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Glial cells of the enteric nervous system (ENS) interact closely with the intestinal epithelium and secrete signals that influence epithelial cell proliferation and barrier formation in vitro. Whether these interactions are important in vivo, however, is unclear because previous studies reached conflicting conclusions (Prochera and Rao, 2023). To better define the roles of enteric glia in steady state regulation of the intestinal epithelium, we characterized the glia in closest proximity to epithelial cells and found that the majority express the gene Proteolipid protein 1 ( PLP1 ) in both mice and humans. To test their functions using an unbiased approach, we genetically depleted PLP1 + cells in mice and transcriptionally profiled the small and large intestines. Surprisingly, glial loss had minimal effects on transcriptional programs and the few identified changes varied along the gastrointestinal tract. In the ileum, where enteric glia had been considered most essential for epithelial integrity, glial depletion did not drastically alter epithelial gene expression but caused a modest enrichment in signatures of Paneth cells, a secretory cell type important for innate immunity. In the absence of PLP1 + glia, Paneth cell number was intact, but a subset appeared abnormal with irregular and heterogenous cytoplasmic granules, suggesting a secretory deficit. Consistent with this possibility, ileal explants from glial-depleted mice secreted less functional lysozyme than controls with corresponding effects on fecal microbial composition. Collectively, these data suggest that enteric glia do not exert broad effects on the intestinal epithelium but have an essential role in regulating Paneth cell function and gut microbial ecology.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections . The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization , but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.

Glial cells of the enteric nervous system (ENS) interact closely with the intestinal epithelium and secrete signals that influence epithelial cell proliferation and barrier formation . Whether these interactions are important however, is unclear because previous studies reached conflicting conclusions [1]. To better define the roles of enteric glia in steady state regulation of the intestinal epithelium, we characterized the glia in closest proximity to epithelial cells and found that the majority express in both mice and humans. To test their functions using an unbiased approach, we genetically depleted PLP1 cells in mice and transcriptionally profiled the small and large intestines. Surprisingly, glial loss had minimal effects on transcriptional programs and the few identified changes varied along the gastrointestinal tract. In the ileum, where enteric glia had been considered most essential for epithelial integrity, glial depletion did not drastically alter epithelial gene expression but caused a modest enrichment in signatures of Paneth cells, a secretory cell type important for innate immunity. In the absence of PLP1 glia, Paneth cell number was intact, but a subset appeared abnormal with irregular and heterogenous cytoplasmic granules, suggesting a secretory deficit. Consistent with this possibility, ileal explants from glial-depleted mice secreted less functional lysozyme than controls with corresponding effects on fecal microbial composition. Collectively, these data suggest that enteric glia do not exert broad effects on the intestinal epithelium but have an essential role in regulating Paneth cell function and gut microbial ecology.

Glial cells of the enteric nervous system (ENS) interact closely with the intestinal epithelium and secrete signals that influence epithelial cell proliferation and barrier formation in vitro. Whether these interactions are important in vivo, however, is unclear because previous studies reached conflicting conclusions (Prochera and Rao, 2023). To better define the roles of enteric glia in steady state regulation of the intestinal epithelium, we characterized the glia in closest proximity to epithelial cells and found that the majority express the gene Proteolipid protein 1 ( PLP1 ) in both mice and humans. To test their functions using an unbiased approach, we genetically depleted PLP1 + cells in mice and transcriptionally profiled the small and large intestines. Surprisingly, glial loss had minimal effects on transcriptional programs and the few identified changes varied along the gastrointestinal tract. In the ileum, where enteric glia had been considered most essential for epithelial integrity, glial depletion did not drastically alter epithelial gene expression but caused a modest enrichment in signatures of Paneth cells, a secretory cell type important for innate immunity. In the absence of PLP1 + glia, Paneth cell number was intact, but a subset appeared abnormal with irregular and heterogenous cytoplasmic granules, suggesting a secretory deficit. Consistent with this possibility, ileal explants from glial-depleted mice secreted less functional lysozyme than controls with corresponding effects on fecal microbial composition. Collectively, these data suggest that enteric glia do not exert broad effects on the intestinal epithelium but have an essential role in regulating Paneth cell function and gut microbial ecology.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections 1 , 2 . The exaggerated colonic inflammation caused by C.   difficile toxins such as toxin B (TcdB) damages tissues and promotes C.   difficile colonization 3 – 6 , but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C.   difficile burden in mice infected with a standard C.   difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI. The molecular mechanism underlying the severe neurogenic inflammation induced by Clostridioides difficile is presented, providing a therapeutic target for treating this infection.

Industrialization and advances in technology over the last half-millennium have led to dramatic changes in human society. The diet of our ancestors, once rich in plant matter of extraordinary chemical diversity, has been replaced with a heavily processed diet. Similarly, the rise of urban centers has resulted in an environment devoid of many microbial and chemical exposures around which our ancestors evolved. This rapid shift in human society is sharply correlated with two facets of modern human health: an extensive restructuring of the human gut microbiome and a dramatic increase in the incidence of chronic inflammatory diseases and infection. The gut microbiome is an incredibly complex ecosystem of microorganisms residing in the chemically complex milieu that is the gut.Diet has emerged as instrumental in driving the composition and dynamics of the gut microbiome, and in the development of diverse human diseases. While the impact of dietary carbohydrates (fiber) on the gut microbiome and human health is well characterized, there is a dearth of information as to how plant small molecules (phytochemicals) and other xenobiotics, many of which are thought to be bioactive, interact with or are transformed by the gut microbiome to affect human health. The dynamic interplay between multiple biotic and abiotic factors in this ecosystem makes deconvoluting molecular mechanism challenging using next-generational community profiling tools that profile community structure alone. This research aims to synergize bottoms-up and top-down approaches to define the landscape of phytochemical metabolism by the gut microbiome as a lever on human health.Chapter 2 describes the characterization of phytochemical glycoside metabolism across diverse gut bacteria, discovery of the genetic and molecular basis for differential glycoside catabolism across both gut bacteria and phytochemical substrates and the identification of a phytochemical-specific bacterial catabolic enzyme, and identification of two phytochemical-derived microbial metabolites with novel bioactivities: protection from experimental colitis and selective antagonism of C. difficile. Chapter 3 describes the identification of a novel small intestinal factor that regulates oral tolerance via modulation of both the microbiome and immune components of the gut, discovery of a dietary microbial tryptophan catabolic pathway that generates food allergy-protective Treg-reactive metabolites, and therapeutic intervention against this factor protecting against loss of tolerance in both early life and adulthood. Together, these findings demonstrate novel microbiome-diet axes wherein gut bacterial catabolism of dietary xenobiotics generates chemically-diverse metabolites that regulate the development of both local (inflammatory bowel disease; enteric C. difficile infection) and global (food allergy) disease processes. Furthermore, these findings emphasize the importance of reducing both the host and microbial components of the gut microbiome to their most basic, tractable parts— microbes, genes, and molecules to mechanistically identify tractable elements for therapeutic intervention.