Gut microbiota-targeted 2’-fucosyllactose and lacto-N-neotetraose ameliorate maternal immune activation-induced autism spectrum disorder via the gut-immune-brain axis

• HMOs alleviated MIA-induced social deficits and repetitive stereotyped behaviors. • HMOs enhanced SCFAs production and related microbes (e.g., Akkermansia) abundance. • HMOs restored Treg/Th17 cell balance and alleviated the inflammation in ASD mice. • The combination of 2’-FL and LNnT showed superior efficacy over individual HMOs. Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder primarily characterized by social deficits and repetitive behaviors. 2’-Fucosyllactose (2’-FL) and Lacto-N-neotetraose (LNnT), the two human milk oligosaccharides (HMOs) approved for infant formulas in China, have exerted potentials to modulate gut microbiota, immune system, and cognitive function. Yet the exact mechanism underpinning the etiology of HMOs on improving neurodevelopment and particularly the ASD-related cognitive impairments remain unclear. In this study, mouse model of ASD was established via maternal immune activation (MIA), specifically by introducing polyinosinic:polycytidylic acid (poly(I:C)) to pregnant mice. Male offspring were selected and received either an individual (2’-FL or LNnT) or a combination (2’-FL+LNnT) interventions for 6 weeks. As expected, ASD mice exhibited significant social deficits and repetitive stereotyped behaviors, accompanied by neuroinflammation and decreased expression of BDNF, and morphological changes in cortex as well as hippocampal area. Regardless of the type of sources, HMOs ameliorated ASD-like behaviors in ASD mice, attenuated neuroinflammatory responses, and elevated BDNF expression. Additionally, the HMOs alleviated intestinal inflammation, enhanced gut barrier, and modulated the gut microbiota structure by upregulating the abundance of beneficial bacteria ( Akkermansia, Lactobacillus , and Butyricicoccus ). Notably, the combination of two HMOs also promoted the production of short-chain fatty acids (SCFAs). In addition, flow cytometry analysis showed a significant increase in the regulatory T cell (Treg) ratio and an decrease in T helper 17 cell (Th17) ratio in the mesenteric lymph nodes of ASD mice following the combination intervention. These findings suggest that the promising efficacy of HMOs, particularly the combination of 2’-FL and LNnT, on counteracting ASD-related pathophysiological changes may be mediated by the gut-immune-brain axis.