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In mammals, males and females show marked differences in immune responses. Males are globally more sensitive to infectious diseases, while females are more susceptible to systemic autoimmunity. X-chromosome inactivation (XCI), the epigenetic mechanism ensuring the silencing of one X in females, may participate in these sex biases. We perturbed the expression of the trigger of XCI, the noncoding RNA Xist , in female mice. This resulted in reactivation of genes on the inactive X, including members of the Toll-like receptor 7 (TLR7) signaling pathway, in monocyte/macrophages and dendritic and B cells. Consequently, female mice spontaneously developed inflammatory signs typical of lupus, including anti-nucleic acid autoantibodies, increased frequencies of age-associated and germinal center B cells, and expansion of monocyte/macrophages and dendritic cells. Mechanistically, TLR7 signaling is dysregulated in macrophages, leading to sustained expression of target genes upon stimulation. These findings provide a direct link between maintenance of XCI and female-biased autoimmune manifestations and highlight altered XCI as a cause of autoimmunity.

Males and females exhibit profound differences in immune responses and disease susceptibility. However, the factors responsible for sex differences in tissue immunity remain poorly understood. Here, we uncovered a dominant role for type 2 innate lymphoid cells (ILC2s) in shaping sexual immune dimorphism within the skin. Mechanistically, negative regulation of ILC2s by androgens leads to a reduction in dendritic cell accumulation and activation in males, along with reduced tissue immunity. Collectively, our results reveal a role for the androgen-ILC2-dendritic cell axis in controlling sexual immune dimorphism. Moreover, this work proposes that tissue immune set points are defined by the dual action of sex hormones and the microbiota, with sex hormones controlling the strength of local immunity and microbiota calibrating its tone.

Toll-like receptor 7 (TLR7) triggers antiviral immune responses through its capacity to recognize single-stranded RNA. TLR7 loss-of-function mutants are associated with life-threatening pneumonia in severe COVID-19 patients. Whereas TLR7-driven innate induction of type I IFN appears central to control SARS-CoV2 virus spreading during the first days of infection, the impact of TLR7-deficiency on adaptive B-cell immunity is less clear. In the present study, we examined the role of TLR7 in the adaptive B cells response to various pathogen-like antigens (PLAs). We used inactivated SARS-CoV2 and a PLA-based COVID-19 vaccine candidate designed to mimic SARS-CoV2 with encapsulated bacterial ssRNA as TLR7 ligands and conjugated with the RBD of the SARS-CoV2 Spike protein. Upon repeated immunization with inactivated SARS-CoV2 or PLA COVID-19 vaccine, we show that Tlr7-deficiency abolished the germinal center (GC)-dependent production of RBD-specific class-switched IgG2b and IgG2c, and neutralizing antibodies to SARS-CoV2. We also provide evidence for a non-redundant role for B-cell-intrinsic TLR7 in the promotion of RBD-specific IgG2b/IgG2c and memory B cells. Together, these data demonstrate that the GC reaction and class-switch recombination to the Myd88-dependent IgG2b/IgG2c in response to SARS-CoV2 or PLAs is strictly dependent on cell-intrinsic activation of TLR7 in B cells., (© 2023 Wiley-VCH GmbH.)

Background: Human endosomal Toll-like receptors TLR7 and TLR8 recognize self and non-self RNA ligands, and are important mediators of innate immunity and autoimmune pathogenesis. TLR7 and TLR8 are, respectively, encoded by adjacent X-linked genes. We previously established that TLR7 evades X chromosome inactivation (XCI) in female immune cells. Whether TLR8 also evades XCI, however, has not yet been explored., Method: In the current study, we used RNA fluorescence in situ hybridization (RNA FISH) to directly visualize, on a single-cell basis, primary transcripts of TLR7 and TLR8 relative to X chromosome territories in CD14 + monocytes and CD4 + T lymphocytes from women, Klinefelter syndrome (KS) men, and euploid men. To assign X chromosome territories in cells lacking robust expression of a XIST compartment, we designed probes specific for X-linked genes that do not escape XCI and therefore robustly label the active X chromosome. We also assessed whether XCI escape of TLR8 was associated with sexual dimorphism in TLR8 protein expression by western blot and flow cytometry., Results: Using RNA FISH, we show that TLR8, like TLR7, evades XCI in immune cells, and that cells harboring simultaneously TLR7 and TLR8 transcript foci are more frequent in women and KS men than in euploid men, resulting in a sevenfold difference in frequency. This transcriptional bias was again observable when comparing the single X of XY males with the active X of cells from females or KS males. Interestingly, TLR8 protein expression was significantly higher in female mononuclear blood cells, including all monocyte subsets, than in male cells., Conclusions: TLR8, mirroring TLR7, escapes XCI in human monocytes and CD4 + T cells. Co-dependent transcription from the active X chromosome and escape from XCI could both contribute to higher TLR8 protein abundance in female cells, which may have implications for the response to viruses and bacteria, and the risk of developing inflammatory and autoimmune diseases., (© 2023. Society for Women's Health Research and BioMed Central Ltd.)

Females have better ability to resolve infections, compared to males, but also, a greater susceptibility to develop autoimmunity. Besides the initial interest on the contribution of sex-steroid hormone signaling, the role of genetic factors linked to X chromosome has recently focused much attention. In human and mouse, the number of X chromosomes, rather than sex-steroid hormones, have been found associated with higher risk or susceptibility to develop autoimmunity, particularly rheumatic diseases, such as SLE, Sjögren's syndrome or Scleroderma. For all of these diseases, the Toll-like receptor TLR7 and TLR8, encoded on the same locus in the human Xp, have been demonstrated to be causal in disease development through gene dosage effect or gain of function mutations. During embryonic development in female mammals, one X chromosome is stochastically inactivated to balance X-linked gene expression between males and females, a process known as X chromosome inactivation (XCI). Nevertheless, some genes including immune related genes can escape XCI to variable degree and penetrance, resulting in a bi-allelic expression in some immune cells, such as TLR7. Because tight regulation of TLR expression is necessary for a healthy, self-tolerant immune environment, XCI escape has been proposed as a mechanism contributing to this sexual dimorphism. In this review, we will summarize general mechanisms of XCI, and describe the known escapee's genes in immune cells, the cellular diversity created by such mechanisms and its potential implication in autoimmune diseases, with a particular focus on the X-linked genes and immune cell populations involved in SLE. Whether dysregulated expression of X-linked genes could contribute to the enhanced susceptibility of females to develop such diseases remains to be proven. Shedding lights onto the X-linked genetic mechanisms contributing to modulation of immune cell functions will undoubtedly provide new insights into the intricate mechanisms underlying sex differences in immunity and autoimmunity., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Males and females differ in their susceptibility to develop autoimmunity and allergy but also in their capacity to cope with infections and cancers. Cellular targets and molecular pathways underlying sexual dimorphism in immunity have started to emerge and appeared multifactorial. It became increasingly clear that sex-linked biological factors have important impact on the development, tissue maintenance and effector function acquisition of distinct immune cell populations, thereby regulating multiple layers of innate or adaptive immunity through distinct mechanisms. This review discusses the recent development in our understanding of the cell-intrinsic actions of biological factors linked to sex, sex hormones and sex chromosome complement, on immune cells, which may account for the sex differences in susceptibility to autoimmune diseases and allergies, and the sex-biased responses in natural immunity and cancer., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Plasmacytoid dendritic cells (pDCs) express TLR7, a ssRNA-sensor encoded on the X chromosome, which escapes X chromosome inactivation (XCI) in females. pDCs are specialized in the production of type 1 interferons (IFN-I) through TLR7 activation which mediates both immune cell activation and also reactivation of latent HIV-1. The effect of HIV-1 infection in women under antiretroviral therapy (ART) on pDC functional responses remains poorly understood. Here, we show that pDCs from HIV/ART women exhibit exacerbated production of IFN-α and TNF-α compared with uninfected controls (UC) upon TLR7 activation. Because TLR7 can escape XCI in female pDCs, we measured the contribution of TLR7 allelic expression using SNP haplotypic markers to rigorously tag the allele of origin of TLR7 gene at single-cell resolution. Herein, we provide evidence that the enhanced functional response of pDCs in HIV/ART women is associated with higher transcriptional activity of the TLR7 locus from both X chromosomes, rather than differences in the frequency of TLR7 biallelic cells. These data reinforce the interest in targeting the HIV-1 reservoir using TLR7 agonists in women., (© 2022 Abbas et al.)

Background: Type I interferon (IFN-I) production by plasmacytoid dendritic cells (pDCs) occurs during viral infection, in response to Toll-like receptor 7 (TLR7) stimulation and is more vigorous in females than in males. Whether this sex bias persists in ageing people is currently unknown. In this study, we investigated the effect of sex and aging on IFN-α production induced by PRR agonist ligands., Methods: In a large cohort of individuals from 19 to 97 years old, we measured the production of IFN-α and inflammatory cytokines in whole-blood upon stimulation with either R-848, ODN M362 CpG-C, or cGAMP, which activate the TLR7/8, TLR9 or STING pathways, respectively. We further characterized the cellular sources of IFN-α., Findings: We observed a female predominance in IFN-α production by pDCs in response to TLR7 or TLR9 ligands. The higher TLR7-driven IFN-α production in females was robustly maintained across ages, including the elderly. The sex-bias in TLR9-driven interferon production was lost after age 60, which correlated with the decline in circulating pDCs. By contrast, STING-driven IFN-α production was similar in both sexes, preserved with aging, and correlated with circulating monocyte numbers. Indeed, monocytes were the primary cellular source of IFN-α in response to cGAMP., Interpretation: We show that the sex bias in the TLR7-induced IFN-I production is strongly maintained through ages, and identify monocytes as the main source of IFN-I production via STING pathway., Funding: This work was supported by grants from Région Occitanie/Pyrénées-Méditerranée (#12052910, Inspire Program #1901175), University Paul Sabatier, and the European Regional Development Fund (MP0022856)., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Tissue-resident innate lymphoid cells (ILCs) regulate tissue homeostasis, protect against pathogens at mucosal surfaces, and are key players at the interface of innate and adaptive immunity. How ILCs adapt their phenotype and function to environmental cues within tissues remains to be fully understood. Here, we show that Mycobacterium tuberculosis (Mtb) infection alters the phenotype and function of lung IL-18Rα + ILC toward a protective interferon-γ-producing ILC1-like population. This differentiation is controlled by type 1 cytokines and is associated with a glycolytic program. Moreover, a BCG-driven type I milieu enhances the early generation of ILC1-like cells during secondary challenge with Mtb. Collectively, our data reveal how tissue-resident ILCs adapt to type 1 inflammation toward a pathogen-tailored immune response., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Type-17 inflammation characterizes psoriasis, a chronic skin disease. Because several inflammatory cytokines contribute to psoriasis pathogenesis, inhibiting the simultaneous production of these cytokines in T H 17 cells may be beneficial in psoriasis. We found that Ca v 1.4, encoded by CACNA1F, was the only Ca v 1 calcium channel expressed in T H 17 cells., Objective: We sought to investigate the role of Ca v 1.4 expression in early T H 17-activation events and effector functions, as well as its association with T H 17 signature genes in lesional psoriatic (LP) skins., Methods: Transcriptional gene signatures associated with CACNA1F expression were examined in LP skins by RT-PCR and in situ hybridization. Ca v 1 inhibitor and/or shRNA lentivectors were used to assess the contribution of Ca v 1.4 in T H 17 activation and effector functions in a 3-dimensional skin reconstruction model., Results: CACNA1F expression correlated with inflammatory cytokine expression that characterizes LP skins and was preferentially associated with RORC expression in CD4 + and CD4 - cells from LP biopsies. Nicardipine, a Ca v 1 channel antagonist, markedly reduced inflammatory cytokine production by T H 17 cells from blood or LP skin. This was associated with decreased TCR-induced early calcium events at cell membrane and proximal signaling events. The knockdown of Ca v 1.4 in T H 17 cells impaired cytokine production. Finally, Ca v 1 inhibition reduced the expression of the keratinocyte genes characteristic of T H 17-mediated psoriasis inflammation in human skin equivalents., Conclusions: Ca v 1.4 channels promote T H 17-cell functions both at the periphery and in inflammatory psoriatic skin., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Voltage-gated calcium (Ca v 1) channels contribute to T-lymphocyte activation. Ca v 1.2 and Ca v 1.3 channels are expressed in Th2 cells but their respective roles are unknown, which is investigated herein., Methods: We generated mice deleted for Ca v 1.2 in T cells or Ca v 1.3 and analyzed TCR-driven signaling. In this line, we developed original fast calcium imaging to measure early elementary calcium events (ECE). We also tested the impact of Ca v 1.2 or Ca v 1.3 deletion in models of type 2 airway inflammation. Finally, we checked whether the expression of both Ca v 1.2 and Ca v 1.3 in T cells from asthmatic children correlates with Th2-cytokine expression., Results: We demonstrated non-redundant and synergistic functions of Ca v 1.2 and Ca v 1.3 in Th2 cells. Indeed, the deficiency of only one channel in Th2 cells triggers TCR-driven hyporesponsiveness with weakened tyrosine phosphorylation profile, a strong decrease in initial ECE and subsequent reduction in the global calcium response. Moreover, Ca v 1.3 has a particular role in calcium homeostasis. In accordance with the singular roles of Ca v 1.2 and Ca v 1.3 in Th2 cells, deficiency in either one of these channels was sufficient to inhibit cardinal features of type 2 airway inflammation. Furthermore, Ca v 1.2 and Ca v 1.3 must be co-expressed within the same CD4 + T cell to trigger allergic airway inflammation. Accordingly with the concerted roles of Ca v 1.2 and Ca v 1.3, the expression of both channels by activated CD4 + T cells from asthmatic children was associated with increased Th2-cytokine transcription., Conclusions: Thus, Ca v 1.2 and Ca v 1.3 act as a duo, and targeting only one of these channels would be efficient in allergy treatment., (© 2021 European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Background: Allergic asthma is more severe and frequent in women than in men. In male mice, androgens negatively control group 2 innate lymphoid cell (ILC2) development and function by yet unknown mechanisms., Objectives: We sought to investigate the impact of androgen on ILC2 homeostasis and IL-33-mediated inflammation in female lungs. We evaluated the role of androgen receptor (AR) signaling and the contribution of the putative inhibitory receptor killer cell lectin-like receptor G1 (KLRG1)., Methods: Subcutaneous pellets mimicking physiological levels of androgen were used to treat female mice together with mice expressing a reporter enzyme under the control of androgen response elements and mixed bone marrow chimeras to assess the cell-intrinsic role of AR activation within ILC2s. We generated KLRG1-deficient mice., Results: We established that lung ILC2s express a functionally active AR that can be in vivo targeted with exogenous androgens to negatively control ILC2 homeostasis, proliferation, and function. Androgen signaling upregulated KLRG1 on ILC2s, which inhibited their proliferation on E-cadherin interaction. Despite evidence that KLRG1 impaired the competitive fitness of lung ILC2s during inflammation, KLRG1 deficiency neither alters in vivo ILC2 numbers and functions, nor did it lead to hyperactive ILC2s in either sexes., Conclusions: AR agonists can be used in vivo to inhibit ILC2 homeostatic numbers and ILC2-dependent lung inflammation through cell-intrinsic AR activation. Although androgen signals in ILC2s to upregulate KLRG1, we demonstrate that KLRG1 is dispensable for androgen-mediated inhibition of pulmonary ILC2s., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Toll-like receptor 7 (TLR7) triggers antiviral immune responses through its capacity to recognize ssRNA. Proteolytic cleavage of TLR7 protein is required for its functional maturation in the endosomal compartment. Structural studies demonstrated that the N- and C-terminal domains of TLR7 are connected and involved in ligand binding after cleavage. Hydroxychloroquine (HCQ), an antimalarial drug, has been studied for its antiviral effects. HCQ increases pH in acidic organelles and has been reported to potently inhibit endosomal TLR activation. Whether HCQ can prevent endogenous TLR7 cleavage in primary immune cells, such as plasmacytoid DCs (pDCs), had never been examined. Here, using a validated anti-TLR7 antibody suitable for biochemical detection of native TLR7 protein, we show that HCQ treatment of fresh PBMCs, CAL-1 leukemic, and primary human pDCs inhibits TLR7 cleavage and results in accumulation of full-length protein. As a consequence, we observe an inhibition of pDC activation in response to TLR7 stimulation with synthetic ligands and viruses including inactivated SARS-CoV2, which we show herein activates pDCs through TLR7-signaling. Together, our finding suggests that the major pathway by which HCQ inhibits ssRNA sensing by pDCs may rely on its capacity to inhibit endosomal acidification and the functional maturation of TLR7 protein., (© 2021 Wiley-VCH GmbH.)

Plasmacytoid dendritic cells (pDCs) produce type I interferon (IFN-I) during HIV-1 infection in response to TLR7 stimulation. However, IFN-I-signaling has been shown to play opposite effects in HIV-1 and SIV infection. TLR7-driven type I interferon production in pDCs is higher in women than in men due to the cell-intrinsic actions of estrogen and X-chromosome complement. Indeed, TLR7 is encoded on the X-chromosome, and the TLR7 gene escapes the X-chromosome inactivation in immune cells of women which express significantly higher levels of TLR7 protein than male cells. Following HIV infection, women have a lower viremia during acute infection and exhibit stronger antiviral responses than men, which has been attributed to the increased capacity of female pDCs to produce IFN-α upon TLR7-stimulation. However, a deleterious functional impact of an excessive TLR7 response on acute viremia in women has been recently revealed by the analysis of the frequent rs179008 c.32A>T SNP of TLR7 . This SNP was identified as a sex-specific protein abundance quantitative trait locus (pQTL) causing a difference in the TLR7 protein dosage and effector function in females only. T allele expression was associated with a lower TLR7 protein synthesis, blunted production of IFN-α by pDCs upon TLR7 stimulation, and an unexpectedly lower viral load during primary HIV-1 infection in women. In the present review, the author will revisit the role of TLR7-driven pDC innate function in the context of HIV-1 infection to discuss at what stage of primary HIV-1 infection the TLR7 rs179008 T allele is likely to be protective in women., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Guéry.)

Innate lymphoid cell (ILC) subsets at barrier surfaces contribute to maintain tissue homeostasis and appropriate responses to infection. ILCs respond to environmental factors produced by non-hematopoietic cells within tissues, but also circulating cytokines or dietary compounds which allow them to adapt to organ milieu. Among these extrinsic signals, evidence is emerging that sex steroid hormones may act in a cell-intrinsic manner to regulate the development, maintenance in tissues and effector functions of specific subsets of ILCs. Understanding the nature and molecular mechanisms of sex steroid hormone actions on ILCs is important to unravel the cause of sexual disparity in human diseases and could lead to new drug development for the treatment of chronic inflammatory diseases or cancers. This review discusses the recent development in our understanding of the cell-intrinsic actions of sex steroid hormones on ILCs and their consequences on tissue-specific immunity with a particular focus on group 2 innate lymphoid cells and NK cells., Competing Interests: Conflicts of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Chang Gung University. Published by Elsevier B.V. All rights reserved.)

Women represent 80% of people affected by autoimmune diseases. Although, many studies have demonstrated a role for sex hormone receptor signaling, particularly estrogens, in the direct regulation of innate and adaptive components of the immune system, recent data suggest that female sex hormones are not the only cause of the female predisposition to autoimmunity. Besides sex steroid hormones, growing evidence points towards the role of X-linked genetic factors. In female mammals, one of the two X chromosomes is randomly inactivated during embryonic development, resulting in a cellular mosaicism, where about one-half of the cells in a given tissue express either the maternal X chromosome or the paternal one. X chromosome inactivation (XCI) is however not complete and 15 to 23% of genes from the inactive X chromosome (Xi) escape XCI, thereby contributing to the emergence of a female-specific heterogeneous population of cells with bi-allelic expression of some X-linked genes. Although the direct contribution of this genetic mechanism in the female susceptibility to autoimmunity still remains to be established, the cellular mosaicism resulting from XCI escape is likely to create a unique functional plasticity within female immune cells. Here, we review recent findings identifying key immune related genes that escape XCI and the relationship between gene dosage imbalance and functional responsiveness in female cells.

Type I IFN (IFN-I) production by plasmacytoid DCs (pDCs) occurs during acute HIV-1 infection in response to TLR7 stimulation, but the role of pDC-derived IFN-I in controlling or promoting HIV-1 infection is ambiguous. We report here a sex-biased interferogenic phenotype for a frequent single-nucleotide polymorphism of human TLR7, rs179008, displaying an impact on key parameters of acute HIV-1 infection. We show allele rs179008 T to determine lower TLR7 protein abundance in cells from women, specifically - likely by diminishing TLR7 mRNA translation efficiency through codon usage. The hypomorphic TLR7 phenotype is mirrored by decreased TLR7-driven IFN-I production by female pDCs. Among women from the French ANRS PRIMO cohort of acute HIV-1 patients, carriage of allele rs179008 T associated with lower viremia, cell-associated HIV-1 DNA, and CXCL10 (IP-10) plasma concentrations. RNA viral load was decreased by 0.85 log10 (95% CI, -1.51 to -0.18) among T/T homozygotes, who also exhibited a lower frequency of acute symptoms. TLR7 emerges as an important control locus for acute HIV-1 viremia, and the clinical phenotype for allele rs179008 T, carried by 30%-50% of European women, supports a beneficial effect of toning down TLR7-driven IFN-I production by pDCs during acute HIV-1 infection.

Asthma and allergy: what about the differences between men and women? Allergic asthma is a chronic pulmonary disease characterized by bronchial hyper responsiveness, hyper production of mucus and remodeling of the airways. Asthma, which often begins before the age of 5, is the most common chronic disease in children and affects approximately 10% of the population in affluent societies. As it is the case for many allergic diseases, asthma affects men and women differently. In childhood, pathology is more common in boys, but this trend reverses at puberty, suggesting a regulation by sex hormones. In this review, we summarize the current knowledge on how sex hormones impact allergic inflammation and particularly describe the protective actions of androgens on the development and function of key immune cell subsets involved in allergic responses., Competing Interests: Les auteurs déclarent que leur recherche est menée indépendamment de toute relation commerciale ou financière, et n’avoir aucun lien d’intérêts.

Plasmacytoid dendritic cells (pDCs) are found in the CNS during neuroinflammation and have been reported to exert regulatory functions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). However, the mechanisms of entry of pDCs into the CNS as well as their phenotype and innate functional properties, once recruited into the CNS, have not been thoroughly examined. Herein, we show that pDCs rapidly accumulate into the brain and spinal cord during the acute phase of EAE, and maintain the expression of numerous phenotypic markers typical of peripheral pDCs. Functionally, CNS-pDCs constitutively expressed IRF7 and were able to rapidly produce type I IFNs and IL-12p40 upon ex vivo TLR-9 stimulation. Using adoptive transfer experiments, we provide evidence that CNS-pDC are recruited from the blood and accumulate into the CNS during the acute phase of EAE. Accumulation of pDCs into the CNS was strongly inhibited in the absence of CD29, but not CD18, suggesting a major role for ß1 but not ß2 integrins. Indeed, blocking the CD49d α4-integrins during acute EAE drastically diminished CNS-pDC numbers. Together, our results demonstrate that circulating pDCs are actively recruited into the CNS during acute EAE through a mechanism largely dependent on CD49d/CD29-integrins., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Women develop stronger immune responses than men, with positive effects on the resistance to viral or bacterial infections but magnifying also the susceptibility to autoimmune diseases like systemic lupus erythematosus (SLE). In SLE, the dosage of the endosomal Toll-like receptor 7 (TLR7) is crucial. Murine models have shown that TLR7 overexpression suffices to induce spontaneous lupus-like disease. Conversely, suppressing TLR7 in lupus-prone mice abolishes SLE development. TLR7 is encoded by a gene on the X chromosome gene, denoted TLR7 in humans and Tlr7 in the mouse, and expressed in plasmacytoid dendritic cells (pDC), monocytes/macrophages, and B cells. The receptor recognizes single-stranded RNA, and its engagement promotes B cell maturation and the production of pro-inflammatory cytokines and antibodies. In female mammals, each cell randomly inactivates one of its two X chromosomes to equalize gene dosage with XY males. However, 15 to 23% of X-linked human genes escape X chromosome inactivation so that both alleles can be expressed simultaneously. It has been hypothesized that biallelic expression of X-linked genes could occur in female immune cells, hence fostering harmful autoreactive and inflammatory responses. We review here the current knowledge of the role of TLR7 in SLE, and recent evidence demonstrating that TLR7 escapes from X chromosome inactivation in pDCs, monocytes, and B lymphocytes from women and Klinefelter syndrome men. Female B cells where TLR7 is thus biallelically expressed display higher TLR7-driven functional responses, connecting the presence of two X chromosomes with the enhanced immunity of women and their increased susceptibility to TLR7-dependent autoimmune syndromes.

Men and women differ in their susceptibility to develop autoimmunity and allergy but also in their capacity to cope with infections. Mechanisms responsible for this sexual dimorphism are still poorly documented and probably multifactorial. This review discusses the recent development in our understanding of the cell-intrinsic actions of biological factors linked to sex, sex hormones and sex chromosome complement, on immune cells, which may account for the sex differences in the enhanced susceptibility of women to develop immunological disorders, such as allergic asthma or systemic lupus erythematosus (SLE). We choose to more specifically discuss the impact of sex hormones on the development and function of immune cell populations directly involved in type-2 immunity, and the role of the X-linked Toll like receptor 7 (TLR7) in anti-viral immunity and in SLE. We will also elaborate on the recent evidence demonstrating that TLR7 escapes from X chromosome inactivation in the immune cells of women, and how this may contribute to endow woman immune system with enhanced responsiveness to RNA-virus and susceptibility to SLE., (© 2019 Elsevier Inc. All rights reserved.)

17β-Estradiol (E2) suppresses the development of experimental autoimmune encephalomyelitis (EAE) through estrogen receptor (ER) α, yet the cellular targets remain elusive. We have used an adoptive transfer model of myelin oligodendrocyte glycoprotein-specific CD4 + T cells from 2D2 TCR transgenic mice. We show that in the recipient mice, ERα expression in bystander CD4 + T cells, rather than in cognate 2D2 T cells, is required for the inhibition of Th17 cell differentiation by E2. Coadministration of estrogen-primed WT, but not ERα-deficient CD4 + T cells, with naive 2D2 T cells lacking ERα inhibited the development of Th17 cell-mediated EAE. Suppression of Th17 cells and protection from EAE were maintained when ERα was deleted in Foxp3 + regulatory T cells. We showed that in vivo PD-L1 blockade alleviated the anti-inflammatory action of E2 and that PD-1 expression on cognate but not bystander T cells was required for the E2-dependent inhibition of Th17 differentiation. In cotransfer experiments, we found that only WT but not PD-1 KO 2D2 T cells were amenable to E2-dependent inhibition of Th17 differentiation. These results support the conclusion that the restriction of Th17 cell development by E2-primed bystander CD4 + T cells requires cell-intrinsic PD-1 signaling within cognate T cells rather than induction of regulatory 2D2 T cells through PD-1 engagement. Altogether, our results indicate that pregnancy-level concentrations of estrogen signal in conventional Foxp3 neg CD4 + T cells to limit the differentiation of cognate Th17 cells through a trans -acting mechanism of suppression that requires a functional PD-1/PD-L1 regulatory axis., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Background: T lymphocytes express not only cell membrane ORAI calcium release-activated calcium modulator 1 but also voltage-gated calcium channel (Ca v ) 1 channels. In excitable cells these channels are composed of the ion-forming pore α1 and auxiliary subunits (β and α2δ) needed for proper trafficking and activation of the channel. Previously, we disclosed the role of Ca v 1.2 α1 in mouse and human T H 2 but not T H 1 cell functions and showed that knocking down Ca v 1 α1 prevents experimental asthma., Objective: We investigated the role of β and α2δ auxiliary subunits on Ca v 1 α1 function in T H 2 lymphocytes and on the development of acute allergic airway inflammation., Methods: We used Ca v β antisense oligonucleotides to knock down Ca v β and gabapentin, a drug that binds to and inhibits α2δ1 and α2δ2, to test their effects on T H 2 functions and their capacity to reduce allergic airway inflammation., Results: Mouse and human T H 2 cells express mainly Ca v β1, β3, and α2δ2 subunits. Ca v β antisense reduces T-cell receptor-driven calcium responses and cytokine production by mouse and human T H 2 cells with no effect on T H 1 cells. Ca v β is mainly involved in restraining Ca v 1.2 α1 degradation through the proteasome because a proteasome inhibitor partially restores the α1 protein level. Gabapentin impairs the T-cell receptor-driven calcium response and cytokine production associated with the loss of α2δ2 protein in T H 2 cells., Conclusions: These results stress the role of Ca v β and α2δ2 auxiliary subunits in the stability and activation of Ca v 1.2 channels in T H 2 lymphocytes both in vitro and in vivo, as demonstrated by the beneficial effect of Ca v β antisense and gabapentin in allergic airway inflammation., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Allergic asthma is a chronic pulmonary inflammatory disease initiated by exposure to normally harmless allergens and marked by bronchial hyperreactivity. It affects more than 300 million people worldwide. Asthma often starts in childhood. Epidemiological studies show that there are sexual disparities in the prevalence and severity of asthma. Before the age of 10, the disease is more common in boys. This tendency reverses at puberty suggesting a regulating role of the sex hormones. In this synthesis, we summarize current knowledge on the role of sex hormones in allergic inflammation, with a particular focus on the impact of androgens on the development and function of recently introduced group 2 innate lymphoid cell subsets (ILC2) as critical actors in the initiation of allergic responses.♢., (© 2018 médecine/sciences – Inserm.)

Toll-like receptor 7 (TLR7) is critical to the induction of antiviral immunity, but TLR7 dosage is also a key pathogenic factor in systemic lupus erythematosus (SLE), an autoimmune disease with strong female bias. SLE prevalence is also elevated in individuals with Klinefelter syndrome, who carry one or more supernumerary X chromosomes, suggesting that the X chromosome complement contributes to SLE susceptibility. TLR7 is encoded by an X chromosome locus, and we examined here whether the TLR7 gene evades silencing by X chromosome inactivation in immune cells from women and Klinefelter syndrome males. Single-cell analyses of TLR7 allelic expression demonstrated that substantial fractions of primary B lymphocytes, monocytes, and plasmacytoid dendritic cells not only in women but also in Klinefelter syndrome males express TLR7 on both X chromosomes. Biallelic B lymphocytes from women displayed greater TLR7 transcriptional expression than the monoallelic cells, correlated with higher TLR7 protein expression in female than in male leukocyte populations. Biallelic B cells were preferentially enriched during the TLR7-driven proliferation of CD27 + plasma cells. In addition, biallelic cells showed a greater than twofold increase over monoallelic cells in the propensity to immunoglobulin G class switch during the TLR7-driven, T cell-dependent differentiation of naive B lymphocytes into immunoglobulin-secreting cells. TLR7 escape from X inactivation endows the B cell compartment with added responsiveness to TLR7 ligands. This finding supports the hypothesis that enhanced TLR7 expression owing to biallelism contributes to the higher risk of developing SLE and other autoimmune disorders in women and in men with Klinefelter syndrome., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Infectious diseases, autoimmune diseases, and also allergy differentially affect women and men. In general, women develop strongest immune responses and thus the proportion of infected individuals and the severity of many viral, bacterial, or parasitic infections are increased in men. However, heightened immunity in women makes them more susceptible than men to autoimmunity and allergy. While sex differences in immunity are well documented, little is known about the cellular and molecular mechanisms underlying these immunological differences, particularly in allergic asthma. Asthma is a chronic inflammation of the airways mediated by exacerbated type 2 immune responses. Sex differences have been reported in the incidence, prevalence, and severity of asthma. While during childhood, males are more susceptible to asthma than females, there is a switch at the onset of puberty as for many other allergic diseases. This decrease of asthma incidence around puberty in males suggests that hormonal mediators could play a protective role in the susceptibility to allergic responses in male. Group 2 innate lymphoid cells (ILC2s) have recently emerged as critical players in the initiation of allergic responses, but also in the resolution of parasitic infection, through their capacity to rapidly and potently produce type 2 cytokines. This review will cover the current understanding of the impact of sex-linked factors in allergic inflammation, with a particular focus on the role of sex hormones on the development and function of tissue-resident ILC2s.

Prevalence of asthma is higher in women than in men, but the mechanisms underlying this sex bias are unknown. Group 2 innate lymphoid cells (ILC2s) are key regulators of type 2 inflammatory responses. Here, we show that ILC2 development is greatly influenced by male sex hormones. Male mice have reduced numbers of ILC2 progenitors (ILC2Ps) and mature ILC2s in peripheral tissues compared with females. In consequence, males exhibit reduced susceptibility to allergic airway inflammation in response to environmental allergens and less severe IL-33-driven lung inflammation, correlating with an impaired expansion of lung ILC2s. Importantly, orchiectomy, but not ovariectomy, abolishes the sex differences in ILC2 development and restores IL-33-mediated lung inflammation. ILC2Ps express the androgen receptor (AR), and AR signaling inhibits their differentiation into mature ILC2s. Finally, we show that hematopoietic AR expression limits IL-33-driven lung inflammation through a cell-intrinsic inhibition of ILC2 expansion. Thus, androgens play a crucial protective role in type 2 airway inflammation by negatively regulating ILC2 homeostasis, thereby limiting their capacity to expand locally in response to IL-33., (© 2017 Laffont et al.)

Autoimmunity, infectious diseases and cancer affect women and men differently. Because they tend to develop more vigorous adaptive immune responses than men, women are less susceptible to some infectious diseases but also at higher risk of autoimmunity. The regulation of immune responses by sex-dependent factors probably involves several non-redundant mechanisms. A privileged area of study, however, concerns the role of sex steroid hormones in the biology of innate immune cells, especially dendritic cells (DCs). In recent years, our understanding of the lineage origin of DC populations has expanded, and the lineage-committing transcription factors shaping peripheral DC subsets have been identified. Both progenitor cells and mature DC subsets express estrogen receptors (ERs), which are ligand-dependent transcription factors. This suggests that estrogens may contribute to the reported sex differences in immunity by regulating DC biology. Here, we review the recent literature and highlight evidence that estrogen-dependent activation of ERα regulates the development or the functional responses of particular DC subsets. The in vitro model of GM-CSF-induced DC differentiation shows that CD11c + CD11b int Ly6c neg cells depend on ERα activation by estrogen for their development, and for the acquisition of competence to activate naive CD4 + T lymphocytes and mount a robust pro-inflammatory cytokine response to CD40 stimulation. In this model, estrogen signaling in conjunction with GM-CSF is necessary to promote early interferon regulatory factor ( Irf ) -4 expression in macrophage-DC progenitors and their subsequent differentiation into IRF-4 hi CD11c + CD11b int Ly6c neg cells, closely related to the cDC2 subset. The Flt3L-induced model of DC differentiation in turn shows that ERα signaling promotes the development of conventional DC (cDC) and plasmacytoid DC (pDC) with higher capability of pro-inflammatory cytokine production in response to TLR stimulation. Likewise, cell-intrinsic ER signaling positively regulates the TLR-driven production of type I interferons (IFNs) in mouse pDCs in vivo . This effect of estrogens likely contributes to the greater proficiency of women's pDCs than men's as regards the production of type I IFNs elicited by TLR7 ligands. In summary, evidence is emerging in support of the notion that estrogen signaling regulates important aspects of cDC and pDC development and/or effector functions, in both mice and humans.

Increased IFN-α production contributes to the pathogenesis of infectious and autoimmune diseases. Plasmacytoid dendritic cells (pDCs) from females produce more IFN-α upon TLR7 stimulation than pDCs from males, yet the mechanisms underlying this difference remain unclear. In this article, we show that basal levels of IFN regulatory factor (IRF) 5 in pDCs were significantly higher in females compared with males and positively correlated with the percentage of IFN-α-secreting pDCs. Delivery of recombinant IRF5 protein into human primary pDCs increased TLR7-mediated IFN-α secretion. In mice, genetic ablation of the estrogen receptor 1 (Esr1) gene in the hematopoietic compartment or DC lineage reduced Irf5 mRNA expression in pDCs and IFN-α production. IRF5 mRNA levels furthermore correlated with ESR1 mRNA levels in human pDCs, consistent with IRF5 regulation at the transcriptional level by ESR1. Taken together, these data demonstrate a critical mechanism by which sex differences in basal pDC IRF5 expression lead to higher IFN-α production upon TLR7 stimulation in females and provide novel targets for the modulation of immune responses and inflammation., (Copyright © 2015 by The American Association of Immunologists, Inc.)

CD4(+) T cells polarize into effector Th subsets characterized by signature transcription factors and cytokines. Although T-bet drives Th1 responses and represses the alternative Th2, Th17, and Foxp3(+) regulatory T cell fates, the role of the T-bet-related transcription factor eomesodermin (Eomes) in CD4(+) T cells is less well understood. In this study, we analyze the expression and effects of Eomes in mouse CD4(+) T lymphocytes. We find that Eomes is readily expressed in activated CD4(+) Th1 T cells in vivo. Eomes(+) CD4(+) T cells accumulated in old mice, under lymphopenic conditions in a T cell transfer model of colitis, and upon oral Ag administration. However, despite its expression, genetic deletion of Eomes in CD4(+) T cells did not impact on IFN-γ production nor increase Th2 or Th17 responses. In contrast, Eomes deficiency favored the accumulation of Foxp3(+) cells in old mice, after in vivo differentiation of Eomes-deficient naive CD4(+) T cells, and in response to oral Ag in a cell-intrinsic way. Enforced Eomes expression during in vitro regulatory T cell induction also reduced Foxp3 transcription. Likewise, bystander Eomes-deficient CD4(+) T cells were more efficient at protecting from experimental autoimmune encephalitis compared with wild-type CD4(+) T cells. This enhanced capacity of Eomes-deficient CD4(+) T cells to inhibit EAE in trans was associated with an enhanced frequency of Foxp3(+) cells. Our data identify a novel role for Eomes in CD4(+) T cells and indicate that Eomes expression may act by limiting Foxp3 induction, which may contribute to the association of EOMES to susceptibility to multiple sclerosis., (Copyright © 2015 by The American Association of Immunologists, Inc.)

A growing body of evidence from basic and clinical studies supports the therapeutic potential of estrogens in multiple sclerosis (MS), originating from the well-established reduction in relapse rates observed among women with MS during pregnancy. The biological effects of estrogens are mediated by estrogen receptors (ERα and ERβ). Estrogens or selective ER-agonists have been shown to exert potent neuroprotective or anti-inflammatory effects in experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. A central question in EAE is to identify the cellular targets that express a functional ER isotype, and the mechanisms underlying the neuroprotective and anti-inflammatory effects of estrogens. Using pharmacological approaches targeting ER-specific functions, and genetic tools such as conditional knockout mice in which ERα or ERβ are selectively deleted in specific cell populations, a clearer picture is now emerging of the various cellular targets and downstream molecules responsible for estrogen-mediated protection against central nervous system autoimmunity.

Human plasmacytoid dendritic cells (pDCs) play a major role in innate immunity through the production of type I IFNs after TLR engagement by pathogens. Sex-based differences in the innate function of human pDCs have been established, with pDCs from women exhibiting enhanced TLR7-mediated IFN-α production as compared with pDCs from males. In mice, we recently provided evidence for a role of estrogens as a positive regulator of pDC innate functions through cell-intrinsic estrogen receptor α signaling, but did not exclude a role for other X-linked factors, particularly in human pDCs. In this study, we investigated the respective contribution of X chromosome dosage and sex hormones using a humanized mouse model in which male or female NOD-SCID-β2m(-/-) were transplanted with human progenitor cells purified from either male or female cord blood cells. We showed that, in response to TLR7 ligands, the frequency of IFN-α- and TNF-α-producing pDCs from either sex was greater in female than in male host mice, suggesting a positive role for estrogens. Indeed, blockade of estrogen receptor signaling during pDC development in vitro inhibited TLR7-mediated IFN-α production by human pDCs, which expressed both ESR1 and ESR2 genes. Interestingly, we also found that X chromosome dosage contributed to this sex bias as female pDCs have an enhanced TLR7-mediated IFN-α response as compared with male ones, irrespective of the sex of the recipient mice. Together, these results indicate that female sex hormones, estrogens, and X chromosome complement independently contribute to the enhanced TLR7-mediated IFN-α response of pDCs in women., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The recognition of multiple ligands by a single TCR is an intrinsic feature of T cell biology, with important consequences for physiological and pathological processes. Polyspecific T cells targeting distinct self-antigens have been identified in healthy individuals as well as in the context of autoimmunity. We have previously shown that the 2D2 TCR recognizes the myelin oligodendrocyte glycoprotein epitope (MOG)35-55 as well as an epitope within the axonal protein neurofilament medium (NF-M15-35) in H-2(b) mice. In this study, we assess whether this cross-reactivity is a common feature of the MOG35-55-specific T cell response. To this end, we analyzed the CD4 T cell response of MOG35-55-immunized C57BL/6 mice for cross-reactivity with NF-M15-35. Using Ag recall responses, we established that an important proportion of MOG35-55-specific CD4 T cells also responded to NF-M15-35 in all mice tested. To study the clonality of this response, we analyzed 22 MOG35-55-specific T cell hybridomas expressing distinct TCR. Seven hybridomas were found to cross-react with NF-M15-35. Using an alanine scan of NF-M18-30 and an in silico predictive model, we dissected the molecular basis of cross-reactivity between MOG35-55 and NF-M15-35. We established that NF-M F24, R26, and V27 proved important TCR contacts. Strikingly, the identified TCR contacts are conserved within MOG38-50. Our data indicate that due to linear sequence homology, part of the MOG35-55-specific T cell repertoire of all C57BL/6 mice also recognizes NF-M15-35, with potential implications for CNS autoimmunity., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Background: In addition to calcium release-activated calcium channel/ORAI calcium channels, the role of voltage-gated calcium (Cav1) channels in T-cell calcium signaling is emerging. Cav1 channels are formed by α1 (CaV1.1 to CaV1.4) and auxiliary subunits. We previously demonstrated that mouse TH2 cells selectively overexpressed CaV1.2 and CaV1.3 channels. Knocking down these channels with Cav1 antisense (AS) oligonucleotides inhibited TH2 functions and experimental asthma., Objective: We investigated the expression profile and role of Cav1 channels in human T-cell subsets, with a focus on TH2 cells., Methods: We compared the profile of CaV1 channel subunit expression in T-cell subsets isolated ex vivo from the blood of healthy donors, as well as in vitro-polarized T-cell subsets, and tested the effect of the Cav1 inhibitors nicardipine and Cav1.2AS on their functions., Results: CaV1.4 expression was detectable in CD4(+) T cells, ex vivo TH1 cells, and TH17 cells, whereas Cav1.2 channels predominated in TH2 cells only. T-cell activation resulted in Cav1.4 downregulation, whereas Cav1.2 expression was selectively maintained in polarized TH2 cells and absent in TH1 or TH9 cells. Nicardipine and CaV1.2AS decreased Ca(2+) and cytokine responses in TH2, but not TH1, cells. Protein kinase C (PKC) α/β inhibition decreased Ca(2+) and cytokine responses, whereas both calcium and cytokine responses induced by PKC activation were inhibited by nicardipine or Cav1.2AS in TH2 cells., Conclusion: This study highlights the selective expression of Cav1.2 channels in human TH2 cells and the role of PKC-dependent Cav1.2 channel activation in TH2 cell function. Blocking PKC or Cav1.2 channel activation in TH2 cells might represent new strategies to treat allergic diseases in human subjects., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

17β-Estradiol (E2) has been shown to regulate GM-CSF- or Flt3 ligand-driven dendritic cell (DC) development through estrogen receptor (ER) α signaling in myeloid progenitors. ERα regulates transcription of target genes through two distinct activation functions (AFs), AF-1 and AF-2, whose respective involvement varies in a cell type- or tissue-specific manner. In this study, we investigated the role of ERα AFs in the development and effector functions of inflammatory DCs, steady-state conventional DCs, and plasmacytoid DCs (pDC), using mouse lacking either AF-1 or AF-2. In agreement with previous works, we showed that E2 fostered the differentiation and effector functions of inflammatory DCs through ERα-dependent upregulation of IFN regulatory factor (IRF)-4 in GM-CSF-stimulated myeloid progenitors. Interestingly, whereas AF-1 was required for early IRF-4 upregulation in DC precursors, it was dispensable to enhance IRF-4 expression in differentiated DCs to a level compatible with the development of the more functional Ly6C(-) CD11b(+) DC subset. Presence of E2 had no effect on progenitors from either knock-in mice with 7-aa deletion in helix 12 of ERα, lacking AF-2, or ERα(-/-) mice. By contrast, in Flt3 ligand-driven DC differentiation, activation of AF-1 domain was required to promote the development of more functionally competent conventional DCs and pDCs. Moreover, lack of ERα AF-1 blunted the TLR7-mediated IFN-α response of female pDCs in vivo. Thus, our study demonstrates that ERα uses AF-1 differently in steady-state and inflammatory DC lineages to regulate their innate functions, suggesting that selective ER modulators could be used to target specific DC subsets.

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 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.

Estrogens influence the incidence and the course of numerous immune or inflammatory diseases in humans and in experimental models. For instance, estrogens prevent the accumulation of granulocytes in acute inflammatory murine models, but the respective actions on neutrophil and eosinophil trafficking remain to be clarified. We demonstrate here that in a model of TGC-induced sterile peritonitis in ovx mice, chronic E2 administration electively and strongly inhibited peritoneal eosinophil accumulation. E2 decreased BM eosinophil number, contributing to a marked prevention of the TGC-induced eosinophil blood mobilization. These effects on eosinophil mobilization and peritoneal accumulation were abolished in ER-α(-/-) mice, demonstrating the crucial role of this nuclear receptor. Grafting ER-α(-/-) mice with ER-α(+/+) BM cells restored the suppressive effect of E2 on peritoneal eosinophilia, although the action on eosinophil blood mobilization was still abrogated. We therefore explored additional mechanisms and found that E2 reduced the peritoneal concentrations of key eosinophil prosurvival factors (IL-5, IL-9, and IL-25) and enhanced eosinophil apoptosis during the inflammatory process. Furthermore, this proapoptotic effect of E2 was abrogated in IL-5-overexpressing Tg mice. To conclude, we demonstrate for the first time that ER-α activation by exogenous E2 administration strongly inhibits eosinophil accumulation during acute inflammation in a nonreproductive target site for estrogen through combined actions on eosinophil mobilization and apoptosis. This specific, suppressive effect of chronic E2 replacement therapy on eosinophils has to be integrated to further understand the evolution of eosinophil-associated diseases in menopausal women.

Sex hormones influence immune responses and the development of autoimmune diseases including MS and its animal model, EAE. Although it has been previously reported that ovariectomy could worsen EAE, the mechanisms implicated in the protective action of endogenous ovarian hormones have not been addressed. In this report, we now show that endogenous estrogens limit EAE development and CNS inflammation in adult female mice through estrogen receptor α expression in the host non-hematopoietic tissues. We provide evidence that the enhancing effect of gonadectomy on EAE development was due to quantitative rather than qualitative changes in effector Th1 or Th17 cell recruitment into the CNS. Consistent with this observation, adoptive transfer of myelin oligodendrocyte glycoprotein-specific encephalitogenic CD4(+) T lymphocytes induced more severe EAE in ovariectomized mice as compared to normal female mice. Finally, we show that gonadectomy accelerated the early recruitment of inflammatory cells into the CNS upon adoptive transfer of encephalitogenic CD4(+) T cells. Altogether, these data show that endogenous estrogens, through estrogen receptor α, exert a protective effect on EAE by limiting the recruitment of blood-derived inflammatory cells into the CNS., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

17Beta-estradiol (E2) has been shown to promote the expression of inflammatory mediators by LPS-activated tissue resident macrophages through estrogen receptor alpha (ERalpha) 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-1beta, IL-6, and TNF-alpha) 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-1beta 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 ERalpha 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 ERalpha in macrophages in vivo, enhance their ability to produce inflammatory mediators and cytokines upon subsequent TLR activation.

Rationale: Th2 cells orchestrate allergic asthma and the cytokines they produce (IL-4, IL-5, and IL-13) are deleterious in allergy. Therefore, it is important to identify key signaling molecules expressed by Th2 cells that are essential for their function. We have previously shown that dihydropyridines selectively modulate Th2 cell functions., Objectives: Because dihydropyridines bind to and modulate voltage-dependent calcium (Ca(v)1) channel in excitable cells, we aimed at showing that Th2 cells selectively express functional Ca(v)1-related channels, the inhibition of which may prevent asthma., Methods: We looked for Ca(v)1 channel expression in Th2 and Th1 cells by real-time polymerase chain reaction and Western blotting. We sequenced the isoforms expressed by Th2 cells and tested whether Ca(v)1 antisense oligodeoxynucleotides (Ca(v)1AS) affected Ca(2+) signaling and cytokine production. Finally, we tested the effect of Ca(v)1AS in the passive asthma model by injection of ovalbumin-specific Th2 cells transfected with Ca(v)1AS into BALB/c mice challenged with intranasal ovalbumin and in the active model of asthma by intranasal delivery of Ca(v)1AS together with soluble ovalbumin in BALB/c mice previously immunized with ovalbumin in alum., Measurements and Main Results: We show that mouse Th2 but not Th1 cells expressed Ca(v)1.2 and Ca(v)1.3 channels. Th2 cells transfected with Ca(v)1AS had impaired Ca(2+) signaling and cytokine production, and lost their ability to induce airway inflammation on adoptive transfer. Furthermore, intranasal administration of Ca(v)1AS suppressed airway inflammation and hyperreactivity in an active model of asthma., Conclusions: These results indicate that Th2 cells selectively express Ca(v)1 channels that may be efficiently targeted in T lymphocytes to prevent experimental asthma.

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

Objective: E2 accelerates reendothelialization through estrogen receptor alpha (ER alpha), and we now aimed at defining the precise local and systemic cellular actors of this process., Methods and Results: The respective roles of endothelial and hematopoietic targets of E2 were investigated in a mouse carotid injury model, using confocal microscopy, to follow endothelium repair. Grafting ER alpha(-/-) mice with ER alpha(+/+) bone marrow (BM) was not sufficient to restore the accelerative effect of E2 on reendothelialization, demonstrating the necessary role of extrahematopoietic ER alpha. Using an endothelial-specific inactivation of ER alpha (Cre-Lox system), we showed that endothelial ER alpha plays a pivotal role in this E2 action. Conversely, in ER alpha(+/+) grafted with ER alpha(-/-) BM, the E2 regenerative effect was abolished, demonstrating that ER alpha-expressing hematopoietic cells are also needed. As eNOS expression in BM was required for this action, both endothelial progenitor cells and platelets could be the hematopoietic targets that participate to this beneficial E2 effect., Conclusions: We demonstrate that endothelial ER alpha plays a pivotal role in E2-mediated reendothelialization. However, endothelial targeting alone is not sufficient because the concomitant stimulation of a subpopulation of BM ER alpha is necessary. This cooperation should be taken into account in strategies aimed at optimizing in-stent reendothelialization.

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.

Short-term exposure to 17beta-estradiol (E2) in vitro has been reported to decrease the production of proinflammatory cytokines by LPS-activated macrophages through estrogen receptor alpha (ERalpha)-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-1beta, 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 ERalpha. 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-kappaB 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 ERalpha and, at least in part, by the down-modulation of the PI3K/Akt pathway, thereby alleviating this negative regulator of TLR4-signaling.

Asthma is a chronic airway disease resulting from inappropriate Th2-cell biased activation. Interleukin (IL)-4, IL-5 and IL-13 produced by Th2 cells contribute to the inflammatory process. Attempts for inhibiting interleukin-4 or IL-5 gave disappointing results. The simultaneous inhibition of several Th2-cytokines could be a more promising issue. Several arguments support the concept that Th2-cells express selective markers that could be targeted in asthma. Our group showed that Th2-cells selectively up-regulated dihydropyridine-sensitive Ca(2+) (DHP-Ca) channels essential for Ca(2+) signaling and type-2 cytokine production. Indeed, DHP-Ca antagonist effectively prevented or even reverted airway inflammation, airway remodeling and airway hyperresponsiveness in experimental models of asthma. Although it remains to be formally demonstrated that human lung infiltrating T-lymphocytes in asthmatic patients express DHP- Ca(2+) channels, we hypothesize that targeting DHP-Ca channels in T-lymphocytes could represent an efficient strategy in the treatment of asthma.This review article also discussed patents relevant to the field.

Dendritic cells (DC) are critical actors in the initiation of primary immune responses and regulation of self-tolerance. The steroid sex hormone 17beta-estradiol (E(2)) 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 ERalpha- or ERbeta-deficient mice, we investigated the role of ER isotypes in DC differentiation and acquisition of effector functions. We report that estrogen-dependent activation of ERalpha, but not ERbeta, 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 ERalpha activation and provide evidence for a cell-autonomous function of ERalpha signaling in DC differentiation. ERalpha-deficient DCs were phenotypically and functionally distinct from wild-type DCs generated in the presence of estrogens. In response to microbial components, ERalpha-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, ERalpha-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 ERalpha is the main receptor regulating estrogen-dependent DC differentiation in vitro and acquisition of their effector functions.