TNFR and LTβR agonists induce follicle-associated epithelium and M cell specific genes in rat and human intestinal epithelial cells

Abstract

M cells assist mucosal immune surveillance by transcytosis of particles to underlying lymphoid tissue, but the mechanisms of M cell differentiation are poorly understood. To develop a better defined cell culture model of M cell differentiation, we treated human (Caco-2BBe) and rat (IEC-6) intestinal epithelial cell lines with lymphotoxin beta receptor (LTβR) and TNF receptor (TNFR) agonists. Treated cells were studied for regulation of genes associated with M cell and FollicleAssociated Epithelium (FAE). We found that LTβR and TNFR agonists induce transcription of FAE specific genes (Ccl20 and Lamb3) in Caco2-BBe cells and IEC-6 cells as well as rodent M cell specific genes such as Sgne-1/Scg5, Cldn4, and Gp2. The cytokines have distinct but complementary effects; TNFR agonists mainly induced FAE specific genes, while the LTβR agonist induced M cell specific genes. The combination of cytokines showed additive induction of the FAE-associated Ccl20, Lamb3 and a surprising induction of CD137/Tnfrsf9. On the other hand TNF agonists appeared to suppress expression of some LTβR-induced genes. Functionally, cytokine treatment led to the reorganization of microvilli and Claudin-4 redistribution. These studies suggest complex interactions between these cytokines in the context of either inflammation or tissue differentiation.

Reduced Regulatory T Cell Diversity in NOD Mice Is Linked to Early Events in the Thymus

“…A function for 4-1BB at earlier thymic differentiation stages has not been reported; however, some studies have shown that 4-1BB can regulate TNF-a expression (46,47) and vice versa (48), suggesting a possible link between the two. Overexpression of TNF-a under control of its natural promoter has been reported to block early thymocyte development at the DN1 to DN2 stage, promoting accelerated thymic atrophy (49) and mirroring our own observations in the NOD thymus.…”

Epithelial Microvilli Establish an Electrostatic Barrier to Microbial Adhesion

“…Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene for all quantitative PCRs (qPCRs). Primers used in qPCR assays were described previously (16). The comparative ⌬⌬C T method was used to determine the relative amount of gene expression.…”

“…Thus, in a broader context, inflammatory processes may also disrupt microvillus formation and the maintenance of the electrostatic barrier even in the absence of effacement effectors and TTSSs. For example, inflammatory cytokines such as lymphotoxin and tumor necrosis factor alpha (TNF-␣) were found to reduce microvillus formation in Caco2BBe cells in vitro (16); since these cytokines are known effectors in inflammatory bowel disease (39,40), a similar effect on microvilli in vivo may be relevant to mucosal barrier function.…”

“…S1A in the supplemental material). Certain genes (CD137, GP2, and ␤1 integrin) have been identified as being important for M cells in the binding and capture of pathogenic microbes ("capture receptor hypothesis") (16,(26)(27)(28); however, MVM cells showed no induction of these genes compared to a cytokine-treated cell model (known to induce these genes) (see Fig. S1B in the supplemental material).…”

Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

“…And HIEC may have a number of different applications (36). For example, they can be used to evaluate the effects of growth or maturation factors on the expression of membrane receptors and antigens, enzymatic activities, transport and secretory functions (37), to study drug metabolism, and to investigate carcinogenic factors and agonists (38).…”