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Introduction: A mutation in the NOD2 gene has been linked to terminal ileal Crohn’s Disease (CD). CD is characterized by repeated mucosal injury and healing. No work has been done on the effect of the NOD2 mutation on wound healing. We have stably transfected the IEC-18 cell line (ileal cells) with wildtype and the c-insertion mutation of the NOD2 gene. We have previously characterized these cells and have found marked disruption of the tight junction proteins in the cells carrying the NOD2 mutation. The hypothesis for this study is that there will be altered wound healing in the cells with the NOD2 mutation.Methods: IEC-18 cells were stably transfected with wild-type human NOD2 (NOD2 cell line) or the c-insertion mutation (3020InsC cell line). Western blot and PCR was used to identify NOD2. Cells were then grown in 6 well plates for 72 hours until confluent. The monolayer was then wounded with a plastic pipette tip in an X formation. 24 or 48 hours later the cells were stained Hema 3 stain kit and viewed with a phase contrast microscope. Duplicate, unwounded, monolayers were used to measure cell proliferation using a coulter counter, n=8.Results: Human NOD2 protein was detected by Western blot and PCR in the NOD2 and 3020InsC cell lines, but not the parental cell line. There was a significant increase in proliferation in the NOD2 cell line compared to the parental cell line (p<0.05). There was no significant change in proliferation in the 3020InsC cell line compared to parental and NOD2 cell lines. The figure shows monolayer wound healing for the three cell lines at 24 hours. The wounds in the NOD2 cell line healed the quickest. They were nearly closed at 24 hours and undectable at 48 hours. The parental cell line had intermediate healing. There was minimal to no wound healing in the 3020InsC cell line at 24 hours or 48 hours. In addition, the 3020InsC cells seemed to bunch up in a “ridge” at the edge of the wound suggesting migration into the wound was impaired. This “ridge” was not seen in the NOD2 or parental cell lines.Conclusions: 1. Wound healing was markedly retarded in the 3020InsC cell line compared to the parental cell line while the NOD2 cell line healed more quickly than the parental cell line. 2. The increase in proliferation seen in the NOD2 cell line may contribute to the improved wound healing in this cell line. However, there was no change in proliferation in the 3020InsC cell line to explain the lack of wound healing in the 3020InsC cell line. 3. The disruption in the tight junction complex we have previously described in the 3020InsC cell line may contribute to the delay in wound healing. 4. Lack of wound healing may have implications in the mucosal injury and repair seen in CD.


Left: IEC-18 parental, Middle: NOD2 Wildtype, Right: 3020InsC mutant