Association of neutrophil extracellular traps with endometriosis-related chronic inflammation

European Journal of Obstetrics & Gynecology and Reproductive Biology 183 (2014) 193–200

Contents lists available at ScienceDirect

European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb

Association of neutrophil extracellular traps with endometriosis-related chronic inflammation Eniko Berkes a,*, Frank Oehmke a, Hans-R. Tinneberg a, Klaus T. Preissner b, Mona Saffarzadeh b,1 a b

Department of Obstetrics and Gynaecology, Justus-Liebig-University, 35392 Giessen, Germany Institute of Biochemistry, Medical School, Justus-Liebig-University, 35392 Giessen, Germany

A R T I C L E I N F O

A B S T R A C T

Article history: Received 17 July 2014 Received in revised form 21 September 2014 Accepted 22 October 2014

Objective: To study if neutrophil extracellular traps (NETs) are present in the peritoneal fluid of endometriosis patients. NETs play a crucial role in fighting against microorganisms. However, exaggerated NET production may lead to tissue damage in their vicinity in pathological conditions. Our study evaluates the presence of NETs in endometriosis peritoneal fluid. Study design: Peritoneal fluid (PF) was collected in a case-control study from 52 women, who underwent either diagnostic or operative laparoscopy. The control group consisted of 17 women with infertility, chronic pelvic pain, simple or functional cysts or irregular bleeding. The endometriosis group, altogether 35 patients, comprised 19 patients with stage I and II and 16 patients with stage III and IV endometriosis. First we tested whether the PF is able to stimulate NET production. Neutrophils from healthy volunteers were treated with the PF of endometriosis patients and controls and NETs were detected with Sytox orange extracellular DNA dye and immunofluorescence microscopy. Then we evaluated if NETs were already present in the collected PF using the specific myeloperoxidase (MPO)-DNA capture ELISA method, based on the MPO associated with the NET scaffold. Results: The PF of endometriosis patients did not stimulate NET release from healthy granulocytes. However, pre-existent NETs could be detected in 17 endometriosis patients out of 35 (49%). In contrary, in the control group NETs were present in only 3 patients out of 17 (18%), (p = 0.03, OR: 4.4). Moreover, the quantification of NETs showed a significantly higher amount of NETs in endometriosis compared to the controls (0.097 vs. 0.02, p = 0.04). Conclusion: This is the first study, which evaluated and described the presence of NETs in the PF of endometriosis patients. Our study shows, that NETs may be involved in the complex pathophysiology of endometriosis. ß 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Endometriosis Neutrophils Neutrophil extracellular trap Inflammation Innate immunity

Introduction Endometriosis affects approximately 10% of women in the reproductive age [1] and negatively influences quality of life. Retrograde menstruation is still the most prevalent pathogenesis hypothesis; however, the formation of endometriotic lesions depends on the attachment, survival, invasion, and proliferation of the respective cells [2]. The ectopic lesions cause a local inflammation of varying intensity, in which activation of leukocytes, macrophages, and natural killer cells are well

* Corresponding author. Tel.: +0049 176 61364497; fax: +0049 64415566127. E-mail address: [email protected] (E. Berkes). 1 Present address: Center for Thrombosis and Hemostasis (CTH), University Medical Center, 55122 Mainz, Germany. http://dx.doi.org/10.1016/j.ejogrb.2014.10.040 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.

documented [3,4]. However, the involvement of neutrophils in the pathomechanism of endometriosis has not been sufficiently explored. Neutrophil granulocytes are considered the first line of defense against microorganisms [5]. Their classical killing mechanisms are the phagocytosis and intracellular killing [6], where the subsequent apoptosis and clearance by macrophages [7–9] protect the surrounding tissue against the noxious components. A decade ago a novel extracellular killing mechanism was described in which activated neutrophils may expel their entire chromatin, which is scattered with intracellular proteins, serving as catch and kill scaffold against microorganisms [10]. The procedure was designated as NETosis and the expelled structure as neutrophil extracellular traps (NETs). More than 20 different proteins were identified in NETs, originating from the granules, cytoplasm,

194

E. Berkes et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 183 (2014) 193–200

cytoskeleton and peroxisome [11,12]. During NETosis the crucial steps are chromatin decondensation, disintegration of intracellular membranes and release of chromatin threads with the associated proteins [13]. NET formation has an unambiguously beneficial role during infection, since deficiency in NET production, e.g. in chronic granulomatosus disease [14,15], or degrading the scaffold by bacterial DNases may lead to severe infections [16–18]. However, the excessive formation of NETs might harm the healthy tissue in their vicinity, as it has been described in acute lung injury [12,19,20], cystic fibrosis [21,22], asthma [23], psoriasis [24], thrombosis [25–27], preeclampsia [28], appendicitis [10], sepsis [29], Crohn’s disease [30], systemic lupus erythematosus (SLE) [31–34] and small vessel vasculitis [35]. Based on the nature of endometriosis, which demonstrates similarities with chronic inflammatory and autoimmune disorders [36], we postulated that NETs might play a role in its pathophysiology. Our study is the first that evaluates the presence of NETs in the peritoneal fluid (PF) of endometriosis patients.

phenol-red free RPMI 1640 medium (Invitrogen, Germany) for further analysis. Treatment of neutrophils and quantification of NETs Approximately 105 neutrophil cells from healthy donors were seeded per well on a 94-well plate and treated with 100 ml PF from either the control or the endometriosis group for 3 h at 37 8C. As a positive control neutrophils were stimulated with phorbol myristate acetate (PMA) as a typical inducer of NETosis [10]. For negative control, RPMI 1640 Medium (Invitrogen) was added to the neutrophils. Each well was stained with 50 ml of 5 mM Sytox Orange (Invitrogen), which is a cell-membrane impermeable DNA stain, in phosphate buffered saline (PBS) for 10 min at room temperature. Afterwards, the excess of Sytox Orange was removed by centrifugation of the plate at 250  g for 10 min, and 50 ml from the supernatants was discarded. The fluorescence intensity was measured at excitation and emission wavelengths of 545 nm and 590 nm, respectively (FLx 800 fluorescence microplate reader; BIO-TEK Instruments).

Materials and methods Immunofluorescence microscopy of neutrophils treated with PF Patients and controls Fifty-five women were included in a case-control study, who underwent either diagnostic or operative laparoscopy between January 2013 and February 2014 at the Justus-Liebig University, Giessen, Germany. The ethical committee approved the study (95/ 09). Laparoscopy was performed and the PF was aspirated immediately after entering the abdominal cavity, collected in EDTA syringes, aliquoted and stored at 20 8C. The control group contained 19 women, on whom either diagnostic laparoscopy due to infertility or chronic pelvic pain or operative laparoscopy, such as tubal sterilization, removal of simple or functional cysts or hysterectomy due to irregular bleeding was performed. One patient was excluded from the control group because of severe hematometra and pelvic inflammation and another one due to pedicle torsion of the adnex and concomitant acute inflammation. The endometriosis group, altogether 36 patients with histologically confirmed endometriosis, was composed of 19 stage I and II and 17 stage III and IV patients. One patient from the stage III and IV group had to be excluded because of Fitz-Hugh-Curtis Syndrome. The patients were scored according to the revised American Society of Reproductive Medicine (rASRM) criteria and deep infiltrating endometriosis (DIE) was described with the ENZIAN System. Within the endometriosis group 22 patients had DIE. We did not include patients in the study groups who had concomitant gynecological conditions, such as cystadenoma, dermoid cysts, borderline or malignant tumors, fibroids or polycystic ovarian syndrome, which may influence the results. Medical history, body mass index (BMI) and white blood cell count (WBC) were recorded and the PF was cultured for microorganisms (Table 1). Neutrophil isolation Human neutrophils from healthy volunteers were isolated using density gradient separation as described previously [12,37]. Briefly, a double gradient was formed by layering an equal volume of histopaque-1077 over histopaque-1119 (SigmaAldrich, Germany). Venous blood was collected in EDTA tubes and layered onto the upper histopaque-1077, by centrifugation at 700  g for 30 min a Granulocytes were concentrated at the 1077/ 1119 interphase. Cell viability was determined to be 98% by trypan blue dye exclusion. The isolated neutrophils were resuspended in

Isolated neutrophils from healthy donors were seeded on coverslips and treated either with the PF of endometriosis patients or kept untreated for 3 h at 37 8C. Afterwards, the cells were fixed with 2% paraformaldehyde and blocked with 3% bovine serum albumin in PBS. For NET detection, the neutrophils were incubated with primary mouse anti-DNA Histone H1 (Millipore, Germany), followed by detection with the secondary antibody coupled to Alexa Fluor 555 donkey anti-mouse IgG (Invitrogen, Germany), and 40 ,6-diamidino-2-phenylindole (DAPI) (Vectashield mounting medium with DAPI; Vector Laboratories, Burlingame, CA, USA) was used for nuclear DNA detection. Images were taken with fluorescence microscope using MetaMorph imaging software version 7.0 (Leica Microsystems, Wetzlar, Germany). Myeloperoxidase (MPO)-DNA ELISA To detect the preexistent NETs in the PF, we used the MPO-DNA capture ELISA method according to Caudrillier and co-workers based on MPO association with NETs [19]. Prior to assessment PF samples were pretreated with 500 U/ml micrococcal nuclease (MNase) from Staphylococcus aureus (Sigma-Aldrich) in order to partially digest NETs and to obtain smaller DNA-MPO particles for the assay. For the capture antibody, 5 mg/ml anti-MPO mAb (Millipore) was coated onto a 96-well plate (dilution 1:500 in 50 ml) overnight at 4 8C. After washing the Plate 3 times (300 ml each), the plate was coated with 1% bovine serum albumin (BSA) in PBS for 1 h. Thereafter, 20 ml of the MNase-treated PF samples was added to the wells with 80 ml incubation buffer containing a peroxidase-labeled anti-DNA mAb (Cell Death ELISAPLUS, Roche; dilution 1:25). The plate was incubated for 2 h, shaking at 300 rpm at room temperature. After 3 washes (300 ml each), 100 ml of peroxidase substrate was added and absorbance at 405-nm wavelength was measured after 20 min of incubation at room temperature in the dark. Statistical analysis The descriptive parameters, e.g. age, BMI, smoking habits and WBC were tested for normality and compared using the Smirnov– Kolmogorov test. In the Sytox Orange analysis the results were expressed as relative absorbance increases compared to the vehicle (sample-vehicle/vehicle) and the not normally distributed data were compared with the Mann–Whitney-U test. In the MPODNA ELISA the percentage of NET positive patients was determined

E. Berkes et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 183 (2014) 193–200

195

Table 1 Characteristics of the patients included in the study, rASRM: revised American Society of Reproductive Medicine score, DIE: deep infiltrating endometriosis, PE: previous endometriosis surgery, range: 10 and 90 percentiles, cig: cigarette, d: day. Control

Endometriosis

Endometriosis rASRM stage I and II

Endometriosis rASRM stage III and IV

DIE

17

35 22 (63%) with DIE 21 (60%) with PE

19 8 (42%) with DIE 10 (53%) with PE

16 14 (88%) with DIE 11 (69%) with PE

22

32 (26–41) 22 (21–26) 7.1 (5.3–9.1)

32 (24–42) 22 (18–28) 7.3 (5.7–11.0)

29 (24–44) 22 (19–26) 7.0 (5.1–10.0)

36 (31–40) 22.5 (18–29) 7.4 (6.0–10.0)

35 (29–43) 22.5 (18–28) 7.4 (5.7–10.0)

10 (59%) 1 (6%) 2 (12%) 4 (23%)

21 (60%) 3 (9%) 4 (11%) 7 (20%)

11 (58%) 2 (11%) 4 (21%) 2 (10%)

10 (63%) 1 (6%) 0 (0%) 5 (31%)

14 (64%) 2 (9%) 0 (0%) 6 (27%)

18 (51%) 13 (72%) 3 (17%) 2 (11%) 6 (17%) 9 (26%) 1 (3%)

12 (63%) 9 (75%) 1 (8%) 2 (17%) 3 (16%) 4 (21%) 0 (0%)

6 (38%)

Not known

11 (64%) 5 (45%) 6 (55%) 0 (0%) 0 (0%) 3 (18%) 1 (6%) (menometrorrhagie) 2 (12%)

1 (3%)

0 (0%)

3 (19%) 5 (31%) 1 (6%) (hysterectomised) 1 (6%)

9 (41%) 6 (67%) 2 (22%) 1 (11%) 5 (23%) 6 (27%) 1 (4.5%) (hysterectomised) 1 (4.5%)

Indication of surgery

8 (47%) pelvic pain

19 (54%) pelvic pain 17 Pelvic pain 2 Pelvic pain and irregular bleeding

11 (58%) pelvic pain

8 (50%) pelvic pain

10 Pelvic pain 1 Pelvic pain and irregular bleeding

7 Pelvic pain 1 Pelvic pain and irregular bleeding

15 (68%) pelvic pain 13 Pelvic pain 2 Pelvic pain and irregular bleeding

7 (20%) Infertility

6 (32%) Infertility

1 (6%) Infertility

1 (4.5%) Infertility

1 (6%) Infertility and pelvic pain

7 (20%) Infertility and pelvic pain

2 (10%) Infertility and pelvic pain

5 (31%) Infertility and pelvic pain

5 (23%) Infertility and pelvic pain

3 (18%) Other 1 Hydrosalpinx

2 (6%) Other 2 Asymptomatic cyst on ultrasound

2 (13%) Other 2 Asymptomatic cyst on ultrasound

1 (4.5%) Other 1 Asymptomatic cyst on ultrasound

Number of Patients

Median (and range) of age Median (and range) of BMI Median (and range) of WBC at sample collection (G/l) Smoking habits No smoking Occasionally (<1 cig/d) Regularly (1-9 cig/d) Heavy smoker (>10 cig/d) Cycle Characteristics regular (cycle 24–32 d) Proliferative Secretory Not known Irregular (>32 d) Hormonal contraception Other

4 Pelvic pain 1 Pelvic pain and irregular bleeding 3 Pelvic pain and cyst on ultrasound 5 (29%) Infertility 4 Infertility 1 Infertility and path.PAP smear

4 (67%) 2 (33%) 0 (0%)

16 (73%) with PE

1 CIN-III 1 Wish after sterilisation Operative characteristics

8 (47%) Diagnostic laparoscopy 7 Diagnostic laparoscopy 1 Diagnostic laparoscopy and cervical biopsy 9 (53%) operative laparoscopy 5 cystectomy 1 Salpingectomy

1 Tubal sterilisation 1 Cystectomy and salpingectomy 1 TLH

11 (31%) Peritoneal endometriosis

11 (58%) Peritoneal endometriosis

0 (0%) Peritoneal endometriosis

0 (0%) Peritoneal endometriosis

1 (3%) Peritoneal and ovarian endometriosis 1 (3%) Ovarian endometriosis

0 (0%) Peritoneal and ovarian endometriosis 0 (0%) Ovarian endometriosis

1 (6%) Peritoneal and ovarian endometriosis 1 (6%) Ovarian endometriosis

0 (0%) Peritoneal and ovarian endometriosis 0 (0%) Ovarian endometriosis

12 (34%) Ovarian endometriosis and DIE

1 (5%) ovarian Endometriosis and DIE

11 (69%) Ovarian endometriosis and DIE

12 (55%) Ovarian endometriosis and DIE

10 (29%) DIE

7 (37%) DIE

3 (19%) DIE

10 (45%) DIE

in each study group and the categorical values were analysed with Pearson’s Chi-Square test. The amounts of detected NETs, as nominal values not following the normal distribution, were compared using the Mann–Whitney-U test. Significance level was set at p  0.05.

Results The study groups were statistically equal regarding age, BMI, WBC and smoking habits. None of the patients had positive peritoneal fluid culture for microorganisms.

196

E. Berkes et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 183 (2014) 193–200

Fig. 1. Increased amounts of extracellular DNA in endometriosis stage III and IV patients with Sytox Orange Intensity Analysis. The isolated human healthy neutrophils have been stimulated with peritoneal fluid of controls and endometriosis (E) stage I and II and stage III and IV patients. The extracellular DNA has been measured with Sytox Orange (SO), a cell membrane impermeable DNA stain. Presented are the relative increases in SO intensity (sample-vehicle/ vehicle) with median, 25 and 75 percentile data. Although the median relative increase of SO intensity, which reflects the amount of extracellular DNA, was higher in the endometriosis stage III and IV group (1.055) compared to the controls (0.87), it did not reach statistical significance (p = 0.31). The endometriosis stage I and II group (0.71) did not differ from controls (0.87). rASRM: revised American Society of Reproductive Medicine Score.

First, we tested whether the PF of endometriosis patients is able to stimulate NET release from healthy neutrophil granulocytes. The results are presented in Fig. 1. Although the median Sytox Orange intensity, which reflects the amount of extracellular DNA, was higher in the endometriosis stage III and IV group (1.055) compared with controls (0.87), it did not reach statistical significance (p = 0.31). The endometriosis stage I and II group did not differ from controls (p = 0.5). The Sytox Orange dye detects extracellular DNA, which might originate either from NETosis of stimulated neutrophils or from other sources of free DNA that could already be present in the PF, including preexistent NETs. Therefore, immunofluorescence microscopy of neutrophils – either treated with PF of endometriosis patients or kept untreated – by using a DNA/ histone antibody was performed. Interestingly the PF of endometriosis patients was not capable to induce NET formation of healthy granulocytes. Fig. 2 shows a representative staining of healthy neutrophils treated with PF of an endometriosis stage III and IV patient compared with the staining pattern of untreated neutrophils and neutrophils treated with PMA, a typical NET inducer. Considering the results of both analyses it appears that either free DNA or NETs may already be present in the PF of endometriosis patients. Thus, PF was assessed by MPO-DNA ELISA, which is specific in detecting NETs based on the association of MPO and DNA in the NET scaffold. The results are presented in Fig. 3. NETs were detected in only 3 patients out of 17 (18%) in the control group, whereas 17 patients out of 35 (49%) contained NETs in the endometriosis PF. Both the frequency of NET positive patients (49% vs. 18%) and the mean amount of the detected NETs (0.097 vs. 0.02)

Fig. 2. Inability of peritoneal fluid from endometriosis patients to induce NET formation in healthy neutrophils. Isolated neutrophils from healthy donors were either treated with peritoneal fluid (PF) from endometriosis patients or kept untreated for 3 h. Immunofluorescence staining of cells was performed for DNA/histone (red), and for nuclear DNA with 40 ,6-diamidino-2-phenylindole (blue). Healthy neutrophils, treated with PF of endometriosis patients did not form NETs. Presented are the untreated neutrophils (A), neutrophils treated with PF of an endometriosis stage III and IV patient (EPF) (B) and neutrophils treated with a typical NET inducer, phorbol-miristate acetate (PMA), as a positive control (C).

E. Berkes et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 183 (2014) 193–200

197

Fig. 3. Neutrophil extracellular traps (NETs) in the peritoneal fluid (PF) of endometriosis patients. NETs in the PF of endometriosis patients and controls were detected using MPO-DNA ELISA. Presented are the percentages of NET positive patients and the quantification (in absolute absorbance increase) of the NET scaffold (mean, 10 and 90 percentile data). (A) Percentage and quantity comparison of NET positive PF in endometriosis stage I to IV patients with controls. The frequency of NET positive patients, as well as the amount of NETs were significantly higher in the endometriosis group. (B) Percentage and quantity comparison of NET positive PF in endometriosis stage I and II, stage III and IV and DIE groups with controls. All the respective endometriosis groups had remarkable higher percentage of NET positive patients and higher amount of NETs in the PF compared to controls. The most abundant results were detected in the stage I and II group, where the comparison with controls reached statistical significance. Statistical significance was set on p  0.05. rASRM: revised American Society of Reproductive Medicine Score, E: endometriosis, DIE: deep infiltrating endometriosis.

198

E. Berkes et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 183 (2014) 193–200

were significantly higher in the endometriosis group (p = 0.03, p = 0.04, respectively). Regarding the specific endometriosis stage I and II and stage III and IV groups we could overall observe a remarkably higher percentage of NET-positive patients (53%, 44%, respectively) compared to controls (18%). Moreover, beside the increased frequency of NET positivity in the endometriosis groups, we detected a higher mean amount of NETs in the stage I and II and stage III and IV groups (0.088, 0.108, respectively) compared to controls (0.02). The presence of NET was most frequent in the endometriosis stage I and II group, significantly more than in controls (p = 0.03). Although the percentage of NET positive patients was clearly higher in the stage III and IV group compared to controls, the statistical comparison was not significant (p = 0.14), presumably because of the sample size. In the endometriosis stage I and II group the mean amount of NETs were significantly higher compared to controls (p = 0.04) and in the stage III and IV group the comparison with controls almost reached statistical significance (p = 0.08). Among the DIE patients the frequency of NET positive patients (45%) as well as the average amount of the NET structure (0.106) were notably higher compared to the controls (p = 0.07 and 0.06, respectively). Comments Our study is the first, which evaluated the NET formation in endometriosis. In 49% of endometriosis patients NETs were present in the PF, whereas the controls had rarely showed NET formation. Neutrophils and their functions have not been widely analysed in endometriosis so far. An increased neutrophil-to-lymphocyte ratio in the blood [38], as well as an increased amount of neutrophils in the PF has been found in endometriosis [39,40]. This might be a consequence of a reduced apoptosis rate, since it has been described that the addition of plasma and PF from endometriosis patients to an in-vitro culture of neutrophils significantly reduces the percentage of apoptotic cells [41]. Moreover, increased amounts of neutrophil chemoattractans, such as IL17A and IL-8, have been detected in endometriosis [40,42,43]. There are even fewer studies focusing on the neutrophil functions in endometriosis. Endometriosis has been found to be associated with increased PF amount of a-defensin [40], myeloperoxidase and lactoferrin, which all of them reflect neutrophil activation [44]. In a mouse model of endometriosis a remarkable infiltration of the ectopic lesions with vascular endothelial growth factor secreting neutrophils and macrophages occurred within the first 5 days after implantation and angiogenesis were initiated consecutively [45]. The authors postulated that in the early stage of endometriosis the infiltration of neutrophils and macrophages is responsible for tissue survival and neoangiogenesis. NET formation plays a crucial role in fighting against infections. However, elevated NET formation may cause tissue damage and impair cell function. SLE patients, carrying auto-antibodies against NET proteins and DNA, are enable to degrade NETs [34]. In transfusion-related acute lung injury, NETs have been found in the lung and in the blood of patients. Inhalation of DNase has been suggested to prevent alveolar NET accumulation in these patients [19,46]. It seems that mainly the most abundant protein in the NET scaffold, the histone component can be held responsible for the cytotoxicity [47]. NETs have also been found to be involved in thrombogenesis [48–50], in ischemia-reperfusion injury syndrome [51] and in preeclampsia, where a massive induction of NETosis by placental IL-8 and microdebris and increased amount of NETs in the placenta has been described [28].

No data are available on NETs in the human endometrium and endometriotic implants. Yet, in the equine endometrium, neutrophils have been detected at estrus, mating or infection, and isolated equine neutrophils, stimulated with bacteria, produced NETs [52]. The authors suggested that enhanced NET production might be involved in equine endometrial fibrosis and impairment of secretory function. In all of the aforementioned pathologies NETs are involved in the initiation or maintenance of chronic inflammation and consecutive tissue damage. Endometriosis is believed to be a chronic inflammatory disease [53] and several facts support the idea that NETs may contribute to its pathogenesis. Reactive oxygen species (ROS) are the major activator of NETosis [54–56]. It is known that endometriosis is associated with increased ROS production [57,58]. Moreover, IL-8 is increased in endometriosis [59], and also promotes NET release [10]. Recent studies have revealed that in SLE or in type-1 diabetes NETs trigger plasmacytoid dendritic cells via the toll-likereceptor-9 to release interferon-a, an important pro-inflammatory cytokine [33,60,61]. In fact, pro-inflammatory cytokines are well known key components in the pathophysiology of endometriosis, causing the activation of nuclear-factor-kappa-b and several transcription factors, resulting in the activation of angiogenesis, matrix metalloproteinases and anti-apoptotic genes [62]. The observed increased amounts of myeloperoxidase, lactoferrin and a-defensins in the endometriosis PF [40,44] – all of them are well known components of NETs – [11,63] may be originated from NETs, although this needs to be further investigated. Another abundant protein of NETs is neutrophil elastase, which degrades the extracellular matrix [63], and as such might be involved in the invasion of the endometrial cells. Interestingly, we detected the highest percentage of NET positive patients in the stage I and II group, whereas NETs were a bit less frequent in the stage III and IV and deep infiltrating patients. We therefore postulate that NETosis may play a role in the initiation of the disease, but later on, other chronic inflammatory processes take over. This hypothesis might be supported with the mouse model of endometriosis, in which neutrophils were present in the ectopic lesions between days 2–5 after implantation and later on disappeared from the implants [45]. Another important result is that NETs were already present in the PF of endometriosis patients, whereas the PF itself could not stimulate NETosis in healthy granulocytes. This suggests that the stimuli of NETosis, as well as the NET formation may take place in the endometriotic implants, whereby these lesions may also secrete NETs into the peritoneal microenvironment. Considering all these hypothetical points, we can postulate that ROS and interleukins, such as IL-8, promote NETosis in the endometriotic lesions with consecutive NET accumulation in the extracellular matrix and secretion to the peritoneal fluid. The NET components may stimulate proinflammatory cytokine secretion, leading to activation of transcriptional factors, which initiate angiogenesis, degradation of the extracellular matrix via matrix metalloproteinases and cell survival through activation of anti-apoptotic genes. Besides the indirect activation, NET components may directly facilitate the degradation of the extracellular matrix and as such support the invasion of the implants. Moreover, exaggerated NET accumulation with its immuno-modulatory properties can initiate and maintain chronic inflammation, causing fibrosis in the endometrial lesions. Similar to SLE, the endometriosis patients may have low amount or less functional DNase, which in turn leads to low resolution of NETs, and the sustainable NET components and enzymes intensifies the inflammatory response.

E. Berkes et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 183 (2014) 193–200

The major strength of our study is a well-defined and thoroughly selected control group and the identification of NET structures with a specific technology. Our study, however, has some limitations, such as the small sample size, especially of the endometriosis sub-groups, causing difficulties in the statistical analysis. Another limitation in interpreting the results is the missing information about NETs in normal functioning human endometrium throughout the menstrual cycle. Yet, our results managed to raise further scientific questions: (i) Why did not all of the endometriosis patients present NETs in the PF? (ii) Which components regulate NETosis? (iii) In which lesions are NETs present? (iv) How exactly do NETs act on the host tissue? Such aspects need to be further explored by in vitro and in vivo investigations. Funding The study was funded by the University-Hospital GiessenMarburg, and by the ‘‘Excellence Cluster Cardio-Pulmonary System (ECCPS) Start-up grant’’ (German Research Foundation, DFG) to M. S. Condensation This study demonstrates the presence of neutrophil extracellular traps in the peritoneal fluid of half of the endometriosis patients, indicative for its contribution to the pathogenesis of the disease. Acknowledgements We would like to thank the OR nurses for their contribution in sample collection. References [1] Giudice L, Kao L. Endometriosis. Lancet 2004;364:1789–99. [2] Burney O, Giudice LC. Pathogenesis and pathophysiology of endometriosis. Fertil Steril 2012;98:511–9. [3] Gazvani R, Templeton A. New considerations for the pathogenesis of endometriosis. Int J Gynaecol Obstet 2002;76:117–26. [4] Oral E, Olive DL, Arici A. The peritoneal environment in endometriosis. Hum Reprod Update 1996;2:385–98. [5] Nathan C. Neutrophils and immunity: challenges and opportunities. Nat Rev Immunol 2006;6:173–82. [6] Brinkmann V, Zychlinsky A. Beneficial suicide: why neutrophils die to make NETs. Nature 2007;5:577–82. [7] Zhang B, Hirahashi J, Cullere X, Mayadas TN. Elucidation of molecular events leading to neutrophil apoptosis following phagocytosis: cross-talk between caspase 8, reactive oxygen species, and MAPK/ERK activation. J Biol Chem 2003;278:28443–54. [8] Serhan CN, Savill J. Resolution of inflammation: the beginning programs the end. Nat Immunol 2005;6:1191–7. [9] Haslett C. Granulocyte apoptosis and inflammatory disease. Br Med Bull 1997;53:669–83. [10] Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacteria. Science 2004;303:1532–5. [11] Urban CF, Ermert D, Schmid M, et al. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense againts Candida albicans. PLoS Pathog 2009;5:e1000639. [12] Saffarzadeh M, Jeunemann C, Queisser MA, et al. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. PLoS One 2012;7:e32366. [13] Fuchs TA, Abed U, Goosmann C, et al. Novel cell death program leads to neutrophil extracellular traps. J Cell Biol 2007;176:231–41. [14] Bianchi M, Hakkim A, Brinkmann V, et al. Restoration of NET formation by gene therapy in CGD controls aspergillosis. Blood 2009;114:2619–22. [15] Bianchi M, Niemiec MJ, Siler U, Urban CF, Reichenbach J. Restoration of antiAspergillus defense by neutrophil extracellular traps in human chronic granulomatosus disease after gene therapy is calprotectin-dependent. J Allergy Clin Immunol 2011;127. 1243.e7-1252.e7. [16] Uchiyama S, Andreoni F, Schuepbach RA, Nizet V, Zinkernagel AS. DNase Sda 1 allows invasive M1T1 Group A Streptococcus to prevent TLR9-dependent recognition. PLoS Pathog 2012;8:e1002736.

199

[17] Chang A, Khemlani A, Kang H, Proft T. Functional analysis of Streptococcus pyogenes nuclease A (SpnA), a novel Group A streptococcal virulence factor. Mol Microbiol 2011;79:1629–42. [18] Berends ET, Horswill AR, Haste NM, Monestier M, Nizet V, von Ko¨ckritzBlickwede M. Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps. J Innate Immun 2010;2:576–86. [19] Caudrillier A, Kessenbrock K, Gilliss BM, et al. Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury. J Clin Invest 2012;122:2661–71. [20] Narasaraju T, Yang E, Samy RP, et al. Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis. Am J Pathol 2011;179:199–210. [21] Manzenreiter R, Kienberger F, Marcos V, et al. Ultrastructural characterization of cystic fibrosis sputum using atomic force and scanning electron microscopy. J Cyst Fibros 2012;11:84–92. [22] Young RL, Malcolm KC, Kret JE, et al. Neutrophil extracellular trap (NET)mediated killing of Pseudomonas aeruginosa: evidence of acquired resistance within the CF airway, independent of CFTR. PLoS One 2011;6:e23637. [23] Dworski R, Simon HU, Hoskins A, Yousefi S. Eosinophil and neutrophil extracellular DNA traps in human allergic asthmatic airways. J Allergy Clin Immunol 2011;127:1260–6. [24] Lin AM, Rubin CJ, Khandpur R, et al. Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis. J Immunol 2011;187:490– 500. [25] von Bru¨hl ML, Stark K, Steinhart A, et al. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J Exp Med 2012;209:819–35. [26] Brill A, Fuchs TA, Savchenko AS, et al. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost 2012;10:136–44. [27] Fuchs TA, Brill A, Duerschmied D, et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci USA 2010;107:15880–85. [28] Gupta AK, Hasler P, Holzgreve W, Hahn S. Neutrophil NETs: a novel contributor to preeclampsia-associated placental hypoxia? Semin Immunopathol 2007;29:163–7. [29] Clark SR, Ma AC, Tavener SA, et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med 2007;13:463–9. [30] Yousefi S, Gold JA, Andina N, et al. Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense. Nat Med 2008;14:949–53. [31] Villanueva E, Yalavarthi S, Berthier CC, et al. Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus. J Immunol 2011;187:538–52. [32] Hakkim A, Fu¨rnrohr BG, Amann K, et al. Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis. Proc Natl Acad Sci USA 2010;107:9813–8. [33] Garcia-Romo GS, Caielli S, Vega B, et al. Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus. Sci Transl Med 2011;3:73ra20. [34] Leffler J, Martin M, Gullstrand B, et al. Neutrophil extracellular traps that are not degraded in systemic lupus erythematosus activate complement exacerbating the disease. J Immunol 2012;188:3522–31. [35] Kessenbrock K, Krumbholz M, Scho¨nermarck U, et al. Netting neutrophils in autoimmune small-vessel vasculitis. Nat Med 2009;15:623–5. [36] Eisenberg VH, Zolti M, Soriano D. Is there an association between autoimmunity and endometriosis. Autoimmun Rev 2012;11:806–14. [37] Costa D, Marques AP, Reis RL, Lima JL, Fernandes E. Inhibition of human neutrophil oxidative burst by pyrazolone derivatives. Free Radic Biol Med 2006;40:632–40. [38] Cho S, Cho H, Nam A, et al. Neutrophil-to-lymphocyte ratio as an adjunct to CA125 for the diagnosis of endometriosis. Fertil Steril 2008;90:2073–9. [39] Tariverdian N, Siedentopf F, Ru¨cke M, et al. Intraperitoneal immune cell status in infertile women with and without endometriosis. J Reprod Immunol 2009;80:80–90. [40] Milewski Ł, Dziunycz P, Barcz E, et al. Increased levels of human neutrophil peptides 1, 2, and 3 in peritoneal fluid of patients with endometriosis: association with neutrophils, T cells and IL-8. J Reprod Immunol 2011;91:64–70. [41] Kwak JY, Park SW, Kim KH, Na YJ, Lee KS. Modulation of neutrophil apoptosis by plasma and peritoneal fluid from patients with advanced endometriosis. Hum Reprod 2002;17:595–600. [42] Takamura M, Osuga Y, Izumi G, et al. Interleukin-17A is present in neutrophils in endometrioma and stimulates the secretion of growth-regulated oncogenea (Gro-a) from endometrioma stromal cells. Fertil Steril 2012;98:1218–24. [43] Arici A, Seli E, Zeyneloglu HB, Senturk LM, Oral E, Olive DL. Interleukin-8 induces proliferation of endometrial stromal cells: a potential autocrine growth factor. J Clin Endocrinol Metab 1998;83:1201–5. [44] Riley CF, Moen MH, Videm V. Inflammatory markers in endometriosis: reduced peritoneal neutrophil response in minimal endometriosis. Acta Obstet Gynecol Scand 2007;86:877–81. [45] Lin YJ, Lai MD, Lei HY, Wing LY. Neutrophils and macrophages promote angiogenesis in the early stage of endometriosis in a mouse model. Endocrinology 2006;147:1278–86. [46] Thomas GM, Carbo C, Curtis BR, et al. Extracellular DNA traps are associated with the pathogenesis of TRALI in humans and mice. Blood 2012;119:6335–43. [47] Saffarzadeh M, Preissner KT. Fighting against the dark side of neutrophil extracellular traps in disease: manoeuvres for host protection. Curr Opin Hematol 2013;20:3–9.

200

E. Berkes et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 183 (2014) 193–200

[48] Fuchs TA, Brill A, Wagner DD. Neutrophil extracellular trap (NET) impact on deep vein thrombosis. Arterioscler Thromb Vasc Biol 2012;120:1777–83. [49] Oehmcke S, Mo¨rgelin M, Herwald H. Activation of the human contact system on neutrophil extracellular traps. J Innate Immun 2009;1:225–30. [50] Massberg S, Grahl L, von Bruehl ML, et al. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases. Nat Med 2010;16: 887–96. [51] De Meyer SF, Suidan GL, Fuchs TA, Monestier M, Wagner DD. Extracellular chromatin is an important mediator of ischemic stroke in mice. Arterioscler Thromb Vasc Biol 2012;32:1884–991. [52] Galvao A, Rebordao MR, Szostek AY, Skarzynski J, Ferreira-Dias G. Cytokins and neutrophil extracellular traps in the equine endometrium. Friends or foes. Pferdeheilkunde 2012;28:4–7. [53] Lousse JC, Van Langendonckt A, Defrere S, Ramos RG, Colette S, Donnez J. Peritoneal endometriosis is an inflammatory disease. Front Biosci 2012; 4:23–40. [54] Kirchner T, Mo¨ller S, Klinger M, Solbach W, Laskay T, Behnen M. The impact of various reactive oxygen species on the formation of neutrophil extracellular traps. Mediators Inflamm 2012;2012:849136. [55] Remijsen Q, Vanden Berghe T, Wirawan E, et al. Neutrophil extracellular trap cell death requires both autophagy and superoxide generation. Cell Res 2011;21:290–304.

[56] Palmer LJ, Cooper PR, Ling MR, Wright HJ, Huissoon A, Chapple IL. Hypochlorous acid regulates neutrophil extracellular trap release in humans. Clin Exp Immunol 2012;167:261–8. [57] Ngoˆ C, Che´reau C, Nicco C, Weill B, Chapron C, Batteux F. Reactive oxygen species controls endometriosis progression. Am J Pathol 2009;175:225–34. [58] Polak G, Wertel I, Barczyn´ski B, Kwas´niewski W, Bednarek W, Kotarski J. Increased levels of oxidative stress markers in the peritoneal fluid of women with endometriosis. Eur J Obstet Gynecol Reprod Biol 2013;168:187–90. [59] Arici A, Tazuke SI, Attar E, Kliman HJ, Olive DL. Interleukin-8 concentration in peritoneal fluid of patients with endometriosis and modulation of interleukin8 expression in human mesothelial cells. Mol Hum Reprod 1996;2:40–5. [60] Mocsai A. Diverse novel functions of neutrophils in immunity, inflammation, and beyond. J Exp Med 2013;210:1283–99. [61] Lande R, Ganguly D, Facchinetti V, et al. Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA–peptide complexes in systemic lupus erythematosus. Sci Transl Med 2011;3(73):73ra19. http://dx.doi.org/10.1126/ scitranslmed.3001180. [62] Soares SR, Martinez-Varea A, Hidalgo-Mora JJ, Pellicer A. Pharmacologic therapies in endometriosis: a systematic review. Fertil Steril 2012;98:529–55. [63] Perl M, Lomas-Neira J, Chung CS, Ayala A. Epithelial cell apoptosis and neutrophil recruitment in acute lung injury—a unifying hypothesis? What we have learned from small interfering RNAs. Mol Med 2008;14:465–75.