Enhanced immunostimulatory activity of cyclic dinucleotides on mouse cells when complexed with a cell‐penetrating peptide or combined with CpG

Abstract

Recognition of pathogen-derived nucleic acids by immune cells is critical for the activation of protective innate immune responses. Bacterial cyclic dinucleotides (CDNs) are small nucleic acids that are directly recognized by the cytosolic DNA sensor STING (stimulator of IFN genes), initiating a response characterized by proinflammatory cytokine and type I IFN production. Strategies to improve the immune stimulatory activities of CDNs can further their potential for clinical development. Here, we demonstrate that a simple complex of cylic-di-GMP with a cell-penetrating peptide enhances both cellular delivery and biological activity of the cyclic-di-GMP in murine splenocytes. Furthermore, our findings establish that activation of the TLR-dependent and TLR-independent DNA recognition pathways through combined use of CpG oligonucleotide (ODN) and CDN results in synergistic activity, augmenting cytokine production (IFN-α/β, IL-6, TNF-α, IP-10), costimulatory molecule upregulation (MHC class II, CD86), and antigen-specific humoral and cellular immunity. Results presented herein indicate that 3 3 -cGAMP, a recently identified bacterial CDN, is a superior stimulator of IFN genes ligand than cyclic-di-GMP in human PBMCs. Collectively, these findings suggest that the immune-stimulatory properties of CDNs can be augmented through peptide complexation or synergistic use with CpG oligonucleotide and may be of interest for the development of CDN-based immunotherapeutic agents. Keywords:Arginine peptide (nona-arginine) r cGAMP r CpG ODN r Cyclic-di-GMP r Immunostimulation See accompanying article by Temizoz et al.Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionDetection of pathogen-derived nucleic acids by innate immune cells is critical for the initiation of protective responses against bacterial, viral, and fungal pathogens. Pathogen-derived nucleic acids are sensed based on their sequence, structure, nucleotide Correspondence: Dr. Mayda Gursel e-mail: mgursel@metu.edu.tr modifications, and their intracellular localization. So far, several nucleic acid sensors have been identified, including the endosomal TLRs TLR3, TLR7/TLR8, and TLR9 that recognize dsRNA, ssRNA, and CpG-containing DNA; cytosolic dsRNA sensors RIG-I, MDA5, LGP2; and a plethora of cytosolic receptors dedicated for dsDNA recognition (DAI, AIM2, RNA polymerase III, IFI16, DEAD-box helicase DDX41) [1]. Signaling in response to cytosolic DNA depends on the expression of the adaptor protein STING (stimulator of IFN genes, also known as TMEM173, MPYS, MITA and ERIS) and proceeds through the TBK1-IRF3 axis, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2015. 45: 1170-1179 Innate immunity 1171 culminating in type I IFN production [2, 3]. STING itself is not a DNA-recognition molecule. However, it can directly recognize cyclic dinucleotides (CDNs) such as the bacteria-derived molecule cyclic-di-GMP (c-di-GMP) [4]. Although CDNs were thought to functio...

Cyclic Dinucleotides Inhibit Osteoclast Differentiation Through STING-Mediated Interferon-β Signaling

“…CDNs released from bacterial biofilm can also activate host immune responses . Previous studies showed that CDNs could be used as novel vaccine adjuvants or immunostimulatory molecules for host defense . Intranasal administration with c‐di‐GMP enhances bacteria clearance and protects mice from Klebsiella pneumoniae infection through stimulation of dendritic cell‐meditated responses .…”

“…(18) Previous studies showed that CDNs could be used as novel vaccine adjuvants or immunostimulatory molecules for host defense. (19)(20)(21)(22) Intranasal administration with c-di-GMP enhances bacteria clearance and protects mice from Klebsiella pneumoniae infection through stimulation of dendritic cell-meditated responses. (23) In addition, c-di-AMP increases autophagy in macrophages and attenuates bacterial virulence through the cytoplasmic surveillance pathway during Mycobacterium tuberculosis infection.…”

Mechanisms of transcriptional activation of the stimulator of interferon genes by transcription factors CREB and c-Myc

“…STING is expressed ubiquitously in multiple human tissues and cell lines and has been observed at the plasma membrane, endoplasmic reticulum (ER), and on the surface of the outer mitochondrial membrane [1, 2]. It is well known that STING controlled TLR-independent cytosolic responses to viruses and nucleic acids, being required for the activation of both IRF3 and NF-κB and for induction of the gene expressing IFN-β [6]. Recent evidence suggested that STING gets involved in STING-TBK1-IRF3 signaling pathway by three distinct cytosolic nucleic acid–sensing pathways, RNA sensing through RLRs, DNA detection by DDX41 and the ALR IFI16, and the sensing of bacterial cyclic dinucleotides, such as c-di-GMP (c[G(3’ -5’)pG(3’ -5’)p] or cyclic di-AMP or cyclic GAMP (cGMP–AMP) [2, 7–9].…”

The Role of Nucleic Acid Sensing in Controlling Microbial and Autoimmune Disorders

“…For example, a current Phase I clinical study is using the synthetic, STING-activating CDN agonist MIW815 (ADU-S100) to study its safety and efficacy in treating patients with advanced/metastatic solid tumors or lymphomas via intratumoral injections (ClinicalTrials.gov Identifier: NCT02675439). Another study has shown that c-di-GMP can be complexed with simple cell-penetrating peptides that enhance cellular delivery and biological activity in murine splenocytes but also that the bacterial 3′−3′-cGAMP is a superior stimulator of IFN genes ligand than c-di-GMP in human peripheral blood mononuclear cells (Yildiz et al, 2015). A more recent study has shown that expression of an inducible c-di-GMP-producing diguanylate cyclase in Klebsiella pneumonia increased endogenous concentrations of c-di-GMP which attenuated its virulence in the lung of mice (Rosen et al, 2017).…”