Sex Differences in Neural – Immune – Epithelial Anatomical Plasticity in the Mouse Small Intestine

Abstract

Background Pathways of neural signaling leading to alterations in gut wall immune – epithelial function are not well understood, particularly in the context of sex. Specific neural factors regulating epithelial or immune cell localization and secretion are emerging. Vasoactive intestinal peptide (VIP), often a neuropeptide, can play a role in goblet cell production (1) and signals bi-directionally with enteric mast cells (2). Immune components like mast cells are key in gut wall defense against pathogens. Epithelial components of host defense include goblet cells secreting mucus to form a physical barrier, and Paneth cells secreting anti-microbial compounds. Neural fibers course intimately with goblet and Paneth cells and may signal with gut mucosal mast cells (2). The present study establishes the anatomic arrangement of immunoreactive VIP (VIP-IR) fibers in relation to mast and Paneth cells in the mouse ileum, and tests the hypothesis that sex is a key variable in neural – immune – epithelial signaling in the gut wall in healthy and perturbed tissue. Methods We used an organotypic gut slice model that maintains the cellular diversity of the gut wall ex vivo. Slices were treated with selected pharmacological reagents that block neuronal function (e.g., tetrodotoxin) or VIP receptors prior to challenge with lipopolysaccharide (LPS) to assess their influence on anatomic plasticity of VIP-IR fibers with Paneth cells and mast cells at 48h ex vivo. Results There were more mast cells in females than males whether drop fixed in vivo (214%) or after 48h of ex vivo culture (53%). The mast cells in tissue from females drop fixed in vivo were ~18% larger than in males, and similarly ~26% larger in slices of ileum maintained ex vivo for 48h (Fig 1A-D). Approximately 70% of mast cells had VIP-IR fibers within 1µm, regardless of sex. However, those mast cells within 1 µm of a fiber were 15% larger than those without a nearby fiber, regardless of sex (Fig 1E-H). A small percentage of mast cells were induced to show substantial VIP-IR ex vivo only in slices treated with a VIP receptor antagonist or tetrodotoxin. Surprisingly, this effect occurred more in female slices (~200% more) than in male (Fig 1I-L). Paneth cells were labelled with the lectin Ulex europaeusagglutinin-1 and >75% were within 1 µm of a VIP-IR neuronal fiber, independent of sex or treatment with LPS. By contrast, Paneth cells in slices from females showed 51% greater fluorescent intensity than males, a difference not found after LPS treatment (Fig 2). Conclusions The present study demonstrates a close anatomical relationship of mast and Paneth cells with VIP-IR neuronal fibers. We extend previous work demonstrating a functional influence of VIP over goblet cell production in mouse ileum. The current results suggest sex differences in neural-immune and neural-epithelial plasticity that could potentially impact function. Additional data is needed to test the hypothesis that VIP has a functional role in regulating mast and Paneth cell secretion of histamine and anti-microbials in healthy and pathogen challenged states. References (1) Schwerdtfeger LA and Tobet SA. Physiol Rep 8:e14363, 2020. (2) Buhner S et al., Front Physiol 8:971, 2017.