Monika Fijak

The testis in immune privilege

Summary:  The production, differentiation, and presence of male gametes represent inimitable challenges to the immune system, as they are unique to the body and appear long after the maturation of the immune system and formation of systemic self‐tolerance. Known to protect germ cells and foreign tissue grafts from autoimmune attack, the ‘immune privilege’ of the testis was originally, and somewhat simplistically, attributed to the existence of the blood–testis barrier. Recent research has shown a previously unknown level of complexity with a multitude of factors, both physical and immunological, necessary for the establishment and maintenance of the immunotolerance in the testis. Besides the blood–testis barrier and a diminished capability of the large testicular resident macrophage population to mount an inflammatory response, it is the constitutive expression of anti‐inflammatory cytokines in the testis by immune and particularly somatic cells, that represents an essential element for local immunosuppression. The role of androgens in testicular immune regulation has long been underestimated; yet, accumulating evidence now shows that they orchestrate the inhibition of proinflammatory cytokine expression and shift cytokine balance toward a tolerogenic environment. Furthermore, the role of the testicular dendritic cells in suppressing antigen‐specific immunity and T‐lymphocyte activation is discussed. Finally, the active role mast cells play in the induction and amplification of immune responses, both in infertile humans and in experimental models, highlights the importance of preventing mast cell activation to maintain the immune‐privileged status of the testis.

Testosterone Replacement Effectively Inhibits the Development of Experimental Autoimmune Orchitis in Rats: Evidence for a Direct Role of Testosterone on Regulatory T Cell Expansion

Despite the immune-privileged status of the male genital tract, infection and inflammation of the male genital tract are important etiological factors in male infertility. A common observation in clinical and experimental orchitis as well as in systemic infection and inflammation are decreased levels of testosterone. Emerging data point to an immunosuppressive role of testosterone. In our study, we substituted testosterone levels in experimental autoimmune orchitis (EAO) in rat by s.c. testosterone implants. EAO development was reduced to 17% when animals were treated with low-dose testosterone implants (3 cm long, EAO+T3) and to 33% when rats were supplied with high-dose testosterone implants (24 cm, EAO+T24) compared with 80% of animals developing disease in the EAO control group. In the testis, testosterone replacement in EAO animals prevented the accumulation of macrophages and significantly reduced the number of CD4+ T cells with a strong concomitant increase in the number of regulatory T cells (CD4+CD25+Foxp3+) compared with EAO control. In vitro testosterone treatment of naive T cells led to an expansion of the regulatory T cell subset with suppressive activity and ameliorated MCP-1–stimulated chemotaxis of T lymphocytes in a Transwell assay. Moreover, expression of proinflammatory mediators such as MCP-1, TNF-α, and IL-6 in the testis and secretion of Th1 cytokines such as IFN-γ and IL-2 by mononuclear cells isolated from testicular draining lymph nodes were decreased in the EAO+T3 and EAO+T24 groups. Thus, our study shows an immunomodulatory and protective effect of testosterone substitution in the pathogenesis of EAO and suggests androgens as a new factor in the differentiation of regulatory T cells.

Uropathogenic Escherichia coli Block MyD88-Dependent and Activate MyD88-Independent Signaling Pathways in Rat Testicular Cells

Uropathogenic Escherichia coli (UPEC) is the most common etiological cause of urogenital tract infections and represents a considerable cause of immunological male infertility. We examined TLR 1–11 expression profiles in testicular cells and the functional response to infection with UPEC. All testicular cell types expressed mRNAs for at least two TLRs and, in particular, synthesis of TLR4 was induced in testicular macrophages (TM), Sertoli cells (SC), peritubular cells (PTC), and peritoneal macrophages (PM) after UPEC exposure. Even though MyD88-dependent pathways were activated as exemplified by phosphorylation of mitogen-activated protein kinases in TM, SC, PTC, and PM and by the degradation of IκBα and the nuclear translocation of NF-κB in PTC and PM, treatment with UPEC did not result in secretion of the proinflammatory cytokines IL-1α, IL-6, and TNF-α in any of the investigated cells. Moreover, stimulated production of these cytokines by nonpathogenic commensal E. coli or LPS in PM was completely abolished after coincubation with UPEC. Instead, in SC, PTC, TM, and PM, UPEC exposure resulted in activation of MyD88-independent signaling as documented by nuclear transfer of IFN-related factor-3 and elevated expression of type I IFNs α and β, IFN-γ-inducible protein 10, MCP-1, and RANTES. We conclude that in this in vitro model UPEC can actively suppress MyD88-dependent signaling at different levels to prevent proinflammatory cytokine secretion by testicular cells. Thus, testicular innate immune defense is shifted to an antiviral-like MyD88-independent response.

Immunoprivileged Sites: The Testis

The testis is an immunological privileged tissue as evidenced by its ability to support grafts with minimal rejection. Immune privilege is essential for the tolerance of neo-antigens from developing germ cells that appear after the constitution of self-tolerance, but imposes the paradoxical task of also providing efficient protection against pathogens and tumor cells. It is becoming increasingly clear that immune privilege cannot be attributed to a single factor such as the sequestration of neo-antigens from the immune system behind the blood-testis barrier, but is based on a complex multifaceted interplay between cells and factors that are essential for the reproductive function of the testis and the testicular immune system. This review summarizes the evidence that has accumulated regarding the role of Sertoli cells, androgens, and selected population of leukocytes in the maintenance of immune privilege and its perturbation in testicular inflammatory sub- and infertility.

Identification of immunodominant autoantigens in rat autoimmune orchitis.

Infection and inflammation of the genital tract are amongst the leading causes of male infertility. Experimental autoimmune orchitis (EAO) in the rat serves as a model for the investigation of inflammatory testicular impairment. In this study, experiments were conducted to identify the molecules that are responsible for eliciting the autoimmune attack on the testis. EAO was induced in in‐bred Wistar rats by active immunization with testis homogenates (EAO group I). Development of disease was observed using histological techniques and a new non‐invasive three‐dimensional (3D) imaging technology for in vivo monitoring, termed flat‐panel volumetric computed tomography (fpvCT). Examination of control and EAO testes demonstrated the superior image quality of high‐resolution fpvCT. A proteomics approach using 2D SDS‐PAGE and immunoblotting analysis with EAO sera identified 12 spots. Seven were subsequently identified by mass spectrometry as heat shock proteins 60 (Hsp60) and 70 (Hsp70), disulphide isomerase ER‐60, alpha‐1‐anti‐trypsin, heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), sperm outer dense fibre major protein 2 (ODF‐2), and phosphoglycerate kinase 1. Hsp70, ODF‐2, hnRNP H1, and ER‐60 were identified by all EAO sera studied. To test the capacity of the identified proteins to elicit testicular autoimmune disease, recombinant proteins were used either individually or in combination to immunize rats (EAO group II). In all groups, the incidence of EAO was 25%. Inflammatory‐type (ED1+) and resident (ED2+) macrophages, lymphocytes (CD45RA+), and dendritic cells (Ox‐62+) were strongly increased in EAO group II animals, comparable to the testes of EAO I rats. Pre‐immunization with a low dose of recombinant Hsp 70, hnRNP H1 or ODF‐2 before induction of EAO with testis homogenate significantly delayed the onset of EAO but could not prevent disease. The identification of testicular autoantigens will allow a better understanding of disease pathogenesis and could provide a basis for the development of novel therapies for inflammation‐based male infertility. Copyright

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.

Testicular innate immune defense against bacteria

Infection and inflammation are relevant entities of male factor infertility. Bacterial infections are mostly the consequence of an ascending infection of the genito-urinary tract which can ultimately lead to epididymo-orchitis. Bacterial toxins and the innate immune responses directed against them may have a significant impact on male reproductive function. Toll-like receptors (TLRs) constitute the major family of pattern recognition receptors that play a pivotal role in innate immunity. In the testis, TLRs are not only found in immune cells such as macrophages and dendritic cells, but also in testicular somatic cells and to a lesser extent in germ cells. In this review we describe relevant bacterial pathogens found in testicular and male reproductive tract infection, new data on the localisation and potential functions of TLRs, recognition and response to bacteria with a special emphasis on uropathogenic Escherichia coli. Mechanisms by which uropathogenic E. coli subvert innate immune responses in the testis are discussed using information derived from animal model studies.

Development of testicular inflammation in the rat involves activation of proteinase-activated receptor-2.

Mast cells are involved in early events crucial to inflammation and autoimmune disease. Recently, proteinase‐activated receptor‐2 (PAR2), a G‐protein coupled receptor important to injury responses, was shown to be activated by mast cell tryptase. To investigate whether mast cells and PAR2 are involved in the development and/or aggravation of testicular inflammation, we studied acute and chronic inflammatory models in the rat. In normal testes, PAR2 was detected immunohistochemically in macrophages, in peritubular cells (PTCs) and in spermatid acrosomes. In experimentally induced autoimmune orchitis (EAO), PAR2 was strongly upregulated in macrophages and peritubular‐like cells, forming concentric layers around granulomas. Mast cells increased 10‐fold in number, were more widely distributed throughout the interstitial tissue, and were partially degranulated. Isolated PTCs expressed functional PAR2, responded to PAR2 activation by phosphorylating extracellular signal‐regulated kinases 1/2 (ERK1/2) and activating protein kinase c, and increased intracellular Ca2+ concentrations as well as monocyte chemoattractant protein‐1 (MCP‐1), transforming growth factor β2 (TGFβ2), and cyclooxygenase‐2 (COX‐2) mRNA expression. Expression of these inflammatory mediators, together with iNOS, also increased significantly in testes 50 days after EAO. In vivo, expression of cytokines and inflammatory mediators was upregulated after injection of recombinant tryptase (MCP‐1, TGFβ2, and COX‐2) and a specific PAR2 peptide agonist (MCP‐1, TGFβ2) in the testis after 5 h. These results suggest that PAR2 activation elicited on PTCs by mast cell tryptase contributes to acute testicular inflammation and that this pathogenetic mechanism may also play a role in autoimmune orchitis. Copyright

Androgen receptor modulates Foxp3 expression in CD4 + CD25 + Foxp3 + regulatory T-cells

CD4+CD25+Foxp3+ Treg cells are crucial for the maintenance of immunological homeostasis. Androgens significantly induce Foxp3 expression in humans and regulate the differentiation of Treg cells. A functional androgen receptor–binding site is identified within the Foxp3 locus leading to epigenetic changes of histone H4.

Testicular infection: microorganisms, clinical implications and host-pathogen interaction

The male reproductive tract harbours effective immune mechanisms to protect the host from invading microorganisms. As the frontline of defence, the innate immune system has evolved receptors that recognise molecular patterns of pathogens to sense infections. These Toll-like receptors discriminate between different pathogen-associated molecules and activate signalling cascades that lead to immune responses, but can also result in tissue destruction leading to fertility disturbances. The testis is unique as it produces a large number of immunogenic cells expressing neo-antigens with no apparent adverse consequences for the majority of men. On the other hand the male gonad appears to be particularly susceptible to tissue damage resulting from infection or inflammatory reactions, a challenge that provides an important task both to clinicians and basic scientists. This review is intended to provide an overview of pathogens relevant in male reproductive tract infection, with a special emphasis on the testis. It also highlights the recent work broadening our understanding of the mechanisms underpinning germ cell loss in the presence of bacteria.