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.

Apelin is expressed in intimal smooth muscle cells and promotes their phenotypic transition.

During atherosclerotic plaque formation, smooth muscle cells (SMCs) switch from a contractile/differentiated to a synthetic/dedifferentiated phenotype. We previously isolated differentiated spindle-shaped (S) and dedifferentiated rhomboid (R) SMCs from porcine coronary artery. R-SMCs express S100A4, a calcium-binding protein. We investigated the role of apelin in this phenotypic conversion, as well as its relationship with S100A4. We found that apelin was highly expressed in R-SMCs compared with S-SMCs. We observed a nuclear expression of apelin in SMCs within experimentally-induced intimal thickening of the porcine coronary artery and rat aorta. Plasmids targeting apelin to the nucleus (N. Ap) and to the secretory vesicles (S. Ap) were transfected into S-SMCs where apelin was barely detectable. Both plasmids induced the SMC transition towards a R-phenotype. Overexpression of N. Ap, and to a lesser degree S. Ap, led to a nuclear localization of S100A4. Stimulation of S-SMCs with platelet-derived growth factor-BB, known to induce the transition toward the R-phenotype, yielded the direct interaction and nuclear expression of both apelin and S100A4. In conclusion, apelin induces a SMC phenotypic transition towards the synthetic phenotype. These results suggest that apelin acts via nuclear re-localization of S100A4, raising the possibility of a new pro-atherogenic relationship between apelin and S100A4.

HIV-1 infection enhances innate function and TLR7 expression in female plasmacytoid dendritic cells.

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.

TLR7 dosage polymorphism shapes interferogenesis and HIV-1 acute viremia in women.

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.

TLR7 escapes X chromosome inactivation in immune cells.

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.