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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. Highlights: TLR8, like TLR7, escapes X chromosome inactivation in immune cells from women and 47,XXY men with Klinefelter syndrome. The frequency of double-positive cells for TLR7 and TLR8 primary transcripts is sevenfold higher in women than in men. TLR7 and TLR8 form a co-regulated gene cluster on the human X chromosome, with sex-specific, divergent transcriptional patterns observable in monocytes and CD4+ T lymphocytes. Co-dependent transcription of the TLR7 and TLR8 genes on the active X was observed in women and in men with Klinefelter syndrome, contrasting with mutually exclusive transcription in euploid men. Blood mononuclear cells, including monocyte subsets, expressed higher levels of TLR8 protein in females than in euploid males. Plain Language summary: Human endosomal Toll-like receptors TLR7 and TLR8, encoded by two adjacent X-linked genes, recognize self and non-self RNA ligands, and are important mediators of innate immunity and autoimmune pathogenesis. We previously reported that TLR7 evades X chromosome inactivation (XCI) in female immune cells, correlating with enhanced functional properties in B cells harboring biallelic expression of this gene. Here, we conducted a comprehensive single-cell resolution analysis of the transcriptional regulation of both TLR7 and TLR8, in CD14+ monocytes and CD4+ T lymphocytes. We unequivocally demonstrated that TLR8, like TLR7, escapes XCI in immune cells from female and Klinefelter syndrome males. When we analyzed TLR7 and TLR8 transcripts together, cells from women and KS men exhibited higher frequencies of cells co-transcribing the two genes. Surprisingly, these differences were attributable not only to the ability of TLR7 and TLR8 to be expressed on the Xi, but also to the joint transcriptional behavior of the TLR7–TLR8 gene pair on the active X chromosome specifically. This contrasted with a striking pattern of mutually exclusive transcription on the single X of euploid men. Corroborating our RNA FISH results, we found higher TLR8 protein expression in female than in male leukocytes, including all monocyte subpopulations. In summary, our data suggest that sex-biased co-regulation of the Toll-like receptor locus and XCI escape of TLR8 contribute to the sexual dimorphism in TLR8 expression, which may have important consequences for the functional make-up of monocyte and T cell populations. [ABSTRACT FROM AUTHOR]

PKC interacts with Cav1.3 calcium channels to promote the Cav1.2/Cav1.3 duo tuning Th2 functions In conclusion, the interaction of PKC with Ca SB v sb 1.3 upon TCR engagement is a new mechanism setting off calcium influx through Ca SB v sb 1 channels in non-excitable cells. Antisense oligonucleotides (ODN) targeting PKC mRNA (AS-PKC ) significantly decreased PKC but not PKC protein levels in Th2 cells (Figure 2A). [Extracted from the article]

Background: Voltage‐gated calcium (Cav1) channels contribute to T‐lymphocyte activation. Cav1.2 and Cav1.3 channels are expressed in Th2 cells but their respective roles are unknown, which is investigated herein. Methods: We generated mice deleted for Cav1.2 in T cells or Cav1.3 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 Cav1.2 or Cav1.3 deletion in models of type 2 airway inflammation. Finally, we checked whether the expression of both Cav1.2 and Cav1.3 in T cells from asthmatic children correlates with Th2‐cytokine expression. Results: We demonstrated non‐redundant and synergistic functions of Cav1.2 and Cav1.3 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, Cav1.3 has a particular role in calcium homeostasis. In accordance with the singular roles of Cav1.2 and Cav1.3 in Th2 cells, deficiency in either one of these channels was sufficient to inhibit cardinal features of type 2 airway inflammation. Furthermore, Cav1.2 and Cav1.3 must be co‐expressed within the same CD4+ T cell to trigger allergic airway inflammation. Accordingly with the concerted roles of Cav1.2 and Cav1.3, the expression of both channels by activated CD4+ T cells from asthmatic children was associated with increased Th2‐cytokine transcription. Conclusions: Thus, Cav1.2 and Cav1.3 act as a duo, and targeting only one of these channels would be efficient in allergy treatment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]