Student Theses and Dissertations

Fanny Matheis

Date of Award

2021

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Mucida Laboratory

Abstract

The intestine is the largest continuous environmental interface of the body. As such, it exerts homeostatic tissue functions, including digestion, sensing and absorption of nutrients, and excretion of waste products. In performing these roles, the intestine faces the unique challenge of remaining tolerant to harmless or beneficial diet- and microbederived stimuli, while simultaneously protecting against pathogen invasion. To tackle these challenges, the intestine houses both the body’s largest immune compartment, as well as a vast neuronal network, the enteric nervous system (ENS). In concert with the commensal intestinal microbiota, the enteric immune and nervous systems communicate with one another, and this crosstalk was the focus of my thesis work. The studies as presented here are divided into two parts: The first part will focus on the influence of gut microbes on the murine ENS and its functions in host physiology. The second part will investigate the dynamic interplay between gut microbes, neurons and immune cells in the murine intestine during homeostasis and upon microbial perturbations. The human intestinal tract is home to ~10 trillion commensal microbes (Sender et al., 2016). The microbiota influences key physiological processes including nutrient absorption and lipid metabolism. Further, it has been demonstrated to influence the basal activity of intestine-associated cells, including the excitability of enteric neurons (Furness et al., 2013). Alterations to the composition of the gut microbiota have a potential role in systemic disorders including obesity and diabetes (Ridaura et al., 2013). Yet, the mechanisms underlying these effects of the microbiota, and whether they are mediated by components of the ENS, are still poorly understood. In addition, the cellular circuits and molecular components that mediate gut-to-enteric neuron or gut-to-brain communication remain largely unknown. We thus aimed to determine how commensal microbes influence enteric neurons and their functions to better characterize their role in tissue function and further sought to investigate how disturbances to the microbial composition – during microbial dysbiosis and enteric infections – impact the ENS and host physiology. Using translating ribosomal affinity purification (TRAP)-sequencing, coupled with confocal microscopy, we found that enteric neurons are functionally adapted to the intestinal segment they occupy. By utilizing germ-free mice, we uncovered a stronger influence of the microbiota on distal intestine neurons, correlating with the region’s higher bacterial density. Chronic antibiotic-mediated microbial depletion reinstated our findings in germ-free mice, establishing that specific subsets of enteric neurons, including those expressing the neuropeptide cocaine and amphetamine-regulated transcript (CART), are dependent on the microbiota for their survival. Notably, these changes were not permanent, as colonization of germ-free mice and replenishment of the microbiota of antibiotic-treated mice restored neuronal numbers and neuropeptide levels. We found that murine enteric infections with different pathogens led to lasting intestinal inflammation, functional disturbances and most notably, rapid and persistent enteric neuron loss driven by a persistent alteration to the microbial composition postinfection; however, restoration of a healthy microbiota was sufficient to induce tissue recovery. Mechanistically, neuronal loss post-infection and following microbial depletion was mediated by a novel form of enteric neuronal cell death, involving the non-canonical inflammasome components NLRP6 and caspase 11. In further characterizing enteric neuronal populations, we identified a subset of intestinal CART+ neurons that were enriched in the distal intestine and modulated by the microbiota. Through microbial modulation strategies and chemogenetic targeting, we found that these enteric CART+ neurons regulate metabolic parameters including blood glucose and insulin levels. Retro- and anterograde tracing studies revealed that a subset of enteric CART+ neurons send axons to the gut sympathetic ganglion and are synaptically connected to the liver and pancreas. Together, we uncovered a gutpancreas- liver circuit that regulates glucose metabolism by sensing microbial cues. This peripherally-restricted circuit offers unique neuronal targets for the treatment of metabolic disorders, such as type 2 diabetes, which would bypass central nervous system effects. We further aimed to better characterize the role of neuro-immune interactions in the context of enteric pathologies, including post-infectious intestinal dysfunction and neuronal damage observed upon enteric infections. We further sought to determine whether a state of tolerance could be induced upon exposure to enteric pathogens, preventing tissue damage during subsequent infections. Finally, we aimed to characterize the role of extrinsic gut-projecting neurons to understand their role in sensing and responding to luminal cues, including enteric infections. Using cell sorting-independent transcriptomics, confocal imaging, genetic gainand loss-of-function approaches, surgical lesioning, chemogenetic manipulations, as well as multiple microbial manipulation strategies, we identified a critical role for enteric neuron-macrophage crosstalk in limiting ENS damage induced by a single enteric infection. A population of tissue-resident macrophages residing in close proximity to enteric neurons responded to luminal cues by upregulating a tissue-protective signature, and mediated enteric neuronal protection through adrenergic receptor signaling, and an arginase 1-polyamine program. Notably, we found engagement of macrophage adrenergic receptor signaling to be dependent on local catecholamine release by gutinnervating sympathetic neurons. We further uncovered that these sympathetic neurons on their end are tuned by enteric microbes and microbial products, in that a healthy microbiota suppresses, and absence of a microbiota, dysbiosis and infection enhance their activity. Finally, we found that previous infection with unrelated pathogens prevented infection-induced neuronal loss during subsequent, heterologous infections, suggesting a form of innate immune memory, or “trained tolerance”. Of note, while enteric bacterial and helminth infections induced distinct immune responses, these converged at the level of tissue-protective intestinal macrophages, which mediated enteric neuronal protection, aiding host fitness. Together, this work identified a functional role for interactions between sympathetic neurons, tissue-resident macrophages and enteric neurons in limiting infection-induced tissue damage. Overall, the research presented in this work uncovered that the ENS relies on the gut-resident microbiota for its homeostatic tissue function, with influence for local intestinal function and systemic metabolism. Furthermore, through communication with gut-extrinsic sympathetic neurons, tissue-resident macrophages upregulate and maintain a tissue-protective program, which protects enteric neurons from excessive damage during primary enteric infections and prevents cumulative damage during subsequent perturbations.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

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