Investigating pathogen-specific TLR signaling of innate immune cells for biosensor applications

The goal of this project is to develop a real-time cell-based biosensor using Toll-like receptors (TLRs) for pathogen detection. Existing biosensors rely on technologies that recognize only specific target analytes, requiring prior knowledge of the possible contaminating agents. Innate immune cells express TLRs that recognize conserved pathogen-associated molecular patterns on bacteria, viruses, parasites and fungi. Using TLRs as the receptor element in this biosensor will eliminate the need for a priori knowledge of the threat. At least 10 different members of the TLR family are expressed on cells of the innate immune system, each responding to different attributes of pathogenic organisms. Through flow cytometry, TLRs 2, 4 and 9 have been identified on THP-1 cells, and TLRs 2, 3, 4, 5 and 9 have been detected on J774 cells. Western blotting has identified Erk activation upon lipopolysacharide (LPS), E. coli and Poly(I):(C) exposure in J774 cells, and upon LPS and E. coli exposure in THP-1 cells. Cellular model systems are being developed to distinguish between bacteria and virus by selective stimulation of TLR3 and TLR5 (TLR3 specifically recognizes double-stranded viral RNA and TLR5 detects bacterial flagellin). A target application of this technology is point-of-care diagnostics. Realtime detection of viruses in nasal or throat swabs could help decrease the inappropriate use of antibiotics.