Rajamouli Pasula

Genetically engineered macrophages expressing IFN-γ restore alveolar immune function in scid mice

Reversal of immunodeficiency in the lung by gene therapy is limited in part by the difficulty of transfecting lung cells in vivo. Many options exist for successfully transfecting cells in vitro, but they are not easily adapted to the in vivo condition. To overcome this limitation, we transduced macrophages in vitro with the murine IFN-γ (mIFN-γ) gene and intratracheally delivered the macrophages to express mIFN-γ in vivo. A recombinant retroviral vector pSF91 system was modified to encode mIFN-γ and enhanced green fluorescent protein (EGFP). A murine macrophage cell line J774A.1 transduced with the retroviral supernatant increased secretion from undetectable levels to 131.6 ± 4.2 μg/ml mIFN-γ at 24 h in vitro. The mIFN-γ-producing macrophages were intratracheally instilled into mechanically ventilated scid mice. mIFN-γ levels in the bronchoalveolar lavage increased from undetectable levels at baseline to 158.8 ± 5.1 pg/ml at 48 h (P < 0.001). Analysis of the lavaged cells for EGFP expression revealed that EGFP expression was directly proportional to the number of transduced macrophages instilled into the lung. Immune function was partially restored in the alveolar spaces of scid mice with evidence of enhanced MHC class II antigen expression and increased phagocytosis (P < 0.05). Tumor necrosis factor α was increased from undetectable at baseline to 103.5 ± 11.4 pg/ml. In contrast, i.p. administration of the engineered macrophages did not enhance IFN-γ levels in the lung. Our study suggests airway delivery of genetically engineered macrophages expressing mIFN-γ gene can partially restore significant immune activity in the lungs of immunodeficient mice.

Fibronectin Facilitates Mycobacterium tuberculosis Attachment to Murine Alveolar Macrophages

ABSTRACT Mycobacterium tuberculosis remains a major cause of pulmonary infection worldwide. Attachment of M. tuberculosis organisms to alveolar macrophages (AMs) represents the earliest phase of primary infection in pulmonary tuberculosis. In this study fibronectin (Fn), an adhesive protein, is shown to bind M. tuberculosis organisms and facilitates attachment of M. tuberculosis to murine AMs. A monoclonal antibody (MAb) specific to the heparin binding domain (HBD) of Fn decreases 125I-Fn binding to M. tuberculosis; whereas MAbs specific to either the cell binding domain (CBD) or the gelatin binding domain (GBD) have no effect on Fn binding to M. tuberculosis. In the presence of exogenous Fn (10 μg/ml) M. tuberculosis attachment to AMs increased significantly from control levels (means ± standard errors of the means) of 11.5% ± 1.1% to 44.2% ± 4.2% (P < 0.05). Fn-enhanced attachment was significantly decreased from 44.2% ± 4.2% to 10.8% ± 1.2% (P < 0.05) in the presence of anti-Fn polyclonal antibodies. The attachment is also inhibited in the presence of MAbs specific for the HBD and CBD, whereas MAbs specific to GBD did not affect the attachment. Further, an Fn cell binding peptide, Arg-Gly-Asp-Ser (RGDS), decreased the attachment from 44.2% ± 4.2% to 15.3% ± 1.2% (P < 0.05), whereas addition of a control peptide, Arg-Gly-Glu-Ser (RGES) did not affect the attachment (40.5% ± 1.8%). These results suggest that Fn-mediated attachment of M. tuberculosis can occur through the binding of Fn to the AM via the CBD and to M. tuberculosis organisms via the HBD.

GALLIUM NITRATE IS EFFICACIOUS IN MURINE MODELS OF TUBERCULOSIS AND INHIBITS KEY BACTERIAL FE-DEPENDENT ENZYMES

ABSTRACT Acquiring iron (Fe) is critical to the metabolism and growth of Mycobacterium tuberculosis. Disruption of Fe metabolism is a potential approach for novel antituberculous therapy. Gallium (Ga) has many similarities to Fe. Biological systems are often unable to distinguish Ga3+ from Fe3+. Unlike Fe3+, Ga3+ cannot be physiologically reduced to Ga2+. Thus, substituting Ga for Fe in the active site of enzymes may render them nonfunctional. We previously showed that Ga inhibits growth of M. tuberculosis in broth and within cultured human macrophages. We now report that Ga(NO3)3 shows efficacy in murine tuberculosis models. BALB/c SCID mice were infected intratracheally with M. tuberculosis, following which they received daily intraperitoneal saline, Ga(NO3)3, or NaNO3. All mice receiving saline or NaNO3 died. All Ga(NO3)3-treated mice survived. M. tuberculosis CFU in the lungs, liver, and spleen of the NaNO3-treated or saline-treated mice were significantly higher than those in Ga-treated mice. When BALB/c mice were substituted for BALB/c SCID mice as a chronic (nonlethal) infection model, Ga(NO3)3 treatment significantly decreased lung CFU. To assess the mechanism(s) whereby Ga inhibits bacterial growth, the effect of Ga on M. tuberculosis ribonucleotide reductase (RR) (a key enzyme in DNA replication) and aconitase activities was assessed. Ga decreased M. tuberculosis RR activity by 50 to 60%, but no additional decrease in RR activity was seen at Ga concentrations that completely inhibited mycobacterial growth. Ga decreased aconitase activity by 90%. Ga(NO3)3 shows efficacy in murine M. tuberculosis infection and leads to a decrease in activity of Fe-dependent enzymes. Additional work is warranted to further define Gas mechanism of action and to optimize delivery forms for possible therapeutic uses in humans.

Morphologic Detection and Functional Assessment of Reconstituted Normal Alveolar Macrophages in the Lungs of SCID Mice

Alveolar macrophages (AMs) from immunocompetent animals were isolated from bronchoalveolar lavage and labeled with the fluorescent marker 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). These AMs were administered intratracheally into mechanically ventilated SCID mice. From 1 to 28 days later, the recipient mice underwent bronchoalveolar lavage to isolate their AMs. To determine whether reconstituted AMs were still immunocompetent, the recovered AMs were assayed for their ability to phagocytose fluorescein-labeled zymosan-coated beads. After incubation with the beads, samples were assayed using a fluorescent-activated cell sorter to identify DiI-labeled reconstituted AMs, unlabeled resident AMs, and the proportion of these two groups undergoing phagocytosis. DiI-labeled AMs accounted for ∼50% of all returned AMs. Additionally, the reconstituted AMs from normal BALB/c mice retained phagocytic activity compared with AMs from immunodeficient SCID mice. Reconstituted AMs demonstrated enhanced phagocytic activity compared with resident SCID AMs for up to 28 days following reconstitution. These results indicate that immunocompetent AMs can be successfully reconstituted into an immunodeficient host to partially restore alveolar host defense.

Inactivation of the potent Pseudomonas aeruginosa cytotoxin pyocyanin by airway peroxidases and nitrite.

Pyocyanin (1-hydroxy-N-methylphenazine, PCN) is a cytotoxic pigment and virulence factor secreted by the human bacterial pathogen, Pseudomonas aeruginosa. Here, we report that exposure of PCN to airway peroxidases, hydrogen peroxide (H(2)O(2)), and NaNO(2) generates unique mononitrated PCN metabolites (N-PCN) as revealed by HPLC/mass spectrometry analyses. N-PCN, in contrast to PCN, was devoid of antibiotic activity and failed to kill Escherichia coli and Staphylococcus aureus. Furthermore, in contrast to PCN, intratracheal instillation of N-PCN into murine lungs failed to induce a significant inflammatory response. Surprisingly, at a pH of ∼7, N-PCN was more reactive than PCN with respect to NADH oxidation but resulted in a similar magnitude of superoxide production as detected by electron paramagnetic resonance and spin trapping experiments. When incubated with Escherichia coli or lung A549 cells, PCN and N-PCN both led to superoxide formation, but lesser amounts were detected with N-PCN. Our results demonstrate that PCN that has been nitrated by peroxidase/H(2)O(2)/NO(2)(-) systems possesses less cytotoxic/proinflammatory activity than native PCN. Yield of N-PCN was decreased by the presence of the competing physiological peroxidase substrates (thiocyonate) SCN(-) (myeloperoxidase, MPO, and lactoperoxidase, LPO) and Cl(-) (MPO), which with Cl(-) yielded chlorinated PCNs. These reaction products also showed decreased proinflammatory ability when instilled into the lungs of mice. These observations add important insights into the complexity of the pathogenesis of lung injury associated with Pseudomonas aeruginosa infections and provide additional rationale for exploring the efficacy of NO(2)(-) in the therapy of chronic Pseudomonas aeruginosa airway infection in cystic fibrosis.

Airway Delivery of Silica Increases Susceptibility to Mycobacterial Infection in Mice: Potential Role of Repopulating Macrophages

Silica exposure results in an increased lifelong risk of developing mycobacterial pulmonary infections. To date, there are no animal models that replicate this finding to permit assessment of the mechanisms underlying susceptibility to mycobacterial infection. To test the hypothesis that prior silica exposure increases risk of mycobacterial infection, we intratracheally (I.T.) administered silica, a control dust (Al2O3) or saline into mechanically ventilated C57BL/6 mice. Later, the mice received Mycobacterium avium or Mycobacterium tuberculosis I.T. Mice were sacrificed at defined time points and mycobacteria in lung homogenates were quantified. M. avium or M. tuberculosis infection was markedly increased in silica-exposed mice compared with mice exposed to either Al2O3 or saline beginning 3 wk after silica exposure. Similarly, lung sections from silica-exposed mice had many more acid fast bacilli+ (AFB+) organisms than from control mice. Alveolar macrophages (AMs) from bronchoalveolar lavage of silica-exposed mice also revealed a higher number of mycobacteria compared with mice treated with Al2O3 or saline. In addition, passive transfer of AMs from silica-exposed mice to control mice increased M. tuberculosis susceptibility. These results indicate that silica exposure converts mycobacteria-resistant mice into mycobacteria-susceptible mice via a process that likely involves a new population of AMs that are more susceptible to mycobacterial infection.

Keratinocyte Growth Factor Administration Attenuates Murine Pulmonary Mycobacterium tuberculosis Infection through Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF)-dependent Macrophage Activation and Phagolysosome Fusion

Background: Mycobacterium tuberculosis is a major cause of morbidity and mortality worldwide, and chemotherapeutic treatments are not always effective. Results: Keratinocyte growth factor (KGF) administration results in an epithelium-dependent, GM-CSF-mediated induction of microbicidal programs in alveolar macrophages. Conclusion: KGF induces GM-CSF and enhances clearance of M. tuberculosis from the lungs of mice. Significance: KGF may be a useful adjunctive strategy for treatment of infection with M. tuberculosis. Augmentation of innate immune defenses is an appealing adjunctive strategy for treatment of pulmonary Mycobacterium tuberculosis infections, especially those caused by drug-resistant strains. The effect of intranasal administration of keratinocyte growth factor (KGF), an epithelial mitogen and differentiation factor, on M. tuberculosis infection in mice was tested in prophylaxis, treatment, and rescue scenarios. Infection of C57BL6 mice with M. tuberculosis resulted in inoculum size-dependent weight loss and mortality. A single dose of KGF given 1 day prior to infection with 105 M. tuberculosis bacilli prevented weight loss and enhanced pulmonary mycobacterial clearance (compared with saline-pretreated mice) for up to 28 days. Similar effects were seen when KGF was delivered intranasally every third day for 15 days, but weight loss and bacillary growth resumed when KGF was withdrawn. For mice with a well established M. tuberculosis infection, KGF given every 3 days beginning on day 15 postinoculation was associated with reversal of weight loss and an increase in M. tuberculosis clearance. In in vitro co-culture experiments, M. tuberculosis-infected macrophages exposed to conditioned medium from KGF-treated alveolar type II cell (MLE-15) monolayers exhibited enhanced GM-CSF-dependent killing through mechanisms that included promotion of phagolysosome fusion and induction of nitric oxide. Alveolar macrophages from KGF-treated mice also exhibited enhanced GM-CSF-dependent phagolysosomal fusion. These results provide evidence that administration of KGF promotes M. tuberculosis clearance through GM-CSF-dependent mechanisms and enhances host defense against M. tuberculosis infection.

Airway delivery of interferon‐γ overexpressing macrophages confers resistance to Mycobacterium avium infection in SCID mice

Mycobacterium avium (M. avium) causes significant pulmonary infection, especially in immunocompromised hosts. Alveolar macrophages (AMs) represent the first line of host defense against infection in the lung. Interferon gamma (IFN‐γ) activation of AMs enhances in vitro killing of pathogens such as M. avium. We hypothesized that airway delivery of AMs into the lungs of immunodeficient mice infected with M. avium will inhibit M. avium growth in the lung and that this macrophage function is in part IFN‐γ dependent. In this study, normal BALB/c and BALB/c SCID mice received M. avium intratracheally while on mechanical ventilation. After 30 days, M. avium numbers increased in a concentration‐dependent manner in SCID mice compared with normal BALB/c mice. Airway delivery of IFN‐γ‐activated BALB/c AMs or J774A.1 macrophages overexpressing IFN‐γ into the lungs of SCID mice resulted in a significant decrease in M. avium growth (P < 0.01, both comparisons) and limited dissemination to other organs. In addition, airway delivery of IFN‐γ activated AMs and macrophages overexpressing IFN‐γ increased the levels of IFN‐γ and TNF‐α in SCID mice. A similar protective effect against M. avium infection using J774A.1 macrophages overexpressing IFN‐γ was observed in IFN‐γ knockout mice. These data suggest that administration of IFN‐γ activated AMs or macrophages overexpressing IFN‐γ may partially restore local alveolar host defense against infections like M. avium, even in the presence of ongoing systemic immunosuppression.

Passive transfer of interferon-γ over-expressing macrophages enhances resistance of SCID mice to Mycobacterium tuberculosis infection

Abstract Infection with Mycobacterium tuberculosis (M.tb) is associated with increased deaths worldwide. Alveolar macrophages (AMs) play a critical role in host defense against infection with this pathogen. In this work we tested the hypothesis that passive transfer of normal AMs, IFN‐&ggr; activated AMs, or macrophages transduced to over‐express IFN‐&ggr; into the lungs of immunosuppressed SCID mice, where resident macrophages are present but not functional, would enhance alveolar immunity and increase clearance of pulmonary M.tb infection. Accordingly, SCID mice were infected with M.tb intratracheally (I.T.), following which they received either control macrophages or macrophages overexpressing IFN‐&ggr; (J774A.1). The extent of M.tb infection was assessed at 30 days post‐M.tb infection. SCID mice administered macrophages over‐expressing IFN‐&ggr; showed a significant decrease in M.tb burden and increased survival compared to J774A.1 control macrophages or untreated mice. This was further associated with a significant increase in IFN‐&ggr; and TNF‐&agr; mRNA and protein expression, as well as NF‐&kgr;B (p65) mRNA, in the lungs. The increase in IFN‐&ggr; and TNF‐&agr; lung levels was inversely proportional to the number of M.tb organisms recovered. These results provide evidence that administration of macrophages overexpressing IFN‐&ggr; inhibit M.tb growth in vivo and may enhance host defense against M.tb infection.