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Deciphering the roles of HDAC6 in the innate immune system

Periodic Reporting for period 1 - IMMUNOACETYL (Deciphering the roles of HDAC6 in the innate immune system)

Reporting period: 2021-02-01 to 2023-01-31

The innate immune system is the first line of defence against invading pathogens. It involves soluble and cellular mediators, such as macrophages, which detect microbial products (e.g. lipopolysaccharide - LPS) via Toll-like Receptors (TLRs). TLR stimulation of macrophages triggers a rapid change in gene expression, leading to the production of cytokines/chemokines and antimicrobial proteins. The histone deactetylase (HDAC) family of enzymes, which deacetylate lysine residues on target proteins, are important regulators of macrophage functions through their capacity to control signalling and gene expression.

HDAC6 is predominantly cytoplasmic and interacts with several proteins, among which alpha tubulin, HSP90 and others, demonstrating a role beyond histone modifications. One recently identified substrate is mitofusin (MFN) 1 that regulates mitochondrial dynamics. Mitochondria constantly shift between a fragmented state and a more tubular state, modulating several physiological aspects in the cell, but the actual functions of this cellular process in immune contexts are still unclear, as are the roles of HDAC6. Furthermore, since mitochondria is one of the main drivers of metabolism and that HDAC6 has been shown to target some enzyme of the TCA cycle, I hypothesised that HDAC6 could play an important, yet still unknown, role in immunometabolism.

This project addresses two health societal needs: it identifies new ways to control mitochondrial function via the manipulation of its dynamics. Indeed, mitochondria are involved in many inflammatory disorders, in particular neuroinflammation. My research also shows that modulating mitochondrial dynamics via HDAC6 inhibition enhances antimicrobial activity. Thus, my results could contribute to the development of novel antimicrobial treatment strategies as alternatives to antibiotics. Indeed, a UK review on Antimicrobial Resistance estimated that by 2050, if nothing is done, up to 10 million people could die annually from AMR. During the course of the project, I have made several contacts within the industry world and, in the future, will strive for translational opportunities in the healthcare and livestock industries

This MSCA-IF project had three major goals: a) to investigate the role of HDAC6 in macrophages, the importance of its catalytic domains and how it can modulate mitochondria and immunometabolism. To answer these questions, I joined the group of Prof. Patrick Matthias– world expert on HDACs, in particular HDAC6 – at the Friedrich Miescher Institute for Biomedical Research (FMI) in Basel, Switzerland.
HDAC6 and mitochondrial dynamics:
A major discovery was that HDAC6 counters mitochondrial fission and that mitochondrial fission triggers a whole transcriptional program via the translocation of the Activating Transcription Factor (ATF) 5. We investigated this pathway using state-of-the-art Cut-&-Run method; this technique allowed us to identify the promotor regions where ATF5 binds in the macrophage chromatin. The sequencing data from this experiment have been uploaded in a public database. One manuscript and one book chapter have been published on mitochondrial dynamics. A large part of these data has been used in a third manuscript that has been submitted for publication.
We also found that the lack of HDAC6 or the mutation of the zinc-binding domain had only a minor effect on inflammasome-dependent cytokine production, notably for interleukin (Il)-1beta. In collaboration with a colleague (Dr Longlong Wang), we used several state-of-the-art systems (PROTAC, DARPIN as well as KO mice, inhibitors) and cell types (macrophages, monocytes, epithelial cells) to confirm our results. This is an important finding as it puts in perspective recent work from another lab on immortalised bone-marrow macrophages showing a role of HDAC6 in inflammasome activation. These data are compiled in a manuscript in preparation.

HDAC6 and bacterial infection:
We discovered that the pathogenic bacterium Salmonella modulates the mitochondrial fission for its own survival: by inhibiting the fragmentation of the mitochondrial network (fission), Salmonellablocks the host antimicrobial response. Pharmacological inhibition or absence of HDAC6 enhance the fission of mitochondrial network, hence, promoting bacterial clearance. More precisely, the deacetylase activity is important and we did not find a clear role of the zinc-finger binding domain on the antibacterial activity of macrophages.

HDAC6 and metabolic screen/Acetylome:
I have first examined the role of HDAC6 on global gene expression using RNA-sequencing. I found that only very few genes were differentially regulated in the absence of HDAC6, as expected, thus confirming that HDAC6 is playing a role mostly at the post-transcriptional level. To identify differences in immunometabolism, I have used mass spectrophotometry to produce a large dataset of metabolites modulated by HDAC6 during inflammatory response. These data will be correlated with the acetylome to confirm which metabolic pathways are controlled by HDAC6.

Dissemination – Networking – Conference:
I have published two manuscripts, one submitted and four in preparation. I have participated in nine meetings during the course of the project where I presented results from this project while always acknowledging the EU funding.
In recent years, there has been great interest in the molecular and cellular mechanisms of mitochondrial dynamics in many biological systems, but no clear picture has emerged as to the relevance of this process to host defense. We characterised an antimicrobial pathway linked to mitochondrial fission and have dissected the steps from bacteria-induced fission to inducible gene expression to lipid droplet production to elimination of intracellular bacteria. Hence, we revealed a novel connection between HDAC6, mitochondrial fission and lipid droplet production in the process that could be of high interest to the international scientific community.

The beneficial role of HDAC6 inhibition during bacterial infection could also lead to potential development of novel antimicrobial treatment strategies as alternatives to antibiotics with translational opportunities in the healthcare and livestock industries. Collaboration with pharmacological and veterinary industries will be initiated in the future.

This project stayed in line with the Horizon 2020 Programme objectives. The EU 2020 funding was focused on three main priorities: 1) carry excellent science, 2) improve Europe industry competitiveness, research and innovation and 3) convert research results to market which will deliver direct benefits to citizens. During the course of this MSCA project, I have produced quality research data (published and submitted work in peer-reviewed journals), I have closely worked with industries research groups within NOVARTIS who are interested in the project, and finally my results could pave the way towards future non-antibiotics antimicrobial treatments.

Another objective of the Individual Fellowships is to support the mobility of researchers as well as helping to attract the researchers who have emigrated outside of EU to come back working in the Europe. This objective was a success as, after my two postdoc positions in Australia and Switzerland, I have now secured a tenured position back in France as a group leader.
The 3 aims of the proposed project address distinct aspects of Hdac6 and innate immunity