Ward, Marc A.

Introduction Visceral obesity is associated with GERD and, traditionally, it has been assumed that visceral fat predisposes to GERD primarily by increasing intra‐abdominal pressure. However, transmission electron microscopy (TEM) of esophageal biopsies from obese individuals without GERD exhibit dilated intercellular spaces (DIS) and, in earlier studies, we found that substances secreted by mature adipocytes in visceral fat from obese patients impair esophageal epithelial barrier function, induce DIS and reduce transepithelial resistance (TER). There is a fat pad at the gastroesophageal junction (GEJ) that is in intimate contact with the distal esophagus. We now explore whether whole GEJ fat tissue of obese individuals secretes substances that impair esophageal barrier function. Methods We obtained GEJ fat from 3 obese (BMI 46, 60, 74) and 3 non‐obese (BMI 24, 26, 27) patients having foregut surgery. Human esophageal squamous cells were seeded into transwells and grown to confluence for 3 days. On day 4, cells were raised to an air‐liquid interface (ALI) to allow for differentiation, stratification, and esophageal barrier formation. ALI cultures were exposed to conditioned media (CM) from GEJ fat or to control medium beginning at day 4. TER was measured on days 4, 6, and 8 using the EVOM2 Epithelial Volt/Ohm Meter. On day 8, ALI cultures were stained with H&E and Ki‐67 and imaged; barrier permeability was assessed by a biotinylation reagent applied to the ALI surface. TEM of ALIs and of esophageal biopsies from 2 non‐obese and 2 obese patients without GERD were assessed. Results CM from all 3 obese patients significantly reduced TER on days 6 and 8; CM from 2 non‐obese patients did not reduce TER whereas CM from 1 non‐obese patient induced a slight reduction in TER at day 8 (Table 1). Marked DIS were found only in ALI cultures grown with CM from obese patients. In ALIs grown with CM from non‐obese patients, the biotinylation reagent stopped at the superficial layers of esophageal cells. In contrast, the biotinylation reagent diffused through all cell layers in ALIs grown with obese patient CM. Increases in basaloid cells and in Ki‐67 were found in ALIs grown with obese patient CM. TEMs of ALIs grown with non‐obese patient CM demonstrated well‐oriented cell‐cell junctions similar to TEMs of non‐obese patient esophageal biopsies. In contrast, ALIs grown with obese patient CM demonstrated cell‐cell junctional structures that were disorganized, aggregated, and floating within the DIS, similar in appearance to TEMs of the obese patients’ esophageal biopsies. Conclusion Substances produced by GEJ fat of obese patients impair esophageal epithelial barrier integrity, cause basal cell hyperplasia and DIS, and disrupt intercellular connections. These findings in ALI cultures recapitulate abnormalities seen by TEM in esophageal biopsies of obese patients.

Gut dysbiosis, host genetics, and environmental triggers are implicated as causative factors in inflammatory bowel disease (IBD), yet mechanistic insights are lacking. Longitudinal analysis of ulcerative colitis (UC) patients following total colectomy with ileal anal anastomosis (IPAA) where >50% develop pouchitis offers a unique setting to examine cause vs. effect. To recapitulate human IPAA, we employed a mouse model of surgically created blind self-filling (SFL) and self-emptying (SEL) ileal loops using wild-type (WT), IL-10 knockout (KO) (IL-10), TLR4 KO (T4), and IL-10/T4 double KO mice. After 5 wk, loop histology, host gene/protein expression, and bacterial 16s rRNA profiles were examined. SFL exhibit fecal stasis due to directional motility oriented toward the loop end, whereas SEL remain empty. In WT mice, SFL, but not SEL, develop pouchlike microbial communities without accompanying active inflammation. However, in genetically susceptible IL-10-deficient mice, SFL, but not SEL, exhibit severe inflammation and mucosal transcriptomes resembling human pouchitis. The inflammation associated with IL-10 required TLR4, as animals lacking both pathways displayed little disease. Furthermore, germ-free IL-10 mice conventionalized with SFL, but not SEL, microbiota populations develop severe colitis. These data support essential roles of stasis-induced, colon-like microbiota, TLR4-mediated colonic metaplasia, and genetic susceptibility in the development of pouchitis and possibly UC. However, these factors by themselves are not sufficient. Similarities between this model and human UC/pouchitis provide opportunities for gaining insights into the mechanistic basis of IBD and for identification of targets for novel preventative and therapeutic interventions.