Claudia Rival

Regulatory T cells control tolerogenic versus autoimmune response to sperm in vasectomy

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.

Autoimmune epididymoorchitis is essential to the pathogenesis of male-specific spondylarthritis in HLA-B27-transgenic rats.

OBJECTIVE Male rats transgenic for HLA-B27 and human β(2) -microglobulin (hβ(2) m) spontaneously develop epididymoorchitis (EO) preceding the development of spondylarthritis (SpA). In the specific B27/hβ(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/hβ(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/hβ(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.

Isocitrate ameliorates anemia by suppressing the erythroid iron restriction response

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.

Egress of sperm autoantigen from seminiferous tubules maintains systemic tolerance

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.

Regulatory T Cells Control Th2-Dominant Murine Autoimmune Gastritis

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.

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 (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.

Genomic Modifiers of Natural Killer Cells, Immune Responsiveness and Lymphoid Tissue Remodeling Together Increase Host Resistance to Viral Infection

The MHC class I Dk molecule supplies vital host resistance during murine cytomegalovirus (MCMV) infection. Natural killer (NK) cells expressing the Ly49G2 inhibitory receptor, which specifically binds Dk, are required to control viral spread. The extent of Dk-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 Dk-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.

Autoimmune Orchitis and Autoimmune Oophoritis

Abstract Autoimmune diseases of the testis [experimental autoimmune orchitis (EAO)] and the ovary [autoimmune ovarian disease (AOD)] are causal to infertility. Sperm autoimmunity also occurs in vasectomized men. In addition, autoantigens expressed in the testis and ovary can express ectopically in numerous human cancers. As cancer/testis antigens, they are subjected to tolerogenic regulation as sperm antigens in normal subjects and confer negative influence on cancer immunity. Alternatively, some cancer/testis antigens (CTA) are highly immunogenic and are desirable as candidate antigens in human cancer vaccines. Study on the spontaneous models of EAO and AOD indicates that regulatory T cell (Treg) is critical in systemic tolerance of the testis and ovarian autoantigens. We discovered two distinct types of sperm autoantigens: one is not hidden and is protected by Treg and the other that is hidden and not protected by Treg. This invalidated the prevailing paradigm of complete testis antigen sequestration. AOD model induced by a novel pZP3 that discovered molecular mimicry at T-cell epitope, epitope spreading as a mechanism of autoantibody response, the retargeting of T cell–mediated injury from one anatomical target to another by autoantibody, and the novel neonatal ovarian autoimmune disease (nAOD) induced by maternal autoantibody in the first 5 days of life. The unique nAOD is a model of human autoimmune disease elicited by maternal autoantibodies including congenital heart block in newborn of lupus mothers with Ro/SSA autoantibodies. nAOD requires neonatal natural killer (NK) cells deficient in inhibitory receptor LY49i. Treg also controls nAOD development. Finally, Th1 CD4 response occurs that adoptively transfer nAOD. The most well-defined clinical autoimmune ovarian and testicular diseases are found in the rare monogenic autoimmune polyendocrine syndrome associated with autoimmune regulator mutations. Contrary to numerous experimental models, the episodic clinical human testis and ovarian autoimmune diseases remain incompletely defined.

Cutting edge: Ly49C/I⁻ neonatal NK cells predispose newborns to autoimmune ovarian disease induced by maternal autoantibody.

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 ∼5% of neonatal and ∼50% of adult NK cells. In this study, we show that the presence of Ly49C/I+ 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.

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−/−Bai1Tg) 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.