The Role of intestinal macrophages in gastrointestinal homeostasis

Gastrointestinal motility is crucial to gut health and has been associated with different disorders such as inflammatory bowel diseases and post-operative ileus. Before investigating the role of intestinal macrophages on gut motility in rodents, it is important to determine which animal model is most suitable. Therefore, this work first aimed to compare colonic motility between two commonly used animal models; mouse and rat. Despite rat and mouse being the two most widely used animal models in gastrointestinal research, minimal studies have investigated gastrointestinal motility in rats. Therefore, this study provides a comparison of colonic motility in the mouse and rat to clarify species differences and assesses the relative effectiveness of each animal model for colonic motility research. We describe the protocol modifications and optimization undertaken to enable video imaging of colonic motility in the rat. Apart from the broad difference in terms of gastrointestinal diameter and length, we identified differences in the fundamental histology of the proximal colon such that the rat had larger villus height-to-width and villus height-to-crypt depth ratios compared with mouse. Since gut motility is tightly regulated by the enteric nervous system (ENS), we investigated how colonic contractile activity within each rodent species responds to modulation of the ENS inhibitory neuronal network. Nitric oxide (NO) is the major inhibitory neurotransmitter within myenteric plexus. Here we used Nω‐nitro‐l‐arginine (NOLA), an inhibitor of nitric oxide synthase (NOS) to assess proximal colon responses to the stimulatory effect of blocking NO. In rats, the frequency of proximal colonic contractions increased in the presence of NOLA (versus control levels) to a greater extent than in mice. This is despite a similar number of NOS-expressing neurons in the myenteric plexus across species. Given this increase in colonic contraction frequency, the rat represents another relevant animal model for investigating how gastrointestinal motility is regulated by the inhibitory neuronal network of the ENS with some similarities and differences to the traditionally used mouse. The second aim of this thesis was to decipher role of intestinal macrophages in colonic motility. Intestinal macrophages are well studied for their conventional roles in the immune response against pathogens while preventing the gut from chronic inflammation. There is growing evidence that intestinal macrophages have both direct and indirect influences on gastrointestinal motility through myenteric neurons. Since the CX3CR1 chemokine receptor is highly expressed in mature intestinal macrophages, we used a conditional knockout Cx3cr1-Dtr (diphtheria toxin receptor) rat model to transiently ablate intestinal macrophages. We verified the animal model by assessing immune cell populations within the myenteric plexus and subsequently utilized ex vivo video imaging to evaluate colonic motility. Our previous studies in brain suggested that microglia start to repopulate 7 days after diphtheria toxin injection, so we performed our experiments at both 48 hr (macrophage depletion) and 7 day (macrophage repopulation) timepoints. In addition, we investigated whether inhibitory neuronal input from the ENS is required for the regulation of colonic motility by intestinal macrophages. Our results demonstrated that CD163 positive resident intestinal macrophages are crucial in regulating colonic motility in the absence of major inhibitory neuronal input. In addition, we found that intestinal macrophages are indispensable in maintaining healthy intestinal structure. Our study provides a novel understanding of how intestinal macrophages regulate colonic motility, thus placing intestinal macrophages as a potential therapeutic target for gastrointestinal motility disorders such as post-operative ileus and Hirschsprung’s disease when inhibitory neuronal input is suppressed. As mentioned, the conventional role of intestinal macrophages is to prevent chronic inflammation from a constant influx of foreign antigens. Although it has been shown in a mouse colitis model that gastrointestinal histology and function are significantly impaired, we have little understanding of how systemic inflammation such as that which occurs with bacterial infection affect gastrointestinal functions. Thus, the third aim of my thesis is to determine how systemic inflammation affects colonic motility. By using the Cx3cr1-Dtr rat model, we further investigate how intestinal macrophages contribute to changes in colonic motility during systemic inflammation. Our data illustrated a shift in the localization of intestinal macrophages during systemic inflammation, resulting in a decrease in Iba-1 positive only macrophages within the myenteric plexus. In these rats, colonic contraction frequency is increased while resting gut diameter and contraction magnitude are decreased during inflammation. We also revealed that when the major inhibitory ENS input is absent, systemic inflammation leads to an inhibition of colonic motility even when intestinal macrophages are depleted. Together, this work shows that intestinal macrophages play a critical role in regulating colonic motility. The data from this thesis provides a detailed account for understanding how colonic motility is regulated through mechanisms other than inhibitory ENS input using the Cx3cr1-Dtr rat model. With further investigation of the molecular mechanisms underlying how specific subtypes of intestinal macrophages communicate with muscle cells, intestinal macrophages have the potential to be used as therapeutic targets for disorders related to gastrointestinal motility.