<i>Clostridioides difficile</i> ‐Derived Extracellular Vesicles Induce Proinflammatory Responses in Macrophages

Clostridioides difficile (CD) is a leading cause of antibiotic-associated diarrhoea in both hospitalized and non-hospitalized patients. This study explores the role of extracellular vesicles (EVs) derived from Clostridioides difficile strain 630 (CD630-EVs), which released spherical EVs during in vitro culture, in modulating pro-inflammatory cytokine production in macrophages. Proteomic analysis identified a total of 1064 proteins within CD630-EVs, including four immune-related proteins: FliC, TrxA, TrxA2 and HtpG. Notably, FliC exhibited the highest expression intensity within immune-related pathways. CD630-EVs significantly stimulated the production of inflammatory factors, such as interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1), IL-1α and TNF-α, in mouse macrophages. However, the addition of TH1020, a flagellin receptor inhibitor, markedly suppressed cytokine production. Protein docking analysis further demonstrated that TH1020 disrupts the interaction between Toll-Like Receptor 5 (TLR5) and FliC, thereby attenuating FliC-mediated inflammatory responses in macrophages. FliC likely plays a pivotal role in mediating the inflammatory response induced by CD630-EVs in macrophages. This effect is potentially driven by the activation of innate immunity through FliC's interaction with TLR5 in the host, which may subsequently influence adaptive immune responses. In conclusion, this study elucidates the immune-modulatory effects of CD630-EVs on host macrophages during Clostridioides difficile infection (CDI). CD630-EVs contain a diverse array of proteins, with FliC emerging as a key mediator of pro-inflammatory responses in macrophages. These findings suggest that FliC may play a critical role in intestinal mucosal injury during CDI, highlighting its potential as a therapeutic target for mitigating inflammation in CDI.