Characterising microbial and neuroimmune interactions in a mouse model of autism

Gastrointestinal dysfunction and changes in the microbial community of the gut are commonly reported in children with autism spectrum disorder (ASD) and have been proposed to contribute to behavioural impairment. Gut microbial communities interact with the immune system and also produce neuro-active molecules that modulate the central and enteric nervous systems. Many rare gene mutations implicated in autism, including the well-studied neuroligin-3 R451C missense mutation, influence neuronal communication. In addition to changes in the nervous system, people with autism are more likely to have immune disorders.  The caecum is involved in generating immune responses and acts as a repository of intestinal microorganisms, but it is unknown whether this organ plays a role in autism. At the blind end of the caecum, gut lymphoid aggregates known as caecal patch (CP) are found which contain various immune cells such as macrophages and dendritic cells, which are crucial for mucosal immunity. To assess whether the autism-associated R451C mutation in the Nlgn3 gene influences the caecum, we first assessed for changes in caecal weight in NL3R451C mice. Using immunofluorescence in wholemount preparations, we quantified the total number of enteric neurons per ganglion in the myenteric and submucosal plexus. In frozen cross sections of the caecal patch, we examined the density and morphology of Iba1-expressing macrophages. We also assessed caecal microbial community composition using Illumina deep sequencing. NL3R451C mice have significantly reduced caecal weight compared to wild-type (0.65±0.02g, 0.54±0.01g, WT and NL3R451C respectively; p=0.0001). NL3R451C caecal myenteric plexus had enlarged ganglia (12.12 ± 0.02 and 17.19 ± 0.05 mm2 per ganglion; WT and NL3R451C respectively; p= 0.008) and more neurons per ganglion (WT:11±1, NL3R451C:15±1; p=0.005). An increase in the number of neurons per ganglion was also observed in the submucosal plexus (WT:5±1 and NL3: 6±1; p=0.04). 3D image analysis revealed a higher density of Iba1-expressing macrophages in the caecal patch (11±1 and 14±1 cells/100 µm2, WT and NL3R451C respectively; p=0.04) and smaller cell volume (946.9 ± 0.4µm3 and 559.7 ± 0.4µm3, WT and NL3R451C respectively; p=0.004) in NL3R451C mice. Moreover, distinctly different caecal microbial community structures were observed in NL3R451C compared to wild-type mice. These findings suggest that the autism-associated R451C mutation in Neuroligin-3 impacts caecal neuronal structure, immune cell properties, and microbial populations.