Increasing indoxyl sulfate induces iNOS expression via aryl hydrocarbon receptor leading to microglia hyperactivation in the prefrontal cortex of autism-like offspring rats

Abstract

The abnormal indole metabolism is associated with the progression of Autism Spectrum Disorder (ASD). Indoxyl sulfate (IS), one of the active products of indole metabolism, still has an unknown role in ASD progression. This study investigates the role of IS/Aryl hydrocarbon receptor (AhR)/iNOS pathway in microglial activation in the prefrontal cortex (PFC) of ASD-like rats. Prenatal LPS-exposed induced autism-like behaviors offspring rats, concomitant with increased IS levels in the PFC. The levels of nuclear-AhR, IBA1, CD16 and iNOS proteins expression were increased in the PFC of LPS-exposed rats, whereas ARG1 protein expression level decreased, indicates microglia hyperactivation coupled with altered microglia morphology. ELISA analysis and further measure of synapses changes showed significantly increased inflammatory factors (TNF-α and IL-1β) and synaptic alterations. In vitro experiments demonstrated that IS treatment significantly upregulated the expression level of nuclear-AhR, enhanced microglia marker (IBA1, CD16 and iNOS) proteins and pro-inflammation factors levels (TNF-α and IL-1β), while concurrently reducing ARG1 protein expression and IL-10 levels in BV2 microglial cells. Moreover, the IS treatment significantly enhanced AhR enrichment in iNOS promoter region by chromatin immunoprecipitation and dual luciferase reporter assays, thereby significantly elevating the iNOS expression. However, the AhR-specific antagonist CH-223191 could block this activation and reverse the above proteins and inflammation factors changes. In a word, increased IS levels in the PFC of ASD-like offspring rats activate the AhR/iNOS pathway, driving microglial hyperresponsiveness and contributing to the development of ASD disease.