Periodic Reporting for period 1 - MATREX (Modulation of tRNA pool and tRNA fragments in obesity and diabetes; focus on their role in macrophage activation and exosome-mediated crosstalk with pancreatic beta cells)
Periodo di rendicontazione: 2021-09-01 al 2023-08-31
Transfer RNAs (tRNAs) have a central role in protein translation. Recently, tRNAs have been discovered to play also a crucial role as mediators of cellular responses to environmental cues. tRNAs molecules are transcribed, post-transcriptionally modified and cleaved by endonucleases giving rise to functionally active tRNA-derived fragments (tRFs): the ensemble of these processes constitutes the tRNA epitranscriptome, strictly regulated by nutrient availability and environmental factors. The important role of tRNA homeostasis and tRF biogenesis in the context of diabetes has recently emerged. Mutations in the tRNA methylase TRMT10A have been linked to tRNA hypomodification and fragmentation contributing to pancreatic β-cells death. β-cells constitute the only cells in the body able to secrete insulin in response to glucose. Moreover, β-cells from newborn and adult rats exhibit peculiar tRF signatures, which are essential for the postnatal maturation of the insulin-secreting cells. However, the effect of environmental factors contributing to the development of diabetes on the tRNA epitranscriptome has not yet been investigated. Obesity represents the strongest risk factor for type 2 diabetes (T2D) and has become a major public health concern due to the diffusion of malnutrition and overnutrition. The rise of glycaemia in T2D derives from the loss of insulin sensitivity of peripheral organs such as muscle, liver, and adipose tissue, combined with defective insulin secretion from pancreatic β-cells, occurring as a result of chronic stress. In this context, the immune cells macrophages (Mφs) resident in pancreatic islets appear to hold a crucial role. In response to obese environment and β-cell distress, islet Mφs proliferate and undergo transcriptional and metabolic changes, to counteract the excess of nutrient intake and the increased need of insulin. The mechanisms underlying the metabolic modulation of islet Mφs and β-cell function during obesity remain elusive. I hypothesized that changes in tRNA epitranscriptome affect the tRF profile and permit a prompt response to environmental cues in islet cells during obesity. The overall objectives of this project were 1) to investigate whether tRNA epitranscriptome is modulated during obesity in macrophages; 2) to study the consequences of tRNA cleavage modulation on macrophage activation and 3) to understand whether tRNA derived fragments could be part of cell-to-cell crosstalk between macrophages and pancreatic beta cells.
In order to pursue my objectives, I have used a mouse model of obesity. The db/db mice hold a mutation in the gene encoding for the Leptin receptor, an hormon controlling nutrient intake and energy espenditure. The lack of this regulation induce the mice to develop obesity and consequently diabetes. I isolated pancreatic islets from db/db mice and lean controls, and purified the populations of islet resident macrophages and β-cells. We used small RNA sequencing in order to identify the tRNA derived fragments (tRFs) present in the different cell types in obesity or phisiological conditions. We found that several tRFs were modulated in obesity conditions. Macrophages are very plastic cells, that respond to environmental stimuli with changing their gene expression and metabolism towards a more pro- or anti-inflammatory profile. We decided then to investigate whether the identified tRFs were important for macrophage activation. In order to understand whether the modulated tRFs were important for macrophage activation, I performed small RNA sequencing also in pro- and anti-inflammatory macrophages that I differentiated from bone marrow cells. With these results I found that part of the tRFs modulated in db/db islet macrophages belonged to an anti-inflammatory profile. The role of specific tRF candidates in macrophage activation was further investigated. To do so, I have used antisense oligonucleotides able to block the specific tRF. With this methodology I was able to demonstrate that some tRFs are essential modulators of macrophage metabolic activation. This was assessed by measuring mitochondrial function which is different between pro- and anti-inflammatory macrophages and by transcriptome analysis of gene expression, followed by bio-informatic analysis. A modulation of tRF signature was also observed in pancreatic β-cells from db/db mice. Only two tRFs were identified to be commonly modulated in the two cell types. Although I obtained some data pointing to a vesicle-mediated transfer of tRFs between macrophages and beta cells, I found that free fatty acids are able to induce an increase of tRFs in both β-cells and islet macrophages, sugesting a common induction mechanism.
The described experiments demonstrated that in db/db obese and hyperglycaemic mice there is a modulation of tRF signature in both islet macrophages and β-cells. Interestingly, based on functional studies, tRFs appeared to be crucial mediators of macrophages activation. This important observation may be exploited not oly in diabetes research but also in different fields in which macrophage dependent inflammatory processes are investigated or targeted for therapeutical purposes. Dissemination of the obtained results has been conducted during the two years fellowship with participation to conferences in the field of diabetes (EASD 2022) and immunometabolism (2023), and it will be further pursued with publications in journal of wide interest.
The described experiments demonstrated that in db/db obese and hyperglycaemic mice there is a modulation of tRF signature in both islet macrophages and β-cells. Interestingly, based on functional studies, tRFs appeared to be crucial mediators of macrophages activation. This important observation may be exploited not oly in diabetes research but also in different fields in which macrophage dependent inflammatory processes are investigated or targeted for therapeutical purposes. Dissemination of the obtained results has been conducted during the two years fellowship with participation to conferences in the field of diabetes (EASD 2022) and immunometabolism (2023), and it will be further pursued with publications in journal of wide interest.
The two-years MSCA fellowship led to major progresses of the current state of research in the field of tRNA modulation in obesity. The MATREX project contributes to identify novel mechanisms of obesity-dependent dynamics happening at the level of pancreatic islets and contributing to shape a dysfunctional environment for diabetes pathogenesis. In this context, the results obtained elucidate the importance of tRNA celavage mechanisms in highly metabolic cells sensitive to environmental changes. Further work is now needed in order to identify the cell specific or common mechansisms of tRFs in macrophages and β-cells, and to untangle the specific molecular mechanisms exerted by these molecules. With the high quality preliminary data generated during the MSCA fellowship, I expect to complete these investigations in the next 2 years. Ongoing experiments aim at elucidating the interactors of tRFs in islet cells, and the biogenesis mechanisms involved. This research will potentially lead to the development of an original and novel research line, with an interest in tRNA biology in the modulation of immune cells metabolisms and β-cells homeostasis. This will be at the center of my future career plan. Discoveries in this context, as well as the set up of techniques to investigate the function and mechanism of action of tRFs has potentially a great impact in the development of technologies to target crucial cellular processes.