Involvement of tumor necrosis factor-α in the pathogenesis of autoimmune orchitis in rats

We studied the testicular macrophages of rats with experimental autoimmune orchitis (EAO) and analyzed whether the tumor necrosis factor-α (TNFα) is involved in germ cell apoptosis and in Leydig cell steroidogenesis. The EAO was induced in adult male Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. In the experimental group, a severe orchitis was observed 80 days after the first immunization. ED1- and ED2-positive macrophages were quantified by immunohistochemistry. The TNFα concentration of conditioned media from testicular macrophages (TMCM) was determined by ELISA. The number of apoptotic TNF receptor 1 (TNFR1)-positive germ cells was identified by combining in situ end labeling of apoptotic DNA and immunohistochemical techniques. The effect of TNFα on Leydig cell testosterone production was determined by RIA. In rats with EAO, we observed a significant increase in the number of TNFα-positive testicular macrophages, the TNFα concentration in TMCM, and the number of TNFR1-positive germ cells. Sixty percent of TNFR1-positive germ cells were apoptotic. These results suggest that TNFα could be involved in the pathogenesis of EAO. Acting together with other local factors such as Fas-FasL, TNFα could trigger germ cell apoptosis. We also demonstrated that TNFα inhibited in vitro testosterone production in basal and hCG-stimulated Leydig cells from rats with orchitis.Facultad de Ciencias Exacta

The Unique Neonatal NK Cells: A Critical Component Required for Neonatal Autoimmune Disease Induction by Maternal Autoantibody

Human maternal autoantibodies can trigger autoimmune diseases such as congenital heart block in the progeny of women with lupus or Sjogren’s disease. The pathogenic effect of early autoantibody exposure has been investigated in a murine neonatal autoimmune ovarian disease (nAOD) model triggered by a unique ZP3 antibody. Although immune complexes are formed in adult and neonatal ovaries, ZP3 antibody triggers severe nAOD only in <7 day-old neonatal mice. Propensity to nAOD is due to the uniquely hyper-responsive neonatal NK cells that lack the inhibitory Ly49C/I receptors. In nAOD, the neonatal NK cells directly mediate ovarian inflammation and oocyte depletion while simultaneously promoting de novo pathogenic ovarian-specific T cell responses. Resistance to nAOD in older mice results from the emergence of the Ly49C/I+ NK cells that regulate effector NK cells and from CD25+ regulatory T cell control. In preliminary studies, the ovarian resident FcgRIII+ macrophages and/or dendritic cells were found to be as indispensable players. Activated by ovarian immune complexes, they migrate to lymphoid organs where NK cell priming occurs. Remarkably, the findings in nAOD are very similar to those reported for neonatal responses to a retrovirus and its cognate antibody that lead to long-lasting immunity. Studies on nAOD therefore provide insights into maternal autoantibody-mediated neonatal autoimmunity, including congenital heart block, while simultaneously uncovering new properties of the neonatal innate and adaptive responses, lethality of premature infant infection, and novel neonatal antiviral vaccine design

Involvement of tumor necrosis factor-α in the pathogenesis of autoimmune orchitis in rats

We studied the testicular macrophages of rats with experimental autoimmune orchitis (EAO) and analyzed whether the tumor necrosis factor-α (TNFα) is involved in germ cell apoptosis and in Leydig cell steroidogenesis. The EAO was induced in adult male Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. In the experimental group, a severe orchitis was observed 80 days after the first immunization. ED1- and ED2-positive macrophages were quantified by immunohistochemistry. The TNFα concentration of conditioned media from testicular macrophages (TMCM) was determined by ELISA. The number of apoptotic TNF receptor 1 (TNFR1)-positive germ cells was identified by combining in situ end labeling of apoptotic DNA and immunohistochemical techniques. The effect of TNFα on Leydig cell testosterone production was determined by RIA. In rats with EAO, we observed a significant increase in the number of TNFα-positive testicular macrophages, the TNFα concentration in TMCM, and the number of TNFR1-positive germ cells. Sixty percent of TNFR1-positive germ cells were apoptotic. These results suggest that TNFα could be involved in the pathogenesis of EAO. Acting together with other local factors such as Fas-FasL, TNFα could trigger germ cell apoptosis. We also demonstrated that TNFα inhibited in vitro testosterone production in basal and hCG-stimulated Leydig cells from rats with orchitis.Facultad de Ciencias Exacta

Expression of co-stimulatory molecules, chemokine receptors and proinflammatory cytokines in dendritic cells from normal and chronically inflamed rat testis

The presentation of self antigens by dendritic cells (DC) plays an important role in the initiation and maintenance of autoimmunity. In a model of experimental autoimmune orchitis (EAO), we have previously characterized dominant testicular autoantigens and shown an increase in DC numbers during the course of disease. In this study, we have developed a protocol for the isolation of a highly pure population of DC (∼97%) from the testis of EAO and control rats to analyse the expression of major histocompatibility complex (MHC) class II and co-stimulatory molecules (CD80, CD86), chemokine receptors (CCR2, CCR7) and cytokines (IL-10, IL-12p70, TNF-α). By flow cytometry, we observed similar percentage and intensity levels of MHC class II, CD80 and CD86 expression in testicular DC in all groups. Moreover, by real-time RT-PCR we have detected significantly higher CCR7 mRNA level in isolated testicular DC from rats with EAO compared to controls, whereas the expression of CCR2 was decreased in orchitis. Transcripts of IL-12p40 were observed in DC from all groups, whereas the expression of IL-10 and the rate limiting IL-12 subunit p35 were detectable exclusively in testicular DC from the inflamed testes. In co-culture experiments, testicular DC isolated from EAO animals significantly enhanced naïve T-cell proliferation compared with control DC. Taken together these results suggest that testicular DC in control testis is not mature and functionally tolerogenic, whereas in EAO testis, IL-12 expression and stimulation of T-cell proliferation points to a mature immunogenic state prior imminent migration to the lymph nodes to amplify immune responses against testicular antigens.Fil: Rival, Claudia. Universidad de Buenos Aires; ArgentinaFil: Guazzone, Vanesa Anabella. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: von Wulffen, Werner. University of Giessen Lung Center; AlemaniaFil: Hackstein, Holger. Institute for Clinical Immunology and Transfusion Medicine; AlemaniaFil: Schneider, Eva. Justus Liebig Universitat Giessen; AlemaniaFil: Lustig, Livia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Meinhardt A.. Justus Liebig Universitat Giessen; AlemaniaFil: Fijak, Monika. Justus Liebig Universitat Giessen; Alemani

Context-dependent compensation among phosphatidylserine-recognition receptors

Phagocytes express multiple phosphatidylserine (PtdSer) receptors that recognize apoptotic cells. It is unknown whether these receptors are interchangeable or if they play unique roles during cell clearance. Loss of the PtdSer receptor Mertk is associated with apoptotic corpse accumulation in the testes and degeneration of photoreceptors in the eye. Both phenotypes are linked to impaired phagocytosis by specialized phagocytes: Sertoli cells and the retinal pigmented epithelium (RPE). Here, we overexpressed the PtdSer receptor BAI1 in mice lacking MerTK (Mertk(-/-) Bai1(Tg)) to evaluate PtdSer receptor compensation in vivo. While Bai1 overexpression rescues clearance of apoptotic germ cells in the testes of Mertk(-/-) mice it fails to enhance RPE phagocytosis or prevent photoreceptor degeneration. To determine why MerTK is critical to RPE function, we examined visual cycle intermediates and performed unbiased RNAseq analysis of RPE from Mertk(+/+) and Mertk(-/-) mice. Prior to the onset of photoreceptor degeneration, Mertk(-/-) mice had less accumulation of retinyl esters and dysregulation of a striking array of genes, including genes related to phagocytosis, metabolism, and retinal disease in humans. Collectively, these experiments establish that not all phagocytic receptors are functionally equal, and that compensation among specific engulfment receptors is context and tissue dependent

Involvement of tumor necrosis factor-α in the pathogenesis of autoimmune orchitis in rats

We studied the testicular macrophages of rats with experimental autoimmune orchitis (EAO) and analyzed whether the tumor necrosis factor-α (TNFα) is involved in germ cell apoptosis and in Leydig cell steroidogenesis. The EAO was induced in adult male Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. In the experimental group, a severe orchitis was observed 80 days after the first immunization. ED1- and ED2-positive macrophages were quantified by immunohistochemistry. The TNFα concentration of conditioned media from testicular macrophages (TMCM) was determined by ELISA. The number of apoptotic TNF receptor 1 (TNFR1)-positive germ cells was identified by combining in situ end labeling of apoptotic DNA and immunohistochemical techniques. The effect of TNFα on Leydig cell testosterone production was determined by RIA. In rats with EAO, we observed a significant increase in the number of TNFα-positive testicular macrophages, the TNFα concentration in TMCM, and the number of TNFR1-positive germ cells. Sixty percent of TNFR1-positive germ cells were apoptotic. These results suggest that TNFα could be involved in the pathogenesis of EAO. Acting together with other local factors such as Fas-FasL, TNFα could trigger germ cell apoptosis. We also demonstrated that TNFα inhibited in vitro testosterone production in basal and hCG-stimulated Leydig cells from rats with orchitis.Facultad de Ciencias Exacta

Phosphatidylserine on viable sperm and phagocytic machinery in oocytes regulate mammalian fertilization

Fertilization is essential for species survival. Although Izumo1 and Juno are critical for initial interaction between gametes, additional molecules necessary for sperm: egg fusion on both the sperm and the oocyte remain to be defined. Here, we show that phosphatidylserine (PtdSer) is exposed on the head region of viable and motile sperm, with PtdSer exposure progressively increasing during sperm transit through the epididymis. Functionally, masking phosphatidylserine on sperm via three different approaches inhibits fertilization. On the oocyte, phosphatidylserine recognition receptors BAl1, CD36, Tim-4, and Mer-TK contribute to fertilization. Further, oocytes lacking the cytoplasmic ELMO1, or functional disruption of RAC1 (both of which signal downstream of BAl1/BAl3), also affect sperm entry into oocytes. Intriguingly, mammalian sperm could fuse with skeletal myoblasts, requiring PtdSer on sperm and BAl1/3, ELMO2, RAC1 in myoblasts. Collectively, these data identify phosphatidylserine on viable sperm and PtdSer recognition receptors on oocytes as key players in sperm: egg fusion

Residency and a broad feeding spectrum are related to extensive spatial exploration in parrots

Resident and nomadic species differ substantially in their mobility, with the former spending most of their lives in a restricted area and the latter encountering many areas while tracking spatiotemporal unpredictable resources. Earlier studies have shown that information gathering differs alongside this mobility axis—resident species pay more attention to changes in their familiar environment than nomadic ones. However, little is known about spatial exploration in resident and nomadic species. We investigated spatial exploration in 10 closely related parrot species that differed in their mobility by giving them access to two unfamiliar aviaries left and right of the familiar aviary. For analyses, mobility and some diet and habitat variables were related to spatial exploration. Nomadic species spent less time exploring the novel aviaries and also started tactile exploration later than resident/nomadic and pure resident species. Furthermore, diet specialists visited more new locations in relation to their overall movements than diet generalists. The variables were not correlated with the molecular phylogeny (mitochondrial cytochrome b) of the species. The results indicate that nomads invest less in spatial exploration than residents possibly because they stay only for limited periods of time in one particular area or use easier-to-assess cues. Residents, in contrast, assess a novel environment in detail as they may collect information about future breeding sites for which they need short- and long-term information. Finally, diet specialists may pay attention to fewer environmental cues than generalists, which allows them to move faster through a novel environment

Estudio de las células presentadoras de antígeno en el testículo normal y durante el desarrollo de la orquitis autoinmune experimental

A pesar de que el testículo es un “órgano inmunoprivilegiado”, pueden generarse reacciones inflamatorias que alteran su función y pueden inducir infertilidad. Un modelo útil para estudiar autoinmunidad específica de órgano e inflamación testicular es la orquitis autoinmune experimental (OAE) que se caracteriza por la presencia de un infiltrado linfomonocitario intersticial y daño de las células germinales que sufren apoptosis y descamación. Nuestro objetivo fue estudiar las características fenotípicas y funcionales de las células presentadoras de antígeno (CPA) en el testículo normal y durante la OAE. En este cuadro, se observó un incremento en el número de macrófagos (residentes y no residentes) y de células dendríticas en el intersticio testicular. Mediante experimentos in vivo, utilizando liposomas conteniendo clodronato, demostramos que los macrófagos desempeñan un importante papel patogénico en la OAE. Detectamos que los macrófagos testiculares secretan un mayor nivel de citoquinas pro-inflamatorias (IL-6, IFNγ y TNFα) durante la orquitis. Utilizando ratones deficientes en la expresión de TNFR1, demostramos, in vivo, que esta molécula está involucrada en el desarrollo de la OAE. Observamos que la IL-6 es capaz de inducir, in vitro, la apoptosis de las células germinales y detectamos, in vivo, un aumento significativo en el número de células germinales IL6R+ en las ratas con orquitis. A su vez, observamos variaciones en la expresión de moléculas del CMH II y co-estimulatorias (CD80 y CD86) en las CPA testiculares. Concluímos que las CPA testiculares están fuertemente involucradas en el desarrollo de la OAE, a través de la secreción de citoquinas pro-inflamatorias y de la presentación antigénica a los linfocitos T.Testicular inflammation with subfertility or infertility can occur despite the fact that the testis is considered an immunoprivileged organ. Experimental autoimmune orchitis (EAO) is a useful model to study organ-specific autoimmunity and testicular inflammation. EAO is characterized by an interstitial inflammatory cell infiltrate and damage of germ cells that undergo apoptosis and sloughing. The aim of this work was to study the phenotype and functions of antigen presenting cells (APC) in the normal testis and during EAO. We observed an increased number of resident and non-resident macrophages and dendritic cells in the testicular interstitium of animals with orchitis. By experiments using liposomes containing clodronate, we showed, in vivo, a pathogenic role for macrophages in EAO. By ELISA, we detected an increased production of pro-inflammatory cytokines (IL-6, IFNγ and TNFα) by testicular macrophages from rats with EAO. TNFR1 deficient mice showed that this molecule is involved in the pathogenesis of EAO. We observed that IL-6 was able to induce germ cell apoptosis in vitro, while an increased number of IL-6R+ germ cells was detected in vivo in EAO. By immunohistochemistry and flow cytometry we detected variations in the expression of MHC II and costimulatory (CD80 and CD86) molecules on testicular APC. We conclude that testicular APC are strongly involved in the development of EAO through the production of pro-inflammatory cytokines and antigen presentation to T cells.Fil: Rival, Claudia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Autoimmune Orchitis and Autoimmune Oophoritis

The etiology of gonadal failure with infertility in men and women may have a genetic, environmental, or autoimmune basis. The major support for an autoimmune basis has come from studies on patients with the autoimmune polyglandular syndrome entity associated with mutation of the AIRE gene. AIRE-null mutant mice are defective in clonal deletion and regulatory T cell production—the two key tolerance mechanisms. When tolerance is disrupted, autoreactive T cells and B cells respond to self antigen and induce immunopathologic organ damage that leads to infertility. Although T cells are pivotal, antibodies are also critically involved. There has been considerable progress in the understanding of pathogenic mechanisms of autoimmune orchitis and oophoritis, as well as the immunologic sequel of vasectomy. In this review we describe both human diseases and the experimental models of autoimmune orchitis and oophoritis, highlighting many aspects, including the relevant role of lymph node-specific regulatory T cells as well as Th1 and Th17 effector cells.Fil: Lustig, Livia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Rival, Claudia. University of Virginia; Estados UnidosFil: Tung, Kenneth S. K.. University of Virginia; Estados Unido