Damien Bertheloot

Total joint arthroplasty is among the most common surgical procedures performed worldwide, with frequency increasing due to demographic changes. Accelerating the diagnostic process using new techniques is crucial for effective therapy. This pilot study aims to test such innovative technology in the context of periprosthetic joint infection (PJI) using Scattered Light Integrating Collector (SLIC) technology. While we wish to evaluate whether SLIC can be used to reliably detect the status of infection within human tissue samples in the future, our current research focused on building its foundation by evaluating steps of sample preparation that allow for heightened growth depiction. It is, to our knowledge, the first study concerning the usage of solid human tissue samples using the SLIC device. Adult patients presenting with native or periprosthetic joint infections were included in this prospective study. Biopsies were obtained using sequential sampling, and bacterial density was optimized through titration series. Cryopreservation and agents influencing coagulation were investigated. Our study demonstrates that simple pretreatment could aid in detecting pathogen growth in infected tissue samples. Findings showed a clear advantage for no addition of agents affecting coagulation. Additionally, our protocols proved reliable after prolonged cryopreservation at −20 °C for up to 8 weeks, showing no significant difference compared to primary testing. AUC comparison showed comparable results for sample storage at −80 °C for up to 8 weeks. Similar outcomes were seen for samples ranging from 25 µL to 300 µL, with biological replicates displaying higher thresholds for larger volumes without significant differences. This study introduces a simple and quick diagnostic tool for detecting bacterial growth using tissue biopsies and develops an SOP for further research with this innovative technique. The suggested SOP enables SLIC to hint at an underlying bacterial infection within 5 h using joint tissue, offering a possible novel approach in diagnosing periprosthetic joint infections and septic arthritis. While not yet designed to compare sensitivity to other culture methods, it provides a solid basis for further clinical research.

Inflammation is an essential response to infection and injury, but unregulated inflammation is damaging and must be limited by negative feedback signalling. Inflammasome signalling drives local inflammation and systemic responses like fever. However, our understanding of how inflammasome signalling is negatively regulated is limited. NLRP3 is activated by a vast number of stimuli and senses perturbations of cytoplasmic homeostasis. As temperature is a fundamental environmental stressor, we hypothesised that NLRP3 inflammasome signalling would be sensitive to increased temperatures and so we investigated the effects of high temperatures on NLRP3 in macrophages. Short-term incubation at high fever range temperatures significantly inhibits NLRP3 activation, while secretion of the inflammasome-independent cytokines TNF and IL-6 are much less affected. High temperature blocks NLRP3 inflammasome formation in a transcription-independent manner, and NLRP3 is highly sensitive to temperature-mediated inhibition relative to the NLRC4, AIM2, and NLRP1 inflammasomes. Using cellular assays and molecular simulations we show that the effect of high temperature on NLRP3 is protein intrinsic. NLRP3 activation is associated with a decrease in the thermal stability of the protein and multiscale molecular dynamics simulations identified a peptide in the C -terminal o f the FI SNA domain (COFI) that is highly flexible and undergoes a significant conformational shift at high temperature. Cellular assays demonstrate that the COFI regulates NLRP3 stability and is required for activation. Furthermore, mice exposed to high temperature display attenuated inflammatory cytokine production upon in vivo LPS challenge. Our studies thus reveal that high temperatures associated with fever limit NLRP3 activity and identify a novel role for NLRP3 as a protein thermosensor.

Osteoclasts (OCs) are important therapeutic targets in the treatment of osteoporosis. The aim of this study was to explore a novel therapeutic approach for osteoporosis using Arcyriaflavin A (ArcyA), a natural compound derived from the marine invertebrate Eudistoma sp. We systematically evaluated the effects of ArcyA on OC differentiation and function in mouse models using molecular biology assays, cellular function analyses and in vivo animal experiments. We also evaluated the efficacy of ArcyA in human cells. The TRAP staining results provide the first clear evidence of the drug’s inhibitory effect, whereby the administration of ArcyA led to a significant reduction in TRAP-positive cells compared to the control group at concentrations that were non-toxic to bone marrow macrophages. Meanwhile, a significant reduction in the number of multinucleated giant cells with more than ten nuclei was observed. Furthermore, similar TRAP staining results were reproduced in human OCs, suggesting that ArcyA has the same effect on OCs derived from human PBMCs. At the molecular level, ArcyA treatment resulted in the downregulation of genes relevant to OC differentiation (NFATc1, cFos and TNFrsf11α), fusion and survival (DCstamp and ATP6v0d2) and resorption function (CTSK, MMP9, integrin β3 and ACP5). A western blot analysis of the corresponding proteins (NFATc1, cFos, CTSK and integrin β3) further confirmed the PCR results. Furthermore, ArcyA-treated OCs produced significantly fewer resorption pits, indicating suppressed bone resorption activity. Consistent with this, in vivo experiments using an ovariectomy (OVX)-induced osteoporosis mouse model showed that ArcyA treatment significantly alleviated bone loss. Mice in the treatment groups had higher BV/TV values, and this therapeutic effect was enhanced in a dose-dependent manner. In addition, our research also showed that IκB could be a potential target for the inhibitory effect of ArcyA. In conclusion, these findings suggest that ArcyA has significant therapeutic potential for the treatment of osteoporosis by inhibiting osteoclastogenesis and bone resorption. Further studies are warranted to explore its clinical applications.

Bacterial antibiotic resistance is an important challenge that the healthcare system is continually battling and a major problem in the treatment of musculoskeletal infections such as periprosthetic joint infections. Current methods to identify infectious microbes and define susceptibility to antibiotics require two to ten days from isolation to the establishment of an antibiogram. This slow process limits advances in antimicrobial drug discovery and, in the clinical context, delays the delivery of targeted treatments, with potentially devastating outcomes for patients. With this in mind, we strived to establish a quicker and more sensitive method to deliver antibiotic susceptibility profiles of clinically relevant microbes using Scattered Light Integrated Collector (SLIC) technology. We established antibiotic panels to obtain an approximate identification of a wide variety of microbes linked to periprosthetic joint infections and determine their susceptibility to antibiotics. We challenged microbes isolated from patients with our tailored antibiotic panels and found that SLIC detects perturbations in bacterial growth accurately and reproducibly within minutes of culture. Indeed, we could show that SLIC can be used to measure the dose-dependent inhibitory or bacteriolytic activity of broad classes of antibiotics. Our panel design enabled us to establish a profile similar to an antibiogram for the tested bacteria within 90 min. Our method can provide information on the class of bacteria tested and potential treatment avenues in parallel. Our proof-of-principle experiments using isolated clinical strains of bacteria demonstrate that SLIC, together with our specifically designed antibiotic panels, could be used to rapidly provide information on the identity of an infecting microbe, such as those associated with periprosthetic joint infections, and guide physicians to prescribe targeted antibiotic treatment early-on. The constant emergence of resistant strains of bacteria pushes the pharmaceutical industry to develop further effective drugs. Our optimized method could significantly accelerate this work by characterizing the efficacy of new classes of compounds against bacterial viability within minutes, a timeframe far shorter than the current standards.

Inflammation models are widely used in the in vitro investigation of new therapeutic approaches for osteoarthritis. TNFα (tumor necrosis factor alpha) plays an important role in the inflammatory process. Current inflammation models lack uniformity and make comparisons difficult. Therefore, this study aimed to systematically investigate whether the effects of TNFα are concentration-dependent and whether chondrocyte expansion has an effect on the inflammatory model. Bovine chondrocytes were enzymatically isolated, expanded to passages 1–3, and transferred into a 3D pellet culture. Chondrocyte pellets were stimulated with recombinant bovine TNFα at different concentrations for 48 h to induce inflammation. Gene expression of anabolic (collagen 2, aggrecan, cartilage oligomeric protein (COMP)), catabolic (matrix metalloproteinases (MMP3, MMP13)), dedifferentiation (collagen 1) markers, inflammation markers (interleukin-6 (IL-6), nuclear factor kappa B (NFkB), cyclooxygenase-2 (COX), prostaglandin-E-synthase-2 (PTGES2)), and the apoptosis marker caspase 3 was determined. At the protein level, concentrations of IL-6, nitric oxide (NO), and sulfated glycosaminoglycans (GAG) were evaluated. Statistical analysis was performed using the independent t-test, and significance was defined as p < 0.05. In general, TNFα caused a decrease in anabolic markers and an increase in the expression of catabolic and inflammatory markers. There was a concentration-dependent threshold of 10 ng/mL to induce significant inflammatory effects. Most of the markers analyzed showed TNFα concentration-dependent effects (COMP, PRG4, AGN, Col1, MMP3, and NFkB). There was a statistical influence of selected gene expression markers from different passages on the TNFα chondrocyte inflammation model, including Col2, MMP13, IL-6, NFkB, COX2, and PTGES2. Considering the expression of collagen 2 and MMP3, passage 3 chondrocytes showed a higher sensitivity to TNFα stimulation compared to passages 1 and 2. On the other hand, MMP13, IL-6, NFkB, and caspase 3 gene expression were lower in P3 chondrocytes compared to the other passages. On the protein level, inflammatory effects showed a similar pattern, with cytokine effects starting at 10 ng/mL and differences between the passages. TNFα had a detrimental effect on cartilage, with a clear threshold observed at 10 ng/mL. Although TNFα effects showed concentration-dependent patterns, this was not consistent for all markers. The selected passage showed a clear influence, especially on inflammation markers. Further experiments were warranted to explore the effects of TNFα concentration and passage in long-term stimulation.

In humans, blood Classical CD14 ⁺ monocytes contribute to host defense by secreting large amounts of pro-inflammatory cytokines. Their aberrant activity causes hyper-inflammation and life-threatening cytokine storms, while dysfunctional monocytes are associated with ‘immunoparalysis’, a state of immune hypo responsiveness and reduced pro-inflammatory gene expression, predisposing individuals to opportunistic infections. Understanding how monocyte functions are regulated is critical to prevent these harmful outcomes. We reveal platelets’ vital role in the pro-inflammatory cytokine responses of human monocytes. Naturally low platelet counts in patients with immune thrombocytopenia or removal of platelets from healthy monocytes result in monocyte immunoparalysis, marked by impaired cytokine response to immune challenge and weakened host defense transcriptional programs. Remarkably, supplementing monocytes with fresh platelets reverses these conditions. We discovered that platelets serve as reservoirs of key cytokine transcription regulators, such as NF-κB and MAPK p38, and pinpointed the enrichment of platelet NF-κB2 in human monocytes by proteomics. Platelets proportionally restore impaired cytokine production in human monocytes lacking MAPK p38α, NF-κB p65, and NF-κB2. We uncovered a vesicle-mediated platelet-monocyte-propagation of inflammatory transcription regulators, positioning platelets as central checkpoints in monocyte inflammation.

Inflammasomes are crucial sentinels of the innate immune system that sense clues of infection, cellular stress, or metabolic imbalances. Upon activation, the inflammasome sensor (e.g., NLRP3) recruits the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). ASC rapidly oligomerizes to form a micron-sized structure termed “ASC speck.” These are crucial for the activation of caspase-1 and downstream inflammatory signals released following a specific form of lytic cell death called pyroptosis. Hence, due to their considerably large size, ASC specks can be easily visualized by microscopy as a simple upstream readout for inflammasome activation. Here, we provide three detailed protocols for imaging ASC specks: (1) live-cell imaging of macrophage cell lines expressing a fluorescent protein fusion form of ASC, (2) imaging of human primary cells using immunofluorescence staining of endogenous ASC, and (3) visualization and quantification of specks on a single-cell level using imaging flow cytometry. Key words Inflammasome ASC Speck Live-cell imaging Immunofluorescence Confocal microscopy Imaging flow cytometry

Inflammasomes sense intracellular clues of infection, damage, or metabolic imbalances. Activated inflammasome sensors polymerize the adaptor ASC into micron‐sized “specks” to maximize caspase‐1 activation and the maturation of IL‐1 cytokines. Caspase‐1 also drives pyroptosis, a lytic cell death characterized by leakage of intracellular content to the extracellular space. ASC specks are released among cytosolic content, and accumulate in tissues of patients with chronic inflammation. However, if extracellular ASC specks contribute to disease, or are merely inert remnants of cell death remains unknown. Here, we show that camelid‐derived nanobodies against ASC (VHH _ASC ) target and disassemble post‐pyroptotic inflammasomes, neutralizing their prionoid, and inflammatory functions. Notably, pyroptosis‐driven membrane perforation and exposure of ASC specks to the extracellular environment allowed VHH _ASC to target inflammasomes while preserving pre‐pyroptotic IL‐1β release, essential to host defense. Systemically administrated mouse‐specific VHH _ASC attenuated inflammation and clinical gout, and antigen‐induced arthritis disease. Hence, VHH _ASC neutralized post‐pyroptotic inflammasomes revealing a previously unappreciated role for these complexes in disease. VHH _ASC are the first biologicals that disassemble pre‐formed inflammasomes while preserving their functions in host defense.

Cell death is a fundamental physiological process in all living organisms. Its roles extend from embryonic development, organ maintenance, and aging to the coordination of immune responses and autoimmunity. In recent years, our understanding of the mechanisms orchestrating cellular death and its consequences on immunity and homeostasis has increased substantially. Different modalities of what has become known as ‘programmed cell death’ have been described, and some key players in these processes have been identified. We have learned more about the intricacies that fine tune the activity of common players and ultimately shape the different types of cell death. These studies have highlighted the complex mechanisms tipping the balance between different cell fates. Here, we summarize the latest discoveries in the three most well understood modalities of cell death, namely, apoptosis, necroptosis, and pyroptosis, highlighting common and unique pathways and their effect on the surrounding cells and the organism as a whole.

Nucleic acid recognition is an important mechanism that enables the innate immune system to detect microbial infection and tissue damage. To minimize the recognition of self-derived nucleic acids, all nucleic acid–sensing signaling receptors are sequestered away from the cell surface and are activated in the cytoplasm or in endosomes. Nucleic acid sensing in endosomes relies on members of the TLR family. The receptor for advanced glycation end-products (RAGE) was recently shown to bind DNA at the cell surface, facilitating DNA internalization and subsequent recognition by TLR9. In this article, we show that RAGE binds RNA molecules in a sequence-independent manner and enhances cellular RNA uptake into endosomes. Gain- and loss-of-function studies demonstrate that RAGE increases the sensitivity of all ssRNA-sensing TLRs (TLR7, TLR8, TLR13), suggesting that RAGE is an integral part of the endosomal nucleic acid–sensing system.