Aged neutrophil populations in menstrual effluent from women with endometriosis. Previous findings using our in vivo SDME model demonstrated neutrophil infiltration peaked after the initiation of endometriosis lesion formation following the injection of minced mouse endometrial pieces into the peritoneal cavity at ~24 hours (14). Therefore, 24 hours was selected for our human studies to establish translational relevance to our SDME model to determine whether neutrophils and/or changes in neutrophil subpopulations were also observed in menstrual effluent of women with endometriosis.

Menstrual effluent from women with and without endometriosis was collected on day 1 of menses for immunophenotyping. WBCs were isolated, stained for cellular surface markers, and quantified using gating strategies to isolate granulocytes, exclude eosinophils, and identify potential unique subpopulations of neutrophils associated with endometriosis (Figure 1A). Identified total neutrophils were normalized as a ratio by: calculating total cells per mL and then neutrophils per mL to total leukocytes per mL. No differences in the total neutrophil numbers were observed between endometriosis and control; consequently, neutrophil subtypes were normalized as a ratio by: calculating cells per mL and then aged or aged angiogenic neutrophils per mL to total neutrophils per mL. These ratios accounted for variability in collection volumes and/or time collected per individual. The ratio of neutrophils (CD45+CD66b+CD193–) to total WBCs (CD45+) was not different between endometriosis and nonendometriosis control samples (Con) (Figure 1B). In contrast, the ratio of aged neutrophils (CD45+CD66b+CD193–CD16–CXCR2+) (expressing CXCR2) to total neutrophils (CD45+CD66b+CD193–) increased significantly by 2-fold in endometriosis compared with control (Figure 1C). Moreover, the ratio of proangiogenic neutrophils (CD45+CD66b+CD193–CD16–CXCR2+VEGFR1+) (additionally expressing VEGFR1) increased by ~2.5-fold in endometriosis samples compared with control (Figure 1D). Together, these observations suggest that aged and proangiogenic neutrophils likely play an important role in endometriosis, potentially by providing cytokines and growth factors for the promotion of endometriosis cell survival, adhesion to the peritoneal cavity, and initiation of lesion development, similar to that observed in our mouse model (14). Aged and proangiogenic neutrophils may also participate in the recruitment of additional immune cells (e.g., macrophages, to promote angiogenesis, which would further lesion development) (14).

Women with endometriosis have higher levels of aged and proangiogenic neutrophils compared with healthy women. (A) Gating strategy of total WBCs isolated from human menstrual effluent. Cells were gated on time, viable cells, single cells, CD45+, CD66b+, and CD193– to identify total neutrophils. Aged neutrophils were defined as CD45+CD66b+CD193–CD16–CXCR2+, and proangiogenic neutrophils are defined as CD45+CD66b+CD193–CD16–CXCR2+VEGFR1+. (B–D) Quantitation of total neutrophils to total WBCs ratio, aged neutrophils to total neutrophil ratio, and proangiogenic aged neutrophils to total neutrophil ratio. Con, healthy controls (n = 13); EMS, endometriosis participants (n = 10). Data represent ± SEM. Statistical significance for each graph was determined by nonparametric, Mann-Whitney, 1-tailed U test. #P < 0.05.

Neutrophil knockdown attenuates endometrial attachment to sites in the peritoneal cavity. To determine the effect of neutrophils on endometrial tissue attachment and initiation of endometriosis-like lesion formation (herein called lesions), donor mice expressing GFP were treated with control IgG or α-CXCR2 antibody (2.5 mg/kg in 1× PBS, bidiurnally) 5 days prior to donor endometrial tissue removal to knock down neutrophil recruitment (Figure 2). Minced endometrial tissue pieces from donor mice were dispersed via surgical incision and injection into the peritoneum of GFP– host mice (14, 40–42), and treated with IgG or α-CXCR2 (2.5 mg/kg in 1× PBS, bidiurnally) with pretreatment beginning 6 days prior to endometriosis induction. The donor-to-host (d:h) transfer groups were treated as follows: (a) IgG to IgG (I:I), (b) IgG to α-CXCR2 (I:α), (c) α-CXCR2 to IgG (α:I), and (d) α-CXCR2 to α-CXCR2 (α:α). Twenty-four hours later, developing lesions, PF, and peritoneal cells were collected. At necropsy, using macroscopic analysis, lesions were categorized into 2 groups — i.e., “attaching” or “unattached” — based on coloration (pink/attached or white/unattached), the presence of an external blood spot (early initiation of angiogenesis), and/or the presence of physical attachment to sites in the peritoneal cavity (Figure 3).

Schematic representation of study treatment and experimental timeline. Donor mice expressing GFP (green) begin treatment bidiurnally with α-CXCR2/IgG 5 days before collection of endometrial tissue (green diamonds). At 41 hours before endometrial collection, donor mice receive an i.p. injection of pregnant mare serum gonadotropin (PMSG) to synchronize uteri. Host mice (black) begin treatment bidiurnally with α-CXCR2/IgG 6 days before surgical induction of endometriosis (black diamonds) with a final dose administered the day of surgery. Mice are euthanized and lesions are collected 24 hours after surgical induction.

Knockdown of neutrophil recruitment in the SDME model decreased the attachment of minced endometrial pieces at 24 hours to form endometriosis lesions. (A) Lesions (24 hours) were derived from GFP minced endometrial pieces and imaged at 488 nm and bright-field from I:I, I:α, α:I, and α:α groups. I, IgG; α, α-CXCR2. Lesions were defined as “attaching” or “unattached” based on color (pink, presence of a blood spot, or attachment for “attaching” versus white and no presence of attachment for “unattached”). Red arrowheads indicate “attaching.” Blue arrowheads indicate “unattached.” Original magnification, 7.5×. (B and C) Quantitation of attaching and unattached lesions. I:I (n = 7), I:α (n = 9), α:I (n = 10), and α:α (n = 11) biological replicates from 2 independent experiments. Data represent ± SEM. Statistical significance for each graph was determined by nonparametric, Kruskal-Wallis followed with 1-tailed Mann-Whitney U tests. Letters different from each other are statistically significant, P ≤ 0.05.

In the control I:I group, 74% of lesions were localized to sites of attachment 24 hours after endometriosis initiation, and endometrial tissue was pink, more firmly attached, and often found with the presence of a blood spot and/or a visual external blood supply, indicating that attachment to peritoneal surfaces and lesion formation had been initiated (Figure 3, A and B). Unattached endometrial tissue (26%) was white in color, found floating in the peritoneal cavity or lying superficially on top of peritoneal sites, and no evidence of physical attachment or formation of a visual external blood supply was observed at 24 hours (Figure 3C). To normalize the number of observed lesions per mouse, a ratio was calculated comparing the number of attached or unattached lesions with the total number of lesions per mouse. In the I:α- and α:I-treated groups, 39% and 54% of endometrial tissue, respectively, was found attaching, and attachment decreased further to 22% in the α:α group when compared with the I:I group (Figure 3B). As expected, the unattached (Figure 3C) to attached lesion ratios were inversely proportional based on treatment.

Together, these observations imply that knocking down neutrophil recruitment in host mice (I:α and α:α) decreased the initiation of lesion formation to a greater degree than host mice treated with control IgG (I:I and α:I), demonstrating that neutrophil recruitment was required for minced endometrial pieces to attach and initiate lesion formation. Treatment of donor mice with α-CXCR2 (α:I) decreased lesion attachment by 27% compared with I:I, suggesting a role for donor uterine neutrophils in attachment where donor endometrial tissue recruits neutrophils lesser than the host (i.e., peritoneum). Treatment of host and donor mice with α-CXCR2 antibody (α:α) further decreased (43%) the ability of endometrial tissue to attach to peritoneum compared with I:α, indicating that neutrophils from both host and donor participate in lesion development. Together, donor endometrial tissue plays a smaller role in the recruitment of neutrophils, accounting for the differences between host groups in attaching lesions (27% and 59%, I:I to α:I and α:I to α: α, respectively).

In addition to the use of α-CXCR2 antibody to knock down neutrophil recruitment, we also investigated neutrophil recruitment in Cxcr2-KO mice (B6.129S2[C]-Cxcr2tm1Mwm/J; gifted by Alex Lentsch, University of Cincinnati) and used a selective CXCR2 inhibitor, SB225002 (data not shown). Unfortunately, Cxcr2-KO mice exhibit failure to thrive when neutrophil recruitment was knocked out and/or there were compensation phenotypes for neutrophil recruitment (43); importantly, surgical procedures were not well tolerated. In addition, neutrophil recruitment was inconsistently reduced in WT mice treated with SB225002. Thus, due to these factors seen in the Cxcr2-KO mouse and SB225002 treatments, all further studies were conducted by acutely knocking down neutrophil recruitment with α-CXCR2 antibody.

α-CXCR2 treatment reduces neutrophil recruitment into PF and endometriosis lesions. Peritoneal lavage obtained from the I:I, α:I, I:α, and α:α treatment groups were analyzed to determine changes in the levels of neutrophils recruited into the peritoneal cavity. In addition, peritoneal lavage obtained from sham surgical mice — i.e., mice having surgery and only given PBS and pretreated with IgG (“I”) or α-CXCR2 (“α”) — served as baseline inflammatory surgical controls. Neutrophils were immunophenotyped as live population, single cells, and Ly6G+ (Figure 4A).

Knockdown of neutrophil recruitment in the SDME model decreased total and proangiogenic neutrophils in peritoneal fluid and lesions. (A) Total neutrophils were gated as live cells, single cells, and Ly6G+ from I sham (n = 10), α sham (n = 9), I:I (n = 7), I:α (n = 9), α:I (n = 10), and α:α (n = 10) groups from biological replicates from 2 independent experiments. I, IgG; α, α-CXCR2. (B) Proangiogenic neutrophils were gated with CXCR4+ from the Ly6G+ population from I sham (n = 9), α sham (n = 10), I:I (n = 10), I:α (n = 10), α:I (n = 10), and α:α (n = 10) groups from biological replicates from 2 independent experiments. (C) Representative images of S100A8 staining scale. (D) Quantitation of S100A8 staining in lesions. I:I (n = 18), I:α (n = 20), α:I (n = 25), and α:α (n = 24) groups from biological replicates from 2 independent experiments from biological replicates from 2 independent experiments. (E–H) Lesions stained with S100A8. Dark purple represents neutrophil infiltration into the lesion. Original magnification, 100×. Representative images from I:I, I:α, α:I, and α:α. Data for each graph represent ± SEM. Statistical significance for each graph was determined by nonparametric, Kruskal-Wallis followed with 1-tailed Mann-Whitney U tests. Letters different from each other are statistically significant. P ≤ 0.05. Scale bar: 100 μm. Original magnification, 100x (C, E–H).

Analysis of neutrophils from the sham surgical I and α groups showed treatment with α-CXCR2 alone significantly decreased recruitment of neutrophils into PF by 10-fold compared with I sham treatment. The addition of endometrial tissue pieces to initiate endometriosis lesion formation increased neutrophil recruitment in control I:I PF 2.5-fold compared with I sham surgical controls. In contrast, I:α and α:α PF showed a 12-fold decrease in neutrophils compared with I:I (Figure 4A), while a modest 1.5-fold decrease in neutrophil recruitment was observed in α:I compared with I:I PF, likely due to the donor endometrial tissue response similarly seen in Figure 3. Together, these findings show that the addition of endometrial tissue pieces is required to promote recruitment of neutrophils into PF, and α-CXCR2 treatment of host mice is sufficient to knock down peritoneal neutrophil levels almost to α sham levels, irrespective of the treatment given to donor animals (e.g., I:α = α:α).

Since external blood spots and early initiation of angiogenesis were observed during tissue attachment and initial lesion formation in I:I mice, we also quantitated levels of proangiogenic neutrophil recruitment into PF. Murine neutrophils expressing CXCR4 in addition to Ly6G are categorized as proangiogenic (32, 44, 45). Analysis of neutrophils (Ly6G+CXCR4+) determined the pattern of proangiogenic neutrophil recruitment similarly mirrored that observed with total neutrophils (Figure 4B), indicating again that presence of minced endometrial pieces was sufficient to increase peritoneal proangiogenic neutrophil numbers with slight differences between host treatment groups (I:I to α:I and I:α to α:α) due to donor tissue interactions and α-CXCR2 treatment of host mice knocked down proangiogenic neutrophil recruitment in PF.

Next, lesion tissue sections were stained with S100A8 to determine whether neutrophils recruited to the peritoneum had infiltrated into the developing endometriosis lesions. Histopathological staining of lesions was characterized based on a staining scale ranging from no staining to very high staining (Figure 4C). Scores for each lesion were quantitated using the staining scale (Figure 4D). Analysis of lesions from all treatment groups exhibited similar histological structure with a mixture of stromal, epithelial cells, and glandular structures 24 hours after endometrial tissue injection (Figure 4, E–H). Moderate to high numbers of neutrophils were observed in I:I control lesions (Figure 4E), while neutrophil recruitment into I:α (Figure 4F) and α:α (Figure 4H) lesions decreased to low numbers, corresponding to the decreases observed in lesion attachment and peritoneal neutrophil recruitment (Figures 3, A and B, and Figure 4, A and B). In addition, neutrophil recruitment into α:I lesions decreased significantly compared with I:I but significantly increased compared with I:α and α:α, suggesting that donor neutrophils, present in minced endometrial tissue, are contributing effector cells in recruitment of host-derived neutrophils.

In summary, the flow cytometry data indicate that neutrophil recruitment was not fully blocked by α-CXCR2 treatment. Not surprisingly, as neutrophils rapidly differentiate and extravasate from the vasculature during inflammation. This increase in neutrophil recruitment into I:I PF, followed by a significant increase of neutrophils into I:I lesions, demonstrates that introduction of minced endometrial pieces into the peritoneum elicits a physiological immune reaction. Moreover, α-CXCR2 treatment knocks down neutrophil recruitment into PF and lesions, correlating with the decrease in lesion attachment following treatment (Figure 3). Concomitantly, the considerable decrease in proangiogenic neutrophils also correlates with the loss of external blood spots or signs of early initiation of angiogenesis (Figure 3). Taken together, these findings propose that neutrophil recruitment is an important factor in tissue survival, attachment, angiogenesis, and initiation of the development of endometriosis lesions.

Neutrophil-associated gene expression in endometriosis lesions decreased with α-CXCR2 treatment. To determine the effect of α-CXCR2 treatment on neutrophils recruited into lesion tissue, we evaluated neutrophil-associated gene expression in I:I, α:I, I:α, and α:α lesions compared with control minced endometrial pieces obtained from IgG-treated (“I”) and α-CXCR2–treated (“α”) donor mice. Neutrophil-associated target genes included Cxcr2, S100a8, S100a9, and Csf3r. We observed that Cxcr2 expression was low in IgG and α-CXCR2 minced endometrial pieces. Thus, given these low levels of expression, Cxcr2 expression in IgG-treated minced endometrial tissue was normalized relative to ribosomal RpL7 gene expression to enable comparisons between treatment and endometriosis groups. Cxcr2 expression increased 8-fold and 9-fold in I:I and α:I lesions compared with I:I, respectively, supporting the increased recruitment of neutrophils observed previously (Figure 4) (14). In addition, Cxcr2 expression in I:α and α:α lesions decreased by > 4.5-fold and 2-fold, respectively, compared with I:I lesions.

S100A8 and S100A9 comprise approximately 45% of the cytoplasmic proteins in neutrophils and are associated with neutrophil recruitment and activation (46–49); CSF3R controls the production, differentiation, and function of granulocytes, including neutrophils (50). Analysis of S100a8, S100a9, and Csf3r gene expression revealed that their expression decreased in parallel with that observed for Cxcr2 gene expression and that the decrease in I:α and α:α lesions was significantly greater compared with I:I and α:I lesions (Figure 5A). Of note, α:I lesions in S100a8 and S100a9 genes showed the overall highest levels of neutrophil-associated target gene expression; although the cause of this was not clear, it may be a result of the host reacting to the α donor tissue.

Knockdown of neutrophil recruitment in the SDME model decreased neutrophil infiltration into lesions and decreased further neutrophil recruitment. (A) Neutrophil-associated gene expression targets from I uterus, α uterus, I:I, I:α, α:I, and α:α groups. Cxcr2: I uterus (n = 3), α uterus (n = 4), I:I (n = 16), I:α (n = 11), α:I (n = 10), and α:α (n = 15). S100a8: I uterus (n = 3), α uterus (n = 4), I:I (n = 16), I:α (n = 12), α:I (n = 11), and α:α (n = 16). S100a9: I uterus (n = 3), α uterus (n = 5), I:I (n = 15), I:α (n = 12), α:I (n = 11), and α:α (n = 16). Csf3r: I uterus (n = 3), α uterus (n = 5), I:I (n = 16), I:α (n = 11), α:I (n = 11), and α:α (n = 15) (B) Knockdown of neutrophil recruitment did not alter a subset of genes associated with endometriosis and inflammation, demonstrating uterine-associated processes in lesion development from I uterus, α uterus, I:I, I:α, α:I, and α:α groups. Il6: I uterus (n = 3), α uterus (n = 5), I:I (n = 7), I:α (n = 10), α:I (n = 12), and α:α (n = 11). Mmp3: I uterus (n = 3), α uterus (n = 5), I:I (n = 16), I:α (n = 12), α:I (n = 10), and α:α (n = 16). Itgb2: I uterus (n = 3), α uterus (n = 5), I:I (n = 16), I:α (n = 12), α:I (n = 11), and α:α (n = 16). Vegfa: I uterus (n = 3), α uterus (n = 5), I:I (n = 17), I:α (n = 12), α:I (n = 11), and α:α (n = 16). For each graph, biological replicates are shown from 2 independent experiments. Data for each graph represent ± SEM. Statistical significance for each graph was determined by nonparametric Kruskal-Wallis followed with 1-tailed Mann-Whitney U tests. Letters different from each other are statistically significant. P ≤ 0.05.

In determining the potential role of endometrial-associated gene expression in lesion formation, we initially hypothesized that integrins and selectins would play a major role in the processes of adhesion of endometrial tissue due to their specialized functions in chemotaxis and extravasation during recruitment of neutrophils (51); however, analysis of a select group of selectins (Sell, Selp) did not show modulation in lesion development in I:I, α:I, I:α, or α:α lesions compared with minced endometrial pieces obtained from I- and α-treated donor mice (data not shown). Interestingly, Il6, Mmp3, Itgb2, and Vegfa, a group of genes known to play a role in endometriosis, increased in the developing lesion tissues in all groups compared with control I and α minced endometrial pieces (Figure 5B). Together, these findings indicate that the expression of factors regulating neutrophil activation and function are primarily modulated via neutrophils that have infiltrated into the lesions.

IL-6 is reported as an important cytokine in both the endometrium and endometriosis lesions in women with endometriosis and is known to be both proinflammatory and antiinflammatory (52). Il6 expression increased by greater than 68-fold in all I:I, α:I, I:α, and α:α lesions compared with I- and α-treated minced endometrial pieces. MMP3 remodels extracellular matrix and is overexpressed in endometriosis, and MMP3 polymorphism increases the risk of developing advanced endometriosis and infertility (53). Mmp3 expression increased by 200-fold in all I:I, α:I, I:α, and α:α lesions compared with I- and α-treated minced endometrial pieces. Integrin β-2 (ITGB2) is an adhesion molecule known to play a role in regulating neutrophil trafficking and other immunological processes (51). Itgb2 expression increased by 4-fold in all I:I, α:I, I:α, and α:α lesions compared with I- and α-treated minced endometrial pieces. VEGFA is highest during menstruation in endometriosis compared with normal controls, is elevated in the PF of women with endometriosis, and promotes angiogenesis (54). Vegfa expression increased by 3-fold in all I:I, α:I, I:α, and α:α lesions compared with I- and α-treated minced endometrial pieces. Additionally, expression of the integrins Itga11 increased by 124-fold (data not shown), and this increase was independent of neutrophil knockdown. Factors regulating endometrial tissue survival, adhesion to peritoneal sites, and angiogenesis during initiation of lesion formation are primarily produced by endometrial cells mediated by signals from the endometrium, and these signals can be modulated by neutrophil-dependent and neutrophil-independent mechanisms.

Vasculature at 24 hours is donor derived. To examine the early initiation of vasculature, lesions from each group were stained with anti-PECAM1 to visualize vasculature and with anti-GFP to validate donor-derived tissue. Histopathological staining of lesions was examined for de novo vasculature since PECAM1 is involved in angiogenesis and is a marker for endothelial junctions. Representative images (Figure 6, B, D, F, and H) from each group are shown. No remarkable differences between the size, location, or formation of vessels at the 24-hour time point was observed. Additionally, staining of lesions with GFP demonstrated that the vasculature in the lesions were donor endometrial tissue derived (Figure 6, A, C, E, and G). This result is comparable with another study that also did not observe de novo angiogenesis in the lesion 72 hours after endometriosis induction (16). Although Vegfa (Figure 5B) was elevated in the endometriosis induction groups, the delay between transcription and translation likely caused the delay in visible neoangiogensis within the lesion.

Endometriosis lesions include existing GFP+ donor-derived blood vessels and not de novo–derived blood vessels, regardless of neutrophil knockdown 24 hours after endometriosis induction. (A, C, E, and G) Developing endometriosis lesions stained for GFP donor-derived tissue are GFP+ (brown stain) and contain endometrial glands from minced uterine tissue. (B, D, F, and H) Developing endometriosis lesions were stained to visualize endothelial cells lining blood vessels for PECAM1 (brown staining). Representative blood vessels (teal arrowheads) found in developing lesions from serial sections stained for GFP and PECAM1. GFP: I:I (n = 12), I:α (n = 16), α:I (n = 18), and α:α (n = 14). PECAM1: I:I (n = 12), I:α (n = 18), α:I (n = 18), and α:α (n = 14). Lesions per group represent biological replicates from 2 independent experiments. Scale bar: 25 μm. Original magnification, 400x.

Neutrophil extracellular traps promote endometrial tissue attachment. Since knocking-down neutrophil recruitment did not alter adhesion factor expression, neutrophil extracellular traps (NETs) were examined to gain further insight into the mechanisms of lesion attachment. NETs are an inflammatory process resulting in the expulsion of decondensed chromatin, histones, and enzymes to produce a viscous and adhesive meshwork intended to contain an inflammatory incident (55, 56). In response to stress, NETs serve as an adhesive substrate for cells (57) and release the byproduct neutrophil elastase (ELA2), which is essential to initiate NET formation and synergizes with another NET byproduct, myeloperoxidase (MPO), the most abundant proinflammatory biomarker present in neutrophilic granulocytes, to accelerate chromatin decondensation during NET formation (i.e., NETosis) (58, 59). Thus, ELA2 and MPO were evaluated in both PF and crushed lesion tissues via ELISA to determine whether NET formation was a potential mechanism involved in the survival and attachment of endometrial tissue.

ELA2 levels decreased by 30% in I:α and by 27% in α:α PF compared with the I:I group, with no changes in α:I compared with I:I (Figure 7A). ELA2 in lesions decreased by 58% in I:α, with no changes in α:I and a 38% decrease in α:α lesions compared with the I:I control group (Figure 7B). MPO decreased by 70% in I:α, with no changes in α:I and a 65% decrease in α:α PF compared with the I:I group (Figure 7C), while MPO decreased by 22% in I:α and by 15% in α:α lesions with no changes in α:I compared with I:I (Figure 7D). Lesions were not compared with eutopic endometrium or sham PF due to reduced neutrophils (i.e., low to no expression of S100A8/A9 in eutopic endometrium). These observations imply that ELA2 likely plays a more prominent role in lesion development, whereas MPO is more important in NET formation in driving the proinflammatory microenvironment provided by the PF.

Knockdown of neutrophil recruitment in the SDME model decreased NET formation in peritoneal fluid and developing lesions. (A–D) ELISA for ELA2 in peritoneal fluid from I:I (n = 4), I:α (n = 11), α:I (n = 5), and α:α (n = 5); ELA2 in lesions from I:I (n = 7), I:α (n = 8), α:I (n = 6), and α:α (n = 11); MPO in peritoneal fluid from I:I (n = 4), I:α (n = 4), α:I (n = 5), and α:α (n = 5); and MPO in lesions from I:I (n = 11), I:α (n = 6), α:I (n = 7), and α:α (n = 15). Each graph contains biological replicates from 2 independent experiments. Data for each graph represent ± SEM. Statistical significance for each graph was determined by nonparametric, Kruskal-Wallis followed with 1-tailed Mann-Whitney U tests. Letters different from each other are statistically significant. P ≤ 0.05.

Neutrophils initiate extracellular matrix remodeling and adhesion sites for lesions. To determine the extracellular matrix (ECM) preferred for lesion adhesion, we used our spontaneously immortalized cell line from mouse endometriosis lesions (mEmLe) (42) in an ECM array assay to delineate lesion attachment at the single-cell level based on their ability to attach to the ECM components fibronectin, collagen I, collagen IV, laminin I, and/or fibrinogen. For this assay, cells were plated in the presence of PF from all groups. Each group was normalized to BSA-coated wells. Fibrinogen is a glycoprotein and component of blood clots that is cleaved by thrombospondin to produce fibrin fibers that provide stability to clots (60). As seen in Figure 8A, mEmLe cells clearly selected fibrinogen as a preferred substrate for adhesion in I:I and α:I PF compared with BSA control. Furthermore, cell attachment decreased by 2.5-fold in both I:α and α:α PF compared with I:I, indicating that decreasing neutrophil recruitment into the peritoneum also decreased attachment at the cellular level. In addition, mEmLe cells attached modestly to fibronectin, a glycoprotein present in plasma that can play a major role in cell adhesion and wound healing (61), in a similar trend seen with fibrinogen. However, mEmLe cells did not attach to collagen I, collagen IV, or laminin over BSA baseline, implying that these ECM components were not essential for lesion adhesion.

mEmLe cells adhere to fibronectin in a neutrophil dependent manner, and the same fibrinogen response is observed in attaching lesion tissue. (A) mEmLe cells were used in an adhesion array with fibronectin, collagen I, collagen IV, laminin I, and fibrinogen. Cells were allowed to adhere in the presence of peritoneal fluid from I:I, I:α, α:I, and α:α groups where I = IgG and α = α-CXCR2. n = 4 representative of 3 independent experiments. (B) Fibrinogen (Fgb) gene expression in minced endometrial pieces, attaching (att), and unattached (unatt) lesions from I uterus, α uterus I:I, I:α, α:I, and α:α groups. I uterus (n = 3), α uterus (n = 5), I:I att (n = 5), I:I unatt (n = 5), I:α att (n = 7), I:α unatt (n = 4), α:I att (n = 5), α:I unatt (n = NA), α:α att (n = NA), and α:α unatt (n = 10) for attaching and unattached lesions of biological replicates from 2 independent experiments. (C) Thrombospondin (Thbs1) gene expression in minced endometrial pieces and lesions from I uterus (n = 3), α uterus (n = 4), I:I (n = 11), I:α (n = 9), α:I (n = 7), and α:α (n = 9) groups. n = 3–5 for minced endometrial pieces, n = 11–17 for lesions of biological replicates from 2 independent experiments. NA = no gene expression. Data represent ± SEM. Statistical significance for each graph was determined nonparametric, Kruskal-Wallis followed with 1-tailed Mann-Whitney U tests. Letters different from each other are statistically significant. P ≤ 0.05.

Next, we determined changes in the expression levels of the fibrinogen α (Fga) and β (Fgb) subunits in attached and unattached lesions in response to neutrophil depletion. Fga was not expressed in lesions or minced endometrial pieces; however, Fgb expression increased > 8-fold in attached I:I, I: α, and α:I lesions compared with I and α minced endometrial pieces, and it decreased in unattached I:I lesions by 2.5-fold compared with I:I attached lesions. Fgb expression significantly decreased in unattached lesions and was almost undetectable in unattached I: α and α:α lesions (Figure 8B). Insufficient lesion numbers were unattached in α:I or attached in α:α groups to measure Fgb expression. In contrast, expression of Thbs1, a glycoprotein involved in hemostasis and cell matrix remodeling (62), was > 10-fold higher in all lesions groups compared with IgG and α-CXCR2 minced endometrial tissue controls (Figure 8C). While other ECM factors may contribute to initiation of lesion attachment, stability, and/or survival at the early 24-hour time point, fibrinogen and, to a lesser extent, fibronectin are most likely key in initiating the lesion attachment process.