The effectiveness of premarriage education is limited by whether couples at high risk of future marital problems attend such education. In the current study, 374 newly married couples were assessed on a range of risk factors for future marital problems as well as whether they had attended marriage education. Couples with certain indices of relationship risk (nonreligious and premarital cohabitation) were underrepresented in premarriage education. Suggestions are offered to attract more couples, particularly those at high risk for future problems, to relationship education.

Background:Despite the importance of inflammation in cancer, the role of the cytokine IL-33, and its receptor ST2, in colon cancer is unclear. The aim of this study was to investigate the role of IL-33, and its receptor isoforms (ST2 and ST2L), in colon cancer.Methods:Serum levels of IL-33 and sST2 were determined with ELISA. ST2 and IL-33 expression was detected with quantitative real-time PCR (qRT–PCR), western blotting and immunohistochemistry. ST2 expression in CT26 cells was stably suppressed using ST2-specific shRNA. Cytokine and chemokine gene expression was detected with qRT–PCR.Results:Human colon tumours showed lower expression of ST2L as compared with adjacent non-tumour tissue (P<0.01). Moreover, the higher the tumour grade, the lower the expression of ST2L (P=0.026). Colon cancer cells expressed ST2 and IL-33 in vitro. Functional analyses showed that stimulation of tumour cells with IL-33 induced the expression of chemokine (C–C motif) ligand 2 (CCL2). Knockdown of ST2 in murine colon cancer cells resulted in enhanced tumour growth (P<0.05) in BALB/c mice in vivo. This was associated with a decrease in macrophage infiltration, with IL-33-induced macrophage recruitment reduced by antagonising CCL2 in vitro.Conclusion:The IL-33/ST2 signalling axis may have a protective role in colon carcinogenesis.

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TLRs play an important role in mediating intestinal inflammation and homeostasis. Fas is best studied in terms of its function in apoptosis, but recent studies demonstrate that Fas signaling may mediate additional functions such as inflammation. The role of Fas, and the Fas ligand (FasL), in the intestine is poorly understood. The aim of this study was to evaluate potential cross-talk between TLRs and Fas/FasL system in intestinal epithelial cells (IECs). IECs were stimulated with TLR ligands, and expression of Fas and FasL was investigated. Treatment with TLR4 and TLR5 ligands, but not TLR2 and 9 ligands, increased expression of Fas and FasL in IECs in vitro. Consistent with this finding, expression of intestinal Fas and FasL was reduced in vivo in the epithelium of TLR4 knockout (KO), 5KO, and germ-free mice, but not in TLR2KO mice. Modulating Fas signaling using agonistic anti-Fas augmented TLR4- and TLR5-mediated TNF-α and IL-8 production by IECs. In addition, suppression of Fas in IECs reduced the ability of TLR4 and TLR5 ligands and the intestinal pathogens Salmonella typhimurium and Listeria monocytogenes to induce the expression of IL-8. In conclusion, this study demonstrates that extensive cross-talk in IECs occurs between the Fas and TLR signaling pathways, with the FasL/Fas system playing a role in TLR-mediated inflammatory responses in the intestine.

The IL-36 cytokines are a recently described subset of the IL-1 family of cytokines, shown to play a role in the pathogenesis of intestinal diseases such as Inflammatory Bowel Disease (IBD). Given the link between IBD and colitis –associated cancer, as well as the involvement of other IL-1 family members in intestinal tumorigenesis, the aim of this work was to investigate whether IL-36 cytokines play a role in the pathogenesis of colon cancer. Whilst research to date has focused on the role of IL-36 family members in augmenting the immune response to induce tumour rejection, very little remains known about IL-36R signalling in tumour cells in this context. In this study we demonstrate that expression of IL-36 family member mRNA and protein are significantly increased in colorectal cancer tissue compared to adjacent non-tumour. In vitro assays showed stimulation of colon cancer cell lines with IL-36R agonists resulted in the activation of the pro-tumorigenic phenotypes of increased cellular migration, invasion and proliferation in both 2D and 3D models. In addition, the IL-36 cytokines induced strong expression of pro-inflammatory chemokines in both human and murine cell lines. Intraperitoneal injection of IL-36Ra significantly reduced tumour burden using the subcutaneous CT26 tumour model in syngeneic Balb/mice, and this was associated with a decrease in Ki-67 expression by tumour cells in the IL-36Ra- treated group relative to untreated, suggesting the inhibition of the pro-proliferative signalling of IL-36 agonists resulted in the decreased tumour size. Moreover, colon cancer cells lacking the IL-36R also showed reduced tumour growth and reduced Ki-67 expression in vivo. Taken together, this data suggests that targeting IL-36R signalling may be a useful targeted therapy for colorectal cancer patients with IL-36R+ tumour cells.

Background & Aims: The IL-1 subfamily, IL-36, is increasingly being implicated as a potent cytokine family involved in chronic inflammatory conditions such as psoriasis and IBD. Recent work has also suggested IL-36 cytokines may act as a potential adjuvant in immunotherapy regimens to augment the anti-cancer immune response. Given the pluripotent nature of IL-1 cytokines and the relationship between inflammation and tumorigenesis, we investigated the effects of IL-36 signalling in colorectal cancer (CRC). Methods: IL-36 family expression in CRC progression was investigated by the NCBI GEO transcriptomic database, and then by immunohistochemistry (IHC) and qRT-PCR. Pro-tumorigenic properties such as cancer cell migration, invasion and proliferation were investigated in 2D and 3D models. In vivo models were performed using Balb-c mice which were subcutaneously inoculated with CT26 cells and treated with IP injections of recombinant IL-36R agonists (IL-36α, β, γ) or antagonist (IL-36RN). Tumour tissues were excised and studied by flow cytometry and IHC analysis. Additionally, CRISPR-Cas9 mediated IL-36R KO CT26 cells were generated and subcutaneously injected into Balb-c mice, with changes in tumour growth and immune cell infiltrate investigated. Results: IL-36R expression was shown to significantly increase with stage of disease in the adenoma-carcinoma sequence, including distant metastases. Additionally, expression of all family members was increased in tumour tissue relative to adjacent normal tissue in a separate colon cancer cohort. In vitro IL-36R signalling on cancer cells augmented a pro-tumorigenic phenotype by induction of pro-tumorigenic cytokines/chemokine production, increased cellular migration, invasion and proliferation of tumour cells. In vivo inhibition of IL-36R signalling by both administration of recombinant IL-36RN and through deletion of the IL-36R gene in CT26 cells, resulted in a significant reduction in tumour growth. This reduction was associated with a significant decrease in tumour cell proliferation, as well as an increase in CD8+ T cell infiltrate in IL-36R KO groups. Of note, administration of IL-36R agonists also resulted in reduced tumour growth, albeit to a lesser extent, associated with an increase in CD4+ and CD8+ T cell infiltration. Conclusions: This data indicates that IL-36 family members, similar to other IL-1 family members, have dual functions in colon cancer. In addition, this data suggests that targeting IL-36R signalling may be a useful targeted therapy for CRC patients with IL-36R+ tumour cells. Citation Format: Kevin J. Baker, Charlotte O'Donnell, Micheal O'Riordain, Elizabeth Brint, Maura Bendix, Aileen Houston. IL-36 signalling enhances a pro-tumorigenic phenotype in colon cancer cells with cancer cell growth restricted by administration of the IL-36R antagonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1324.

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