Periodic Reporting for period 1 - MeRGeR (Physiological significance of the glucocorticoid and mineralocorticoid receptor signalling in the innate immune system)
Okres sprawozdawczy: 2021-01-01 do 2022-12-31
The purpose of the MeRGeR project was to unravel the role of the mineralocorticoid receptor (MR) in the immune response. This receptor is important in mediating the effects of cortisol, a key stress hormone in humans. However, prior to this project, the effects of cortisol on the immune system were primarily attributed to the other receptor of cortisol, the glucocorticoid receptor (GR) and very little was known about the immunomodulatory effects of MR. Determining how these receptors modulate the immune system, both independently and together is essential to our understanding of how stress impacts our immunity. Given the ubiquitous nature of stress, and the impact that it has on our society at both an individual and population level understanding the interaction between stress and the immune system is of strategic importance.
To determine the role of MR in the immune system, we designed an interdisciplinary approach that combined advanced microscopy with novel in vivo models. The first objective was to determine the role of MR in modulating the inflammatory response. This objective was completed using the zebrafish animal model were I characterized the role of MR both during immune system development and in response to an inflammatory stimulus. The overall conclusion of this objective was that MR promotes the immune response, which was in contrast to the activation of GR, which was primarily anti-inflammatory.
The aim of the second objective was to determine how MR and GR functionally interact to alter gene expression. Here we also used zebrafish as a model, as they are the only major model organism that can harbour a complete loss of GR and MR. Therefore, we were provided with a unique opportunity to assess the interaction of GR and MR in the whole animal. To do this we introduced mutated forms of GR and MR that could either only interact with themselves (homodimer) or each other (heterodimer). The conclusions of this objective were that GR and MR can indeed interact, and that this interaction has physiological and immunological significance.
Overall, the publications that will be generated from this project will have considerable impact on the current paradigm of cortisol action in the immune system, how we understand stress, and in extending our understanding of the molecular actions of immunomodulating glucocorticoid drugs.
To determine the role of MR in the immune system, we designed an interdisciplinary approach that combined advanced microscopy with novel in vivo models. The first objective was to determine the role of MR in modulating the inflammatory response. This objective was completed using the zebrafish animal model were I characterized the role of MR both during immune system development and in response to an inflammatory stimulus. The overall conclusion of this objective was that MR promotes the immune response, which was in contrast to the activation of GR, which was primarily anti-inflammatory.
The aim of the second objective was to determine how MR and GR functionally interact to alter gene expression. Here we also used zebrafish as a model, as they are the only major model organism that can harbour a complete loss of GR and MR. Therefore, we were provided with a unique opportunity to assess the interaction of GR and MR in the whole animal. To do this we introduced mutated forms of GR and MR that could either only interact with themselves (homodimer) or each other (heterodimer). The conclusions of this objective were that GR and MR can indeed interact, and that this interaction has physiological and immunological significance.
Overall, the publications that will be generated from this project will have considerable impact on the current paradigm of cortisol action in the immune system, how we understand stress, and in extending our understanding of the molecular actions of immunomodulating glucocorticoid drugs.
The MeRGeR project covered a period of two years (January 2021-Decemeber 2023), where nearly all the initial objectives were achieved with modifications designed to increase the novelty and scope of the findings. Below is an overview of the key findings:
In the first objective we postulated that activation of receptors for cortisol, MR and GR, would have opposite effects on the inflammatory response, where MR promotes, and GR suppresses the innate immune system. As outlined in the proposed research, the model organism we used to test this hypothesis was zebrafish. We used this model because immune cells can be visualized with fluorescent tags, and the stress response, including cortisol signalling is well characterized. All proposed research in this objective was achieved. Key results include the findings that: i) MR is involved in immune system development and ii) MR activation promotes the immune response to inflammation.
As part of this objective, we also sought to determine the potential targets of MR in immune cells. To achieve this we isolated macrophages from zebrafish using fluorescence activated cell sorting. Analysis of these cells revealed that: i) Macrophages lacking MR had altered expression in key genes involved in migration during development and ii) Macrophages treated with an MR antagonist had altered expression of key genes involved in migration during inflammation. The data from this objective has been presented at several international meetings and will culminate in the publication of at least one publication.
In objective 2 we initially proposed cell models of inflammation to determine the functional interaction of GR and MR. However, we modified the experimental design to use in vivo models, thereby increasing the novelty and scope of the findings. Instead of the proposed cell experiments, which limits our findings to a single cell type, we instead established whole animal models to receptor interaction. To do this we designed mutated forms of GR and MR that could either only interact with themselves (homodimer) or each other (heterodimer). We then injected these plasmids into zebrafish embryos lacking both GR and MR and observed whether there was a change in the response to inflammation. To determine the potential target genes of MR and GR in immune cells we performed next generation RNA-sequencing. Overall, this objective demonstrated that there was a physiological and immunological significance to MR and GR interaction. The data from this objective has been presented at local symposia at Leiden University and will culminate in the publication of at least one publication.
In the first objective we postulated that activation of receptors for cortisol, MR and GR, would have opposite effects on the inflammatory response, where MR promotes, and GR suppresses the innate immune system. As outlined in the proposed research, the model organism we used to test this hypothesis was zebrafish. We used this model because immune cells can be visualized with fluorescent tags, and the stress response, including cortisol signalling is well characterized. All proposed research in this objective was achieved. Key results include the findings that: i) MR is involved in immune system development and ii) MR activation promotes the immune response to inflammation.
As part of this objective, we also sought to determine the potential targets of MR in immune cells. To achieve this we isolated macrophages from zebrafish using fluorescence activated cell sorting. Analysis of these cells revealed that: i) Macrophages lacking MR had altered expression in key genes involved in migration during development and ii) Macrophages treated with an MR antagonist had altered expression of key genes involved in migration during inflammation. The data from this objective has been presented at several international meetings and will culminate in the publication of at least one publication.
In objective 2 we initially proposed cell models of inflammation to determine the functional interaction of GR and MR. However, we modified the experimental design to use in vivo models, thereby increasing the novelty and scope of the findings. Instead of the proposed cell experiments, which limits our findings to a single cell type, we instead established whole animal models to receptor interaction. To do this we designed mutated forms of GR and MR that could either only interact with themselves (homodimer) or each other (heterodimer). We then injected these plasmids into zebrafish embryos lacking both GR and MR and observed whether there was a change in the response to inflammation. To determine the potential target genes of MR and GR in immune cells we performed next generation RNA-sequencing. Overall, this objective demonstrated that there was a physiological and immunological significance to MR and GR interaction. The data from this objective has been presented at local symposia at Leiden University and will culminate in the publication of at least one publication.
Overall, the results generated from this project were within the experimental parameters of the initially described objectives. However, there was one key instance where there was progress made beyond the expected results. While this project primarily focused on the role of MR, it was placed within the broader context of stress-immune interaction. This is important because cortisol is a key stress hormone, and it is well known that chronic stress has negative effects on our immunity. To this end when we observed that a disruption in MR resulted in changes to immune cell distribution, we next asked the question: does stress and the attendant rise in cortisol also result in redistribution of immune cells during stress? This led to the realization, and experimental validation, that stress, in particular acute stress, could be immune enhancing rather than immune-suppressive. Using our unique zebrafish models developed during this project, we further characterized the involvement of the receptors for cortisol and the potential targets in immune cells. Scientifically, the results generated from this project provide a significant contribution to our current understanding of the mechanisms of action that dictate stress-immune interactions. From a societal perspective, understanding how stress modulates the immune system is important as nearly every individual experiences stress and when a stressor persists it can have negative effects both psychologically and physiologically. Therefore, discovering how we might temper the negative side effects of stress can potentially benefit both the individual and by extension have a wider socio-economic impact.