Introduction
Ovulation displays features that resemble inflammation, which can disrupt the normal follicular dynamics and lead to reduced oocyte quality and infertility [
1]. According to the World Health Organization (WHO), infertility is a condition that affects 15% of reproductive-aged couples worldwide, with many requiring the support of assisted reproductive techniques (ART) [
2,
3]. Several studies aimed to characterize potential biomarkers of oocyte competence with pregnancy outcomes. Genes expressed by human granulosa cells (hGCs), such as cyclooxygenase-2 (COX-2) and 5-, 12-, and 15-lipoxygenase (LOX), may be useful as effective biomarkers to identify oocytes with high development potential [
4,
5].
Ovulation is initiated by the onset of a luteinizing hormone (LH) surge that is accompanied by inflammatory signals, such as inflammatory mediator’s production, blood flow increase, leukocyte infiltration, swelling, and tissue degradation and repair. Pre-ovulatory follicles are comprised of an oocyte surrounded by hGCs, follicular fluid (FF), and a highly vascularized theca cells (TCs) compartment, in which resident leukocytes are found [
6].
GCs play a remarkable role in oocyte development, being interconnected with their plasma membrane by gap junctions that sustain them at an arresting stage and transport nutrients. These cells can exhibit distinct phenotypes: mural GCs (MGCs) covering the follicle wall and participating in the physical barrier, estradiol production, and follicle growth; and cumulus cells (CCs) that directly surround the oocyte, providing them with nutrients and protection from harmful signals that can influence their quality [
7].
The LH surge activates signalling pathways in GCs of pre-ovulatory follicles associated with inflammation, such as the expression of COX-2 and 5-, 12-, and 15-LOX. These activation leads to the production of inflammatory mediators, such as eicosanoids and specialized pro-resolving mediators (SPMs), which can be found at high levels in human FF impacting on oocyte growth and quality [
6‐
8]. In the ovaries, PGE
2 contributes to tissue remodelling, follicular wall proteolysis, corpus luteum formation, and regulating vascular changes. All these events are considered as final players of ovulation that coordinate the overall response, which results in the release of the cumulus-enclosed oocyte [
6].
In general, inflammation is a defensive response triggered by stimuli that aims to restore homeostasis. The initial response consists of a progression of events from leukocytes recruitment to pro-inflammatory lipids production. A successful acute inflammatory response leads to the elimination of the stimuli and suspension of pro-inflammatory lipids production, initiating the resolution and repair phases with the production of SPMs [
9]. The influx of neutrophils is replaced by macrophages that undergo reprogramming from classically activated M1 macrophages to alternatively activated M2 macrophages [
10].
Macrophages are among the predominant immune cells in the acute inflammatory response and ovulation, being found at high levels in FF [
11]. The molecular pathways in which these cells are involved in regulation ovulation are not yet fully elucidated. However, evidence suggests that macrophages are implicated in leukocyte recruitment, secretion of immune mediators, breakdown of the extracellular matrix component, and regulation of tissue remodelling after ovulation [
12]. Since macrophages act as drivers and regulators of diseases, therapeutic strategies that either decrease the amount and function of M1 or increase M2 activity are undergoing extensive pre-clinical and clinical research [
13].
However, uncontrolled or unsuccessful immune responses can lead to continued recruitment of inflammatory cells. The persistence of tissue damage results in organ dysfunction and, consequently, the establishment of chronic inflammatory diseases [
14]. An enhanced understanding of the role of inflammation in ovulation and the control of this process may lead to exploiting this knowledge to treat anovulatory infertility.
Since the crosstalk between hGCs and macrophages shows issues relevant to the production of key factors during oocyte development, this relationship requires further investigation. Therefore, the present study aimed to simulate the ovarian microenvironment and investigate how the production of pro- and anti-inflammatory mediators generated by M1 and M2 macrophages, respectively, might activate inflammatory pathways in GCs isolated from women undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) techniques.
Discussion
Macrophages are the most dominant ovarian leukocytes that can be found in the theca layers of growing follicles and in human FF [
22]. Over the last few decades of investigation, it is becoming increasingly evident that macrophages are phenotypically and functionally heterogeneous. In fact, macrophages display high degrees of plasticity in their roles when exposed to various environments. M1 macrophages are differentiated by Th1 cytokines, such as IFN-γ, or by recognition of LPS, producing high levels of pro-inflammatory cytokines (TNF-α and IL-1β). Functionally, the pro-inflammatory M1 macrophages are implicated in the clearance of pathogens in the course of infection by generating reactive oxygen/nitrogen species (ROS/RNS), which can induce tissue damage. In contrast, M2 macrophages are differentiated by Th2 cytokines, such as IL-4 and/or IL-13, secreting pro-resolving mediators (CCL18, CCL22, and SPMs). Functionally, pro-resolving M2 macrophages show the ability to phagocytose and promote tissue remodelling and wound healing. The heterogeneous and dynamic nature of macrophages in the ovaries implies their proactive involvement in ovarian homeostasis and hormonal control [
23].
Considering that M1 and M2 macrophages are associated with inflammatory processes, macrophages polarization may be an important aspect of their interaction with hGCs. Thus, the first aim of this study was to obtain M1 and M2 macrophages phenotypes from the THP-1 human monocyte cell line. The THP-1 is an immortalized cell line isolated from the peripheral blood of a 1-year-old male patient with monocytic leukemia. Resting THP-1 cells retain most of the inflammatory monocytes signalling pathways and exhibit the ability to differentiate into macrophages when stimulated with PMA. For these reasons, this cell line was chosen as an appropriate cell model for obtaining macrophages derived from human monocyte [
24].
However, although the THP-1 cell line is widely recognized as a suitable model for assessing macrophage functions and responses to foreign stimuli in vitro, there is currently no standardized protocol for the differentiation of THP-1 monocyte into macrophages using PMA. The lack of a standard protocol has a significant impact on the interpretation of results and comparison of studies. The differentiation protocol used in this study was based on the one previously published by our research group. We initiated THP-1 differentiation by exposing the cells to PMA (25 ng/mL) for 48 h, followed by a 24 h resting period [
18]. Differentiation of monocytes into resting M0 was confirmed by observation that macrophage-like cells adhered to tissue culture flask, adopting a stellate morphology. After that, resting M0 were primed for 24 h with fresh medium supplemented with IFN-γ (20 ng/mL) + LPS (10 pg/mL) to differentiate into M1 and by IL-13 (20 ng/mL) for M2. Transcriptional markers previously used to characterize distinct subsets of macrophages were identified in the literature and used in the present study [
24,
25]. As stated earlier, a distinctive hallmark of M1 polarization is the high production of pro-inflammatory cytokines, while M2 polarization regulates the production of pro-resolving mediators. Thus, M1 and M2 macrophages have been shown to be distinguished by distinct chemokine arrays. Studies conducted in ex vivo human systems reported that IL-1β and TNF-α genes were expressed in the M1 macrophages [
26]. Our results showed a very similar array of up-regulated genes in M1 macrophages, suggesting a successful differentiation. In turn, the chemokines CCL18 and CCL22 are categorized according to the M2 state [
25,
26]. Our findings show that M2 macrophages expressed all the chemokines mentioned above (Fig.
1).
Inflammatory conditions can severely disrupt normal ovarian function and oocyte quality. In vitro models that adequately simulate the ovarian microenvironment may provide a helpful tool to study the mechanisms by which pro-inflammatory M1 and pro-resolving M2 macrophages may affect the hGCs functions. At this point, we established an interaction model between CMs obtained from culture of M1 or M2 macrophages and GCs to mimic the ovary in inflamed states. This model of interaction has long been widely used to study fundamental cellular interactions of any kind. These systems are highly relevant for drug discovery as they provide a more representative in vivo model of human tissue than animal models [
27]. Culture models using CMs obtained from another cell type incorporate a physical barrier between cell types, allowing only signalling through the cell secretome (CS). The CM is commonly employed, where the medium is used first to culture one cell type and then transferred to the second cell type. The CM is constituted by the CS of the first cell type that contains soluble factors and can affect the behaviour of the second cell type in a positive and/or negative way [
28].
In the culture model adopted in this study, we decided to use only primary human GCs isolated from women who had male factor as a cause of infertility. Exclusion criteria for women with endometriosis or PCOS, for example, were based on eliminating cells obtained from inflammatory-associated pathologies and, therefore, investigating only the contribution of macrophages to physiological ovarian inflammation. An immortalized hGCs line could be helpful in studying many of the processes that are implicated in human follicle development [
29]. We chose to employ the COV434 cell line due to its ability to synthesize estradiol from FSH stimulation, presence of specific markers of apoptosis enabling the induction of follicular atresia, and capacity to form intercellular connections with cells surrounding an oocyte [
29,
30].
Regarding cell viability, we observed that CMs obtained from culture of M1 and M2 macrophages affected the viability of both hGCs and COV434 cells, when compared to the resting macrophage (M0), which was used as a control. However, no morphological changes were observed in both cells. As seen in Fig.
2, when hGCs and COV434 cells are exposed to CM of M1 macrophages, a significant decrease in cell viability occurs, suggesting that the pro-inflammatory environment may be influencing cells’ viability. However, it is possible to notice a greater significant difference in the decrease of hGCs viability (
p < 0.05) in comparison to COV434 (
p < 0.001), which in part can be explained by the fact that hGCs are originated from a previous inflammatory environment, such as ovaries. Furthermore, it is known that pro-inflammatory M1 macrophages can produce ROS/RNS, which can induce apoptosis in hGCs [
31]. However, this observation requires further investigation. Thus, the pro-inflammatory environment can affect the ovarian oxidative balance. Regarding cell viability when hGCs and COV434 cells are exposed to the CM of M2 macrophages, it can be observed that it is affected in a similar statistical proportion for both cells (
p < 0.0001) when compared to the M0 control, suggesting that the anti-inflammatory environment provided by M2 macrophages can, to some extent, contribute to cell proliferation.
In the present study, the interaction between CM obtained from culture of M2 macrophages and hGCs and COV434 cells for 72 h induced a significant increase in mRNA and protein levels of COX-2. However, no significant increase was observed when cells were exposed to CM obtained from culture of M1 macrophages (Figs.
4a and
5a). Narki et al. [
32] previously reported that the expression of COX-2 in GCs isolated from women undergoing ART was induced by the pro-inflammatory cytokine IL-1β, contrasting our results. From these findings, it is possible to suggest that the induction of COX-2 expression by the CM of M2 macrophages may increase the production of pro-resolving mediators by hGCs and COV434 cells, leading to the resolution of inflammation.
Feldam et al. [
33] demonstrated for the first time the expression of 5-, 12-, and 15-LOX in GCs isolated from women undergoing ART through the characterization of specific products derived from the metabolism of arachidonic acid (AA), such as 5-, 12-, and 15-hydroxyeicosatetraenoic acids (HETEs). In a recent study, Zhang et al. [
8] reported that the pro-resolving mediator resolvin E1 (RvE1) improved oocyte quality by decreasing apoptosis rate of CCs and increasing cell viability and proliferation. Considering that COX-2 and LOXs, both involved in RvE1 production, when stimulated can produce pro-inflammatory and pro-resolving mediators, we also investigated their expression in hGCs and COV434 cells by CMs of M1 and M2 macrophages. From our data, it is possible to observe that neither CM of M1 nor that of M2 macrophages were effective in inducing 5-LOX expression in hGCs, both in terms of RNA and protein levels, when compared to the positive control (PC, placenta homogenate) (Fig.
4b). Regarding COV434 cells, a significant increase in 5-LOX mRNA levels is observed when exposed to CMs of M1 and M2 macrophages. However, the same was not observed according to the results of western blotting, where a significant increase in 5-LOX protein expression was observed only when the cells were exposed to the CM of M2 macrophages (Fig.
5b). Concerning 12-LOX expression, a significant increase in its expression is only observed when hGCs are exposed to the CM of M1 macrophages (Fig.
4c). However, 12-LOX expression is not observed when COV434 cells are exposed to CMs of M1 and M2 macrophages (Fig.
5c). These results can be explained, at least to some extent, by the fact that the hGCs are originated from an inflammatory environment per se. Thus, increased expression of 12-LOX may be a compensatory way in which cells produce pro-resolving mediators to overcome inflammation. Finally, CMs of M1 and M2 macrophages, despite 72 h of interaction with hGCs, were not able to significantly affect the 15-LOX expression when compared to control (M0) (Fig.
4d). This result contrasts with the one published by Liao et al. [
34], in which the authors demonstrated that 15-LOX expression was up-regulated in GCs isolated from women with PCOS. In regard to COV434 cells, a significant increase in 15-LOX expression is observed when these cells are also exposed to CMs of M1 and M2 macrophages (Fig.
5d).
Conclusions
In conclusion, it can be inferred that M2 macrophages with anti-inflammatory characteristics were able to significantly influence the expression of COX-2 and 5-, 12-, and 15-LOX in hGCs. Therefore, it is possible to suggest that the production of pro-resolving mediators by hGCs can, at least in part, reverse the physiological inflammation present in the ovaries. As far as we know, this model of interaction between CMs obtained from culture of M0, M1 and M2 macrophages and hGCs and COV434 cells that aims to investigate the expression of COX-2 and 5-, 12-, and 15-LOX had not been investigated earlier, which enabled us to contribute with new findings related to the role of differentiated macrophages found in the ovaries. However, the extent to which pro-resolving concentrations affect oocyte quality, maturation, fertilization potential, and embryonic development is unclear and requires further investigation.
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