Part 2: Cytosolic DNA sensing

December 07, 2016

Many pathogens can be recognised in the intracellular compartment of the host cells. In particular, non-self DNA and RNA are the main target of cytosolic PRRs, which trigger the innate immune response after the detection of viral and bacterial components.

Several putative cytosolic receptors for DNA have been found in the last decade [Figure 1.3] and DNA-dependent activator of IFN-regulatory factors (DAI) was the first sensor to be discovered. It can bind DNA molecules and signal downstream to induce type I IFN via NF-κB and IRF3 after poly(dA:dT) stimulation. In 2009, studies showed the ability of RNA polymerase III to convert AT-rich dsDNA, such as poly(dA:dT) which might be the only synthetic DNA ligand sensed, to 5’triphosphate containing RNA, which can lead to the activation of the RIG-I pathway and the production of type I IFNs and other inflammatory cytokines. In the last 6 years, many other proteins showed a possible role as DNA sensors, for example cGAS, LRRFIP1, DHX9, DHX36, DDX41, IFI16, AIM2, Ku70 and DNA-PK. Interestingly, two members of the human PYHIN family, AIM2 and IFI16, were proposed to be cytosolic DNA sensors thanks to their ability to bind DNA through their HIN domain (discussed later) [4].

STING, the key intracellular adaptor protein
Microbial cytosolic DNA is a potent trigger of the innate immune response. Since the discovery of the immune adaptor protein STimulator of INterferon Genes (STING, also known as TMEM173, MPYS, MITA and ERIS) in 2008, pivotal steps forward have been made in the field of DNA sensing. Human and mouse STING amino-acid sequences share 68% identity and 81% similarity and orthologs were found in other species. The first study showed the function of STING in innate immunity as a downstream effector in anti-viral signalling that regulated the activation of both NF-ƘB and IRF3 transcription pathways and the induction of type I IFNs and pro-inflammatory cytokines. Indeed, STING was required for the induction of type I IFN in murine embryonic fibroblasts, macrophages and dendritic cells when infected with the DNA virus HSV-1 or bacteria Listeria monocytogenes. Subsequent studies highlighted the key role of this adaptor protein in responses to DNA and bacterial nucleic acids called “cyclic dinucleotides”. These nucleic acids are involved in the regulation of several bacterial processes, such as virulence, stress survival, motility, antibiotic production, metabolism, biofilm formation, and differentiation. STING can also be activated by viral events of membrane-fusion, independent from DNA, RNA or viral capsid detection. Moreover, recent studies revealed the important role of this adaptor protein in the control of cancer development and autoimmune diseases [4].

The cGAS-cGAMP-STING pathway
An important contribution in the understanding of the innate immune response to microbial DNA has been provided by the results obtained from the Chen team in 2013 [5]. Their first study showed how a novel mammalian cyclic dinucleotide, called cyclic GMP-AMP (cGAMP), was able to trigger IFNβ production through STING and IRF3 activation. cGAMP is an endogenous second messenger in innate immune signalling by cytosolic DNA. Further analysis allowed the identification of cGAS, the cytoplasmic protein responsible for cGAMP production, which is a member of the nucleotidyl transferase (NTase) family. cGAS can recognize and bind nucleic acids in the cytoplasm through its DNA-binding residues, starting the production of cGAMP from ATP and GTP due to DNA induced opening of active site. cGAMP has a high-affinity for STING and is able to bind directly to this adaptor protein. The activation of STING leads to the recruitment of TBK1 and the consequent phosphorylation of IRF3 and activation of NF-κB. IRF3 and NF-κB are then able to translocate into the nucleus to induce the production of type I IFNs and other pro-inflammatory cytokines. cGAMP has a similar structure to c-di-GMP, the bacterial second messenger and this fact helped explain why STING was activated by both DNA stimulation and bacterial infections [5].

Figure 3: DNA sensing overview