RESUMO
The MHC class I D(k) molecule supplies vital host resistance during murine cytomegalovirus (MCMV) infection. Natural killer (NK) cells expressing the Ly49G2 inhibitory receptor, which specifically binds D(k), are required to control viral spread. The extent of D(k)-dependent host resistance, however, differs significantly amongst related strains of mice, C57L and MA/My. As a result, we predicted that relatively small-effect modifier genetic loci might together shape immune cell features, NK cell reactivity, and the host immune response to MCMV. A robust D(k)-dependent genetic effect, however, has so far hindered attempts to identify additional host resistance factors. Thus, we applied genomic mapping strategies and multicolor flow cytometric analysis of immune cells in naive and virus-infected hosts to identify genetic modifiers of the host immune response to MCMV. We discovered and validated many quantitative trait loci (QTL); these were mapped to at least 19 positions on 16 chromosomes. Intriguingly, one newly discovered non-MHC locus (Cmv5) controlled splenic NK cell accrual, secondary lymphoid organ structure, and lymphoid follicle development during MCMV infection. We infer that Cmv5 aids host resistance to MCMV infection by expanding NK cells needed to preserve and protect essential tissue structural elements, to enhance lymphoid remodeling and to increase viral clearance in spleen.
Assuntos
Infecções por Citomegalovirus/imunologia , Genes MHC Classe I/genética , Células Matadoras Naturais/imunologia , Muromegalovirus/imunologia , Locos de Características Quantitativas/genética , Receptores Imunológicos/genética , Animais , Mapeamento Cromossômico , Infecções por Citomegalovirus/patologia , Feminino , Genes MHC Classe I/imunologia , Loci Gênicos , Genótipo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunidade Celular , Tecido Linfoide/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Polimorfismo Genético , Receptores Imunológicos/metabolismo , Baço/imunologia , Baço/patologiaRESUMO
Pernicious anemia and gastric carcinoma are serious sequelae of autoimmune gastritis (AIG). Our study indicates that in adult C57BL/6-DEREG mice expressing a transgenic diphtheria toxin receptor under the Foxp3 promoter, transient regulatory T cell (Treg) depletion results in long-lasting AIG associated with both H(+)K(+)ATPase and intrinsic factor autoantibody responses. Although functional Tregs emerge over time during AIG occurrence, the effector T cells rapidly become less susceptible to Treg-mediated suppression. Whereas previous studies have implicated dysregulated Th1 cell responses in AIG pathogenesis, eosinophils have been detected in gastric biopsy specimens from patients with AIG. Indeed, AIG in DEREG mice is associated with strong Th2 cell responses, including dominant IgG1 autoantibodies, elevated serum IgE, increased Th2 cytokine production, and eosinophil infiltration in the stomach-draining lymph nodes. In addition, the stomachs exhibit severe mucosal and muscular hypertrophy, parietal cell loss, mucinous epithelial cell metaplasia, and massive eosinophilic inflammation. Notably, the Th2 responses and gastritis severity are significantly ameliorated in IL-4- or eosinophil-deficient mice. Furthermore, expansion of both Th2-promoting IFN regulatory factor 4(+) programmed death ligand 2(+) dendritic cells and ILT3(+) rebounded Tregs was detected after transient Treg depletion. Collectively, these data suggest that Tregs maintain physiological tolerance to clinically relevant gastric autoantigens, and Th2 responses can be a pathogenic mechanism in AIG.
Assuntos
Doenças Autoimunes/imunologia , Mucosa Gástrica/imunologia , Gastrite/imunologia , Células Parietais Gástricas/imunologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Células Cultivadas , Humanos , Imunoglobulina G/sangue , Imunomodulação , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
NK cells are critical in immune responses against pathogens. However, their role in autoimmunity is still controversial. In this study, we demonstrate that neonatal NK cells render newborns more susceptible to neonatal autoimmunity induced by maternal autoantibodies (neonatal autoimmune ovarian disease); thus, neonatal but not adult NK cells are pathogenic after transfer into NK cell-deficient pups. The inhibitory receptors Ly49C/I are expressed in â¼5% of neonatal and â¼50% of adult NK cells. In this study, we show that the presence of Ly49C/I⺠adult NK cells inhibits neonatal autoimmune ovarian disease induction. Thus, the ontogenetic regulation of Ly49C/I expression determines the propensity to autoantibody-induced autoimmunity. In summary, this study provides definitive evidence of a pathogenic role of NK cells in neonatal autoimmunity and also elucidates a novel mechanism by which neonatal NK cells render newborns more susceptible to autoantibody-induced autoimmunity.
Assuntos
Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Células Matadoras Naturais/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Doenças Ovarianas/imunologia , Animais , Animais Recém-Nascidos , Autoimunidade/imunologia , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , GravidezRESUMO
Vasectomy is a well accepted global contraceptive approach frequently associated with epididymal granuloma and sperm autoantibody formation. To understand the long-term sequelae of vasectomy, we investigated the early immune response in vasectomized mice. Vasectomy leads to rapid epithelial cell apoptosis and necrosis, persistent inflammation, and sperm granuloma formation in the epididymis. Vasectomized B6AF1 mice did not mount autoimmune response but instead developed sperm antigen-specific tolerance, documented as resistance to immunization-induced experimental autoimmune orchitis (EAO) but not experimental autoimmune encephalomyelitis. Strikingly, tolerance switches over to pathologic autoimmune state following concomitant CD4(+)CD25(+)Foxp3(+) regulatory T cell (Treg) depletion: unilaterally vasectomized mice produce dominant autoantibodies to an orchitogenic antigen (zonadhesin), and develop CD4 T-cell- and antibody-dependent bilateral autoimmune orchitis. Therefore, (i) Treg normally prevents spontaneous organ-specific autoimmunity induction by persistent endogenous danger signal, and (ii) autoantigenic stimulation with sterile autoinflammation can lead to tolerance. Finally, postvasectomy tolerance occurs in B6AF1, C57BL/6, and A/J strains. However, C57BL/6 mice resisted EAO after 60% Treg depletion, but developed EAO after 97% Treg reduction. Therefore, variance in intrinsic Treg function--a possible genetic trait--can influence the divergent tolerogenic versus autoimmune response to vasectomy.
Assuntos
Autoimunidade/imunologia , Tolerância Imunológica/imunologia , Espermatozoides/imunologia , Linfócitos T Reguladores/imunologia , Vasectomia , Animais , Autoanticorpos/imunologia , Western Blotting , Proliferação de Células , Eletroforese em Gel de Poliacrilamida , Masculino , Proteínas de Membrana/imunologia , Camundongos , Camundongos Mutantes , Estatísticas não ParamétricasRESUMO
IgE antibodies against the mammalian oligosaccharide allergen galactose-α-1,3-galactose (αGal) can result in a severe allergic disease known as alpha-gal syndrome (AGS). This syndrome, acquired by tick bites that cause αGal sensitization, leads to allergic reactions after ingestion of non-primate mammalian meat and mammalian-derived products that contain αGal. Allergen-specific immunotherapies for this tickborne allergic syndrome are understudied, as are the immune mechanisms of allergic desensitization that induce clinical tolerance to αGal. Here, we reveal that prophylactic administration of αGal glycoprotein-containing nanoparticles to mice prior to tick protein-induced αGal IgE sensitization blunts the production of Th2 cytokines IL-4, IL-5, and IL-13 in an αGal-dependent manner. Furthermore, these effects correlated with suppressed production of αGal-specific IgE and hypersensitivity reactions, as measured by reduced basophil activation and histamine release and the systemic release of mast cell protease-1 (MCPT-1). Therapeutic administration of two doses of αGal-containing nanoparticles to mice sensitized to αGal had partial efficacy by reducing the Th2 cytokine production, αGal-specific IgE production, and MCPT-1 release without reducing basophil activation or histamine release. These data identify nanoparticles carrying encapsulated αGal glycoprotein as a potential strategy for augmenting αGal-specific immune tolerance and reveal diverse mechanisms by which αGal nanoparticles modify immune responses for established αGal-specific IgE-mediated allergic reactions.
RESUMO
B cells and the antibodies they produce are critical in host defense against pathogens and contribute to various immune-mediated diseases. B cells responding to activating signals in vitro release extracellular vesicles (EV) that carry surface antibodies, yet B cell production of EVs that express antibodies and their function in vivo is incompletely understood. Using transgenic mice expressing the Cre recombinase in B cells switching to IgG1 to induce expression of fusion proteins between emerald green fluorescent protein (emGFP) and the EV tetraspanin CD63 as a model, we identify emGFP expression in B cells responding to foreign antigen in vivo and characterize the emGFP+ EVs they release. Our data suggests that emGFP+ germinal center B cells undergoing immunoglobulin class switching to express IgG and their progeny memory B cells and plasma cells, also emGFP+, are sources of circulating antigen-specific IgG+ EVs. Furthermore, using a mouse model of influenza virus infection, we find that IgG+ EVs specific for the influenza hemagglutinin antigen protect against virus infection. In addition, crossing the B cell Cre driver EV reporter mice onto the Nba2 lupus-prone strain revealed increased circulating emGFP+ EVs that expressed surface IgG against nuclear antigens linked to autoimmunity. These data identify EVs loaded with antibodies as a novel route for antibody secretion in B cells that contribute to adaptive immune responses, with important implications for different functions of IgG+ EVs in infection and autoimmunity.
Assuntos
Linfócitos B , Vesículas Extracelulares , Imunoglobulina G , Camundongos Transgênicos , Animais , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Camundongos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Infecções por Orthomyxoviridae/imunologia , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Antígenos/imunologia , Switching de Imunoglobulina , Camundongos Endogâmicos C57BL , Centro Germinativo/imunologia , Centro Germinativo/metabolismoRESUMO
OBJECTIVE: Male rats transgenic for HLA-B27 and human ß(2) -microglobulin (hß(2) m) spontaneously develop epididymoorchitis (EO) preceding the development of spondylarthritis (SpA). In the specific B27/hß(2) m-transgenic rat cross-strain (21-3 × 382-2)F(1) , only the males develop SpA, and neither sex develops gut inflammation. This study was undertaken to determine whether EO and SpA in male (21-3 × 382-2)F(1) rats are causally related. In addition, the primary characteristics of EO in this rat arthritis model were assessed. METHODS: Male B27/hß(2) m-transgenic (21-3 × 382-2)F(1) rats underwent bilateral, unilateral, or sham epididymoorchiectomy between ages 36 and 125 days. The castrated rats were given testosterone replacement. Alternatively, the 21-3 and 283-2 transgene loci were crossed with a transgene inducing aspermatogenesis. Rats were observed for the development of EO, arthritis, and spondylitis. RESULTS: In unmanipulated transgenic rats, inflammation was first evident in the ductuli efferentes (DE; ducts linking the rete testis to epididymis) as early as age 30 days. The inflammation was initially neutrophilic, and later became granulomatous. Antisperm and anti-testis cell antibodies appeared in the rat serum after age 70 days. Cells infiltrating the testes were predominantly CD4+ T cells and CD68+ or CD163+ macrophages. Quantitative polymerase chain reaction of the DE, epididymis, and testis showed elevations in the levels of interferon-γ, interleukin-10 (IL-10), and IL-17A. In addition, levels of IL-12A, IL-22, IL-23A, and IL-23 receptor were found to be elevated in the DE. Remarkably, castration of the rats before age 91 days completely prevented the subsequent onset of arthritis and spondylitis, as did transgene-induced azospermia. CONCLUSION: Autoimmune EO develops spontaneously in HLA-B27/hß(2) m-transgenic (21-3 × 283-2)F(1) rats at age 30 days, the age when antigen-positive meiotic germ cells first exit the testis. Persistent testicular inflammation and/or antigenic stimulation are essential prerequisites for the subsequent development of SpA. Thus, dysregulated innate immunity at immune-privileged sites may be an essential mechanism triggering the onset of SpA.
Assuntos
Doenças Autoimunes/complicações , Epididimite/complicações , Antígeno HLA-B27/genética , Orquite/complicações , Caracteres Sexuais , Espondilartrite/etiologia , Espondilartrite/genética , Animais , Doenças Autoimunes/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Epididimo/metabolismo , Epididimo/cirurgia , Epididimite/imunologia , Feminino , Imunidade Inata/imunologia , Masculino , Orquite/imunologia , Fenótipo , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Espondilartrite/imunologia , Testículo/metabolismo , Testículo/cirurgia , Transgenes/genéticaRESUMO
Continuous exposure of tissue antigen (Ag) to the autoantigen-specific regulatory T cells (Treg) is required to maintain Treg-dependent systemic tolerance. Thus, testis autoantigens, previously considered as sequestered, may not be protected by systemic tolerance. We now document that the complete testis antigen sequestration is not valid. The haploid sperm Ag lactate dehydrogenase 3 (LDH3) is continuously exposed and not sequestered. It enters the residual body (RB) to egress from the seminiferous tubules and interact with circulating antibody (Ab). Some LDH3 also remains inside the sperm cytoplasmic droplets (CD). Treg-depletion in the DEREG mice that express diphtheria toxin receptor on the Foxp3 promoter results in spontaneous experimental autoimmune orchitis (EAO) and Ab to LDH3. Unlike the wild-type male mice, mice deficient in LDH3 (wild-type female or LDH3 NULL males) respond vigorously to LDH3 immunization. However, partial Treg depletion elevated the wild-type male LDH3 responses to the level of normal females. In contrast to LDH3, zonadhesin (ZAN) in the sperm acrosome displays properties of a sequestered Ag. However, when ZAN and other sperm Ag are exposed by vasectomy, they rapidly induce testis Ag-specific tolerance, which is terminated by partial Treg-depletion, leading to bilateral EAO and ZAN Ab response. We conclude that some testis/sperm Ag are normally exposed because of the unique testicular anatomy and physiology. The exposed Ag: 1) maintain normal Treg-dependent systemic tolerance, and 2) are pathogenic and serve as target Ag to initiate EAO. Unexpectedly, the sequestered Ags, normally non-tolerogenic, can orchestrate de novo Treg-dependent, systemic tolerance when exposed in vasectomy.
Assuntos
Orquite , Vasectomia , Animais , Autoantígenos , Feminino , Humanos , Tolerância Imunológica , Masculino , Camundongos , Linfócitos T ReguladoresRESUMO
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 BAI1, 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 BAI1/BAI3), also affect sperm entry into oocytes. Intriguingly, mammalian sperm could fuse with skeletal myoblasts, requiring PtdSer on sperm and BAI1/3, 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.
Assuntos
Oócitos/metabolismo , Fagócitos/metabolismo , Fosfatidilserinas/metabolismo , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Angiogênicas/metabolismo , Animais , Antígenos CD36/metabolismo , Proteínas do Citoesqueleto/metabolismo , Epididimo , Feminino , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Mioblastos Esqueléticos , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , Fosfatidilserinas/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , c-Mer Tirosina Quinase/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
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 ( approximately 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-alpha). 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.
Assuntos
Citocinas/imunologia , Células Dendríticas/imunologia , Inflamação/imunologia , Receptores de Quimiocinas/imunologia , Testículo/imunologia , Animais , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Proliferação de Células , Citocinas/metabolismo , Células Dendríticas/metabolismo , Citometria de Fluxo , Expressão Gênica , Inflamação/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-12/genética , Interleucina-12/metabolismo , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Complexo Principal de Histocompatibilidade/genética , Masculino , Ratos , Ratos Wistar , Receptores CCR2/genética , Receptores CCR2/metabolismo , Receptores CCR7/genética , Receptores CCR7/metabolismo , Receptores de Quimiocinas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermatozoides/citologia , Espermatozoides/imunologia , Espermatozoides/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Testículo/citologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Apoptose , Células Germinativas/metabolismo , Fagocitose , Epitélio Pigmentado da Retina/metabolismo , Células de Sertoli/metabolismo , c-Mer Tirosina Quinase/metabolismo , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/metabolismo , Animais , Células Germinativas/patologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Epitélio Pigmentado da Retina/patologia , Células de Sertoli/patologia , c-Mer Tirosina Quinase/genéticaRESUMO
Autoimmune responses to meiotic germ cell antigens (MGCA) that are expressed on sperm and testis occur in human infertility and after vasectomy. Many MGCA are also expressed as cancer/testis antigens (CTA) in human cancers, but the tolerance status of MGCA has not been investigated. MGCA are considered to be uniformly immunogenic and nontolerogenic, and the prevailing view posits that MGCA are sequestered behind the Sertoli cell barrier in seminiferous tubules. Here, we have shown that only some murine MGCA are sequestered. Nonsequestered MCGA (NS-MGCA) egressed from normal tubules, as evidenced by their ability to interact with systemically injected antibodies and form localized immune complexes outside the Sertoli cell barrier. NS-MGCA derived from cell fragments that were discarded by spermatids during spermiation. They egressed as cargo in residual bodies and maintained Treg-dependent physiological tolerance. In contrast, sequestered MGCA (S-MGCA) were undetectable in residual bodies and were nontolerogenic. Unlike postvasectomy autoantibodies, which have been shown to mainly target S-MGCA, autoantibodies produced by normal mice with transient Treg depletion that developed autoimmune orchitis exclusively targeted NS-MGCA. We conclude that spermiation, a physiological checkpoint in spermatogenesis, determines the egress and tolerogenicity of MGCA. Our findings will affect target antigen selection in testis and sperm autoimmunity and the immune responses to CTA in male cancer patients.
Assuntos
Autoantígenos/imunologia , Tolerância Imunológica , Túbulos Seminíferos/imunologia , Espermatogênese/imunologia , Espermatozoides/imunologia , Linfócitos T Reguladores/imunologia , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células de Sertoli/imunologiaRESUMO
Experimental autoimmune orchitis (EAO) is an organ-specific model of autoimmunity characterized by an interstitial lymphomononuclear cell infiltrate as well as sloughing and apoptosis of germ cells. EAO was induced in adult male Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. Rats injected with saline solution and adjuvants were used as control group. The aim of this work was to study the expression of interleukin-6 (IL-6) and its receptor (IL-6R) in the testis of rats with EAO and analyze whether IL-6 could be involved in germ cell apoptosis. By immunohistochemistry, we detected IL-6 expression in testicular macrophages and Leydig cells of control and EAO rats. Sertoli cells showed IL-6 immunoreactivity in most of the seminiferous tubules of control rats, while a few IL-6+ Sertoli cells were found in the testis of rats with EAO. IL-6R immunoreactivity was observed in macrophages, Leydig and germ cells. A significant increase was noted in the number of IL-6R+ germ cells in rats with EAO compared to control rats. The content of IL-6 (ELISA) in the conditioned media obtained from testicular macrophages of rats with orchitis was significantly higher than in the control group. By immunofluorescence performed on isolated testicular macrophages, IL-6 was shown to be expressed by monocytes recently arrived from circulation (ED1+ cells), while resident macrophages (ED2+ cells) were negative. In vitro experiments (trypan blue and MTS assays) showed that IL-6 (50 ng/ml) reduced germ cell viability. We demonstrated also using the TUNEL technique that IL-6 added to cultures of seminiferous tubule segments induced apoptosis of germ cells. Our results suggest that IL-6 and IL-6R may be involved in the pathogenesis of autoimmune orchitis by promoting testicular inflammation and germ cell apoptosis.
Assuntos
Doenças Autoimunes/imunologia , Interleucina-6/biossíntese , Orquite/imunologia , Receptores de Interleucina-6/biossíntese , Testículo/imunologia , Animais , Apoptose/imunologia , Doenças Autoimunes/patologia , Ensaio de Imunoadsorção Enzimática/métodos , Imunofluorescência , Imuno-Histoquímica , Interleucina-6/imunologia , Macrófagos/imunologia , Masculino , Orquite/patologia , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-6/imunologia , Espermatozoides/imunologia , Espermatozoides/patologia , Testículo/patologiaRESUMO
AIM: To evaluate the immunohistopathological changes in the contralateral testis of rats after an experimental spermatic cord torsion. METHODS: Male Sprague-Dawley rats of 45-50 days old were subjected to a 720 degree unilateral spermatic cord torsion for 10, 30 and 80 days (experimental group, E), respectively or sham operation (control group, C). Histopathology of the contralateral testis as well as germ cell apoptosis were studied using the Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) technique. The number of testicular lymphocytes, mast cells and macrophages, and the expression of tumor necrosis factor-alpha (TNF-alpha) and its receptor (TNFR1) in testicular cells of the contralateral testis were quantified by histochemistry and immunohistochemistry. TNF-alpha concentration in testicular fluid was determined by ELISA. RESULTS: In the contralateral testis of rats from the E group, the maximal degree of damage of the germinal epithelium was seen 30 days after torsion. At this time we observed in the E group vs. the C group increases: (i) the number of testicular T-lymphocytes; (ii) the number of testicular mast cells and macrophages; (iii) the percentage of macrophages expressing TNF-alpha; (iv) TNF-a concentration in testicular fluid; (v) the number of apoptotic germ cells; and (vi) the number of TNFR1+ germ cells. CONCLUSION: Experimental spermatic cord torsion induces, in the contralateral testis, a focal damage of seminiferous tubules characterized by apoptosis and sloughing of germ cells. Results suggest humoral and cellular immune mediated testicular cell damage in which macrophages and mast cells seem to be involved in the induction of germ cell apoptosis through the TNF-alpha/TNFR1 system and in the modulation of the inflammatory process.
Assuntos
Torção do Cordão Espermático/patologia , Torção do Cordão Espermático/cirurgia , Testículo/patologia , Animais , Apoptose , Modelos Animais de Doenças , Lateralidade Funcional , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Ratos , Ratos Sprague-Dawley , Túbulos Seminíferos/patologia , Fator de Necrose Tumoral alfa/análiseRESUMO
Experimental autoimmune orchitis (EAO) is characterized by an interstitial mononuclear cell infiltrate and a severe lesion of seminiferous tubules with germ cells that undergo apoptosis and sloughing. The mechanism by which immune cells migrate and extravasate in the testicular interstitium is poorly understood. The aim of this study was to detect the variations in the expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and its receptor in the testis of rats undergoing autoimmune orchitis. EAO was induced in Sprague-Dawley adult rats by active immunization with an emulsion of testicular homogenate and complete Freund adjuvant using Bordetella pertussis as co-adjuvant. Control rats injected with saline and adjuvants and normal untreated rats were also studied. By ELISA we observed a significant increase of MCP-1 in the testicular fluid (TF) and in the conditioned medium obtained from cultures of testicular macrophages of rats with EAO compared with control groups. By immunohistochemistry, an increase in MCP-1 expression was observed in mononuclear, endothelial, Leydig and peritubular cells. MCP-1 immunoreactivity was also detected in Sertoli cell cytoplasm of rats with severe orchitis. A 2-fold increase in the number of mononuclear cells that express CCR2 was also found in rats with orchitis compared with controls. In conclusion, we demonstrated in vivo that MCP-1 is highly expressed in testicular interstitial cells suggesting that this chemokine has an important role in recruiting immune cells to the testis in rats undergoing autoimmune orchitis.
Assuntos
Doenças Autoimunes/imunologia , Quimiocina CCL2/metabolismo , Orquite/imunologia , Receptores de Quimiocinas/metabolismo , Animais , Doenças Autoimunes/etiologia , Doenças Autoimunes/patologia , Imuno-Histoquímica , Macrófagos/imunologia , Masculino , Orquite/etiologia , Orquite/patologia , Ratos , Ratos Sprague-Dawley , Receptores CCR2RESUMO
Human maternal autoantibodies can trigger autoimmune diseases such as congenital heart block (CHB) in the progeny of women with lupus or Sjogren's disease. The pathogenic effect of early autoantibody (autoAb) exposure has been investigated in a murine neonatal autoimmune ovarian disease (nAOD) model triggered by a unique ZP3 antibody. Although immune complexes (IC) 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 natural killer (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, FcγRIII(+) NK cells as well as the ovarian resident FcγRIII(+) macrophages and/or dendritic cells were found to be as indispensable players. Activated by ovarian IC, 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 autoAb-mediated neonatal autoimmunity, including CHB, while simultaneously uncovering new properties of the neonatal innate and adaptive responses, lethality of premature infant infection, and novel neonatal antiviral vaccine design.
RESUMO
The unique sensitivity of early red cell progenitors to iron deprivation, known as the erythroid iron restriction response, serves as a basis for human anemias globally. This response impairs erythropoietin-driven erythropoiesis and underlies erythropoietic repression in iron deficiency anemia. Mechanistically, the erythroid iron restriction response results from inactivation of aconitase enzymes and can be suppressed by providing the aconitase product isocitrate. Recent studies have implicated the erythroid iron restriction response in anemia of chronic disease and inflammation (ACDI), offering new therapeutic avenues for a major clinical problem; however, inflammatory signals may also directly repress erythropoiesis in ACDI. Here, we show that suppression of the erythroid iron restriction response by isocitrate administration corrected anemia and erythropoietic defects in rats with ACDI. In vitro studies demonstrated that erythroid repression by inflammatory signaling is potently modulated by the erythroid iron restriction response in a kinase-dependent pathway involving induction of the erythroid-inhibitory transcription factor PU.1. These results reveal the integration of iron and inflammatory inputs in a therapeutically tractable erythropoietic regulatory circuit.
Assuntos
Anemia/tratamento farmacológico , Células Eritroides/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Deficiências de Ferro , Isocitratos/farmacologia , Aconitato Hidratase/metabolismo , Anemia/metabolismo , Anemia/patologia , Animais , Células Cultivadas , Células Eritroides/enzimologia , Feminino , Humanos , Interferon gama/fisiologia , Isocitratos/uso terapêutico , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Ratos Endogâmicos Lew , Transdução de Sinais , Transativadores/metabolismo , Ativação TranscricionalRESUMO
CD4+ CD25+ regulatory T cells (Tregs) strongly influence the early and late autoimmune responses to meiotic germ cell antigens (MGCA) and the gonadal immunopathology in vasectomized mice. This is supported by the published and recently acquired information presented here. Within 24h of unilateral vasectomy (uni-vx) the ipsilateral epididymis undergoes epithelial cell apoptosis followed by necrosis, severe inflammation, and granuloma formation. Unexpectedly, vasectomy alone induced MGCA-specific tolerance. In contrast, uni-vx plus simultaneous Treg depletion resulted in MGCA-specific autoimmune response and bilateral autoimmune orchitis. Both tolerance and autoimmunity were strictly linked to the early epididymal injury. We now discovered that testicular autoimmunity in uni-vx mice did not occur when Treg depletion was delayed by one week. Remarkably, this delayed Treg depletion also prevented tolerance induction. Therefore, tolerance depends on a rapid de novo Treg response to MGCA exposed after vasectomy. Moreover, tolerance was blunted in mice genetically deficient in PD-1 ligand, suggesting the involvement of induced Treg. We conclude that pre-existing natural Treg prevents post-vasectomy autoimmunity, whereas vasectomy-induced Treg maintains post-vasectomy tolerance. We further discovered that vasectomized mice were still resistant to autoimmune orchitis induction for at least 12-16 months; thus, tolerance is long-lasting. Although significant sperm autoantibodies of low titers became detectable in uni-vx mice at 7 months, the antibody titers fluctuated over time, suggesting a dynamic "balance" between the autoimmune and tolerance states. Finally, we observed severe epididymal fibrosis and hypo-spermatogenesis at 12 months after uni-vx: findings of highly critical clinical significance.
Assuntos
Epididimo/patologia , Orquite/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Vasectomia , Animais , Autoanticorpos/sangue , Autoimunidade/genética , Antígenos CD4/metabolismo , Fibrose/etiologia , Humanos , Tolerância Imunológica/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Depleção Linfocítica , Masculino , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Orquite/etiologia , Receptor de Morte Celular Programada 1/genética , Vasectomia/efeitos adversosRESUMO
OBJECTIVE: 12/15-lipoxygenase (12/15-LO), one of a family of fatty acid oxidoreductase enzymes, reacts with polyenoic fatty acids to produce proinflammatory lipids. 12/15-LO is expressed in macrophages and pancreatic beta-cells. It enhances interleukin 12 production by macrophages, and several of its products induce apoptosis of beta-cells at nanomolar concentrations in vitro. We had previously demonstrated a role for 12/15-LO in beta-cell damage in the streptozotocin model of diabetes. Since the gene encoding 12/15-LO (gene designation Alox15) lies within the Idd4 diabetes susceptibility interval in NOD mice, we hypothesized that 12/15-LO is also a key regulator of diabetes susceptibility in the NOD mouse. RESEARCH DESIGN AND METHODS: We developed NOD mice carrying an inactivated 12/15-LO locus (NOD-Alox15(null)) using a "speed congenic" protocol, and the mice were monitored for development of insulitis and diabetes. RESULTS: NOD mice deficient in 12/15-LO develop diabetes at a markedly reduced rate compared with NOD mice (2.5 vs. >60% in females by 30 weeks). Nondiabetic female NOD-Alox15(null) mice demonstrate improved glucose tolerance, as well as significantly reduced severity of insulitis and improved beta-cell mass, when compared with age-matched nondiabetic NOD females. Disease resistance is associated with decreased numbers of islet-infiltrating activated macrophages at 4 weeks of age in NOD-Alox15(null) mice, preceding the development of insulitis. Subsequently, islet-associated infiltrates are characterized by decreased numbers of CD4(+) T cells and increased Foxp3(+) cells. CONCLUSIONS: These results suggest an important role for 12/15-LO in conferring susceptibility to autoimmune diabetes in NOD mice through its effects on macrophage recruitment or activation.
Assuntos
Araquidonato 12-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/genética , Diabetes Mellitus Tipo 1/prevenção & controle , Animais , Araquidonato 12-Lipoxigenase/deficiência , Araquidonato 15-Lipoxigenase/deficiência , Mapeamento Cromossômico , DNA/genética , DNA/isolamento & purificação , Primers do DNA , Predisposição Genética para Doença , Genoma , Glicosúria/genética , Fígado/enzimologia , Ativação de Macrófagos/genética , Camundongos , Camundongos Endogâmicos NOD , Deleção de Sequência , CaudaRESUMO
Experimental autoimmune orchitis (EAO) in the rat is the primary chronic animal model for the investigation of one of the main causes of male infertility, viz., testicular inflammation. Dendritic cells (DC) are potent antigen-presenting cells that play a fundamental role in autoimmune disease. We investigated the number of DC in normal testis and examined whether DC infiltrated the testis during the development of EAO. EAO was induced by active immunization with testis homogenate and adjuvants in two strains of rat (Wistar and Sprague Dawley). The presence of DC in testis was determined, 50 and 80 days after the first immunization, by immunohistochemical staining with specific antibodies (OX-62 and CD11c), and then the total number of DC was measured by stereological analysis. Labeled cells were found only in the interstitial compartment and within granulomas of EAO animals. The number of DC in EAO testes increased compared with control rats in both strains, whereas the number of OX-62+ and CD11c+ cells in adjuvant controls remained unchanged compared with untreated rats. Interspecies variations in the quantity of DC were found, with the total number of DC per testis in untreated and adjuvant control Sprague-Dawley rats being about three times higher than that seen in Wistar rats. Moreover, the increase in DC numbers at 80 days was less prominent in EAO testes of Sprague-Dawley rats than in the Wistar strain in which EAO was more severe and showed a higher number of granulomae. Thus, we have identified the DC population in normal and chronically inflamed testis. The increase in DC observed in EAO suggests that, under inflammatory conditions, the modified action(s) of these cells is a factor in the induction of the autoimmune response in testis.