Periodic Reporting for period 1 - STIMULATE (STING signalling modulation via the Electron Transport Chain)
Berichtszeitraum: 2021-07-01 bis 2023-06-30
This project, STIMULATE, was carried out in the Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, under the supervision of Dr. David Sancho. STIMULATE began in an attempt to characterise the metabolic changes that occur in macrophages upon stimulation with different immune stimuli, in particular focusing on the Stimulator of Interferon Genes (STING)-dependent cytoplasmic DNA sensing pathway. STING is an adapter protein essential for the innate immune response to cytosolic double stranded DNA (dsDNA) and has been shown to be important in a wide range of clinical scenarios, in the context of viral and bacterial infection, cancer, and autoimmunity. Upon activation of STING, there is strong downstream activation of several inflammatory cytokines, including Type I interferons (IFN-I) and IFN stimulated genes important in the immune response against viruses and other pathogens.
Despite the growing interest in immunometabolism and the potential for targeting these axes in human disease, the relationship between STING signalling and cellular metabolism is not known. Our objectives were to use a variety of techniques to study cellular metabolism in cells with or without STING stimulation. Then based on our findings, to try and find a link between the immune signalling pathway and the metabolic machinery focusing on mitochondrial targets. The mitochondria is the main metabolic hub in the cell, important not just for energy production but for the supply of building blocks required for cellular function and effective immune responses. To find these interaction partners our aim was to stimulate cells with STING agonists and perform proteomics of the mitochondria to determine interaction partners.
Then, based on the direction of the data, we planned to use inhibitors and genetic approaches to characterise the effect of inhibiting upregulated metabolic pathways on STING signalling. Macrophages have various functions within the body both in homeostasis and inflammation. We aimed to investigate their cytokine production, particularly the IFN-I response, and their ability to perform phagocytosis, one of their key functions.
Despite the growing interest in immunometabolism and the potential for targeting these axes in human disease, the relationship between STING signalling and cellular metabolism is not known. Our objectives were to use a variety of techniques to study cellular metabolism in cells with or without STING stimulation. Then based on our findings, to try and find a link between the immune signalling pathway and the metabolic machinery focusing on mitochondrial targets. The mitochondria is the main metabolic hub in the cell, important not just for energy production but for the supply of building blocks required for cellular function and effective immune responses. To find these interaction partners our aim was to stimulate cells with STING agonists and perform proteomics of the mitochondria to determine interaction partners.
Then, based on the direction of the data, we planned to use inhibitors and genetic approaches to characterise the effect of inhibiting upregulated metabolic pathways on STING signalling. Macrophages have various functions within the body both in homeostasis and inflammation. We aimed to investigate their cytokine production, particularly the IFN-I response, and their ability to perform phagocytosis, one of their key functions.
During our investigation of STING-mediated metabolic changes, we found that adding STING agonist to cells caused a metabolic change not at early time points (1 hour or 4 hours after stimulation), but at later timepoints (20 hours post stimulation). The changes that we observed were an active mitochondrial metabolism characterised by increased oxygen consumption and a mild increase in glycolysis, accompanied by a strong decrease in mitochondrial membrane potential and mitochondrial fission. The loss of mitochondrial membrane potential and fission is not typically linked to increased mitochondrial function, and for us this was a very interesting and unique response to stimulation.
Since the effect was only observed at later timepoints, we suspected that it was not due to intrinsic STING signalling but due to a second-wave factor produced upon activation. The first potential candidate for this effect upon STING activation was IFN-I. To test this, we tested if IFN-I alone would have the same effect, and if blocking IFN-I would block this effect. Indeed, addition of recombinant IFN-I to macrophages showed the same metabolic effect at early timepoints. And the effect upon STING agonist or IFN-I could be blocked by blocking the IFN-I receptor. We then dedicated our study to determining the metabolic changes upon IFN-I stimulation rather than the use of direct STING agonists.
As we observed more mitochondrial fission upon STING/IFN, we used a genetic model to determine if fission was required for this phenotype. However, we found that the metabolic changes, excluding fission, still occurred in this model. Based on literature about downstream effects of IFN-I, we then tested some IFN stimulated genes and identified one that mediates the effects of STING/IFN stimulation.
To then study the effects of these changes, we tested primary cytokine production in both the fission-defective model and cells deficient in the target IFN-stimulated gene to differentiate between effects of decreased mitochondrial fission vs the other metabolic phenotypes observed. We found that in our genetic models, primary cytokine production upon STING agonist stimulation was not affected. We then tested the response to IFN-I stimulation and found an increase in IFN stimulated genes in fission-deficient cells. This indicated that the induction of mitochondrial fission by IFN-I acts as a negative feedback loop.
We then tested the effect on phagocytosis, focussing on the uptake of dead cells, efferocytosis. We found that IFN-I stimulation increased the efferocytosis rate of macrophages and this increase could be inhibited in cells deficient in the identified target IFN-stimulated gene. IFN-I can be inflammatory and is important for viral clearance, but it also serves a homeostatic function in the resolution of inflammation. Part of the resolution of inflammation is the clearance of dead cells and debris by macrophages, and we found that the metabolic changes induced by IFN-I were important for this resolutive function.
Since the effect was only observed at later timepoints, we suspected that it was not due to intrinsic STING signalling but due to a second-wave factor produced upon activation. The first potential candidate for this effect upon STING activation was IFN-I. To test this, we tested if IFN-I alone would have the same effect, and if blocking IFN-I would block this effect. Indeed, addition of recombinant IFN-I to macrophages showed the same metabolic effect at early timepoints. And the effect upon STING agonist or IFN-I could be blocked by blocking the IFN-I receptor. We then dedicated our study to determining the metabolic changes upon IFN-I stimulation rather than the use of direct STING agonists.
As we observed more mitochondrial fission upon STING/IFN, we used a genetic model to determine if fission was required for this phenotype. However, we found that the metabolic changes, excluding fission, still occurred in this model. Based on literature about downstream effects of IFN-I, we then tested some IFN stimulated genes and identified one that mediates the effects of STING/IFN stimulation.
To then study the effects of these changes, we tested primary cytokine production in both the fission-defective model and cells deficient in the target IFN-stimulated gene to differentiate between effects of decreased mitochondrial fission vs the other metabolic phenotypes observed. We found that in our genetic models, primary cytokine production upon STING agonist stimulation was not affected. We then tested the response to IFN-I stimulation and found an increase in IFN stimulated genes in fission-deficient cells. This indicated that the induction of mitochondrial fission by IFN-I acts as a negative feedback loop.
We then tested the effect on phagocytosis, focussing on the uptake of dead cells, efferocytosis. We found that IFN-I stimulation increased the efferocytosis rate of macrophages and this increase could be inhibited in cells deficient in the identified target IFN-stimulated gene. IFN-I can be inflammatory and is important for viral clearance, but it also serves a homeostatic function in the resolution of inflammation. Part of the resolution of inflammation is the clearance of dead cells and debris by macrophages, and we found that the metabolic changes induced by IFN-I were important for this resolutive function.
Beyond the state of the art, we looked in unprecedented detail into mitochondrial function in primary immune cells, bone marrow derived macrophages. Some of these experiments required the processing of a large number of macrophages in order to acquire enough cells to have sufficient mitochondrial proteins. With these macrophages, we performed proteomics to identify altered mitochondrial proteins. The information resulting from these experiments can reveal new targets in the response to infection or auto-inflammatory disease. As of now, post-translational modifications of mitochondrial proteins is a relatively new field. We have identified a new post-translational modification upon IFN-I stimulation which results in enhanced macrophage function in a disease context. We looked both in vitro and in vivo at their function to find physiological relevance. This study is now in preparation for publication and we hope that this work will contribute to our wider understanding of inflammation resolution and immune regulation.