Irinotecan-Induced gastrointestinal dysfunction and pain are mediated by common TLR4-dependent mechanisms

Strong epidemiological data indicate that chemotherapyinduced gut toxicity and pain occur in parallel, indicating common underlying mechanisms. We have recently outlined evidence suggesting that TLR4 signaling may contribute to both side effects. We therefore aimed to determine if genetic deletion of TLR4 improves chemotherapy-induced gut toxicity and pain. Forty-two female wild-type (WT) and 42 Tlr4 null (-/-) BALB/c mice weighing between 18 and 25 g (10-13 weeks) 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 hours. Bacterial sequencing was conducted on cecal samples of control animals to determine the gut microbiome profile. Gut toxicity was assessed using validated clinical and histopathologic markers, permeability assays, and inflammatory markers. Chemotherapy-induced pain was assessed using the validated rodent facial grimace criteria, as well as immunologic markers of glial activation in the lumbar spinal cord. TLR4 deletion attenuated irinotecan-induced gut toxicity, with improvements in weight loss (P= 0.0003) and diarrhea (P < 0.0001). Crypt apoptosis was significantly decreased in BALB/c-Tlr4 -/-^billy mice (P < 0.0001), correlating with lower mucosal injury scores (P < 0.005). Intestinal permeability to FITCdextran (4 kDa) and LPS translocation was greater in WT mice than in BALB/c-Tlr4 -/-^billy (P= 0.01 and P < 0.0001, respectively). GFAP staining in the lumbar spinal cord, indicative of astrocytic activation, was increased at 6 and 72 hours in WT mice compared with BALB/c-Tlr4 -/-^billy mice (P= 0.008, P= 0.01). These data indicate that TLR4 is uniquely positioned to mediate irinotecan-induced gut toxicity and pain, highlighting the possibility of a targetable gut/CNS axis for improved toxicity outcomes.