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.

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.

Suicidal B cells: In response to an infection, immunological B cells undergo a maturation process that results in the production of immunoglobulin (Ig) that is better able to bind and clear the invading pathogen. This occurs through somatic cell hypermutation and class switch recombination of the Ig gene and requires activation-induced deaminase (AID). Péron et al. (p. 931 , published online 26 April) observed that the 3' cis-regulatory region of the heavy-chain locus is transcribed and undergoes AID-mediated mutation and recombination. The resulting deletion of the Ig heavy gene cluster generates B cells that are no longer able to express Ig on the cell surface. Because cell-surface Ig expression is essential for B cell survival, this process is termed “locus suicide recombination” (LSR) and may be important in shaping the dynamics of B effector cell differentiation and homeostasis.

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-α (ERα) in the atheroprotective effect of estradiol, because this action was completely abolished in mice deficient in both the low-density lipoprotein receptor (LDLr) and ERα. Second, using chimeric mice with an ERα deficiency in the hematopoietic lineage, we showed the persistence of the protective action of estradiol, which suggests the involvement of extrahematopoietic ERα. Third, we showed that loxP-flanked ERα mice (ERα ^flox/flox ) bred with Tie2-Cre ⁺ mice on an LDLr ^−/− background had complete inactivation of ERα 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 ⁺ ERα ^flox/flox LDLr ^−/− mice transplanted with either Tie2-Cre ⁺ ERα ^flox/flox or ERα ^−/− bone marrow, whereas it was present in analogous chimeric Tie2-Cre ⁻ ERα ^flox/flox LDLr ^−/− receivers expressing endothelial ERα. Conclusions— We demonstrate directly and for the first time that endothelial ERα represents a key target of the atheroprotective effect of estradiol, whereas hematopoietic ERα is dispensable. Selective estrogen receptor modulators that mimic the endothelial action of estradiol should now be considered in atheroprotection.

Dendritic cells (DC) are critical actors in the initiation of primary immune responses and regulation of self-tolerance. The steroid sex hormone 17β-estradiol (E2) has been shown to promote the differentiation of DCs from bone marrow (BM) precursors in vitro. However, the estrogen receptor (ER) involved in this effect has not yet been characterized. Using recently generated ERα- or ERβ-deficient mice, we investigated the role of ER isotypes in DC differentiation and acquisition of effector functions. We report that estrogen-dependent activation of ERα, but not ERβ, is required for normal DC development from BM precursors cultured with GM-CSF. We show that reduced numbers of DCs were generated in the absence of ERα activation and provide evidence for a cell-autonomous function of ERα signaling in DC differentiation. ERα-deficient DCs were phenotypically and functionally distinct from wild-type DCs generated in the presence of estrogens. In response to microbial components, ERα-deficient DCs failed to up-regulate MHC class II and CD86 molecules, which could account for their reduced capacity to prime naive CD4+ T lymphocytes. Although they retained the ability to express CD40 and to produce proinflammatory cytokines (e.g., IL-12, IL-6) upon TLR engagement, ERα-deficient DCs were defective in their ability to secrete such cytokines in response to CD40–CD40L interactions. Taken together, these results provide the first genetic evidence that ERα is the main receptor regulating estrogen-dependent DC differentiation in vitro and acquisition of their effector functions.

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 IgG2a^b (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.