RESUMO
Experimental autoimmune orchitis (EAO) is a useful model to study organ-specific autoimmunity and chronic testicular inflammation. This model reflects testicular pathological changes reported in immunological infertility in men. Progression of EAO in rodents is associated with a significantly increased percentage of testicular endothelial cells and interstitial testicular blood vessels, indicating an ongoing angiogenic process. Vascular endothelial growth factor A (VEGFA), the main regulator of physiological and pathological angiogenesis, can stimulate endothelial cell proliferation, chemotaxis and vascular permeability. The aim of this study was to explore the role of VEGFA in the pathogenesis of testicular inflammation. Our results found VEGFA expression in Leydig cells, endothelial cells and macrophages in testis of rats with autoimmune orchitis. VEGFA level was significantly higher in testicular fluid and serum of rats at the end of the immunization period, preceding testicular damage. VEGF receptor (VEGFR) 1 is expressed mainly in testicular endothelial cells, whereas VEGFR2 was detected in germ cells and vascular smooth muscle cells. Both receptors were expressed in testicular interstitial cells. VEGFR2 increased after the immunization period in the testicular interstitium and VEGFR1 was downregulated in EAO testis. In-vivo-specific VEGFA inhibition by Bevacizumab prevented the increase in blood vessel number and reduced EAO incidence and severity. Our results unveil relevance of VEGFA-VEGFR axis during orchitis development, suggesting that VEGFA might be an early marker of testicular inflammation and Bevacizumab a therapeutic tool for treatment of testicular inflammation associated with subfertility and infertility.
Assuntos
Doenças Autoimunes/patologia , Neovascularização Patológica , Testículo/irrigação sanguínea , Testículo/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/prevenção & controle , Bevacizumab/farmacologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Orquite/imunologia , Orquite/metabolismo , Orquite/prevenção & controle , Codorniz/embriologia , Ratos Wistar , Transdução de Sinais , Testículo/efeitos dos fármacos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Doenças Autoimunes/patologia , Epididimite/patologia , Antagonistas dos Receptores Histamínicos H1/farmacologia , Cetotifeno/farmacologia , Mastócitos/efeitos dos fármacos , Orquite/patologia , Torção do Cordão Espermático/patologia , Testículo/efeitos dos fármacos , Animais , Doenças Autoimunes/imunologia , Contagem de Células , Epididimo/efeitos dos fármacos , Epididimo/imunologia , Epididimo/patologia , Epididimite/imunologia , Hipersensibilidade Tardia , Imunidade Celular/efeitos dos fármacos , Masculino , Mastócitos/imunologia , Mastócitos/patologia , Orquite/imunologia , Ratos , Índice de Gravidade de Doença , Torção do Cordão Espermático/imunologia , Testículo/imunologia , Testículo/patologia , VacinaçãoRESUMO
AIMS: We previously reported that recombinant human Secretory Leukocyte Protease Inhibitor (SLPI) inhibits mitogen-induced proliferation of human peripheral blood mononuclear cells. To determine the relevance of this effect in vivo, we investigated the immuno-regulatory role of SLPI in an experimental autoimmune orchitis (EAO) model. MAIN METHODS: In order to increase SLPI half life, poly-ε-caprolactone microspheres containing SLPI were prepared and used for in vitro and in vivo experiments. Multifocal orchitis was induced in Sprague-Dawley adult rats by active immunization with testis homogenate and adjuvants. Microspheres containing SLPI (SLPI group) or vehicle (control group) were administered s.c. to rats during or after the immunization period. KEY FINDINGS: In vitro SLPI-release microspheres inhibited rat lymphocyte proliferation and retained trypsin inhibitory activity. A significant decrease in EAO incidence was observed in the SLPI group (37.5%) versus the control group (93%). Also, SLPI treatment significantly reduced severity of the disease (mean EAO score: control, 6.33±0.81; SLPI, 2.72±1.05). In vivo delayed-type hypersensitivity and ex vivo proliferative response to testicular antigens were reduced by SLPI treatment compared to control group (p<0.05). SIGNIFICANCE: Our results highlight the in vivo immunosuppressive effect of released SLPI from microspheres which suggests its feasible therapeutic use.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Imunidade Celular/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Orquite/tratamento farmacológico , Inibidor Secretado de Peptidases Leucocitárias/farmacologia , Animais , Doenças Autoimunes/imunologia , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Composição de Medicamentos , Hipersensibilidade Tardia/tratamento farmacológico , Imunidade Celular/imunologia , Terapia de Imunossupressão , Linfócitos/efeitos dos fármacos , Masculino , Microesferas , Orquite/imunologia , Ratos , Ratos Sprague-Dawley , Proteínas RecombinantesRESUMO
Experimental autoimmune orchitis (EAO) is a useful model to study chronic testicular inflammation and infertility. EAO is characterized by severe damage of seminiferous tubules with germ cells that undergo apoptosis and sloughing. We previously reported an increase in CD4+ and CD8+ effector T cells in the testes of rats with EAO. Since cytokine patterns determine T cell effector functions, in the present work we analyzed the cytokines expressed by these cells during disease development. By flow cytometry, we detected an increase in the number of tumor necrosis factor-α (TNF) and interferon -γ (IFNG)-producing CD4+ T cells in the testis at EAO onset. As the severity of the disease progressed, these cells declined while CD8+ T cells producing TNF and IFNG increased, with the predominance of IFNG expression. As a novel finding, we identified by immunofluorescence CD4+ interleukin 17 (IL17)+ and CD8+ IL17+ cells in the testes of EAO rats, with CD4+ and CD8+ T cells predominating at the onset and in the chronic phase of EAO respectively. Moreover, IL17 (western blot) and IL23 content (ELISA) increased in EAO, with maximum levels in the chronic phase. These results suggest the involvement of CD4+ T helper (Th) 1 and Th17 subsets as co-effector cells governing EAO onset, as well as the central contribution of CD8+ T cells producing Th1 and Th17 cytokines in the maintenance of chronic inflammation. The expression of T-bet and RORγt (western blot) in the testis over the course of disease also supports the presence of Th1 and Th17 cells in the testes of EAO rats.
Assuntos
Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Interleucina-17/metabolismo , Interleucina-1/metabolismo , Orquite/imunologia , Animais , Doenças Autoimunes/metabolismo , Western Blotting , Doença Crônica , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Técnicas Imunoenzimáticas , Masculino , Orquite/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas com Domínio T/metabolismoRESUMO
Although the testis is an immunoprivileged organ, infection and inflammation may overwhelm immunosuppressor mechanisms inducing autoimmune reactions against spermatic antigens which result in aspermatogenesis and infertility. Autoimmune orchitis is a model of chronic inflammation useful for elucidating pathogenic mechanisms involved in testicular damage. We developed experimental autoimmune orchitis (EAO) in rats by active immunization with spermatic antigens and adjuvants characterized by interstitial inflammatory cell infiltrate, apoptosis and sloughing of germ cells. Quantitative and phenotypic analysis of testis-infiltrating cells revealed an increased number of macrophages, dendritic cells and T cell subsets that include effector Th1 and Th17 cells as well as Foxp3+ regulatory T cells (T(regs)). Immune cells secrete pro-inflammatory cytokines, TNF-α, IFN-γ, IL-6, IL-12, IL-17 and IL-23, which disrupt the normal testicular immunosuppressor microenvironment. As a consequence, increased numbers of germ cells expressing TNFR1, IL-6R and Fas undergo apoptosis. Functional analysis shows that dendritic cells in EAO testis have a mature immunogenic status and are able to induce immune responses to testicular antigens. We also observed that T(regs) accumulated in the inflamed testis are functionally suppressive but are unable to downregulate inflammation, probably due to the function limiting effect of pro-inflammatory cytokines.
Assuntos
Autoimunidade , Orquite/imunologia , Testículo/imunologia , Células Apresentadoras de Antígenos/imunologia , Apoptose/genética , Apoptose/imunologia , Barreira Hematotesticular/imunologia , Células Germinativas/patologia , Células Germinativas/fisiologia , Humanos , Masculino , Subpopulações de Linfócitos T/imunologia , Testículo/patologiaRESUMO
A wide spectrum of data in the literature shows the relevance of cytokines as paracrine regulators of spermatogenesis and steroidogenesis in the normal testis. In this brief review, we highlight the relevance of cytokines in the testis during inflammation. This phenomenon involves complex and multiple interactions among immune and germ cells generally resulting in the alteration of spermatogenesis. The complexity of these cell interactions is multiplied because Sertoli and Leydig cells are also producers of pro- and anti-inflammatory cytokines and chemokines. Also, cytokines are pleiotropic and they exert opposite and/or redundant effects in different conditions. However, in spite of this bidirectional immunoregulatory function of cytokines, the mass of the data, reported from experiments of acute testicular inflammation, shows upregulation of interleukin (IL)-1beta, IL-1alpha, IL-6, and tumor necrosis factor alpha (TNF-alpha), which induce adverse effects on germ cells. In autoimmune orchitis, a chronic testicular inflammation, chemokines such as CCL2, CCL3, and CCL4 induce attraction and extravasation of immune cells within the testicular interstitium. These cells alter the normal immunosuppressor microenvironment principally through the secretion of proinflammatory cytokines, interferon-gamma initially, and IL-6 and TNF-alpha thereafter. Germ cells expressing TNFR1, IL-6R, and Fas increase in number and undergo apoptosis, through the TNF-alpha/TNFR1, IL-6/IL-6R, and Fas/Fas L systems. The knowledge of immune-germ and somatic testicular cell interactions will contribute to the understanding of the mechanisms by which chronic inflammatory conditions of the testis can disrupt the process of spermatogenesis.