Periodic Reporting for period 1 - MY MITOCOMPLEX (Functional relevance of mitochondrial supercomplex assembly in myeloid cells)
Período documentado: 2021-11-01 hasta 2023-10-31
The emerging field of immunometabolism has a strong potential to uncover novel targets for the manipulation of immune cell function. Myeloid cells are involved in innate and adaptive immunity and tolerance, therefore the identification of pathways that regulate their activity may have implications in many diseases. Research in the host laboratory has focused on how sensing of innate stimuli (infections and tissue damage) lead to mitochondrial adaptations in myeloid cells. These mitochondrial adaptations can influence the electron transport chain (ETC), resulting in differences in reactive oxygen species (ROS) production, ATP synthesis, redox balance and metabolites. The ETC consists of four respiratory complexes (CI-CIV), which can, excluding CII, form super complexes. The formation of these super complexes is regulated and this regulation has been shown to have biological relevance. However, whether mitochondrial SC organization couples to regulation of immune cell function and the molecular mechanisms involved is not known. Therefore, we propose to investigate how mitochondrial SC formation affects macrophage and dendritic cell function. Identification of the mechanisms connecting mitochondrial adaptations and myeloid cell function could potentially unveil therapeutic targets. Much immunometabolism studies could be improved by in vivo models, therefore we aim at studying the effects of SC formation regulation in vivo.
We intend to use targeted and non-targeted approaches to address this question. A mouse model that exhibits a non-active SC assembly factor (SCAF1) will be a key tool to address this question in vivo. The non-independent approach includes state-of-the-art metabolomics and transcriptomics.
We intend to use targeted and non-targeted approaches to address this question. A mouse model that exhibits a non-active SC assembly factor (SCAF1) will be a key tool to address this question in vivo. The non-independent approach includes state-of-the-art metabolomics and transcriptomics.
We discovered that there are few differences in immune cell compartments due to differential SC assembly. We aimed to elucidate whether regulation of supercomplex (SC) formation affects innate and adaptive immunity and the molecular mechanisms involved.
This project was based on two different sets of preliminary data obtained in the host lab. The functional differences that were observed were considered reliable and worthy to pursue based on various reasons, as previously described. Previous data indicated a difference in both innate and adaptive immune responses. This gave us two interesting paths to investigate that would probably not rely on one another. They were not contingent, therefor it would be less risky; either one of them would have been an interesting project to dissect. Quickly after starting the project, it became clear that the mouse lines needed to be refreshed, this resulted in a significant delay at the start of the project.
At this point the decision was made to start a contingency plan in parallel, by adapting the project in a way that the genetic mouse models were not necessary.
Aim 1 was to characterize the effect of the different SCAF1 isoforms in the immune compartment development. We found some minor differences in thymus and bone marrow populations that were not consistent between males and females.
For the second aim of the project, the difference in the innate immune response presented in the project proposal were reproduced in female mice, but not in male mice.
There was another drawback of the model organism; namely that the different isoforms were present in all cells of the body, not limited to the immune compartment. However, this could be addressed by using cell-culture methods or by using bone-marrow transplanted (BMT) models. I was un-able to confirm data obtained in the whole body transgenic mice by using BMT chimeric mice. This indicated that the difference in innate immune response upon influenza virus infection in mice with SCAF1 isoforms did not rely heavily on the myeloid cell compartment.
As an important contingency plan of this proposal we had various research lines to pursue. First, the basal differences in immune composition and respiration with a focus on dendritic cells (DCs) and macrophages. We did not find mayor differences. Second, differences in innate immune responses, as described above. And third (Aim 3), differences in cross-presentation by DCs. For this part we also needed to refresh the mouse lines before being able to begin, during the delay that this caused, a parallel contingency plan was started within the scope of this third aim. In the comparison of cross-presentation capabilities by DCs derived from mice with different SCAF1 isoforms we also found sex-differences.
The results from the contingency project are the following: Several innate immune cells have been described to harbor memory traits, a process called trained immunity. However, the induction of trained immunity in conventional DCs is poorly characterized. (Contingency Aim1: characterization of the physiological effects of beta-glucan training of DCs) Here, we explored the effects of β-glucan treatment during dendritic cell differentiation in trained immunity hallmarks. (Contingency aim 2: Innate immunity in DCs upon beta-glucan training) Transcriptomics analysis of β-glucan-treated cDC2s showed an increase in glycolysis and HIF1α pathway in β-glucan compared to untreated Flt3L bone marrow derived cDC2s (Flt3L-cDC2s). Upon different secondary stimuli, β-glucan- treated Flt3L-cDC2s produced increased amounts of TNFα compared to untreated Flt3L-cDC2s, dependent on Dectin-1 expression. (COntingency Aim4: Discribing an underlying mechanism for increased responses by DCs after innate immune training by beta-glucan). ATACseq analysis of Flt3L-cDC2s treated with β-glucan showed increased open chromatin. (Contingency Aim 3: Elucidating how innat eimmune training might affect DC activation and antigen presenting capacity) Moreover, treatment with β-glucan in vivo resulted in increased dendritic-cell-mediated cross-presentation. Overall, β-glucan can result in epigenetic changes in dendritic cells that lead to enhanced secondary responses.
This project was based on two different sets of preliminary data obtained in the host lab. The functional differences that were observed were considered reliable and worthy to pursue based on various reasons, as previously described. Previous data indicated a difference in both innate and adaptive immune responses. This gave us two interesting paths to investigate that would probably not rely on one another. They were not contingent, therefor it would be less risky; either one of them would have been an interesting project to dissect. Quickly after starting the project, it became clear that the mouse lines needed to be refreshed, this resulted in a significant delay at the start of the project.
At this point the decision was made to start a contingency plan in parallel, by adapting the project in a way that the genetic mouse models were not necessary.
Aim 1 was to characterize the effect of the different SCAF1 isoforms in the immune compartment development. We found some minor differences in thymus and bone marrow populations that were not consistent between males and females.
For the second aim of the project, the difference in the innate immune response presented in the project proposal were reproduced in female mice, but not in male mice.
There was another drawback of the model organism; namely that the different isoforms were present in all cells of the body, not limited to the immune compartment. However, this could be addressed by using cell-culture methods or by using bone-marrow transplanted (BMT) models. I was un-able to confirm data obtained in the whole body transgenic mice by using BMT chimeric mice. This indicated that the difference in innate immune response upon influenza virus infection in mice with SCAF1 isoforms did not rely heavily on the myeloid cell compartment.
As an important contingency plan of this proposal we had various research lines to pursue. First, the basal differences in immune composition and respiration with a focus on dendritic cells (DCs) and macrophages. We did not find mayor differences. Second, differences in innate immune responses, as described above. And third (Aim 3), differences in cross-presentation by DCs. For this part we also needed to refresh the mouse lines before being able to begin, during the delay that this caused, a parallel contingency plan was started within the scope of this third aim. In the comparison of cross-presentation capabilities by DCs derived from mice with different SCAF1 isoforms we also found sex-differences.
The results from the contingency project are the following: Several innate immune cells have been described to harbor memory traits, a process called trained immunity. However, the induction of trained immunity in conventional DCs is poorly characterized. (Contingency Aim1: characterization of the physiological effects of beta-glucan training of DCs) Here, we explored the effects of β-glucan treatment during dendritic cell differentiation in trained immunity hallmarks. (Contingency aim 2: Innate immunity in DCs upon beta-glucan training) Transcriptomics analysis of β-glucan-treated cDC2s showed an increase in glycolysis and HIF1α pathway in β-glucan compared to untreated Flt3L bone marrow derived cDC2s (Flt3L-cDC2s). Upon different secondary stimuli, β-glucan- treated Flt3L-cDC2s produced increased amounts of TNFα compared to untreated Flt3L-cDC2s, dependent on Dectin-1 expression. (COntingency Aim4: Discribing an underlying mechanism for increased responses by DCs after innate immune training by beta-glucan). ATACseq analysis of Flt3L-cDC2s treated with β-glucan showed increased open chromatin. (Contingency Aim 3: Elucidating how innat eimmune training might affect DC activation and antigen presenting capacity) Moreover, treatment with β-glucan in vivo resulted in increased dendritic-cell-mediated cross-presentation. Overall, β-glucan can result in epigenetic changes in dendritic cells that lead to enhanced secondary responses.
Beyond the state of the art, we looked in unprecedented detail into innate immune training of DC by beta-glucan both in vitro and in vivo. Some of these experiments required the processing of a large number of DCs in order to acquire enough cells for state-of-the art techniques such as ATAC-seq and Seahorse on ex vivo DCs. The information resulting from these experiments can reveal new targets in the response to infection or auto-inflammatory disease. Epigenetic remodelling of DCs is a relatively new field and recent developments in tools altering epigenetic changes in vivo provide interesting translation possibilities of these results. We have identified new epigenetic modification upon beta-glucan stimulation which results in enhanced DC function highly relevant for vaccine developement. 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 immune regulation.