Experimental Autoimmune Orchitis Research Articles

Seminal plasma S100A8/A9 as a potential biomarker of genital tract inflammation

Abstract Infections and inflammatory reactions in the male genital tract are the leading causes of male infertility with a prevalence of 6%–10%, primarily affecting testicular and epididymal function and ultimately compromising sperm quality. However, most infertile patients with genital infection/inflammation are asymptomatic and easily overlooked. Traditional indicators, including white blood cells, elastase, and other components in semen, can reflect inflammation of the genital tract, but there is still a lack of a uniform standard method of detection. Therefore, it is necessary to explore reliable markers in semen that reflect the inflammatory status of the genital tract. Using the experimental autoimmune orchitis (EAO) model to simulate noninfectious chronic orchitis, we successfully collected ejaculated seminal fluid from EAO rats using optimized electrical stimulation devices. Proteomic analysis was performed using isobaric tags for relative and absolute quantification (iTRAQ). Compared to the control group, 55 upregulated and 105 downregulated proteins were identified in seminal plasma samples from the EAO group. In a preliminary screening, the inflammation-related protein S100A8/A9 was upregulated. We further verified that S100A8/A9 was increased in seminal plasma and highly expressed in testicular macrophages of the EAO model. In patients with oligoasthenospermia and genital tract infections, we also found that S100A8/A9 levels were remarkably increased in seminal plasma and testicular macrophages. S100A8/A9 in semen may be a potential biomarker for chronic genital inflammation. Our study provides a new potential biomarker for early diagnosis and further understanding of male infertility caused by genital inflammation.

Impaired Spermatogenesis in Infertile Patients with Orchitis and Experimental Autoimmune Orchitis in Rats.

Experimental autoimmune orchitis (EAO) is a well-established rodent model of organ-specific autoimmunity associated with infertility in which the testis immunohistopathology has been extensively studied. In contrast, analysis of testis biopsies from infertile patients associated with inflammation has been more limited. In this work, testicular biopsies from patients with idiopathic non-obstructive azoospermia diagnosed with hypospermatogenesis (HypoSp) [mild: n = 9, and severe: n = 11], with obstructive azoospermia and complete Sp (spermatogenesis) (control group, C, n = 9), and from Sertoli cell-only syndrome (SCOS, n = 9) were analyzed for the presence of immune cells, spermatogonia and Sertoli cell (SCs) alterations, and reproductive hormones levels. These parameters were compared with those obtained in rats with EAO. The presence of increased CD45+ cells in the seminiferous tubules (STs) wall and lumen in severe HypoSp is associated with increased numbers of apoptotic meiotic germ cells and decreased populations of undifferentiated and differentiated spermatogonia. The SCs showed an immature profile with the highest expression of AMH in patients with SCOS and severe HypoSp. In SCOS patients, the amount of SCs/ST and Ki67+ SCs/ST increased and correlated with high serum FSH levels and CD45+ cells. In the severe phase of EAO, immune cell infiltration and apoptosis of meiotic germ cells increased and the number of undifferentiated and differentiated spermatogonia was lowest, as previously reported. Here, we found that orchitis leads to reduced sperm number, viability, and motility. SCs were mature (AMH-) but increased in number, with Ki67+ observed in severely damaged STs and associated with the highest levels of FSH and inflammatory cells. Our findings demonstrate that in a scenario where a chronic inflammatory process is underway, FSH levels, immune cell infiltration, and immature phenotypes of SCs are associated with severe changes in spermatogenesis, leading to azoospermia. Furthermore, AMH and Ki67 expression in SCs is a distinctive marker of severe alterations of STs in human orchitis.

Unraveling the effect of the inflammatory microenvironment in spermatogenesis progression.

Experimental autoimmune orchitis (EAO) is a chronic inflammatory disorder that causes progressive spermatogenic impairment. EAO is characterized by high intratesticular levels of nitric oxide (NO) and tumor necrosis factor alpha (TNFα) causing germ cell apoptosis and Sertoli cell dysfunction. However, the impact of this inflammatory milieu on the spermatogenic wave is unknown. Therefore, we studied the effect of inflammation on spermatogonia and preleptotene spermatocyte cell cycle progression in an EAO context and through the intratesticular DETA-NO and TNFα injection in the normal rat testes. In EAO, premeiotic germ cell proliferation is limited as a consequence of the undifferentiated spermatogonia (CD9+) cell cycle arrest in G2/M and the reduced number of differentiated spermatogonia (c-kit+) and preleptotene spermatocytes that enter in the meiotic S-phase. Although inflammation disrupts spermatogenesis in EAO, it is maintained in some seminiferous tubules at XIV and VII-VIII stages of the epithelial cell cycle, thereby guaranteeing sperm production. We found that DETA-NO (2mM) injected in normal testes arrests spermatogonia and preleptotene spermatocyte cell cycle; this effect reduces the number of proliferative spermatogonia and the number of preleptotene spermatocytes in meiosis S-phase (36h after). The temporal inhibition of spermatogonia clonal amplification delayed progression of the spermatogenic wave (5days after) finally altering spermatogenesis. TNFα (0.5 and 1µg) exposure did not affect premeiotic germ cell cycle or spermatogenic wave. Our results show that in EAO the inflammatory microenvironment altered spermatogenesis kinetics through premeiotic germ cell cycle arrest and that NO is a sufficient factor contributing to this phenomenon.

Activin A and CCR2 regulate macrophage function in testicular fibrosis caused by experimental autoimmune orchitis

Experimental autoimmune-orchitis (EAO), a rodent model of chronic testicular inflammation and fibrosis, replicates pathogenic changes seen in some cases of human spermatogenic disturbances. During EAO, increased levels of pro-inflammatory and pro-fibrotic mediators such as TNF, CCL2, and activin A are accompanied by infiltration of leukocytes into the testicular parenchyma. Activin A levels correlate with EAO severity, while elevated CCL2 acting through its receptor CCR2 mediates leukocyte trafficking and recruits macrophages. CCR2 + CXCR4 + macrophages producing extracellular matrix proteins contribute widely to fibrogenesis. Furthermore, testicular macrophages (TMs) play a critical role in organ homeostasis. Therefore, we aimed to investigate the role of the activin A/CCL2-CCR2/macrophage axis in the development of testicular fibrosis. Following EAO induction, we observed lower levels of organ damage, collagen deposition, and leukocyte infiltration (including fibronectin+, collagen I+ and CXCR4+ TMs) in Ccr2−/− mice than in WT mice. Furthermore, levels of Il-10, Ccl2, and the activin A subunit Inhba mRNAs were lower in Ccr2−/− EAO testes. Notably, fibronectin+ TMs were also present in biopsies from patients with impaired spermatogenesis and fibrotic alterations. Overexpression of the activin A antagonist follistatin reduced tissue damage and collagen I+ TM accumulation in WT EAO testes, while treating macrophages with activin A in vitro increased the expression of Ccr2, Fn1, Cxcr4, and Mmp2 and enhanced migration along a CCL2 gradient; these effects were abolished by follistatin. Taken together, our data indicate that CCR2 and activin A promote fibrosis during testicular inflammation by regulating macrophage function. Inhibition of CCR2 or activin A protects against damage progression, offering a promising avenue for therapeutic intervention.

OP-2 - HSPA4L and GIT1 as possible biomarkers in testicular autoimmunity

Clinically, 60–75% of male infertility cases are categorized as idiopathic spermatogenic disturbance. In previous studies, lymphocytic infiltration and immune deposits were present in several testis biopsy specimens, indicating that immunological factors contribute to the occurrence of the idiopathic spermatogenic disturbance. We recently found that immunization of A/J male mice with heat shock protein family A member 4 like (HSPA4L) and G protein-coupled receptor kinase interacting protein-1 (GIT1) induces experimental autoimmune orchitis (EAO). In this study, we detected autoantibodies and lymphocyte activity to investigate whether these autoimmunogenic antigens could be biomarkers for EAO. A/J male mice, 8 weeks old, were subcutaneously injected twice with TGC, to induce EAO and injected with HBSS alone were used as controls. The spleen cells and sera of control mice and TGC-immunized mice were analyzed by ELISA assay and lymphocyte stimulation test. In the result, lymphocyte proliferation was significantly stimulated in vitro by GIT1 (100 μg) and HSPA4L (10, 50, 100, and 200 μg) in TGC immunized mice. In addition, the sera of TGC-immunized mice contained more anti-GIT1 and anti-HSPA4L antibodies than did the serum of control mice. Therefore, the detection of immune responses against HSPA4L and GIT1 may be useful parameters of immunologic male infertility.

Evidence of pyroptosis and ferroptosis extensively involved in autoimmune diseases at the single-cell transcriptome level

BackgroundApproximately 8–9% of the world’s population is affected by autoimmune diseases, and yet the mechanism of autoimmunity trigger is largely understudied. Two unique cell death modalities, ferroptosis and pyroptosis, provide a new perspective on the mechanisms leading to autoimmune diseases, and development of new treatment strategies.MethodsUsing scRNA-seq datasets, the aberrant trend of ferroptosis and pyroptosis-related genes were analyzed in several representative autoimmune diseases (psoriasis, atopic dermatitis, vitiligo, multiple sclerosis, systemic sclerosis-associated interstitial lung disease, Crohn’s disease, and experimental autoimmune orchitis). Cell line models were also assessed using bulk RNA-seq and qPCR.ResultsA substantial difference was observed between normal and autoimmune disease samples involving ferroptosis and pyroptosis. In the present study, ferroptosis and pyroptosis showed an imbalance in different keratinocyte lineages of psoriatic skinin addition to a unique pyroptosis-sensitive keratinocyte subset in atopic dermatitis (AD) skin. The results also revealed that pyroptosis and ferroptosis are involved in epidermal melanocyte destruction in vitiligo. Aberrant ferroptosis has been detected in multiple sclerosis, systemic sclerosis-associated interstitial lung disease, Crohn’s disease, and autoimmune orchitis. Cell line models adopted in the study also identified pro-inflammatory factors that can drive changes in ferroptosis and pyroptosis.ConclusionThese results provide a unique perspective on the involvement of ferroptosis and pyroptosis in the pathological process of autoimmune diseases at the scRNA-seq level. IFN-γ is a critical inducer of pyroptosis sensitivity, and has been identified in two cell line models.

Exposed and Sequestered Antigens in Testes and Their Protection by Regulatory T Cell-Dependent Systemic Tolerance.

Continuous exposure of tissue antigen (Ag) to the autoantigen-specific regulatory T cells (Treg) is required to maintain Treg-dependent systemic tolerance. Thus, testis autoantigens, previously considered as sequestered, may not be protected by systemic tolerance. We now document that the complete testis antigen sequestration is not valid. The haploid sperm Ag lactate dehydrogenase 3 (LDH3) is continuously exposed and not sequestered. It enters the residual body (RB) to egress from the seminiferous tubules and interact with circulating antibody (Ab). Some LDH3 also remains inside the sperm cytoplasmic droplets (CD). Treg-depletion in the DEREG mice that express diphtheria toxin receptor on the Foxp3 promoter results in spontaneous experimental autoimmune orchitis (EAO) and Ab to LDH3. Unlike the wild-type male mice, mice deficient in LDH3 (wild-type female or LDH3 NULL males) respond vigorously to LDH3 immunization. However, partial Treg depletion elevated the wild-type male LDH3 responses to the level of normal females. In contrast to LDH3, zonadhesin (ZAN) in the sperm acrosome displays properties of a sequestered Ag. However, when ZAN and other sperm Ag are exposed by vasectomy, they rapidly induce testis Ag-specific tolerance, which is terminated by partial Treg-depletion, leading to bilateral EAO and ZAN Ab response. We conclude that some testis/sperm Ag are normally exposed because of the unique testicular anatomy and physiology. The exposed Ag: 1) maintain normal Treg-dependent systemic tolerance, and 2) are pathogenic and serve as target Ag to initiate EAO. Unexpectedly, the sequestered Ags, normally non-tolerogenic, can orchestrate de novo Treg-dependent, systemic tolerance when exposed in vasectomy.

GIT1 is an untolerized autoantigen involved in immunologic disturbance of spermatogenesis.

Male haploid cells, spermatids and spermatozoa, that appear after the establishment of immune tolerance express novel cell surface and intracellular proteins that can be recognized as foreign antigens by the self-immune system. However, these germ cells do not normally evoke a pathological immune response. The immune-privileged micro-circumstance in testis involving the blood-testis-barrier formed by Sertoli cells protects these germ cells from autoimmune attack. We recently found that immunization with heat shock protein family A member 4-like (HSPA4L), one of the new differentiation antigens of haploid cells, induced experimental autoimmune orchitis (EAO) in A/J male mice. In this study, we focused on G protein-coupled receptor kinase interacting protein-1 (GIT1), another haploid cell-specific differentiation antigen, to investigate whether GIT1 is a target autoantigen for EAO induction. GIT1 emulsified with complete Freund's adjuvant was injected subcutaneously into the mice inguinal region once on day 0 and again on day 14, and the optimum condition of EAO induction was determined. Mice immunized with 200μg GIT1 showed significantly higher incidence of EAO than that of immunization with other concentrations. In particular, significant lymphocytic inflammation and extensive aspermatogenesis were observed in these mice at 120days after the first immunization. These findings indicate that GIT1 is also a target antigen that induces EAO, like HSPA4L.

High Throughput scRNA-Seq Provides Insights Into Leydig Cell Senescence Induced by Experimental Autoimmune Orchitis: A Prominent Role of Interstitial Fibrosis and Complement Activation.

Leydig cells (Lc), located in the interstitial space of the testis between seminiferous tubules, produce 95% of testosterone in male individuals, which is pivotal for male sexual differentiation, spermatogenesis, and maintenance of the male secondary sex characteristics. Lc are prone to senescence in aging testes, resulting in compromised androgen synthesis capability upon aging. However, little is known about whether Lc undergo senescence in a chronic inflammatory environment. To investigate this question, mouse models of experimental autoimmune orchitis (EAO) were used, and Lc were analyzed by high throughput scRNA-Seq. Data were screened and analyzed by correlating signaling pathways with senescence, apoptosis, androgen synthesis, and cytokine/chemokine signaling pathways. EAO did induce Lc senescence, and Lc senescence in turn antagonized androgen synthesis. Based on the correlation screening of pathways inducing Lc senescence, a plethora of pathways were found to play potential roles in triggering Lc senescence during EAO, among which the Arf6 and angiopoietin receptor pathways were highly correlated with senescence signature. Notably, complement and interstitial fibrosis activated by EAO worsened Lc senescence and strongly antagonized androgen synthesis. Furthermore, most proinflammatory cytokines enhanced both senescence and apoptosis in Lc and spermatogonia (Sg) during EAO, and proinflammatory cytokine antagonism of the glutathione metabolism pathway may be key in inducing cellular senescence during EAO.

Experimental autoimmune orchitis induced by immunization with GIT1 in mice

Experimental autoimmune orchitis induced by immunization with GIT1 in mice

Bone Status in a Mouse Model of Experimental Autoimmune-Orchitis.

Investigations in male patients with fertility disorders revealed a greater risk of osteoporosis. The rodent model of experimental autoimmune-orchitis (EAO) was established to analyze the underlying mechanisms of male infertility and causes of reduced testosterone concentration. Hence, we investigated the impact of testicular dysfunction in EAO on bone status. Male mice were immunized with testicular homogenate in adjuvant to induce EAO (n = 5). Age-matched mice were treated with adjuvant alone (adjuvant, n = 6) or remained untreated (control, n = 7). Fifty days after the first immunization specimens were harvested. Real-time reverse transcription-PCR indicated decreased bone metabolism by alkaline phosphatase and Cathepsin K as well as remodeling of cell-contacts by Connexin-43. Micro computed tomography demonstrated a loss of bone mass and mineralization. These findings were supported by histomorphometric results. Additionally, biomechanical properties of femora in a three-point bending test were significantly altered. In summary, the present study illustrates the induction of osteoporosis in the investigated mouse model. However, results suggest that the major effects on bone status were mainly caused by the complete Freund’s adjuvant rather than the autoimmune-orchitis itself. Therefore, the benefit of the EAO model to transfer laboratory findings regarding bone metabolism in context with orchitis into a clinical application is limited.

Heat shock protein A4L is a potent autoantigen for testicular autoimmunity in mice

Experimental autoimmune orchitis (EAO) may be used as a model to investigate immunological infertility in men. Murine EAO is induced via immunization with auto-immunogenic antigens (AIAgs) from testicular germ cells (TGCs). CD4 + T cells play a crucial role in EAO induction. However, whether AIAgs induce an immune response remains unclear. We aimed to identify self-antigens that induce EAO by screening a phage display library of random TGC peptides using IgG from EAO-induced A/J mice. Twenty TGC-specific AIAgs were detected, and G protein-coupled receptor kinase 2 interacting protein-1 (GIT1) and heat shock protein A4L (HSPA4L) were identified as candidate AIAgs that induce EAO. Immunization with GIT1 or HSPA4L, emulsified in complete Freund’s adjuvant, resulted in 66 % or 100 % incidence of EAO, respectively, indicating that HSPA4L is a most potent AIAg that induces EAO in mice. These findings may expectedly help improve the diagnostic procedures and treatment of immunological infertility in men.

Autoimmune experimental orchitis and chronic glomerulonephritis with end stage renal disease are controlled by Cgnz1 for susceptibility to end organ damage

Cgnz1 on chromosome 1 mapped into a 1.34 Mb region of chromosome 1 in NZM2328 confers the progression of immune complex (IC)-mediated glomerulonephritis (GN) from acute GN (aGN) to chronic GN (cGN) with severe proteinuria and end stage renal disease in female mice. This genetic locus mediates podocyte susceptibility to IC-mediated damage. Taking advantage of the published observation that Cgnz1 is derived from NZW and that NZW is susceptible to orchitis, epididymitis and vasitis while C57L/J is resistant to these diseases, the possibility that this genetic region also confers germ cells susceptible to damage with aspermatogenesis and sterility in an active experimental autoimmune orchitis (EAO) model was investigated. Male mice from multiple intrachromosome (chromosome 1) recombinant strains were subjected to immunization with a sperm homogenate in CFA with concomitant administration of Bordetella pertussis toxin. There was concordance of the progression from aGN to cGN, severe proteinuria and end stage renal disease with susceptibility of EAO in NZM2328 and its congenic strains with various chromosome 1 genetic intervals introgressed from C57L/J to NZM2328. Both resistant and susceptible strains made comparable anti-testis and anti-sperm Abs. Thus the genetic interval that determines susceptibility to EAO is identical to that of Cgnz1 and mapped to the 1.34 Mb region in chromosone 1. This region likely confers germ cells in the male gonad susceptible to damage by immunologically mediated inflammation. This region has been tentatively renamed Cgnz1/Eaoz1. These observations further emphasize the importance of end organ susceptibility to damage in the pathogenesis of both systemic and organ specific autoimmune diseases.

Experimental autoimmune orchitis induced by immunization with HSP in mice

A series of far-red-emitting Ca2M3(SiO4)2(PO4)O (M = La, Y) (CMSPO):xEu3+ (x = 0.05, 0.10, 0.20, 0.30, 0.40 and 0.50) phosphors were successfully synthesized by high-temperature solid-phase reaction. The fabricated samples were characterized by X-ray powder diffraction (XRD), scanning electron microscopy (SEM), fourier transform infrared (FT-IR), ultraviolet–visible diffuse reflectance (UV–Vis DR), photoluminescence excitation (PLE) and emission (PL) spectra. Under 394 nm excitation, the samples show red emission locating at 618 nm, which is attributed to the 5D0 → 7F2 electric dipole (ED) transition. Also, a deep red-emitting originating from 5D0 → 7F4 transition matches well with the absorption spectra of PR and PFR regions. It is also found that the PL intensity of CLSPO:0.30Eu3+ is enhanced about 1.6 times compared with the CYSPO:0.20Eu3+. Additionally, the results demonstrated that the phosphors can provide reference for the development of Eu3+-doped phosphors that can be used for plant cultivation LEDs.

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.

Fluoride Induces Autoimmune Orchitis Involved with Enhanced IL-17A Secretion in Mice Testis

Fluoride (F) widely exists in the water and food. Recent studies reported that F induced testicular toxicity via inflammation reaction. This study was aimed to explore the mechanism of F-induced inflammation in testis. 100 healthy male mice (BALB/cJ strain) were randomly divided into five groups including: control, experimental autoimmune orchitis (EAO), and three F groups (25, 50, and 100 mg/L sodium fluoride (NaF)). After 150 d, the results showed a significant increase in testicular cytokines levels including of IL-17A, IL-6, IFN-γ, and TNF-α in NaF and EAO groups compared with control group. Interestingly, the presence of specific antisperm autoantibodies in antitesticular autoantibodies and the notable recruitment of immunocyte (T cells and dendritic cells) were also observed in NaF and EAO groups. In addition, findings showed that in NaF and EAO groups macrophages and T cells both significantly secreted IL-17A, and the protein and mRNA levels of cytokines (IL-6 and TGF-β) were significantly increased. From these results, it can be concluded that autoimmune orchitis and IL-17A are implicated in F-induced testicular inflammation.

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.

Investigation of activin A in inflammatory responses of the testis and its role in the development of testicular fibrosis.

Does activin A contribute to testicular fibrosis under inflammatory conditions? Our results show that activin A and key fibrotic proteins are increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis and in murine experimental autoimmune orchitis (EAO) and that activin A stimulates fibrotic responses in peritubular cells (PTCs) and NIH 3T3 fibroblasts. Fibrosis is a feature of EAO. Activin A, a regulator of fibrosis, was increased in testes of mice with EAO and its expression correlated with severity of the disease. This is a cross-sectional and longitudinal study of adult mice immunized with testicular homogenate (TH) in adjuvant to induce EAO, collected at 30 (n= 6), 50 (n= 6) and 80 (n= 5) days after first immunization. Age-matched mice injected with adjuvant alone (n= 14) and untreated mice (n= 15) were included as controls. TH-immunized mice with elevated endogenous follistatin, injected with a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of follistatin (rAAV-FST315; n= 3) or vector with an empty cassette (empty vector controls; n= 2) 30days prior to the first immunization, as well as appropriate adjuvant (n= 2) and untreated (n = 2) controls, were also examined.Human testicular biopsies showing focal inflammatory lesions associated with impaired spermatogenesis (n= 7) were included. Biopsies showing intact spermatogenesis without inflammation, from obstructive azoospermia patients, served as controls (n= 7).Mouse primary PTC and NIH 3T3 fibroblasts were stimulated with activin A and follistatin 288 (FST288) to investigate the effect of activin A on the expression of fibrotic markers. Production of activin A by mouse primary Sertoli cells (SCs) was also investigated. Testicular RNA and protein extracts collected from mice at days 30, 50 and 80 after first immunization were used for analysis of fibrotic marker genes and proteins, respectively. Total collagen was assessed by hydroxyproline assay and fibronectin; collagen I, III and IV, α-smooth muscle actin (α-SMA) expression and phosphorylation of suppressor of mothers against decapentaplegic (SMAD) family member 2 were measured by western blot. Immunofluorescence was used to detect fibronectin. Fibronectin (Fn), αSMA (Acta2), collagen I (Col1a2), III (Col3a1) and IV (Col4a1) mRNA in PTC and NIH 3T3 cells treated with activin A and/or FST288 were measured by quantitative RT-PCR (qRT-PCR). Activin A in SC following tumour necrosis factor (TNF) or FST288 stimulation was measured by ELISA. Human testicular biopsies were analysed by qRT-PCR for PTPRC (CD45) and activin A (INHBA), hydroxyproline assay and immunofluorescence. Production of activin A by SC was stimulated by 25 and 50ng/ml TNF (P< 0.01, P< 0.001, respectively) as compared to untreated cells. INHBA mRNA was increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis, compared with control biopsies (P< 0.05), accompanied by increased total collagen (P< 0.01) and fibronectin deposition. Total testicular collagen (P< 0.0001) and fibronectin protein expression (P< 0.05) were also increased in EAO, and fibronectin expression was correlated with the severity of the disease (r= 0.9028). In animals pre-treated with rAAV-FST315 prior to immunization with TH, protein expression of fibronectin was comparable to control. Stimulation of PTC and NIH 3T3 cells with activin A increased fibronectin mRNA (P< 0.05) and the production of collagen I (P< 0.001; P< 0.01) and fibronectin (P< 0.05). Moreover, activin A also increased collagen IV mRNA (P< 0.05) in PTC, while αSMA mRNA (P< 0.01) and protein (P< 0.0001) were significantly increased by activin A in NIH 3T3 cells. N/A. A limited number of human testicular specimens was available for the study. Part of the study was performed in vitro, including NIH 3T3 cells as a surrogate for testicular fibroblasts. Resident fibroblasts and PTC may contribute to the progression of testicular fibrosis following inflammation, and activin A is implicated as a key mediator of this process. This work was supported by the National Health and Medical Research Council of Australia, the Victorian Government's Operational Infrastructure Support Program and the International Research Training Group between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK 1871/1-2) on `Molecular pathogenesis on male reproductive disorders' funded by the Deutsche Forschungsgemeinschaft and Monash University. The authors declare no competing financial interests.

Effect of ketotifen fumarate on experimental autoimmune orchitis and torsion of the spermatic cord.

The aim of this work was to study effects of ketotifen fumarate (KF) on prevention of tissue damage in testes of rats with experimental autoimmune orchitis (EAO) and on the contralateral testis in a model of prolonged testicular cord torsion (TCT). Rats with EAO or TCT were injected intraperitoneally once daily with KF or saline solution (vehicle group). Incidence and severity of testicular damage were evaluated by histopathology using an EAO score or a Johnsen score. Mast cells (MC) were identified by histochemistry and quantified. In EAO model, KF significantly reduced severity of histopathological testicular damage compared to rats in the vehicle group. KF also reduced the number of testicular MC compared to vehicle group. Similarly, in TCT model, multifocal damage of the contralateral testis was observed 30 days after testicular torsion characterized by sloughing of the germinal epithelium, seminiferous tubule atrophy, and interstitial edema. Focal signs of inflammation and fibrosis of seminiferous tubular walls were also observed. In contrast, sections of contralateral testis of rats injected with KF and killed 30 days after surgery showed normal histological features. A significant decrease in the number of MC was observed in rats treated with KF compared to untreated animals. In conclusion, we demonstrated that treatment with KF reduced testicular inflammatory process and MC infiltrates in both EAO and TCT models. The results suggest a promising treatment for infertile male patients with testicular pathologies associated with inflammation and germ cell loss.

The role of regulatory T Cells in autoimmune orchitis.

Regulatory T cells (Tregs) mediate tolerance to self-antigens maintaining immune homeostasis. Defects in the number and function of Tregs lead to aberrant immune responses to autologous components, thereby causing autoimmune diseases. Male infertility as a result of immune testicular damage follows through auto-reactive T-cell activation by antigens or pathogens that disrupt testis tolerance mechanisms. In this review we summarise the main evidence on Treg behaviour in inflammatory testicular pathologies focusing on reports on experimental autoimmune orchitis. Increased numbers of different Treg phenotypes are observed in the chronically inflamed testis and in lymph nodes draining to it; however these cells are outnumbered by effector T cells. Distortion of the effector/regulatory cell balance in favour of a pro-inflammatory response is suspected to contribute to exacerbation of autoimmune disease. Under inflammatory conditions, effector T-cell subsets can overwhelm the inhibitory effect of Tregs, and pro-inflammatory cytokines may directly or indirectly affect the ability of Tregs to control autoimmunity. Therefore, Tregs alone may not be sufficient to limit excessive T-cell activation in autoimmune settings. Treg immunotherapy for autoimmune disease treatment aims to restore the normal balance of effector and Tregs in the inflamed tissue. Therapies combining the transfer of Tregs with Treg-stabilising drugs are expected to be the most effective to restrain autoimmune diseases.