The structure of the adult mammalian testis is highly organized and complex consisting of two distinct regions: the seminiferous tubules and the interstitial spaces between tubules (Figure 1). The testis has two major functions: the generation of sperms (spermatogenesis), and the synthesis of sex steroid hormones (steroidogenesis). Spermatogenesis takes place within the seminiferous tubules, and steroidogenesis is fulfilled by Leydig cells located in the interstitial spaces. The sex steroid hormones, mainly testosterone in the testis, are critical for normal spermatogenesis.

The seminiferous tubules, which are highly coiled, originate and terminate at the rete testis. The tubules are surrounded by myoid peritubular cells (MPCs), which, together with Sertoli cells (SCs), secrete components of the basement membranes that enclose the seminiferous epithelium. Columnar SCs extend from the basal lamina to the lumen of the tubules. These SCs are responsible for physical support of the germ cells, providing them with essential nutrients and growth factors.

Besides Leydig cells in the testicular interstitial spaces, there are also blood and lymphatic vessels, as well as various immune cells including macrophages, mast cells, dendritic cells (DCs), and lymphocytes (Figure 1). The interstitium represents the first line of testicular defense against pathogens from the bloodstream. The rete testis, which is a transition zone between the testis and the epididymitis, is the exit of the testis. The BTB is terminated in the rete testis and spermatozoa are no longer protected from autoimmune attacks in the rete testis. Thus, certain forms of autoimmune orchitis are primarily observed in the rete testis (Itoh et al., 2005).

The BTB is created near the basement membrane by various junctions including tight junctions (TJs), basal ectoplasanic specializations, gap junctions, and demosome-like junctions between two adjacent SCs (Su et al., 2011). The BTB is anatomically much more complex than other blood–tissue barriers, such as the blood–brain and blood–retina barriers, which are constructed exclusively of TJs between endothelial cells. Moreover, the BTB can be comprised of three components, namely anatomical, physiological, and immunological barriers (Mital et al., 2011). The junctions that restrict passage of molecules and cells into or out of the BTB form the anatomical barrier. The physiological barrier comprises transporters that regulate the passage of substances, creating a microenvironment for spermatogenesis. The immunological barrier limits the access of systemic immunity and sequesters the majority of the auto-antigenic germ cells. A functional BTB relies on the complex interaction between the three components.

The immune privilege status of the testis was initially believed to be mainly based on the sequestration of antigens and antibodies by the BTB. This view has been challenged by the observation that the testicular interstitial spaces, which are located outside the BTB, are also immune-privileged (Setchell, 1990). Moreover, although the BTB sequesters most of the auto-antigenic germ cells in the adluminal compartment of the testis, preleptotene spermatocytes and spermatogonia located outside the BTB also express antigenic molecules (Yule et al., 1988). Therefore, the BTB is partially responsible for the testicular immune privilege. A growing body of evidence shows that the actively immunosuppressive mechanisms in the testis play important roles in maintaining the testicular immune privilege status (Meinhardt and Hedger, 2011). Thus, both the BTB and local immunoregulatory mechanisms contribute to the testicular immune privilege.

The testicular immune privilege depends on the species. Allografts and xenografts can survive in the testes of some species, such as rat and mouse (Head and Billingham, 1985). By contrast, similar studies on ram and monkey have not been successful (Setchell et al., 1995). Moreover, the testes of some species do not have privilege properties as donor tissues. Mouse testis allografts under the kidney capsule survive long time (Bellgrau et al., 1995), but adult rat testes grafted to the kidney were rejected (Statter et al., 1988). These differences suggest that the factors controlling testicular immune privilege are variable among species.

An intact testis is not necessary for the immune privilege. In fact, SCs display an inherent immunosuppressive role that supports the survival of cells from other tissues, such as pancreatic islet cells, when they were co-transplanted (Selawry and Cameron, 1993; Suarez-Pinzon et al., 2000). While these previous studies support that the genetic elements play roles in the maintenance of testicular immune privilege, some common mechanisms underlying the immune privilege of the testis should be existed in different species. Notably, the immune privilege of the testis does not mean the absence of immune responses in this tissue, rather, such responses are only reduced.

While the testis is a remarkable immune privilege site, it is well connected to afferent lymph nodes. Therefore, the testis has most types of immune cells, including macrophages, T lymphocytes, DCs, and mast cells. These immune cells are important in the maintenance of the special testicular immune environment.

Aside from the immune cells, growing evidence shows that the testis tissue-specific cells exhibit immunological functions and contribute to the maintenance of the testicular immune environment.

Macrophages and Leydig cells represent major immune and endocrine cells in the interstitial spaces, these two cell populations regulate each other’s development (Hedger, 2002). Luteinizing hormone may control macrophage expansion in the testis during puberty and maintenance of macrophages in the adult testis by acting on Leydig cells (Raburn et al., 1993). Evidence shows that follicle-stimulating hormone regulates maturation of testicular macrophages via SCs (Duckett et al., 1997). Moreover, the testicular endocrine environment can affect other type of immune cells in the testis (Hedger and Meinhardt, 2000). The immune cells in the testis should be regulated by the function of a mature testis, rather than the direct effects of the hormones on the immune cells that lack hormone receptors (Meinhardt and Hedger, 2011).

Production of androgens (mainly testosterone) is a major function of Leydig cells. Androgens exhibit immunosuppressive activities that contribute to different immune responses between the sexes (Cutolo et al., 2004). Testosterone reduces TLR4 expression in macrophages (Rettew et al., 2008). Administration of testosterone suppresses autoimmune diseases (Cutolo, 2009; Gold and Voskuhl, 2009). Accordingly, gonadotropin-releasing hormone antagonists significantly reduce the percentage of Treg cells, and increase the proportion of NK cells in human males (Page et al., 2006). These data suggest that androgens play roles in maintaining the balance between autoimmunity and tolerance. Substantial evidence of the link between androgens and testicular immune privilege was found in investigations using mice conditional knockout androgen receptors in SCs. SC-specific deletion of the androgen receptor in mice disrupts testicular immune privilege (Meng et al., 2011), possibly because androgens regulate SC TJs (McCabe et al., 2010). In agreement with these the data above, an earlier study indicated that androgens regulate the permeability of the BTB by regulating the expression of a SC TJ protein, CLDN3 (Meng et al., 2005). Taken together, androgens play critical roles in maintaining the integrity of testicular immune privilege by regulating local microenvironments.

FasL-induced lymphocyte apoptosis was once thought as a critical mechanism underlying the testicular immune privilege. This concept was contradicted by different studies. FasL suppresses immune responses by inducing apoptosis of Fas-bearing activated lymphocytes (Dhein et al., 1995). The testis is a major source of FasL mRNA in rodents (Suda et al., 1993), and the Fas/FasL system was demonstrated to be important in the maintenance of testicular immune privilege based on observations that SCs expressing FasL induce apoptosis of Fas-bearing lymphocytes (Bellgrau et al., 1995; Takeda et al., 1998; Figure 2). This conclusion was challenged by other studies. Genetically modified islets expressing FasL did not protect the islets from immune rejection (Allison et al., 1997). Neutralizing antibodies against FasL did not significantly reduce the survival of islets in non-obese diabetic mice that received co-grafts of SCs and islets (Korbutt et al., 2000).

In fact, convincing evidence showed that testicular FasL is expressed not in SCs but in meiotic and post-meiotic germ cells (D’Alessio et al., 2001). However, it remains to be clarified whether the germ cells expressing FasL contribute to the immune privileged status within the seminiferous tubules. Discrepant distribution of FasL as indicated in these previous studies might be caused by cell contamination and usage of non-specific antibodies. The involvement of Fas/FasL in the immunosuppressive properties of SCs should be reconsidered.

While the role of Fas/FasL system in maintaining the testicular immune privilege has been in doubt, other immunosuppressive molecules have been demonstrated to play roles in suppressing immune responses in the testis. A recent study shows that lyso-glycerophosphatidylcholines (lyso-GPCs) in interstitial fluid inhibit T cell activity, contributing to testicular immunosuppression (Foulds et al., 2008). However, the mechanisms underlying the lyso-GPC-mediated immune suppression remain unclear. Programmed death receptor-1 (PD-1) is a transmembrane protein, and PD-L1 (also named B7-H1) is a functional ligand of PD-1. PD-1 is expressed in T cells, and its activation by PD-L1 mediates T cell tolerance (Keir et al., 2006). A recent study demonstrated that PD-L1 is constitutively expressed in the testis and contributes to the long-term survival of islet allografts transplanted in the testis, suggesting that the PD-1/PD-L1 system may contribute to testicular immune privilege (Cheng et al., 2009).

Another attention deserves to be paid to the role of Gas6/ProS-TAM system in maintaining testicular immune environment. TAM receptors are the latest identified subfamily of receptor tyrosine kinases, which include three members, namely, Tyro3, Axl and Mer (TAM; Hafizi and Dahlback, 2006). Two close relative vitamin K-dependent proteins, the product of Gas6 and ProS (a blood anticoagulant cofactor), are biological ligands of TAM receptors (Hafizi and Dahlback, 2006). TAM triple knockout mice are male infertile due to progressive loss of germ cells (Lu et al., 1999; Chen et al., 2009). Gene targeting mutation studies have shown that TAM receptors are essential regulators of the immune homeostasis by regulating negatively TLR-initiated innate immune responses in DCs and macrophages (Rothlin et al., 2007; Lemke and Rothlin, 2008). We previously demonstrated that TAM receptors are abundantly expressed in mouse SCs and Leydig cells, whereas Gas6 and ProS are prominently expressed in Leydig cells (Wang et al., 2005). We also provided recently evidence that TAM signaling suppresses TLR-initiated testicular innate immune responses in SCs and Leydig cells (Sun et al., 2010; Shang et al., 2011). Based on these findings, we speculate that the Gas6/TAM system may be critical in regulating the immune privileged status of the testis. It is worthwhile to investigate this possibility.

An immune privileged status does not indicate that the site has no effective immune response. Actually, the testis tolerates invading antigens. TLR-mediated innate immune responses in testicular cells play a critical role in the protection of the testis from infections.

Toll-like receptors are pattern recognition receptors that recognize pathogen-associated molecular patterns (PAMPs). The activation of TLRs by PAMPs represents one of the most important mechanisms of immune responses against pathogens (Takeda and Akira, 2005). TLRs can be also recognized and activated by endogenous ligands that can be released from damaged tissues and necrotic cells, which are termed damage-associated molecular patterns (DAMPs; Seong and Matzinger, 2004). The expression and significance of TLRs in the testicular functions have recently been recognized (Hedger, 2011). Various TLRs are expressed in the testis of different species including humans (Fujita et al., 2011) and murines (Bhushan et al., 2008). Most studies on TLR functions in the testis have been performed using murine models. Recent studies demonstrated that mouse SCs express functional TLR2 to TLR6 (Riccioli et al., 2006; Starace et al., 2008; Wu et al., 2008), which were confirmed in rat SCs (Bhushan et al., 2008; Winnall et al., 2011). Using isolated testicular cells, we also demonstrated in vitro that TLR3 and TLR4 in mouse Leydig cells, and TLR3 in spermatogenic cells trigger innate immunity in response to ligand stimulation (Shang et al., 2011; Tao et al., 2012). TLR2 and TLR4 were observed in human sperms, and responded to bacterial endotoxins (Fujita et al., 2011). The observations imply that TLRs in non-immune testicular cells may play important roles for the testis to mount appropriate local innate immune responses to invading microbial pathogens. Notably, SCs are relatively less sensitive to ultra-pure LPS or Poly(I:C) compared with macrophages based on the expression levels of inflammatory cytokines, although both cell types express comparable levels of TLR3 and TLR4. Inhibitory effect of Gas6/ProS-TAM system on TLR signaling in both SCs and Leydig cells should be responsible, at least in part, for the less sensitive to TLR stimulation. Therefore, the interplay of TLRs and TAM receptors could be important in the maintenance of immunological balance in the testis. We are collecting more evidence to confirm this hypothesis. It should be note that most of the studies on expression and function of TLRs in the testicular cells are performed in vitro using primary cells and purified ligands. Role of TLRs in defense against invading pathogens in the testis should be confirmed in vivo when animals are infected with microbes using TLR knockout mice.

Numerous paracrine and autocrine cytokines in the testis are critical for normal testicular functions. Most of these cytokines belong to immunoregulatory factors including those with immunosuppressive or pro-inflammatory activities, such as TGF-β family (Avallet et al., 1994; O’Bryan et al., 2005). TGF-βs contribute to testicular immune privilege through their immunosuppressive activities (Pollanen et al., 1993). TGF-β1 facilitates SCs to support graft survival in co-transplantation experiments (Suarez-Pinzon et al., 2000).

Major pro-inflammatory factors, including IL-1, IL-6, and TNF-α, are expressed in the testis and regulate testicular functions under physiological conditions. IL-1 is an important mediator of inflammation in the testis. A recent study showed that IL-1α facilitates BTB opening by affecting the actin cytoskeleton (Sarkar et al., 2008). IL-1β is feebly produced by the testis under physiological conditions. However, an upregulation of IL-1β has been observed under inflammatory conditions in the testis, which may be harmful to spermatogenesis (Guazzone et al., 2009). IL-6 promotes potently inflammatory events via the expansion and activation of T cells. Most testicular cells, including somatic cells and germ cells in normal rats, produce IL-6 (Potashnik et al., 2005). IL-6 production is upregulated in LPS-induced acute testicular inflammation model. Moreover, IL-6 expression in testicular macrophages is significantly increased in EAO (Rival et al., 2006). Circulating monocytes (ED1+) that arrive at the testis notably express IL-6 at high levels compared with resident testicular macrophages (ED2+), suggesting the distinct roles of the two types of macrophages in mediating inflammatory responses. An in vitro study has shown that exogenous IL-6 induces germ cell apoptosis (Theas et al., 2003). TNF-α is the most potent proinflammatory cytokine. TNF-α is prominently synthesized by germ cells within normal seminiferous tubules (De et al., 1993). Various types of interstitial cells, such as macrophages and mast cells, synthesize TNF-α (Xiong and Hales, 1993). TNF-α protects germ cells from apoptosis at a physiologically low concentrations in normal testes. In oppositely, TNF-α behaves as an apoptotic factor that induces germ cell death under inflammatory conditions (Theas et al., 2008). These three pro-inflammatory cytokines can be induced by TLR activation in testicular cells, including SCs, Leydig, and germ cells. Moreover, antiviral cytokines including IFN-α, -β are produced by these testis-specific cells through TLR activation (Wu et al., 2008; Sun et al., 2010; Shang et al., 2011; Wang et al., 2012). These cytokines could participate in the host defense against pathogens through the regulation of immune responseshfilneg or the direct killing of invading pathogens in vivo. They may also impair spermatogenesis under the inflammatory conditions.

Various chemokines may regulate immune responses in the testis. Chemokines are a large family of small cytokines with chemoattractive activities. They can be grouped into the two major subfamilies, namely, CC ligands (CCLs) and CXC ligands (CXCLs). Among the CCL subfamilies, CCL2 (MCP-1) is present in the testis at physiologically low levels (Gerdprasert et al., 2002b). MCP-1 is expressed by Leydig and MPCs and can be regulated by IL-1, TNF-α, and IFN-β. In response to TLR activation, MCP-1 is upregulated in SCs (Riccioli et al., 2006). Injection of LPS in vivo induces elevated MCP-1 levels in the testicular fluid of rats (Gerdprasert et al., 2002b), increased MCP-1 levels have been observed in the testicular fluid of and EAO model (Guazzone et al., 2003). Among the CXCL subfamilies, CXCL10 is expressed in rat Leydig cells and upregulated by IL-1α, TNF-α, and IFN-γ (Hu et al., 1998). In addition, Sendai viruses induce CXCL1 and CXCL10 in rat testicular macrophages, SCs, Leydig cells, and MPCs (Aubry et al., 2000). Chemokines are not detected in rat germ cells. Increased chemokines could facilitate leukocyte infiltrations and promote the inflammatory reactions in the testis.

In contrast to inflammatory cytokines, IL-10, another anti-flammatory cytokine, may reduce inflammation, autoimmunity, and spermatogenic damages in the mouse model of EAO (Watanabe et al., 2005). During inflammation, testicular resident macrophages produce IL-10. Production of immunosuppressive cytokines supports the speculation that testicular immune cells contribute to the testicular immune privileged status.

While the testis is a remarkable immune privileged site, chronic orchitis and autoimmunity are important etiological factors of male infertility. Further investigation on the immunoregulation of the testis and the link between testicular inflammatory disorders and male infertility will have important implications for interventions of inflammatory disorder-related male infertility.

Mechanisms underlying testicular immune privilege remain largely misunderstood. A local innate immune system of the testis apparently plays an important role in the initiation of the testicular immune responses. Negative regulation of innate immunity in the testis must contribute to testicular immune privilege, which could be the focus of future research. TLR expression and function have recently been revealed in many testis-specific cells. These findings suggest that both immune cells and local tissue-specific cells possess innate immune protective roles against pathogens. Therefore, local cell-initiated inflammatory responses represent a topic that is worthy of further investigation. Endogenous TLR ligand-triggered non-infectious inflammatory conditions in the testis must be considered, because a large portion of spermatogenic cells undergo apoptosis under physiological and pathological conditions, which may release endogenous TLR ligands if the apoptotic cells are not removed timely. The manner by which systemic and local inflammations impair male fertility is another issue that remains unclear. Understanding of the fundamental aspects described above may provide novel insights into the development of prevention and treatment approaches for testicular inflammation-related male infertility.