Abstract
Objective. TLR7 ligation in plasmacytoid dendritic cells is promising for the treatment of cancer, allergy, and infectious diseases; however, high doses of ligands are required. We hypothesized that the combination of structurally different TLR7 ligands exponentiates the resulting immune response. Methods. CAL-1 (human pDC line) cells were incubated with the TLR7-specific adenine analog CL264 and single-stranded 9.2s RNA. Protein secretion was measured by ELISA. Microarray technique was used to detect modified gene expression patterns upon synergistic stimulation, revealing underlying functional groups and networks. Cell surface binding properties were studied using FACS analysis. Results. CL264 in combination with 9.2s RNA significantly enhanced cytokine and interferon secretion to supra-additive levels. This effect was due to a stronger stimulation of already regulated genes (by monostimulation) as well as to recruitment of thus far unregulated genes. Top scoring canonical pathways referred to immune-related processes. Network analysis revealed IL-1β, IL-6, TNF, and IFN-β as major regulatory nodes, while several minor regulatory nodes were also identified. Binding of CL264 to the cell surface was enhanced by 9.2s RNA. Conclusion. Structurally different TLR7 ligands act synergistically on gene expression patterns and on the resulting inflammatory response. These data could impact future strategies optimizing TLR7-targeted drug design.
Highlights
Immune cells utilize toll-like receptors (TLRs) to sense pathogen associated molecular patterns (PAMPs), which represents the starting point of the innate immune response [1]
CAL-1 cells were stimulated with the specific TLR7 ligand CL264 and the TLR7/8 ligand 9.2s RNA complexed to PLarg
CAL-1 cells do not express TLR8, so response to 9.2s RNA is limited to TLR7 stimulation [11]
Summary
Immune cells utilize toll-like receptors (TLRs) to sense pathogen associated molecular patterns (PAMPs), which represents the starting point of the innate immune response [1]. PDCs are the major source of type 1 interferon (IFN) in humans and are, of particular importance for antiviral immunity and autoimmune diseases [2]. In pDCs, TLR7 recognizes these ligands in specialized endolysosomes, resulting in the subsequent activation of NF-κB and interferon regulatory factor (IRF) 7 via MyD88, and induces production of inflammatory cytokines and type 1 IFN, respectively [1]. Studying TLR7 in human pDCs is of particular interest. Preclinical studies utilizing TLR7 ligation revealed promising results in the treatment of cancer, allergy, and infectious diseases [6, 7]. We hypothesized that the simultaneous use of two structurally
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