Animal Models for the Study of Nucleic Acid Immunity: Novel Tools and New Perspectives

Abstract

Unresolved inflammation fosters and supports a wide range of human pathologies. There is growing evidence for a role played by cytosolic nucleic acids in initiating and supporting pathological chronic inflammation. In particular, the cGAS-STING pathway has emerged as central to the mounting of nucleic acid-dependent type I interferon responses, leading to the identification of small-molecule modulators of STING that have raised clinical interest. However, several new challenges have emerged, representing potential obstacles to efficient clinical translation. Indeed, the current literature underscores that nucleic acid-induced inflammatory responses are subjected to several layers of regulation, further suggesting complex coordination at the cell-type, tissue or organism level. Untangling the underlying processes is paramount to the identification of specific therapeutic strategies targeting deleterious inflammation. Herein, we present an overview of human pathologies presenting with deregulated interferon levels and with accumulation of cytosolic nucleic acids. We focus on the central role of the STING adaptor protein in these pathologies and discuss how in vivo models have forged our current understanding of nucleic acid immunity. We present our opinion on the advantages and limitations of zebrafish and mice models to highlight their complementarity for the study of inflammatory human pathologies and the development of therapeutics. Finally, we discuss high-throughput screening strategies that generate multi-parametric datasets that allow integrative analysis of heterogeneous information (imaging and omics approaches). These approaches are likely to structure the future of screening strategies for the treatment of human pathologies.

Keywords: STING; drug screening; inflammatory models; innate immunity; interferon signaling.

Figure 1. STING-dependent and STING-independent signaling.

(a) Nucleic acid ligands, in particular dsDNA, are recognized by a broad array of receptors. Among these, in mammalian cells (human, murine), cGAS has been shown to be the major receptor. In addition to dsDNA, cGAS has been shown to be stimulated by ssDNA and RNA:DNA hybrids. Activation of cGAS leads to STING dependent activation of IRF3 and NF-kB. In zebrafish, cGAS has been shown to be dispensable for STING activation. STING is rather activated by DHX9 and DDX41, leading to activation of NF-kB- dependent cytokine production. (b) Double-stranded DNA (dsDNA) can elicit a STING independent through recognition by the DNA-PK DNA repair complex. dsDNA can also inhibit STING following recognition by AIM2. In addition, LysRS is activated by RNA:DNA hybrids to inhibit STING. TF: transcription factors.

Figure 2. Evolutionary and structural study of STING.

(a) Left: A comprehensive phylogenetic tree of STING across the tree of life (arrows pinpoint Homo sapiens, HS; Mus musculus, MM; and Danio rerio, DR). Right: The sequence alignment among the human, mouse and the zebrafish STING sequences (arrow points at V155 and N154). (b) Top left: Molecular modeling of the zebrafish STING (magenta ribbon) superposed on the human STING (green ribbon). Top right: Superposition of the mouse STING (blue ribbon) superposed on the human STING (green ribbon). Bottom: Hydrophobicity surface representation of the superposed human, mouse and zebrafish STING proteins. The arrow point the entry point of the STING binding site, which is more hydrophobic. (c) Superposition of the human, mouse and zebrafish STING proteins with V155 and Q155 showing in ball and stick representation in the insert. The color coding follows the conventions of (b).

Figure 3

Comparison of experimental models used for discovery of new drugs. The drug development pipeline can take more than 10 years. The figure shows the different steps of drug development. Appropriate use of experimental models for studying nucleic acid immunity is of major importance for selection of lead drug candidates. In vitro, ex vivo or in vivo models can be used. While there are all amenable to genome editing, there are important differences regarding the feasibility of live imaging and high-throughput screening, physiological relevance and immune system complexity. Live imaging in mouse models can be performed at high resolution to a limited deepness using imaging window and two-photon microscopy or at low resolution using bioluminescence imaging. Complexity and variability of 3D organoids culture have been problematic for establishment of high-throughput screening, but different screening strategy has already been well implemented with zebrafish larvae. Relevance of the model for the disease studied has to be examine carefully. Zebrafish larvae are useful to study innate immunity as adaptive immunity is functionally mature at 4 weeks post-fertilization.