Investigating caecal changes in mouse models of immune and nervous system dysfunction

Gut-brain interactions have emerged as a focal point in the study of neurodevelopmental disorders, including autism spectrum disorder (ASD; autism), given the frequent co-occurrence of gastrointestinal symptoms alongside core behavioural features. Despite its significance in neuro-immune-microbe functions, the caecum, a segment of the gastrointestinal tract analogous to the human appendix, has often been overlooked in gastrointestinal research. This dissertation aims to elucidate the impact of the Nlgn3R451C autism-associated mutation on neuro-immune interactions and functional changes within the caecum. The Nlgn3R451C mutation, identified in two Swedish brothers with ASD, is a missense substitution of Arg to Cys at position 451 (R451C) of the Neuroligin-3 protein (Jamain et al., 2003). Intriguingly, these brothers exhibit comorbid gastrointestinal disorders, including chronic gut pain and diarrhoea (Hosie et al., 2019). Consistent with these clinical observations, Nlgn3R451C knock-in mice also display disrupted gastrointestinal function and altered structure of the enteric nervous system (ENS) (Hosie et al., 2019). Previous research from the current laboratory has demonstrated distinct differences in caecal weight, enteric neuronal proportions, and intestinal macrophages between Nlgn3R451C mice and WT littermates (Sharna et al., 2020). However, the understanding of how neuro-immune circuitry influences caecal function, particularly in terms of motility and intestinal barrier function, remains limited. To address this, I first developed an ex vivo motility assay specifically tailored to accommodate the unique anatomical shape and size of the mouse caecum. Building upon existing protocols widely employed in colon and small intestine segments to study intestinal motility, I integrated modifications to incorporate a tri-cannulation technique previously applied only in rabbit caecum. Through the utilization of this new assay, I identified a novel neurogenic motility pattern in the mouse caecum, now termed Caecal Motor Complexes (CaeMCs). CaeMCs represent clusters of forward/pro-peristaltic and reverse/anti-peristaltic contractions that travel along the caecal body, interspersed with quiescent periods exhibiting minimal or no contractile activity. Subsequently, I investigated caecal function in the Nlgn3R451C mouse model of autism. Notably, I observed increased caecal motility in Nlgn3R451C mice compared to WT littermates, which correlated with reduced caecal content. I propose that this dysmotility contributes to the previously reported reduction in caecal weight in these mice. While no differences were detected in mucus content, transepithelial resistance, or permeability across caecal tissue preparations, Nlgn3R451C caecum samples exhibited decreased neurally-evoked secretion following stimulation with a nicotinic acetylcholine receptor agonist (1,1-dimethyl-4-phenylpiperazinium, DMPP). As the ENS plays a vital role in regulating gastrointestinal function, particularly within the submucosal plexus to modulate intestinal secretory function, immunostaining for key enteric neuronal markers, including the pan-neuronal marker Hu, vasoactive intestinal peptide (VIP), and choline acetyltransferase (ChAT) was undertaken. Surprisingly, no differences in the proportions of these enteric neuronal subpopulations in the Nlgn3R451C caecum were observed, suggesting that an alternative subpopulation of enteric neurons could be involved in the reduced secretion phenotype, or the expression of the relevant nicotinic acetylcholine receptor could be altered in these mice. The altered caecal motility and secretory function observed in Nlgn3R451C mice highlight that the relatively unexplored caecal region is impacted by this autism-associated gene mutation. The caecum plays a crucial role in immunological processes. The caecum contains gut-associated lymphoid tissue (GALT) known as caecal patches that are responsible for generating IgA-secreting plasma cells. Novel findings in this dissertation indicated an elevated number of caecal patches in the Nlgn3R451C mouse model of autism compared to WT littermates. Pre-term brain injury is also known to be associated with neurodevelopmental disorders such as ASD, and here, potential changes in caecal tissue structure were studied in a mouse model of moderate perinatal systemic inflammation of intraperitoneal IL-1β injections from post-natal Day 1 to 5. Interestingly, a prominent phenotype of increased caecal patch formation was observed in IL-1β-treated mice. Notably, the investigation of ENS neuronal subtypes and specific immune cell populations, including dendritic cells and gut macrophages, revealed no significant changes in subpopulation numbers, density, or cell morphology, indicating resilience or recovery of the ENS following IL-1β insult. An important finding from this work is that GALT neogenesis occurs in response to inflammatory insult during a critical window of vulnerability in development. To investigate the role of the caecum in Nlgn3R451C mice in the context of inflammation, we generated a unique mouse model of genetic and environmental insult by combining the Nlgn3R451C autism-associated nervous system mutation with the type-1 interferon alpha receptor-1 (Ifnar1) immune system mutation. Ablation of the type-1 interferon (IFN) pathway alleviates neuro-inflammation and ameliorates neuro-behavioural symptoms in various neurological diseases (Main et al., 2016; Minter et al., 2016; Zhang et al., 2017). The current study revealed that Nlgn3R451C×Ifnar1-/- mice are resistant to dextran sodium sulphate (DSS)-induced intestinal inflammation compared to sham-treated controls. This was evidenced by the improvement and recovery from colitis symptoms on Day 8 of the treatment regimen, the preservation of colon length, improved intestinal barrier integrity, and resilience to sickness behaviours in Nlgn3R451C×Ifnar1-/- mice. To investigate the role of the caecum in gastrointestinal function and its response to inflammation, we report several significant caecum-specific findings. Notably, Ifnar1 knockout mice demonstrate resistance to DSS-induced disruptions in caecal motility patterns. Intriguingly, no discernible differences in the proportions of enteric subpopulations were observed in response to DSS suggesting no disproportionate effects of DSS on neuronal survival for subpopulations assessed in the current study. Collectively, these findings highlight the potential for targeting the type-I interferon pathway to provide beneficial outcomes for individuals with autism and related conditions characterized by elevated levels of inflammation. In summary, this study contributes to the growing body of research highlighting the role of the caecum in modulating neuro-immune interactions in autism. This research included the generation of a new experimental protocol using ex vivo motility assays in mouse caecal preparations, an approach which offers valuable insights into caecal function in preclinical models. In addition to identifying atypical caecal motility and secretory function in the Nlgn3R451C mouse model of autism, the expansion of caecal patch lymphoid aggregates (GALT) in both autism and preterm brain injury models is suggestive of common mechanisms across different mouse models of neurodevelopmental disorders. Importantly, we provide compelling evidence suggesting that targeting the type-I interferon pathway through IFNAR1 holds promise for alleviating aberrant inflammatory responses specifically in the caecum which may be relevant in the context of gastrointestinal dysfunction in autism. Overall, this research underscores a significant role of the caecum in several preclinical models of neurodevelopmental disorders, highlights its potential as a therapeutic target for improving communications at the interface between luminal commensal bacteria and host physiology.