TLR3 mediated Type I Interferon response in mammalian cell defense against Cryptosporidium parvum infection.

Abstract

Cryptosporidium parvum is a major cause of a diarrheal disease which affects many children in the USA as well as the rest of the world, yet the contribution of its double stranded RNA (dsRNA) viral symbiont, Cryspovirus (CSpV1) to host innate immunity remains unclear. This study investigated whether CSpV1 activates Toll Like Receptor 3 (TLR3) signaling pathways and downstream Interferon type I (IFN I) responses in mammalian intestinal epithelial cells. Confluent Human Ileocecal Colorectal Adenocarcinoma cells (HCT8) were exposed to CSpV1, and the expression levels of Tumor Necrosis Factor Receptor-Associated Factor 3 (TRAF3), Nuclear Factor--light chain enhancer of activated B-cells (NF-B), Interferon Regulatory Factor (IRF3) and IFN- were measured using Indirect Enzyme-Linked Immunosorbent Assay (ELISA), Time Resolved Förster Resonance Energy Transfer (TR-FRET), Cell-based colorimetric-ELISA, sandwich-ELISA, respectively, and compared with uninfected controls. CSpV1 exposure induced a time dependent increase in TRAF3 with a peak at 3h, enhanced NF-κB phosphorylation, and a reduction in total IRF3 signal consistent with activation and nuclear translocation. These changes were accompanied by a significant increase in IFN- production compared with controls. Together these findings support a model in which CSpV1 dsRNA activated epithelial TLR3, initiating TRIF-dependent activation of TRAF3, NF-κB and IRF3 activation, culminating in the induction of IFN I responses