Gasdermin D in macrophages drives orchitis by regulating inflammation and antigen presentation processes.

Inflammation in the testes induced by infection and autoimmunity contributes significantly to male infertility, a public health issue. Current therapies using antibiotics and broad-spectrum anti-inflammatory drugs are ineffective against non-bacterial orchitis and induce side effects. This highlights the need to explore the pathogenesis of orchitis and develop alternative therapeutic strategies. In this study, we demonstrated that Gasdermin D (GSDMD) was activated in the testes during uropathogenic Escherichia coli (UPEC)-induced acute orchitis, and that GSDMD in macrophages induced inflammation and affected spermatogenesis during acute and chronic orchitis. In testicular macrophages, GSDMD promoted inflammation and antigen presentation, thereby enhancing the T-cell response after orchitis. Furthermore, the pharmacological inhibition of GSDMD alleviated the symptoms of UPEC-induced acute orchitis. Collectively, these findings provide the first demonstration of GSDMD's role in driving orchitis and suggest that GSDMD may be a potential therapeutic target for treating orchitis.

Insights into Canine Infertility: Apoptosis in Chronic Asymptomatic Orchitis.

Chronic asymptomatic orchitis (CAO) is a common cause of acquired non-obstructive azoospermia in dogs. To understand the impact and mode of action of apoptosis, we investigated TUNEL, Bax, Bcl-2, Fas/Fas ligand, and caspase 3/8/9 in testicular biopsies of CAO-affected dogs and compared the results to undisturbed spermatogenesis in healthy males (CG). TUNEL+ cells were significantly increased in CAO, correlating with the disturbance of spermatogenesis. Bcl-2, Bax (p < 0.01 each), caspase 9 (p < 0.05), Fas, caspase 8 (p < 0.01 each), and caspase 3 (p < 0.05) were significantly increased at the mRNA level, whereas FasL expression was downregulated. Cleaved caspase 3 staining was sporadic in CAO but not in CG. Sertoli cells, some peritubular (CAO/CG) and interstitial immune cells (CAO) stained Bcl-2+, with significantly more immunopositive cells in both compartments in CAO compared to CG. Bcl-2 and CD20 co-expressing B lymphocytes were encountered interstitially and in CAO occasionally also found intratubally, underlining their contribution to the maintenance of CAO. Our results support the crucial role of the intrinsic and extrinsic apoptotic pathways in the pathophysiology of canine CAO. Autoprotective Bcl-2 expression in Sertoli cells and B lymphocytes seems to be functional, however, thereby also maintaining and promoting the disease by immune cell activation.

Immune cell characterization in spontaneous autoimmune orchitis in dogs.

With a prevalence of up to 35% in dogs with reproductive problems, azoospermia is one of the most important reasons for male infertility. Non-obstructive azoospermia, without clinical symptoms, but histopathological damage of the testicular tissue and immune cell infiltration is referred to as spontaneous autoimmune orchitis (AIO) in the literature. Published cases in dogs describe immune cell infiltration; however, there is no consent about the involved immune cell types. We aimed to characterize immune cells in testicular biopsies of dogs with AIO (n = 9) and to compare them to those in testicular specimens from healthy control dogs with normospermic ejaculates (CG; n = 5). Immunohistochemistry was performed using specific antibodies against CD3, PAX5, MAC387, IgG and IgM to proof the presence of T lymphocytes, B lymphocytes, macrophages and early and late plasma cells, respectively. Presence of immune cells in healthy testicular tissue was low and restricted to T lymphocytes and macrophages in the interstitium with the latter also being found within the blood vessels. Different to this, AIO samples revealed presence of all investigated immune cells, underlining lymphoplasmacytic nature of chronic asymptomatic immune-mediated orchitis. Canine spontaneous AIO is characterised by a significantly increased number of immune cells, namely ≥33 immune cells/mm2 (sensitivity/specificity: 100% based on our data). The pathogenesis of canine AIO is hypothesized to be as follows: 1. Macrophages initiate AIO via T lymphocyte activation. 2. T lymphocytes lead to a "delayed type immunological response" and development of AIO. 3. Invaded B lymphocytes later differentiating to plasma cells are responsible for the second humoral immunological response and cause progression of AIO. Different to the situation in CG, T lymphocytes and plasma cells were identified within the seminiferous tubules indicating that disruption of spermatogenesis in AIO might be related to invading immune cells. Testicular biopsies provide an essential tool in the diagnosis of spontaneous AIO.

Testicular pathology in fatal COVID-19: A descriptive autopsy study.

BACKGROUND: Multi-organ damage is a common feature of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, going beyond the initially observed severe pneumonia. Evidence that the testis is also compromised is growing. OBJECTIVE: To describe the pathological findings in testes from fatal cases of COVID-19, including the detection of viral particles and antigens, and inflammatory cell subsets. MATERIALS AND METHODS: Postmortem testicular samples were obtained by percutaneous puncture from 11 deceased men and examined by reverse-transcription polymerase chain reaction (RT-PCR) for RNA detection and by light and electron microscopy (EM) for SARS-CoV-2. Immunohistochemistry (IHC) for the SARS-CoV-2 N-protein and lymphocytic and histiocytic markers was also performed. RESULTS: Eight patients had mild interstitial orchitis, composed mainly of CD68+ and TCD8+ cells. Fibrin thrombi were detected in five cases. All cases presented congestion, interstitial edema, thickening of the tubular basal membrane, decreased Leydig and Sertoli cells with reduced spermatogenesis, and strong expression of vascular cell adhesion molecule (VCAM) in vessels. IHC detected SARS-Cov-2 antigen in Leydig cells, Sertoli cells, spermatogonia, and fibroblasts in all cases. EM detected viral particles in the cytoplasm of fibroblasts, endothelium, Sertoli and Leydig cells, spermatids, and epithelial cells of the rete testis in four cases, while RT-PCR detected SARS-CoV-2 RNA in three cases. DISCUSSION AND CONCLUSION: The COVID-19-associated testicular lesion revealed a combination of orchitis, vascular changes, basal membrane thickening, Leydig and Sertoli cell scarcity, and reduced spermatogenesis associated with SARS-CoV-2 local infection that may impair hormonal function and fertility in men.

The ameliorative effect of ellagic acid on di-(2-ethylhexyl) phthalate-induced testicular structural alterations, oxidative stress, inflammation and sperm damages in adult mice.

BACKGROUND: Phthalates such as di (2-ethylhexyl) phthalate (DEHP) are well known exogenous substances, disrupting reproductive system function and structure. The current research demonstrated the effect of ellagic acid (EA) on DEHP-induced testicular injury in mice. METHODS: Thirty-five healthy adult male mice were randomly divided to five groups; normal saline receiving group, DEHP (2 g/kg/day, dissolved in corn oil, p.o.) receiving group, DEHP (2 g/kg/day, dissolved in corn oil, p.o.) and EA receiving groups (25, 50 and 100 mg/kg/day, p.o.). Treatment duration of animals was 14 days. Body and testes weights and sperm characteristics and histological changes of testes were evaluated. Serum testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were analyzed. In the testicular tissue, oxidative/nitrosative stress markers and inflammatory cytokine levels were measured. RESULTS: Ellagic acid significantly reduced DEHP-induced reduction of body and testes weights. The DEHP-induced reduction of spermatogonia, primary spermatocyte and sertoli cells numbers as well as reduction of sperm vitality and progressive motility were reversed by EA. Furthermore, EA inhibited DEHP-induced alterations in serum hormone levels. These effects were associated with the reduction of DEHP-induced increased level of oxidative stress and inflammatory responses. CONCLUSIONS: Ellagic acid considerably inhibits testicular toxicity of DEHP through reducing oxidative/nitrosative stress and inflammatory responses. Our data suggest that EA may be considered as a promising agent to inhibit male reproductive toxicity induced by endocrine disrupting chemicals such as DEHP.

Hard bilateral syphilitic testes with vasculitis: a case report and literature review.

BACKGROUND: We report the case of a patient with syphilitic testicular gumma and vasculitis with adrenal failure due to chronic steroid use. CASE PRESENTATION: A 63-year-old male presented with hard right eye swelling and very firm bilateral testes on palpation, which he had for 2 years. Testicular tumor markers were negative; syphilis test was positive. Radiological examination suggested aortitis and bilateral testicular malignancy. The patient received ampicillin for the infection and prednisolone for vasculitis. Left orchidectomy was performed to confirm the presence of testicular tumor; histological examinations revealed granulomatous orchitis. The prednisolone doses were adjusted because of relapses and adverse effects of steroid use. Unfortunately, the patient died in the intensive care unit because of uncontrolled blood pressure and pneumonia. CONCLUSIONS: This is a rare case of syphilis with testicular involvement and vasculitis. This report shows the importance of broadening the differential diagnoses of testicular firmness.

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.

cDC1 Dependent Accumulation of Memory T Cells Is Required for Chronic Autoimmune Inflammation in Murine Testis.

As an immune privilege site, there are various types of immune cells in the testis. Previous research has been focused on the testicular macrophages, and much less is known about the T cells in the testis. Here, we found that T cells with memory phenotypes were the most abundant leukocyte in the testis except for macrophages. Our results showed that the proportion of testicular T cells increases gradually from birth to adulthood in mice and that the primary type of T cells changed from γδTCR+ T cells to αßTCR+ T cells. In addition, under homeostatic conditions, CD8+ T cells are the dominant subgroup and have different phenotypic characteristics from CD4+ T cells. We found that cDC1, but not cDC2, is necessary for the presence of T cells in the testis under physiological state. A significant decrease of T cells does not have a deleterious effect on the development of the testis or spermatogenesis. However, cDC1-dependent T cells play an indispensable role in chronic autoimmune orchitis of the testis. Collectively, our multifaceted data provide a comprehensive picture of the accumulation, localization, and function of testicular T cells.

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.

Monolateral chronicon orchitis in a stallion.

Orchitis and epididymo-orchitis are inflammatory lesions of the testicle. We herein describe a case of monolateral chronic orchitis which occurred in a Tiro Pesante Rapido (TPR) stallion, born in 2002, with a history of good fertility. The stallion was healthy and asymptomatic although the left testis was found to be smaller as compared with the right one and was hard in consistency. Histopathology examination revealed tubular atrophy and parenchymal sclerosis. Scattered foci of calcification and chronic inflammation, the latter dominated by macrophages and lymphocytes, were also observed. Although lesions were clearly present, the semen was demonstraed to be of good quality. This study highlights the need for periodic clinical and ultrasound evaluation of stallions, in order to preserve their reproductive performance.

A Case of Testicular Granulomatous Inflammation Mistaken for Malignancy: Tuberculosis Identified Post Orchiectomy.

We describe the case of a 41-year-old Hispanic male, inconsistently adherent to visits and workup due to socioeconomic challenges, who presented with a right testicular mass. Because of the overriding concern that this was malignant, he underwent a right orchiectomy. Pathology revealed granulomatous disease with no evidence of malignancy. No specific diagnosis was made histologically or microbiologically on primary laboratory investigation. Six months later, he developed swelling of the left testicle and was subsequently seen in consultation at the Infectious Disease Clinic Kern Medical. An extensive evaluation for granulomatous inflammation was undertaken without a positive result. A clinical diagnosis of tuberculous epididymal orchitis was made and the patient was initiated on standard 4-drug antituberculous therapy. There was a gradual resolution of pain and swelling. After 6 months of therapy, there was no evidence of residual disease. The patient remains asymptomatic after 8 months of post-therapy follow-up.

Interleukin-18 levels and mouse Leydig cell apoptosis during lipopolysaccharide-induced acute inflammatory conditions.

Interleukin (IL)-18 is an inflammasome-mediated cytokine produced by germ cells, Leydig cells, and resident macrophages that is indispensable in the maintenance of homeostasis in the testis. We previously demonstrated that endogenous IL-18 induces testicular germ cell apoptosis during acute inflammation when plasma IL-18 levels are very high. However, the impact of acute inflammation and IL-18 on Leydig cells remained unclear. TM3 cells, a mouse Leydig cell line, and RAW264.7 cells, a mouse macrophage cell line, were stimulated with lipopolysaccharide (LPS) or recombinant IL-18 (rIL-18). We assessed the expression of inflammatory cytokines, caspase cleavage, and markers of apoptotic pathways. In Leydig cells, caspase 3 cleavage was increased and death-receptor-mediated apoptotic pathways were activated after LPS stimulation. However, LPS stimulation did not increase IL-18 expression in the Leydig cell line. When high-dose rIL-18 was administered to the Leydig cell line to mimic levels seem after inflammation, rIL-18 upregulated Tnf-α mRNA, Fadd mRNA, and Fas protein, promoted cleavage of caspase-8 and caspase-3, and induced apoptosis. Low-dose rIL-18 did not stimulate apoptosis. To determine if the high level of IL-18 seen in the testes after inflammation was derived from immune cells, we examined IL-18 protein expression in a macrophage cell line, RAW264.7. In contrast to the TM3 cells, IL-18 was significantly increased in RAW264.7 cells after LPS stimulation. These results suggest that high-dose IL-18 derived from macrophages is harmful to Leydig cells. Reducing the overexpression of IL-18 could be a new therapeutic approach to prevent Leydig cell apoptosis as a result of acute inflammation.

Differential tissue-specific damage caused by bacterial epididymo-orchitis in the mouse.

Ascending bacterial urinary tract infections can cause epididymo-orchitis. In the cauda epididymidis, this frequently leads to persistent tissue damage. Less coherent data is available concerning the functional consequences of epididymo-orchitis on testis and caput epididymidis. This in vivo study addresses the functional and spatial differences in responsiveness of murine epididymis and testis to infection with uropathogenic Escherichia coli (UPEC). Whole transcriptome analysis (WTA) was performed on testis, caput, corpus and cauda epididymidis of adult C57BL/6 J wildtype mice. Following UPEC-induced epididymo-orchitis in these mice, epididymal and testicular tissue damage was evaluated histologically and semi-quantitatively at 10 days and 31 days post-inoculation. Expression of inflammatory markers and candidate antimicrobial genes were analysed by RT-qPCR. WTA revealed distinct differences in gene signatures between caput and cauda epididymidis, particularly amonst immunity-related genes. Cellular and molecular signs of testicular inflammation and disruption of spermatogenesis were noticed at day 10, but recovery was observed by day 31. In contrast to the cauda, the caput epididymidis did not reveal any signs of gross morphological damage or presence of pro-inflammatory processes despite confirmed infection. In contrast to beta-defensins, known UPEC-associated antimicrobial peptides (AMP), like Lcn2, Camp and Lypd8, were inherently highly expressed or upregulated in the caput following infection, potentially allowing an early luminal protection from UPEC. At the time points investigated, the caput epididymidis was protected from any obvious infection/inflammation-derived tissue damage. Studies addressing earlier time-points will conclude whether in the caput epididymidis a pro-inflammatory response is indeed not essential for effective protection from UPEC.

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.

Evaluating the effects of acacetin versus a low dose of cisplatin drug on male reproductive system and kidney in mice: With emphasis on inflammation process.

This study aimed to compare acacetin adverse effect besides a cisplatin low dose on male reproductive and urinary systems on mice. In this study, 36 male Balb/c mice were received Dimethyl sulfoxide, cisplatin (1 mg/kg) or acacetin (10, 25, 50 mg/kg) for 3 days, while the sixth group, treated with acacetin (50 mg/kg) for 10 days. All treatments were done consequence daily and intraperitoneally. Histological and biochemical factors to male reproductive and urinary systems were assayed. Only in cisplatin exposed group, significant differences were seen with the others. So that, some reproductive criteria were significantly decreased; serum levels of follicle-stimulating hormone, luteinizing hormone, testosterone, sperm parameters, the diameters of seminiferous tubules, Johnsen's score and from the urinary system; renal corpuscles' space, Mg2+ concentration (p < .01). Moreover, significant increases were seen in serum levels of tumour necrosis factor-alpha, Blood urea nitrogen, creatinine, testis myeloperoxidase activity and tumour necrosis factor-alpha expression, kidney histological damage and renal corpuscle diameter (p < .01). Cisplatin exposure disrupts histological and functional characteristics of either male reproductive or urinary systems, suggesting by inflammation-promoting. Acacetin does not induce any harmful impact on these two systems and could be considered as a safe flavonoid by high dose and prolonged usage.

Effect of ketotifen fumarate on experimental autoimmune orchitis and torsion of the spermatic cord / 亚洲男科学杂志(英文版)

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.

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.

Hematogenous dissemination of Chlamydia muridarum from the urethra in macrophages causes testicular infection and sperm DNA damage†.

The incidence of Chlamydia infection, in both females and males, is increasing worldwide. Male infections have been associated clinically with urethritis, epididymitis, and orchitis, believed to be caused by ascending infection, although the impact of infection on male fertility remains controversial. Using a mouse model of male chlamydial infection, we show that all the major testicular cell populations, germ cells, Sertoli cells, Leydig cells, and testicular macrophages can be productively infected. Furthermore, sperm isolated from vas deferens of infected mice also had increased levels of DNA damage as early as 4 weeks post-infection. Bilateral vasectomy, prior to infection, did not affect the chlamydial load recovered from testes at 2, 4, and 8 weeks post-infection, and Chlamydia-infected macrophages were detectable in blood and the testes as soon as 3 days post-infection. Partial depletion of macrophages with clodronate liposomes significantly reduced the testicular chlamydial burden, consistent with a hematogenous route of infection, with Chlamydia transported to the testes in infected macrophages. These data suggest that macrophages serve as Trojan horses, transporting Chlamydia from the penile urethra to the testes within 3 days of infection, bypassing the entire male reproductive tract. In the testes, infected macrophages likely transfer infection to Leydig, Sertoli, and germ cells, causing sperm DNA damage and impaired spermatogenesis.