We previously showed that activation of TLR4 signaling by γ-PGA leading to induction of IFN was dependent on TLR4-associated accessary factors CD14 and MD26. In the present study, results from Q-dot-conjugated γ-PGA confirm the necessity of CD14 and MD2 for sensing γ-PGA. Dependence on CD14 in MyD88-dependent pathway-mediated induction of TNF-α was marginal in LPS-stimulated macrophages53,54. However, our results indicate the absolute requirement of CD14 and its prominent role over MD2 in sensing γ-PGA. Our findings suggesting a substantial stimulatory effect of γ-PGA in the lamina propria raises interest on the possibility of differential expression of MD2, CD14, and TLR4 in macrophages and dendritic cells in the lamina propria and PPs. Currently, requirement of these accessory factors in sensing various non-LPS TLR4 ligands remains largely unknown. Irrespective of the mechanisms involved in γ-PGA entry and sensing by TLR4-positive cells in the ileum, both, results from ex vivo culture indicating stimulation of the ileum with γ-PGA and the serum cytokine profile of mice receiving peroral γ-PGA, provide clues as to how γ-PGA activates the TRIF-dependent TLR4 signaling pathway in the ileum.