Project description:Analyses of gene alterations of human Treg cells treated with or without TLR8 ligand using the Illumina whole-genome Human HT-12 BeadChips. The study will provide important information for the molecular signature changes during the reversal of human Treg suppression mediated by activation of TLR8 signaling.
Project description:The innate immune system is equipped with multiple receptors to detect microbial nucleic acids and induce type I interferon (IFN) to restrict viral replication. When dysregulated these receptor pathways induce inflammation in response to host nucleic acids and promote development and persistence of autoimmune diseases like Systemic Lupus Erythematosus (SLE). IFN production is regulated by the Interferon Regulatory Factor (IRF) transcription factor family of proteins that function downstream of several innate immune receptors such as Toll-like receptors (TLRs) and Stimulator of Interferon Genes (STING). Although both TLRs and STING activate the same downstream molecules, the pathway by which TLRs and STING activate IFN response are thought to be independent. Here we show that STING plays a previously undescribed role in human TLR8 signaling. Stimulation with the TLR8 ligands induced IFN secretion in primary human monocytes, and inhibition of STING reduced IFN secretion from primary monocytes from 8 healthy donors. We demonstrate that TLR8-induced IRF activity was reduced by STING inhibitors. Moreover, TLR8-induced IRF activity was blocked by inhibition or loss of IKKε, but not TBK1. Bulk RNA transcriptomic analysis supported a model where TLR8 induces transcriptional responses associated with SLE that can be downregulated by inhibition of STING. These data demonstrate that STING is required for full TLR8-to-IRF signaling and provide evidence for a new framework of crosstalk between cytosolic and endosomal innate immune receptors, which could be leveraged to treat IFN driven autoimmune diseases.
Project description:To gain a comprehensive understanding of gene regulation in CXCL4 and TLR8 signaling crosstalk, we treated primary human blood monocytes with CXCL4 and TLR8 ssRNA ligand ORN8L for 6 h and performed transcriptomic analysis via RNA-seq. We observed that CXCL4 interacted with TLR8 ssRNA ligand and triggered inflammatory cytokine storm including IL6, IL12p40, TNF and IFNβ, and pro-fibrotic gene expression and activated NLRP3 inflammasome leading to interleukin-1β (IL-1β) secretion and pyroptosis in human blood monocytes.
Project description:Goal was to detect differences in response to TLR7 versus TLR8 agonists in human monocytes from healthy donors 3 deidentified donors from the Red Cross, monocytes from each donor incubated overnight with either vehicle, TLR7 agonist or TLR8 agonist
Project description:Mechanistic studies have revealed that TLR8 senses single-stranded RNA (ssRNA) fragments, processed via synergistic cleavage by ribonucleases (RNase) T2 and RNase A family members. Processing by these RNases releases uridines and purine-terminated residues resulting in TLR8 activation. Monocytes show high expression of RNase 6, yet this RNase has not been analyzed for its physiological contribution to recognition of bacterial RNA by TLR8. Herein, we characterized molecular RNase 6 cleavage mechanisms. BLaER1 RNASE6-/- cells showed a dampened TLR8-dependent response upon stimulation with isolated bacterial RNA (bRNA) but also upon infection with live whole bacteria. Pretreatment of bacterial RNA with recombinant RNase 6 generated fragments that induced stimulation in RNASE6 knockout cells. 2’O-RNA methyl modification, when introduced at the first uridine in the UA dinucleotide, impaired upstream processing by RNase 6 and dampened TLR8 stimulation. In summary, this data shows that RNase 6 plays a critical role in the processing of bacterial RNA by generating uridine-terminated breakdown products that ultimately activate TLR8.
Project description:To gain a comprehensive understanding of gene regulation in CXCL4 and TLR8 signaling crosstalk, we treated primary human blood monocytes with CXCL4 and TLR8 ssRNA ligand ORN8L for 6 h and performed transcriptomic analysis via RNA-seq. We observed that CXCL4 interacted with TLR8 ssRNA ligand and triggered inflammatory cytokine storm including IL6, IL12p40, TNF and IFNβ, and pro-fibrotic gene expression and activated NLRP3 inflammasome leading to interleukin-1β (IL-1β) secretion and pyroptosis in human blood monocytes.
Project description:Notch receptors direct the differentiation of T helper (Th) cell subsets, but their influence on regulatory T (TR) cell responses is obscure. Interruption of Notch signaling in TR cells resulted in a super-regulatory phenotype, with suppression of TR cell Th1 programming and apoptosis as well as Th1 cell responses in systemic inflammation. In contrast, gain of function Notch1 signaling in TR cells resulted in lymphoproliferation, dysregulated Th1 responses and autoimmunity. To determine mechanisms by which Notch signaling may alter TR cell function, we compared the transcriptional profiles of splenic TR cells of Foxp3EGFPCre mice with those of Foxp3EGFPCreR26N1c/N1c (gain of function Notch signaling), Foxp3EGFPCreRBPJ∆/∆ (loss of function canonical Notch signaling), and Foxp3EGFPCreR26N1c/N1cRBPJ∆/∆ mice (gain of function/canonical loss of function Notch signaling). Regulatory T cells are isolated from the spleen of 6 weeks old males, based on the expression of CD3, TCRbeta, CD4 and GFP (Foxp3), after Red blood cell lysis by ACK and gate on lineage negative (CD8, B220, CD11b, CD11c, Gr1) cells. To reduce variability and increase cell number, cells from multiple mice were pooled for sorting and at least three replicates were generated for all groups. RNA from 4.0-5.0x104 cells was amplified, labeled and hybridized to Affymetrix Mouse Gene 1.0 ST Arrays