Immunoregulatory mechanisms between epithelial clear cells and mononuclear phagocytes in the epididymis

Abstract

Introduction

One of the most intriguing aspects of male reproductive physiology is the ability of the epididymis to prevent the mounting of immune responses against the onslaught of foreign antigens carried by spermatozoa while initiating very efficient immune responses versus stressors. Epithelial clear cells (CCs) are strategically positioned to work in a concerted manner with region-specific heterogeneous subsets of mononuclear phagocytes (MPs) to survey the epididymal barrier and regulate the balance between inflammation and immune tolerance in the post-testicular environment.

Objective

To summarize and critically analyze available data on the role of CCs and MPs in the epididymal mucosa. This review aims to describe how CCs communicate with MPs to contribute to the unique immune environment in which sperm mature and are stored in the epididymis.

Materials/methods

A comprehensive systematic review was performed. PubMed was searched for articles specific to clear cells, mononuclear phagocytes, and the epididymal mucosa. Articles that did not specifically address the target material were excluded.

Results

In this review, we discuss the unexpected roles of CCs, including the transfer of new proteins to sperm via extracellular vesicles and nanotubes as they transit along the epididymal tubule; and we summarize the immune phenotype, morphology, and antigen capturing, processing, and presenting abilities of MPs. Moreover, we present the current knowledge of immunoregulatory mechanisms by which CCs and MPs may contribute to the immune-privileged environment optimal for sperm maturation and storage.

Discussion and conclusion

Notably, we provide an in-depth characterization of CC-MP communication networks in the steady state epididymis and in the presence of injury. This review highlights crucial concepts of mucosal immunology and cell–cell interactions, all of which are critical but understudied facets of human male reproductive health.

Introduction

The epididymis is a single convoluted duct that presents a high level of functional complexity (1–9). It is organized into several segments with distinct key functions: the initial segments (IS), the caput, the corpus, and the cauda. Each segment possesses distinct gene expression profiles as well as discrete morphological features (2–4, 7, 10–12). The epididymal epithelium is composed of three main cell types: clear cells (CCs), principal cells, and basal cells. There is a fourth epithelial cell type, known as the narrow cell, with similar characteristics to CCs, which is exclusively found in the IS (10, 13, 14).

Epithelial cells in the epididymis form the blood-epididymis barrier where tight junctions and specific cellular transporters ensure the maintenance of a unique luminal immune-privileged environment that is essential for sperm maturation, protection, and storage (10, 11, 15–18). This barrier is a critical structure for male fertility (8, 19–30). The epididymis depends on a delicate and carefully regulated interaction between the endocrine and immune systems. What is unique and fascinating about this reproductive organ is its ability to produce and nurture spermatozoa that appear long after the establishment of self-tolerance without invoking a damaging immune response. Therefore, the epididymis relies on immune mechanisms of sperm tolerance, similar to the system that controls oral tolerance to food antigens and commensal bacteria in the digestive tract (31–33).

The epididymal mucosa is a particularly dynamic environment that constantly interacts with numerous spermatozoa to prevent autoimmune responses and when necessary, initiates immune defense reactions against pathogenic microorganisms or stressors (4, 5, 12, 25, 34–39). These paradoxical immune responses poise the epididymis as a unique and complex immunological site. The appropriate balance between sperm tolerance and immune activation is crucial to preserve epididymal function, and disruption of this homeostasis is responsible for a large proportion of male infertility cases (8, 19–30). The remarkable organization of epithelial and immune cells contributes to the formation of the immune-privileged environment in the epididymis. A dense heterogeneous network of mononuclear phagocytes (MPs) is strategically situated to play a significant role in epididymal mucosal immunity (4, 12, 34, 35, 40). These myeloid cells, consisting of dendritic cells, monocytes, and macrophages, are located in the epithelium as well as the interstitium. They are responsible for tissue homeostatic maintenance and help orchestrate the innate immune response following infection or injury in the epididymis (5, 8, 36, 40) as well as in other organs (41–43). Among other functions, epididymal MPs detect and capture molecules that are perceived as a threat, and they process and present antigens to T cells (4, 12, 34). Different subsets of epididymal MPs present overlapping functional and molecular profiles in the epididymis (4, 8, 12, 40, 44).

It is still unclear how sperm antigens are sampled and recognized as “self” to establish immune tolerance, while foreign antigens initiate immune activation in the epididymis. However, understanding this dynamic is of high clinical relevance. Deeper insights into how the epididymis senses and mounts defense responses could significantly impact our management of common disorders such as epididymitis, a condition that causes significant scrotal pain (26). This condition can provoke irreversible ductal obstruction and fibrosis and ultimately induces male infertility (19–21, 25, 26, 28, 29, 45). Despite the deleterious effects of epididymitis on male fertility and well-being, most patients receive inadequate early diagnostic and therapeutic interventions, highlighting the need for more basic and clinical research (46, 47). Escherichia coli (E. coli) is among the most prevalent causative pathogens and colonizes the epididymis by retrograde ascent through the urethra (26, 37, 48). An animal model that mimics this situation is the retrograde injection of the bacteria or lipopolysaccharide (LPS), a component of the outer membrane of the gram-negative bacteria, such as E. coli, into the vas deferens/epididymal lumen (5, 29, 37, 40, 45). Both challenges increase cytokine and chemokine production and initiate immune cell infiltration, epithelial damage, and fibrotic tissue remodeling in the mouse and rat epididymis (5, 8, 29, 37, 49).

The blood-epididymis barrier is at the interface between spermatozoa and potentially harmful immune cells and highly depends on epithelial integrity (50, 51). It has been shown that CCs are strategically positioned to interact with MPs to survey the epididymis barrier and regulate the balance between inflammation and immune tolerance in the post-testicular environment (5, 40). Importantly, region-specific heterogeneous subsets of MPs establish intimate contact with CCs in the epididymis (5). Our review aims to describe how CCs communicate with MPs to contribute to the unique immune environment in which sperm mature and are stored in the epididymis. Here, we address crucial concepts of mucosal immunology and cell–cell interactions, all of which are critical but understudied facets of human male reproductive health. So-called mucosal immune systems reside in other organ systems such as the digestive tract and the airways, and despite differences among these organs, all of them are populated by strikingly similar networks of epithelial and immune cells that can exert niche-specific tasks (31–33, 52).

Clear cells

Epithelial CCs are located along the epididymis, where they regulate and maintain acid-base balance by adjusting proton secretion through a critical enzyme, the proton-pumping V-ATPase, located in their apical membrane (10, 14, 53–55). Luminal acidification in the epididymis is essential for the maintenance of sperm in a quiescent state. The crucial role of CCs in luminal acidification is well-discussed in previous reviews (10, 14, 54).

How CCs respond to extracellular cues to modulate their functions, and their communication with neighboring cells remains incompletely characterized. To gain insight into the molecular mechanisms by which CCs interact with other epididymal cells, their gene expression profile was analyzed using bulk RNA sequencing (RNAseq) (5). To visualize and isolate CCs by fluorescence-activated cell sorting, transgenic mice that express an enhanced green fluorescent protein (EGFP) driven by the promoter of the B1 subunit of V-ATPase (B1-EGFP mice) were used (5, 56). RNAseq identified distinct CC transcriptomic signatures depending on their location in the three epididymal regions examined (proximal (IS and caput), middle (corpus), and distal (cauda)) (5). Region-specific transcriptomic and functional signatures were observed in CCs isolated from the proximal epididymis versus CCs from the middle and distal epididymal regions. These findings support previous global transcriptomic characterization of human epididymis showing that the caput is functionally divergent from the corpus and cauda (6, 57). More recently, a single-cell atlas of the murine epididymis revealed two subpopulations of CCs that are distinguished by the expression of Amylin (7). Amylin labels a subset of V-ATPase-positive CCs in the caput and the corpus epididymis, and these cells are undetectable in the cauda (7). It seems unlikely that Amylin-positive CCs correspond to narrow cells since the latter are uniquely found in the IS. Moreover, the single-cell RNAseq data shows the regional specialization of principal cells across the length of the epididymis (7).

Interestingly, CC transcriptomics data uncovered unexpected roles of these professional acidifying cells in the epididymis, including the maturation and protection of sperm as they transit along the epididymal tubule (5). It is now recognized that CCs are critical sensors and mediators of epididymal immune responses. These complex functions of CCs lining the tubule lumen are in addition to their important acid-base physiological role (10, 14, 54, 58, 59). In this review, RNAseq data from Battistone and collaborators (5) was re-analyzed. We provide lists of the most differentially expressed genes related to sperm maturation and immune regulation in CCs from the proximal, middle and distal epididymal regions (Fig. 1).

Figure 1. Top differentially expressed genes related to sperm maturation and immune regulation in CCs from the proximal, middle, and distal epididymis.

Most up-regulated genes related to sperm maturation (including genes reported to be expressed in epididymosomes) and immune regulation in proximal CCs (Fold change (FC) >2 vs middle CCs and distal CCs (P<0.05)); in middle CCs (FC>2 vs proximal CCs and distal CCs (P<0.05)); and in distal CCs (FC>2 vs proximal CCs and middle CCs (P<0.05)). RNAseq data from proximal, middle and distal B1-EGFP CCs (5) was re-analyzed for this figure. Genes with asterisks are reportedly involved in both sperm maturation and immune regulation.

CCs and sperm maturation: transfer of new proteins to sperm

While sperm transit through the epididymal lumen, they interact with the epithelial surface and different components of the luminal fluid, which promote substantial molecular, biochemical, and functional changes in the spermatozoa (10, 60–64). The epididymal fluid contains proteins, RNA, and ions secreted by epithelial cells, which are regulated by lumicrine factors and hormones. The epididymal lumen has also extracellular vesicles (EVs), also known as epididymosomes (60–62, 65–69), which play an essential role in modifying the molecular cargo of sperm, a transcriptionally and translationally silent cell.

Principal cells are conventionally considered to produce EVs, through apocrine secretion, which involves the formation of a cytoplasmic bleb at the apical pole of cells (66). However, confocal and electron microscopy images provided evidence for the presence of apical membrane protrusions, such as apical blebs and long membrane extensions, like tunneling nanotubes (TNTs), in epididymal proton-secreting cells (narrow cells and CCs) (5, 70, 71) (Fig. 2A–B). These nanotubes may allow the exchange of substances, vesicles, and organelles between non-adjacent cells, like proton-secreting cells and spermatozoa, in the proximal epididymis. Moreover, not only using confocal and electron microscopies but also performing imaging flow cytometry (AMNIS), it was shown the presence of luminal EGFP-positive vesicles originating from CCs that are in close contact with spermatozoa in the epididymis of the transgenic B1-EGFP mice (5). Additionally, these vesicles are positive for CD63 and CD9, markers of EVs (5, 72). Interestingly, it was reported that CCs express several transcripts encoding proteins that have been described in the epididymal EVs (65) and are also acquired by sperm during the epididymal maturation (73–77). Therefore, it is plausible that CC-sperm interaction occurs through TNTs (nanotubes) and/or via EVs deriving from CC apical blebbing. CCs may transfer to sperm proteins such as Src, CRISP1, ADAM7, serpine 2, and numerous Wnt proteins, which are known to be essential for male fertility (73–77). Indeed, CC apical blebs, CC-derived EVs, and CC nanotubes all contain Src (5), a protein acquired by sperm during epididymal transit (77). The fact that the V-ATPase subunit a4, specifically expressed in epididymal CCs (78), was detected in EVs (65), provides an additional indication that CCs produce EVs. More recent studies further support the novel role of CCs in the transfer of proteins to sperm during epididymal transit. Of note, SLC27A2 (the very long-chain acyl-CoA synthetase), EDDM3B (epididymal secretory protein E3-beta), KRT19 (keratin type I cytoskeletal 19), and WFDC8 (WAP four-disulfide core domain protein 8) are sperm proteins with an epididymis-specific origin that are expressed in EVs as well as CCs, in both the human and mouse epididymis (79).

Figure 2. Confocal microscopy images showing CC-spermatozoa and CC-MP interactions.

A. EGFP-positive narrow/CCs in the initial segments (IS) of the B1-EGFP mouse epididymis, in which EGFP is expressed in CCs, showing the presence of apical blebs (i, arrow) and apical membrane protrusions similar to nanotubes that reach out into the lumen and come into contact with spermatozoa (ii, arrows). A small EGFP-positive vesicle is shown in contact with the mid-piece of a sperm cell (arrowhead). Bars = 5 μm. Nuclei are labeled with DAPI (blue). Panel i is another example of the images published in Figure 3A in Battistone et al., 2019 (5). Panel ii is an image modified from Figure 3A in Battistone et al., 2019 (5). B. Spermatozoa - vesicle interaction in the lumen of B1-EGFP mouse epididymis. Large luminal EGFP-positive vesicles appear in the lumen of the IS (arrowheads). EGFP-positive vesicles (green) are attached to spermatozoa (blue) (double-head arrows). EGFP-positive CCs are shown with stars. Bar = 10 μm. Nuclei are labeled with DAPI (blue). Panel B is an image capture of Movie 9 in Battistone et. al., 2019 (5). C. 3D reconstruction of a confocal microscopy image showing multiple projections of CD11c EYFP-positive MPs (green), in very close proximity to CCs (V-ATPase-positive cells, red) in the IS of CD11c-EYFP mouse, in which EYFP is expressed in CD11c⁺ MPs. Nuclei are labeled with DAPI (blue). Bar = 5 μm. This figure is an image capture of Movie 11 in Battistone et. al., 2019 (5).

Together, these findings indicate that, in addition to principal cells, CCs also participate in cargo transmission to sperm via EVs and nanotubes, and, therefore, play a potential role in sperm maturation. However, it is still unclear how these proteins are transferred to spermatozoa, and further studies related to this are needed. In addition, CC-derived EVs and nanotubes may allow the exchange of substances, vesicles, and organelles from CCs to other epididymal cells. In summary, CCs use distinct mechanisms to sense and decode extracellular signals and communicate with surrounding cells via cell-cell crosstalk.

CCs modulate mucosal immunity in the epididymis.

The epididymal mucosal barrier is not only physical; it is the source of potent immune mediators such as chemokines, cytokines, antioxidants, and antimicrobial peptides. The transcriptomic characterization of CCs revealed that these proton-secreting cells express pro-inflammatory as well as anti-inflammatory genes, including sperm tolerance mediators, in all epididymal regions (5). At steady state, epididymal CCs express components involved in pro-inflammatory responses, such as IL-17, IFN-γ and TNF receptors, and several chemokines including Ccl17, Cxcl10, Cxcl17, Cxcl1, and Cx3cl1. In addition, the CC transcriptome uncovered the specific regional distribution of several immune response mediators. For example, proximal CCs show high expression of IL-34, which promotes the release of chemokines and macrophage infiltration in other epithelia (80). The danger-associated molecular pattern molecule (S100a8), which primes immune cell recruitment (81), is expressed only in distal CCs, in agreement with its expression in human cauda epididymis (6). Interestingly, among mediators of immune activation, CCs are an important source of several β-defensins (Defbs), a major subfamily of host defense peptides that are abundant in the epididymis (2, 3, 82). β-defensins protect epididymal spermatozoa against LPS or Gram-negative bacteria. However, how β-defensins communicate with other key innate immunity effectors such as MPs in the maintenance of the epididymal function, in both normal and non-homeostatic conditions, is still poorly understood.

Immunofluorescence (IF) revealed that the intraepithelial projections of MPs are near CCs (5, 40) (Fig. 2C). Importantly, CCs participate in innate immunity against bacterial-associated antigens (5). B1-EGFP mice were subjected to an intravasal-epididymal injection with LPS or saline as control (5). LPS increased pro-inflammatory associated gene expression by cauda CCs, such as Cxcl10, Cxcl1, Cxcl2, Ccl5, and Il6. These factors play crucial roles in MP recruitment to the epididymis upon infection (5). The crosstalk between CCs and immunocytes will be discussed later in this review.

Additionally, CCs express CRES proteins that form the epididymal amyloid matrix (5, 72), which play important biological roles in host defense, including bacterial trapping and membrane disruption to protect against pathogens that commonly cause epididymal infections (83). Furthermore, CCs express several antioxidant molecules (5) that are involved in the protection of the epididymal epithelium and spermatozoa (84). All these pieces of evidence support the very likely involvement of CCs in immune defense.

Under physiological conditions, CCs also express well-known anti-inflammatory response-associated genes, such as IL-4 and IL-13 receptors, and mediators involved in the maintenance of immune tolerance. For example, Indoleamine 2,3-dioxygenase (Ido1), Galectin-1 (Lgals1), High-mobility group box 1 (Hmgb1), Transforming growth factor-beta (TGFβ) ligands (TGFb1, TFGb2, and TFGb3) and Interleukin-18-Binding Protein (Il18bp) (5, 85). The maintenance of immune tolerance against self-antigens is essential to prevent autoimmune responses. The breakdown of sperm tolerance causes autoimmune-associated male infertility (19, 20). IDO1 is a well-known tolerogenic molecule that is expressed in the epididymis and is a key mediator of sperm tolerance (86). The absence of IDO1 promotes epididymal inflammation, with an increase of different inflammatory cytokines (IFN-γ, TNF-α, IL-6, IL-1ß) and chemokines (CCL3, CCL5, CXCL9, and CX3CL1) (86), and CCs express several of these mediators (5). Notably, the ablation of IDO1 in the epididymis causes male autoimmune infertility (86). Moreover, other epithelial cells, immunocytes, and/or cell types (such as fibroblasts) might be involved in maintaining the blood-epididymis barrier. Indeed, it has been reported that principal cells and other epididymal cells express immunomodulatory proteins such as TGFβ or IDO (85, 130–132).

Interestingly, Whey-acidic protein four-disulfide core domain (WFDC) genes display putative roles in innate immunity and fertility (87–89). It was recently shown that LPS-induced inflammation differentially regulated the Wfdc locus in the proximal and distal epididymis. This indicates the region-specific roles of the Wfdc family in innate immune responses during epididymitis (49). Importantly, several of these mediators are highly expressed in proximal and distal CCs, such as Wfdc2, Wfd15a, Wfdc15b, Wfdc6b, and Wfdc10 (5).

Transcriptomic data, confocal microscopy studies, and functional assays showed the expression of some purinergic receptors in CCs, such as the adenosine ADORA2B and the ATP P2X4 receptors (5, 53). The activation of both receptors has been proposed to protect against bacterial infections and injury in the urogenital tract (90). It is noteworthy that similar proton-secreting cells in the kidney, the so-called intercalated cells, also play a role in immune responses by the activation of another purinergic receptor, P2Y14, which contributes to renal tubule damage following renal ischemia-reperfusion injury (91). Remarkably, proximal CCs of the mouse epididymis show P2Y14 expression under physiological conditions (5). Furthermore, it was recently reported that P2Y14 is expressed in a subpopulation of CCs in the human epididymis (92). In vasectomized men, an increase in P2RY14 mRNA and the presence of numerous leukocytes were observed in the epididymis compared to non-vasectomized men (92). The potential role of P2Y14 in the generation of an inflamed-prone environment in the epididymis following vasectomy was proposed.

Together, these findings suggest that the functional diversity adopted by CCs could represent a generalized phenomenon by which similar epithelial cells decode signals, communicate with neighbors, and mediate mucosal immunity in the epididymis as well as in other organs. Further studies are needed to investigate the molecular mechanisms by which CCs respond to a stressful environment and how they orchestrate the early innate immune response. Interestingly, a comparative single-cell RNA-seq analysis between human nasal, bronchial, and epididymis epithelia showed that human CCs present a strong similarity with ionocytes in the nasal and bronchial epithelia (93).

Mononuclear phagocytes

Tissue-resident MPs are involved in the maintenance of tissue homeostasis, inflammation, and activation of immune responses against injury and infections (4, 8, 12, 34–36, 38–43, 94). MP subsets, including dendritic cells, monocytes, and macrophages, are all essential components of the immune system that connect innate and adaptive immunity (4, 12, 36, 39, 41–43, 94–97).

In the epididymis, there is a dense network of heterogenous MPs, with distinct morphology, phenotype, and function, in the epithelium as well as in the interstitial compartment (4, 8, 12, 34–36, 38–40, 42, 43, 94). Moreover, similar to what was observed in the lumen of the intestine (32, 98, 99), in the proximal regions of the epididymis, MPs show elongation of thin projections between epithelial cells toward the lumen (Fig. 2) (4, 12, 34, 35, 38, 40). These cells simply cover the base of the epithelium in the distal regions of the epididymis. Studies in reproductive immunology have recently focused on elucidating the unique roles of MPs in sperm protection and understanding how the epididymis faces its immunological challenges (4, 8, 12, 34–36, 38–40). Tissue-resident MPs are optimal for regulating the balance between effector immune response and sperm tolerance (4, 8, 12, 23, 34–36, 38–40). Some studies have examined the functional and morphological characteristics of region-specific MPs (4, 8, 12, 39, 40), revealing the differences between immune cells populating the most proximal or most distal segments of the epididymis. These regions play drastically different roles in the maturation, storage, and protection of sperm cells.

Using different transgenic mice expressing fluorescent markers specific for immunocytes, such as CX3CR1 (chemokine (C-X3-C motif) receptor 1) and CD11c (integrin alpha X) (4, 12, 34, 35, 38, 40), it was found that CX3CR1⁺ MPs and CD11c⁺ MPs represent two subsets of epididymal myeloid cells that express region-specific surface markers and present morphological and functional differences. Importantly, the CX3CR1⁺ cell population partially overlaps with the CD11c⁺ MP subset at different proportions among the epididymis (4, 12, 35). These MPs can partake in the capture, processing, and presentation of antigens in the epididymis (4, 12, 34, 35, 38, 40). The transcriptomic profiles of CX3CR1⁺ MPs, isolated by fluorescence-activated double-cell sorting from the proximal, middle, and distal epididymis of CX3CR1^EGFP+/− transgenic mice, which express EGFP exclusively in CX3CR1⁺ MPs, revealed their heterogeneous region-specific gene signature (4). More recently, single-cell RNAseq of epididymal immune cells identified several other subgroups of MPs within the epididymis (8). In this review, we will describe the heterogeneous region-specific gene and functional signatures of MPs, and their role in protecting the fine-tuned balance between sperm tolerance and immune activation, which is essential for epididymal sperm maturation.

Region-specific transcriptomic and functional signatures of epididymal MPs

The epididymis is populated by subsets of region-specific MPs which express a combination of surface molecules, showing dendritic cell or macrophage-like phenotypes (4, 12). MPs that are positive for both the high-affinity IgG gamma chain FcγRI surface receptor (CD64) and F4/80 are considered macrophages, and dendritic cells are usually defined as CD11c⁺CD64⁺, together with their MHCII expression (100). Remarkably, most of the epididymal MPs express CD64, and this cell population decreases from the proximal to the distal epididymis (4, 12, 39). In contrast, MPs with a dendritic cell phenotype progressively increase toward the more distal regions (4, 8, 12, 39).

The diversity in the MP population was also observed by confocal microscopy: in the IS, most of the CX3CR1⁺ and CD11c⁺ MPs are co-labeled with the F4/80 macrophage marker, including the intraepithelial and the interstitial cells (4, 12, 34, 35, 38). However, in the more distal part of the epididymis, the abundance of these double-positive cells decreases. In contrast, MPs with the dendritic cell signature (CD11c⁺MHCII⁺) are located either at the base of the epithelium or in the interstitium (Fig. 3). They are found in the highest percentages in the cauda and decrease in percentage in the caput/corpus and IS, which was consistent with the flow cytometry analysis (4, 12). The same trends were observed for the population of CX3CR1⁺ cells with a dendritic cell profile (4). These results indicate a higher concentration of dendritic cells in the distal epididymis versus the proximal regions. Remarkably, in the IS, MHCII⁺ cells do not extend thin luminal-reaching projections between epithelial cells as was observed for F4/80⁺, CD11c^+, and CX3CR1⁺ MPs (4, 12, 34, 35, 38). However, MHCII⁺ cells do send projections along the base of the epithelium. MHCII is used to present processed antigens suggesting that while these cells are not involved in sampling luminal antigens, they might capture and process antigens that are present in the interstitial space.

Figure 3. Regional diversity of the epididymal MPs.

Confocal microscopy images showing CD11c⁺ MPs (green) and MHCII⁺ cells (red) in the proximal and distal regions. Double-positive MPs are shown in yellow (arrowheads), and CD11c-positive only MPs are shown in green (arrows). The colocalization of CD11c and MHCII occurs more frequently among cells in the distal than IS. Nuclei are labeled with DAPI (blue). Low magnification scale bars = 100 μm; Inset scale bars = 20 μm. The figure was modified from Figure 1 in Mendelsohn et. al., 2020 (12).

Interestingly, a reduction in the number of MP dendrites reaching toward the epididymal proximal lumen was observed in epididymal MPs from CX3CR1-deficient mice (CX3CR1^EGFP/EGFP KO mice), in which both copies of the Cx3cr1 gene are substituted by the Egfp gene. This morphological alteration indicates that the formation of the luminal reaching projections depends on CX3CR1 signaling pathway activation. It has been reported that a reduction in the abundance of macrophage luminal projections impairs the oral tolerance to food antigens in the intestine (33, 99), and induces alterations in adult neurogenesis in the olfactory bulb (101, 102). In addition, in the colon, these membrane protrusions decrease in the absence of the CX3CR1 ligand: the chemokine CX3CL1 (33). In the epididymis, CX3CL1 is expressed by CCs, which are in close interaction with CX3CR1⁺ MPs (5, 40). Despite the CX3CR1-deficient epididymal MPs presenting fewer luminal reaching projections, 3D confocal microscopy showed that these projections are still in close interaction with the CCs (40) (Fig. 4). This indicates that both cell types would be strategically positioned to play an important role in epididymal mucosa immunity. Flow cytometry revealed a switch in the immunophenotypic profile of CX3CR1-deficient MPs: they present a reduced monocytic phenotype under physiological conditions, indicating a critical role of this receptor on tissue-resident monocyte homeostasis that contributes to the optimal epididymal microenvironment (40). Indeed, CX3CR1 has previously been found to be involved in the migration of blood monocytes into other tissues (103). Furthermore, CX3CR1 deletion downregulates the expression of the gap junction protein connexin 43 in epididymal MPs, which induces potential defective cell-cell communication between MPs and other epididymal cells (40). This cell-cell crosstalk will be discussed in the following section.

Figure 4. CX3CR1-deficient MPs in close interaction with the CCs.

3D reconstruction of a confocal microscopy image showing CX3CR1-deficient MPs (green) in close interaction with CCs (V-ATPase-positive cells, red) in the IS epididymis of CX3CR1^{EGFP/ EGFP} mouse. Nuclei are labeled with DAPI (blue). Bar = 5 μm. The figure is an image capture of Video 1 in Barrachina et. al., 2022 (40).

The transcriptomic profiles of CX3CR1⁺ MPs revealed their specific gene expression signature in each of the evaluated epididymal regions. A more diverse subpopulation of MPs is present in the proximal regions versus more distal regions, in agreement with the distinct morphology of the epididymal MPs. As aforementioned, while CX3CR1⁺ MPs in the IS present numerous luminal reaching projections, they only cover the base of the epithelium in the cauda (4, 34, 35, 38, 40). Moreover, proximal MPs express more components of the cell adhesion and leukocyte transendothelial migration pathways (i.e. Cldn10, Mag, Cadm1, Itga6, Cldn2, Itga9, Comp, Itgb5, Tln2 and Vcam1) than distal MPs (4).

MPs have immune functional specialization depending on their location within the epididymis (4, 8, 12). As expected, genes related to phagocytic activity and antigen processing and presentation are highly expressed in MPs from all regions (4, 8, 12). However, specific gene sets involved in each of these pathways are expressed differently depending on the location of the MPs. As anticipated from the function of MPs in other tissues (41, 104, 105), epididymal CX3CR1⁺ MPs express several pro-inflammatory and anti-inflammatory (potentially involved in sperm tolerance) gene subsets depending on their epididymal location (4). For example, TNF receptors (Tnfrsf1a, Tnfrsf21, Tnfrsf13b) are predominant in caput/corpus and cauda MPs, and Tnfrsf11a is abundant in IS MPs. The same was observed for some chemokines: Ccl6, Cxcl13, Ccl9, Ccl22, and Ccl17 are up-regulated in distal MPs; and Ccl12, CxCl2, Cxcl10, Cxcl14, CxCl1, and Cxcl16 are enriched in proximal MPs. While proximal MPs express high levels of anti-inflammatory receptors (Il10ra, Il10rb, Il21r, Il4ra, Tgfbr1, Tgfbr2), MPs from the other regions are enhanced in ligands involved in immune tolerance, such as Ido1 and Tgfb1 (caput/corpus) and Lgals1 (cauda).

Pleuger and collaborators, using single-cell RNAseq, identified other subgroups of epididymal MPs that populate different niches according to their homeostatic, reparative, and inflammatory properties (8). Their study confirmed that CX3CR1⁺ MPs are situated within and around the epididymal epithelium (4, 34). The transcriptional signature of proximal CX3CR1⁺ MPs (4, 8) combined with their known phagocytic activity (4, 38) suggests their crucial function in immune surveillance to maintain the epithelial integrity and the luminal microenvironment efficiently. This is required for proper sperm maturation and storage. Indeed, the high density of CX3CR1⁺ MPs (4, 34), which express sperm tolerance-associated genes (4), in combination with the narrow proximal epididymal lumen (10) suggests that in the IS, the luminal content is constantly monitored to promote tolerance toward sperm antigens and to eliminate pathogens/stressors. However, the molecular mechanisms by which immune cells eliminate pathogens or maintain tolerance to sperm, thus conserving the epididymal homeostatic state, are still unknown.

The distal epididymal regions (corpus, cauda) are also populated by heterogeneous MP subpopulations (4, 8). As mentioned above, in these segments, MPs do not extend intraepithelial luminal projections, but there is a high abundance of interstitial MPs that show double labeling for MHCII and CD11c (12) (Fig. 3). Previous studies showed the presence of interstitial F4/80⁺ MPs that do not express CX3CR1 receptor in the cauda region (4, 34, 35). Pleuger and collaborators further characterized the distal interstitium and showed that it is populated with pro-inflammatory monocyte-derived CCR2⁺MHCII⁺ MPs, vasculature-associated talactoferrin positive (TLF⁺) macrophages, as well as CX3CR1⁻TLF⁻CCR2⁻ macrophages (8). Moreover, the authors revealed that TLF⁺ macrophages are self-renewing and CX3CR1⁺ macrophages are partially dependent on monocyte differentiation in the distal, but not in the proximal regions (8). These findings support the hypothesis that local environmental factors regulate monocyte entrance and replacement of distinct MP populations in different regions of the epididymis.

The capture of circulatory antigens by epididymal MPs.

Epididymal CX3CR1⁺ MPs can both capture and process antigens in all regions (4, 12, 40). To study the ability of MPs to take up circulatory antigens, mice were injected (i.v.) with fluorescent ovalbumin-Alexa647 (OVA), and to study antigen processing, animals were inoculated with Prosense 680 (Pro), which fluoresces upon processing by intracellular proteases (4, 12, 40). Antigen uptake occurs quickly, within 1 h, but processing activity requires a longer time (24 h) (4). Interestingly, a higher percentage of MPs that had captured and processed antigens was observed in the proximal region compared to the other epididymal segments by confocal microscopy and flow cytometry (4, 12, 40) (Fig 5), in agreement with the presence of a large number of fenestrated blood vessels in the IS (18, 106–108). In this region, intraepithelial and interstitial MPs are positive for OVA and Pro, confirming their capture and processing activities (4). However, the MP intraepithelial luminal projections were positive only for OVA, indicating that while capture and processing occur in these cells, antigen processing may not take place within these structures (4). In contrast, in the distal region, only MPs located in the interstitium were positive for OVA and Pro (4, 12, 40), showing that they provide an efficient protective barrier, and prevent them from reaching the epithelial lining. These findings suggest that the epithelium of the proximal regions is more likely to be invaded by blood-borne pathogens, compared to more distal regions.

Figure 5. The capture of circulatory antigens by epididymal MPs.

Circulatory antigens (Alexa 647-labeled OVA) were administered by tail vein injection (4 mg/kg) into CX3CR1-EGFP mice. A. Confocal microscopy images showing CX3CR1⁺ MPs (green) and OVA⁺ (red) cells in IS (initial segments). Interstitial and intraepithelial MPs internalized OVA. Several CX3CR1⁺OVA⁺ MPs are in close proximity to the epithelium, and their intraepithelial projections also contain OVA (arrows). Nuclei are labeled with DAPI (blue). Bars = 20 μm. Panel A is another example of the images published in Figure 6 in Battistone et. al., 2020 (4). B. Flow cytometry showed CX3CR1⁺ cells that internalized fluorescent OVA in IS, caput/corpus and cauda epididymis. A higher percentage of CX3CR1⁺OVA⁺ cells was detected in the IS. ****P<0.0001. Data are shown as means ± SEM. Panel B was modified from Figure 4 in Battistone et. al. 2020 (4).

Surprisingly, in mice lacking CX3CR1, while intraepithelial epididymal MPs from the proximal regions are devoid of antigens upon OVA challenge compared to control MPs, no impairment in the internalization of OVA was observed in interstitial MPs (40). Moreover, CX3CR1-deficient MPs that internalized OVA displayed phenotypic switches on their macrophage profile (CD64⁺CD11b⁺CD45⁺ cells) in the IS, suggesting the involvement of the CX3CR1 receptor in the cellular plasticity of MPs. Furthermore, the majority of interstitial MPs that internalized OVA in the corpus and cauda epididymis were negative for CX3CR1, but positive for F4/80 (4, 40). These data indicate that MPs show distinct antigen-capture activities and immune phenotypes depending on their localization within the epididymis.

Similar to CX3CR1⁺ cells, CD11c⁺ MPs from all epididymal regions, take up circulatory antigens, and most of these cells expressed the surface macrophage marker CD64 (12). A high percentage of OVA-positive CD11c⁺ MPs was observed in the proximal region compared to other segments. The CD11c⁺OVA⁺ cells, which are CD64⁻MHCII⁺, exist at a significantly higher percentage in the cauda than in other regions, demonstrating the capture activity of dendritic cells in the distal regions (12). In addition, epididymal CD11c⁺ MPs can process and present peptides to naïve CD4⁺ T-cells in vitro (12).

Overall, these results support the hypothesis that MPs are gatekeepers of the immunological blood-epididymis barrier by surveying antigens. Moreover, these studies revealed new differences in the immune responses depending on the epididymal segments (4, 12, 40). It is still unclear if other recently described subpopulations of MPs (CCR2⁺MHCII⁺, TLF⁺, and CX3CR1⁻TLF⁻CCR2⁻ MPs) have capture, processing, and presenting capacities in the epididymis (8). It is also possible that these “new” TRF and CCR2 expressing subpopulations have overlapped markers with the subsets described previously by our group (4).

The role of MPs during epididymitis

Several studies clearly showed that the different epididymal regions develop distinct immune responses following bacterial infection or LPS-associated epididymitis (5, 8, 25, 26, 28, 29, 40); however, the underlying mechanisms remained unclear. Most of the immunopathological injury following epididymitis occurs almost exclusively in the distal epididymis (5, 8, 26, 28, 29, 40) with loss of epithelial integrity, interstitial fibrosis, and duct obstruction. The tissue damage is accompanied by a massive influx of neutrophils and macrophages with concurrent loss of epithelial integrity (5, 26, 28, 29, 40). Specifically, intravasal-epididymal injection of LPS or bacteria induces early recruitment of macrophage-like MPs into the distal epididymis (5, 8, 29). The MP infiltration in the interstitial space persisted for several weeks after the injection (8, 29). Bacterial infection in CCR2-deficient epididymis showed a reduction in monocyte infiltration that was associated with less tissue damage, indicating the crucial role of pro-inflammatory monocyte-derived MPs during epididymitis (8). In addition, CX3CR1⁺ MPs are involved in the pathophysiology of epididymitis, and the lack of functional CX3CR1 alters the ability of MPs to initiate immune responses after LPS injection (40). In that study, attenuation of epithelial cell damage and reduction in the number of apoptotic cells were observed in CX3CR1-deficient mice compared to control mice exposed to a luminal LPS challenge. It was proposed that the lack of CX3CR1 protects the epithelium by allowing repair mechanisms to take place and/or by attenuating further damage by reducing inflammation (40).

Systemic inflammation induced by intraperitoneal injection of LPS also resulted in a stronger pro-inflammatory response in the cauda of the mouse epididymis compared to the other regions (48). As mentioned above, the region-specific epididymal inflammatory response could be attributed to the varied composition of immune cell populations present in the different regions under steady-state and disease conditions (4, 8, 12, 18, 36). However, in the proximal segments, immunocytes and epithelial cells can also respond to inflammatory stimuli (5, 8, 28, 29, 40). In the IS, CX3CR1⁺ MP luminal-reaching dendrites sense bacterial antigens and severely shorten their membrane extrusions (40). Similar changes were observed after efferent duct ligation and vasectomy (38, 109). Importantly, most of the CX3CR1⁺ and CD11c⁺ MPs in the IS returned to their normal dendriform morphology four days after efferent duct ligation, indicating that the presence of sperm and testicular factors in the lumen is not required for the formation and extension of MP intraepithelial projections (38). Interestingly, intravenous LPS injection in rats resulted in the activation of the transcription factor nuclear factor kappa B (NFKB), which up-regulates the expression of inflammatory mediators, in both caput and cauda epididymis (110). Moreover, it was reported that CCs, located in the proximal and middle epididymis, up-regulated the expression of the CXCL10 chemokine after LPS intravasal-epididymal challenge (5), supporting the notion that LPS or bacterial inoculation impacts not only distal segments but also more proximal epididymal regions. The molecular mechanisms of these regional differences remain poorly understood.

Together, these findings support the region-specific immunomodulatory roles of MPs and confirm that these heterogeneous cell populations exhibit features that strongly depend on their location and the microenvironment of the epididymis. The different epididymal regions play considerably distinctive functions in sperm maturation, storage, sperm tolerance, and defense against stressors (4–6, 8, 10, 12, 25, 28, 29, 36, 37, 111).

Cell-cell communication networks between MPs and CCs in the epididymis.

Our group provided evidence that CCs and MPs are in close interaction in the epididymis at the steady state as well as during epididymitis (5, 40). Comparison of their transcriptomic profiles revealed several ligand-receptor pairs related to pro-inflammatory (Fig. 6Ai) and anti-inflammatory immune responses between MPs and CCs (Fig. 6Bi) (4, 5). In particular, RNAseq and IF showed that CX3CL1 is highly expressed in CCs (5); and this chemokine binds CX3CR1 expressed by epididymal MPs (4, 5, 34, 40). This ligand-receptor pair is expressed in all epididymal regions (Fig. 6Aii). There is also regional distribution of pro-inflammatory ligands in CCs and their receptors in MPs, including Cxcl10 - Cxcr3 and Il34 - Csf1r that are highly expressed in the proximal region, and S100a8 - Tlr4 that are up-regulated in the cauda (Fig. 6Aiii–v). Interestingly, the comparison between CC and MP transcriptomic data also revealed many enriched ligands in MPs and their matching receptors expressed in CCs. Most of these pairs are involved in pro-inflammatory immune responses such as ll1b – Ir1b1, Tnf – Tnfrsf21, and Il4 – Il13ra2 (4, 5).

Figure 6. Transcriptomic analysis revealed ligand-receptor pairs between CCs and MPs.

We reanalyzed published CC and MP transcriptomic data (4–5). A. i. Several ligand-receptor pairs related to pro-inflammatory responses: Cx3cl1-Cx3cr1 is expressed in all the epididymis (ii), Cxcl10-Cxcr3 (iii) and Il34-Csf1r (iv) are highly expressed in the proximal region. S100a8-Tlr4 (v) is up-regulated in the cauda. B. i. Several ligand-receptor pairs related to tolerance responses: in particular related to sperm tolerance: Tgfb-Tgfbr pathway (ii).

Supporting the CC-MP crosstalk, as we previously mentioned, CCs would participate in innate immunity against bacterial-associated antigens by up-regulating the expression of pro-inflammatory mediators, ultimately contributing to macrophage infiltration (5). Intriguingly, similar proton-secreting cells in the kidney also release pro-inflammatory molecules that induce myeloid cell infiltration during renal injury (91). This implies that the proposed CC-MP crosstalk in the epididymis might reflect a more widespread, but under-appreciated cell-cell communication that involves cell acidification.

Bacterial antigens, such as LPS, induce IFN-γ release in the epididymis and other organs (37, 112, 113). Immune cells express toll-like receptors (TLRs) and produce IFN-γ after activation by LPS (114). Epididymal CX3CR1⁺ MPs express Tlr2, Tlr4, and Tlr6, among other TLRs, in all epididymal regions (4). IFN-γ receptors are present in CCs, and there is a dramatic increase in the expression of Cxcl10 by these cells following intravasal-epididymal injection of IFN-γ (5). It has been reported in other organs that this mediator is implicated in the recruitment and differentiation of pro-inflammatory macrophage-like MPs under different physio-pathological situations (115–117). Thus, these data suggest that CCs respond to pathogens and initiate crosstalk with MPs during LPS-induced epididymitis, involving IFN-γ and CXCL10 as well as other mediators (5). In addition, under physiological conditions, TLRs are expressed in the epididymal epithelium (possibly in principal cells) and on epididymal spermatozoa, indicating potential crosstalk between CCs and other epididymal cells (118–120).

In addition, ligand-receptor pairs potentially related to sperm tolerance are found between CCs and MPs (4, 5) (Fig. 6Bi). TGFβ1, TFGβ2, and TFGβ3 are expressed in CCs throughout the epididymis, indicating their potential participation in crosstalk with MPs, which express TGFβ type 1 and type 2 receptors (Fig. 6Bii). The ablation of TGFβ type 2 receptor in dendritic cells, a subset of MPs, disrupts epididymal sperm tolerance (23). Another sperm tolerance mechanism involves IDO1 release (86); and both MPs and CCs highly express this mediator in all epididymal regions (4, 5). As previously mentioned, the lack of IDO1 promotes epididymal inflammatory responses and causes autoimmune infertility (86). Together, these observations suggest that the TGFβ and IDO1 pathways would be involved in the CC–MP communication networks to maintain and establish the protective immune-privileged epididymal environment where sperm maturation and storage occurs. The unique distribution of several of the ligand-receptor pairs also suggests the specificity of their role in protecting the epididymis.

Upon infection, epididymal cells such as CCs would communicate through pro-inflammatory chemokines/cytokines with strategically located MPs to mount a bacterial-associated response (5, 37, 48, 49). This would then be followed by rapid infiltration of monocytes that ultimately differentiate into macrophages and CD103⁺ dendritic cells as it was described in other organs (5, 8, 36, 121–123). Epididymal CX3CR1⁺ MPs capture antigens (4, 40) and may quickly transfer them to adjacent CD103⁺ dendritic cells, which would migrate to lymph nodes and facilitate a local inflammatory response that contributes to the injury, similarly to CX3CR1 cells in the intestine, skin and lung (32, 124–127). This cell-cell communication between CX3CR1⁺ MPs and CD103⁺ cells is mediated by the gap junction protein connexin 43 in the intestine (32, 33). CX3CR1 deletion downregulates the expression of connexin 43 in epididymal MPs and probably induces defective communication between these MPs and dendritic cells (40). In addition, intraepithelial CX3CR1-deficient MPs showed impaired capture activity (40). Thus, the combined reduction of antigen uptake by intraepithelial CX3CR1-deficient MPs and the impaired antigen transfer from MPs to dendritic cells would affect the ability of CD103⁺ cells to migrate to the lymph nodes. These dendritic cells would then accumulate in the epididymal interstitium, as was observed by Barrachina and collaborators (40). Moreover, epididymal tubule damage was significantly attenuated in CX3CR1-deficient mice compared to control mice, most likely by blunting the release of inflammatory molecules from CX3CR1-deficient MPs.

Summary

Infertility is a growing public health problem affecting 17.5% of couples worldwide (128). Male factors are involved in 40–50% of these cases, half of which are classified as “idiopathic”, illustrating our poor knowledge of male reproductive physiology (111, 129). A critical but understudied aspect of male reproductive health is the close interaction between the reproductive system and the immune system in the post-testicular environment. Importantly, ~15% of male infertility cases originate from infections, inflammation, or autoimmunity in the reproductive tract (19–21, 24).

The remarkable communication between epithelial CCs and MPs would contribute to the establishment of a unique milieu that protects the epididymal immune-privileged environment from invading pathogens while protecting spermatozoa from destructive autoimmune reactions. Both types of cells might be gatekeepers of the blood-epididymis barrier by surveying antigens and activating immune responses.

We provide new insights into major immunoregulatory mechanisms by which CCs communicate with MPs to maintain mucosal homeostasis in the epididymis and cell-cell crosstalk during inflammation and infection. However, future research is needed to further investigate the role of CCs together with MPs in healthy epididymis, as well as during inflammation. Identifying immunoregulatory mechanisms in the male excurrent duct also has a high potential to lead to new and targeted therapies for pathologies arising from autoimmune or inflammatory dysfunctions, including epididymitis and infertility; and to identify potential targets for immuno-contraception.

Data availability

The datasets generated during this study are available from the corresponding author upon reasonable request.