Paneth cell granule dynamics on secretory responses to bacterial stimuli in enteroids

Abstract

Paneth cells at the base of small intestinal crypts secrete granules containing α-defensins in response to bacteria and maintain the intestinal environment by clearing enteric pathogens and regulating the composition of the intestinal microbiota. However, Paneth cell secretory responses remain debatable and the mechanisms that regulate the secretion are not well understood. Although enteroids, three-dimensional cultures of small intestinal epithelial cells, have proven useful for analyzing intestinal epithelial cell functions including ion transport, their closed structures have imposed limitations to investigating interactions between Paneth cells and the intestinal microbiota. Here, we report that microinjection of bacteria or lipopolysaccharide (LPS) into the enteroid lumen provides an ex vivo system for studying Paneth cell secretion in real-time. The results show that Paneth cells released granules immediately when the apical surfaces of enteroid epithelial cells were exposed to LPS or live bacteria by microinjection. However, Paneth cells did not respond to LPS delivered in culture media to enteroid exterior basolateral surface, although they responded to basolateral carbamyl choline. In addition, Paneth cells replenished their granules after secretion, enabling responses to second stimulation. These findings provide new insight for apically-induced Paneth cell secretory responses in regulating the intestinal environment.

Establishing Boundaries: The Relationship That Exists between Intestinal Epithelial Cells and Gut-Dwelling Bacteria

“…A plethora of microbes inhabiting the gut luminal area interact with Paneth cells, ensuring sufficient protection from otherwise transient microorganisms. Apical stimulation of PRRs such as TLRs primed by LPS has been described by the immediate degranulation and secretion of AMPs by Paneth cells, indicating that Paneth cells directly sense LPS [25]. Gong et al describe how the deletion of the signaling adaptor protein myeloid differentiation primary-response gene 88 (MyD88) results in decreased production of RegIIIγ, RELMβ, and RegIIIβ by intestinal epithelium, increasing susceptibility to Salmonella enterica serovar Typhimurium [26].…”

Mycotoxin Deoxynivalenol Has Different Impacts on Intestinal Barrier and Stem Cells by Its Route of Exposure

“…Using enteroids as an in vitro model to test the effects of luminal toxin exposure is difficult because of their closed structures. Thus, we used microinjection technique to deliver substances to the enteroid lumen as previously described [ 20 ] (see Supplementary Materials and Methods: 1 ). Between before and after microinjection ( Figure 1 B, Video S1 ), the microinjection solution concentration was diluted by the enteroid lumen volume.…”

“…Thus, we first addressed if luminal or basolateral DON exposure affects intestinal barrier functions using our enteroid model, which mimics most features of the native intestine. Recently, Yokoi et al [ 20 ] devised a novel microinjection method that quantitatively luminally delivers substances to enteroids. This method allowed us to compare between the effects of luminal and basolateral DON treatment at equal concentrations ( Figure 1 ).…”

“…In contrast, luminal DON exposure was conducted through DON microinjection (28.3 µM in DPBS) into enteroids. Enteroid lumen microinjection and volume measurement of enteroid lumen and microinjection were done as previously described [ 20 ]. The final concentration was equivalent to 1 µM in microinjected enteroids ( Table S1 ).…”

An Update Review on the Paneth Cell as Key to Ileal Crohn's Disease

“…TLR (toll like receptor) signals are transferred through MyD88, limiting microbial adherence and invasion through Paneth cell direct sensing and antibacterial secretions (21). Interestingly, as shown in organoids only the apical and not the basolateral surface of Paneth cells was responsive to lipopolysaccharide or live bacteria (22). However, even simple molecules like butyric acid or leucine may induce Paneth cell α-defensin secretion (23).…”