Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors.

Environmental factors contribute to the initiation, progression, and maintenance of type 1 diabetes (T1D), although a single environmental trigger for disease has not been identified. Studies have documented the contribution of immunity within the gastrointestinal tract (GI) to the expression of autoimmunity at distal sites. Intestinal epithelial cells (IECs) regulate local and systemic immunologic homeostasis through physical and biochemical interactions with innate and adaptive immune populations. We hypothesize that a loss in the tolerance-inducing nature of the GI tract occurs within T1D and is due to altered IECs’ innate immune function. As a first step in addressing this hypothesis, we contrasted the global immune microenvironment within the GI tract of individuals with T1D as well as evaluated the IEC-specific effects on adaptive immune cell phenotypes. The soluble and cellular immune microenvironment within the duodenum, the soluble mediator profile of primary IECs derived from the same duodenal tissues, and the effect of the primary IECs’ soluble mediator profile on T-cell expansion and polarization were evaluated. Higher levels of IL-17C and beta-defensin 2 (BD-2) mRNA in the T1D-duodenum were observed. Higher frequencies of type 1 innate lymphoid cells (ILC1) and CD8+CXCR3+ T-cells (Tc1) were also observed in T1D-duodenal tissues, concomitant with lower frequencies of type 3 ILC (ILC3) and CD8+CCR6+ T-cells (Tc17). Higher levels of proinflammatory mediators (IL-17C and BD-2) in the absence of similar changes in mediators associated with homeostasis (interleukin 10 and thymic stromal lymphopoietin) were also observed in T1D-derived primary IEC cultures. T1D-derived IEC culture supernatants induced more robust CD8+ T-cell proliferation along with enhanced polarization of Tc1 populations, at the expense of Tc17 polarization, as well as the expansion of CXCR3+CCR6+/− Tregs, indicative of a Th1-like and less regulatory phenotype. These data demonstrate a proinflammatory microenvironment of the T1D-duodenum, whereby IECs have the potential to contribute to the expansion and polarization of innate and adaptive immune cells. Although these data do not discern whether these observations are not simply a consequence of T1D, the data indicate that the T1D-GI tract has the capacity to foster a permissive environment under which autoreactive T-cells could be expanded and polarized.

The host-environment dialogue at the intestinal interface has been strongly implicated in the development of inflammatory diseases. We are interested in understanding the role of this dialogue in autoinflammatory and autoimmune diseases such as type 1 diabetes (T1D). Although the gastrointestinal tract, a dynamic and highly regulated immunologic organ, has been demonstrated to be impaired in T1D, the mechanisms of any (mis)communications taking place remain unknown. Here we describe the global intestinal inflammatory environment and the phenotype of intestinal lymphocytes in human T1D. In addition, we evaluated the intestinal epithelial cell (IEC) innate immune responses using primary cell culture. Our data indicate a whole organ reduction in tolerogenic mediators concomitant with elevated IEC-induced pro-inflammatory mediators. Significant alterations in intestinal leukocyte populations were observed, whereby increased frequencies of pathogenic T cells and alterations in tolerogenic DCs were noted. Finally, T1D-derived primary IEC cultures demonstrated a lack of tolerogenic responses concomitant with induction of inflammatory response to microbial ligand stimulation as compared to non-T1D derived cultures. Together these data suggest a loss of gastrointestinal homeostasis in T1D potentially as a result of a dysfunctional IEC-mediated host-environment dialogue, though whether this is a cause of consequence of disease remains to be defined.