P-321 Regulation of Innate Immunity in Intestinal Epithelial Cells

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Abstract

Background:

The goal of this study is to determine how intestinal epithelial cells (IECs) control innate immune functions of toll-like-receptor 5 (TLR5), an important regulator of inflammatory bowel diseases (IBD). Dysregulation of innate immune receptors is thought to be an important driver of inflammation in IBD. Of note, the most common antigenic response in patients with Crohn's disease is directed at bacterial flagellin, the ligand for the innate receptor, TLR5. TLR5 is expressed on IECs and is thought to signal exclusively from the basolateral surface. Based on this, localization of TLR5 to the basolateral membrane is considered the primary mechanism controlling where and when this receptor can sense flagellins. However, several groups have challenged this idea and have presented data showing TLR5 on the apical surface of IECs and even within endosomes. TLR5 was suggested to induce signal transduction from these locations. Thus, the precise subcellular sites of TLR5 signaling in IECs and what mechanisms regulate TLR5 signaling in general, are issues that remain unresolved. Here we explore TLR5 signaling from various subcellular locations and examine the signal transduction pathways responsible for initiating responses to flagellin in IECs. We hypothesize that TLR5 signaling occurs from multiple subcellular locations where specific responses are elicited in a location-dependent manner.

Methods:

To assess location specific responses in IECs, primary mouse IECs or immortal human IECs were stimulated with flagellin from the apical or basolateral membrane and assessed for gene expression patterns using qPCR. Cells deficient in adaptor proteins, MyD88 or TRIF were used to assess involvement of specific innate pathways. To examine TLR5-dependent gene expression from endosomes, cells were treated with dynamin inhibitors to inhibit receptor endocytosis prior to stimulation with flagellin. Localization of GFP-labeled TLR adaptors, which may define the sites of TLR5 signaling, were assessed using fluorescence microscopy.

Results:

Examination of gene expression in response to flagellin revealed that type-I interferon expression was initiated from the apical domain of IECs. Furthermore, these responses were induced from the endosomal compartment and required TRIF and TLR5 for activation. Next, we monitored localization of TLR-family adaptor proteins in IECs. MyD88-dependent pathway adaptor protein TIRAP localized to the basolateral membrane of epithelial cells, consistent with previous studies showing that most MyD88-dependent gene expression occurs from this membrane.

Conclusions:

We find classes of inflammatory genes are differentially upregulated from the apical or basolateral sides of IECs, including interferon family members. Thus, TLR5 signaling may be an unappreciated source of interferon, a regulator of epithelial barrier function, in the intestine. Furthermore, TLR5-induced gene expression from various locations within the cell is likely directed by location of intracellular TLR adaptors. Collectively, TLR5 signaling from various subcellular compartments may function to support immune homeostasis in response to normal flora in addition to its role in promoting inflammation during infections.

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