Characterising Changes in the Gut-Brain Axis in Mice With Modified Neuro-Immune Pathways

Individuals with autism spectrum disorder (ASD; autism) exhibit deficits in social communication and repetitive behaviours, which are associated with changes in the nervous system. Individuals with autism are also more susceptible to inflammatory disorders and show increased systemic and brain inflammation. Gastrointestinal symptoms, increased intestinal permeability, and changes in the composition of microbial communities in the gut are also commonly experienced by these individuals. Growing evidence suggests atypical interactions between microbes, immune cells, and the brain occur in autism, however, how changes in the nervous system impact inflammatory responses remain to be clarified. Disruptions in signalling pathways between the brain and gut may alter gastrointestinal structure and function as well as behaviour, and these changes could contribute to increased neuroinflammation and gastrointestinal dysfunction in individuals with autism. This thesis investigated changes to the gut-brain axis following immune stimulation in transgenic mice relevant to autism and immune dysfunction. To understand cross talk between immune and neural pathways, the impact of the chemical inflammatory stimulus, dextran sulphate sodium (DSS), a well-established mouse model of intestinal injury and inflammation, was studied in three mouse models. Specifically, the Neuroligin-3R451C mouse model of autism expressing a missense mutation modifying a cell adhesion protein expressed at neuronal synapses (Nlgn3R451C mice) and mice lacking an important receptor in the type-I interferon immune response (Interferon α/β receptor subunit 1 (IFNAR1-/-) mice) were studied. We also bred Nlgn3R451C mice with IFNAR1-/- to generate a novel strain of mice with a combined neuro-immune mutation (Nlgn3R451CxIFNAR1- /- mice). The interactions of these genetic mutations and inflammatory pathways were assessed using a model of acute experimental colitis in which mice were given 3% DSS in drinking water for a period of 7 days. We hypothesised that Nlgn3R451C mice exhibit increased susceptibility to DSS-induced colitis symptoms, and that this increase is attenuated by deletion of IFNAR1. Mice were monitored and scored during each day of treatment to calculate an overall colitis- relevant disease activity index, including weight loss, faecal pellet consistency, the presence of blood in faeces, and rectal bleeding. Gut and brain tissue including colon and small intestine length, caecal and spleen weight, and the presence of gut-associated lymphoid tissue (Peyer’s and caecal patch numbers) were assessed. To determine changes in structural and functional gastrointestinal function, distal colon histopathology, faecal pellet production, and permeability of ileal and colonic tissue were assessed. To examine whether intestinal inflammation affects the brain, glial cells were studied by immunofluorescence to compare the density and morphology of microglia and astrocytes in the cortex and dentate gyrus of the hippocampus. Finally, to assess how altering neuroimmune function affects behaviour, locomotor function was examined in the open field test, and anxiety-like behaviour was observed in the open field, elevated plus maze, and light-dark box. Social behaviours were also considered in a reciprocal social interaction test where mice faced an unfamiliar mouse in the open field arena. We found that Nlgn3R451C, IFNAR1-/-, and Nlgn3R451CxIFNAR1-/- mice exhibit resilience to DSS-induced colitis compared to WT mice across a number of measures. Mice harbouring these mutations impacting the nervous and immune system exhibited decreased severity of colitis from day 0 to 8, particularly in Nlgn3R451C and Nlgn3R451CxIFNAR1-/- mice. Furthermore, all three groups exhibited faster recovery one day after treatment cessation when compared to WT mice. Anatomically, colon shortening, a hallmark of DSS-induced colitis, persists in Nlgn3R451C and IFNAR1-/- mice. However, Nlgn3R451CxIFNAR1-/- mice were resistant to this decrease in colon length. Moreover, DSS-induced splenomegaly was reduced in Nlgn3R451C mice and absent in IFNAR1-/- and Nlgn3R451CxIFNAR1-/- mice, demonstrating a reduced immune response to the toxin. DSS-treated WT mice had increased ileal and colonic permeability compared to SHAM-treated controls, as demonstrated by Ussing chamber experiments. DSS-treated Nlgn3R451C mice exhibited an increase in colonic permeability, whereas IFNAR1-/- and Nlgn3R451CxIFNAR1-/- mice were resilient to DSS-induced intestinal barrier dysfunction. Immunofluorescent staining of brain glial cells showed morphological changes indicative of increased reactivity of microglia in DSS-treated WT mice, but not in mice with mutations affecting NLGN3 and/or IFNAR1. Behavioural testing revealed significant locomotor dysfunction in DSS-treated WT mice, which was prevented in Nlgn3R451C, IFNAR1-/-, and Nlgn3R451CxIFNAR1-/- mice. Mice with acute experimental colitis did not exhibit increased anxiety-like behaviour. Instead, DSS- treated Nlgn3R451CxIFNAR1-/- mice exhibit increased exploratory behaviour in the elevated plus maze, while both IFNAR1-/- and Nlgn3R451CxIFNAR1-/- mice demonstrate increased social behaviour when faced with a novel mouse. Interestingly, mice expressing the autism-relevant Nlgn3R451C mutation demonstrated decreased severity of experimental colitis, and exhibit improvements in splenomegaly and ileal barrier dysfunction compared to WT mice. Similarly, mice lacking IFNAR1 exhibited less severe colitis outcomes, together with complete rescue from splenomegaly and ileal and colonic barrier dysfunction, as well as reduced evidence of glial cell reactivity. Finally, mice possessing both mutations (Nlgn3R451CxIFNAR1-/- mice) showcased a profound and additive mitigation of physiological impairments associated with DSS-induced colitis, including the absence of colon shortening, the absence of signs indicating hunching or discomfort, and a notable enhancement in exploratory and social behaviour. These findings suggest that alteration to the expression of Nlgn3 results in impairments to immune function and the ability to mount an inflammatory response to a foreign toxin. Given evidence for expression of Nlgn3 in immune cell populations, we propose that the R451C mutation directly impacts the innate immune response. We suggest that Nlgn3R451C-evoked immune impairments operate via differing mechanisms to the dysfunction caused by the disruption to type-I IFN signalling given that we observed an additive effect in double mutant mice. This study contributes to a growing body of research highlighting a role for Nlgn3 in immune function. This work enhances the understanding of immune and gut abnormalities under pathophysiological conditions with relevance to autism.