Small molecule Toll-like receptor 7 agonists localize to the MHC class II loading compartment of human plasmacytoid dendritic cells

Abstract

TLR7 and TLR8 are intracellular sensors activated by single-stranded RNA species generated during viral infections. Various synthetic small molecules can also activate TLR7 or TLR8 or both through an unknown mechanism. Notably, direct interaction between small molecules and TLR7 or TLR8 has never been shown. To shed light on how small molecule agonists target TLRs, we labeled 2 imidazoquinolines, resiquimod and imiquimod, and one adenine-based compound, SM360320, IntroductionSince the discovery of type I IFN (IFN-I) in 1957 and its potential for treatment of viral infections and cancer, 1 several small molecule inducers of this factor have been identified. Many of these compounds are now known to be TLR7 or TLR8 or both agonists. Among them are imidazoquinolines, 2,3 such as resiquimod (R848) that activates both TLR7 and TLR8 in humans, and imiquimod (R837) that activates TLR7 only. 4,5 Imidazoquinolines have been largely tested in humans, and R837, formulated as a cream (Aldara), is licensed for topical treatment of genital warts, basal cell carcinoma, and actinic keratosis. 6 Similar to R837, purinelike molecules, such as 9-benzyl-8-hydroxy-2-(2-methoxyethoxy) adenine (SM360320 or 1V136), are TLR7 agonists. 7 TLR7 and TLR8 are phylogenetically related, located on the X chromosome in most mammals, and probably derive from a gene duplication event. TLR7 and TLR8 are both activated by various singlestranded RNAs (ssRNAs) from viruses, 8 synthetic guanosine-or uridine-rich ssRNAs, 9 and synthetic small molecules. TLR9, together with TLR7 and TLR8, form a subfamily of TLRs that is based on genomic structure and sequence homology. 10 The natural agonist of TLR9 is unmethylated, CpG-containing DNA of bacterial or viral origin that can be mimicked by synthetic ssCpG oligonucleotides that can be classified as A-type (CpG-A), B-type (CpG-B), or C-type (CpG-C) on the basis of their different sequence motifs and biologic activities. 11 Several reports have suggested that TLR7, TLR8, and TLR9 can interact directly with nucleic acids [12][13][14][15] ; however, structural evidence confirming this hypothesis is not available yet. No convincing evidence of direct binding of imidazoquinolines or purine-like molecules to TLR7 or TLR8 exists either.TLR7 and TLR9 are the only TLRs expressed by plasmacytoid dendritic cells (pDCs), which are a rare subset of circulating DCs that are considered as a frontline defense against viral infections. pDCs produce most of the systemic IFN-I (ie, IFN-␣ and -␤) after viral infection and rapidly activate T cells with the use of presynthesized MHC class I and II molecules stored in their early and late endosomal compartments, respectively. 16,17 Although nucleic acid sensing provides protection from intracellular infection, nucleic acids are not exclusively from pathogens; therefore, a balance between self versus foreign responsiveness is necessary. Indeed, recognition of self nucleic acids by TLR7 and TLR9 and the resultant IFN-I production have been linked to autoimmune disorders such as lupu...

Bioconjugation of Small Molecules to RNA Impedes Its Recognition by Toll-Like Receptor 7

“…The same structure suggested that a PEG chain might bridge the two identified binding sites in this receptor, one for resiquimod, and the other for RNA, potentially causing a cooperative effect from a bidentate ligand made of RNA and a small molecule derivative of resiquimod. Since RNAs bearing terminal alkyne groups are readily accessible, we synthesized azide derivatives with 1 H -imidazo-[4,5- c ]-purine structure (52–54), i.e., derivatives of imidazoquinolines of the quimod series and produced the respective bioconjugates for testing. While the anticipated cooperativity could not be evidenced, we observed that covalent modifications of RNA effectively decrease TLR7-mediated signaling.…”

Evaluation of novel synthetic TLR7/8 agonists as vaccine adjuvants

“…TLR7/8 recognize single-stranded viral RNA [7, 8]. Upon ligation of the receptor-ligand pair, intracellular signalling is initiated and downstream transcription factors activate genes of proinflammatory cytokines (TNF, IL-1), type I interferons (IFNα) and co-stimulatory molecules (CD80, CD86) [9–11]. The activation of these genes promotes maturation of dendritic cells (DCs), facilitating the presentation of antigen and stimulation of the ensuing adaptive immune response.…”

Imiquimod: the biochemical mechanisms of immunomodulatory and anti-inflammatory activity

“…Совсем недавно был поднят вопрос о способах проникновения имиквимода внутрь клетки [1]. Исследования на плазмацитоидных дендритных клетках (для которых TLR7 и TLR8 являются единственными экспрессируемыми молекулами из семейства TLR) показало, что имидазохинолины концентрируются в LAMP1+СD63+HLA-DR+ эндосомах.…”

“…У плазмацитоидных ДК эндолизосомальный компартмент специализирован на антигенной презентации через MHC II, поэтому можно сказать, что имиквимод локализуется в MIIC компартменте. Но, так как имиквимод представляет собой слабое основание, способное к пассивной диффузии сквозь клеточную мембрану, в данной работе высказано предположение, что в начале имиквимод пассивно проникает в клетку, после чего аккумулируется в MIIC компартменте, где протонируется и уже не может выйти наружу [1].…”