The Yin and Yang of Bile Acid (BA) Action on Tight Junctions (TJ) in Colonic Epithelia: A Putative Role for Pro‐inflammatory Cytokine (PiC)
Authors: Detloff Sally Jo , Khan N , Ao M , Movva B , Nair T , Sirajuddin H , Domingue J , Rao M C , Sarathy J 
Published: November 2023

Epithelial barrier loss due to TJ dysfunction and BA induced‐diarrhea are common occurrences in patients with Crohn's disease and colitis. We reported (FASEB J: 29, 998.1, ‘15) that the primary BA, chenodeoxycholic acid (CDCA; 500 μM) and its 7α‐dehydroxylated derivative, lithocholic acid (LCA; 50μM) had opposite effects on epithelial integrity in human colonic T84 cells. CDCA decreased transepithelial resistance (TER) and increased paracellular permeability, effects that were enhanced by a cocktail of pro‐inflammatory cytokines (cPiC [ng/ml]: TNFα [10]+IL‐1β [10]+IFNγ [30]). In contrast, LCA restored barrier integrity that was disrupted by cPiC ± CDCA. In this study, we further delineated these opposing roles of CDCA, cPiC and LCA on TJs. Confluent T84 cells (TER >1000Ω.cm2) were apically treated with DMSO (control), 500μM CDCA or 50μM LCA. Junctional charge selectivity was measured at 5 min intervals over 30 mins, after a 50% dilution of mucosal buffer. Similar to other colonic epithelia, control T84 cells have a cation‐selective paracellular barrier with a dilution potential (DΨ) of 7.5 ± 0.5 mV. While LCA did not alter DΨ (8.0 ± 1.3mV; n=7), CDCA dramatically reversed the DΨ to −6.5 ± 2.2 mV (n=7), suggesting altered pore charge selectivity. Next, we determined if PiCs played a role in BA action. We have shown, in T84 cells, that the major colonic PiC, TNFα, induced the release of IL‐8, another PiC known to alter epithelial integrity (J. Agric. Food Chem. ‘08, 56, 3777–84). To probe the role of IL‐8 in BA action, confluent T84 cells were serum‐starved, treated overnight (O/N) with 500μM CDCA ± 50μM LCA ± cPiCs or the positive control, TNFα (100 ng/ml). IL‐8 released in the media, assessed by ELISA in pg/ml, were as follows: control: 727±46; TNFα: 2597±170; CDCA: 3460±384; LCA: 395±28; CDCA+LCA: 614±30; n≥6). Thus, while TNFα and CDCA respectively caused 3‐ and 4‐fold increases in IL‐8 production, LCA caused a 50% reduction in basal and an 83% decrease in CDCA‐induced IL‐8 production. These results parallel the inhibitory effects of LCA on CDCA‐induced increases in 10kDa dextran flux (FASEB J: ibid). The cPiCs caused a robust ~ 20 fold increase in IL‐8 release, which was not further enhanced by CDCA but reduced 50% by LCA (pg/ml, control: 777±52; cPiC: 15123±362; CDCA+cPiC: 16730±82; LCA+cPiC: 8072±511; n=6). To assess if TNFα is an intermediary in BA‐induced IL‐8 release, T84 cells were exposed O/N to varying amounts of CDCA, LCA, after which TNFα release was measured by ELISA. Neither CDCA nor LCA increased TNFα release (n=4), suggesting that the latter is not involved in CDCA‐induced IL‐8 release. Preliminary confocal microscopy and Western blotting studies indicate that CDCA redistributes TJ occludin protein and alters the expression of the pore‐forming TJ protein, claudin‐2. In conclusion, our findings suggest that, in T84 cells, CDCA plays a role in the inflammatory response, causing barrier dysfunction and enhancing the release of IL‐8, while LCA restores barrier integrity by suppressing the release of IL‐8. LCA could potentially be a therapeutic strategy for inflammatory and diarrheal diseases.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 and Institutional Funds, Ben. U. to JS Institutional Funds, UIC to MR