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.

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.

Reproductive biology research down under: highlights from the Australian and New Zealand Annual Meeting of the Society for Reproductive Biology, 2021.

Against the backdrop of a global pandemic, the Society for Reproductive Biology (SRB) 2021 meeting reunited the Australian and New Zealand reproductive research community for the first time since 2019 and was the first virtual SRB meeting. Despite the recent global research disruptions, the conference revealed significant advancements in reproductive research, the importance of which span human health, agriculture, and conservation. A core theme was novel technologies, including the use of medical microrobots for therapeutic and sperm delivery, diagnostic hyperspectral imaging, and hydrogel condoms with potential beyond contraception. The importance of challenging the contraceptive status quo was further highlighted with innovations in gene therapies, non-hormonal female contraceptives, epigenetic semen analysis, and in applying evolutionary theory to suppress pest population reproduction. How best to support pregnancies, particularly in the context of global trends of increasing maternal age, was also discussed, with several promising therapies for improved outcomes in assisted reproductive technology, pre-eclampsia, and pre-term birth prevention. The unique insights gained via non-model species was another key focus and presented research emphasised the importance of studying diverse systems to understand fundamental aspects of reproductive biology and evolution. Finally, the meeting highlighted how to effectively translate reproductive research into policy and industry practice.

Evaluation of microRNA expression in a sheep model for lung fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibroproliferative disorder that has one of the poorest prognoses amongst interstitial lung diseases. Recently, the finding of aberrant expression levels of miRNAs in IPF patients has drawn significant attention to the involvement of these molecules in the pathogenesis of this disease. Clarification of the differential expression of miRNAs in health and disease may identify novel therapeutic strategies that can be employed in the future to combat IPF. This study evaluates the miRNA expression profiles in a sheep model for lung fibrosis and compares them to the miRNA profiles of both IPF patients and the mouse bleomycin model for pulmonary fibrosis. Pathway enrichment analyses were performed on differentially expressed miRNAs to illustrate which biological mechanisms were associated with lung fibrosis. RESULTS: We discovered 49 differentially expressed miRNAs in the sheep fibrosis model, in which 32 miRNAs were significantly down regulated, while 17 miRNAs were significantly upregulated due to bleomycin-induced lung injury. Moreover, the miRNA families miR-29, miR-26, miR-30, let-7, miR-21, miR-19, miR-17 and miR-199 were aberrantly expressed in both sheep and mouse models, with similar differential miRNAs expression observed in IPF cases. Importantly, 18 miRNAs were aberrantly expressed in both the sheep model and IPF patients, but not in mice. CONCLUSION: Together with pathway enrichment analyses, these results show that the sheep model can potentially be used to characterize previously unrecognized biological pathways associated with lung fibrosis.

Region-specific immune responses to autoimmune epididymitis in the murine reproductive tract.

Epididymitis is a common pathology of the male reproductive tract, potentially leading to infertility. Studies on bacterial epididymitis indicate that the cauda epididymis is more susceptible to inflammatory damage than the caput. These regional differences in immunoregulation are further investigated using an experimental autoimmune epididymo-orchitis model. Adult mice were immunized against testicular antigens and tissues were collected at 30 and 50 days following the first immunization. Epididymitis developed progressively; 70% of the mice developed disease at 30 days after the initial immunization and 93% at 50 days. Epididymitis was characterized by epithelial damage, immune cell infiltrates and fibrosis in the cauda, with minimal changes in the corpus, while the caput was unaffected. The incidence of epididymitis was greater than that of orchitis but similar to vasitis. The severity of epididymitis was positively correlated with the orchitis severity. Expression of key genes implicated in epididymal immunoregulation, inflammation and fibrosis, such as Ido1, Tnf, Tgfb1, Ccl2, Il1b, Il10, Cx3cl1 and Col1a1, was unchanged in the caput but increased in proportion to damage severity in the cauda at 50 days. Activin receptor mRNA expression in the cauda was negatively correlated with disease severity. These data suggest that the cauda is highly susceptible to inflammatory damage following an autoimmune challenge but the caput is minimally affected. This may be because the cauda is required to combat ascending infections through a robust inflammatory response, while the caput provides a more tolerogenic environment in order to protect the auto-antigenic sperm released from the testis.

Dexamethasone improves therapeutic outcomes in a preclinical bacterial epididymitis mouse model.

STUDY QUESTION: Can dexamethasone improve infertility-related cauda epididymidal tissue damage caused by bacterial epididymitis? SUMMARY ANSWER: Dexamethasone in addition to anti-microbial treatment effectively reduces long-term deleterious epididymal tissue damage by dampening the host's adaptive immune response. WHAT IS KNOWN ALREADY: Despite effective anti-microbial treatment, ~40% of patients with epididymitis experience subsequent sub- or infertility. An epididymitis mouse model has shown that the host immune response is mainly responsible for the magnitude of epididymal tissue damage that is fundamentally causative of the subsequent fertility issues. STUDY DESIGN, SIZE, DURATION: Bacterial epididymitis was induced in male mice by using uropathogenic Escherichia coli (UPEC). From Day 3 after infection onwards, mice were treated with daily doses of levofloxacin (20 mg/kg, total n = 12 mice), dexamethasone (0.5 mg/kg, total n = 9) or both in combination (total n = 11) for seven consecutive days. Control animals were left untreated, i.e. given no interventional treatment following UPEC infection (total n = 11). Half of the animals from each group were killed either at 10 or 31 days post-infection. PARTICIPANTS/MATERIALS, SETTING, METHODS: A mouse model of induced bacterial epididymitis was applied to adult male C57BL/6J mice. At the respective endpoints (10 or 31 days post-infection), epididymides were collected. Effectiveness of antibiotic treatment was assessed by plating of epididymal homogenates onto lysogeny broth agar plates. Overall tissue morphology and the degree and nature of tissue damage were assessed histologically. Quantitative RT-PCR was used to assess local cytokine transcript levels. Blood was drawn and serum analysed for systemic IgG and IgM levels by ELISA. In addition, correlation analyses of clinical data and serum-analyses of IgG and IgM levels in patients with epididymitis were performed. MAIN RESULTS AND THE ROLE OF CHANCE: The addition of dexamethasone to the standard anti-microbial treatment did not further worsen epididymal tissue integrity. In fact, an obviously dampened immune response and reduced tissue reaction/damage was observed at both 10 and 31 days post-infection following combined treatment. More specifically, epididymal duct continuity was preserved, enabling sperm transit. In contrast, in untreated or antibiotic-treated animals, damage of the epididymal duct and duct constrictions were observed, associated with a lack of cauda spermatozoa. In line with the bacteriostatic/bactericidal effect of levofloxacin (alone as well as in combination), local cytokine transcript levels were significantly and similarly reduced in animals treated with levofloxacin alone (P < 0.01) or in combination with dexamethasone (P < 0.05) compared to UPEC-infected untreated animals. Interestingly, the addition of dexamethasone to the anti-microbial treatment induced a unique dampening effect on adaptive immunity, since systemic IgG and IgM levels as well as the pan-T cell marker CD3 were reduced at both 10 and 31 days post-infection. LIMITATIONS, REASONS FOR CAUTION: Breeding studies to address the fertility-protecting effect of the combined treatment were not possible in the experimental animals because the vas deferens was ligated (model specific). WIDER IMPLICATIONS OF THE FINDINGS: Whereas innate immunity is necessary and involved in acute bacterial clearance, adaptive immunity seems to be responsible for long-term, subclinical immunological activities that may negatively affect the pathogenesis of bacterial epididymitis even after effective bacterial eradication. These effects can be reduced in mice by the additional treatment with dexamethasone. This immunological characteristic of bacterial epididymitis shows similarities to the Jarisch-Herxheimer reaction known from other types of bacterial infection. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by grants from the Deutsche Forschungsgemeinschaft, Monash University and the Medical Faculty of Justus-Liebig University to the International Research Training Group on 'Molecular pathogenesis of male reproductive disorders' (GRK 1871). R.W., K.L.L. and M.P.H. were supported by grants from the National Health and Medical Research Council of Australia (ID1079646, ID1081987, ID1020269 and ID1063843) and by the Victorian Government's Operational Infrastructure Support Program. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: No clinical trial involved.

Comparative analysis of activins A and B in the adult mouse epididymis and vas deferens.

Activin A regulates testicular and epididymal development, but the role of activin B in the epididymis and vas deferens is unknown. Mouse models with reduced activin A (Inhba+/- and InhbaBK/+), or its complete absence (InhbaBK/BK), were investigated to identify specific roles of activins in the male reproductive tract. In 8-week-old Inhba+/- mice, serum activin A decreased by 70%, with a 50% reduction of gene expression and protein in the testis, epididymis and vas deferens. Activin B and the activin-binding protein, follistatin, were similar to wild-type. Testis weights were slightly reduced in Inhba+/- mice, but the epididymis and vas deferens were normal, while the mice were fertile. Activin A was decreased by 70% in the serum, testis, epididymis and vas deferens of InhbaBK/+ mice and was undetectable in InhbaBK/BK mice, but activin B and follistatin levels were similar to wild-type. In 6-week-old InhbaBK/BK mice, testis weights were 60% lower and epididymal weights were 50% lower than in either InhbaBK/+ or wild-type mice. The cauda epididymal epithelium showed infoldings and less intra-luminal sperm, similar to 3.5-week-old wild-type mice, but at 8 weeks, no structural differences in the testis or epididymis were noted between InhbaBK/BK and wild-type mice. Thus, Inhbb can compensate for Inhba in regulating epididymal morphology, although testis and epididymal maturation is delayed in mice lacking Inhba Crucially, reduction or absence of activin A, at least in the presence of normal activin B levels, does not lead to major defects in the adult epididymis or vas deferens.

New aspects of activin biology in epididymal function and immunopathology.

The activins (A and B) and their binding protein, follistatin, play crucial roles in development, immunoregulation and inflammation throughout the body. In the male reproductive tract of the mouse, activin A and B production is largely confined to the initial segment and proximal caput of the epididymis and the efferent ducts, under normal conditions, with very low expression in the corpus, cauda and vas deferens. However, activin A protein is present throughout the epididymis and vas deferens and is largely associated with the epithelium and interstitial macrophages. Conversely, the activin-binding protein follistatin is produced in the distal epididymis, with very high expression in the vas deferens. Activin activity in the distal tract is inhibited by follistatin, and the activin-follistatin balance is important for regulating coiling of the duct during epididymal development. In further experiments, as described in this report, in situ hybridisation was used to localise activin A mRNA principally to cells in the periductal zone and interstitium in the efferent ducts and proximal caput. Activin B mRNA, on the other hand, was localised to periductal cells in the efferent ducts and proximal epididymis and, most notably, to epithelial cells in the initial segment. Activin A is implicated in the regulation of mononuclear phagocyte function and immune responses in the caput and stimulates the expression of the key immunoregulatory protein, indoleamine 2,3-dioxygenase in this region. Activin A production in the corpus and cauda increases dramatically during bacterial epididymitis in mice, promoting inflammation and fibrosis and causing damage to the epithelium and obstruction of the epididymal duct. Consequently, it appears that the activin-follistatin axis is crucial for maintaining normal epididymal structure and function, but disruption of this balance during inflammation has deleterious effects on male fertility. Follistatin has therapeutic potential in ameliorating the proinflammatory and profibrotic effects of activins.

Regulation of macrophage number and gene transcript levels by activin A and its binding protein, follistatin, in the testes of adult mice.

The cytokine activin A is expressed throughout testicular development and is a critical regulator of macrophage function, but its effects on the testicular macrophages are not well-defined. Macrophage distribution and gene transcript levels were examined in testes of adult mice with reduced levels of either activin A (Inhba+/-), or its binding protein, follistatin (TghFST315). Macrophages were identified using F4/80 immunohistochemistry and enumerated by morphometry. Transcript levels were measured in testis extracts by qRT-PCR and Fluidigm ™ analyses. Interstitial macrophages were twice as numerous as peritubular macrophages in Inhba+/- and TghFST315 mice and their littermate controls. Macrophage numbers were significantly reduced in all regions of the Inhba+/- testis, and the volume density of peritubular and subcapsular macrophages was significantly reduced compared to littermate controls (by 52.9% and 36.3% respectively). Transcripts encoding macrophage chemokines, Csf1 and Ccl2, and receptor Csf1r, were elevated (by 35%, 44% and 27% respectively) in Inhba+/- testes, but Cx3cl1 and their receptors, Cx3cr1 and Ccr2, were not altered. Transcripts encoding MHC class II antigens and the co-stimulatory molecule Cd86, also increased (by 32% and 60% respectively), but other co-stimulatory molecules Cd80 and Cd274, and the scavenger receptor Mrc1 (CD206), were unaffected. In the follistatin-deficient testes, macrophage numbers and most macrophage-specific transcripts were not significantly affected, but Mrc1 expression was reduced by 35%. These data indicate that activin A maintains macrophage numbers, but selectively inhibits the levels of key transcripts associated with macrophage antigen-presentation, recruitment and differentiation in the adult mouse testis.

Examination of testicular lumicrine regulation of activins and immunoregulatory genes in the epididymal caput.

BACKGROUND: Immunoregulatory genes encoding activin A (Inhba) and B (Inhbb), and indolamine 2,3-dioxygenase-1 (Ido1) are highly expressed in the murine caput epididymidis, which also has a network of intraepithelial mononuclear phagocytes. This environment is postulated to promote immunological tolerance to epididymal sperm. The factors regulating the immunoregulatory agents in the epididymal caput are poorly understood. OBJECTIVES: This study aimed to investigate the potential role of testicular lumicrine factors in regulating activin and other immune-related genes in the caput epididymidis. MATERIALS AND METHODS: The efferent ducts in adult C57/Bl6 mice were exposed and ligated bilaterally. Serum and tissues were collected seven days later. Animals with bilateral sham ligation and animals with no ligations (collectively referred to as the "intact" group) were used as controls. RESULTS: Pressure-induced seminiferous epithelial damage due to intratubular fluid accumulation was observed in all ligated testes. Testicular inhibin was significantly increased and testosterone was elevated in some animals following bilateral ligation, but serum testosterone, serum LH, and serum inhibin were normal. Ligation caused epithelial regression in the initial segment, with similar but less severe effects in other caput segments. Activin A staining by immunohistochemistry in the epithelium was reduced in bilateral ligation, particularly in the initial segment, with moderately reduced staining intensity in the rest of the caput. Inhba expression within the caput was not significantly affected by bilateral ligation, but Inhbb was reduced by more than 60%. Transcripts encoding the macrophage-specific receptor Cx3cr1 were significantly reduced following bilateral ligation, but other immune cell markers, Ido1, and inflammatory genes were unaffected. CONCLUSION: These data indicate that testicular lumicrine secretion regulates several genes that are preferentially expressed in the initial segment, but has marginal effects on genes such as those encoding activin A and IDO1, which are expressed more widely in the caput.

Activin over-expression in the testis of mice lacking the inhibin α-subunit gene is associated with androgen deficiency and regression of the male reproductive tract.

Regionalised interaction of the activins, follistatin and inhibin was investigated in the male reproductive tract of mice lacking the inhibin α-subunit (Inha-/-). Serum and intratesticular activin B, although not activin A and follistatin, were increased in Inha-/- mice at 25 days of age, but all three proteins were elevated at 56 days. None of these proteins were altered within the epididymis and vas deferens at either age. At 25 days, histology of the epididymis and vas deferens was similar to wild-type. At 56 days, the testis contained extensive somatic cell tumours, leading to Leydig cell regression and testosterone deficiency. The epididymis and vas deferens showed epithelial regression and increased prominence of the interstitial stroma. Immunoregulatory and fibrotic gene expression in the epididymis and vas deferens were unchanged. Thus, absence of the inhibin α-subunit has marginal effects on activins in the epididymis and vas deferens, and regression of these tissues is associated with androgen deficiency.