14 ). Neutrophils can recruit other immune cells to the TME, including M1 macrophages with pro-inflammatory and anti-tumor activity ( 14 , 22 , 23 ). Neutrophils are able to inhibit metastasis through cytotoxicity towards tumor cells in the circulation or in the pre-metastatic niche and by stimulating T cells proliferation ( 14 , 22 ). They also have the ability to present antigens to T cells and to produce interferon- gamma (IFN-γ) ( 14 ). In the presence of cytokines such as TGF-β, available in high concentrations at the primary tumor site, neutrophils acquire a pro-tumor phenotype - N2 ( 24 ). The neutrophil N2 phenotype is shaped by the premetastatic microenvironment and may promote tumor cell dissemination and progression ( 20 ). Protumorigenic neutrophils actively support metastasis through various mechanisms, including the formation of a premetastatic niche, attraction of tumor cells and direct promotion of tumor cell proliferation ( 22 ). The influence on the immunosuppressive environment in the pre-metastatic niche is related to the ability to secrete arginase to degrade arginine, which is crucial for the effectiveness of tumor killing by T cells ( 20 ). Protumor neutrophils can release MMP-9, which promotes angiogenesis and tumor cell proliferation and can suppress NK cells function ( 14 ). Neutrophils recruit other immune cells that can have dual effects on the TME, for example, anti-inflammatory and pro-tumor M2 macrophages and regulatory T cells ( 14 , 23 ). The role of pro-tumor and anti-tumor neutrophils is shown in Figure 3 . The role of pro-tumor and anti-tumor neutrophils The figure presents two neutrophil phenotypes: the anti-tumor phenotype (N1) and the pro-tumor phenotype (N2), and the role they may play in carcinogenesis. The discovered role of neutrophils in the following diseases is described below: breast, ovarian, cervical cancer, corpus uteri cancers, premature ovarian failure and endometriosis. Yin et al. ( 25 ) detected TAN-related genes and investigated their association with breast cancer. Patients with these genes showed tumor immunosuppression and adverse therapeutic effects ( 25 ). TANs correlate with poor breast cancer prognosis ( 25 , 26 ). High TAN density correlates with unfavorable prognostic factors in breast cancer such as: large tumor size, type and unfavorable histological grade, high rate of lymph node metastasis, advanced stage of disease, breast cancer subtype and selected mutations: MAP3K1, ERBB2 and TP53 ( 26 ). TANs are able to secrete oncostatin M, which promotes the secretion of VEGF in the environment of human breast cancer cells and increases their ability to invade ( 27 ). G-CSF secreted by tumor cells stimulates pro-tumorigenic neutrophils functions in invasive breast cancer ( 21 ). Breast cancer patients with circulating tumor cell and neutrophil aggregates showed worse progression-free survival than patients without such aggregates ( 21 ). Improved survival has been demonstrated in patients with breast cancer and other cancers who experience neutropenia during chemotherapy, which may be due to a reduction in pro-metastatic neutrophils ( 28 ). In luminal A and luminal B subtypes, researchers found no association between neutrophil-to-lymphocyte ratio (NLR) and overall survival in breast cancer patients ( 29 ). However, a correlation was detected in the analyses of HER2-positive breast cancer and triple negative breast cancer (TNBC) ( 29 ). NLR is currently used as a predictor of overall mortality and cancer-free survival ( 30 ). However, it should be noted that the increase in the number of neutrophils is not specific to the cancer process, it is also observed in infections and inflammation ( 31 , 32 ). NLR therefore reflects well the inflammation that plays an important role in the progression of some cancers and the formation of metastases ( 29 ). In ovarian cancer, it has been shown that neutrophils can have a deregulating effect on the immune system, potentially contributing to the progression and metastatic potential of cancer cells ( 33 ). Elevated NLR ratio in ovarian cancer patients before treatment may be a predictor of poor disease prognosis ( 31 ). NLR was significantly higher in patients with ovarian cancer compared to patients with benign ovarian tumor, other gynecological diseases and healthy individuals ( 34 – 36 ). Elevated TAN levels are associated with poor prognosis and immune tolerance in ovarian cancer ( 34 ). Mayer et al. ( 35 ) demonstrated that neutrophils in the ovarian cancer microenvironment can shift tumor cells toward a mesenchymal and migratory phenotype. Interestingly, this effect was observed after incubation of cancer cells with neutrophil elastase, which is also a component of NETs ( 35 ). In cervical cancer, neutrophils have been shown to possibly contribute to the progression and metastatic potential of cancer cells ( 33 ). A significant association has been demonstrated between neutrophilia and advanced cervical cancer ( 37 ). More than 10% of cervical cancer patients experienced tumor-related leukocytosis (TRL) detected at initial diagnosis ( 38 ). Carus et al. ( 39 ) demonstrated that the number of TAN is an independent prognostic factor for short recurrence-free survival in localized cervical cancer. Elevated NLR was associated with worse overall patient survival and shorter progression-free survival in patients with cervical cancer ( 40 ). NLR can therefore be used as a prognostic indicator in patients with cervical cancer ( 41 ). However, its prognostic value may be higher in locally advanced and/or advanced cervical cancer compared to patients with early stage disease ( 33 ). High density of infiltrating neutrophils in cervical cancer tissues was associated with poor prognosis ( 42 ). Srisutha et al. ( 43 ) showed that in patients diagnosed with uterine leiomyosarcoma, the NLR was significantly higher than in patients diagnosed with uterine leiomyoma. NLR is therefore an effective marker of prediction the presence of uterine leiomyosarcoma in patients preoperatively diagnosed with a uterine tumor ( 43 ). Premature ovarian insufficiency (POI) is caused by a decline in ovarian function due to premature depletion of follicles ( 44 ). The NLR ratio was statistically higher in the POI group compared to the control group and also correlated with follicle-stimulating hormone (FSH) and Anti-Müllerian Hormone (AMH) ( 45 ). Ilhan et al. ( 45 ) proved that it can be a marker for POI diagnosis. It has been shown that in the circulatory system and peritoneal fluid of patients with endometriosis there is an increased number of neutrophils and cytokines released by them, which promotes endometrial cell proliferation and invasion ( 46 – 48 ). Comparison of NLR values ​​with CA125, endometriosis stage and painful menstruation, after taking into account previous therapy, did not show any significant association ( 46 ). However, an association between NLR and chronic pelvic pain has been demonstrated ( 46 ).

Initially, numerous observations indicated undoubtedly positive aspects of NETs formation, however, as it results from the studies conducted so far, their formation may also accompany the pathogenesis of many diseases, including diseases of the female reproductive organs, in which excessive or chronic NETs formation or their improper/abnormal removal has been demonstrated. Moreover, the conducted studies do not fully explain the causes of interactions between cancer cells and NETs, which may prove helpful in explaining and understanding many aspects of the body’s immune response against cancer cells, as well as in developing new diagnostic and therapeutic strategies in patients with breast cancer and gynecological cancers. The few studies on the role of NETs in the course of other reproductive organ diseases also indicate their participation in the pathogenesis of these diseases, which requires further, more detailed research taking into account the importance of NETs as potential biomarkers and their use in therapy.