H. Denny Liggitt

Thymic stromal lymphopoietin as a key initiator of allergic airway inflammation in mice

The cytokine thymic stromal lymphopoietin (TSLP) has been linked to human allergic inflammatory diseases. We show here that TSLP expression was increased in the lungs of mice with antigen-induced asthma, whereas TSLP receptor–deficient mice had considerably attenuated disease. Lung-specific expression of a Tslp transgene induced airway inflammation and hyperreactivity characterized by T helper type 2 cytokines and increased immunoglobulin E. The lungs of Tslp-transgenic mice showed massive infiltration of leukocytes, goblet cell hyperplasia and subepithelial fibrosis. TSLP was capable of activating bone marrow–derived dendritic cells to upregulate costimulatory molecules and produce the T helper type 2 cell–attracting chemokine CCL17. These findings suggest that TSLP is an important factor necessary and sufficient for the initiation of allergic airway inflammation.

Cutting edge: Myeloid differentiation factor 88 is essential for pulmonary host defense against Pseudomonas aeruginosa but not Staphylococcus aureus

Myeloid differentiation factor 88 (MyD88) is an adapter molecule required for signal transduction via Toll-like receptors (TLRs) and receptors of the IL-1 family. Consequently, MyD88-deficient mice are highly susceptible to bacterial infections, including systemic infection with Staphylococcus aureus. To determine the role of MyD88 in innate immunity to bacterial pneumonia, we exposed MyD88-deficient and wild-type mice to aerosolized Pseudomonas aeruginosa or S. aureus. As predicted, MyD88-deficient mice failed to mount an early cytokine or inflammatory response or to control bacterial replication after infection with P. aeruginosa, which resulted in necrotizing pneumonia and death. By contrast, MyD88-deficient mice controlled S. aureus infection despite blunted local cytokine and inflammatory responses. Thus, whereas MyD88-dependent signaling is integral to the initiation of cytokine and inflammatory responses to both pathogens following infection of the lower respiratory tract, MyD88 is essential for innate immunity to P. aeruginosa but not S. aureus.

The mSin3A chromatin-modifying complex is essential for embryogenesis and T-cell development.

ABSTRACT The corepressor mSin3A is the core component of a chromatin-modifying complex that is recruited by multiple gene-specific transcriptional repressors. In order to understand the role of mSin3A during development, we generated constitutive germ line as well as conditional msin3A deletions. msin3A deletion in the developing mouse embryo results in lethality at the postimplantation stage, demonstrating that it is an essential gene. Blastocysts derived from preimplantation msin3A null embryos and mouse embryo fibroblasts (MEFs) lacking msin3A display a significant reduction in cell division. msin3A null MEFs also show mislocalization of the heterochromatin protein, HP1α, without alterations in global histone acetylation. Heterozygous msin3A +/− mice with a systemic twofold decrease in mSin3A protein develop splenomegaly as well as kidney disease indicative of a disruption of lymphocyte homeostasis. Conditional deletion of msin3A from developing T cells results in reduced thymic cellularity and a fivefold decrease in the number of cytotoxic (CD8) T cells, while helper (CD4) T cells are unaffected. We show that CD8 development is dependent on mSin3A at a step downstream of T-cell receptor signaling and that loss of mSin3A specifically decreases survival of double-positive and CD8 T cells. Thus, msin3A is a pleiotropic gene which, in addition to its role in cell cycle progression, is required for the development and homeostasis of cells in the lymphoid lineage.

Gene therapy for donor hearts: ex vivo liposome-mediated transfection.

OBJECTIVE Liposomes may be an appropriate transfection vehicle for transplanted hearts, avoiding the use of viruses in immunosuppressed hosts and allowing transfection of nondividing cells. To study whether liposome-mediated transfection could be accomplished during transplantation, we used a liposome-reporter gene system in a rabbit model of allograft cardiac transplantation. METHODS After aortic crossclamping, Stauffland donor hearts were injected with 10 ml Stanford cardioplegic solution; then a 1.3 to 2.0 mg/kg dose of chloramphenicol acetyl transferase in 1:1 deoxyribonucleic acid-liposome complexes was injected proximal to the aortic crossclamp for coronary artery perfusion. The hearts were transplanted into New Zealand White rabbit recipients in the heterotopic cervical position (n = 11 transplants). Recipients were sacrificed at 24 hours. Myocardial specimens (right and left ventricles) and vascular specimens (epicardial coronary artery, aortic root, and coronary sinus) from both the transplanted and native hearts were analyzed for chloramphenicol acetyl transferase protein by means of the enzymatic liquid scintillation assay (counts per minute per milligram of total protein). RESULTS In the recipient, myocardial chloramphenicol acetyl transferase activity was significantly greater in treated donor hearts (mean 4.6 x 10(5) cpm/mg +/- 1.1 x 10(5) [standard error]) than in native hearts (mean 4.1 x 10(2) cpm/mg +/- 72 [standard error], p < 0.01, Mann-Whitney U test). In treated donor hearts, right and left ventricular specimens, as well as apical and basal myocardial specimens, were transfected equally. Chloramphenicol acetyl transferase activity in vascular specimens also indicated transfection (mean 5.4 x 10(5) cpm/mg +/- 2.5 x 10(5) [standard error]). Chloramphenicol acetyl transferase activity in the coronary sinus was comparable with that in the coronary arteries, which suggests that liposomes can transverse the coronary capillary beds. CONCLUSIONS These findings demonstrate that ex vivo transfection of donor hearts with a liposome-reporter gene system results in significant in vivo expression of the transfected gene product after cardiac transplantation. Genetic alteration of myocardium and cardiac vasculature has potential clinical applications in the prevention of posttransplantation rejection, ischemia-reperfusion injury, and both transplant and nontransplant coronary artery disease.

Role of Toll-like receptor 5 in the innate immune response to acute P. aeruginosa pneumonia

Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and an important pathogen in patients with chronic lung disease, such as cystic fibrosis and bronchiectasis. The contribution of Toll-like receptor 5 (TLR5) to the innate immune response to this organism is incompletely understood. We exposed wild-type and TLR5-deficient (Tlr5(-/-)) mice to aerosolized P. aeruginosa at low and high inocula and assessed bacterial clearance, lung inflammation, and cytokine production 4 and 24 h after infection. Bacterial clearance was impaired in Tlr5(-/-) mice after low-inoculum, but not high-inoculum, infection. Early bronchoalveolar accumulation of neutrophils was reduced in Tlr5(-/-) mice after low- and high-dose infection. Cytokine responses, including markedly impaired monocyte chemoattractant protein-1 production 4 h after low- and high-inoculum challenge, were selectively altered in Tlr5(-/-) mice. In contrast, there was no impairment of bacterial clearance, neutrophil recruitment, or monocyte chemoattractant protein-1 production in Tlr5(-/-) mice after infection with a nonflagellated isotypic strain of P. aeruginosa. Thus TLR5-mediated recognition of flagellin is involved in activating pulmonary defenses against P. aeruginosa and contributes to antibacterial resistance in a manner that is partially inoculum dependent. These data are the first to demonstrate a unique role for TLR5 in the innate immune response to P. aeruginosa lung infection.

Aerosol Delivery of Lipid:DNA Complexes to Lungs of Rhesus Monkeys

AbstractPurpose. The potential use of aerosol delivery for non-viral gene therapy was tested by nebulization of lipid:DNA complexes to the lungs of rhesus monkeys. Methods. Four female rhesus monkeys were dosed with lipid:DNA formulations via aerosol inhalation, where the DNA coded for the human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR) protein. Delivery of DNA was determined in lung samples by polymerase chain reaction (PCR) by qualitative and quantitative methods. Transgene specific messenger RNA was measured by reverse transcriptase PCR (RT-PCR) and protein expression and localization were evaluated by immunohistochemistry (IHC). Results. Approximately four mg of DNA, complexed with cationic lipid 1,2-dimyristoyl-sn-glycero-3-ethylphosphatidylcholine (EDMPC) and cholesterol were delivered to the lungs of animals by air jet nebulizer. Three days after dosing, tissue samples from the lung were collected and shown to have vector specific DNA, RNA and the presence of CFTR protein. Specifically, the hCFTR protein was distributed widely, although non-uniformly, throughout airway epithelium being located on the apical surface of epithelial cells. Importantly, no adverse clinical effects were observed and the lungs showed no histological abnormalities or signs of acute inflammation. Conclusions. This study shows that lipid:DNA formulations based on EDMPC and cholesterol can be administered to primates by nebulization resulting in measurable expression of the hCFTR protein. The absence of inflammation is also encouraging and such systems may have utility for delivery of genes to the lungs for the treatment of a variety of pulmonary diseases including cystic fibrosis.

In utero exposure to diesel exhaust air pollution promotes adverse intrauterine conditions, resulting in weight gain, altered blood pressure, and increased susceptibility to heart failure in adult mice.

Exposure to fine particulate air pollution (PM2.5) is strongly associated with cardiovascular morbidity and mortality. Exposure to PM2.5 during pregnancy promotes reduced birthweight, and the associated adverse intrauterine conditions may also promote adult risk of cardiovascular disease. Here, we investigated the potential for in utero exposure to diesel exhaust (DE) air pollution, a major source of urban PM2.5, to promote adverse intrauterine conditions and influence adult susceptibility to disease. We exposed pregnant female C57Bl/6J mice to DE (≈300 µg/m3 PM2.5, 6 hrs/day, 5 days/week) from embryonic day (E) 0.5 to 17.5. At E17.5 embryos were collected for gravimetric analysis and assessed for evidence of resorption. Placental tissues underwent pathological examination to assess the extent of injury, inflammatory cell infiltration, and oxidative stress. In addition, some dams that were exposed to DE were allowed to give birth to pups and raise offspring in filtered air (FA) conditions. At 10-weeks of age, body weight and blood pressure were measured. At 12-weeks of age, cardiac function was assessed by echocardiography. Susceptibility to pressure overload-induced heart failure was then determined after transverse aortic constriction surgery. We found that in utero exposure to DE increases embryo resorption, and promotes placental hemorrhage, focal necrosis, compaction of labyrinth vascular spaces, inflammatory cell infiltration and oxidative stress. In addition, we observed that in utero DE exposure increased body weight, but counterintuitively reduced blood pressure without any changes in baseline cardiac function in adult male mice. Importantly, we observed these mice to have increased susceptibility to pressure-overload induced heart failure, suggesting this in utero exposure to DE ‘reprograms’ the heart to a heightened susceptibility to failure. These observations provide important data to suggest that developmental exposure to air pollution may strongly influence adult susceptibility to cardiovascular disease.

Inhalation of Burkholderia thailandensis results in lethal necrotizing pneumonia in mice: a surrogate model for pneumonic melioidosis

Burkholderia thailandensis is closely related to B. pseudomallei, the causative agent of melioidosis, a lethal tropical disease. Burkholderia thailandensis is largely avirulent in humans and, unlike B. pseudomallei, does not require strict biocontainment conditions for study. Because it may be a useful research surrogate for B. pseudomallei, we developed a murine model of airborne B. thailandensis infection. In both C57BL/6 and BALB/c mice, deposition of 103 colony-forming units (CFU)/lung or less of B. thailandensis was non-lethal and infection was readily controlled. Compared with C57BL/6 mice, BALB/c mice exhibited modest resistance to infection after deposition of 10(4) CFU/lung. Deposition of 10(5) CFU/lung resulted in disseminated infection and was universally fatal by 3d. This dose induced robust pulmonary neutrophilia, production of inflammatory cytokines, and elevated serum markers of distant organ injury. Histology demonstrated multiple small foci of necrotizing pneumonia but lung architecture was otherwise preserved, suggesting that respiratory failure is not the cause of death. These findings demonstrate that airborne B. thailandensis infection in mice provides an accessible surrogate model of melioidosis.

Choriodecidual Group B Streptococcal Inoculation Induces Fetal Lung Injury without Intra-Amniotic Infection and Preterm Labor in Macaca nemestrina

Background Early events leading to intrauterine infection and fetal lung injury remain poorly defined, but may hold the key to preventing neonatal and adult chronic lung disease. Our objective was to establish a nonhuman primate model of an early stage of chorioamnionitis in order to determine the time course and mechanisms of fetal lung injury in utero. Methodology/Principal Findings Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118–125 days gestation (term = 172 days) received one of two treatments: 1) choriodecidual and intra-amniotic saline (n = 5), or 2) choriodecidual inoculation of Group B Streptococcus (GBS) 1×106 colony forming units (n = 5). Cesarean section was performed regardless of labor 4 days after GBS or 7 days after saline infusion to collect fetal and placental tissues. Only two GBS animals developed early labor with no cervical change in the remaining animals. Despite uterine quiescence in most cases, blinded review found histopathological evidence of fetal lung injury in four GBS animals characterized by intra-alveolar neutrophils and interstitial thickening, which was absent in controls. Significant elevations of cytokines in amniotic fluid (TNF-α, IL-8, IL-1β, IL-6) and fetal plasma (IL-8) were detected in GBS animals and correlated with lung injury (p<0.05). Lung injury was not directly caused by GBS, because GBS was undetectable in amniotic fluid (∼10 samples tested/animal), maternal and fetal blood by culture and polymerase chain reaction. In only two cases was GBS cultured from the inoculation site in low numbers. Chorioamnionitis occurred in two GBS animals with lung injury, but two others with lung injury had normal placental histology. Conclusions/Significance A transient choriodecidual infection can induce cytokine production, which is associated with fetal lung injury without overt infection of amniotic fluid, chorioamnionitis or preterm labor. Fetal lung injury may, thus, occur silently without symptoms and before the onset of the fetal systemic inflammatory response syndrome.

Focused Ultrasound to Expel Calculi from the Kidney: Safety and Efficacy of a Clinical Prototype Device

PURPOSE Focused ultrasound has the potential to expel small stones or residual stone fragments from the kidney, or move obstructing stones to a nonobstructing location. We evaluated the efficacy and safety of ultrasonic propulsion in a live porcine model. MATERIALS AND METHODS Calcium oxalate monohydrate kidney stones and laboratory model stones (2 to 8 mm) were ureteroscopically implanted in the renal pelvicalyceal system of 12 kidneys in a total of 8 domestic swine. Transcutaneous ultrasonic propulsion was performed using an HDI C5-2 imaging transducer (ATL/Philips, Bothell, Washington) and the Verasonics® diagnostic ultrasound platform. Successful stone relocation was defined as stone movement from the calyx to the renal pelvis, ureteropelvic junction or proximal ureter. Efficacy and procedure time was determined. Three blinded experts evaluated histological injury to the kidney in the control, sham treatment and treatment arms. RESULTS All 26 stones were observed to move during treatment and 17 (65%) were relocated successfully to the renal pelvis (3), ureteropelvic junction (2) or ureter (12). Average ± SD successful procedure time was 14 ± 8 minutes and a mean of 23 ± 16 ultrasound bursts, each about 1 second in duration, were required. There was no evidence of gross or histological injury to the renal parenchyma in kidneys exposed to 20 bursts (1 second in duration at 33-second intervals) at the same output (2,400 W/cm(2)) used to push stones. CONCLUSIONS Noninvasive transcutaneous ultrasonic propulsion is a safe, effective and time efficient means to relocate calyceal stones to the renal pelvis, ureteropelvic junction or ureter. This technology holds promise as a useful adjunct to surgical management for renal calculi.