Conditional expression of interferon-gamma to enhance host responses to pulmonary bacterial infection.

Strategies to augment host defense against pulmonary infection run the risk of inducing excess pulmonary inflammation and tissue injury. To address this problem, we investigated conditional expression in lung tissue of the murine interferon-gamma (IFN-gamma) transgene. A recombinant adenoviral vector (AdTetIFN) was constructed by placing a murine IFN-gamma cDNA downstream of a tetracycline (Tet)-responsive promoter, inserted into a replication-defective adenoviral vector. Co-infection of target cells with AdTetIFN and a second vector encoding a reverse tetracycline controlled transactivator allowed doxycycline (Dox)-regulated IFN-gamma production. We then administered 10(8) plaque-forming units (PFU) of AdTetIFN to mice by intratracheal injection. When the mice were provided with Dox in drinking water (0.5mg/ml in 5% sucrose), there was significant release of IFN-gamma in lavage fluid by ELISA in comparison to mice on water/sucrose alone (399+/-74 pg/ml vs undetectable, p<0.01). IFN-gamma in lavage fluid was associated with upregulation of Class II Major histocompatibility complex markers on alveolar macrophages by flow cytometry, suggesting macrophage activation. We then injected AdTetIFN into mice three days prior to challenge with 10(4) CFU Klebsiella pneumoniae. Test mice were maintained on water+Dox and control mice on water+sucrose. Bacterial burden was assayed in lung tissue at serial intervals. At 24h after challenge, mice on doxycycline had significantly lower infection burden in comparison to mice on water/sucrose (0.77+/-0.05 colony forming units/lung for 10(8) PFU AdTetIFN plus Dox compared to 1.4+/-0.11 colony-forming units/lung for AdTetIFN without Dox, p<0.05). Survival of the vector treated mice given doxycycline in drinking water was also enhanced. Microscopic examination of lavaged cells showed a significant increase in pulmonary neutrophils in the AdTetIFN+Dox mice in comparison to AdTetIFN+sucrose mice (16+/-1.0 x 10(5) vs 10+0.8 cells/lung, p<0.05). We conclude that local release of IFN-gamma can be selectively activated to enhance neutrophil recruitment and host resistance to bacterial pneumonia.

Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters.

BACKGROUND: The goal of this study was to design improved regulatable lentivirus vector systems. The aim was to design tetracycline (tet)-regulatable lentivirus vectors based on the Tet-on system displaying low background expression in the absence of the doxycycline (DOX) inducer and high transgene expression levels in the presence of DOX. METHODS: We constructed a binary lentivirus vector system that is composed of a self-inactivating (SIN) lentivirus vector bearing inducible first- or second-generation tet-responsive promoter elements (TREs) driving expression of a transgene and a second lentivirus vector encoding a reverse tetracycline-controlled transactivator (rtTA) that activates transgene expression from the TRE in the presence of DOX. RESULTS: We evaluated a number of different rtTAs and found rtTA2S-M2 to induce the highest levels of transgene expression. Regulated transgene expression was stable in human breast carcinoma cells implanted into nude mice for up to 11 weeks. In an attempt to minimize background expression levels, the chicken beta-globin cHS4 insulator element was cloned into the 3' long terminal repeat (LTR) of the transgene transfer vector. The cHS4 insulator element reduced background expression but expression levels following DOX addition were lower than those observed with vectors lacking an insulator sequence. In a second strategy, vectors bearing second-generation TREs harboring repositioned tetracycline operator elements were used. Such vectors displayed greatly reduced leakiness in the absence of DOX and induced transgene expression levels were up to 522-fold above those seen in the absence of DOX. CONCLUSIONS: Inducible lentivirus vectors bearing insulators or second-generation TREs will likely prove useful for applications demanding the lowest levels of background expression.