Abstract
Activation of TLR7 and TLR9 by endogenous RNA- or DNA-containing ligands, respectively, is thought to contribute to the complicated pathophysiology of systemic lupus erythematosus (SLE). These ligands induce the release of type-I interferons by plasmacytoid dendritic cells and autoreactive antibodies by B-cells, both responses being key events in perpetuating SLE. We recently described the development of inhibitory oligonucleotides (INH-ODN), which are characterized by a phosphorothioate backbone, a CC(T)XXX3–5GGG motif and a chemical modification of the G-quartet to avoid the formation of higher order structures via intermolecular G-tetrads. These INH-ODNs were equally or significantly more efficient to impair TLR7- and TLR9-stimulated murine B-cells, macrophages, conventional and plasmacytoid dendritic cells than the parent INH-ODN 2088, which lacks G-modification. Here, we evaluate the inhibitory/therapeutic potential of our set of G-modified INH-ODN on human immune cells. We report the novel finding that G-modified INH-ODNs efficiently inhibited the release of IFN-α by PBMC stimulated either with the TLR7-ligand oligoribonucleotide (ORN) 22075 or the TLR9-ligand CpG-ODN 2216. G-modification of INH-ODNs significantly improved inhibition of IL-6 release by PBMCs and purified human B-cells stimulated with the TLR7-ligand imiquimod or the TLR9-ligand CpG-ODN 2006. Furthermore, inhibition of B-cell activation analyzed by expression of activation markers and intracellular ATP content was significantly improved by G-modification. As observed with murine B-cells, high concentrations of INH-ODN 2088 but not of G-modified INH-ODNs stimulated IL-6 secretion by PBMCs in the absence of TLR-ligands thus limiting its blocking efficacy. In summary, G-modification of INH-ODNs improved their ability to impair TLR7- and TLR9-mediated signaling in those human immune cells which are considered as crucial in the pathophysiology of SLE.
Highlights
Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disorder involving different organs such as skin, joints, kidneys, lung and nervous system
Before we examined the inhibitory potential of G-modified INH-ODNs for human immune cells, we evaluated the stimulatory activity of TLR7- and TLR9-ligands, since their activity counteracts the inhibitory potency of INH-ODNs
Since only human plasmacytoid dendritic cells (pDCs) and B-cells express high levels of TLR7 and TLR9 [28] and both cell types are involved in the pathophysiology of SLE, we explored the inhibitory potential of G-modified INH-ODNs on human B-cells stimulated via TLR7 or TLR9
Summary
Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disorder involving different organs such as skin, joints, kidneys, lung and nervous system. A defective clearance of cytosolic DNA was observed in DNase II deficient mice, resulted in an IFN-β-mediated apoptosis of liver erythrocyte precursors and death in utero and points to the possibility that nucleic acids are the driving force for autoimmune inflammation [2] These initial steps activate dendritic cells, which in turn stimulate resting autoreactive T- and B-cells to produce autoantibodies forming complexes with DNA or RNA [1,3]. Type I interferons, play a central role in this scenario and it is not surprising that SLE patients display an interferogenic signature, i.e. many type I interferon induced genes are expressed [1] These complex events lead to a self-augmenting circle of inflammation, which leads to organ damage and failure
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