B cell-intrinsic TLR7 signaling is required for neutralizing antibody responses to SARS-CoV-2 and pathogen-like COVID-19 vaccines.

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.

The Influence of Sex Hormones and X Chromosome in Immune Responses.

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.

Hydroxychloroquine inhibits proteolytic processing of endogenous TLR7 protein in human primary plasmacytoid dendritic cells.

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.

Influence of X chromosome in sex-biased autoimmune diseases.

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.

Targeting androgen signaling in ILC2s protects from IL-33-driven lung inflammation, independently of KLRG1.

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.

Cav1.4 calcium channels control cytokine production by human peripheral TH17 cells and psoriatic skin-infiltrating T cells.

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 TH17 cells may be beneficial in psoriasis. We found that Cav1.4, encoded by CACNA1F, was the only Cav1 calcium channel expressed in TH17 cells. OBJECTIVE: We sought to investigate the role of Cav1.4 expression in early TH17-activation events and effector functions, as well as its association with TH17 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. Cav1 inhibitor and/or shRNA lentivectors were used to assess the contribution of Cav1.4 in TH17 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 Cav1 channel antagonist, markedly reduced inflammatory cytokine production by TH17 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 Cav1.4 in TH17 cells impaired cytokine production. Finally, Cav1 inhibition reduced the expression of the keratinocyte genes characteristic of TH17-mediated psoriasis inflammation in human skin equivalents. CONCLUSIONS: Cav1.4 channels promote TH17-cell functions both at the periphery and in inflammatory psoriatic skin.

Long non-coding RNA Xist contribution in systemic lupus erythematosus and rheumatoid arthritis.

Growing evidence points towards the role of the long non-coding (lnc)-RNA Xist expressed in female cells as a predominant key actor for the sex bias observed in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Indeed, in female cells, lnc-Xist controls transcription directly by spreading across the inactivated X chromosome (Xi) and indirectly by sequestring miRNAs as a sponge. The inactivation process at Xi is altered in lymphocytes from SLE women and associated with important variations in ribonucleoproteins (RNP) associated with lnc-Xist. In fibroblast-like synoviocytes (FLS) and osteoclasts from RA women, proinflammatory and proliferative pathways are upregulated due to the sequestration effect exerted by lnc-Xist overexpression on miRNAs. The key role played by lnc-Xist in SLE and RA is further supported by it's knock down that recapitulates the SLE B cell extrafollicular profile and controls RA associated FLS proinflammatory cytokine production and proliferation.

Separation of the CaV 1.2-CaV 1.3 calcium channel duo prevents type 2 allergic airway inflammation.

BACKGROUND: Voltage-gated calcium (Cav 1) channels contribute to T-lymphocyte activation. Cav 1.2 and Cav 1.3 channels are expressed in Th2 cells but their respective roles are unknown, which is investigated herein. METHODS: We generated mice deleted for Cav 1.2 in T cells or Cav 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 Cav 1.2 or Cav 1.3 deletion in models of type 2 airway inflammation. Finally, we checked whether the expression of both Cav 1.2 and Cav 1.3 in T cells from asthmatic children correlates with Th2-cytokine expression. RESULTS: We demonstrated non-redundant and synergistic functions of Cav 1.2 and Cav 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, Cav 1.3 has a particular role in calcium homeostasis. In accordance with the singular roles of Cav 1.2 and Cav 1.3 in Th2 cells, deficiency in either one of these channels was sufficient to inhibit cardinal features of type 2 airway inflammation. Furthermore, Cav 1.2 and Cav 1.3 must be co-expressed within the same CD4+ T cell to trigger allergic airway inflammation. Accordingly with the concerted roles of Cav 1.2 and Cav 1.3, the expression of both channels by activated CD4+ T cells from asthmatic children was associated with increased Th2-cytokine transcription. CONCLUSIONS: Thus, Cav 1.2 and Cav 1.3 act as a duo, and targeting only one of these channels would be efficient in allergy treatment.

Escape from X Chromosome Inactivation and the Female Predominance in Autoimmune Diseases.

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.

CD49d/CD29-integrin controls the accumulation of plasmacytoid dendritic cells into the CNS during neuroinflammation.

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.

Estrogen Signaling in Bystander Foxp3neg CD4+ T Cells Suppresses Cognate Th17 Differentiation in Trans and Protects from Central Nervous System Autoimmunity.

17ß-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-1KO 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 Foxp3neg 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.

The ß and α2δ auxiliary subunits of voltage-gated calcium channel 1 (Cav1) are required for TH2 lymphocyte function and acute allergic airway inflammation.

BACKGROUND: T lymphocytes express not only cell membrane ORAI calcium release-activated calcium modulator 1 but also voltage-gated calcium channel (Cav) 1 channels. In excitable cells these channels are composed of the ion-forming pore α1 and auxiliary subunits (ß and α2δ) needed for proper trafficking and activation of the channel. Previously, we disclosed the role of Cav1.2 α1 in mouse and human TH2 but not TH1 cell functions and showed that knocking down Cav1 α1 prevents experimental asthma. OBJECTIVE: We investigated the role of ß and α2δ auxiliary subunits on Cav1 α1 function in TH2 lymphocytes and on the development of acute allergic airway inflammation. METHODS: We used Cavß antisense oligonucleotides to knock down Cavß and gabapentin, a drug that binds to and inhibits α2δ1 and α2δ2, to test their effects on TH2 functions and their capacity to reduce allergic airway inflammation. RESULTS: Mouse and human TH2 cells express mainly Cavß1, ß3, and α2δ2 subunits. Cavß antisense reduces T-cell receptor-driven calcium responses and cytokine production by mouse and human TH2 cells with no effect on TH1 cells. Cavß is mainly involved in restraining Cav1.2 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 TH2 cells. CONCLUSIONS: These results stress the role of Cavß and α2δ2 auxiliary subunits in the stability and activation of Cav1.2 channels in TH2 lymphocytes both in vitro and in vivo, as demonstrated by the beneficial effect of Cavß antisense and gabapentin in allergic airway inflammation.

Eomesodermin Expression in CD4+ T Cells Restricts Peripheral Foxp3 Induction.

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.

Sex Differences in Plasmacytoid Dendritic Cell Levels of IRF5 Drive Higher IFN-α Production in Women.

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.

X-Chromosome complement and estrogen receptor signaling independently contribute to the enhanced TLR7-mediated IFN-α production of plasmacytoid dendritic cells from women.

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-ß2m(-/-) 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.

Bispecificity for myelin and neuronal self-antigens is a common feature of CD4 T cells in C57BL/6 mice.

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.

Estradiol promotes functional responses in inflammatory and steady-state dendritic cells through differential requirement for activation function-1 of estrogen receptor α.

17ß-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.

Protein kinase C-dependent activation of CaV1.2 channels selectively controls human TH2-lymphocyte functions.

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/ß 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.

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/ß) 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 17ß-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 17ß-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.