We thus sought to address the question of whether γ-PGA can trigger TLR4 signaling-mediated IFN production in the ileum (composed of epithelial cells plus diverse immune cells in the laminar propria and the PPs) to exert its antiviral activity against norovirus. To this end, distal part of the ileum with or without PPs was retrieved from BALB/c mice. These were treated with γ-PGA in culture dishes for 18 h ex vivo. After treatment, the villi released into the culture media and the remaining basal body of the ileum (ileal submucosa and lamina propria) were collected separately and the IFN-β mRNA levels were quantified in these ileal compartments by reverse-transcription quantitative PCR (RT-qPCR) (Fig. 3a). Interestingly, we found induction of IFN-β production by γ-PGA only in the villi (compartments II and IV) and not in PPs and basal body of the ileum (compartments III and I) (Fig. 3b). This induction was more substantial in the villi derived from the ileum with PPs (compartment II) than from the ileum without PPs (compartment IV). TLR4-stained cells were more abundant in compartment II than in compartment IV (Fig. 3c). These results suggest that to activate TLR4 signaling, γ-PGA transverses the epithelial cell layer of the villi to reach the laminar propria bearing TLR4-positive macrophages and dendritic cells. Despite the abundance of TLR4-positive cells in PPs, γ-PGA treatment did not significantly induce IFN-β mRNA expression, suggesting non-responsiveness of PPs to γ-PGA by an as-yet-unknown mechanism.Figure 3Induction of IFN-β expression by γ-PGA and MNV in mouse ileum. (a) Experimental schemes for ex vivo stimulation of the ileum with γ-PGA. (b) Mouse (n = 4) ileum with or without PPs was treated with γ-PGA ex vivo. After 18 h incubation, IFN-β mRNA levels in the ileal compartments depicted in (a) were determined by RT-qPCR. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant; by two-tailed unpaired Student’s t-test. (c) Microscopic analysis of TLR4-expressing cells in the ileum with or without PPs. Intestinal microvilli stained with a villin-specific antibody. (d) Microscopic analysis of the entry of MNV. Mouse ileum with or without PPs was infected with Qdot fluorophore-tagged MNV ex vivo in serum-free DMEM. At 2 h post-infection, cryosection images were taken after extensively washing the infected tissues. Scale bar, 50 μm.