Critical Roles of Inflammation and Apoptosis in Improved Survival in a Model of Hyperoxia-Induced Acute Lung Injury in Pneumocystis murina-Infected Mice

Pneumocystis infections increase host susceptibility to additional insults that would be tolerated in the absence of infection, such as hyperoxia. In an in vivo model using CD4-depleted mice, we previously demonstrated that Pneumocystis murina pneumonia causes significant mortality following an otherwise nonlethal hyperoxic insult. Infected mice demonstrated increased pulmonary inflammation and alveolar epithelial cell apoptosis compared to controls. To test the mechanisms underlying these observations, we examined expression of components of the Fas-Fas ligand pathway in P. murina-infected mice exposed to hyperoxia. Hyperoxia alone increased expression of Fas on the surface of type II alveolar epithelial cells; conversely, infection with P. murina led to increased lung expression of Fas ligand. We hypothesized that inhibition of inflammatory responses or direct inhibition of alveolar epithelial cell apoptosis would improve survival in P. murina-infected mice exposed to hyperoxia. Mice were depleted of CD4⁺ T cells and infected with P. murina and then were exposed to >95% oxygen for 4 days, followed by return to normoxia. Experimental groups received vehicle, dexamethasone, or granulocyte-macrophage colony-stimulating factor (GM-CSF). Compared with the vehicle-treated group, treatment with dexamethasone reduced Fas ligand expression and significantly improved survival. Similarly, treatment with GM-CSF, an agent we have shown protects alveolar epithelial cells against apoptosis, decreased Fas ligand expression and also improved survival. Our results suggest that the dual stresses of P. murina infection and hyperoxia induce lung injury via activation of the Fas-Fas ligand pathway and that corticosteroids and GM-CSF reduce mortality in P. murina-infected mice exposed to hyperoxic stress by inhibition of inflammation and apoptosis.Pneumocystis jirovecii infections remain a significant cause of morbidity and mortality in immunosuppressed individuals, including individuals with human immunodeficiency virus (HIV) infection and those receiving immunosuppressive therapy (27). Despite prompt institution of appropriate antibiotic therapy, a significant number of patients experience clinical deterioration and respiratory failure after hospital admission. The pathophysiologic mechanisms leading to deterioration in some patients have not been defined. Treatment with corticosteroids has become the standard of care for moderate to severe P. jirovecii pneumonia in patients with AIDS and has been found to decrease the rate of posthospitalization progression to respiratory failure (2, 7, 13, 22). Presumably, the beneficial effects of corticosteroids are mediated by blunting of the patients'' pulmonary inflammatory responses, but the mechanisms by which this improvement occurs are not understood.In previous work, we hypothesized that a secondary stress triggers respiratory failure in patients with P. jirovecii pneumonia (5). This secondary insult likely triggers an inflammatory response that results in detrimental effects to the host. Among potential triggers (acting alone or in combination) are drug effects, coincident bacterial infections, and supplemental oxygen therapy. We reasoned that oxidant stress might be a common mechanism precipitating progression to respiratory failure. To date, prospective or retrospective analysis of oxygen therapy as an independent predictor or respiratory failure has not been possible in patients with P. jirovecii pneumonia, with or without corticosteroid therapy. Therefore, we have used an animal model to test this concept. We previously developed a model in which mice were depleted of CD4⁺ T cells, infected with P. murina, and then exposed to a limited period of hyperoxia (5). This period of hyperoxia, by itself, caused minimal lung injury and no lethality. However, we found that P. murina-infected mice exposed to hyperoxia succumbed to this dual stress, whereas either individual insult alone did not result in death. We demonstrated that mice exposed to sublethal hyperoxia did not have an increased burden of organisms compared to mice maintained in normoxia. However, the dual stress of hyperoxia and P. murina infection resulted in significantly increased apoptosis of lung cells and in exuberant inflammatory responses. Histologically, we have identified increased apoptosis in alveolar macrophages and alveolar epithelial cells, but we have not examined the cells ex vivo or investigated the specific mechanisms by which this combined stress leads to alveolar epithelial cell apoptosis.Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a product of normal alveolar epithelial cells that has important effects on pulmonary innate immunity and surfactant homeostasis. We previously demonstrated that GM-CSF is critical for host defense against P. murina (25). GM-CSF is also an antiapoptotic factor for alveolar epithelial cells (24). In mice exposed to sustained hyperoxia, overexpression of GM-CSF conferred significant protection from alveolar epithelial cell injury and mortality (24). These effects were associated with reduced alveolar epithelial cell apoptosis, an effect that was reproduced with pharmacologic treatment with recombinant murine GM-CSF.In the present experiments, we hypothesized that the combination of inflammation due to P. murina infection and hyperoxic stress results in increased vulnerability of alveolar epithelial cells to Fas-mediated apoptosis, leading to lung injury and death. We determined that the dual insults of P. murina infection and hyperoxic stress induce components of the Fas-Fas ligand apoptotic pathway and found that treatment with GM-CSF improves survival in this immunologically relevant model of Pneumocystis pneumonia. Furthermore, treatment with corticosteroids improves survival in this model, recapitulating the benefit of treatment in human disease and providing a possible explanation for the benefits humans derive from treatment with an anti-inflammatory medication in the setting of acute infection.     

Transgenic overexpression of granulocyte macrophage-colony stimulating factor in the lung prevents hyperoxic lung injury

Granulocyte macrophage-colony stimulating factor (GM-CSF) plays an important role in pulmonary homeostasis, with effects on both alveolar macrophages and alveolar epithelial cells. We hypothesized that overexpression of GM-CSF in the lung would protect mice from hyperoxic lung injury by limiting alveolar epithelial cell injury. Wild-type C57BL/6 mice and mutant mice in which GM-CSF was overexpressed in the lung under control of the SP-C promoter (SP-C-GM mice) were placed in >95% oxygen. Within 6 days, 100% of the wild-type mice had died, while 70% of the SP-C-GM mice remained alive after 10 days in hyperoxia. Histological assessment of the lungs at day 4 revealed less disruption of the alveolar wall in SP-C-GM mice compared to wild-type mice. The concentration of albumin in bronchoalveolar lavage fluid after 4 days in hyperoxia was significantly lower in SP-C-GM mice than in wild-type mice, indicating preservation of alveolar epithelial barrier properties in the SP-C-GM mice. Alveolar fluid clearance was preserved in SP-C-GM mice in hyperoxia, but decreased significantly in hyperoxia-exposed wild-type mice. Staining of lung tissue for caspase 3 demonstrated increased apoptosis in alveolar wall cells in wild-type mice in hyperoxia compared to mice in room air. In contrast, SP-C-GM mice exposed to hyperoxia demonstrated only modest increase in alveolar wall apoptosis compared to room air. Systemic treatment with GM-CSF (9 micro g/kg/day) during 4 days of hyperoxic exposure resulted in decreased apoptosis in the lungs compared to placebo. In studies using isolated murine type II alveolar epithelial cells, treatment with GM-CSF greatly reduced apoptosis in response to suspension culture. In conclusion, overexpression of GM-CSF enhances survival of mice in hyperoxia; this effect may be explained by preservation of alveolar epithelial barrier function and fluid clearance, at least in part because of reduction in hyperoxia-induced apoptosis of cells in the alveolar wall.     

Neonatal oxygen increases sensitivity to influenza A virus infection in adult mice by suppressing epithelial expression of Ear1

Oxygen exposure in premature infants is a major risk factor for bronchopulmonary dysplasia and can impair the host response to respiratory viral infections later in life. Similarly, adult mice exposed to hyperoxia as neonates display alveolar simplification associated with a reduced number of alveolar epithelial type II cells and exhibit persistent inflammation, fibrosis, and mortality when infected with influenza A virus. Because type II cells participate in innate immunity and alveolar repair, their loss may contribute to oxygen-mediated sensitivity to viral infection. A genomewide screening of type II cells identified eosinophil-associated RNase 1 (Ear1). Ear1 was also detected in airway epithelium and was reduced in lungs of mice exposed to neonatal hyperoxia. Electroporation-mediated gene delivery of Ear1 to the lung before infection successfully reduced viral replication and leukocyte recruitment during infection. It also diminished the enhanced morbidity and mortality attributed to neonatal hyperoxia. These findings demonstrate that novel epithelial expression of Ear1 functions to limit influenza A virus infection, and its loss contributes to oxygen-associated epithelial injury and fibrosis after infection. People born prematurely may have defects in epithelial innate immunity that increase their risk for respiratory viral infections.     

Latency is not an inevitable outcome of infection with Pneumocystis carinii.

Severe combined immunodeficiency (SCID) mice resolve naturally acquired Pneumocystis carinii pneumonia after reconstitution with immunocompetent spleen cells and can therefore be used as a model to study latent P. carinii infection. Neither P. carinii nor amplified P. carinii DNA was detected in the lungs of SCID mice killed 21 days after spleen cell reconstitution. Furthermore, SCID mice that recovered from P. carinii infection failed to reactivate the infection after they were either depleted of CD4+ cells for up to 84 days or depleted of CD4+ cells and treated with corticosteroid for 35 days. These results indicate that an immune response to P. carinii can completely clear the organism from the host. This supports the hypothesis that P. carinii pneumonia that develops in immunocompromised patients may be a new infection resulting from exposure to an exogenous source of P. carinii and not necessarily from reactivation of latent infection.     

Up-regulation of surfactant protein production in a mouse model of secondary pulmonary alveolar proteinosis

Although Pneumocystis infection might be one of the causes of secondary pulmonary alveolar proteinosis (PAP), the mechanism of its pathogenesis is uncertain. We analyzed a mouse model of secondary PAP resulting from Pneumocystis infection using mice deficient in CD40 (CD40KO), and evaluated the mechanism of the pathogenesis of secondary PAP from the viewpoint of surfactant-associated protein (SP) homeostasis, the overproduction of SP by type II alveolar epithelial cells, and the phagocytic function of alveolar macrophages (AMs). The effect of CD40 on SP production was also investigated in vitro using the H441 cell line, which has a phenotype similar to type II alveolar epithelial cells and primary alveolar epithelial cells. After long-term exposure to ovalbumin, CD40KO mice showed Pneumocystis infection and accumulation of surfactants in the alveoli (ApCD40KO). The amounts of SP production were up-regulated in ApCD40KO mice compared with wild-type mice treated using the same procedure. On the other hand, AMs from ApCD40KO mice did not show either phagocytic dysfunction or down-regulation of PU.1 expression. Furthermore, the stimulation of CD40-CD40 ligand (CD154) pathway regulated the production of SPs in H441 cells or primary alveolar epithelial cells. These results suggested that CD40KO mice could be one of the models useful for developing secondary PAP resulting from Pneumocystis infection. Surfactant accumulation was due to the overproduction in our model of secondary PAP. The CD40-CD154 interaction plays an important role in the regulation of surfactant-associated protein production.     

Relationship of alveolar epithelial injury and repair to the induction of pulmonary fibrosis.

Explants of mouse lung were cultured at various stages of injury after exposure to hyperoxia for determination of whether endothelial or epithelial injury alone could stimulate fibrosis in a blood-free environment. Mice were exposed to 95% O2 for periods up to 6 days. Then one lobe of lung was prepared for organ culture, and others were used for assessment of lung damage by morphologic studies and by the protein and cellular content of bronchoalveolar lavage (BAL) fluid. Explants cultured when the lung showed endothelial injury only were not different from air-exposed controls. As alveolar damage, particularly to Type 1 epithelial cells, increased at 6 days, more protein was found by lavage; and after culture, overall DNA synthesis in explants was reduced. Autoradiography showed that epithelial cell proliferation was preferentially retarded while fibroblast growth became predominant. Collagen production was also significantly increased after 3 and 6 days of culture. In these explants there were few macrophages and no white blood cells or other blood components. Some mice, returned to air after hyperoxia, showed prompt epithelial repair, and cultures of these lungs were not different from controls. The results suggest that severe injury and retarded repair of the alveolar epithelium disturbs normal epithelial-fibroblast interactions and is sufficient to promote the fibrotic process. Less severe injury involving the endothelium only is not associated with fibrosis.     

NOX1 is responsible for cell death through STAT3 activation in hyperoxia and is associated with the pathogenesis of Acute Respiratory Distress Syndrome

Reactive oxygen species (ROS) contribute to alveolar cell death in Acute Respiratory Distress Syndrome (ARDS) and we previously demonstrated that NOX1-derived ROS contributed to hyperoxia-induced alveolar cell death in mice. The study investigates whether NOX1 expression is modulated in epithelial cells concomitantly to cell death and associated to STAT3 signaling in the exudative phase of ARDS. In addition, the role of STAT3 activation in NOX1-dependent epithelial cell death was confirmed by using a lung epithelial cell line and in mice exposed to hyperoxia. NOX1 expression, cell death and STAT3 staining were evaluated in the lungs of control and ARDS patients by immunohistochemistry. In parallel, a stable NOX1-silenced murine epithelial cell line (MLE12) and NOX1-deficient mice were used to characterize signalling pathways. In the present study, we show that NOX1 is detected in alveolar epithelial cells of ARDS patients in the exudative stage. In addition, increased alveolar epithelial cell death and phosphorylated STAT3 are observed in ARDS patients and associated with NOX1 expression. Phosphorylated STAT3 is also correlated with TUNEL staining. We also confirmed that NOX1-dependent STAT3 activation participates to alveolar epithelial cell death. Silencing and acute inhibition of NOX1 in MLE12 led to decreased cell death and cleaved-caspase 3 induced by hyperoxia. Additionally, hyperoxia-induced STAT3 phosphorylation is dependent on NOX1 expression and associated with cell death in MLE12 and mice. This study demonstrates that NOX1 is involved in human ARDS pathophysiology and is responsible for the damage occurring in alveolar epithelial cells at least in part via STAT3 signalling pathways.     

Memory CD8+ T Cells Are Sufficient To Alleviate Impaired Host Resistance to Influenza A Virus Infection Caused by Neonatal Oxygen Supplementation

Supplemental oxygen administered to preterm infants is an important clinical intervention, but it is associated with life-long changes in lung development and increased sensitivity to respiratory viral infections. The precise immunological changes caused by neonatal oxygen treatment remain poorly understood. We previously reported that adult mice exposed to supplemental oxygen as neonates display persistent pulmonary inflammation and enhanced mortality after a sublethal influenza A virus infection. These changes suggest that neonatal hyperoxia impairs the cytotoxic CD8(+) T cell response required to clear the virus. In this study, we show that although host resistance to several different strains of influenza A virus is reduced by neonatal hyperoxia, this treatment does not impair viral clearance, nor does it alter the magnitude of the virus-specific CD8(+) T cell response to primary infection. Moreover, memory T cells are sufficient to ameliorate the increased morbidity and mortality and alleviate the excessive lung damage observed in mice exposed to high oxygen levels as neonates, and we attribute this sufficiency principally to virus-specific memory CD8(+) T cells. Thus, we show that neonatal hyperoxia reduces host resistance to influenza virus infection without diminishing the function of cytotoxic T lymphocytes or the generation of virus-specific memory T cells and that CD8(+) memory T cells are sufficient to provide protection from negative consequences of this important life-saving intervention. Our findings suggest that vaccines that generate robust T cell memory may be efficacious at reducing the increased sensitivity to respiratory viral infections in people born prematurely.     

Rat lung alveolar type I epithelial cell injury and response to hyperoxia.

Hyperoxia has been shown to cause extensive lung injury, which involves all components of the alveolar septum, although the type I epithelium has generally been reported to be resistant to significant injury. Electron microscopic morphometry was performed to define changes in volumes of subcellular components of alveolar epithelial cells in rats exposed to 85% O2 for 0, 7, and 14 d. Because of their large size, type I cells in control animals actually contain a greater volume of most of the organelles involved in cell metabolism than do type II cells. Hyperoxic exposure causes a dramatic change in the subcellular composition of the average type I cell, suggesting significant injury and/or response. Injury was suggested by the finding that lysosomes plus peroxisomes increased 1,250% after 7 d in hyperoxia and remained elevated by 200% after 14 d of exposure. Volumes of mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum, and Golgi apparatus increased by 100%, 51%, 91%, and 500%, respectively, after hyperoxia. Qualitative analysis showed an altered, ruffled air border with focal areas of cytoplasmic translucency (suggesting injury) and focal areas of subcellular hypertrophy. Exposure to hyperoxia was associated with more organelles being found in peripheral or attenuated portions of type I alveolar cells. Since the increase in type I organelles exceeds the volume of these organelles in its progenitor, the type II cell, it is likely that hyperoxia causes hypertrophy of the type I alveolar epithelium itself, independent of simple type II cell differentiation. Because of the large size and wide distribution of the type I cell, dramatic shifts in cell substructure caused by hyperoxia are more difficult to detect and require quantitative analysis to fully ascertain the extent of cell alterations.     

Apoptosis in neonatal murine lung exposed to hyperoxia

Exposure to high concentrations of oxygen in the neonatal period may impair lung growth and is a major contributing factor to the development of bronchopulmonary dysplasia. Cell death from hyperoxic injury may occur through either an apoptotic or nonapoptotic pathway, and we were interested in determining the type of cell death that occurs in the lung of neonatal mice exposed to hyperoxia. We found increased levels of Bax messenger RNA, a gene associated with apoptosis, in the lungs of neonatal mice born and raised in 92% hyperoxia. We next determined the extent of apoptosis taking place in the lungs of neonatal mice exposed to hyperoxia using terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling in 3.5-, 4.5-, and 5.5-d-old neonatal lung. The number of apoptotic cells in peripheral lung was significantly higher in the 3.5-, 4.5-, and 5.5-d-old mice treated with oxygen compared with that in the room-air control mice. Further, the number of apoptotic cells in the lung increased with longer exposure duration. In murine lung bronchus cells exposed to hyperoxia, growth arrest occurred after 48 h of oxygen exposure. Using annexin V binding, necrotic cell death was found to be the major form of cell death in these cells after 72 h of hyperoxic exposure. We conclude that 92% hyperoxia causes significant lung injury in neonatal mice exposed to hyperoxia, and that the number of apoptotic cells in the lung increases the longer the duration of exposure. The increase in apoptosis from hyperoxic exposure during a critical period of lung development may be an important factor in the impaired lung growth and remodeling that occur in animals exposed to high oxygen concentrations. Finally, it appears that hyperoxic injured cells in neonatal lung undergo both apoptotic and nonapoptotic cell death.     

Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis

Complications of acute respiratory distress syndrome (ARDS) are common among critically ill patients infected with highly pathogenic influenza viruses. Macrophages and neutrophils constitute the majority of cells recruited into infected lungs, and are associated with immunopathology in influenza pneumonia. We examined pathological manifestations in models of macrophage- or neutrophil-depleted mice challenged with sublethal doses of influenza A virus H1N1 strain PR8. Infected mice depleted of macrophages displayed excessive neutrophilic infiltration, alveolar damage, and increased viral load, later progressing into ARDS-like pathological signs with diffuse alveolar damage, pulmonary edema, hemorrhage, and hypoxemia. In contrast, neutrophil-depleted animals showed mild pathology in lungs. The brochoalveolar lavage fluid of infected macrophage-depleted mice exhibited elevated protein content, T1-α, thrombomodulin, matrix metalloproteinase-9, and myeloperoxidase activities indicating augmented alveolar-capillary damage, compared to neutrophil-depleted animals. We provide evidence for the formation of neutrophil extracellular traps (NETs), entangled with alveoli in areas of tissue injury, suggesting their potential link with lung damage. When co-incubated with infected alveolar epithelial cells in vitro, neutrophils from infected lungs strongly induced NETs generation, and augmented endothelial damage. NETs induction was abrogated by anti-myeloperoxidase antibody and an inhibitor of superoxide dismutase, thus implying that NETs generation is induced by redox enzymes in influenza pneumonia. These findings support the pathogenic effects of excessive neutrophils in acute lung injury of influenza pneumonia by instigating alveolar-capillary damage.     

The effect of neonatal hyperoxia on the lung of p21Waf1/Cip1/Sdi1-deficient mice

Hyperoxia is an important factor in the development of bronchopulmonary dysplasia and is associated with growth arrest and impaired alveolar septal development in the neonatal lung. p21(Waf1/Cip1/Sdi1) (p21), a cyclin-dependent kinase inhibitor, acts as a checkpoint regulator in the cell cycle during periods of stress and is induced in neonatal lung during hyperoxia exposure. To determine if p21 protects against lung injury during neonatal lung development, we placed newborn p21 knockout (p21(-/-)) and p21 wild-type (p21(+/+)) mice in 85-90% O(2) for 4 d. We found that newborn p21(-/-) mice exposed to O(2) had decreased survival in hyperoxia compared with p21(+/+) mice (P < 0.01). At 2 and 6 wk after exposure to neonatal hyperoxia, p21(-/-) O(2) lung had significantly larger alveoli then p21(-/-) control lung, as assessed by mean alveolar size and mean linear intercept. Pulmonary function tests at 6 wk demonstrated increased lung volume in both p21(-/-) and p21(+/+) O(2) mice consistent with altered lung growth from neonatal exposure to hyperoxia. Antibodies to nitrotyrosine, a marker for oxidative stress revealed that at 2 and 6 wk of age, p21(-/-) O(2) lung had significantly more oxidative stress than p21(-/-) and p21(+/+) control and p21(+/+) O(2) lung. We therefore conclude that p21 confers some additional protection to the lung during exposure to neonatal hyperoxia. Furthermore, p21 may be important during recovery from lung injury because it is associated with lower levels of oxidative stress and increased oxidative stress may contribute to alveolar growth abnormalities in the p21(-/-) O(2) lung.     

Increased synthesis and mRNA of surfactant protein A in oxygen-exposed rats

Surfactant protein A (SP-A) is an abundant glycoprotein in surfactant that is synthesized and secreted by alveolar type II cells and likely has important roles in mediating surfactant function and metabolism. In the present study, we demonstrate that exposure to 85% oxygen increased alveolar lavage and lung SP-A, and that these increases were related to increased SP-A synthesis and mRNA. Adult rats were exposed to room air or to 85% oxygen for 3, 5, or 7 days. Continuous exposure to hyperoxia progressively increased SP-A content, with a 20-fold increase in alveolar lavage and a 10-fold increase in lung SP-A content observed after 7 days. SP-A-specific mRNA increased in the lungs of rats exposed to oxygen, occurring with a time course similar to the increase in tissue SP-A. SP-A mRNA was increased 7-fold after 7 days of oxygen exposure. Synthesis of SP-A was increased 2- to 3-fold and secretion was increased 6- to 7-fold by type II epithelial cells isolated from oxygen-exposed rats. We conclude that exposure to hyperoxia increased lung and alveolar SP-A pool sizes. Increased expression of SP-A was related, at least in part, to increased SP-A mRNA and increased SP-A synthesis and secretion by type II epithelial cells.     

一氧化氮及一氧化氮合酶在急性吸入高浓度氧大鼠肺泡上皮细胞凋亡中的作用

目的: 观察一氧化氮及其合酶在急性吸入高浓度氧大鼠肺泡上皮细胞凋亡中的作用。方法: 60只大鼠随机分为空气对照组(21%O₂)和高氧实验组4 h组、8 h 组、12 h组和16 h组(85%~100%O₂),每组12只,雌雄各半。比色法测定血浆、肺组织匀浆中丙二醛(MDA)、一氧化氮(NO)和一氧化氮合酶(NOS)活性。Western blotting检测肺组织中eNOS和iNOS蛋白表达。采用TUNEL染色法检测肺泡表面凋亡细胞,HE染色观察肺组织病理改变。结果: 与对照组比较,高氧各时相组血浆及肺组织匀浆MDA、NO、NOS均升高,差异显著(P<0.01)。对照组eNOS明显表达,高氧4 h组表达开始升高,8 h组eNOS蛋白质表达升高明显。对照组iNOS蛋白微量表达,但表达量远低于eNOS,16 h组表达略增强。与对照组(2.17%±1.80%)比较,4 h组肺泡上皮细胞凋亡数量增加9.13%±3.20%,8 h组、12 h组及16 h组凋亡细胞的数量增加达17.47%±3.50%、19.22%±4.50%和11.03%±2.80%。高氧各组血浆及肺组织各指标与细胞凋亡呈明显的正相关。结论: NO及eNOS在急性高氧诱导的肺泡上皮细胞凋亡的过程中可能发挥介导作用。     

The host response of CD28-deficient mice to Pneumocystis infection

Pneumocystis infection leads to a life threatening pneumonia in susceptible individuals. While depletion or dysfunction of CD4+T cells is a key determinant of susceptibility to Pneumocystis, the host response that leads to resolution of infection or lung injury is less well understood. We had previously shown that mice deficient in the T cell costimulatory molecule CD28 are susceptible to infection with Pneumocystis. A detailed analysis revealed that they clear Pneumocystis with delayed kinetics. This is associated with an influx of naïve CD8+T cells. Depletion of CD8+T cells did not alter organism burden, suggesting these cells are not responsible for clearance. Analysis of the cytokine milieu demonstrated a consistent increase in mRNA for IL-10 and IFN-γ in the CD28-deficient mice. These data suggest that CD28 function in important in the efficiency of the host response to Pneumocystis pneumonia.     

Resistance of human regulatory Foxp3+ T cells to normobaric hyperoxia exposure under resting and stimulating conditions

Oxygen tension levels may modulate immune responses. Evidence shows that hyperoxia influences the risk of infection, autoimmunity and alloreactivity and hence is a possible therapeutic option in a number of disorders. Regulatory T cells (Tregs) play a central role in tolerance maintenance, but their behaviour under hyperoxia is largely unknown. We investigated in vitro the impact of normobaric hyperoxia on human Tregs and their cellular network. Peripheral blood mononuclear cells isolated from six healthy men were cultured under normoxia and escalating duration of normobaric hyperoxia (10 min, 1, 16, 88 h) under resting conditions and at the presence of anti-CD3/CD28 beads. Foxp3+ Tregs' and other T cell subsets' survival, proliferation, activation, maturation and Th1/Th2 markers were assessed by flow cytometry. We observed decreasing CD4+ cell survival with increasing duration of hyperoxia irrespectively of the presence of stimulators. The prevalence of CD4+ CD45RA+ cells increased under stimulation (P=0.001). In stimulated samples, the proliferation and induced Foxp3 expression decreased after 88 h of hyperoxia (both P=0.001). In conclusion, normobaric hyperoxia up to 16 h does not induce significant changes in basic human T cell subsets, including the prevalence naturally occurring Tregs. Prolonged exposure to hyperoxia likely affects all unstimulated T cell subsets in a similar way. In stimulated T lymphocytes, the proliferation is hampered and cell death increases more evidently after prolonged hyperoxia (several days). Inducible Foxp3 expression is likely closely related to these processes. Naive CD4+ T cells are maintained by stimulation during exposure to hyperoxia.     

Enhanced lung injury and delayed clearance of Pneumocystis carinii in surfactant protein A-deficient mice: attenuation of cytokine responses and reactive oxygen-nitrogen species

Surfactant protein A (SP-A), a member of the collectin family, selectively binds to Pneumocystis carinii and mediates interactions between pathogen and host alveolar macrophages in vitro. To test the hypothesis that mice lacking SP-A have delayed clearance of Pneumocystis organisms and enhanced lung injury, wild-type C57BL/6 (WT) and SP-A-deficient mice (SP-A(-/-)) with or without selective CD4(+)-T-cell depletion were intratracheally inoculated with Pneumocystis organisms. Four weeks later, CD4-depleted SP-A-deficient mice had developed a more severe Pneumocystis infection than CD4-depleted WT (P. carinii pneumonia [PCP] scores of 3 versus 2, respectively). Whereas all non-CD4-depleted WT mice were free of PCP, intact SP-A(-/-) mice also had evidence of increased organism burden. Pneumocystis infection in SP-A-deficient mice was associated histologically with enhanced peribronchial and/or perivascular cellularity (score of 4 versus 2, SP-A(-/-) versus C57BL/6 mice, respectively) and a corresponding increase in bronchoalveolar lavage (BAL) cell counts. Increases in SP-D content, gamma interferon, interleukin-4, interleukin-5, and tumor necrosis factor alpha in BAL fluid occurred but were attenuated in PCP-infected SP-A(-/-) mice compared to WT mice. There were increases in total BAL NO levels in both infected groups, but nitrite levels were higher in SP-A(-/-) mice, indicating a reduction in production of higher oxides of nitrogen that was also reflected in lower levels of 3-nitrotyrosine staining in the SP-A(-/-) group. We conclude that despite increases in inflammatory cells, SP-A-deficient mice infected with P. carinii exhibit an enhanced susceptibility to the organism and attenuated production of proinflammatory cytokines and reactive oxygen-nitrogen species. These data support the concept that SP-A is a local effector molecule in the lung host defense against P. carinii in vivo.