MODE‐K Cells as a Model to Study the Gut Epithelial Response: An Outside‐in and Inside‐out Approach

The gut epithelium serves as a barrier between the intestinal lumen and immune cells residing in gut‐associated lymphoid tissues. These epithelial cells are required to respond to stimuli from the outside‐in (e.g., microbiome and dietary components), as well as from the inside‐out (e.g., cytokines and growth mediators). This study evaluated the utility of a gut epithelial cell model and its response to inside‐out (tumor necrosis factor; TNF‐α) or outside‐in ( E. coli lipopolysaccharide; LPS) immune challenges. Experiments were designed to evaluate alterations in cell viability, proliferation, and gene expression using the MODE‐K cell line which was derived from the small intestine of C3H/HeJ mice. For all experiments, MODE‐K cells were grown at 37°C with 5% CO 2 /95% humidified air in RPMI GlutMAX media supplemented with 10% FBS, 1% HEPES, 1% NEAA, 1% sodium pyruvate, and 0.1% β‐mercaptoethanol. Cell viability and proliferation were assessed using trypan blue exclusion and MTT assays at 12, 24, and 48 hours. Doses of TNF‐α and LPS were selected based on MTT results and cell viability (≥90%). MODE‐K cells were cultured in 12‐well plates and treated with TNF‐α (0, 0.1 or 1.0 ng/mL) or LPS (0, 0.1, 1.0, or 10 ug/mL). Cells were harvested after 2 or 6 hours and total RNA was extracted, followed by qRT‐PCR using Cyclo as the invariant control. Both doses TNF‐α up‐regulated ( p<0.05 ) the relative abundance of interleukin‐6 ( Il6), a pro‐inflammatory cytokine, at 2 hrs, whereas only the higher dose of TNF‐α up‐regulated Il6 at 6 hrs. The relative abundance of Tgfb , a cytokine involved in T‐cell differentiation, was elevated with the 1.0 TNF‐α dose at 2 hrs, but not by 6 hrs. The gene expression of the anti‐inflammatory cytokines, interleukin‐10 ( Il10 ) and ‐4 ( Il4 ) , was not detectable at either time point. Expression of toll‐like receptor 4 ( Tlr4 ), a pattern recognition receptor, was suppressed ( p< 0.05) at 2 hrs with all doses of TNF‐α and LPS. This response was normalized by 6 hrs, with the exception of the 1.0 TNF‐α treatment. All doses of LPS, representing the outside‐in stimulus, up‐regulated Il6 at both time points. Likewise, all doses of LPS up‐regulated Tnfa ( p <0.05) at 2 hrs, while only the two highest doses increased ( p <0.05) Tgfb. No alterations in these responses were observed after 6 hrs. These findings demonstrate that the MODE‐K cells respond to the pro‐inflammatory cytokine, TNF‐α, from the gut mucosa, as well as endotoxins present in the intestinal lumen (i.e., LPS), demonstrating the importance of both the mucosal immune system and the microbiome in initiating epithelial cell signaling. This cell culture model can potentially serve as a system to study the gut epithelial cells' in‐side out and outside‐in responses and to understand the modulating effects of dietary bioactive components. Support or Funding Information Oklahoma Center for the Advancement of Science and Technology (HR14‐126)