Monocytes expressing activin A and CCR2 exacerbate chronic testicular inflammation by promoting immune cell infiltration.

STUDY QUESTION: Does the chemokine/chemokine receptor axis, involved in immune cell trafficking, contribute to the pathology of testicular inflammation and how does activin A modulate this network? SUMMARY ANSWER: Testicular chemokines and their receptors (especially those essential for trafficking of monocytes) are elevated in orchitis, and activin A modulates the expression of the chemokine/chemokine receptor network to promote monocyte/macrophage and T cell infiltration into the testes, causing extensive tissue damage. WHAT IS KNOWN ALREADY: The levels of CC motif chemokine receptor (CCR)2 and its ligand CC motif chemokine ligand (CCL)2 are increased in experimental autoimmune orchitis (EAO) compared with healthy testes, and mice deficient in CCR2 are protected from EAO-induced tissue damage. Activin A induces CCR2 expression in macrophages, promoting their migration. Moreover, there is a positive correlation between testicular activin A concentration and the severity of autoimmune orchitis. Inhibition of activin A activity by overexpression of follistatin (FST) reduces EAO-induced testicular damage. STUDY DESIGN, SIZE, DURATION: EAO was induced in 10-12-week-old male C57BL/6J (wild-type; WT) and B6.129P2-Ccr2tm1Mae/tm1Mae (Ccr2-/-) mice (n = 6). Adjuvant (n = 6) and untreated (n = 6) age-matched control mice were also included. Testes were collected at 50 days after the first immunization with testicular homogenate in complete Freund's adjuvant. In another experimental setup, WT mice were injected with a non-replicative recombinant adeno-associated viral vector carrying a FST315-expressing gene cassette (rAAV-FST315; n = 7-9) or an empty control vector (n = 5) 30 days prior to EAO induction. Appropriate adjuvant (n = 4-5) and untreated (n = 4-6) controls were also examined. Furthermore, human testicular biopsies exhibiting focal leukocytic infiltration and impaired spermatogenesis (n = 17) were investigated. Biopsies showing intact spermatogenesis were included as controls (n = 9). Bone-marrow-derived macrophages (BMDMs) generated from WT mice were treated with activin A (50 ng/ml) for 6 days. Activin-A-treated or untreated BMDMs were then co-cultured with purified mouse splenic T cells for two days to assess chemokine and cytokine production. PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative real-time PCR (qRT-PCR) was used to analyze the expression of chemokines in total testicular RNA collected from mice. Immunofluorescence staining was used to detect activin A, F4/80, and CD3 expression in mouse testes. The expression of chemokine/chemokine-receptor-encoding genes was examined in human testicular biopsies by qRT-PCR. Correlations between chemokine expression levels and either the immune cell infiltration density or the mean spermatogenesis score were analyzed. Immunofluorescence staining was used to evaluate the expression of CD68 and CCR2 in human testicular biopsies. RNA isolated from murine BMDMs was used to characterize these cells in terms of their chemokine/chemokine receptor expression levels. Conditioned media from co-cultures of BMDMs and T cells were collected to determine chemokine levels and the production of pro-inflammatory cytokines tumor necrosis factor (TNF) and interferon (IFN)-γ by T cells. MAIN RESULTS AND THE ROLE OF CHANCE: Induction of EAO in the testes of WT mice increased the expression of chemokine receptors such as Ccr1 (P < 0.001), Ccr2 (P < 0.0001), Ccr3 (P < 0.0001), Ccr5 (P < 0.0001), CXC motif chemokine receptor (Cxcr)3 (P < 0.01), and CX3C motif chemokine receptor (Cx3cr)1 (P < 0.001), as well as that of most of their ligands. Ccr2 deficiency reversed some of the changes associated with EAO by reducing the expression of Ccr1 (P < 0.0001), Ccr3 (P < 0.0001), Ccr5 (P < 0.01), Cxcr3 (P < 0.001), and Cx3cr1 (P < 0.0001). Importantly, the biopsies showing impaired spermatogenesis and concomitant focal leukocytic infiltration exhibited higher expression of CCL2 (P < 0.01), CCR1 (P < 0.05), CCR2 (P < 0.001), and CCR5 (P < 0.001) than control biopsies with no signs of inflammation and intact spermatogenesis. The gene expression of CCR2 and its ligand CCL2 correlated positively with the immune cell infiltration density (P < 0.05) and negatively with the mean spermatogenesis score (P < 0.001). Moreover, CD68+ macrophages expressing CCR2 were present in human testes with leukocytic infiltration with evidence of tubular damage. Treatment of BMDMs, as surrogates for testicular macrophages, with activin A increased their expression of Ccr1, Ccr2, and Ccr5 while reducing their expression of Ccl2, Ccl3, Ccl4, Ccl6, Ccl7 Ccl8, and Ccl12. These findings were validated in vivo, by showing that inhibiting activin A activity by overexpressing FST in EAO mice decreased the expression of Ccr2 (P < 0.05) and Ccr5 (P < 0.001) in the testes. Interestingly, co-culturing activin-A-treated BMDMs and T cells reduced the levels of CCL2 (P < 0.05), CCL3/4 (P < 0.01), and CCL12 (P < 0.05) in the medium and attenuated the production of TNF (P < 0.05) by T cells. The majority of cells secreting activin A in EAO testes were identified as macrophages. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: BMDMs were used as surrogates for testicular macrophages. Hence, results obtained from the in vitro experiments might not be fully representative of the situation in the testes in vivo. Moreover, since total RNA was extracted from the testicular tissue to examine chemokine expression, the contributions of individual cell types as producers of specific chemokines may have been overlooked. WIDER IMPLICATIONS OF THE FINDINGS: Our data indicate that macrophages are implicated in the development and progression of testicular inflammation by expressing CCR2 and activin A, which ultimately remodel the chemokine/chemokine receptor network and recruit other immune cells to the site of inflammation. Consequently, inhibition of CCR2 or activin A could serve as a potential therapeutic strategy for reducing testicular inflammation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the International Research Training Group in 'Molecular pathogenesis on male reproductive disorders', a collaboration between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK1871/1-2) funded by the Deutsche Forschungsgemeinschaft and Monash University, a National Health and Medical Research Council of Australia Ideas Grant (1184867), and the Victorian Government's Operational Infrastructure Support Programme. The authors declare no competing financial interests.

Varicocele and testicular cord torsion: immune testicular microenvironment imbalance.

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

Salidroside Ameliorates Furan-Induced Testicular Inflammation in Relation to the Gut-Testis Axis and Intestinal Apoptosis.

Furan is a heat-induced food contaminant, and it causes damage to visceral organs, including the testis. To determine the mechanism of the damage to the testis, a mouse model treated with furan (8 mg/kg bw/day) and salidroside (SAL, 10/20/40 mg/kg bw/day) was established, and levels of testicular functional markers and changes of morphology were investigated in furan-induced mice treated with SAL. The change in related proteins and genes suggested that SAL restored the furan-mediated leaky tight junction and triggered the TLR4/MyD88/NF-κB pathway and NLRP3 inflammasome together with inflammation. To find out the gut-testis axis, microbiota PICRUSt analysis and correlation analysis were conducted to investigate the core microbiota and metabolites. The endoplasmic reticulum stress (ERS)-related key protein levels and the result of transmission electron microscopy suggested that SAL inhibited the furan-induced intestinal ERS. The result of TUNEL and levels of apoptosis-related proteins suggested that furan-induced intestinal apoptosis was alleviated by SAL. Collectively, SAL inhibited furan-induced ERS-mediated intestinal apoptosis through modulation of intestinal flora and metabolites, thus strengthening the gut barrier. It inhibited LPS from entering the circulatory system and suppressed the testicular TLR4/MyD88/NF-κB pathway and NLRP3 inflammasome, which alleviated testicular inflammation.

Chronic alcohol-induced dysbiosis of the gut microbiota and gut metabolites impairs sperm quality in mice.

Studies have indicated that the ethanol exposure impairs the gut microbiota, At the same time, high levels of alcohol exposure damage sperm in mice. However, whether the gut microbiota is involved in mediating the effects of alcohol on sperm quality remains unclear. This study aimed to assess the effect of chronic alcohol consumption on intestinal microbiota in mice and analyze the potential pathophysiological effect of altered intestinal microbiota on sperm quality. We established a mouse model of chronic alcohol consumption by allowing male C57 mice to freely ingest 10% ethanol for 10 weeks, and collected the fecal microbiota of the male mice in the chronic drinking group (alcohol) and the control group (control) and transplanted the specimens into the transplant groups (the alcohol-fecal microbiota transplantation [FMT] group and the control-FMT group). Sperm quality was significantly decreased in the alcohol-FMT group compared with the control-FMT group. Gut microbiota analysis revealed that the abundance of 11 operational taxonomic units (OTUs) was altered in the alcohol-FMT group. Nontargeted metabolomics identified 105 differentially altered metabolites, which were mainly annotated to amino acids, lipids, glycerophosphoethanolamine, organic oxygenic compounds, organic acids and their derivatives, steroids, and flavonoids. In particular, the oxidative phosphorylation pathway, which is the key to spermatogenesis, was significantly enriched in the alcohol-FMT group. Moreover, compared with the control-FMT group, the alcohol-FMT group presented significantly higher serum endotoxin and inflammatory cytokine levels, with more pronounced T cell and macrophage infiltration in the intestinal lamina propria and elevated levels of testicular inflammatory cytokines. In addition, RNA sequencing showed significant differences in the expression of testis-related genes between the alcohol-FMT group and the control-FMT group. In particular, the expression of genes involved in gamete meiosis, testicular mitochondrial function, and the cell division cycle was significantly reduced in alcohol-FMT mice. In conclusion, these findings indicated that intestinal dysbiosis induced by chronic alcohol consumption may be an important factor contributing to impaired sperm quality. Chronic alcohol consumption induces intestinal dysbiosis, which then leads to metabolic disorders, elevated serum endotoxin and inflammatory cytokine levels, testicular inflammation, abnormal expression of related genes, and ultimately, impaired sperm quality. These findings are potentially useful for the treatment of male infertility.

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.

Hadh deficiency induced oligoasthenoteratozoospermia through the TNF-α/Bcl-2 pathway in male mice.

The process of spermatogenesis is a complex and delicate process that is still not fully understood. In this study, we examined the role of fatty acid oxidase 3-hydroxy acyl CoA dehydrogenase (HADH) in maintaining normal spermatogenesis in mice. In male mice, ablation of the Hadh gene using CRISPR/Cas9 technology arrested spermatocyte meiosis, increased multinucleated giant germ cells and vacuoles in seminiferous tubules, and accompanied with acrosomal dysplasia. Hadh-/- male mice showed the typical features of oligoasthenoteratozoospermia (OAT), including decreased sperm concentration and motility and increased sperm abnormalities. Next, we explored the molecular events in the testes of the mutant mice. We found fatty acids accumulated in the testis of Hadh-/- mice. And also, inflammatory factors TNF-α, IL-1ß, and IL-6 were significantly increased, apoptosis-related protein Bcl-2 was decreased, and Bax and cleaved-Caspase3 were increased in Hadh-/- male mice testis. After using etanercept, a specific inhibitor of TNF-α, testis injury caused by Hadh knockout was significantly alleviated, the sperm quality and motility were improved, and germ cell apoptosis was reduced. So our study demonstrated that Hadh deletion caused an increase in fatty acids. The accumulated fatty acids further induced testicular inflammation and germ cell apoptosis through the TNF-α/Bcl-2 signaling pathway, finally resulting in OAT in the Hadh-/- mice. Inhibiting TNF-α may be used as a new treatment approach for testicular inflammation and OAT.

Mechanism of Inflammatory Associated Impairment of Sperm Function, Spermatogenesis and Steroidogenesis.

Infection and inflammation are relevant entities of male reproductive disorders that can lead to sub-/infertility. Associated damage of the testis of affected men and in rodent models include leukocytic infiltration, edema formation, fibrosis, germ cell loss and reduced androgen levels. Negative effects on spermatogenesis are thought to be elicited by oxidative stress sustained mostly by increased levels of ROS and pro-inflammatory cytokines. Under normal conditions these cytokines have physiological functions. However, increased levels as seen in inflammation and infection, but also in obesity and cancer are harmful for germ cells and impair steroidogenesis. As a summary, there is mounting evidence that the activation of inflammatory pathways is a rather common feature in various forms of male testicular disorders that extends beyond established infectious/inflammatory cues. This mini review will focus on relevant entities and the mechanisms of how a dysbalance of local testicular factors contributes to disturbances of spermatogenesis and steroidogenesis.

NLRP3 inflammasome is involved in testicular inflammation induced by lipopolysaccharide in mice.

PROBLEM: Systemic inflammation induced by infection, which is associated with testicular inflammation, predisposes males to subfertility. Recently, the nucleotide-binding oligomerization domain, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) inflammasome was identified as a key mediator of inflammation, and excessive activation of the NLRP3 inflammasome was shown to contribute to the pathogenesis of a wide variety of diseases. However, the mechanisms underlying infectious inflammation in the testis remain unclear. We investigated the effect of lipopolysaccharide (LPS)-induced systemic inflammation on the role of the NLRP3 inflammasome in murine testes. METHOD OF STUDY: We performed in vivo and in vitro studies using an LPS-induced model of NLRP3 inflammasome activation and testicular inflammation. RESULTS: Intraperitoneal administration of LPS significantly impaired sperm motility in the epididymis of wild type (WT) and NLRP3-knockout (KO) mice. LPS administration stimulated interleukin (IL)-1ß production and secretion in the testes of WT mice, and these adverse effects were improved in the testes of NLRP3-KO mice. LPS administration also stimulated neutrophil infiltration as well as its chemoattractant C-C motif chemokine ligand 2 (CCL2) in WT testes, which were suppressed in NLRP3-KO testes. In in vitro cell culture, treatment with LPS and NLRP3 inflammasome activation significantly induced IL-1ß and CCL2 secretion from WT but not NLRP3-KO testicular cells. CONCLUSIONS: Taken together, our results suggest that testicular cells have the potential to secrete IL-1ß and CCL2 in an NLRP3 inflammasome-dependent manner and that these cytokines from the testis may further exacerbate testicular function, resulting in subfertility during infectious diseases.

Anti-testicular Inflammation Mechanism of Wuzi Yanzongwan Based on Network Pharmacology / 中国实验方剂学杂志

ObjectiveTo explore the anti-testicular inflammation mechanism of Wuzi Yanzongwan through network pharmacological analysis and experimental verification. MethodThe active ingredients of Wuzi Yanzongwan were searched in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）， and their targets were predicted via SwissTargetPredicition. GeneCards and Online Mendelian Inheritance in Man （OMIM） were used to obtain the known targets for the treatment of testicular inflammation. STRING 11.0 was employed to construct the protein-protein interaction （PPI） network， and Cytoscape 3.7.1 to construct the “herbal medicine-active ingredient-target” network of Wuzi Yanzongwan and “active ingredient-disease target” network. Metascape was used for gene ontology （GO） annotation and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment of signaling pathways. ResultA total of 72 active ingredients and 624 potential targets of Wuzi Yanzongwan were screened out， and 196 key targets were identified for the treatment of testicular inflammation by Wuzi Yanzongwan. The results of GO annotation showed that the anti-testicular inflammation targets of Wuzi Yanzongwan were involved in the biological processes such as the response to molecule of bacterial origin， the positive regulation of response to external stimulus， and the response to extracellular stimulus. KEGG pathway enrichment revealed that the major pathways associated with the treatment （P<0.01） included the pathways in cancer， epidermal growth factor receptor （EGFR） tyrosine kinase inhibitor resistance， transcriptional misregulation in cancer， calcium signaling pathway， apelin signaling pathway， and NOD-like receptor signaling pathway. Wuzi Yanzongwan may exert the anti-testicular inflammation effect by inhibiting the activation of the NLR family pyrin domain-containing 3 （NLRP3） inflammasome pathway to alleviate the inflammatory response， which verifies the prediction results based on network pharmacology to a certain extent. ConclusionThe inhibitory effect of Wuzi Yanzongwan on testicular inflammation is multi-target， multi-pathway， and multi-mechanism. The findings of this study provide evidence support for the clinical application of Wuzi Yanzongwan.

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.

Ameliorative effect of nerolidol on cyclophosphamide-induced gonadal toxicity in Swiss Albino mice: Biochemical-, histological- and immunohistochemical-based evidences.

Cyclophosphamide (CP) is commonly used as antineoplastic and immunosuppressant drug with noticeable gonadotoxic profile. Nerolidol (NER) is a sesquiterpene with potent antioxidant and anti-inflammatory properties. Thus, the present study was designed to explore its possible gonadal protective potential against cyclophosphamide-induced testicular, epididymal, seminal and spermatozoal toxicities. Animals were divided into five groups: control (normal saline for 14 days), treatment group (NER 200 and 400 mg/kg, p.o) for 14 days along with a single dose of cyclophosphamide (200 mg/kg, i.p) on 7th day, toxic and Per se groups (cyclophosphamide 200 mg/kg i.p) on 7th day and NER 400 mg/kg for 14 days respectively. Animals were sacrificed on the 15 day, and body weight, weight of reproductive organs, testosterone level, sperm count, biochemical parameters, histopathological and immunohistochemical studies were performed in the testes, epididymis and in the serum. CP administration induced oxidative stress, nitrative stress, inflammation, reduced testosterone level, sperm count, increased expression of MPO and caused histological aberrations in the testes, epididymis and seminal vesicles. CP caused reduced sperm count, sperm motility and testosterone level which got reversed upon treatment with nerolidol in a dose-dependent manner. Nerolidol thus acted as a gonadoprotective molecule and prevented the gonadotoxicity of CP.

Protective mechanism of saponins of Panax Japonicus on high-fat diet induced reproductive dysfunction in mice / 中国药理学通报

Aim To investigate the effects of saponins of Panax japonicus (SPJ) on high-fat diet induced reproductive dysfunction in mice and its underlying mechanisms. Methods Sperm viability and sperm count were assessed using hemocytometer and eosin Y staining under light microscope. Testicular morphology was evaluated by HE staining. Germ cell apoptosis was detected by deoxyribonucleotide terminal transferasemediated (TUNEL). The relative protein expression levels of p-NF-κB, TNF-α and IL-1β were detected by Western blot. The numbers of testicular macrophages were assessed by immunofluorescence. Results Compared with high-fat model group, SPJ significantly reduced the body mass of mice, significantly increased the weight and index of testis and epididymis, elevated sperm viability and sperm count, improved the morphology of seminiferous tubules, and reduced germ cell the apoptosis. In addition, SPJ markedly decreased the relative protein expression levels of p-NF-κB, TNF-α and IL-1β, and reduced the numbers of testicular macrophages. Conclusions SPJ significantly improves high-fat diet induced reproductive dysfunction in male mice, and its mechanism may be related to the reduction of macrophage infiltration and inhibition of NF-ΚB pathway-mediated testicular inflammation.

[Establishment of animal models of orchitis: Advances in studies].

Male infertility is becoming a concern of the world. Studies show that testicular inflammation is closely related to male infertility, which often manifests itself in low sperm count and motility and even the loss of fertility. In recent years, testicular inflammation-induced male infertility is arousing more and more attention, which adds to the significance of its study. It is imperative to establish stable and reliable animal models for further research on orchitis-induced spermatogenetic dysfunction and the mechanisms of spermatogenesis. This article presents an overview of recent studies on the establishment of animal models of orchitis to provide some reference for researchers in the relevant fields.

Perspective in Pediatric Pathology, Chapter 24. Testicular Inflammatory Processes in Pediatric Patients.

Acute scrotal pain in children represents a major diagnostic and therapeutic challenge. An important initial differentiation should be made between epididymitis and other processes that cause acute scrotal pain, such as testicular torsion and tumor. Infectious agents disseminating through the blood flow can damage the testis by causing orchitis. On the other hand, infections ascending via spermatic pathways typically lead to epididymitis [ 1 ].

Predomination of IL-17-producing tryptase-positive/chymase-positive mast cells in azoospermic chronic testicular inflammation.

Chronic testicular inflammation and infection have been regarded as important factors in the pathogenesis of azoospermia. As key effector cells in innate and adaptive immune system, mast cells (MCs) were observed in inflammation and autoimmune disease. Furthermore, increased expression of tryptase-positive MCs has been reported in testicular disorders associated with male infertility/subfertility. However, little is known about the potential relationship between MCs and chronic testicular inflammation in azoospermic patients. Moreover, the preferential expression of MCs' subtypes in testis of these patients is still far from being understood. Thus, this study aimed to investigate characteristics of testicular MCs as well as their subtypes in azoospermic men with chronic testicular inflammation (AZI, n = 5) by immunohistochemical techniques. Our results showed significant increase of MCs in AZI, and more importantly, considerable numbers of tryptase-positive/chymase-positive MCs could also be demonstrated in AZI, when compared to control groups representing azoospermia without chronic testicular inflammation (AZW, n = 5) and normal spermatogenesis (NT, n = 5) respectively. Most interestingly, immunofluorescence staining revealed autoimmune-associated interleukin (IL)-17-producing MCs in AZI, whereas co-expression of MC markers with tumour necrosis factor (TNF)-α, IL-10 and IL-1ß could not be detected. In conclusion, AZI is associated with significant increase of tryptase-positive/chymase-positive MCs expressing IL-17, and these MCs might contribute to the pathogenesis of AZI.

Experimental Escherichia coli epididymitis in rats: assessment of testicular involvement in a long-term follow-up.

The objective of this study was to investigate spermatogenesis and testicular inflammation in a rat model of unilateral Escherichia coli epididymitis in a long-term follow-up. Unilateral epididymitis was induced in 30 Sprague-Dawley rats by injecting E. coli into the right ductus deferens. Oral antimicrobial treatment with sparfloxacin (50 mg kg(-1) body weight/7 days) was administered in half of the animals 24 h after infection. Five treated and five untreated rats were killed at 2 weeks, 3 months and 6 months after infection. Spermatogenesis was investigated using a histological semi-quantitative score. The presence of inflammatory cells (B- and T lymphocytes, macrophages and granulocytes) in the testicular tissues was evaluated by immunohistochemistry. The testes were sterile at all times. Over the course of 6 months, spermatogenesis underwent significant incremental impairment on the inoculated side as compared to the contralateral side (P < 0.001). However, overall spermatogenesis scores were not significantly different between treated and untreated animals (P > 0.3 at each time point). Finally, loss of testicular architecture on the inoculated side was not associated with any cellular inflammatory response. Thus, adjuvant therapies need to be studied, and research is necessary on how to prevent deterioration of testicular function in bacterial epididymitis.

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

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

Autoantibodies against protein disulfide isomerase ER-60 are a diagnostic marker for low-grade testicular inflammation.

STUDY QUESTION: Is there a non-invasive biomarker for the diagnosis of testicular inflammatory lesions? SUMMARY ANSWER: In sera from infertile azoospermic patients with histologically confirmed low-grade testicular inflammation, significantly elevated titers of autoantibodies against disulfide isomerase family A, member 3 (ER-60) were found. WHAT IS KNOWN ALREADY: Infection and inflammation of the genital tract are supposed to be responsible for up to 15% of cases among infertile males. However, specific seminal or serological markers are not available to assess subacute or chronic inflammatory conditions in the testis. STUDY DESIGN, SIZE, DURATION: This study consisted of the identification of autoantibodies for testicular antigens in sera of patients with low-grade testicular inflammation, validation of candidates, development of an ELISA for the most promising target antigen and measurement of autoantibodies titers in healthy normozoospermic men (n = 20); male blood donors (n = 14); men with impaired semen quality without (n = 14) or with (n = 26) symptoms of genital tract infection/inflammation; azoospermic men with histologically confirmed testicular inflammatory lesions (n = 16); men after pharmacotherapy of genital tract infection/inflammation (n = 15) and men with acute epididymo-orchitis (n = 30). PARTICIPANTS/MATERIALS, SETTING, METHODS: Proteins in lysates of normal testicular tissue were separated by high-resolution 2D gel electrophoresis and probed with sera of 13 patients with histologically confirmed chronic testicular inflammation. There were 14 proteins that immunoreacted with a majority of these sera and could be identified by mass spectrometry. Of these 14 proteins, disulfide isomerase family A, member 3 (ER-60), transferrin and chaperonin containing TCP1 complex, subunit 5 (epsilon) (CCT5) were considered as specific. Since ER-60 reacted with 92% of patient sera, an ER-60-autoantibody ELISA was developed. MAIN RESULTS AND THE ROLE OF CHANCE: The newly established ELISA detected significantly elevated titers of autoantibodies against ER-60 in the sera from infertile men with histologically confirmed chronic testicular inflammation (median 8.6; P < 0.01) compared with the control groups. Moreover, elevated levels of anti-ER-60 titers were detected in patients suffering from acute epididymo-orchitis (median 3.3; P < 0.05) as compared with healthy normozoospermic men (median 2.13; P < 0.001), male blood donors with unknown fertility status (median 2.72; P < 0.01), patients with impaired semen quality but no infection/inflammation (median 2.59; P < 0.001) and patients with symptoms of genital tract infections and/or inflammation (median 2.18; P < 0.001). Significantly lower levels of anti-ER-60 antibodies were measured in sera from patients after application of anti-inflammatory pharmacotherapy (median 1.9; P < 0.01) compared with those with histologically confirmed chronic testicular inflammation. The cut-off value of the assay was set to 6.6 U/ml based on a calculated sensitivity of 100% and a specificity of 81.2%. LIMITATIONS, REASONS FOR CAUTION: The results obtained in this study showed statistically significant elevated titers of ER-60 antibodies in sera from patients with histologically confirmed testicular inflammatory lesions and from a few patients with acute epididymo-orchitis. However, the number of serum samples tested was limited. Severe testicular damage seen in azoospermic patients could represent a bias towards ER-60 reactivity, while the assay does not allow for different etiologies of the lesions to be distinguished. Due to ethical reasons, the prevalence of testicular inflammatory lesions among controls and non-azoospermic men cannot be studied at the histological level. WIDER IMPLICATIONS OF THE FINDINGS: Measurement of ER-60 autoantibody titers in serum could be a novel non-invasive marker for the diagnosis of asymptomatic testicular inflammation causing male fertility disturbances. STUDY FUNDING/COMPETING INTERESTS: This study was supported by a grant of the Deutsche Forschungsgemeinschaft (ME 1323/4-4) and the Translational Science Fund (Wirtschafts-und Strukturbank Hessen-WI Bank). M.F., A.P., W.W., H.-C.S. and A.M. are supported by the LOEWE focus group 'MIBIE' (Male infertility during infection and inflammation). The ER-60 ELISA is protected by a patent to the Justus-Liebig-University of Giessen with A.M. and M.F. as inventors (patent no. DE 10 2008 053 503). T.Z. as employee of the DRG Company was responsible for the ELISA development.

Synergistic effect of interferon-gamma and tumor necrosis factor-alpha on coxsackievirus and adenovirus receptor expression: an explanation of cell sloughing during testicular inflammation in mice.

Coxsackievirus and adenovirus receptor (CAR) is a junction molecule that expresses on Sertoli and germ cells. It mediates Sertoli-germ cell adhesion and facilitates migration of preleptotene/leptotene spermatocytes across the blood-testis barrier, suggesting that CAR-based cell adhesion and migration are crucial for spermatogenesis. Interferon-gamma (IFNG) and tumor necrosis factor alpha (TNF) are two major cytokines that are elevated during testicular inflammation and cause reduced fertility. We investigated the mechanism by which IFNG and TNF exert their disruptive effects on testicular cell adhesion. We have demonstrated that combined treatment with IFNG and TNF (IFNG+TNF) exerts a synergistic effect by downregulating CAR mRNA and protein levels. Immunofluorescence staining revealed that IFNG+TNF treatment effectively removes CAR from the site of cell-cell contact. Using inhibitor and co-immunoprecipitation, we confirmed that IFNG+TNF mediates CAR protein degradation via ubiquitin-proteasome and NFKB pathways. Blockage of ubiquitin-proteasome pathway significantly inhibits CAR degradation, as indicated by the reappearance of CAR at the site of cell-cell contact. Additionally, IFNG+TNF reduces CAR mRNA via transcriptional regulation. Mutational studies have shown that IFNG+TNF-induced CAR repression is achieved by suppression of the basal transcription. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays further confirmed that IFNG+TNF treament not only inhibits binding of the basal transcription factors but also promotes binding of NFKB subunits and Sp1 (negative regulators) to the CAR promoter region. Taken together, IFNG+TNF treatment significantly downregulates CAR expression, which provides an explanation of how cell sloughing in the epithelium mediates, by loss of CAR-based cell adhesion, during testicular inflammation.