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

Can dexamethasone improve infertility-related cauda epididymidal tissue damage caused by bacterial epididymitis? Dexamethasone in addition to anti-microbial treatment effectively reduces long-term deleterious epididymal tissue damage by dampening the host's adaptive immune response. 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. 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. 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. 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. 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). 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. 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. No clinical trial involved.

Klein B ，Pant S ，Bhushan S ，Kautz J ，Rudat C ，Kispert A ，Pilatz A ，Wijayarathna R ，Middendorff R ，Loveland KL ，Hedger MP ，Meinhardt A ... -  《-》

Epididymitis: ascending infection restricted by segmental boundaries.

Is the regionalization of epididymitis related to epididymal segmentation? We show for the first time that luminal ascent of bacteria is strictly gated by epididymal segment boundaries, involving ductal constriction adjacent to the infected area. The epididymal duct is a continuous, unbranched tube, coiled into segments that are divided by connective tissue septa. Sonographic analysis indicates that swelling associated with epididymitis is predominant in the cauda region. Epididymal segmentation has never been investigated in the context of pathological alterations. We analyzed segment-specific changes in the epididymal duct in a mouse model and in men. In the mouse epididymitis model (3 days post-infection, injection of bacteria into the lumen of the vas deferens), two Escherichia coli strains were tested: a uropathogenic strain CFT073 (UPEC, n = 7) and a fecal non-pathogenic strain NPEC470 (NPEC, n = 5). Two control groups: phosphate-buffered saline, sham-treated animals (n = 4) and untreated mice (n = 8). In addition, segmentation was verified by ex vivo injection of dye into the interstitial spaces of untreated mouse epididymides. Histological findings were compared with specimens from epididymitis patients (n = 10, age range 14-78, median 60 years) who underwent surgical intervention; control: samples from patients without epididymitis (n = 16, age range 38-87, median 73 years). We investigated the ascending infections by detailed histological analysis in correlation with local infection status in a mouse epididymitis model. As a proof of concept, rare patient material from two archives was analyzed: epididymides from patients who underwent surgical intervention for persisting epididymitis, and for control, histologically normal epididymides from men who underwent orchiectomy for therapy of prostatic carcinoma. Luminal ascent of E. coli in mice was strictly gated by epididymal segment boundaries. In the mouse model, both strains of E. coli were detected exclusively in the distal cauda segment associated with damage of the epithelium and muscle layer. Ductal constriction occurred in the non-infected upstream segments of infected area, putatively blocking further luminal ascent of bacteria in UPEC-infected animals. Corresponding histological and morphological changes were found in epididymitis patients. The caput region was found to be unaffected in patients and the mouse model. Patient samples represented advanced cases of epididymitis that made surgical intervention necessary. Our data demonstrate the impact of epididymal segmentation, presumably a protective response mechanism against infectious invasion and bacterial ascent, during epididymitis and affirm the importance of rapid intervention. This work was supported by grants from the State of Hessen (LOEWE-MIBIE) and the DFG (KFO 181). The authors have no conflicts of interest to declare. No clinical trial involved.

Stammler A ，Hau T ，Bhushan S ，Meinhardt A ，Jonigk D ，Lippmann T ，Pilatz A ，Schneider-Hüther I ，Middendorff R ... -  《-》

Specific immune cell and cytokine characteristics of human testicular germ cell neoplasia.

Which immune cells and cytokine profiles are characteristic for testicular germ cell neoplasia and what consequences does this have for the understanding of the related testicular immunopathology? The unique immune environment of testicular germ cell neoplasia comprises B cells and dendritic cells as well as high transcript levels of IL-6 and other B cell supporting or T helper cell type 1 (Th1)-driven cytokines and thus differs profoundly from normal testis or inflammatory lesions associated with hypospermatogenesis. T cells are known to be the major component of inflammatory infiltrates associated with either hypospermatogenesis or testicular cancer. It has previously been reported that B cells are only involved within infiltrates of seminoma samples, but this has not been investigated further. Immunohistochemical characterisation (IHC) of infiltrating immune cells and RT-qPCR-based analysis of corresponding cytokine microenvironments was performed on different testicular pathologies. Testicular biopsies, obtained from men undergoing andrological work-up of infertility or taken during surgery for testicular cancer, were used in this study. Samples were grouped as follows: (i) normal spermatogenesis (n = 18), (ii) hypospermatogenesis associated with lymphocytic infiltrates (n = 10), (iii) samples showing neoplasia [germ cell neoplasia in situ (GCNIS, n = 26) and seminoma, n = 18]. IHC was performed using antibodies against T cells (CD3+), B cells (CD20cy+), dendritic cells (CD11c+), macrophages (CD68+) and mast cells (mast cell tryptase+). Degree and compartmental localisation of immune cells throughout all groups analysed was evaluated semi-quantitatively. RT-qPCR on RNA extracted from cryo-preserved tissue samples was performed to analyse mRNA cytokine expression, specifically levels of IL-1β, IL-6, IL-17a, tumour necrosis factor (TNF)-α (pro-inflammatory), IL-10, transforming growth factor (TGF)-β1 (anti-inflammatory), IL-2, IL-12a, IL-12b, interferon (IFN)-γ (Th1-driven), IL-4, IL-5, IL-13, IL-23a (Th2-driven), CXCL-13, CXCL-10 and CCL-5 (chemokines). This is the first study showing a direct linkage between the distribution pattern of immune cells in hypospermatogenesis versus testicular cancer and analysis of a wide range of 17 related cyto- and chemokines. A fundamental difference between testicular inflammation patterns associated with different testicular inflammatory conditions either containing or lacking neoplastic cells was demonstrated. In hypospermatogenesis, T cells were detected, whereas B cells and dendritic cells were almost absent. Within GCNIS and seminoma, in addition to T cells, high numbers of dendritic cells and B cells were found, the latter additionally organised in cell clusters, whereas mast cells were absent. Transcripts encoding pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α), anti-inflammatory cytokines (TGF-β1), Th1-driven cytokines (IL-2 and IFN-γ) as well as chemokines (CXCL-13, CXCL-10 and CCL-5) were all significantly increased in testicular germ cell neoplasia (P ≤ 0.01), suggesting the presence of a pro-tumorigenic environment. In contrast, Th2-related cytokines (IL-5, IL-13 and IL-23a) characterised the environment within samples showing normal spermatogenesis as well as hypospermatogenesis. One of the most important outcomes is the pivotal role of IL-6 in testicular cancer that opens potential novel diagnostic and/or immune-therapeutic perspective for testis cancer. Testicular tissue composed of immune cells as well as other somatic cells and germ cells does not allow identification of specific cytokine sources or single cell types, being responsible for establishing the overall cytokine environment. In this study, laser-assisted microdissection did not reach the required efficiency for RT-qPCR analyses. Therefore, in vitro models would be suggested for addressing the above-mentioned issue. Conclusions about cytokine levels in testes with GCNIS are based on a small number of samples. The unique B cell presence and the significantly increased expression level of IL-6 in testicular germ cell neoplasia (P < 0.001) strengthen its special role in this disease. In line with current knowledge on other types of cancer, these results underline the relevance of further investigating the potential of IL-6 as early biomarker and target for therapeutic intervention in testicular germ cell neoplasia. This study (and B.K. in person) was supported by the Deutsche Forschungsgemeinschaft (DFG) as part of the International Research Training Group between Justus Liebig University of Giessen and Monash University, Melbourne (GRK 1871/1) on 'Molecular pathogenesis on male reproductive disorders'. T.H., H.-C.S. and M.B. were supported by the LOEWE focus group 'MIBIE' (male infertility during infection & inflammation)-an excellence initiative of the German state government of Hessen. From the Australian side, K.L. was supported by NHMRC grants (Fellowship, ID1079646 and Project, ID1081987); K.L., S.I. and M.H. received scholarship (S.I.) and research funding (K.L., M.H.) from Monash University. The project also drew support from the Victorian Government's Operational Infrastructure Support Program. The authors have no competing interests to declare.

Klein B ，Haggeney T ，Fietz D ，Indumathy S ，Loveland KL ，Hedger M ，Kliesch S ，Weidner W ，Bergmann M ，Schuppe HC ... -  《-》

Uropathogenic Escherichia coli causes fibrotic remodelling of the epididymis.

Despite antibiotic treatment, up to 40% of patients have impaired fertility after epididymitis due to serovars of Escherichia coli, a frequent pathogen. The reasons for infertility are unclear, but it may result from epididymal duct obstruction. To determine whether E. coli infection of the epididymis causes obstruction due to fibrosis, and to identify the key mediators, tissues from patients with epididymitis were assessed. Additionally, epididymitis was induced with uropathogenic E. coli (UPEC) or commensal serovars in wild-type and MyD88(-/-) mice, which are relatively unresponsive to bacterial pathogens. Epididymal organ cultures were treated with activin A and bacteria and their histology and levels of cytokines and fibrosis markers were analysed. Patients with epididymitis showed severe fibrosis of the epididymal duct. In mice, UPEC infection also caused fibrosis and ductal obstruction in the cauda epididymis. Levels of mRNA for fibrotic markers (α-smooth muscle actin, fibronectin) and cytokines (activin A, TNFα, IL-1α, IL-1β, IL-6) and total collagen levels were significantly elevated. This fibrotic response was blunted by the loss of MyD88. Activin A induced fibrosis in cultured epididymis, which was inhibited by the activin-binding protein follistatin. In summary, bacterial epididymitis causes fibrosis and obstruction. The milder tissue damage in Myd88(-/-) UPEC epididymitis highlights the importance of the host response to infection in causing epididymal damage. Elevated levels of activin A in vivo and fibrotic remodelling elicited by activin A in vitro indicate that this cytokine is a potential target for supplementary treatment to antibiotic therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Michel V ，Duan Y ，Stoschek E ，Bhushan S ，Middendorff R ，Young JM ，Loveland KL ，Kretser DM ，Hedger MP ，Meinhardt A ... -  《-》

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

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

Kauerhof AC ，Nicolas N ，Bhushan S ，Wahle E ，Loveland KA ，Fietz D ，Bergmann M ，Groome NP ，Kliesch S ，Schuppe HC ，Pilatz A ，Meinhardt A ，Hedger MP ，Fijak M ... -  《-》