Escherichia coli A0 34/86 (EcO83) is a probiotic strain used in newborns to prevent nosocomial infections and diarrhoea. This bacterium stimulates both pro- and anti-inflammatory cytokine production and its intranasal administration reduces allergic airway inflammation in mice. Despite its benefits, there are concerns about the use of live probiotic bacteria due to potential systemic infections and gene transfer. Extracellular vesicles (EVs) derived from EcO83 (EcO83-EVs) might offer a safer alternative to live bacteria. This study characterizes EcO83-EVs and investigates their interaction with host cells, highlighting their potential as postbiotic therapeutics. EcO83-EVs were isolated, purified, and characterised following the Minimal Information of Studies of Extracellular Vesicles (MISEV) guidelines. Ex vivo studies conducted in human nasal epithelial cells showed that EcO83-EVs increased the expression of proteins linked to oxidative stress and inflammation, indicating an effective interaction between EVs and the host cells. Further in vivo studies in mice demonstrated that EcO83-EVs interact with nasal-associated lymphoid tissue, are internalised by airway macrophages, and stimulate neutrophil recruitment in the lung. Mechanistically, EcO83-EVs activate the NF-κΒ signalling pathway, resulting in the nitric oxide production. EcO83-EVs demonstrate significant potential as a postbiotic alternative to live bacteria, offering a safer option for therapeutic applications. Further research is required to explore their clinical use, particularly in mucosal vaccination and targeted immunotherapy strategies.

Department of Clinical Medicine Aarhus University Aarhus Denmark

Department of Infectious Diseases Aarhus University Hospital Aarhus Denmark

Department of Medical Physics M Smoluchowski Institute of Physics Faculty of Physics Astronomy and Applied Computer Science Jagiellonian University Krakow Poland

Division of Chemistry 1 Department of Medical Biochemistry and Biophysics Karolinska Institutet Stockholm Sweden

Division of Neonatology Paediatric Intensive Care and Neuropediatric Department of Paediatrics and Adolescent Medicine Comprehensive Centre for Paediatrics Medical University of Vienna Vienna Austria

Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy of Sciences Wroclaw Poland

Institute of Immunology and Microbiology 1st Faculty of Medicine Charles University and General University Hospital Prague Czech Republic

Institute of Nanostructured Materials CNR ISMN Bologna Italy

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science Prague Czech Republic

Institute of Specific Prophylaxis and Tropical Medicine Centre for Pathophysiology Infectiology and Immunology Medical University of Vienna Vienna Austria

Laboratory of Gnotobiology Institute of Microbiology of the Czech Academy of Sciences Novy Hradek Czech Republic

Proteomics Biomedicum Division of Chemistry 1 Department of Medical Biochemistry and Biophysics Karolinska Institutet Stockholm Sweden

Research Support Facilities Imaging Unit CIUS Faculty of Life Sciences University of Vienna Vienna Austria

Research Unit for Molecular Medicine Department of Clinical Medicine Aarhus University Aarhus Denmark

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