Periodic Reporting for period 1 - PathAutoBIO (Uncovering the pathway of DNA-induced autophagy and its biological functions in viral central nervous system infection)
Reporting period: 2018-05-01 to 2020-04-30
The host mobilizes a series of responses against invading pathogens. However, pathogens have developed a wide range of strategies to counteract or hijack these responses. Hence, a deep understanding of the different defense mechanisms against infection and their regulation by pathogens is critical for development of new therapeutic strategies. The innate immune system constitutes the first line of defense against infections and is rapidly activated after sensing of pathogen- and abnormal self-derived molecules. For instance, cytoplasmic DNA can be detected by a variety of immune sensors, notably the key sensor cGAS. In most cases, these sensors require the function of the adaptor protein STING to lead to the production of interferons and other molecules playing important roles in inflammation and defense against pathogens. It is well established that sensing of foreign DNA and STING functions play a central role in the defense against a wide range of bacterial and viral infections. On the other hand, excessive STING activation is linked to disproportionate inflammatory responses and could lead to inflammation-driven carcinogenesis or autoimmune disease. A fine-tuning of STING functions is thus absolutely required to ensure both protective immunity and limitation of uncontrolled inflammation. Interestingly, our lab and others showed that DNA sensing activates autophagy, an ancient and conserved catabolic process involved in metabolic recycling but also degradation of pathogens and positive and negative regulation of immune responses.
Importantly, our knowledge of the cross-regulations between autophagy and innate immunity and their modulation by pathogens, including viruses, is fragmented. In particular, a comprehensive view of the connections between STING and autophagy is missing. Our project aim at deciphering the pathway of this newly discovered autophagy-activating function of the cGAS-STING axis. Especially, we aim at defining the involvement of known molecules of the autophagy pathway in this mechanism, identify STING-interacting proteins involved in STING-mediated autophagy and uncover their mode of action. This project allowed us to uncover that STING pathways leading to autophagy and to immune responses are at least partially distinct. We also found that STING-mediated autophagy represent a previously unsuspected non-canonical autophagy pathway.
Importantly, our knowledge of the cross-regulations between autophagy and innate immunity and their modulation by pathogens, including viruses, is fragmented. In particular, a comprehensive view of the connections between STING and autophagy is missing. Our project aim at deciphering the pathway of this newly discovered autophagy-activating function of the cGAS-STING axis. Especially, we aim at defining the involvement of known molecules of the autophagy pathway in this mechanism, identify STING-interacting proteins involved in STING-mediated autophagy and uncover their mode of action. This project allowed us to uncover that STING pathways leading to autophagy and to immune responses are at least partially distinct. We also found that STING-mediated autophagy represent a previously unsuspected non-canonical autophagy pathway.
1. Importance of STING regions in autophagy induction
We generated STING deletion mutants encompassing conserved regions as well as the C terminal tail, important for the activation of STING immune response. We characterized the effect of STING deletions on autophagy induction by co-expressing these mutants with cGAS in HEK293T cells, in which we found STING-cGAS co-expression being sufficient to stimulate autophagy. Our results showed that STING C terminal tail is dispensable for autophagy activation. This data, and other confidential data we generated, unraveled that STING-mediated immune response and autophagy are at least partly distinct pathways. We also found that deletion of a small region before the C terminal tail was sufficient to impair both STING functions as well as STING dimerization. This led us to hypothesize that STING-mediated autophagy occurs after STING dimerization, which happens at the endoplasmic reticulum (ER) prior STING trafficking from the ER to the Golgi.
2. Impact of STING trafficking on autophagy induction
STING trafficking from ER to Golgi is an important step of STING activation leading to STING-mediated immune response. To test the importance of this mechanism in STING-mediated autophagy, we used the drug Brefeldin A, known to block ER exit. Experiments done in both HaCat and THP1 cells showed that ER exit of STING was important for STING-mediated autophagy.
3. Involvement of key players of autophagy
By using siRNA downregulation and pharmacological inhibition, we surprisingly found that the key autophagy regulator VPS34 was not involved in STING-mediated autophagy. Moreover, we failed to detect activation of another important autophagy regulator, Beclin-1. These results suggest that STING-mediated autophagy is a non-canonical autophagy pathway, that is activated independently of Beclin-1 and VPS34.
We also performed a mass spectrometry analysis of STING-interacting proteins using WT and mutants STING in order to identify new proteins involved in STING-mediated autophagy. Unfortunately, and after testing several candidates, we so far did not uncover any new factor involved in this pathway.
4. Technical developments and establishment of datasets
During this project, I have developed assays of Imaging Flow Cytometry, a powerful cutting-edge imaging technique allowing high throughput analysis of diverse parameters. Notably, I optimized protocols to quantitatively study various STING functions, such as STING trafficking from ER to Golgi, autophagy activation and nuclear translocation of transcription factors downstream of STING. These assays were used by me and several people in the lab and I believe that they will benefit future studies of STING functions. I plan to prepare a method paper describing those assays, in order to disseminate these methods with the scientific community.
Moreover, I generated in collaboration with Pr. Robert Fenton from Aarhus University a STING interactome, by analyzing by mass spectrometry proteins interacting with STING WT and mutants before and after activation. This dataset is already exploited by lab members to find new factors important for STING functions and will be profitable for the follow up of my own project as well as for the design of new projects in the lab.
During the course of this project, we uncovered that STING-mediated autophagy and STING-mediated immune response are at least partially distinct, as STING-mediated autophagy does not require the C terminal tail, a region that is instrumental for STING-mediated immune response. We also found that STING-mediated autophagy represent a previously unsuspected non-canonical autophagy pathway that is independent of the key autophagy factors VPS34 and Beclin 1. Finally, this project lead to important technical advancements and generation of datasets that will benefit the host lab in the long term.
We generated STING deletion mutants encompassing conserved regions as well as the C terminal tail, important for the activation of STING immune response. We characterized the effect of STING deletions on autophagy induction by co-expressing these mutants with cGAS in HEK293T cells, in which we found STING-cGAS co-expression being sufficient to stimulate autophagy. Our results showed that STING C terminal tail is dispensable for autophagy activation. This data, and other confidential data we generated, unraveled that STING-mediated immune response and autophagy are at least partly distinct pathways. We also found that deletion of a small region before the C terminal tail was sufficient to impair both STING functions as well as STING dimerization. This led us to hypothesize that STING-mediated autophagy occurs after STING dimerization, which happens at the endoplasmic reticulum (ER) prior STING trafficking from the ER to the Golgi.
2. Impact of STING trafficking on autophagy induction
STING trafficking from ER to Golgi is an important step of STING activation leading to STING-mediated immune response. To test the importance of this mechanism in STING-mediated autophagy, we used the drug Brefeldin A, known to block ER exit. Experiments done in both HaCat and THP1 cells showed that ER exit of STING was important for STING-mediated autophagy.
3. Involvement of key players of autophagy
By using siRNA downregulation and pharmacological inhibition, we surprisingly found that the key autophagy regulator VPS34 was not involved in STING-mediated autophagy. Moreover, we failed to detect activation of another important autophagy regulator, Beclin-1. These results suggest that STING-mediated autophagy is a non-canonical autophagy pathway, that is activated independently of Beclin-1 and VPS34.
We also performed a mass spectrometry analysis of STING-interacting proteins using WT and mutants STING in order to identify new proteins involved in STING-mediated autophagy. Unfortunately, and after testing several candidates, we so far did not uncover any new factor involved in this pathway.
4. Technical developments and establishment of datasets
During this project, I have developed assays of Imaging Flow Cytometry, a powerful cutting-edge imaging technique allowing high throughput analysis of diverse parameters. Notably, I optimized protocols to quantitatively study various STING functions, such as STING trafficking from ER to Golgi, autophagy activation and nuclear translocation of transcription factors downstream of STING. These assays were used by me and several people in the lab and I believe that they will benefit future studies of STING functions. I plan to prepare a method paper describing those assays, in order to disseminate these methods with the scientific community.
Moreover, I generated in collaboration with Pr. Robert Fenton from Aarhus University a STING interactome, by analyzing by mass spectrometry proteins interacting with STING WT and mutants before and after activation. This dataset is already exploited by lab members to find new factors important for STING functions and will be profitable for the follow up of my own project as well as for the design of new projects in the lab.
During the course of this project, we uncovered that STING-mediated autophagy and STING-mediated immune response are at least partially distinct, as STING-mediated autophagy does not require the C terminal tail, a region that is instrumental for STING-mediated immune response. We also found that STING-mediated autophagy represent a previously unsuspected non-canonical autophagy pathway that is independent of the key autophagy factors VPS34 and Beclin 1. Finally, this project lead to important technical advancements and generation of datasets that will benefit the host lab in the long term.
Both STING and autophagy pathways are now recognized as therapeutic targets in a variety of infections and pathologies such as cancers or autoimmune diseases. Our work thus contributes to a broader understanding of mechanisms regulating a wide range of diseases that present global threats for societies. Notably, our findings will help the research in therapeutic targeting of the different STING functions. The proposed action also led to the development of cutting-edge assays and tools to study autophagy and innate immunity as well as the generation of a STING interactome that will benefit the whole lab for future discovery of factors important for STING functions. Our findings thus led to a better understanding of the mechanisms governing the balance between protective responses and deleterious inflammation and will help the design of broad-spectrum innovative therapies as well as future scientific advancements in the field of immunology and cellular biology.