A55 INVESTIGATING THE ROLE OF ENDOGENOUS INTERLEUKIN-10 IN INTESTINAL EPITHELIAL CELLS AND HOMEOSTASIS

Background IL-10 is appreciated for its potent anti-inflammatory effects on leukocytes in mucosal immunity. However, far less attention has been paid to the impact of IL-10 on epithelial cells, which make up the crucial barrier interface between the host mucosa and the external environment. Furthermore, most studies examine the effects of exogenous IL-10, disregarding the possible presence and function of autocrine or paracrine IL-10 in the epithelium. Aims Using ex vivo organoids we aimed to examine the small intestinal epithelium for IL-10 and dissect any role for endogenously produced cytokine. Methods We growed small intestinal organoids (enteroids) from crypts isolated from C57BL/6 mice (WT) and IL-10-gene knockout mice (IL-10KO). Cellular markers were characterized through qpCR, while IL-10 and IL-10 receptor localization was characterized though immunofluorescence. Results We discovered that cells in WT enteroids expressed IL-10 and IL-10R1 constitutively throughout development. Immunofluorescent staining revealed that IL-10 localizes to Paneth cells and appears to be secreted apically. Having established that IL-10 is secreted in enteroids, we compared enteroids from IL-10KO versus WT mice. IL-10KO enteroids developed to morphologically resemble WT enteroids; however, we detected an imbalance with lower secretory cell markers over absorptive cell types in the IL-10KO enteroids, measured as less mRNA for lysozyme, cryptdins and mucin-2. Addition of IL-10 to IL-10KO enteroids did not correct these defects, but did ameliorate the lineage balance by reducing absorptive cell lineage markers (sucrose isomaltase). IL-10R1 was localized on both apical and basolateral side of cell in enteroids. We suspect that epithelial-derived IL-10 likely acts on apical IL-10R, which may conduct a different response from basolateral receptor stimulation. Conclusions In conclusion, IL-10 is present in the small intestinal epithelium; more remains to be determined regarding the role this cytokine plays in gut development and homeostasis. Funding Agencies NSERC

Beta 2-microglobulin co-distributes with the heavy chain of the intestinal IgG-Fc receptor throughout the transepithelial transport pathway of the neonatal rat

Maternal IgG crosses the proximal small intestine of the suckling rat by receptor-mediated endocytosis and transepithelial transport. The Fc receptor resembles the major histocompatibility complex class I antigens in that it consists of two subunits: a transmembrane glycoprotein (gp50) in association with beta 2-microglobulin. We used immunofluorescence microscopy and quantitative immunogold cytochemistry to study the subcellular distribution of the two subunits. In mature absorptive cells both subunits were colocalized in each of the membrane compartments that mediate transcytosis of IgG. IgG administered in situ apparently caused both subunits to concentrate within endocytic pits of the apical plasma membrane, suggesting that ligand causes redistribution of receptors at this site. These results support a model for transport in which IgG is transferred across the cell as a complex with both subunits. During absorptive cell differentiation, gp50 and beta 2-microglobulin showed nearly identical patterns of increased expression that accompanied the development of the apical endocytic apparatus and terminal web. However, absorptive cells in weanling rats expressed no detectable gp50 and only low levels of beta 2-microglobulin in the Golgi region and on the basolateral plasma membrane where class I antigens would likely reside. Thus, beta 2-microglobulin has a novel distribution unrelated to its function as a subunit of the class I antigens. The co-expression of the two receptor subunits is restricted to neonatal epithelial cells engaged in IgG transport and is coordinately regulated during absorptive cell differentiation and during postnatal intestinal development.