TRIF-dependent pathway of TLR4 signaling is activated in endosomes upon internalization of TLR4-ligand complexes1–4. Accordingly, in the present study, γ-PGA delivered orally seemed to be internalized into IECs or transverses the tight junctions between enterocytes to be internalized into the sentinel cells in the lamina propria to induce IFN-β production. Thus, we further investigated as to how γ-PGA reached a site where functional interaction with TLR4 occurred in the ileum. The first barrier for the entry of γ-PGA was the mucus layer, where access of γ-PGA to the small intestine is more likely to occur as it is covered by a thinner mucus layer compared to the colon51. For induction of IFN production, γ-PGA needs to be sensed by TLR4 in and/or on villi. Immunostaining revealed lack of overlapping between TLR4 staining and villin, a cytoskeletal protein specific to villi in the ileum, indicating a barely detectable level of TLR4 at the apical site of IECs. We therefore hypothesized a model in which γ-PGA transverses the tight junctions between enterocytes to be sensed by sentinel cells such as dendritic cells within villi or is directly uptaken by transepithelial dendrites (Supplementary Fig. 5). Expression of IFN-β mRNA was significantly enhanced solely in the villi with lack of noticeable IFN-β induction in PPs. Absence of response to γ-PGA in PPs resulting in lack of inflammation was consistent with the previous findings suggesting anergy of intestinal macrophages. Anergy was mediated by their inability to activate NF-κB, and low-level expression of TLR4 and its accessory factors CD14 and MD252.