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GENEtic DiSsection of Innate Immune Sensing and Signalling

Periodic Reporting for period 3 - GENESIS (GENEtic DiSsection of Innate Immune Sensing and Signalling)

Reporting period: 2018-10-01 to 2020-03-31

Background - The innate immune system
One of the main functions of the immune system is to maintain the organism’s well-being against invading pathogens. To perceive potentially harmful signals as non-self, the innate branch of the immune system has evolved a conserved set of receptors that can detect microbial pathogens through the presence of so called microbe associated molecular patterns (MAMPs). These germ-line encoded receptors are commonly referred to as pattern recognition receptors (PRRs). In our research we are trying to understand how PRRs sense the presence of non-self and how these signals are integrated by cells of our innate immune system.

Nucleic Acid Sensing by the Innate Immune System
A common theme in antimicrobial immunity is the recognition of nucleic acids (NA). In fact, most microbes expose some type of nucleic acid or degradation products thereof at some point during their life cycle (e.g. viral genomic DNA/RNA or RNA transcripts). To this effect, the host has evolved a number of PRRs that are specialized to sense certain components of ‘non-self’ DNAs or RNAs. These NA-sensing PRRs can also play important roles in the initiation of autoimmune diseases. Indeed, most of these NA-receptors are not PRRs in sensu stricto, as they can also respond to endogenous NAs under certain conditions. Here, the principle of compartmentalization plays a pivotal role. For example, all NA-sensing toll-like receptors (TLRs) reside within the endolysosomal compartment that is usually devoid of endogenous NAs. Along similar lines, double stranded DNA (dsDNA) of both exogenous and endogenous sources is readily sensed as a sign of ‘danger’ within the cytoplasm. As soon as dsDNA is translocated into the cytoplasm, it is readily detected by certain PRRs that do not discriminate self from non-self. Again, in light of the fact that the cytoplasm is usually devoid of dsDNA, this sensing system is silent under homeostatic conditions.
While recent advances in the field of innate immunity have delineated the core set of PRRs involved in microbial nucleic acid recognition. This includes certain members of the Toll-like receptor family sampling the endolysosomal compartment, as well as cytoplasmic PRRs such as RIG-I-like helicases and the cGAS-STING axis. While, the cGAS-STING axis appears to play a predominant role in driving antiviral gene expression upon DNA recognition, a number of publications point to the existence of additional DNA sensing mechanisms. This could be due to cell type specificity, ligand specificity, specificity for a certain signaling cascade or readout under study or cooperative effects between signaling pathways that appear like linear signaling cascades upon perturbation. Most importantly, despite their strong evolutionary conservation, there are numerous examples that have highlighted species-specific differences in otherwise homologous innate sensing pathways and such phenomena could also be relevant for this field. Apart from this, a number of interesting candidates cannot be studied in murine knockout models, given the fact that no clear homologies to the human system exist or because knocking them out would be embryonic lethal.

Objectives of GENESIS
GENESIS will elucidate how intracellular nucleic acids are sensed by the human innate immune system and how this sensing is hardwired to the induction of antimicrobial immunity. This will be achieved by employing the powerful technology of genome engineering, which will allow us to obtain insight into innate immune signaling pathways at unprecedented precision, depth and breadth. More specifically, GENESIS will define the role of nucleic acid sensing receptors in the detection of microbial pathogens and synthetic ligands in a systematic and exhaustive fashion (1). This will be achieved by knocking out putative PRRs in a human macrophage model that closely resembles primary macrophages. (2) In addition, using an unbiased, genome-wide knockout approach
The human ‘DNA inflammasome’
One important aim of our proposal was to genetically dissect the role and mechanisms of DNA recognition within the human innate immune system. To this end, we have established a knockout cell generation pipeline that allows to conduct hypothesis-driven perturbation studies at high throughput. Doing so, we were able to establish a critical role for the cGAS-STING axis in NLRP3 inflammasome activation, a pathway that plays a predominant role in human, but not in murine cells. As such, re-evaluating the role of AIM2 in human cells (compare objective 1), we made the surprising finding that AIM2 is completely dispensable for DNA-mediated inflammasome activation (Gaidt et al. Cell 2017). Instead, in human myeloid cells the ‘DNA inflammasome’ was completely dependent on NLRP3. We identified a cGAS-STING-dependent signaling cascade that resulted in a lytic form of cell death upstream of NLRP3 activation. Interestingly, this signaling cascade was independent of the canonical function of STING to trigger de novo gene expression. Conducting a forward-genetic screen (compare objective 2a), our attention was directed towards lysosomes as potential modulators of this cGAS-STING-dependent cell death cascade leading to NLRP3 activation. Indeed, following up on these results we found that STING was recruited to lysosomes upon activation, resulting in membrane permeabilization und thus the induction of lysosomal cell death (LCD). This type of cell death triggered potassium efflux and thus NLRP3 activation. Validating these data in primary cells revealed that this signaling cascade was fully operational in human monocytes, as well as in bone marrow cells. All in all, these results uncovered a new type of signaling cascade in human myeloid cells that functions to connect the predominant DNA recognition module (cGAS-STING axis) to its membrane perturbation sensing module (NLRP3 inflammasome). The connecting link between these two modules is the induction of lysosomal cell death, which we speculate to be an ancient functionality of STING. From an evolutionary perspective, these results would also explain why AIM2 is a rather ‘novel invention’ in the repertoire of innate immune defense mechanisms that is not stringently conserved across different mammalian lineages. Indeed, some mammalian species have completely lost AIM2, despite the fact that they are facing the same microbial pathogens as AIM2-sufficient species.

Novel Nucleic Acid Sensing Modalities
As initially planned, we have gene targeted most of the previously reported nucleic acid sensing pathways that are amenable to genetic deletion (‘non-essential’ genes). Respective knockout cell lines are currently being characterized for defects in nucleic acid sensing pathways in a systematic fashion.
CRISPaint – a versatile gene tagging system
We established a new gene tagging system (CRISPaint) that allows us to tag endogenous genes using CRISPR/Cas in a simple and versatile fashion (Schmid-Burgk et al. Nature Communications 2016): While homologous recombination-based integration techniques using designer nucleases have been improved during the last years, e.g. by shortening homology arms or by using ssDNA oligonucleotides as donor templates, a second route of DNA integration based on NHEJ has also been reported: For this mechanism, only few bases or even no homologies at all are required, promising the development of a fully modular gene tagging method that does not require laborious template construction. Considering this highly attractive approach, we have set out to develop and iteratively optimize a modular gene tagging toolbox based on CRISPR/Cas9 (CRISPR assisted insertion tagging = CRISPaint), whose mechanism of action we genetically deciphered to depend on canonical NHEJ ligation. Despite the wide notion of NHEJ being a generally error-prone repair mechanism, we found that depending on the donor design high fidelity in-frame integration could be achieved at total cellular frequencies of up to 10% without further selection steps. This resource allows the adding of a broad variety of C-terminal tags to endogenously expressed human proteins, designed for expression quantification, visualization, purification, controllable destabilization and interaction measurement. This new methodology has been and will be a valuable methodology for our future studies in the context of this grant.