Exploring the role of mononuclear phagocytes in the epididymis.

The onslaught of foreign antigens carried by spermatozoa into the epididymis, an organ that has not demonstrated immune privilege, a decade or more after the establishment of central immune tolerance presents a unique biological challenge. Historically, the physical confinement of spermatozoa to the epididymal tubule enforced by a tightly interwoven wall of epithelial cells was considered sufficient enough to prevent cross talk between gametes and the immune system and, ultimately, autoimmune destruction. The discovery of an intricate arrangement of mononuclear phagocytes (MPs) comprising dendritic cells and macrophages in the murine epididymis suggests that we may have underestimated the existence of a sophisticated mucosal immune system in the posttesticular environment. This review consolidates our current knowledge of the physiology of MPs in the steady state epididymis and speculates on possible interactions between auto-antigenic spermatozoa, pathogens and the immune system by drawing on what is known about the immune system in the intestinal mucosa. Ultimately, further investigation will provide valuable information regarding the origins of pathologies arising as a result of autoimmune or inflammatory responses in the epididymis, including epididymitis and infertility.

MONONUCLEAR PHAGOCYTES

The murine epididymis, which is the primary site of sperm maturation and storage, contains an intricate network of mononuclear phagocytes (MPs) comprising dendritic cells (DCs) and macrophages (Mφs), which are key regulators of innate and adaptive immunity in numerous organ systems.12345 The ontogeny and functions of MPs have been studied extensively in lymphoid and nonlymphoid organs, particularly in the small intestine mucosa.6789 In contrast, the epididymis remains poorly studied and, therefore, the role of MPs in this largely uncharacterized mucosal system remains mostly speculative. This review will describe what is currently known about MPs in the epididymal mucosa and explore the possible roles of epididymal MPs (eMPs) based on comparisons with the small intestine.

Dendritic cells and Mφs are phagocytes and professional antigen-presenting cells (APCs). These two fundamental functions give them the ability to orchestrate innate and adaptive immune responses, by sampling self and nonself antigens, presenting processed peptides to naïve T cells, and secreting immunomodulatory factors. The first characteristic of the MP system is the tremendous heterogeneity of Mφs and DCs.610 The phenotyping of MP subsets is essentially based on the detection of multiple surface markers using flow cytometry-based techniques. describes a selection of immune cell surface markers that will frequently be mentioned in this review. As most markers are expressed by multiple subsets of cells rather than being cell type-specific, the clear identification of phenotypically and functionally distinct populations of immune cells has become increasingly complicated. For instance, the coexpression of CD11c (integrin alpha X chain) and major histocompatibility complex (MHC) class II has been largely used to identify DCs in lymphoid tissues. However, since Mφs from peripheral nonlymphoid tissues also express CD11c and MHC class II, the distinction between DCs and Mφs has been at the center of a long and passionate debate in the immunology community. Recent reviews are available to better appreciate the complexity of the MP system.61011

MONONUCLEAR PHAGOCYTES IN THE SMALL INTESTINE

In order to better understand of the putative roles of MPs in the epididymis, we will briefly recapitulate the current knowledge of the murine small intestine, which is a well-characterized mucosal system.11121314151617 DCs and Mφs perform complementary functions to maintain epithelial homeostasis and regulate the delicate balance between immune defense and tolerance in the steady state intestine. As potent phagocytes, they clear defective surrounding epithelial cells and sample the luminal compartment by extending dynamic intraepithelial dendrites across the apical tight junction barrier. Intestinal Mφs are sedentary and mostly derived from circulating Ly 6C (lymphocyte antigen 6C)-high monocytes that enter the lamina propria and ultimately differentiate into CX3CR1+ F4/80+ cells, F4/80 being the archetypal murine Mϕ marker.11 Recent results have suggested that some resident tissue Mφs arise from the local self-renewal of fetal precursors that were seeded in organs early during development.18 The precise mechanisms of MP replenishment in peripheral tissues are still under intense investigation. In contrast, intestinal DCs are replenished by myeloid precursors via a Flt3L-dependent mechanism and ultimately represent at least four subsets of cells expressing various levels of integrins including CD11b, CD11c and CD103, in addition to other leukocyte markers.11 Unlike bona fide Mφs, DCs do not express F4/80. Located in the lamina propria of the intestinal epithelium, DCs have been shown to acquire luminal antigens directly,1516171920 via interaction with goblet cells14 and/or via interaction with intraepithelial CX3CR1+ Mφs.13 In contrast to Mφs, DCs limit the degradation of phagocytozed particles to preserve antigenicity and are, therefore, specialized in antigen presentation.212223 Following antigen uptake and processing, they migrate from the lamina propria to lymph nodes (LNs) to prime and polarize naïve T cells via MHC class II. By inducing the development of Foxp3+ regulatory T cells (Treg), CD103+ DCs initiate the tolerogenic response to food antigens and commensal bacteria.24 Disruption of the equilibrium between tolerance and defense can result in inflammatory conditions of the gastrointestinal tract tract such as inflammatory bowel disease.1125

To summarize, the steady state small intestinal mucosa contains two major families of phenotypically distinct and functionally complementary MPs. On one hand, Mφs are in charge of maintaining the integrity of the epithelial barrier and scavenging materials. On the other hand, DCs populate the lamina propria and constitutively acquire luminal antigens directly or indirectly via cell-cell interactions with Mφs or epithelial cells, and finally migrate into mesenteric LNs (MLNs) to prime naïve T cells and induce either a tolerogenic or an immunogenic response.

FROM AN IMMUNOLOGICAL POINT OF VIEW, IS THE EPIDIDYMIS A SMALL INTESTINE ANALOGUE26?

Although the small intestine and the epididymis constitute two radically disparate environments, their respective mucosa may face surprisingly similar immunological challenges. Using the intestine as a model, it seems reasonable that the MPs lining the epididymal duct could be in charge of maintaining the integrity of the epithelial barrier and orchestrating peripheral tolerance to auto-antigenic spermatozoa undergoing maturation; simultaneously, they may fight the pathogens that constantly ascend and threaten the reproductive tract and monitor pathogens circulating in the vasculature ().

Localization, phenotype and possible functions of MPs in the steady state murine epididymis. The epididymal epithelium contains principal cells (gray), clear or narrow cells (orange), BCs (red), lymphocytes or halo cells (blue) and MPs (green). In this model, circulating Ly6Chi monocytes and DC precursors constitutively enter the epididymis and differentiate into mature CX3CR1+ CD11c+ F4/80+ Mφs, and CD11c+ CD103+ DCs, respectively. Resident Mφs may also self-renew in situ. MPs could acquire luminal soluble antigens and/or particles directly via extension of intraepithelial processes, or indirectly using conduits created by principal, clear and BCs. Following antigen uptake, migratory CD103+ DCs may migrate to lymph nodes to induce the development of FoxP3+ regulatory T cells (Tregs) in order to regulate tolerance to luminal antigens, or interact locally with resident lymphocytes. In addition, MPs play a major role in the maintenance of the BEB by rapidly removing apoptotic epithelial cells and debris. DCs and Mφs are also involved in the regulation of innate and adaptive immunity when a danger signal is sensed (infection, inflammation), and are also ideally positioned to survey circulating antigens. Our schematic diagram depicts observations made on the MP system in the IS; however, some MP functions might be segment specific. These putative MP functions in the steady state epididymis are largely inspired by studies of the gastrointestinal tract.1113141620 MPs: mononuclear phagocytes; BEB: blood-epididymis barrier; DC: dendritic cell; Mφs: macrophages; BCs: basal cells; IS: initial segment.

Sperm are postmeiotic cells that display antigens not expressed anywhere else in the body. In addition, they are abundant (1000 to 2000 sperm enter each human epididymis every second) and appear at puberty, long after the establishment of central tolerance.2728 Consequently, male reproductive function relies on strong immunoregulatory mechanisms that prevent sperm from being identified as nonself-cells and rejected by the host's immune system, both in the testis and in the posttesticular environment. In order to control interactions between immune cells and sperm antigens, the reproductive tract is lined by barriers named the “blood-testis barrier” (BTB) and the “blood-epididymis barrier” (BEB).29 The physiology of the BEB and BTB has been extensively studied, and our readers are kindly advised to consult recent reviews and articles that highlight fundamental differences between the testis and the epididymis.303132 Unlike the epididymis, the testis is often considered an “immunologically privileged organ” (i.e., foreign tissues implanted in a testis are not rejected) and, despite their anatomical proximity and their functional complementarity, these two organs seem to constitute two fundamentally distinct immunological environments.27283334 Simplistically, the term “barrier” suggests that a physical separation consisting of epithelial cells joined by tight junctions is sufficient to partition sperm from the immune system, thus averting the development of autoimmunity against sperm antigens. In light of recent research, it has become evident that the BTB and the BEB are complex and dynamic structures that do not fully prevent interactions between the immune system and germ cells, but provide support for finely tuned cellular and molecular crosstalk.313235

In humans and rodents, the pseudostratified epithelium that lines the epididymal duct is composed of several types of cells, including principal cells, clear/narrow cells, and basal cells (BCs). Altogether, these cells are in charge of creating a succession of microenvironments in which sperm mature and are stored.363738394041 Sperm maturation and storage involves a finely tuned sequence of complex interactions between the epithelium, the luminal fluid, and spermatozoa. Alongside epithelial cells, cells of the immune system were identified in the epididymis several decades ago, including lymphocytes (most likely referred to as “halo cells”) and Mφs.442434445 Both Mφs and lymphocytes have been shown to respond to changes in the epididymis initiated by aging, vasectomy and infections.346474849 However, these discoveries provide not more than a superficial view of the increasingly complex mucosal immune system composed of many phenotypically, morphologically and functionally distinct subsets of lymphoid and myeloid cells, including MPs, which maintain constant interactions. In order to unravel the immunophysiology of the epididymis, it is now vital to unambiguously identify each subset of immune cell and determine how they interact with their environment. Fortunately, immunologists have developed a battery of powerful tools to study immune cells in other organ systems, thus introducing new perspectives to reproductive biologists. Among those tools, two lines of transgenic mice (CD11c-EYFP50 and CX3CR1-GFP51) have allowed us to illuminate an extensive network of DCs and Mφs in the mouse epididymis1 (). CD11c (integrin alpha X) and CX3CR1 (a G-protein coupled chemokine receptor) are expressed by several subsets of Mφs and DCs.

MPs in the proximal mouse epididymis. CD11c+ and CX3CR1+ MPs are abundant in the murine epididymis. This partial view of the most proximal region of a section of CD11c-EYFP mouse epididymis reveals CD11c+ DCs and Mφs. CD11c+ MPs are present in the peritubular region as well as in the interstitium and the capsule (arrowhead) that wraps the epididymis. In the IS (brightest area), which is the most proximal epididymal segment, CD11c+ (and CX3CR1+) intraepithelial Mφs extend dendritic processes toward the apical side of the epithelium, suggesting that this particular subset of MPs is in charge of directly surveying the luminal content, possibly in a segment-specific manner. Scale bars = 50 μm, L: lumen; MPs: mononuclear phagocytes; DCs: dendritic cell; Mφs: macrophages; IS: initial segment.

Although Mφs have been described in the murine epididymis in the past, these mouse models revealed the density of the MP network and its complex interactions with the duct, highlighting the fact that the mucosal immune system present in the epididymis is more sophisticated than generally documented in the literature. MPs line the excurrent duct in all epididymal segments (as well as efferent ducts and the vas deferens), and are also present in the interstitial space and the connective tissue capsule that covers the epididymis. Several studies have suggested that BCs of the rodent epididymis express Mφ markers and exert an immunological function.2364445 This long-standing confusion between epididymal BCs and intraepithelial Mφs illustrates the importance of characterizing each subset of cell by capitalizing on tools and technologies recently introduced and newly discovered markers specific for each cell type. Indeed, BCs and Mφs are both located in the basal region of the epithelium and share apparent morphological similarities. However, using powerful imaging techniques in combination with specific markers and flow cytometry-based analyses, Shum et al. have demonstrated that BCs and Mφs represent two unequivocally distinct cell types.5 Both occupy the basolateral compartment of the epididymal epithelium and, in the initial segment (IS), extend slender processes toward the lumen. Only intraepithelial Mφs express F4/80, CX3CR1, and CD11c, while BCs express the widely acknowledged BC marker keratin5 (KRT5). Although BCs do not belong to the immune system, a contribution to local immunoregulatory mechanisms (possibly via crosstalk with neighboring MPs) cannot be excluded.

In the gut, one of the primary functions of epithelial Mφs is to maintain the homeostasis of the epithelial barrier. The intestinal lumen is an extremely harsh environment; the thin epithelium is constantly challenged by trillions of microorganisms that make up the natural intestinal flora, pathogenic microbes, digestive acids and enzymes, as well as ingested materials that must be either assimilated or rejected. Comparatively, the epididymal luminal compartment may seem quiescent: during the entire reproductive life, the duct is mostly occupied by a complex yet stable fluid, radically changing just once with the introduction of spermatozoa at puberty. However, the physical integrity of the pseudostratified epithelium should constitute an essential prerequisite to the establishment of an immunoprotective environment for spermatozoa.

HOMEOSTASIS OF THE BLOOD-EPIDIDYMIS BARRIER

In addition to its immunological role, the BEB is an anatomical and physiological barrier that controls the exchange of molecules between the lumen and the interstitial space containing blood and lymph, thus allowing the formation of the specialized luminal environment in which sperm transit and mature.31 Both immunoregulatory and physiological roles of the BEB are strictly dependent on the proper maintenance of the single layer of epithelial cells that lines the entire duct. Over the past decade, work from many laboratories has revealed the crucial importance of apoptotic cell clearance in the maintenance of all epithelia. The rapid removal of defective epithelial cells is critical for maintaining both the physical integrity of epithelial barriers and the tolerance to autoantigens. Failed clearance of apoptotic cells can lead to the development of various pathogenic states, including autoimmune diseases.52535455 Apoptotic cell clearance is a finely regulated multistep process that involves various molecular and cellular interactions, from the detection of apoptotic cells using “find-me” signals to their destruction. The professional effectors of phagocytic clearance are Mφs and DCs, nevertheless neighboring epithelial cells may be involved in the process. Interestingly, apoptotic cells are rarely detected in the epididymal epithelium; however, MPs are conveniently abundant, suggesting the presence of highly efficient clearance mechanisms. In addition, intraepithelial Mφs express CX3CR1, the receptor for the chemokine CX3CL1 (fractalkine), which has been identified as a find-me signal released by dying cells.

Efferent duct ligation (EDL) is a simple surgical procedure that provides an interesting opportunity to study apoptotic clearance in the epididymis; by preventing testicular factors from entering the IS, EDL induces a massive yet transient wave of epithelial apoptosis in the proximal epididymis.455657 Smith et al. have recently reported that epididymal intraepithelial CD11c+ CX3CR1+ Mφs respond to EDL by rapidly engulfing apoptotic cells and debris, thus preserving the integrity of the apical tight junction network.58 Contrasting with Mφs, BCs do not seem to be involved in apoptotic cell clearance (); however, recent data have demonstrated that the absence of testis-derived luminal factors reduces their projections and furthermore, BCs could play a role in the post-EDL epithelial regeneration.59 While the extreme situation caused by EDL is never encountered in physiological conditions, these results suggest that one of the primary functions of eMPs, and more specifically intraepithelial CX3CR1+ CD11c+ Mφs, is to actively maintain the integrity of the BEB. However, Mφs are much more than the janitors of the epithelium; by engulfing apoptotic cells, intraepithelial Mφs could play an active role in the maintenance of peripheral tolerance to local self-antigens, including antigens displayed on or released by maturing spermatozoa.

MP and BC response to the loss of luminal factors caused by EDL in the proximal mouse epididymis. In the steady state epididymis (a), CX3CR1+ CD11c+ intraepithelial Mφs (green) and KRT5+ BCs (red) sit in the basal region of the epithelium and occasionally project extensions (arrows) toward the luminal compartment. After 48 h EDL (b), MPs engulf apoptotic cells and debris and exhibit a phagocytic appearance, while BCs are not visibly involved in the apoptotic cell clearance process. Scale bars = 5 μm, L: lumen; MPs: mononuclear phagocytes; Mφs: macrophages; BCs: basal cells; EDL: efferent duct ligation.

EPIDIDYMAL MONONUCLEAR PHAGOCYTES AND TOLERANCE

In rodents as well as in humans, immunological tolerance is the result of fundamentally distinct mechanisms that involve both innate and adaptive immunity. Central tolerance is established in the bone marrow and thymus during the early stages of the immune system development. T and B cells that recognize self-antigens are removed or silenced, thus preventing the development of an immune response against the host's antigens. Germ cells and mature sperm constitute a rather unique challenge for the immune system; they start populating the reproductive system at puberty (long after the establishment of central tolerance), are abundant and express antigens that are not expressed elsewhere in the body. Accordingly, autoimmunity against sperm antigen is a direct cause of male infertility and underscores the fact that robust tolerogenic mechanisms must take place in the reproductive system during the entire reproductive life.2728 These mechanisms are known as peripheral tolerance,60 which takes place after the initial maturation of B and T cells. Peripheral tolerance results from multiple complementary mechanisms that involve a variety of cellular and molecular actors, including MPs.

In the gut, CD103+ DCs acquire antigens directly or indirectly (via epithelial cells or intraepithelial Mφs), and then migrate to the MLNs via a CCR7-dependent mechanism.13141624 Once in the MLN, they induce the development of a specialized subset of CD4+ T cells known as regulatory T cells (Treg), primarily distinguishable by their expression of the transcription factor FoxP3. Induced Tregs exert a suppressor activity on effector T cells, thus preventing the development of an immune response against self or innocuous antigens. eMPs are ideally positioned to have a similar activity in the BEB, although experimental evidence remains to be provided. Strikingly similar to the small intestine, the proximal region of the epididymal duct contains CX3CR1+ intraepithelial Mφs that project numerous dendrites toward the luminal compartment. We have recently demonstrated that CX3CR1+ CD11c+ eMPs are potent phagocytes and occasionally penetrate the luminal compartment, suggesting that they have the ability to directly sample soluble antigens and particles from the lumen (Smith et al., 2014 American Society of Andrology meeting, Atlanta, USA). It is therefore tempting to speculate that some Mφs may directly acquire material originating from spermatozoa in order to initiate a tolerogenic response, mediated by the CD11c+ CD103+ DCs that are also present in the epididymis.1 Acquisition of sperm components might also occur via indirect mechanisms involving epithelial cells, or by modulating the permeability of apical tight junctions.35 In addition, CD11c+ MPs isolated from the whole murine epididymis have potent antigen-presenting capabilities in vitro.1 The current challenge is to characterize the function of clearly defined subsets of Mφs and DCs in the context of a complex epididymal architecture, where MPs are likely to have region-specific and possibly segment-specific activities. Importantly, despite their abundance and their heterogeneity, MPs represent only a few pieces in the immunological puzzle that is the epididymis. Several factors involved in immunosuppression have been identified in the epididymis, including interleukin-10, cyclo-oxygenase 1 and 2, transforming growth factor beta, and indoleamine 2,3-dioxygenase (Ido). Ido is an enzyme that exerts immunosuppressive effects by regulating the catabolism of tryptophan. In the gut, Ido is synthesized by CD11c+ CD103+ DCs and controls the development of Tregs, thus regulating oral tolerance.61 The disruption of Ido in a mouse model modified the immunological equilibrium of the epididymis and, ultimately, impacted sperm quality and quantity.6263 While current data indicate that CD103+ DCs are not the primary source of epididymal Ido, the influence of Ido on local DCs and Mφs should be further investigated.

EPIDIDYMAL MONONUCLEAR PHAGOCYTES, CAPILLARIES AND LYMPHATIC VESSELS

The architecture of blood and lymphatic vasculature is expected to have a direct impact on eMP function by influencing their replenishment and migration toward LNs. Interestingly, several groups have observed that blood and lymphatic capillaries are mutually exclusive in the rodent epididymis: in the proximal regions, blood vessels and capillaries are extremely abundant, and lymphatic vessels are scarce. In contrast, the cauda contains a dense network of lymphatic vessels and few capillaries. In addition, capillaries from the IS are fenestrated and establish close interactions with the duct, thus facilitating a very active exchange of fluid, solutes and cells between the epididymal duct and the blood flow.646566 While the abundance of fenestrated capillaries in the IS might be predominantly related to the high rate of fluid reabsorption characteristic of this segment,4041 it should be noted that these capillaries are in close proximity with CX3CR1+ Mφs. Mucosal antigen sampling is not strictly unidirectional: in the gut, lamina propria CX3CR1+ DCs closely interact with the fenestrated capillary network to facilitate surveillance of circulatory antigens, consequently modulating the activation of mucosal T cells.67 The strikingly similar network of vessels and MPs in the proximal epididymis leads us to believe that a subset of MPs might be in charge of surveying the blood compartment, thus creating another conduit in the BEB.

EPIDIDYMAL MONONUCLEAR PHAGOCYTES IN ACTIVE IMMUNE RESPONSES

The immunophysiology of the epididymis receives relatively little attention partly because this organ is not affected by major medical conditions. The most common pathology is epididymitis (inflammation of the epididymis), often in response to a bacterial infection of the urogenital tract. However, the immunological environment of the epididymis is also affected by other events, including aging, surgical procedures (such as vasectomy), physical trauma, as well as experimentally induced autoimmune disorders.327284968 Vasectomy is often associated with the development of an autoimmune response against sperm antigens, as well the appearance of sperm granulomas in the epididymis.46496970 Epididymal Mφs and DCs are likely to be directly involved in the complex immune disturbance caused by vasectomy,49 from the early sensing of danger signals in the distal epididymis to the late development of granulomas (masses of extravasated spermatozoa surrounded by immune cells) and sperm autoantibodies. It is worth noting that epididymal tumors are extremely rare.71 We postulate that the dense network of MPs occupying the epididymis, including Mφs that have been shown to be impressively efficient in the clearance of abnormal cells,525458 may play a role in the low incidence of epididymal cancer.

CONCLUSION

Despite an intense global effort to understand the immunophysiology of the intestinal mucosa over many decades, new information is continually revealed. Our discovery of a sophisticated network of DCs and Mφs in the mouse epididymis has put the spotlight on one of the best-kept secrets of mammalian physiology, one that is challenging to study. Fortunately, the previously outlined similarities between these two organs, both functional and morphological, allow us to apply discoveries from the research effort in the intestine to postulate on the function of the epididymal mucosal immune system. Historically it has been accepted that the BEB physically separates the immune system from spermatozoa and the intricate and abundant blood vasculature unique to the IS of the epididymis unveils an “immunologically fragile site.”64 Contrarily, recent observations suggest that the IS may be a primary site for immunoregulation by virtue of a dense network of MPs projecting long intraepithelial dendrites exclusively in the IS. The IS lumen represents an opportunity for sperm leaving the testis to interact directly with the components of the epithelium, including intraepithelial MPs. In this respect, the IS might be seen as an “in vivo flow cytometer.” Notwithstanding these findings and requiring extensive research, the IS is also a region that is comparatively poor in lymphatic vessels, which might suggest that intense trafficking of migratory DCs is not a primary feature of this segment. On the other hand, this might also suggest that the process of migration and antigen presentation is particularly potent, and a sparse network of lymphatic vessels is sufficient for maintaining immune homeostasis in this region. In the remaining segments, MPs line the basal region of the epithelium in addition to being scattered throughout the interstitium. Dendrites extending toward the lumen are not observed; however, thus suggesting that direct acquisition of luminal antigen does not occur, at least in the steady state epididymis, beyond the IS. Rather, any antigens acquired from the lumen must be shuttled through the epithelium. Interestingly, the lymphatic system is comparatively more generous in the distal epididymis, suggesting that either migration of APCs to LNs is not exclusive to the IS, or that the distal epididymis has a large requirement for clearing extravasated fluid. Speculation aside, the segmentation of the epididymal epithelium and the various morphologies and distribution of APCs must, therefore, be taken into consideration when exploring the function of this unique mucosa. Finally, if a germ cell “quality control” mechanism does take place in the epididymis as per previous suggestions,62637273 eMPs are ideally positioned and equipped to play a role in the detection (and possible removal) of defective gametes in the IS.

Unraveling the mysteries of the epididymal mucosa will require an extensive basic research effort in favor of gaining a greater understanding of destructive inflammatory responses in the epididymis contributing to epididymitis and infertility, and ultimately exploiting the system in pursuit of an effective immunocontraceptive. Beyond the scope of reproductive immunology, the epididymis provides a clean site free of commensal bacteria that we anticipate will provide a fruitful tool for the broad study of mucosal immunology.

COMPETING INTERESTS

The author declares that they have no competing interests.

Table 1

Most common cell-surface markers used to identify murine MPs