Periodic Reporting for period 4 - DCRIDDLE (A novel physiological role for IRE1 and RIDD..., maintaining the balance between tolerance and immunity?)
Período documentado: 2023-08-01 hasta 2024-01-31
Dendritic cells (DCs) play a crucial role as gatekeepers of the immune system, coordinating the balance between protective immunity and tolerance to self-antigens. They enter peripheral tissues as immature (resting) DCs, looking for antigens to grab. Once they’ve acquired an antigen, they mature and migrate to the lymph node to present the antigen to naïve T cells. Depending on how a DC perceives the antigen (i.e. as danger or self-antigen), they will mature in an immunogenic respectively tolerogenic manner, which will steer the ensuing T cell response into protective immunity or tolerance. The signals driving immunogenic maturation of DCs are fairly well characterized thanks to the discovery of Toll like receptors and other pattern recognition receptors. On the contrary, the signals driving tolerogenic maturation remain as yet unknown. This is a highly relevant question as the proper maturation state of the DC (immunogenic versus tolerogenic) is absolutely essential to avoid autoimmunity or enable tumor cell killing.
By serendipity, our lab found a role for a sensor of the unfolded protein response, IRE1, in tolerogenic DC maturation. This made us wonder what are the pathways driving tolerogenic DC maturation and how an evolutionary conserved protein like IRE1 could fit in this process. It led us to uncover an unexpected link between apoptotic cell engulfment, regulation of cholesterol metabolism, tolerogenic DC maturation and IRE1, and clearly expanded the function of IRE1 beyond its well-established role in protein folding.
The work done in frame of this ERC grant has laid the foundation of several follow-up studies in my lab. We are currently trying to understand how IRE1 at the structural level coordinates these different outputs, i.e. how does it balance protein folding versus lipid metabolism. Besides, we became interested in understanding in great detail how dendritic cell maturation pathways are being regulated. Why are cholesterol levels differentially regulated in tolerogenic versus immunogenic responses? What happens during infection? How does the cell avoid that autoantigens are being presented in these conditions? And how does a tumor microenvironment affect dendritic cell maturation and tweaks how tumor antigens are being presented to naïve T cells?
These questions will be the focus of my lab in the coming years and will learn us more on the quintessential question in immunology, i.e. how do dendritic cells balance protective immunity and tolerance and why do things go wrong in autoimmune diseases or during tumor immune escape?
By serendipity, our lab found a role for a sensor of the unfolded protein response, IRE1, in tolerogenic DC maturation. This made us wonder what are the pathways driving tolerogenic DC maturation and how an evolutionary conserved protein like IRE1 could fit in this process. It led us to uncover an unexpected link between apoptotic cell engulfment, regulation of cholesterol metabolism, tolerogenic DC maturation and IRE1, and clearly expanded the function of IRE1 beyond its well-established role in protein folding.
The work done in frame of this ERC grant has laid the foundation of several follow-up studies in my lab. We are currently trying to understand how IRE1 at the structural level coordinates these different outputs, i.e. how does it balance protein folding versus lipid metabolism. Besides, we became interested in understanding in great detail how dendritic cell maturation pathways are being regulated. Why are cholesterol levels differentially regulated in tolerogenic versus immunogenic responses? What happens during infection? How does the cell avoid that autoantigens are being presented in these conditions? And how does a tumor microenvironment affect dendritic cell maturation and tweaks how tumor antigens are being presented to naïve T cells?
These questions will be the focus of my lab in the coming years and will learn us more on the quintessential question in immunology, i.e. how do dendritic cells balance protective immunity and tolerance and why do things go wrong in autoimmune diseases or during tumor immune escape?
The main objective of this research project was to decipher the function of the unfolded protein sensor IRE1 in dendritic cells (cDC). Earlier studies from my lab had revealed that IRE1 shows a high basal activity in cDC1s, one of the two major subsets of conventional DCs that play an important role in tolerance and anti-viral and anti-tumor immunity. Of note, IRE1 activity was not reflected by activation of XBP1, the transcription factor that is typically activated downstream of IRE1 signaling. This made us wonder how and why IRE1 was active specifically in cDC1s.
A transcriptional profiling analysis performed before the start of the ERC revealed that absence of IRE1 in cDC1 led to a decrease in expression of maturation genes, genes associated with efferocytosis and genes implicated in cholesterol metabolism. This led to the hypothesis that IRE1 could be a regulator of apoptotic cell engulfment and homeostatic DC maturation, which formed the basis of the current DC-RIDDLE project. More concrete, we formulated 4 main working hypotheses: In WP1, we wanted to test the assumption that IRE1 main’s actions in dendritic cells were not explained by XBP1-dependent transcriptional activity, but rather by IRE1-dependent RIDD activation and degradation of miRNAs, in WP2, we postulated that IRE1 would be important for apoptotic cell engulfment in DCs, in WP3, we postulated that IRE1 might play a role in tolerogenic antigen presentation, and finally, in WP4, we wanted to investigate the role of IRE1 in viral infections.
Early during the DC-RIDDLE project, we noticed that cDC1s that are deficient for IRE1 no longer matured in homeostatic conditions, while immunogenic maturation induced by pIC did not appear to be affected.
For many years, the upstream signals driving the homeostatic maturation of DCs have remained enigmatic and seeing the crucial role of IRE1 in homeostatic not immunogenic DC maturation gave us a tool to find out the mechanisms underlying this homeostatic maturation program. The results of this study can be found in (Bosteels et al., Sci Imm, 2023). In brief, we showed that apoptotic cell engulfment and concomitant lipid influx is a major driver for cDC1 homeostatic maturation. Homeostatic maturation is associated with cholesterol efflux, while in immunogenic conditions cholesterol levels are retained, showing a tight link between cholesterol metabolism and the balance between immunity and tolerance.
Later, we found that IRE1 in cDC1s is triggered by the cholesterol influx during engulfment in cDC1s, and that in response, IRE1 regulates the cholesterol efflux. In absence of IRE1, free cholesterol accumulates at the ER, inducing lipotoxicity and cell death of the late immature and mature cDC1 substages (Bosteels et al., in revision).
A transcriptional profiling analysis performed before the start of the ERC revealed that absence of IRE1 in cDC1 led to a decrease in expression of maturation genes, genes associated with efferocytosis and genes implicated in cholesterol metabolism. This led to the hypothesis that IRE1 could be a regulator of apoptotic cell engulfment and homeostatic DC maturation, which formed the basis of the current DC-RIDDLE project. More concrete, we formulated 4 main working hypotheses: In WP1, we wanted to test the assumption that IRE1 main’s actions in dendritic cells were not explained by XBP1-dependent transcriptional activity, but rather by IRE1-dependent RIDD activation and degradation of miRNAs, in WP2, we postulated that IRE1 would be important for apoptotic cell engulfment in DCs, in WP3, we postulated that IRE1 might play a role in tolerogenic antigen presentation, and finally, in WP4, we wanted to investigate the role of IRE1 in viral infections.
Early during the DC-RIDDLE project, we noticed that cDC1s that are deficient for IRE1 no longer matured in homeostatic conditions, while immunogenic maturation induced by pIC did not appear to be affected.
For many years, the upstream signals driving the homeostatic maturation of DCs have remained enigmatic and seeing the crucial role of IRE1 in homeostatic not immunogenic DC maturation gave us a tool to find out the mechanisms underlying this homeostatic maturation program. The results of this study can be found in (Bosteels et al., Sci Imm, 2023). In brief, we showed that apoptotic cell engulfment and concomitant lipid influx is a major driver for cDC1 homeostatic maturation. Homeostatic maturation is associated with cholesterol efflux, while in immunogenic conditions cholesterol levels are retained, showing a tight link between cholesterol metabolism and the balance between immunity and tolerance.
Later, we found that IRE1 in cDC1s is triggered by the cholesterol influx during engulfment in cDC1s, and that in response, IRE1 regulates the cholesterol efflux. In absence of IRE1, free cholesterol accumulates at the ER, inducing lipotoxicity and cell death of the late immature and mature cDC1 substages (Bosteels et al., in revision).
Several important breakthroughs were made in the DC-RIDDLE project.
The signals driving the homeostatic DC maturation program have remained for a long time enigmatic. While it was known that DCs engulf apoptotic cells, the general dogma was that this would not induce maturation, because otherwise T cells might recognize and react to self-antigens. This concept turned out to be wrong and we now showed that uptake of apoptotic cells is sufficient to drive cDC1 maturation. This implies that also in steady state conditions, DC do migrate and carry self-antigens to the LN. However, we found that these homeostatic DCs are under tight control by Tregs, thereby preventing the activation of naive effector T cells.
Secondly, we found that IRE1 in DCs becomes activated by influx of lipids, most likely cholesterol. In yeast, it has been described that IRE1 can be activated by so-called aberrant lipid stress, like the accumulation of cholesterol at the ER. In mammals this is less established. The fact that we see activation of IRE1 in DCs in absence of canonical UPR genes being activated and triggered by a different signal, makes us wonder what other functions the UPR could harbor beyond its canonical role in folding.
Finally, the role of IRE1 dependent RIDD or IRE1 dependent degradation of mRNAs or miRNAs has mostly been observed in XBP1-deficient conditions, which lead to strong hyperactivation of IRE1 endonuclease activity. This has cast some doubt on the physiological role of RIDD. We now describe, for the very first time, a physiological role for RIDD in degradation of mIRNAs.
In conclusion, the DC-RIDDLE project has provided us several new insights both in the field of DC biology, efferocytosis, and UPR biology. Our findings will have implications in the field of tolerance and autoimmunity, and will help the emerging lipid nanoparticle field for safer design of mRNA vaccins.
In the coming years my lab will pursue several lead findings that came out from this study and start implementing some of them in more applied settings together with industrial partners.
The signals driving the homeostatic DC maturation program have remained for a long time enigmatic. While it was known that DCs engulf apoptotic cells, the general dogma was that this would not induce maturation, because otherwise T cells might recognize and react to self-antigens. This concept turned out to be wrong and we now showed that uptake of apoptotic cells is sufficient to drive cDC1 maturation. This implies that also in steady state conditions, DC do migrate and carry self-antigens to the LN. However, we found that these homeostatic DCs are under tight control by Tregs, thereby preventing the activation of naive effector T cells.
Secondly, we found that IRE1 in DCs becomes activated by influx of lipids, most likely cholesterol. In yeast, it has been described that IRE1 can be activated by so-called aberrant lipid stress, like the accumulation of cholesterol at the ER. In mammals this is less established. The fact that we see activation of IRE1 in DCs in absence of canonical UPR genes being activated and triggered by a different signal, makes us wonder what other functions the UPR could harbor beyond its canonical role in folding.
Finally, the role of IRE1 dependent RIDD or IRE1 dependent degradation of mRNAs or miRNAs has mostly been observed in XBP1-deficient conditions, which lead to strong hyperactivation of IRE1 endonuclease activity. This has cast some doubt on the physiological role of RIDD. We now describe, for the very first time, a physiological role for RIDD in degradation of mIRNAs.
In conclusion, the DC-RIDDLE project has provided us several new insights both in the field of DC biology, efferocytosis, and UPR biology. Our findings will have implications in the field of tolerance and autoimmunity, and will help the emerging lipid nanoparticle field for safer design of mRNA vaccins.
In the coming years my lab will pursue several lead findings that came out from this study and start implementing some of them in more applied settings together with industrial partners.