These findings demonstrate that TLR3 activation by poly(I:C) modulates the local inflammatory response in the lung and suggest a critical role of TLR3 activation in driving lung function impairment. Thus, TLR3 activation may be one mechanism through which viral infections contribute toward exacerbation of respiratory disease.

SummaryHaemophilus ducreyi causes chancroid, a sexually transmitted genital ulcer disease implicated in increased heterosexual transmission of HIV. As part of an effort to identify H. ducreyi gene products involved in virulence and pathogenesis, we created random TnphoA insertion mutations in an H. ducreyi 35 000 library cloned in Escherichia coli. Inserts encoding exported or secreted PhoA fusion proteins were characterized by DNA sequencing. One such clone encoded a Cu-Zn superoxide dismutase (SOD) enzyme. The CuZn SOD was periplasmic in H. ducreyi and accounted for most of the detectable SOD activity in whole-cell lysates of H. ducreyi grown in vitro. To investigate the function of the Cu-Zn SOD, we created a Cu-Zn SOD-deficient H. ducreyi strain by inserting a cat cassette into the sodC gene. The wild-type and Cu-Zn SOD null mutant strains were equally resistant to excess cytoplasmic superoxide induced by paraquat, demonstrating that the Cu-Zn SOD did not function in the detoxification of cytoplasmic superoxide. However, the Cu-Zn SOD null strain was significantly more susceptible to killing by extracellular superoxide than the wild type. This result suggests that the H. ducreyi Cu-Zn SOD may play a role in bacterial defence against oxidative killing by host immune cells during infection.

To gain global pathway perspective of ex vivo viral infection models using human peripheral blood mononuclear cells (PBMCs), we conducted expression analysis on PBMCs of healthy donors. RNA samples were collected at 3 and 24 h after PBMCs were challenged with the Toll-like receptor-3 (TLR3) agonist polyinosinic acid-polycytidylic acid [poly(I:C)] and analyzed by internally developed cDNA microarrays and TaqMan PCR. Our results demonstrate that poly(I:C) challenge can elicit certain gene expression changes, similar to acute viral infection. Hierarchical clustering revealed distinct immediate early, early-to-late, and late gene regulation patterns. The early responses were innate immune responses that involve TLR3, the NF-kappaB-dependent pathway, and the IFN-stimulated pathway, whereas the late responses were mostly cell-mediated immune response that involve activation of cell adhesion, cell mobility, and phagocytosis. Overall, our results expanded the utilities of this ex vivo model, which could be used to screen molecules that can modulate viral stress-induced inflammation, in particular those mediated via TLRs.

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Toll-like receptor 3 (TLR3) recognizes dsRNA and initiates an innate immune response through the formation of a signaling unit (SU) composed of one double-stranded RNA (dsRNA) and two TLR3 molecules. We report the crystal structure of human TLR3 ectodomain (TLR3ecd) in a quaternary complex with three neutralizing Fab fragments. Fab15 binds an epitope that overlaps the C-terminal dsRNA binding site and, in biochemical assays, blocks the interaction of TLR3ecd with dsRNA, thus directly antagonizing TLR3 signaling through inhibition of SU formation. In contrast, Fab12 and Fab1068 bind TLR3ecd at sites distinct from the N- and C-terminal regions that interact with dsRNA and do not inhibit minimal SU formation with short dsRNA. Molecular modeling based on the co-structure rationalizes these observations by showing that both Fab12 and Fab1068 prevent lateral clustering of SUs along the length of the dsRNA ligand. This model is further supported by cell-based assay results using dsRNA ligands of lengths that support single and multiple SUs. Thus, their antagonism of TLR3 signaling indicates that lateral clustering of SUs is required for TLR3 signal transduction.