Carol M. Mason

Pulmonary Interleukin-23 Gene Delivery Increases Local T-Cell Immunity and Controls Growth of Mycobacterium tuberculosis in the Lungs

ABSTRACT Interleukin-23 (IL-23) is a heterodimeric cytokine that shares IL-12 p40 but contains a unique p19 subunit similar to IL-12 p35. Previous studies indicate a greater importance for intact IL-12/23 p40 expression than IL-12 p35 for immunity against Mycobacterium tuberculosis, suggesting a role for IL-23 in host defense. The effects of IL-23 on the outcome of pulmonary infection with M. tuberculosis have not been described. Here, we show that local delivery of replication-defective adenovirus vectors encoding IL-23 (AdIL-23) greatly stimulated expression of both gamma interferon (IFN-γ) and IL-17 in lung tissues of otherwise normal mice. When given 72 h prior to infection with M. tuberculosis, AdIL-23 significantly reduced the bacterial burden at 14, 21, and 28 days. Markedly lower levels of lung inflammation were observed at 28 days than in control mice pretreated with control adenovirus (AdNull) or vehicle controls. AdIL-23 pretreatment resulted in increased numbers of CD4+ CD25+ activated T cells in lungs and draining lymph nodes compared to control groups and more CD4+ T cells bearing surface memory markers in lung lymph nodes. IL-23 gene delivery also significantly enhanced host anti-mycobacterial T-cell responses, as shown by elevated levels of IFN-γ and IL-17 secreted in vitro following restimulation with M. tuberculosis purified protein derivative. Overall, our data show that transient IL-23 gene delivery in the lung is well tolerated, and they provide the initial demonstration that this factor controls mycobacterial growth while augmenting early pulmonary T-cell immunity.

Decreased Expression of CD3ζ and Nuclear Transcription Factor κB in Patients with Pulmonary Tuberculosis: Potential Mechanisms and Reversibility with Treatment

BACKGROUND The protective immune response against Mycobacterium tuberculosis relies both on antigen-presenting cells and on T lymphocytes. In patients with different forms of tuberculosis, varying degrees of T cell function--ranging from positive delayed-type hypersensitivity, in asymptomatic infected healthy individuals, to the absence of the response, in patients with miliary or pulmonary tuberculosis (PTB)--have been reported. The decreased expression of CD3zeta reported in T cells from patients with either cancer or leprosy has provided possible explanations for the altered immune response observed in these diseases. METHODS The present study aimed to compare the expression of CD3zeta , nuclear transcription factor- kappa B (NF- kappa B), arginase activity, and cytokine production in 20 patients with PTB, in 20 tuberculin-positive asymptomatic subjects, and in 14 tuberculin-negative control subjects. RESULTS Compared with those in tuberculin (purified protein derivative)-negative control subjects, peripheral-blood T lymphocytes from patients with active PTB had significantly (P < .001) decreased expression of CD3zeta and absence of the p65/p50 heterodimer of NF- kappa B. These alterations were reversed only in patients who responded to treatment. Also reported here for the first time is that the presence of arginase activity in peripheral-blood mononuclear-cell lysates of patients with PTB parallels high production of interleukin-10. CONCLUSIONS The presence of arginase could, in part, explain the decreased expression of CD3zeta . These findings provide a novel mechanism that may explain the T cell dysfunction observed in patients with PTB.

Keratinocyte growth factor attenuates lung leak induced by alpha-naphthylthiourea in rats.

OBJECTIVE To investigate the effect of pretreatment with keratinocyte growth factor on acute permeability pulmonary edema. DESIGN Prospective, randomized, controlled animal study. SETTING University research laboratory. SUBJECTS Specific pathogen-free Sprague-Dawley rats. INTERVENTION Acute permeability pulmonary edema was induced with an injection of alpha-naphthylthiourea, and lung leak was assessed in an isolated perfused lung model over 180 mins. Leak was confirmed with wet/dry lung weight ratios, and the alveolar fluid protein concentration was measured after bronchoalveolar lavage. The effect of pretreatment with keratinocyte growth factor (injected intratracheally 48 hrs before the experiment) on alpha-naphthylthiourea-induced pulmonary edema was assessed (keratinocyte growth factor/alpha-naphthylthiourea group). Control groups (Control and keratinocyte growth factor/Control) were also studied. Histopathology was performed for each of the four groups. MEASUREMENTS AND MAIN RESULTS The alpha-naphthylthiourea produced an acute permeability pulmonary edema detected by lung leak over the 180-min ex vivo period of monitoring the isolated perfused lung (leak = 8+/-mL; wet/dry weight ratio 14.7+/-2; lavage protein 3.1+/-1 mg/mL). Pretreatment with keratinocyte growth factor significantly attenuated these parameters (leak = 2.3+/-0.4 mL; wet/dry weight ratio 7.1 +/- 0.5; lavage protein 0.28 +/-0.03 mg/mL), which were not significantly different from the control group and the keratinocyte growth factor/control group. Histopathology showed abundant type II pneumocyte hyperplasia in the lungs of animals pretreated with keratinocyte growth factor, and marked pulmonary edema in animals pretreated with alpha-naphthylthiourea. Less edema was apparent in the keratinocyte growth factor/alpha-naphthylthiourea group. All data are expressed as mean +/- SEM. CONCLUSIONS Pretreatment with keratinocyte growth factor significantly attenuates pulmonary edema induced by alpha-naphthylthiourea. The mechanisms of this protection are likely related to type II pneumocyte hyperplasia, but remain to be specifically elucidated.

Ventilation-induced lung injury is associated with an increase in gut permeability.

Mechanical ventilation is associated with several harmful effects mainly related to high tidal volumes (Vt). Ventilator-induced lung injury can be responsible for an increased production of inflammatory mediators. We evaluated remote consequences on the gut of lung triggered inflammatory response, neutralizing anti-tumor necrosis factor (TNF) antibody was administered to assess the role of TNF in lung and gut permeability changes. Rats were anesthetized and ventilated for 2 h. A control group (Con: Vt = 10 mL/kg) was compared with a high Vt group (HV: Vt = 30 mL/kg). One &mgr;Ci of I125-labeled human serum albumin was injected to measure extravascular albumin space. Gut permeability was evaluated by plasma-to-lumen ratio leakage of I125 human serum albumin. Extravascular albumin space increased in the HV group from 446 ± 50 &mgr;L to 2783 ± 887 &mgr;L. Gut index of permeability increased from 5.1 ± 1.2 to 14.2 ± 4.9. Anti-TNF antibody prevented both lung and gut increase in permeability. High tidal volume ventilation resulted in an increase in lung edema and gut permeability, antagonism of TNF with neutralizing antibodies abrogated the increase in gut permeability as well as lung edema.

Alcohol Exacerbates Murine Pulmonary Tuberculosis

ABSTRACT Alcohol consumption has been described as a risk factor for infection with Mycobacterium tuberculosis, but its contribution to tuberculosis has been difficult to isolate from other adverse socioeconomic factors. Our objective was to evaluate the impact of alcohol consumption on pulmonary infection with M. tuberculosis in a murine model. BALB/c mice were maintained on the Lieber-DeCarli liquid ethanol diet or a liquid control diet and infected intratracheally with low-dose M. tuberculosis H37Rv. Lung organism burdens, lung and lung-associated lymph node CD4+- and CD8+- lymphocyte numbers and rates of proliferation, and CD4+-lymphocyte cytokine production levels were compared between the groups. The alcohol-consuming mice had significantly higher lung organism burdens than the control mice, and the CD4+- and CD8+-lymphocyte responses to pulmonary infection with M. tuberculosis were blunted in the alcohol group. Lymphocyte proliferation and production of gamma interferon were decreased in the CD4+ lymphocytes from the alcohol-consuming mice. Additionally, lung granulomas were significantly smaller in the alcohol-consuming mice. In conclusion, murine alcohol consumption is associated with decreased control of pulmonary infection with M. tuberculosis, which is accompanied by alterations in the region-specific CD4+- and CD8+-lymphocyte responses and defective lung granuloma formation.

Intraportal Lipopolysaccharide Suppresses Pulmonary Antibacterial Defense Mechanisms

Translocation of enteric bacteria or their components (or both) has been postulated to play a role in precipitating sepsis or the systemic inflammatory response syndrome. To simulate the effects of translocation on pulmonary host defenses, lipopolysaccharide was injected into the portal vein of normal rats that were subsequently challenged by aerosol inoculation with Pseudomonas aeruginosa. Injection of LPS into the portal vein resulted in increased serum tumor necrosis factor (TNF)-alpha levels and reduction in lung clearance of P. aeruginosa after aerosol challenge. There were corresponding reductions in alveolar neutrophil recruitment, diminished alveolar macrophage phagocytosis and superoxide anion (O2-) production, and diminished lung TNF recovered by bronchoalveolar lavage. Furthermore, prior intravenous injection of recombinant TNF-alpha reproduced the defective bacterial clearance, the altered recruitment of airspace neutrophils, and the defective alveolar macrophage phagocytosis. Thus, systemic TNF-alpha is important in altering pulmonary defenses, and this work supports the concept that bacterial translocation may adversely affect host defenses in distant organs.

HOST DEFENSE IN RESPIRATORY INFECTIONS

Respiratory defenses against infection involve a diverse and complex system. Mechanical barriers limit exposure of the respiratory tract to potential pathogenic organisms, whereas the mucociliary apparatus and cough reflexes work to expel any microbes that may bypass the initial defenses. When microorganisms have gained entry to the lower respiratory tract, the alveolar macrophage and recruited phagocytes may eliminate the culprits before active infection can be established. Only after the failure of the innate immune defenses is a specific immune response mounted. Examination of clinical defects in host defense allows one to understand the importance of the multitude of components of the lungs immune defense system.

Phase 1 safety trial of Filgrastim (r-metHuG-CSF) in non-neutropenic patients with severe community-acquired pneumonia

The objectives of the present study were to: (1) evaluate the safety of Filgrastim therapy in non-neutropenic patients with severe community-acquired pneumonia; (2) determine the absolute neutrophil count (ANC) response to various dosages of Filgrastim in non-neutropenic patients with active infection; and (3) describe the impact of therapy with Filgrastim in combination with antibiotics on selected pneumonia-related clinical parameters. The study design was an open-label, dose-ranging, clinical trial, set in the General Clinical Research Unit of a large, public community hospital. The study population consisted of 30 patients who had presented to the Emergency Department with severe, community-acquired pneumonia. One of five dosages (75, 150, 300, 450 or 600 micrograms day-1) of Filgrastim (r-metHuG-CSF) was given subcutaneously daily for 10 days, until discharge or until the absolute neutrophil count > 75 x 10(9) l(-1), whichever was earlier. Vital signs, pulse oximetry, arterial blood gases, daily complete blood counts with differential, serum chemistries, coagulation profiles, electrocardiograms, chest radiographs, plasma G-CSF concentrations and duration of hospitalization were measured. There was no evidence of Filgrastim-related lung injury or evidence of extra-pulmonary toxicity. There was no apparent dose-response effect of Filgrastim on pneumonia-related clinical variables. Dosages of Filgrastim between 150 and 600 micrograms day-1 had similar effects on increasing the ANC. Filgrastim appeared to be safe in non-neutropenic patients with severe, community-acquired pneumonia when given in dosages of 75-600 micrograms day-1 in combination with appropriate antibiotic therapy. Further study is needed to determine the effect of Filgrastim on morbidity, mortality and duration of symptoms in this patient population.

CD4+ CD25+ transforming growth factor‐β‐producing T cells are present in the lung in murine tuberculosis and may regulate the host inflammatory response*

CD4+ CD25+ regulatory T cells produce the anti‐inflammatory cytokines transforming growth factor (TGF)‐β or interleukin (IL)‐10. Regulatory T cells have been recognized to suppress autoimmunity and promote self‐tolerance. These cells may also facilitate pathogen persistence by down‐regulating the host defence response during infection with Mycobacterium tuberculosis. We evaluated TGF‐β+ and IL‐10+ lung CD4+ CD25+ T cells in a murine model of M. tuberculosis. BALB/c mice were infected with ∼50 colony‐forming units of M. tuberculosis H37Rv intratracheally. At serial times post‐infection, lung cells were analysed for surface marker expression (CD3, CD4, CD25) and intracellular IL‐10, TGF‐β, and interferon (IFN)‐γ production (following stimulation in vitro with anti‐CD3 and anti‐CD28 antibodies). CD4+ lung lymphocytes were also selected positively after lung digestion, and stimulated in vitro for 48 h with anti‐CD3 and anti‐CD28 antibodies in the absence and presence of anti‐TGF‐β antibody, anti‐IL‐10 antibody or rmTGF‐β soluble receptor II/human Fc chimera (TGFβsrII). Supernatants were assayed for elicited IFN‐γ and IL‐2. Fluorescence activated cell sorter analyses showed that TGF‐β‐ and IL‐10‐producing CD4+ CD25+ T cells are present in the lungs of infected mice. Neutralization of TGF‐β and IL‐10 each resulted in increases in elicited IFN‐γ, with the greatest effect seen when TGFβsrII was used. Elicited IL‐2 was not affected significantly by TGF‐β neutralization. These results confirm the presence of CD4+ CD25+ TGF‐β+ T cells in murine pulmonary tuberculosis, and support the possibility that TGF‐β may contribute to down‐regulation of the host response.

Pulmonary Stress Injury Within Physiological Ranges of Airway and Vascular Pressures

PURPOSE The aim of this study was to assess the respective role of a small elevation in pulmonary capillary pressure, airway pressure, or both on alveolar capillary barrier permeability in an isolated perfused rat lung model. MATERIALS AND METHODS Four groups were studied with low or high airway pressure (LA: 10 mL/kg (tidal volume); HA: 20 mL/kg), low or high pulmonary artery pressure (LP: 9 mm Hg; HP: 12 mm Hg): LALP, HALP, LAHP, and HAHP. The lungs were ventilated and perfused ex vivo for 30 minutes. Quantification of fluorescein isothiocyanate-labeled (FITC) dextran in bronchoalveolar lavage (BAL) fluid and radiolabeled tracers assessed alveolar capillary barrier permeability. RESULTS BALF FITC-dextran was similar in the three groups with either one or two low-pressure parameters (LALP, LAHP, HALP), but high amounts were found in the HAHP group (375.2 x 10(-6) mg/mL v, respectively, 21.4, 26.2, and 30 x 10(-6) mg/mL, P = .0001). These results were consistent with the albumin space and extravascular lung water: higher values only in the HAHP group statistically different from the other groups (P < .002). Interalveolar pore examined with scanning electron microscopy showed an increase in diameters between LALP and HAHP (P < .0001). CONCLUSIONS We can conclude that elevation of either the pulmonary artery pressure from 8 to 11 mm Hg or the alveolar pressure from 10 to 15 mm Hg alone does not change the permeability of the alveolar capillary membrane; however, there is an additive effect of these pressures.