**7. Changes in gut barrier defense following PN**

The epithelial barrier of the intestine, which turns over rapidly due to highly proliferative pluripotent Lgr5+ stem cells in the intestinal crypts, serve as the first line of defense against the external environment of the gut. The epithelium turns over every 3–5 days [66]. One cell type that does not turn over rapidly are small intestinal Paneth cells, that turn over every 20–30 days. The selective barrier allows absorption of water, electrolytes, and some macromolecules via tight-junction proteins, including zonulins, occludins, and claudins between enterocytes. Epithelial permeability increases significantly during PN feeding [67, 68], in parallel with a loss of tight-junction proteins [69]. In addition to the physical barrier along the gut lining, subepithelial dendritic cells extend dendrites between epithelial cells, which are hypothesized to sample luminal antigens and augment barrier responses [70]. A smaller proportion (10%) of intestinal cells secretory cells, including enteroendocrine cells and mucous secreting goblet cells [47].

The epithelium's contribution to defense includes not only physical barrier formation, but release of antimicrobial molecules that influence microbial community composition and membership. Enterocytes comprise 90% of total epithelial cells in the gut and release β-defensins and RegIIIγ enzymes that limit microbial growth at the mucous barrier [71]. The far less abundant Paneth cells, found at the crypt bases, produce a large array of antimicrobial peptides and enzymes, including lysozyme, RegIIIγ, secretory PLA2, Angiogenin4, and α-defensins (murine cryptdins) [72]. These cationic antimicrobials localize to the negatively-charged mucous surface and work by targeting conserved aspects of microbial cell walls and membranes [67]. Studies demonstrate Paneth cell antimicrobial molecules reach 15–100 mg/mL within intestinal crypts, exceeding antimicrobial concentrations [73]. The colon exhibits two layers of mucous, an inner sterile layer and an outer more loosely colonized layer.

Paneth cell antimicrobial release is regulated by Th2 cytokines, including IL-4, IL-9, and IL-13, GLP-2, and insulin [37, 74, 75]. Stimulatory triggers include the ligands TLRs and NOD2 and parasympathetic cholinergic stimulation [76]. PN decreases antimicrobial production through lower levels of intestinal IL-4 and IL-13 [77, 78]. Enterocyte release of RegIIIγ is also lost during PN [79]. Exogenous administration of IL-25 stimulates production of IL-4 and IL-13 cytokines and levels of Lysozyme, sPLA2, and RegIIIγ compared with PN feeding alone [80]. Following PN, mucosal secretions contain less Paneth cell antimicrobial products, leading to decreased killing of bacteria in vitro [76]. Tissue explants from PN fed animals have been demonstrated to be more susceptible to enteroinvasive *E. coli* compared with controls [81].

The function of goblet cells is to produce the mucous barrier, composed of glycoprotein mucins. These proteins have numerous carbohydrate residues including *O*-glycosylation, *N*-actyl-galactosamine, galactose, and *N*-actyl-glucosamines [82]. Functionally, mucins mucous serves as a selective physical barrier that allows for movement of luminal digesta, absorption of nutrients, and limiting bacterial access to the gut wall. Mucins also help concentrate Paneth cell antimicrobial molecules and sIgA through charge interactions [83]. The importance of mucins is demonstrated by in mutant animals lacking the more abundant mucin, MUC2, which leads to inflammatory enteritis and increased risk of tumor formation [45].

PN decreases the release of MUC2, RELMβ, and trefoil factor 3 (TFF3) in neonatal piglets and adult mice [75, 84]. TFF3 promotes epithelial response to injury. Animals deficient in RELMβ display increased susceptibility to *Citrobacter rodentium* challenge. The release of goblet cell products are influenced by Th2 cytokines, including IL-4 and IL-13, which are decreased under PN feeding [85]. Exogenous administration of the Th2 stimulating cytokine, IL-25, elevates luminal MUC2 levels [77].

Although only 1% of the total intestinal epithelial cells are enteroendocrine cells (EECs), they collectively make up the largest endocrine organ, expressing almost 2 dozen peptide hormones that shape metabolism, immunity, and behavior [86, 87]. EECs respond to enteral nutrients as well as microbial ligands [88]. EECs are also intricately linked to innate immunity, indirectly activating and recruiting immune cells by producing the chemokines CXCL-1, CXCL-3, and the cytokine IL-32. EEC hormones can also influence epithelial cell function in the gut, including Paneth cell release of antimicrobial molecules. GLP-2 mutant animals are at increased susceptibility to gut infection than wild-type littermates [37].
