Department of Bioengineering, Carnegie Mellon University, McGowan Institute of Regenerative Medicine

Immunomodulators and macrophages in endometriosis pathogenesis and progression

Hilda Jafaraha, Isabelle Chickanoskyb, Alexis Nolfia,c , Mangesh Kulkarnia,c, Clint Skillena,c, Bryan Browna,c

aDepartment of Bioengineering, bCarnegie Mellon University, cMcGowan Institute of Regenerative Medicine

Hilda Jafarah Hilda Jafarah is a senior bioengineering student who was born and raised in Jeddah, Saudi Arabia. She’s motivated to combine her passion for immunomodulation, tissue engineering, and drug delivery to advance medicine especially in women’s health. After graduation, she aspires to pursue an Md/PhD.

Dr. Bryan Brown is an Associate Professor in the Department of Bioengineering with secondary appointments in the Department of Obstetrics, Gynecology, and Reproductive Sciences and the Clinical and Translational Science Institute at the University of Pittsburgh. He is also Bryan Brown, Ph.D. a core faculty member of the McGowan Institute for Regenerative Medicine where he serves as the Director of Educational Outreach. Dr. Brown is also an Adjunct Assistant Professor of Clinical Sciences at the Cornell University College of Veterinary Medicine and Chief Technology Officer of Renerva, LLC, a Pittsburgh-based start-up company.

Significance Statement

Endometriosis, affecting 200 million women worldwide, causes chronic pelvic pain, infertility, and increases the risk of cancer. This research found that endometriosis development is caused by dysregulated macrophage involvement and subsequent cytokine cascade. This knowledge opens doors to understanding disease identification, potential disease biomarkers, and immunotherapies for diagnosis and treatment.

Category: Review Paper

Keywords: Endometriosis, Cytokines, Macrophages,

Pathophysiology, Immune response

Abstract

A comprehensive literature review was conducted focusing on recent findings on the topic of the pathogenesis of endometriosis which is characterized by the ectopic growth of endometrial cells within the peritoneal cavity. Women with endometriosis display a dysfunctional innate and adaptive immune response where immune cells including natural killer cells (NK cells), dendritic cells (DC cells), cytotoxic T cells (T cells), and, specifically, macrophages behave differently. An overpowering imbalance of immunosuppressive factors play a role in the immunoescape of endometrial cells, and the upregulation in angiogenic and neurogenic factors promote the formation and growth of ectopic lesions. With the lack of efficient models, the pathogenesis of endometriosis remains to be unclear; however, current research emphasizes the role of a dysfunctional immune response to refluxed endometrial cells in women with endometriosis including the differential response of immune cells and the expression of various hormones, immunomodulatory cytokines, growth factors, prostaglandins, and angiogenic and neurogenic factors.

1. Introduction

Endometriosis (EMS) is a gynecological disease characterized by the endometrial lining binding outside of the uterine cavity, forming “lesions”. Typically lesions form on the lining of the pelvic cavity (peritoneum) or the organs of the cavity (e.g., ovaries). It is associated with chronic pelvic pain, infertility, and fatigue. EMS affects about 1015% of women of reproductive age, up to 50% of infertile women, and is prevalent in 71–97% of women with chronic pelvic pain [1]; meanwhile, diagnosis is usually delayed by an average of 10 years from the onset of symptoms. Sampson’s theory of retrograde menstruation, defined as the pathogenic reflux of endometrial cells during menses through the fallopian tubes, is commonly accepted to explain the mechanism through which endometrial tissue travels into the peritoneal cavity; however, it fails to explain the discrepancy between the 76-90% of women who experience retrograde menstruation and the 10-15% of women affected by EMS [1]. This literature review allows for a comprehensive understanding of immune malfunction in macrophage and cytokine recruitment in EMS. The macrophage is a unique subset of monocytes with vital roles in both innate and adaptive immunity as it can differentiate into a wide variety of phenotypes ranging from the two extremes: M1 and M2. Pro-inflammatory, “M1-like,” macrophages perpetuate inflammation, secrete pro-inflammatory signaling molecules, and destroy tissues while anti-inflammatory, “M2-like,” macrophages aid in tissue healing and restorative processes. Some studies propose that M1-like macrophages initiate EMS, while others suggest that a localized tissue-level M2-like macrophage population enhances the growth and development of ectopic lesions [2,3]. The hormonal and immune involvement of M1-like and M2-like macrophages during the pathogenesis of EMS recruit various immunomodulators to the peritoneal cavity and result in a cytokine

cascade. An imbalance of immunomodulators and a dysfunctional immune response could aid in the immunoescape, the evasion of phagocytosis or immune triggered apoptosis, of endometrial cells. In this literature review, the immune dysfunction in EMS was outlined to illustrate its involvement in the pathogenesis of the disease. This work opens doors to understanding stage-specific disease identification, potential biomarkers of the disease, and further immunotherapies that could replace laparoscopic surgery as the gold standard of EMS diagnosis and treatment.

2. Methods

Using various search engines and data bases such as PubMed and ScienceDirect, a comprehensive literature review was conducted focusing on 120 peer-reviewed EMS research papers published from 1997-2020. This range of research included the disease pathogenesis, immune cell involvement in EMS, and the role of key biomolecules involved in immunomodulation. Qualitative figures were made illustrating the role of immunomodulators at each stage of pathogenesis based on the results of the 120 papers studied.

3. Endometriosis and the Immune System

3.1 Macrophages in Endometriosis

Women with EMS display a dysfunctional innate and adaptive immune response where immune cells including natural killer cells, dendritic cells, cytotoxic T cells, and macrophages behave differently. Among the dysfunctional immune responses, macrophages are the most critical in EMS. In fact, EMS has been termed a “disease of the macrophage.” Macrophages contribute to the pathogenesis of EMS by creating an environment that favors implantation, promotion of the formation, growth, and development of lesions, and augmentation of pain sensitivity and inflammation.

In EMS patients, macrophages display various dysfunctions including a loss of scavenging ability [4] and an obstruction of phagocytosis through the upregulation of CD200 [5] while continuing the release of inflammatory, oxidative, and nociceptive mediators. This augments the immunoescape and implantation of endometrial cells and inflammation. Furthermore, macrophages in EMS abnormally downregulate IL-24 in the endometriotic milieu causing an increase in the invasiveness and proliferation of endometrial stromal cells through various cytokines [6]. In addition to dysfunctional macrophages, EMS patients present a phenotypic imbalance where the concentration of M2-like to M1-like ratio increases through the Smad2/ Smad3 pathway, and macrophages experience an M1 to M2 polarization through various upregulated cytokines, including IL-17A, which can mobilize granulocytes and stimulate the production of cox-2, in the endometriotic milieu [7, 8, 9]. Therefore, there is a deviation towards M2-like polarization which results in the secretion of growth factors and angiogenic factors, such as VEGF and TGF, aiding in endometriosis grafting, development, and maintenance. An M1 to M2 transition results in the functional shift from an acute inflammatory response to the promotion of tissue repair and regeneration including extracellular matrix synthesis and angiogenesis. Finally, macrophages in endometriosis also promote neurogenesis and sensitization [10], and it was shown that macrophage depletion alleviates abnormal pain in mice models with induced EMS.

Figure 1. In EMS, macrophages (MO) experience various dysfunctions including the loss of scavenging ability (A) due to the lack of adherence to the peritoneal cavity. In MO and endometrial stromal cells co-cultures, MO upregulate IL-10 and TGF-�� which inhibits the cytotoxicity of NK cells permitting the immunoescape of refluxed endometrial cells (B). (C)Finally, ectopic lesions upregulate estrogen which directly (black) affects the MO resulting in an increase in the production of MMP’s and VEGF and indirectly (red) limits the phagocytosis ability of MO’s by inducing an increase of CD200 production in ESC’s. Both promote the growth of lesions.

3.2 The Role of Immunomodulators in Endometriosis Pathogenesis

An overpowering imbalance of immunomodulators including cytokines, matrix metalloproteinases, and growth factors are involved in the progression of EMS. The development of EMS is multifaceted and can be simplified into seven different factors or stages: the priming of the

microenvironment, the initial lesion formation, a dysregulated immune response, the evasion of apoptosis, the promotion of angiogenesis, the continuation of cell proliferation, and the induction of chronic inflam-

mation. The involvement of cytokines in each of these stages shown in Figure 2, as well as the immune malfunction, facilitates the establishment and development of ectopic lesions.

During the onset of EMS, an abnormal priming of the microenvironment allows for the implantation and survival of the initial endometriosis lesion. The priming takes place through various hormones and cytokines which continue to be involved in later stages of EMS. The initial endometriotic lesion forms shortly after retrograde menstruation occurs. As endometrial fragments, including endometrial cells characterized by plasticity and cells with reduced differentiation status, begin to leave the uterus through the fallopian tubes and ovaries, specific immunomodulators promote the initial lesion formation. The endometriotic lesion formed has an altered estrogen signal and progesterone resistance. This causes genetic and epigenetic changes, an altered immune response and immunoescape, and aberrantly activated signaling pathways [23]. During the priming of the microenvironment and the formation of the initial lesion, many matrix metalloproteinases (MMPs) were shown to be elevated including MMP-2, which can promote the breakdown of the basement membrane in eutopic endometrium as well as aid in the adhesion and angiogenesis in the ectopic endometrium [11]. In addition to MMPs, interleukins including IL-15, IL-8, and IL-6 are also involved in this stage [14-16]. Most importantly, IL-8 acts as an autocrine growth factor in the endometriotic milieu [15].

Retrograde menstruation occurs in 90% of menstruators, though only 10-15% of these patients develop EMS. The discrepancy between the population of patients who experience retrograde menstruation and those who develop EMs occurs because of the dysfunctional immune response to the initial lesion formation. The dysfunctional immune response results in lesion growth, neurogenesis, inflammation, and angiogenesis. This stage is carried out by a plethora of immunomodulators including MMPs, EOTAXIN, growth factors, interferons, and interleukins. This dysfunctional immune response initiates the evasion of apoptosis, vascularization, increased proliferation, and the chronic inflammation.

Apoptosis is a programmed cell death mechanism that acts as a safety measure and is induced by local immunomodulators. In EMS, the dysfunctional immune response results in the evasion of apoptosis. This step is crucial to the development of EMS, and many hypothesize that it could be the cause of the statistical discrepancy mentioned before. Some important immunomodulators inhibiting the apoptosis of endometrial cells include IP-10, EGF, and IFN-g [19,20,22]. This reflects the parallelism between ectopic endometrial cells and tumorigenic cells.

Vascularization is crucial for the sustainability of the invasive lesions. Without blood flow and oxygenation, endometriotic cells cannot survive in the peritoneal environment. Current research has shown a significant elevation in angiogenic factors most importantly VEGF [25]. IL-15 also induces angiogenesis through the elevation of VEGF secretion [14].

Through the immune dysfunction, endometriotic cells continue to proliferate and create more lesions aided by angiogenesis and the evasion of apoptosis. During this step of pathogenesis, the lesions spread creating more severe stages of disease development such as Stages III and IV or Deep Infiltrating EMS (DIE) as categorized by the American Society of Reproductive Medicine. Many of the immunomodulators involved in this step of pathogenesis are remnants from the previous steps including IL-15 [14] and EGF [20]. One unique immunomodulator is IL-1b which has been shown to increase the proliferation and migration of endometriotic cells as well as the induction of VEGF through the COX-2 cascade [24,25].

Figure 2. Endometriosis pathogenesis begins with a primed peritoneal microenvironment which supports the implantation of ectopic endometrial stromal cells during initial lesion formation. The lesion causes a dysregulated immune response, resulting in the evasion of apoptosis and increased angiogenesis. This begins the cycle of survival for the endometriotic tissue as the malfunctioning of the immune system prevents the removal of the harmful tissue. With access to vasculature, the lesion promotes cell proliferation and spreads throughout the peritoneal cavity. This spread leads to chronic inflammation which continues for the duration of disease presence.

Chronic inflammation in the peritoneum is a key indicator of EMS. Even though inflammation exists throughout the development of EMS, the chronic inflammation which occurs illustrates the systemic role that inflammation plays in the continuation of endometriotic growth and the preparation of the surrounding microenvironment for other lesion development.

4. Clinical Significance

Despite the prevalence of EMS, there is little known about the involvement of the immune system and the general pathogenesis of the disease. Understanding the role of macrophages and immunomodulators in the progression of EMS can aid in the development of early detection and possibly less invasive treatments. An effective pre-clinical study proved that trichostatin A had anti-proliferative activity on endometrial stromal cells by reducing the expression of COX-2, which is elevated due to high expression of IL-1b and other inflammatory cytokines, and its administration in mice significantly decreased the size of endometriotic implants [26]. Additionally, human recombinant TNFa antagonists, TNFRSF1A and c5N, demonstrated inhibitory activity on endometriotic lesions in baboons without affecting their menstrual cycle [27, 28]. Finally, this knowledge can help in the development of in vitro model systems of EMS for further investigation of the disease pathogenesis as well as for drug testing.

5. Conclusion

The pathogenesis of EMS remains to be unclear due to the lack of models; however, current research emphasizes the role of a dysfunctional immune response to retrograde menstruation in EMS patients. Moreover, the aberrant expression of hormones, immune cells, immunomodulatory cytokines, growth factors, prostaglandins, and angiogenic and neurogenic factors contribute to the development of EMS. Understanding the immunomodulators and their trends in the stages of EMS development could allow for better disease detection and assessment as well as immunotherapeutic treatments. However, the question remains whether the differential immune response results from the refluxed endometrial cells and formed lesions or is the differential immune response innate to EMS patients.

6. Acknowledgments

This work was funded by the Swanson School of Engineering, the Office of the Provost, the Department of Bioengineering and was conducted under the mentoring of Dr. Brown and Alexis Nolfi with the help of Dr. Kulkarni and Clint Skillen.

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