TLR3–TICAM1–p38 signalling partially mediates IL-6 induction during human adenovirus type 26 infection in epithelial cells with inhibited clathrin-mediated endocytosis

Abstract Human adenovirus type 26 (HAdV-D26) is a clinically important vector, but the connection between its entry pathway, intracellular trafficking, and innate immune sensing remains unclear. As innate immune sensors are distributed throughout the cell, the virus entry route and subcellular localization likely shape immune activation. Here, we investigated how altered entry routes and endosomal retention of HAdV-D26 influence Toll-like receptor (TLR)-mediated signalling in epithelial cells. Using A549 cells with inhibited clathrin-mediated endocytosis (CME) or de novo αvβ3 integrin expression, we analysed IL-6 and IL-8 expression following HAdV-D26 infection and examined the underlying signalling mechanisms. In CME-inhibited A549 cells, where HAdV-D26 persisted longer in early endosomes, HAdV-D26 infection induced IL-6 expression partially through a TLR3–TICAM1–p38-dependent mechanism. Inhibition of TLR3 or p38, as well as knockdown of TICAM1, reduced the elevated IL-6 response observed under CME inhibition. In contrast, inhibition of TLR4 or knockdown of MyD88 increased HAdV-D26-induced IL-6 and IL-8 expression in A549 cells, indicating a negative regulatory role of TLR4–MyD88 signalling. Furthermore, MyD88 knockdown reduced IL-8 expression in A549 cells with de novo αvβ3 integrin expression, linking integrin-mediated entry to MyD88-dependent signalling. In conclusion, these findings highlight how entry pathway and intracellular localization shape innate immune sensing of adenoviral vectors.