Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Deficiency of cannabinoid receptors enhances host susceptibility to bacterial infection

ABSTRACT Host resilience to bacterial infection depends on tightly regulated immune responses, which can be shaped by metabolic cues, including the contribution from bioactive lipids. The endocannabinoid system (ECS), a lipid signaling network known for its neuromodulatory roles, also influences immunity; however, the receptor-specific contributions of cannabinoid receptor 1 (CB1R) and cannabinoid receptor 2 (CB2R) in host–pathogen interactions remain incompletely defined in this context. Using receptor-deficient mouse models, we investigated how CB1R and CB2R modify immune responses to Salmonella Typhimurium. CB1R-deficient (CB1R-KO) mice exhibited heightened systemic inflammation, impaired bacterial clearance, and reduced survival in systemic infection, associated with dysregulated macrophage polarization and diminished neutrophil recruitment. In contrast, CB2R-KO mice showed increased susceptibility in both systemic and mucosal infection models, marked by a pro-inflammatory macrophage profile, enhanced neutrophilia, and microbiota dysbiosis. Shotgun metagenomic analysis revealed a reduced abundance of specific protective commensals and altered microbial metabolic pathway profiles in CB2R-KO mice, suggesting a role for CB2R in maintaining mucosal immune–microbiota homeostasis. Collectively, these findings highlight non-redundant roles for CB1R and CB2R in regulating immune dynamics and salmonellosis disease severity, and they point to the ECS as a potential target for host-directed immunomodulatory therapies.IMPORTANCEEffective immunity against bacterial pathogens requires a delicate balance between microbial clearance and the containment of inflammatory damage encountered during many infections. The molecular pathways that regulate this equilibrium remain incompletely defined, and the involvement of bioactive lipid signaling mechanisms also needs to be better described. Here, we show that the endocannabinoid receptors CB1R and CB2R play non-redundant roles in host defense against Salmonella infection. CB1R deficiency results in exacerbated systemic inflammation, defective bacterial clearance, and dysregulated macrophage polarization. In contrast, CB2R deficiency leads post-infection to gut dysbiosis and has been found to negatively affect the outcome for the host in both systemic and mucosal infection with Salmonella. By describing cannabinoid receptor-specific contributions to immune regulation and microbiota dynamics, our findings reveal a previously underappreciated axis of host–pathogen interaction. This study broadens our understanding of lipid-mediated immune modulation and identifies CB1R and CB2R as potential targets for therapies aimed at restoring immune homeostasis and improving infectious disease outcomes

Targeting deubiquitinating enzymes and ubiquitin pathway modulators to enhance host defense against bacterial infections

ABSTRACT The rise of antibiotic-resistant bacterial pathogens poses a critical global health challenge, necessitating innovative therapeutic strategies. This study explores host-targeted therapies by focusing on deubiquitinating enzymes (DUBs), key regulators of the ubiquitin-proteasome system (UPS) that mediate host-pathogen interactions. Using Salmonella-infected macrophages, we screened a UPS-targeted compound library and identified several compounds that enhanced bacterial clearance without affecting host cell viability. Among these, the dual USP25/USP28 inhibitor AZ-1 emerged as one of the top candidates. Transcriptomic profiling revealed infection-induced upregulation of DUBs, particularly USP25, USP46, and OTUD7B. USP25 knockdown significantly reduced intracellular Salmonella, confirming its role as a critical host factor. AZ-1 also exhibited broad-spectrum intracellular activity against multidrug-resistant Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. In vivo, AZ-1 reduced fecal bacterial loads, clinical scores, and infection-induced weight loss, though it did not extend survival. AZ-1 had no direct antibacterial activity in axenic culture, indicating a host-targeting mechanism. Transcriptomic and signaling analyses revealed AZ-1 suppressed key immune pathways, including nuclear factor-kappa B (NF-κB) signaling. These findings establish DUBs as promising targets for host-directed therapies and support further development of UPS-targeted agents to combat antimicrobial resistance.IMPORTANCEAntibiotic-resistant infections, particularly those caused by intracellular pathogens, represent an urgent public health threat due to their ability to evade immune responses and resist conventional antibiotics. This study identifies the ubiquitin-proteasome system, specifically deubiquitinating enzymes, as viable targets for host-directed therapy. We demonstrate that the USP25/USP28 inhibitor AZ-1 enhances intracellular bacterial clearance without compromising host cell viability and is effective against several multidrug-resistant gram-negative pathogens. Knockdown of USP25 alone also reduced intracellular Salmonella, stressing out its proposed role in bacterial persistence. AZ-1 improved early infection outcomes in vivo but was insufficient as monotherapy. These findings support a novel therapeutic approach that targets host pathways to enhance bacterial clearance, offering a promising adjunct to traditional antibiotics in the fight against antimicrobial resistance