Biology subjects

Boucher, D.

Publications and source records attributed to Boucher, D..

9 recordsLinked to original sources

bioRxiv2025

Evolutionary convergence of a Pyroptosis-Apoptosis crosstalk drives non-canonical inflammasome signalling.

The coordination of efficient immune responses to infections is essential to enable pathogen clearance and host survival. Cell death modalities are a crucial component of the innate immune system and are key to controlling intracellular and extracellular infections. Human caspase-4 and -5 are cysteine proteases activated upon intracellular detection of bacterial lipopolysaccharide (LPS) and triggers pyroptosis, a lytic and pro-inflammatory form of cell death. However, the interaction between these caspases and other cell death modalities remains poorly understood. Here, we build on evidence that caspase-4 can engage in crosstalk with the apoptotic executioner caspases-3 and -7. Our work validated caspase-3 and -7 as direct substrates of caspase-4 and -5, and demonstrated that this specificity was regulated by exosite interactions as opposed to the tetrapeptide. Using AlphaFold, we generated an interaction model between caspase-4 and executioner caspases-3 and -7. From this, we experimentally validated the importance of a hydrophobic interface on caspase-3 and -7 that engaged the caspase-4 exosite, enabling their recognition and cleavage. Importantly, this interface is not used by the apoptotic initiator caspase-8, which cleaved caspase-3 in a tetrapeptide-dependent manner. Our work highlights that inflammatory caspases have evolved a novel mechanism to coordinate crosstalk between pyroptotic and apoptotic signalling, and suggests that these pathways may synergise for the efficient amplification of cell death pathways.

biochemistry↗

bioRxiv2025

Non-canonical inflammasome drives intrinsic anti-microbial responses in human Natural Killer (NK) cells.

Inflammasome pathways are critical for detecting pathogens and initiating inflammatory responses, yet how individual cell types configure these signalling platforms remains poorly understood. Here, we demonstrate that human Natural Killer cells possess distinct inflammasome machinery, lacking canonical components and innate sensors (NLRP3, NLRC4, TLR4) but harbour a functional non-canonical inflammasome (NCI). Using Salmonella enterica Typhimurium infection of primary human NK cells, we show that cytosolic bacterial access triggers caspase-4-dependent pyroptosis and IL-18 secretion, without canonical inflammasome activation or IL-1{beta} production. Strikingly, murine NK cells express inflammasome transcripts but lack functional caspase-11 protein and do not undergo pyroptosis upon infection, revealing species-specific post-transcriptional regulation. We further identify IL-12, but not IFN{gamma}, as a priming signal for NCI components in human NK cells, a functional distinction from macrophages and epithelial cells. Together, these findings reveal that human NK cells have evolved a specialised inflammasome with unique regulatory inputs, establishing these cells as autonomous sensors of intracellular Gram-negative infection.

immunology↗

bioRxiv2025

Caspase-1 self-terminates protease activity to enforce homeostasis and prevent inflammasome-driven diseases

Signal shutdown mechanisms must exist to silence the potent inflammatory programs initiated by the caspase-1 (CASP1) protease, to allow inflammation to resolve and reinstate tissue homeostasis. It is unknown how CASP1 terminates its activity in vivo. Here, we use a knock-in mouse model in which the CASP1 CARD domain linker (CDL) is mutated to prevent self-cleavage (Casp1.CDL mice) to show that CASP1 CDL autoproteolysis terminates CASP1 activity in vivo. We examined these mice under homeostatic conditions and in response to major physiological challenges. In the brain, CASP1 CDL mutation caused anxiety-like behaviour under homeostatic conditions, and exacerbated hippocampal spatial learning deficits in the APP23 genetic model of amyloid-induced neurodegeneration. In the bone marrow, CASP1 CDL mutation promoted steady-state granulopoiesis. In a model of diet-induced liver disease, CASP1 CDL mutation accelerated liver steatosis and promoted liver immune cell infiltration, inflammation and damage. In a liver healing model, CASP1 CDL mutation delayed disease resolution, indicating that CASP1 autocleavage is required to restore homeostasis after a major challenge to organ function. Our data reveal that CASP1 CDL self-cleavage terminates CASP1 inflammatory programs in vivo to maintain homeostasis in steady-state, restore homeostasis after a major challenge to organ function, and suppress inflammasome-driven diseases. These data identify CASP1 as a prime anti-inflammatory drug target, as CASP1 inhibitors may enforce homeostasis and prevent inflammasome-driven diseases.

immunology↗

bioRxiv2025

A Rosella-PLIN2 Knock-in Mouse Reveals Lipophagy and Immunometabolic Interplay in Atherosclerosis

Cytosolic lipid droplets (LDs) regulate lipid homeostasis, with abnormal LD dynamics linked to metabolic diseases like atherosclerosis. In macrophage foam cells, LDs undergo autophagic degradation via lipophagy, but the extent of this process in vascular smooth muscle cell (VSMC) foam cells remains unclear. To track lipophagy in real time, we developed a Rosella-PLIN2 biosensor by tagging perilipin 2 (PLIN2) with the fluorescent pH-biosensor Rosella. We show that proatherogenic lipoproteins and autophagy activators stimulate lipophagy in human macrophages. Targeting LDs with an LC3 fusion protein or LD-autophagy tethering compounds (LD-ATTECs) selectively enhanced lipophagy, promoting foam cell LD clearance. In an atherosclerosis model, Rosella-PLIN2 accurately tracked lipophagy in arterial foam cells, revealing distinct PLIN2 expression patterns in macrophage and non-leukocyte foam cells. We identified a lipophagy deficiency in VSMC foam cells and demonstrate that enhancing lipophagy promotes LD catabolism in primary VSMC foam cells. TREM2+ macrophages exhibited high lipid content and low lipophagy flux, whereas TREM2- macrophages had low lipid content and high lipophagy flux. Our findings highlight a cell-specific interplay between lipophagy and immunometabolism in arterial foam cells, unveiling novel therapeutic avenues for atherosclerosis. Additionally, the Rosella-PLIN2 model provides a powerful tool for studying LD metabolism, offering new insights into lipid homeostasis and disease mechanisms.

cell biology↗

bioRxiv2024

Distinct roles for the Staphylococcus aureus POT transporter DtpT in di/tripeptide uptake and glutathione utilisation inside human macrophages

Peptides available in biological niches inhabited by the human pathogen Staphylococcus aureus serve as a rich source of amino acids required for growth and successful host colonisation. Uptake of peptides by S. aureus involves at least two transport systems: the di/tripeptide permease DtpT and the oligopeptide ABC transporter Opp3. Here we study the individual and combined functions of DtpT and Opp3 in enabling utilisation of diverse di-/tripeptides via a high-throughput phenotypic screen. We reveal that DtpT is the primary route of uptake for dipeptides, and although many peptides can be utilised via either of the two transport systems, we demonstrate a clear preference for Asp/Glu-containing peptides among DtpT substrates. To better understand the substrate preferences of DtpT, the protein was purified and reconstituted into proteoliposomes. Active transport of diverse di- and tripeptides was demonstrated, supporting the conclusions of the phenotypic screen. During this in vitro analysis, we discovered that DtpT could transport the biologically prevalent tripeptide glutathione (GSH). Bacterial growth assays demonstrate that dtpT is essential for GSH utilisation in the absence of the known glutathione transporter, Gis, identifying DtpT as the second GSH uptake system of S. aureus. We demonstrate that GSH transport is required by S. aureus for complete fitness during in vitro macrophage infection experiments. Finally, based on analysis of the DtpT structure and identification of key residues needed for GSH binding and transport, we suggest that GSH transport may be conserved in the DtpT orthologue of Listeria monocytogenes. Together, these data reveal important new functions for DtpT in the utilisation of diverse peptides and point toward a novel role for DtpT (and, potentially, other bacterial POT proteins) in glutathione acquisition during intracellular infection. Author summaryThe environments where bacterial pathogens thrive are often rich in proteins and their degradation products, including oligopeptides, which can be taken up by the bacterium and used as nutrients. Understanding how this occurs could help us find ways to tackle the growth of these pathogens during infection. Here we examine how the major human pathogen Staphylococcus aureus takes up oligopeptides. We demonstrate that a membrane transporter protein, DtpT, is the major route of dipeptide uptake and reveal over 100 new di-/tripeptide targets for this protein. Our findings highlight a defined role for DtpT in the accumulation of Aspartate- and Glutamate-containing peptides, which may serve as relevant nitrogen sources during infection. We also provide the first evidence that DtpT transports the prevalent human metabolite reduced glutathione, and demonstrate that DtpT functions alongside the previously identified Gis glutathione transport system to support intracellular survival of this pathogen inside macrophages. Overall, our findings provide a clear example of how substrate selectivity allows DtpT to fulfil specific biological roles in S. aureus, and this functional specialisation may be a common feature of homologous peptide transporters in other bacterial pathogens and across the Tree of Life.

microbiology↗

bioRxiv2024

Trained innate immunity attenuates macrophage efferocytosis of cancer cells

Macrophage phagocytosis has been implicated in regulating anti-tumour immunity. Trained innate immunity (TII), induced via modulation of mature myeloid cells or their bone marrow progenitors, mediates sustained increased responsiveness to secondary challenges. Despite the advances in the study of TII-mediated anti-tumour activity, the impact of TII on the orchestration of phagocytosis in the tumour setting requires further elucidation. Here, we investigated whether macrophage phagocytosis of tumour cells can be modulated through induction of TII. To this end, mice were pre-treated with {beta}-glucan, a fungal-derived agonist of TII, and bone marrow was isolated for macrophage differentiation. Macrophages were then co-cultured with tumour cells that were either apoptotic or opsonised with an antibody recognising a tumour antigen, to mimic efferocytosis and antibody-dependent cellular phagocytosis (ADCP), respectively. While TII did not have any impact in the modulation of ADCP, efferocytosis was decreased in trained macrophages. Along the same line, gene expression analysis demonstrated that mRNA levels of molecules promoting efferocytosis were downregulated in trained macrophages. Trained macrophages exerted decreased levels of active caspase-1 and produced decreased levels of interleukin-1{beta} upon efferocytosis of tumour cells. Our findings reveal a hitherto unknown role of TII in the regulation of anti-tumour immunity and may set the stage for designing new cancer immunotherapeutic approaches targeting macrophage efferocytosis.

immunology↗

bioRxiv2023

Caspase-4 dimerisation and D289 auto-processing elicits an interleukin-1β converting enzyme

The non-canonical inflammasome is a signalling complex critical for cell defence against cytosolic Gram-negative bacteria. A key step in the human non-canonical inflammasome pathway involves unleashing the proteolytic activity of caspase-4 within this complex. Caspase-4 induces inflammatory responses by cleaving gasdermin-D (GSDMD) to initiate pyroptosis, although the molecular mechanisms that activate caspase-4 and govern its capacity to cleave substrates are poorly defined. Caspase-11, the murine counterpart of caspase-4, acquires protease activity within the non-canonical inflammasome by forming a dimer that self-cleaves at D285 to directly cleave GSDMD. These cleavage events trigger signalling via the NLRP3-ASC-caspase-1 axis, leading to downstream cleavage of the pro-interleukin-1{beta} (pro-IL-1{beta}) cytokine precursor. Here, we show that caspase-4 first dimerises then self-cleaves at two sites - D270 and D289 - in the interdomain linker to acquire full proteolytic activity, cleave GSDMD and induce cell death. Surprisingly, caspase-4 dimerisation and self-cleavage at D289 generates a caspase-4 p34/p9 protease species that directly cleaves pro-IL-1{beta}, resulting in its maturation and secretion independently of the NLRP3 inflammasome in primary human myeloid and epithelial cells. Our study thus elucidates the key molecular events that underpin signalling by the caspase-4 inflammasome, and identifies IL-1{beta} as a natural substrate of caspase-4.

immunology↗

bioRxiv2021

Mathematical modelling of activation-induced heterogeneity reveals cell state transitions underpinning macrophage responses to LPS

Despite extensive work on macrophage heterogeneity, the mechanisms driving activation induced heterogeneity (AIH) in macrophages remain poorly understood. Here, we use two in vitro cellular models of LPS-induced tolerance (bone marrow-derived macrophages or BMDMs and RAW 264.7 cells), single-cell protein measurements, and mathematical modelling to explore how AIH underpins primary and secondary responses to LPS. We measure expression of TNF, IL-6, pro-IL-1{beta}, and NOS2 and demonstrate that macrophage community AIH is dependent on LPS dose. We show that altered AIH kinetics in macrophages responding to a second LPS challenge underpin hypo-responsiveness to LPS. These empirical data can be explained by a mathematical 3-state model including negative, positive, and non-responsive states (NRS), but they are also compatible with a 4-state model that includes distinct reversibly NRS and non-responsive permanently states (NRPS). Our mathematical model, termed NoRM (Non-Responsive Macrophage) model identifies similarities and differences between BMDM and RAW 264.7 cell responses. In both cell types, transition rates between states in the NoRM model are distinct for each of the tested proteins and, crucially, macrophage hypo-responsiveness is underpinned by changes in transition rates to and from NRS. Overall, our findings provide support for a critical role for phenotypically negative macrophage populations as an active component of AIH and primary and secondary responses to LPS. This reveals unappreciated aspects of cellular ecology and community dynamics associated with LPS-driven training of macrophages.

immunology↗

bioRxiv2020

Inflammatory and oxidative status in European captive black rhinoceroses: a link with Iron Overload Disorder?

Iron Overload Disorder (IOD) is a syndrome developed by captive browsing rhinoceroses like black rhinoceroses (Diceros bicornis) in which hemosiderosis settles in vital organs while free iron accumulates in the body, potentially predisposing to various secondary diseases. Captive grazing species like white rhinoceroses (Ceratotherium simum) do not seem to be affected. The pro-oxidant and pro-inflammatory properties of iron, associated with the poor antioxidant capacities of black rhinoceroses, could enhance high levels of inflammation and oxidative stress leading to rapid ageing and promoting diseases. In this prospective study, 15 black (BR) and 29 white rhinoceroses (WR) originating from 22 European zoos were blood-sampled and compared for their iron status (serum iron), liver/muscle biochemical parameters (AST, GGT, cholesterol), inflammatory status (total proteins, protein electrophoresis) and oxidative stress markers (SOD, GPX, dROMs). Results showed higher serum iron and liver enzyme levels in black rhinoceroses (P<0.01), as well as higher GPX (P<0.05) and dROM (P<0.01) levels. The albumin/globulin ratio was lower in black rhinoceroses (P<0.05) due to higher α2-globulin levels (P<0.001). The present study suggests a higher inflammatory and oxidative profile in captive BR than in WR, possibly in relation to iron status. This could be either a consequence or a cause of iron accumulation, potentially explaining rapid ageing and various diseases. Further investigations are needed to assess the prognostic value of the inflammatory and oxidative markers in captive black rhinoceroses, particularly for evaluating the impact of reduced-iron and antioxidant-supplemented diets.View Full Text

zoology↗