Reactive Nitrogen and Oxygen Species (RNS and ROS) Play a Role in Bile Acid (BA)‐Induced Barrier Dysfunction and Proinflammatory Cytokines (PiC) Release in Human Colon Carcinoma T84 Cells
Authors: Dinsmonaite Ugne , Haq M. , Couri T. , Pierzchanowski A. , Gornick E. , Khan S. , Donner F. , Whitman P. , Rao MC. , Rubush DM. , Sarathy J. 
Published: June 2021

Dysfunction of mucosal immune response and tight junctions (TJ) play an important role in the pathogenesis of inflammatory bowel diseases (IBD) and diarrhea. We reported that excess BA, namely chenodeoxycholic acid (CDCA; 500μM) induced PiC release and altered T84 TJ by increasing ROS. The effect on leak function of TJ was attenuated by CDCA's derivative, lithocholic acid (LCA, 50 μM) and ROS inhibitors (Physiol Rep, ′17, 5: e13294). In patients with IBD, PiC upregulate inducible nitric oxide synthase (iNOS). Thus, we hypothesize a role for RNS in mucosal damage and studied the involvement of oxidative/nitrosative stress in BA‐induced TJ dysfunction and cytokine release in T84 cells.

We previously described the synthesis of fluorescein amine‐tagged CDCA (CDCA‐FA) and its use to track transepithelial (TE) BA movement. Confluent T84 cells (TE Resistance; TER >1000Ωcm2) were treated apically with DMSO, 500μM CDCA‐FA, 50μM LCA, CDCA+LCA, ± PiC ([ng/ml]: TNFα[10]+IL‐1β[10]+IFNγ[30]), ± 50μM L‐NAME ( L‐NG‐Nitroarginine methyl ester, NOS inhibitor), ± 1mM NAC, (N‐acetyl cysteine, ROS scavenger), for 0.5–18 H. [NO2/NO3] was measured by the Griess assay. We examined the role of RNS/ROS in BA action by studying the effect of BAs±L‐NAME± NAC on: a. Apoptosis (Annexin V, Flow cytometry); b. Paracellular permeability (Pore function as TER; Leak function as TE CDCA‐FA movement); and c. IL‐8 release (ELISA).

CDCA (18 H), but not LCA, increased [NO2/NO3] 3‐fold and this was enhanced by PiC (μmol/mg protein; DMSO: 12±1; CDCA: 36±1; LCA: 16± 4, PiC: 18±1; PiC+CDCA: 55±2, n=3). LCA decreased CDCA±PiC‐induced [NO2/NO3] (CDCA+LCA: 25±1; PiC+LCA: 13±2, PiC+CDCA+LCA: 27±4; p<0.05). Inhibiting RNS did not alter CDCA‐induced apoptosis. In pore function, L‐NAME reduced the initial rate of CDCA‐FA‐induced decrease in TER (Ωcm2/sec; 1H CDCA‐FA: 12±1; CDCA‐FA+L‐NAME: 7±1, n>3; p<0.05 ), with no statistical difference at 4 and 18H. L‐NAME altered leak function, reducing CDCA‐FA flux by ~30% at 18 H, (Apparent permeability: Papp ×10−9 cm/sec: CDCA‐FA: 57±2; CDCA‐FA+L‐NAME: 30±1; p<0.05, n=3). LCA decreased CDCA‐FA flux by ~58%, and LCA+L‐NAME completely attenuated it (CDCA‐FA+LCA: 24±2; CDCA‐FA+LCA+L‐NAME: 3±1; p<0.05, n>3). NAC+LCA caused only a ~85% decrease in CDCA‐FA flux (CDCA‐FA+LCA+NAC: 8±2).

Like CDCA, CDCA‐FA±PiC stimulated IL‐8 release, which was decreased by LCA. Inhibiting RNS, but not ROS, caused ~40% reduction in CDCA‐induced IL‐8 release (ng/ml; CDCA: 4.6±0.5; CDCA+L‐NAME: 2.7±0.1; CDCA+NAC: 4.2±0.3; n=3), suggesting a role for NO in BA‐induced inflammatory process.

We demonstrate a novel role for RNS in CDCA‐induced TJ dysfunction in T84 cells. Inhibiting RNS in the presence of LCA reverses CDCA action on leak function and reduces PiC release. Equally important, its roles overlap with but are distinct from ROS. Understanding the role of ROS/RNS in BA action can lead to novel therapeutic strategies for IBD.

Support or Funding Information

NSF ‐ MRI: DBI‐1427937 to JS and Ben U Funds to JS and DMR; UIC Funds to MCR; APS‐STRIDE National Heart, Lung and Blood Institute (Grant #1 R25 HL115473‐01) to UD; APS‐UGSRF to MH

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.