P. M. Smith1, A. Means1, S. Novitskiy2, R. D. Beauchamp1 1Vanderbilt University Medical Center,Department Of Surgery,Nashville, TN, USA 2Vanderbilt University Medical Center,Department Of Medicine,Nashville, TN, USA
Introduction: Evidence suggests that colorectal carcinogenesis is promoted by a combination of microbiota- and host-dependent mechanisms that trigger epithelial cell inflammatory signaling. This is particularly true in ulcerative colitis (UC)-associated cancer (UCAC) in which longstanding UC predisposes to colorectal cancer. Our lab has discovered that TGFβ signaling via SMAD4 has a central inhibitory role in colon epithelial innate inflammatory signaling. Furthermore, by three months after DSS-induced colitis, 100% of mice with adult-onset deletion of the Smad4 gene in intestinal epithelium developed invasive carcinomas of the colon compared to 0% of control mice. Based on these preliminary observations, we hypothesize that SMAD4 functions as an immune-modulator in colonic epithelial cells through inhibition of pro-inflammatory cytokine production, and loss of SMAD4 results in altered immune cell infiltration.
Methods: The Smad4 gene was conditionally and selectively knocked out (KO) of intestinal epithelium in adult mice using transgenic mice expressing intestinal epithelial cell selective and inducible Cre-recombinase (Cre) and crossed with mice with LoxP sites inserted into the Smad4 gene locus. Activation of Cre results in excision of the Smad4 gene selectively in the intestinal epithelial cells while retaining it elsewhere, including in leukocytes. The submucosal colonic stroma was isolated from KO and control mice and processed for either RNA-sequencing or for immunophenotyping by flow cytometry. KO and control mouse colon was also dissected and examined histologically by H&E staining for quantity and size of Gut-Associated Lymphoid Tissue aggregates (GALTs).
Results: Compared to control mice, KO mice exhibited a striking increase in stromal gene expression consistent with inflammatory cell signaling with increased expression of multiple pro-inflammatory genes including those related to T cell activation/differentiation, B cell development/signaling, myeloid cell differentiation/proliferation/chemotaxis, and communication between innate and adaptive immune cells (all p<0.0001). Correspondingly, KO of Smad4 in the colon epithelium resulted in a significant and robust increase in leukocyte (CD45+ cell) infiltration into the sub-epithelial compartment (34.0 vs 9.8%, p=0.03). GALT size and number did not vary significantly between KO and control mice (p=0.69 and p=0.91, respectively).
Conclusion: Epithelial SMAD4 modulates the immune system through inhibition of pro-inflammatory cytokines, and adult-onset loss of SMAD4 in intestinal epithelium is associated with increased immune cell infiltration. These results strongly suggest that the SMAD4 tumor suppressor role is associated with its inflammation inhibitory function.
