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

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, TLR4 is critical in tight junction regulation. The current study aimed to (1) determine the molecular characteristics of intestinal tight junctions in wild-type and Tlr4-/- BALB/c mice, and (2) characterize the secretory profile of the distal colon. Forty-two female wild-type and 42 Tlr4-/- BALB/c mice weighing between 18-25 g received a single 270 mg/kg (i.p.) dose of irinotecan hydrochloride or vehicle control and were killed at 6, 24, 48, 72 and 96 h. 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 h, 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 wild-type 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 h (53.19{plus minus}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 h (wild-type 100.35{plus minus}18.37 μA/cm2; p=0.022; Tlr4-/- 102.72{plus minus}18.80 μA/cm2; p=0.023). Results suggest TLR4-dependent claudin-1 internalization and secondary anion secretion contribute to irinotecan-induced diarrhea.