Innate Immunity

In our research projects, we are trying to understand what mechanisms are employed by our innate immune system to distinguish self from non-self. Central to this complex task is a repertoire of pattern recognition receptors (PRRs) that have evolved to detect the presence of microorganisms. The ligands or targets of these PRRs are usually referred to as pathogen-associated molecular patterns (PAMPs). PAMPs are typically molecular structures that are unique to the physiological processes of microorganisms but not the host. In addition, PAMPs usually constitute products that are essential to microorganisms and thus cannot be changed or lost by the respective microorganism via adaptive evolution. In addition to the recognition of PAMPs, some PRRs can also detect endogenous danger and stress situations. Cell stress or tissue damage can lead to the release of normally compartmentalized molecules or the chemical modification of self-molecules. These and other endogenous inflammatory signals that appear after cellular damage or due to metabolic derangements are collectively known as danger-associated molecular patterns (DAMPs), and their ability to trigger inflammation is mediated by the PRRs of our innate immune system.

THE INFLAMMASOME

The inflammasome is a large multimeric pro-caspase-1 activating platform that is essential for the processing and thus activation of the proinflammatory cytokines IL-1β and IL-18. Initial biochemical characterization by the pioneering work of Jürg Tschopp’s group led to the term inflammasome, which emphasizes its role in inflammation and describes the fact of a large protein complex forming (σώμα = soma; ancient Greek for body). Activation of inflammasomal sensors, like NLRP proteins or AIM2, triggers the cleavage of pro-caspase-1 to active caspase-1 that then processes, amongst a variety of other targets, the highly proinflammatory cytokine pro-IL-1β to mature IL-1β. In the case of IL-1β, three distinct events are required for secretion of the bioactive cytokine: Firstly, activation of the cell by PRRs, like TLRs, leads to transcription of pro-IL-1β. Secondly, pro-IL-1β is cleaved into biologically active IL-1β by an active caspase-1 complex. Thirdly, mature IL-1β is secreted into the extracellular space. The inflammasome complexes play an important role in the second step and are probably also instrumental in initiating the third and final step of secretion. Next to its prominent role in processing of proinflammatory cytokine targets, caspase-1 also cleaves many other cytosolic proteins, the role of those is only starting to be understood. In our current projects we are mainly focusing on two inflammasome complexes: on the one hand, AIM2, a very specific sensor for cytosolic DNA and on the other hand NLRP3, a very important, yet up to now mechanistically poorly understood NLR sensor that responds to a large variety of cytoplasmic damage signals. Using various complementary approaches, we are currently trying to understand their molecular mechanisms of activation and we aim at deciphering their functional roles at the cellular and organismic level in health and disease.

CYTOPLASMIC NUCLEIC ACID SENSING

A common theme in antimicrobial immunity is the recognition of pathogen-derived nucleic acids. In fact, most microbial pathogens expose some type of nucleic acid or degradation products thereof at some point during their life cycle (e.g. 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. Within the cytoplasm, members of the RIG-I-like helicase family play a pivotal role in sensing RNAs from viruses or bacteria. RIG-I is engaged in the course of negative-strand RNA virus infection and we and others have discovered that RIG-I senses the 5’ phosphorylation status of the viral RNA. MDA5, which is closely related to RIG-I and also shares its domain architecture, detects long double-stranded RNA, which is often found during single-stranded plus-strand RNA virus infection. RIG-I is also indirectly involved in the recognition of DNA. To this effect, we have found that RNA Pol III can convert certain DNA molecules into 5’ triphosphate dsRNA, which is in turn sensed by RIG-I.


A major focus of our current work is to elucidate the cytosolic mechanisms of DNA sensing. With AIM2 we have recently identified a novel, non-redundant DNA sensing receptor that activates caspase-1 activation (see above). However, AIM2 is not involved in the activation of pro-inflammatory gene expression. Indeed, ground-breaking studies by James Chen and colleagues led to the identification of the predominant cytosolic DNA sensing pathway that orchestrates the induction of antiviral and pro-inflammatory gene expression. To this end, it was shown that the presence of DNA in the cytosol is sensed by the cytoplasmic nucleotidyltransferase cGAS, which produces the cyclic dinucleotide second messenger molecule cGAMP upon DNA contact. cGAMP activates the ER-resident receptor molecule STING, which in turn leads to the activation of antiviral immunity within the sensing cell. Following the seminal work by Chen and colleagues, we went on to establish a LC-MS based detection method to measure cGAMP in various cell types. To our surprise, analyzing cGAS-derived cGAMP we observed that cGAS produces a cyclic GMP-AMP dinucleotide, which comprises a 2'-5' and a 3'-5' phosphodiester linkage >Gp(2'-5')Ap(3'-5')> / cGAMP(2’-5’). Moreover, we could show that cGAMP(2’-5’) is synthesized in two steps that are both catalyzed by cGAS. Interestingly, unlike bacterial cyclic dinucleotides the metazoan second messenger cGAMP(2’-5’) turned out to be a potent agonist for human STING, whereas its 3’-5’ linked counterpart only exerted minimal activity. The cGAS-STING axis plays a pivotal role in the detection of DNA viruses and moreover, it also seems to play an important role in the response towards endogenous DNA species that arise during tissue damage and cell death. Moreover, we have recently identified in vitro that the initial cell intrinsic antiviral immune response is horizontally transmitted into neighboring cells by transfer of cGAMP(2’-5’). Upon DNA sensing, cGAS-produced cGAMP(2’-5’) is transferred to bystander cells via gap junctions, where it also confers antiviral immunity in a STING-dependent manner. In ongoing projects, we are delineating the molecular mechanisms and role of the cGAS-STING axis in various infectious and sterile inflammatory conditions and study the role of horizontal cGAMP signaling in vivo.

 

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