In conclusion, our results suggest the existence of an immune-triggering mechanism mediated by S100A9 released by CECs that helps to scavenge invading microorganisms when epithelial barriers are impaired. This mechanism may also induce further most undesirable destruction in epithelial linings, leading to the exacerbation of colitis symptoms. Further studies should investigate how this mechanism can be controlled without interfering with immune surveillance in CECs. Our findings suggest that drugs inhibiting STAT3 signals may be good candidates for controlling disease activities in patients with UC while avoiding CAC development. Materials and Methods Cell Culture A Caco-2 human IEC line was obtained from the Korean Cell Line Bank (Seoul, Republic of Korea).

Human embryonic kidney cell line 293T and human colon cancer cell line HCT116 were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in appropriate media with 10% heat-inactivated fetal bovine serum (Lonza, Walkersville, MD, USA). Reagents and Inhibitors DSS (molecular weight, 35,000�C50,000 Da; United States Biochemical, Cleveland, OH, USA) was used to induce experimental colitis in mice. IL-6 (PeproTech, Rocky Hill, NJ, USA) was used to stimulate Caco-2 cells. The following inhibitors were used to treat Caco-2 cells throughout IL-6 stimulation: S3I and STAT3 inhibitor peptide (Merck) to block phosphorylation by targeting a tyrosine residue and to dimerize STAT3, respectively [55], [56].

To prepare small interfering (si) RNA-incorporated chitosan nanoparticles (CH-NPs), siRNAs for STAT3 and S100A9 were purchased from Bioneer Corporation (Daejeon, Republic of Korea). siRNA-incorporated CH-NPs were prepared according to a previously described method [44] by ionic gelation of anionic tripolyphosphate (TPP; Sigma Chemical, St. Louis, MO, USA) and Batimastat siRNA with cationic chitosan (Sigma Chemical). The weight ratio of chitosan to TPP was set to 31 to generate particles about 200nm in size. The prepared siRNA/CH-NPs (si-STAT3/CH-NPs and si-S100A9/CH-NPs) were injected intravenously twice into mice with DSS-induced colitis. Establishment of an Experimental DSS-Induced Colitis Mouse Model Six-week-old male C57BL/6 mice were purchased from Joongang Laboratory Animal Co. (Seoul, Republic of Korea) and maintained under specific pathogen-free conditions. As described previously [57], colitis was induced with water containing 3% DSS. The disease activity index (DAI) represents the combined scores of weight loss, stool consistency, and bleeding, according to scoring criteria described previously [58], [59].