Xylo-oligosaccharides (XOS) have been shown to improve the immune system of weaned piglets, but the molecular mechanism of their action remains unclear. Therefore, this study aimed to investigate the impact of XOS on intestinal and thymic immune function in weaned piglets challenged with lipopolysaccharide (LPS) and elucidate the underlying mechanism. In a 2 × 2 factorial arrangement, consisting of diet treatment (basal diet vs 0.02% XOS diet) and immunological challenge [saline vs LPS], 24 piglets were randomly divided into 4 groups (n = 6): CON group, basal diet + saline
LPS group, basal diet + LPS
XOS group, 0.02% XOS diet + saline
XOS_LPS group, 0.02% XOS diet + LPS. Piglets were fed either the basal or XOS diet for 21 d, followed by intraperitoneal injections of normal saline or LPS on the 22nd day. Ileum, thymus, and colon samples were collected 4 h after the intraperitoneal saline or LPS injection. The piglets fed the XOS diet had higher average daily feed intake and average daily weight gain (P <
0.05). The XOS diet increased ileal villus height and decreased crypt depth. XOS also enhanced ileal and thymic antioxidant enzyme activities, anti-inflammatory cytokine expression, and decreased malondialdehyde levels and mRNA abundance of pro-inflammatory cytokines in piglets (P <
0.05). The XOS diet also downregulated the ileal and thymic NOD-like receptor family pyrin domain containing 3 and gasdermin-D gene and protein expression associated with pyroptosis (P <
0.05). Moreover, The XOS diet increased the mRNA abundance of forkhead box P3, signal transducer and activator of transcription 5, and transforming growth factor beta 1 while decreasing signal transducer and activator of transcription 3 and retinoid-related orphan receptor-gammat mRNA abundance (P <
0.05). The XOS diet enhanced forkhead box P3 protein expression and reduced retinoid-related orphan receptor-gammat protein expression following the LPS challenge (P <
0.05). At the same time, The XOS diet affected the gut microbiota and increased levels of short-chain fatty acids (P <
0.05). In conclusion, XOS may modulate ileal and thymic immune function in weaned piglets following a 4-h LPS challenge by affecting gut microbiota, pyroptosis, and Th17/Treg immune responses.