Varicocele and testicular cord torsion: immune testicular microenvironment imbalance.

The main functions of the testis, steroidogenesis and spermatogenesis, depend on the endocrine axis and systemic and local tolerance mechanisms. Infectious or non-infectious diseases may disturb testicular immune regulation causing infertility. Literature has illustrated that bacterial and viral infections lead to autoimmune infertility: either sperm antibodies or autoimmune epidydimo-orchitis. However, little is known about the association between non-infectious testicular pathologic diseases and autoimmunity. Here we review the novel aspect of varicocele and testicular cord torsion pathology linked to inflammation and discuss how immune factors could contribute to or modulate autoimmunity in ipsi- and contralateral testis.

Pathomechanisms of Autoimmune Based Testicular Inflammation.

Infection and inflammation of the male reproductive tract are relevant causes of infertility. Inflammatory damage occurs in the special immunosuppressive microenvironment of the testis, a hallmark termed testicular immune privilege, which allows tolerance to neo-antigens from developing germ cells appearing at puberty, long after the establishment of systemic immune tolerance. Experimental autoimmune orchitis (EAO) is a well-established rodent model of chronic testicular inflammation and organ specific autoimmunity that offers a valuable in vivo tool to investigate the pathological and molecular mechanisms leading to the breakdown of the testicular immune privilege. The disease is characterized by the infiltration of the interstitium by immune cells (mainly macrophages, dendritic cells, and T cells), formation of autoantibodies against testicular antigens, production of pro-inflammatory mediators such as NO, MCP1, TNFα, IL6, or activins and dysregulation of steroidogenesis with reduced levels of serum testosterone. EAO leads to sloughing of germ cells, atrophic seminiferous tubules and fibrotic remodeling, parameters all found similarly to changes in human biopsies from infertile patients with inflammatory infiltrates. Interestingly, testosterone supplementation during the course of EAO leads to expansion of the regulatory T cell population and inhibition of disease development. Knowledge of EAO pathogenesis aims to contribute to a better understanding of human testicular autoimmune disease as an essential prerequisite for improved diagnosis and treatment.

Immune Cell Subtypes and Their Function in the Testis.

Immunoregulation in the testis is characterized by a balance between immuno-suppression (or immune privilege) and the ability to react to infections and inflammation. In this review, we analyze the phenotypes of the various immune cell subtypes present in the testis, and how their functions change between homeostatic and inflammatory conditions. Starting with testicular macrophages, we explore how this heterogeneous population is shaped by the testicular microenvironment to ensure immune privilege. We then describe how dendritic cells exhibit a tolerogenic status under normal conditions, but proliferate, mature and then stimulate effector T-cell expansion under inflammatory conditions. Finally, we outline the two T-cell populations in the testis: CD4+/CD8+ αß T cells and CD4+/CD8+ Foxp3+ regulatory T cells and describe the distribution and function of mast cells. All these cells help modulate innate immunity and regulate the immune response. By improving our understanding of immune cell behavior in the testis under normal and inflammatory conditions, we will be better placed to evaluate testis impairment due to immune mechanisms in affected patients.

Role of indoleamine 2,3-dioxygenase in testicular immune-privilege.

Male meiotic germ cell including the spermatozoa represent a great challenge to the immune system, as they appear long after the establishment of normal immune tolerance mechanisms. The capacity of the testes to tolerate autoantigenic germ cells as well as survival of allogeneic organ engrafted in the testicular interstitium have led to consider the testis an immunologically privileged site. Disruption of this immune privilege following trauma, tumor, or autoimmune orchitis often results in male infertility. Strong evidence indicates that indoleamine 2,3-dioxygenase (IDO) has been implicated in fetal and allograft tolerance, tumor immune resistance, and regulation of autoimmune diseases. IDO and tryptophan 2,3-dioxygenase (TDO) catalyze the same rate-limiting step of tryptophan metabolism along a common pathway, which leads to tryptophan starvation and generation of catabolites collectively known as kynurenines. However, the relevance of tryptophan metabolism in testis pathophysiology has not yet been explored. Here we assessed the in vivo role of IDO/TDO in experimental autoimmune orchitis (EAO), a model of autoimmune testicular inflammation and immunologically impaired spermatogenesis. EAO was induced in adult Wistar rats with testicular homogenate and adjuvants. Control (C) rats injected with saline and adjuvants and normal untreated rats (N) were also studied. mRNA expression of IDO decreased in whole testes and in isolated Sertoli cells during EAO. TDO and IDO localization and level of expression in the testis were analyzed by immunostaining and Western blot. TDO is expressed in granulomas from EAO rats, and similar protein levels were observed in N, C, and EAO groups. IDO was detected in mononuclear and endothelial cells and reduced IDO expression was detected in EAO group compared to N and C rats. This phenomenon was concomitant with a significant reduction of IDO activity in EAO testis measured by tryptophan and kynurenine concentrations (HPLC). Finally, in vivo inhibition of IDO with 1-methyl-tryptophan increased severity of the disease, demonstrating down regulation of IDO-based tolerance when testicular immune regulation was disrupted. We present evidence that an IDO-based mechanism is involved in testicular immune privilege.

Exploring the role of antigen presenting cells in male genital tract.

Antigen presenting cells (APCs) are a critical mediator between innate and adaptive immune response. APCs have diverse functions in physiological and pathological conditions, such as maintenance of tissue homoeostasis, prevention of autoimmunity and defence against pathogenic microorganisms and cancer cells. Dendritic cells (DCs) and macrophages (Mϕs) are "professional" APCs that internalise and process allo- and autoantigens; then, resulting peptides are exhibited together with major histocompatibility complex (MHC) molecules expressed at the cell surface. MHC-antigen complexes are presented to "naïve" T cells, thereby stimulating proliferation and differentiation of effector and regulatory T cells. The aim of this review was to summarise current understanding of DCs and Mϕs in testis and epididymis. Male reproductive tract environment is characterised by contradictory needs for tolerance against autoantigenic germ cells that appear after the establishment of central tolerance, and the capacity to mount pro-inflammatory innate immune responses against a wide array of sexually transmitted pathogens. Therefore, exploration of the role of APCs in male reproductive organs is helpful to understand mechanisms of male infertility associated with disruption of the delicate equilibrium between immune privilege and inflammation.

Impaired male fertility and abnormal epididymal epithelium differentiation in mice lacking CRISP1 and CRISP4.

Epididymal Cysteine Rich Secretory Proteins 1 and 4 (CRISP1 and CRISP4) associate with sperm during maturation and play different roles in fertilization. However, males lacking each of these molecules individually are fertile, suggesting compensatory mechanisms between these homologous proteins. Based on this, in the present work, we generated double CRISP1/CRISP4 knockout (DKO) mice and examined their reproductive phenotype. Our data showed that the simultaneous lack of the two epididymal proteins results in clear fertility defects. Interestingly, whereas most of the animals exhibited specific sperm fertilizing ability defects supportive of the role of CRISP proteins in fertilization, one third of the males showed an unexpected epididymo-orchitis phenotype with altered levels of inflammatory molecules and non-viable sperm in the epididymis. Further analysis showed that DKO mice exhibited an immature epididymal epithelium and abnormal luminal pH, supporting these defects as likely responsible for the different phenotypes observed. These observations reveal that CRISP proteins are relevant for epididymal epithelium differentiation and male fertility, contributing to a better understanding of the fine-tuning mechanisms underlying sperm maturation and immunotolerance in the epididymis with clear implications for human epididymal physiology and pathology.

Dual roles of endogenous and exogenous galectin-1 in the control of testicular immunopathology.

Galectin-1 (Gal-1), a proto-type member of galectin family, is highly expressed in immune privileged sites, including the testis. However, in spite of considerable progress the relevance of endogenous and exogenous Gal-1 in testis pathophysiology have not yet been explored. Here we evaluated the in vivo roles of Gal-1 in experimental autoimmune orchitis (EAO), a well-established model of autoimmune testicular inflammation associated with subfertility and infertility. A significant reduction in the incidence and severity of EAO was observed in mice genetically deficient in Gal-1 (Lgals1(-/-)) versus wild-type (WT) mice. Testicular histopathology revealed the presence of multifocal testicular damage in WT mice characterized by an interstitial mononuclear cell infiltrate and different degrees of germ cell sloughing of seminiferous tubules. TUNEL assay and assessment of active caspase-3 expression, revealed the prevalence of apoptotic spermatocytes mainly localized in the adluminal compartment of seminiferous tubules in EAO mice. A significant increased number of TUNEL-positive germ cells was detected in EAO testis from WT compared with Lgals1(-/-) mice. In contrast, exogenous administration of recombinant Gal-1 to WT mice undergoing EAO attenuated the severity of the disease. Our results unveil a dual role of endogenous versus exogenous Gal-1 in the control of autoimmune testis inflammation.

Targeting high mobility group box protein 1 ameliorates testicular inflammation in experimental autoimmune orchitis.

STUDY QUESTION: Does high mobility group box protein 1 (HMGB1) regulate inflammatory reactions in a rat model of experimental autoimmune orchitis (EAO)? SUMMARY ANSWER: HMGB1 appears to be involved in regulating inflammatory reactions in testes, as HMGB1 is translocated from testicular cells during the course of EAO and blocking its action by ethyl pyruvate (EP) reduces disease progression and spermatogenic damage. WHAT IS KNOWN ALREADY: Despite its immune privileged status, the human testis is prone to inflammatory lesions associated with male factor infertility. Accumulating evidence shows that HMGB1 plays an important role in onset and progression of autoimmune diseases. STUDY DESIGN, SIZE, DURATION: This is a cross sectional and longitudinal study involving Wistar male rats immunized with testicular homogenates to induce EAO 50 (EAO50; n = 10) and 80 (EAO80; n = 10) days after first immunization. Control adjuvant animals received saline instead of testicular homogenate (n = 16). Untreated animals (n = 10) were also studied. An interventional study was performed to block the action of HMGB1 starting 20 days after first immunization in EAO animals and respective controls (n = 17). Rats were treated i.p. with EP and the effect of EP treatment on testicular pathogenesis was evaluated 30 days later. Moreover, human testicular biopsies from infertile men with focal lymphocytic infiltrates (n = 7) and sections with intact spermatogenesis (n = 6) were probed with antibodies against HMGB1. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular RNA and protein extracts from EAO animals, EAO animals treated with EP and relevant controls were used for analysis of cytokine expression by real-time RT-PCR and enzyme-linked immunosorbent assay. HMGB1 was co-localized on rat testicular cross sections with antibodies against testicular macrophages (TM), peritubular cells (PTC) and Sertoli cells (SC). Interaction of HMGB1 and its receptors (RAGE, TLR4) as well signaling pathways after HMGB1 stimulation were studied in isolated TM, PTC and SC by proximity ligation assay and western blot, respectively. Furthermore, HMGB1 immunofluorescence on human testicular biopsies was performed. MAIN RESULTS AND THE ROLE OF CHANCE: HMGB1 was translocated from the nuclei in EAO testes and testes of infertile men with impaired spermatogenesis and lymphocytic infiltrates. Elevated HMGB1 levels were observed during late phase of EAO. In testicular somatic cells HMGB1 receptors Toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE) were differentially expressed: HMGB1-TLR4 binding was predominant in TM, while HMGB1-RAGE interaction was prevalent in SC and PTC. In support, HMGB1 triggered extracellular signal regulated kinase (ERK)1/2 and cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) activation in SC and PTC, while TM responded to HMGB1 stimulation with p38 mitogen-activated protein kinase (MAPK) and p65 nuclear factor Kappa B (NF-ĸB) phosphorylation followed by increased tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) mRNA levels. In vivo treatment of EAO animals with EP 20 days after induction of disease revealed beneficial effects, as documented by reduced disease progression and spermatogenic damage, lower macrophage numbers, as well as decreased concentrations of HMGB1 and IL-6 in the testis compared with EAO controls. LIMITATIONS, REASONS FOR CAUTION: The ability of HMGB1 to bind to a wide range of receptors makes it difficult to prevent its action by blockade of a specific receptor; therefore we applied EP, a drug preventing HMGB1 release from cells. Due to its mode of action EP decreases also the secretion of some other pro-inflammatory cytokines. Using isolated primary cells imposes limitations for cell transfection studies. As a compromise between purity and yield primary cells need to be isolated from animals of different age, which has to be considered when comparing their responses. WIDER IMPLICATIONS OF THE FINDINGS: HMGB1 could be a promising target in attenuating testicular damage caused by inflammatory reactions.

CD4+ Foxp3+ regulatory T cells in autoimmune orchitis: phenotypic and functional characterization.

PROBLEM: The phenotype and function of regulatory T (Treg) cells in rats with experimental autoimmune orchitis (EAO) was evaluated. METHOD OF STUDY: Distribution of Treg cells in draining lymph nodes from the testis (TLN) and from the site of immunization (ILN) was analysed by immunohistochemistry. The number, phenotype and proliferative response (5-bromo-2'-deoxyuridine incorporation) of Treg cells were evaluated by flow cytometry and Treg cell suppressive activity by in vitro experiments. TGF-ß expression was evaluated by immunofluorescence. RESULTS: Absolute numbers of Treg cells and BrdU+ Treg cells were increased in LN from experimental compared to normal and control rats. These cells displayed a CD45RC(-), CD62L(-), Helios(+) phenotype. CD4(+) CD25(bright) T cells from TLN of experimental rats were able to suppress T cell-proliferation more efficiently than those derived from normal and control rats. Cells isolated from TLN and ILN expressed TGF-ß. CONCLUSION: Our results suggest that Treg cells with a memory/activated phenotype proliferate extensively in the inflamed testis and LN of rats with EAO exhibiting an enhanced suppressive capacity. TGF-ß may be involved in their suppressive mechanism.

Dual role of immune cells in the testis: Protective or pathogenic for germ cells?

The purpose of this review is to describe how the immune cells present in the testis interact with the germinal epithelium contributing to survival or apoptosis of germ cells (GCs). Physiologically, the immunosuppressor testicular microenvironment protects GCs from immune attack, whereas in inflammatory conditions, tolerance is disrupted and immune cells and their mediators respond to GC self antigens, inducing damage of the germinal epithelium. Considering that experimental models of autoimmune orchitis have clarified the local immune mechanisms by which protection of the testis is compromised, we described the following topics in the testis of normal and orchitic rats: (1) cell adhesion molecule expression of seminiferous tubule specialized junctions and modulation of blood-testis barrier permeability by cytokines (2) phenotypic and functional characteristics of testicular dendritic cells, macrophages, effector and regulatory T cells and mast cells and (3) effects of pro-inflammatory cytokines (TNF-α, IL-6 and FasL) and the nitric oxide-nitric oxide synthase system on GC apoptosis.

Expression of co-stimulatory molecules, chemokine receptors and proinflammatory cytokines in dendritic cells from normal and chronically inflamed rat testis.

The presentation of self antigens by dendritic cells (DC) plays an important role in the initiation and maintenance of autoimmunity. In a model of experimental autoimmune orchitis (EAO), we have previously characterized dominant testicular autoantigens and shown an increase in DC numbers during the course of disease. In this study, we have developed a protocol for the isolation of a highly pure population of DC ( approximately 97%) from the testis of EAO and control rats to analyse the expression of major histocompatibility complex (MHC) class II and co-stimulatory molecules (CD80, CD86), chemokine receptors (CCR2, CCR7) and cytokines (IL-10, IL-12p70, TNF-alpha). By flow cytometry, we observed similar percentage and intensity levels of MHC class II, CD80 and CD86 expression in testicular DC in all groups. Moreover, by real-time RT-PCR we have detected significantly higher CCR7 mRNA level in isolated testicular DC from rats with EAO compared to controls, whereas the expression of CCR2 was decreased in orchitis. Transcripts of IL-12p40 were observed in DC from all groups, whereas the expression of IL-10 and the rate limiting IL-12 subunit p35 were detectable exclusively in testicular DC from the inflamed testes. In co-culture experiments, testicular DC isolated from EAO animals significantly enhanced naïve T-cell proliferation compared with control DC. Taken together these results suggest that testicular DC in control testis is not mature and functionally tolerogenic, whereas in EAO testis, IL-12 expression and stimulation of T-cell proliferation points to a mature immunogenic state prior imminent migration to the lymph nodes to amplify immune responses against testicular antigens.

Interleukin-6 and IL-6 receptor cell expression in testis of rats with autoimmune orchitis.

Experimental autoimmune orchitis (EAO) is an organ-specific model of autoimmunity characterized by an interstitial lymphomononuclear cell infiltrate as well as sloughing and apoptosis of germ cells. EAO was induced in adult male Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. Rats injected with saline solution and adjuvants were used as control group. The aim of this work was to study the expression of interleukin-6 (IL-6) and its receptor (IL-6R) in the testis of rats with EAO and analyze whether IL-6 could be involved in germ cell apoptosis. By immunohistochemistry, we detected IL-6 expression in testicular macrophages and Leydig cells of control and EAO rats. Sertoli cells showed IL-6 immunoreactivity in most of the seminiferous tubules of control rats, while a few IL-6+ Sertoli cells were found in the testis of rats with EAO. IL-6R immunoreactivity was observed in macrophages, Leydig and germ cells. A significant increase was noted in the number of IL-6R+ germ cells in rats with EAO compared to control rats. The content of IL-6 (ELISA) in the conditioned media obtained from testicular macrophages of rats with orchitis was significantly higher than in the control group. By immunofluorescence performed on isolated testicular macrophages, IL-6 was shown to be expressed by monocytes recently arrived from circulation (ED1+ cells), while resident macrophages (ED2+ cells) were negative. In vitro experiments (trypan blue and MTS assays) showed that IL-6 (50 ng/ml) reduced germ cell viability. We demonstrated also using the TUNEL technique that IL-6 added to cultures of seminiferous tubule segments induced apoptosis of germ cells. Our results suggest that IL-6 and IL-6R may be involved in the pathogenesis of autoimmune orchitis by promoting testicular inflammation and germ cell apoptosis.

Identification of a dendritic cell population in normal testis and in chronically inflamed testis of rats with autoimmune orchitis.

Experimental autoimmune orchitis (EAO) in the rat is the primary chronic animal model for the investigation of one of the main causes of male infertility, viz., testicular inflammation. Dendritic cells (DC) are potent antigen-presenting cells that play a fundamental role in autoimmune disease. We investigated the number of DC in normal testis and examined whether DC infiltrated the testis during the development of EAO. EAO was induced by active immunization with testis homogenate and adjuvants in two strains of rat (Wistar and Sprague Dawley). The presence of DC in testis was determined, 50 and 80 days after the first immunization, by immunohistochemical staining with specific antibodies (OX-62 and CD11c), and then the total number of DC was measured by stereological analysis. Labeled cells were found only in the interstitial compartment and within granulomas of EAO animals. The number of DC in EAO testes increased compared with control rats in both strains, whereas the number of OX-62+ and CD11c+ cells in adjuvant controls remained unchanged compared with untreated rats. Interspecies variations in the quantity of DC were found, with the total number of DC per testis in untreated and adjuvant control Sprague-Dawley rats being about three times higher than that seen in Wistar rats. Moreover, the increase in DC numbers at 80 days was less prominent in EAO testes of Sprague-Dawley rats than in the Wistar strain in which EAO was more severe and showed a higher number of granulomae. Thus, we have identified the DC population in normal and chronically inflamed testis. The increase in DC observed in EAO suggests that, under inflammatory conditions, the modified action(s) of these cells is a factor in the induction of the autoimmune response in testis.

Monocyte chemoattractant protein-1 (MCP-1/CCL2) in experimental autoimmune orchitis.

Experimental autoimmune orchitis (EAO) is characterized by an interstitial mononuclear cell infiltrate and a severe lesion of seminiferous tubules with germ cells that undergo apoptosis and sloughing. The mechanism by which immune cells migrate and extravasate in the testicular interstitium is poorly understood. The aim of this study was to detect the variations in the expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and its receptor in the testis of rats undergoing autoimmune orchitis. EAO was induced in Sprague-Dawley adult rats by active immunization with an emulsion of testicular homogenate and complete Freund adjuvant using Bordetella pertussis as co-adjuvant. Control rats injected with saline and adjuvants and normal untreated rats were also studied. By ELISA we observed a significant increase of MCP-1 in the testicular fluid (TF) and in the conditioned medium obtained from cultures of testicular macrophages of rats with EAO compared with control groups. By immunohistochemistry, an increase in MCP-1 expression was observed in mononuclear, endothelial, Leydig and peritubular cells. MCP-1 immunoreactivity was also detected in Sertoli cell cytoplasm of rats with severe orchitis. A 2-fold increase in the number of mononuclear cells that express CCR2 was also found in rats with orchitis compared with controls. In conclusion, we demonstrated in vivo that MCP-1 is highly expressed in testicular interstitial cells suggesting that this chemokine has an important role in recruiting immune cells to the testis in rats undergoing autoimmune orchitis.