Jean-Charles Guéry

Estradiol enhances primary antigen-specific CD4 T cell responses and Th1 development in vivo. Essential role of estrogen receptor α expression in hematopoietic cells

It is widely accepted that females have superior immune responses than males, but the ways by which sex hormones may enhance T cell responses are still poorly understood. In the present study,we analyzed the effect of estrogens on CD4 T cell activation and differentiation after immunization with exogenous antigens. We show that administration of low doses of 17ß‐estradiol (E2) to castrated female mice results in a striking increase of antigen‐specific CD4 T cell responses and in the selective development of IFN‐γ‐producing cells. Quantitative assessment of the frequency of T cells bearing a public TCR ß chain CDR3 motif demonstrated that the clonal size of primary antigen‐specific CD4 T cells was dramatically increased in immune lymph nodes from E2‐treated mice. By usingmice with disrupted estrogen receptor (ER) α or ß genes, we show that ERα, but not ERβ, was necessary for the enhanced E2‐driven Th1 cell responsiveness. Furthermore, ERα expressionin hematopoietic cells was essential, since E2 effects on Th1 responses were only observed in mice reconstituted with bone marrow cells from ERα+/+, but not ERα‐deficient mice. These results demonstrate that estrogen administration promotes strong antigen‐specific Th1 cell responses in a mechanism that requires functional expression of ERα in hematopoietic cells.

17Beta-estradiol promotes TLR4-triggered proinflammatory mediator production through direct estrogen receptor alpha signaling in macrophages in vivo.

17β-estradiol (E2) has been shown to promote the expression of inflammatory mediators by LPS-activated tissue resident macrophages through estrogen receptor α (ERα) signaling. However, it remained to be determined whether E2 similarly influences macrophages effector functions under inflammatory conditions in vivo, and whether this action of E2 resulted from a direct effect on macrophages. We show in this study that chronic E2 administration to ovariectomized mice significantly increased both cytokine (IL-1β, IL-6, and TNF-α) and inducible NO synthase mRNA abundance in thioglycolate (TGC)-elicited macrophages. The proinflammatory action of E2 was also evidenced at the level of released IL-1β and IL-6 by ex vivo LPS-activated macrophages. E2 concomitantly inhibited PI3K activity as well as Akt phosphorylation in TGC-elicited macrophages, suggesting that E2 promoted TLR-dependent macrophage activation by alleviating this suppressive signaling pathway. Indeed, this effect was abolished in the presence of the inhibitor wortmannin, demonstrating a key functional link between inhibition of PI3K activity and the E2 action on macrophage functions. Endogenous estrogens levels circulating in ovary-intact mice were sufficient to promote the above described actions. Finally, thanks to a CreLox strategy, targeted disruption of ERα gene in macrophages totally abolished the effect of E2 on the expression of inflammatory mediators by both resident and TGC-elicited peritoneal macrophages. In conclusion, we demonstrate that estrogens, through the activation of ERα in macrophages in vivo, enhance their ability to produce inflammatory mediators and cytokines upon subsequent TLR activation.

Selective Activation and Expansion of High-Affinity CD4+ T Cells in Resistant Mice upon Infection with Leishmania major

Using multimers of MHC class II molecules linked to a peptide derived from the Leishmania LACK antigen, we have compared the fate of parasite-specific CD4+ T cells in resistant and susceptible mice transgenic for the beta chain of a LACK-specific TCR. Activated T cells were readily detected in the draining lymph nodes of infected animals. Although the kinetics of activation and expansion were similar in both strains, T cells from susceptible and resistant mice expressed low- and high-affinity TCR, respectively. As T cells from resistant mice produced more IFN-gamma and less IL-4 than those from susceptible animals, our results suggest that differences in TCR usage between MHC-matched animals may influence the development of the antiparasite immune response.

Estrogen Receptor α Signaling in T Lymphocytes Is Required for Estradiol-Mediated Inhibition of Th1 and Th17 Cell Differentiation and Protection against Experimental Autoimmune Encephalomyelitis

Estrogen treatment exerts a protective effect on experimental autoimmune encephalomyelitis (EAE) and is under clinical trial for multiple sclerosis therapy. Estrogens have been suspected to protect from CNS autoimmunity through their capacity to exert anti-inflammatory as well as neuroprotective effects. Despite the obvious impacts of estrogens on the pathophysiology of multiple sclerosis and EAE, the dominant cellular target that orchestrates the anti-inflammatory effect of 17β-estradiol (E2) in EAE is still ill defined. Using conditional estrogen receptor (ER) α-deficient mice and bone marrow chimera experiments, we show that expression of ERα is critical in hematopoietic cells but not in endothelial ones to mediate the E2 inhibitory effect on Th1 and Th17 cell priming, resulting in EAE protection. Furthermore, using newly created cell type-specific ERα-deficient mice, we demonstrate that ERα is required in T lymphocytes, but neither in macrophages nor dendritic cells, for E2-mediated inhibition of Th1/Th17 cell differentiation and protection from EAE. Lastly, in absence of ERα in host nonhematopoietic tissues, we further show that ERα signaling in T cells is necessary and sufficient to mediate the inhibitory effect of E2 on EAE development. These data uncover T lymphocytes as a major and nonredundant cellular target responsible for the anti-inflammatory effects of E2 in Th17 cell-driven CNS autoimmunity.

Chronic Estradiol Administration In Vivo Promotes the Proinflammatory Response of Macrophages to TLR4 Activation: Involvement of the Phosphatidylinositol 3-Kinase Pathway

Short-term exposure to 17β-estradiol (E2) in vitro has been reported to decrease the production of proinflammatory cytokines by LPS-activated macrophages through estrogen receptor α (ERα)-dependent activation of the PI3K pathway. In the present study, we confirm that in vitro exposure of mouse peritoneal macrophages to E2 enhanced Akt phosphorylation and slightly decreased LPS-induced cytokine production. In striking contrast, we show that chronic administration of E2 to ovariectomized mice markedly increases the expression of IL-1β, IL-6, IL-12p40, and inducible NO synthase by resident peritoneal macrophages in response to LPS ex vivo. These results clearly indicate that short-term E2 treatment in vitro does not predict the long-term effect of estrogens in vivo on peritoneal macrophage functions. We show that this in vivo proinflammatory effect of E2 was mediated through ERα. Although the expression of components of the LPS-recognition complex remained unchanged, we provided evidences for alterations of the TLR4 signaling pathway in macrophages from E2-treated mice. Indeed, E2 treatment resulted in the inhibition of PI3K activity and Akt phosphorylation in LPS-activated macrophages, whereas NF-κB p65 transcriptional activity was concomitantly increased. Incubation of macrophages with the PI3K inhibitor wortmanin enhanced proinflammatory cytokine gene expression in response to TLR4 activation, and abolishes the difference between cells from placebo- or E2-treated mice, demonstrating the pivotal role of the PI3K/Akt pathway. We conclude that the macrophage activation status is enhanced in vivo by E2 through ERα and, at least in part, by the down-modulation of the PI3K/Akt pathway, thereby alleviating this negative regulator of TLR4-signaling.

Natural killer cells recruited into lymph nodes inhibit alloreactive T-cell activation through perforin-mediated killing of donor allogeneic dendritic cells

Natural killer (NK)-cell alloreactivity is exploited in bone marrow transplantation to improve clinical outcome. Likewise, in solid organ transplantation, it has been recently shown that recipient NK cells may limit alloreactive T-cell responses through their capacity to prevent the persistence of graft-derived allogeneic dendritic cells (DCs). In a model of CD4(+) T cell-mediated allogeneic skin graft rejection, we show that the absence of host NK-cell alloreactivity was characterized by enhanced expansion of alloreactive effector T lymphocytes, including Th2 cells, and massive eosinophilic infiltrates in the rejected tissues. In CD8(+) T cell-deficient C57BL/6 (H-2(b)) recipients injected with allogeneic BALB/c (H-2(d)) DCs, we demonstrated that NK cells expressing the H-2D(d)-specific Ly49D activating receptor were implicated in the regulation of alloreactive CD4(+) T-cell responses. Moreover, we showed that Ly49D(+) CD127(-) NK cells were recruited within DC draining lymph nodes and rapidly eliminated allogeneic H-2(d) DCs through the perforin pathway. In normal mice, we further demonstrated that NK cells by quickly eliminating allogeneic DCs strongly inhibited alloreactive CD8(+) T-cell responses. Thus, NK cells act as early regulators of alloreactive T-cell priming in allotransplantation through their capacity to kill allogeneic DCs in draining lymph nodes.

The TLR-mediated response of plasmacytoid dendritic cells is positively regulated by estradiol in vivo through cell-intrinsic estrogen receptor α signaling.

Plasmacytoid dendritic cells (pDCs) produce large amounts of type I interferons (IFN-α/β) in response to viral or endogenous nucleic acids through activation of their endosomal Toll-like receptors (TLR-7 and TLR-9). Enhanced TLR-7-mediated IFN-α production by pDCs in women, compared with men, has been reported, but whether sex hormones, such as estrogens, are involved in this sex-based difference is unknown. Here we show, in humanized mice, that the TLR-7-mediated response of human pDCs is increased in female host mice relative to male. In a clinical trial, we establish that treatment of postmenopausal women with 17β-estradiol markedly enhances TLR-7- and TLR-9-dependent production of IFN-α by pDCs stimulated by synthetic ligands or by nucleic acid-containing immune complexes. In mice, we found exogenous and endogenous estrogens to promote the TLR-mediated cytokine secretion by pDCs through hematopoietic expression of estrogen receptor (ER) α. Genetic ablation of ERα gene in the DC lineage abrogated the enhancing effect of 17β-estradiol on their TLR-mediated production of IFN-α, showing that estrogens directly target pDCs in vivo. Our results uncover a previously unappreciated role for estrogens in regulating the innate functions of pDCs, which may account for sex-based differences in autoimmune and infectious diseases.

Estrogen Enhances Susceptibility to Experimental Autoimmune Myasthenia Gravis by Promoting Type 1-Polarized Immune Responses

Myasthenia gravis (MG) is an organ-specific autoimmune disease caused in most cases by autoantibodies against the nicotinic acetylcholine receptor (AChR). It is now well documented that many autoimmune diseases, including MG, are more prevalent in women than in men, and that fluctuations in disease severity occur during pregnancy. These observations raise the question of the potential role of sex hormones, such as estrogens, as mediators of sex differences in autoimmunity. In the present study, we have analyzed the effect of 17β-estradiol (E2) on the pathogenesis of experimental autoimmune myasthenia gravis (EAMG), an animal model of MG. We show that treatment with E2 before Ag priming is necessary and sufficient to promote AChR-specific Th1 cell expansion in vivo. This time-limited exposure to E2 enhances the production of anti-AChR IgG2ab (specific for b allotype; e.g., B6) and IgG2b, but not IgG1, and significantly increases the severity of EAMG in mice. Interestingly, the E2-mediated augmentation in AChR-specific Th1 response correlates with an enhanced production of IL-12 by splenic APCs through the recruitment of CD8α+ dendritic cells. These data provide the first evidence that estrogen enhances EAMG, and sheds some light on the role of sex hormones in immune responses and susceptibility to autoimmune disease in women.

Estrogen Receptor α Signaling in Inflammatory Leukocytes Is Dispensable for 17β-Estradiol-Mediated Inhibition of Experimental Autoimmune Encephalomyelitis

Estrogen treatment has been shown to exert a protective effect on experimental autoimmune encephalomyelitis (EAE), and is under clinical trial for multiple sclerosis. Although it is commonly assumed that estrogens exert their effect by modulating immune functions, we show in this study that 17β-estradiol (E2) treatment can inhibit mouse EAE without affecting autoantigen-specific T cell responsiveness and type 1 cytokine production. Using mutant mice in which estrogen receptor α (ERα) has been unambiguously inactivated, we found that ERα was responsible for the E2-mediated inhibition of EAE. We next generated irradiation bone marrow chimeras in which ERα expression was selectively impaired in inflammatory T lymphocytes or was limited to the radiosensitive hemopoietic compartment. Our data show that the protective effect of E2 on clinical EAE and CNS inflammation was not dependent on ERα signaling in inflammatory T cells. Likewise, EAE development was not prevented by E2 treatment in chimeric mice that selectively expressed ERα in the systemic immune compartment. In conclusion, our data demonstrate that the beneficial effect of E2 on this autoimmune disease does not involve ERα signaling in blood-derived inflammatory cells, and indicate that ERα expressed in other tissues, such as CNS-resident microglia or endothelial cells, mediates this effect.

Endothelial Estrogen Receptor-α Plays a Crucial Role in the Atheroprotective Action of 17β-Estradiol in Low-Density Lipoprotein Receptor–Deficient Mice

Background— The prevention of early atheroma by estrogens has been clearly demonstrated in all animal models and appears to be mediated through a direct action on the arterial wall rather than through an effect on the lipoprotein profile. The goal of the present study was to evaluate which cellular target is crucial in this beneficial action of estradiol. Methods and Results— We first confirmed the key role of estrogen receptor-&agr; (ER&agr;) in the atheroprotective effect of estradiol, because this action was completely abolished in mice deficient in both the low-density lipoprotein receptor (LDLr) and ER&agr;. Second, using chimeric mice with an ER&agr; deficiency in the hematopoietic lineage, we showed the persistence of the protective action of estradiol, which suggests the involvement of extrahematopoietic ER&agr;. Third, we showed that loxP-flanked ER&agr; mice (ER&agr;flox/flox) bred with Tie2-Cre+ mice on an LDLr−/− background had complete inactivation of ER&agr; in most hematopoietic and all endothelial cells. Remarkably, in this mouse model, the atheroprotective effect of estradiol was completely abolished. Fourth, the atheroprotective effect of estradiol remained abolished in Tie2-Cre+ ER&agr;flox/flox LDLr−/− mice transplanted with either Tie2-Cre+ ER&agr;flox/flox or ER&agr;−/− bone marrow, whereas it was present in analogous chimeric Tie2-Cre− ER&agr;flox/flox LDLr−/− receivers expressing endothelial ER&agr;. Conclusions— We demonstrate directly and for the first time that endothelial ER&agr; represents a key target of the atheroprotective effect of estradiol, whereas hematopoietic ER&agr; is dispensable. Selective estrogen receptor modulators that mimic the endothelial action of estradiol should now be considered in atheroprotection.