TLR4-dependent claudin-1 internalization and secretagogue-mediated chloride secretion regulate irinotecan-induced diarrhea

Abstract

We have previously shown increased intestinal permeability, to 4-kDa FITC-dextran, in BALB/c mice treated with irinotecan. Importantly, genetic deletion of Toll-like receptor 4 (TLR4; Tlr4-/-) protected against loss of barrier function, indicating that TLR4 is critical in tight junction regulation. The current study aimed (i) to determine the molecular characteristics of intestinal tight junctions in wild-type and Tlr4-/- BALB/c mice and (ii) to characterize the secretory profile of the distal colon. Forty-two female wild-type and 42 Tlr4-/- BALB/c mice weighing between 18 and 25 g received a single 270 mg/kg [intraperitoneal (i.p.)] dose of irinotecan hydrochloride or vehicle control and were killed at 6, 24, 48, 72, and 96 hours. The secretory profile of the distal colon, following carbachol and forksolin, was assessed using Ussing chambers at all time points. Tight junction integrity was assessed at 24 hours, when peak intestinal permeability and diarrhea were reported, using immunofluorescence, Western blotting, and RT-PCR. Irinotecan caused internalization of claudin-1 with focal lesions of ZO-1 and occludin proteolysis in the ileum and colon of wildtype mice. Tlr4-/- mice maintained phenotypically normal tight junctions. Baseline conductance, a measure of paracellular permeability, was increased in irinotecan-treated wild-type mice at 24 hours (53.19 + 6.46 S/cm2; P = 0.0008). No change was seen in Tlr4-/- mice. Increased carbachol-induced chloride secretion was seen in irinotecan-treated wild-type and Tlr4-/- mice at 24 hours (wild-type: 100.35 + 18.37 mA/cm2; P = 0.022; Tlr4-/-: 102.72 + 18.80 mA/cm2; P = 0.023). Results suggest that TLR4-dependent claudin-1 internalization and secondary anion secretion contribute to irinotecan-induced diarrhea.